Using Saccharomyces Cerevisiae for the Biosynthesis of Tetracycline Antibiotics

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Using Saccharomyces cerevisiae for the Biosynthesis of Tetracycline Antibiotics

Ehud Herbst

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences

COLUMBIA UNIVERSITY 2019

ABSTRACT

Using Saccharomyces cerevisiae for the biosynthesis of tetracycline antibiotics

Ehud Herbst

Developing treatments for antibiotic resistant bacterial infections is among the most urgent public health challenges worldwide. Tetracyclines are one of the most important classes of antibiotics, but like other antibiotics classes, have fallen prey to antibiotic resistance. Key small changes in the tetracycline structure can lead to major and distinct pharmaceutically essential improvements.

Thus, the development of new synthetic capabilities has repeatedly been the enabling tool for powerful new tetracyclines that combatted tetracycline-resistance. Traditionally, tetracycline antibiotics were accessed through bacterial natural products or semisynthetic analogs derived from these products or their intermediates. More recently, total synthesis provided an additional route as well. Importantly however, key promising antibiotic candidates remained inaccessible through existing synthetic approaches.

Heterologous biosynthesis is tackling the production of medicinally important and structurally intriguing natural products and their unnatural analogs in tractable hosts such as Saccharomyces cerevisiae. Recently, the heterologous biosynthesis of several tetracyclines was achieved in

Streptomyces lividans through the expression of their respective biosynthetic pathways. In addition, the heterologous biosynthesis of fungal anhydrotetracyclines was shown in S. cerevisiae.

This dissertation describes the use of Saccharomyces cerevisiae towards the biosynthesis of target tetracyclines that have promising prospects as antibiotics based on the established structure- activity relationship of tetracyclines but have been previously synthetically inaccessible.

Chapter 1 provides an introduction to the pursuit of tetracycline antibiotics using S. cerevisiae.

Following an overview of tetracycline drugs, the chapter describes the methods for making

tetracyclines and their limitations in accessing the tetracycline analogs targeted in this study. The desirability of making these target analogs as well as key desired properties are then exemplified by natural products, totally synthetic and semisynthetic derivatives. The target tetracycline analogs pursued in this study are then outlined and the considerations in choosing their desired properties are discussed, as well as the reasons for employing S. cerevisiae in their synthesis.

Chapter 2 describes the use of Saccharomyces cerevisiae for the final steps of tetracycline biosynthesis, setting the stage for total biosynthesis of tetracyclines in Saccharomyces cerevisiae.

Chapter 3 describes the work towards biosynthesis of the target tetracycline analogs using

Saccharomyces cerevisiae, utilizing successful expression optimization and gene biomining approaches. Chapter 4 describes the work towards the target tetracycline analogs from fungal anhydrotetracyclines in Saccharomyces cerevisiae.

The challenge of enzyme evolution towards unnatural substrates and the complex environment of cells require metabolic engineering efforts to be performed in libraries, as it is currently impossible to predetermine which modifications will prove beneficial. Traditional methods in DNA mutagenesis and increasingly, advances in DNA synthesis, DNA assembly and genome engineering are enabling high throughput strain construction. Thus, there is a need for a general, high-throughput, versatile and readily implemented assay for the detection of target molecule biosynthesis. The development of such an assay is described in Chapter 5. The assay is demonstrated to detect tetracycline derivatives, and differentiate a producer and a nonproducer strain of the fungal anhydrotetracycline TAN-1612. The yeast three hybrid assay for metabolic engineering of tetracycline derivatives described in this chapter could be used in the next steps towards the heterologous biosynthesis of the target tetracycline analogs in S. cerevisiae and beyond.

Table of Contents

List of Figures……………………………………………………………………………………vii

List of Schemes……………………………………………………………………………………x

List of Tables……………………………………………………………………………………...xi

List of Abbreviations……………………………………………………………………………xiii

1 An introduction to tetracycline biosynthesis using Saccharomyces cerevisiae ...... 1

1.1 Chapter outline ...... 2

1.2 An antibiotic crisis ...... 2

1.3 Tetracycline antibiotics ...... 3

1.3.1 General historical context for tetracycline antibiotics and tetracycline-resistance ...... 3

1.3.2 Tetracycline analogs – small structural changes can lead to major pharmaceutical

improvements ...... 3

1.3.3 A structural overview of the FDA-approved tetracyclines ...... 4

1.4 Making tetracycline antibiotics ...... 8

1.4.1 Biosynthesis and semisynthesis of tetracyclines using the original microbial hosts .... 9

1.4.2 Total synthesis of tetracycline antibiotics ...... 12

1.4.3 Heterologous biosynthesis and semisynthesis of tetracyclines ...... 14

1.5 Tetracycline analogs in the 6-position ...... 17

1.5.1 Dactylocyclines – natural product 6-epiglycotetracyclines ...... 17

1.5.2 6-demethyl-6-deoxy-6α-aryltetracyclines – totally synthetic tetracyclines ...... 18

1.5.3 6-demethyl-6-deoxy-6α-methylenemercaptantetracyclines – semisynthetic

tetracyclines ...... 19

1.6 Design of the biosynthesis of 6-demethyl-6-epiglycotetracyclines using S. cerevisiae 21

1.6.1 Targeting 6-demethyl derivatives of 6-epiglycotetracyclines for acid stability ...... 21

1.6.2 Targeting glycoside libraries on 6-demethyl-6-epiglycotetracyclines for broad

spectrum activity...... 23

1.6.3 Targeting the 6-epi forms of 6-demethylglycotetracyclines for antibiotic efficacy ... 25

1.6.4 S. cerevisiae as the heterologous host of choice for 6-demethyl-6-epiglycotetracycline

biosynthesis ...... 26

1.7 Research progress and future goals in the biosynthesis of 6-demethyl-6-

epiglycotetracyclines using S. cerevisiae ...... 29

1.7.1 Chapter 2 – Using Saccharomyces cerevisiae for the final steps of tetracycline

biosynthesis ...... 29

1.7.2 Chapter 3 – Towards the biosynthesis of 6-demethyl-6-epitetracyclines using S.

cerevisiae ...... 30

1.7.3 Chapter 4 – Towards the biosynthesis of 6-demethyl-6-epitracyclines from TAN-1612

in Saccharomyces cerevisiae ...... 30

1.7.4 Chapter 5 – a yeast three hybrid system for the metabolic engineering of tetracycline

derivatives ...... 31

1.8 References ...... 32

2 Using Saccharomyces cerevisiae for the final steps of tetracycline biosynthesis ...... 41

2.1 Chapter outline ...... 42

2.2 Introduction ...... 42

2.3 Materials and methods ...... 43

2.3.1 General protocol for hydroxylation/reduction assay in cell lysate ...... 43

2.3.2 Protocol for hydroxylation/reduction assay in whole cells ...... 44

2.4 Results ...... 45

2.4.1 6β-hydroxylation of anhydrotetracycline in S. cerevisiae ...... 45

2.4.2 5a(11a)-dehydrotetracycline reduction in S. cerevisiae ...... 50

2.5 Discussion ...... 53

2.5.1 6β-hydroxylation of anhydrotetracycline in S. cerevisiae ...... 53

2.5.2 5a(11a)-dehydrotetracycline reduction in S. cerevisiae ...... 55

2.6 Conclusion ...... 56

2.7 Appendix ...... 58

2.8 References ...... 64

3 Towards the biosynthesis of 6-demethyl-6-epitetracyclines using S. cerevisiae ...... 70

3.1 Chapter outline ...... 71

3.2 Introduction ...... 72

3.3 Materials and methods ...... 72

3.3.1 General protocol for high throughput assay of hydroxylation/reduction in whole cells

in microtiter plates ...... 72

iii

3.3.2 General protocol for western blots ...... 73

3.4 Results ...... 74

3.4.1 Optimizing DacO1 expression in S. cerevisiae – rational design ...... 74

3.4.2 Searching the bacterial hydroxylase space for 6α-hydroxylation of

anhydrotetracyclines ...... 77

3.4.3 Developing a microtiter plate assay for anhydrotetracycline hydroxylation ...... 79

3.4.4 Evolving a 6α-hydroxylase of anhydrotetracyclines – DacO1 random mutagenesis . 80

3.4.5 Testing DacO1 expression optimization constructs and other bacterial hydroxylases 82

3.5 Discussion ...... 97

3.5.1 Optimizing DacO1 expression in S. cerevisiae – rational design ...... 97

3.5.2 Searching the bacterial hydroxylase space for 6α-hydroxylation of

anhydrotetracyclines ...... 98

3.5.3 Developing a microtiter plate assay for anhydrotetracycline hydroxylation ...... 98

3.5.4 Evolving a 6α-hydroxylase of anhydrotetracyclines – DacO1 random mutagenesis . 99

3.5.5 Testing DacO1 expression optimization constructs and other hydroxylases...... 100

3.6 Conclusion ...... 102

3.7 Appendix ...... 105

3.8 References ...... 127

4 Towards the biosynthesis of 6-demethyl-6-epitracyclines from TAN-1612 in Saccharomyces cerevisiae ...... 131

4.1 Chapter outline ...... 132

4.2 Introduction ...... 132

4.3 Materials and methods ...... 133

4.3.1 General protocol for high throughput assay of TAN-1612 hydroxylation in whole cells

in microtiter plates ...... 133

4.3.2 Protocol for selecting amino acids for mutagenesis in hypothesized binding pockets of

monooxygenases ...... 134

4.4 Results ...... 135

4.4.1 Testing PgaE and other bacterial hydroxylases for TAN-1612 hydroxylation...... 135

4.4.2 Searching the fungal hydroxylase space for 6α-hydroxylation of TAN-1612 ...... 145

4.4.3 Evolving monooxygenases for 6α-hydroxylation of anhydrotetracyclines – OxyS

example ...... 149

4.4.4 Library screening for TAN-1612 hydroxylation in S. cerevisiae ...... 152

4.5 Discussion ...... 154

4.5.1 Testing PgaE and other bacterial hydroxylases for TAN-1612 hydroxylation...... 154

4.5.2 Searching the fungal hydroxylase space for 6α-hydroxylation of TAN-1612 ...... 159

4.5.3 Evolving monooxygenases for 6α-hydroxylation of anhydrotetracyclines ...... 159

4.5.4 Library screening for TAN-1612 hydroxylation in S. cerevisiae ...... 160

4.6 Conclusion ...... 161

4.7 Appendix ...... 163

4.8 References ...... 179

5 A yeast three hybrid assay for metabolic engineering of tetracycline derivatives ...... 181

5.1 Chapter outline ...... 182

5.2 Introduction ...... 183

5.3 Materials and methods ...... 185

5.4 Results ...... 192

5.4.1 Y3H system design and CID design and synthesis ...... 192

5.4.2 Initial characterization of the Y3H assay for tetracycline derivatives ...... 194

5.4.3 Applying the Y3H assay to the detection of target molecule biosynthesis ...... 198

5.5 Discussion ...... 200

5.6 Conclusion ...... 203

5.7 Appendix ...... 204

5.7.1 Supplementary figures ...... 204

5.7.2 Supplementary methods ...... 205

5.7.3 Supplementary tables ...... 206

5.8 References ...... 211

List of Figures

Figure 1-1 Structural comparison of FDA-approved tetracyclines...... 5

Figure 1-2 Comparison of the tetracycline lipophilic and zwitterionic forms ...... 7

Figure 1-3 Key steps in the Myers convergent total synthesis of tetracyclines ...... 14

Figure 1-4 Structures of the dactylocycline family of natural products ...... 18

Figure 1-5 6-demethyl-6-deoxy-6α-methylenemercaptantetracyclines and their antibiotic activities

...... 20

Figure 1-6 Two possible C-O bond cleavage pathways in glycoside hydrolysis ...... 22

Figure 1-7 Retrosynthetic analysis of 6-demethyl-6-epiglycotetracyclines ...... 27

Figure 1-8 Structural Comparison between anhydrotetracycline, chloranhydrotetracycline, TAN-

1612 and anhydrotdactylocyclinone ...... 28

Figure 2-1 Mass spectrometry analysis of anhydrotetracycline hydroxylation in cell lysate expressing OxyS ...... 47

Figure 2-2 UV/Vis spectroscopy analysis of the reaction of anhydrotetracycline in whole cells expressing OxyS ...... 49

Figure 2-3 Mass spectrometry of anhydrotetracycline hydroxylation and reduction in cell lysate expressing OxyS in the presence of G6P ...... 52

Figure 3-1 Western Blot of DacO1 and OxyS ...... 75

Figure 3-2 A simplified outline of the microtiter plate assay for anhydrotetracycline hydroxylation.

...... 79

Figure 3-3 DacO1 error-prone mutagenesis screen excerpt ...... 81

Figure 3-4 ΔExcitation spectrum for DacO1 and DacO1 error-prone PCR mutant ...... 82

vii

Figure 3-5 Western blot analysis of DacO1 fusion proteins and other bacterial hydroxylases in

FY251 ...... 86

Figure 3-6 Western blot analysis of bacterial hydroxylases in BJ-5464-NpgA ...... 87

Figure 3-7 Western blot analysis of DacO1-DacO4 and DacO4-DacO1 fusion proteins ...... 88

Figure 3-8 Western blot analysis of DacO1 and its fusion proteins in BJ5464-NpgA ...... 90

Figure 3-9 Mass spectrometry analysis of anhydrotetracycline hydroxylation and reduction by cell lysates of strains expressing DacO1, its fusion proteins and other bacterial hydroxylases ...... 93

Figure 3-10 Mass spectrometry analysis of anhydrotetracycline hydroxylation and reduction in cell lysates of strains expressing DacO1, its fusion proteins and other bacterial hydroxylases ...... 96

Figure 3-11 Mass spectrometry analysis of anhydrotetracycline hydroxylation and reduction in cell lysates of strains expressing OxyS and PgaE ...... 97

Figure 4-1 UV/Vis analysis of hydroxylation attempt of TAN-1612 by bacterial hydroxylases 137

Figure 4-2 UV/Vis chromatogram of the HPLC separation of extracts from +/- PgaE strains encoding the TAN-1612 pathway...... 139

Figure 4-3 Mass spectrometry analysis of isolate from +/-PgaE strain encoding the TAN-1612 pathway...... 142

Figure 4-4 MSMS analysis of isolate from +PgaE strain encoding the TAN-1612 pathway..... 142

Figure 4-5 1H-NMR spectra of isolates from +/-PgaE strains encoding the TAN-1612 pathway.

...... 143

Figure 4-6 COSY NMR spectrum of isolate from +PgaE strain encoding the TAN-1612 pathway.

...... 144

Figure 4-7 Western blot analysis of fungal hydroxylase expression in BJ5464-NpgA ...... 148

viii

Figure 4-8 PyMOL illustrations of amino acids in proximity to the tetracycline substrate in aklavinone-11-Hydroxylase and in OxyS ...... 151

Figure 4-9 Library screening for TAN-1612 hydroxylation in S. cerevisiae ...... 153

Figure 4-10 Possible moieties in the product isolated from +PgaE strain coexpressing the TAN-

1612 bioysnthetic pathway ...... 155

Figure 4-11 Possible intermediates in the biosynthesis of TAN-1612 ...... 156

Figure 4-12 xanthurenic acid and its derivative that could correspond to experimental mass and

NMR spectra ...... 158

Figure 5-1 Detection of a target molecule biosynthesis by the yeast three hybrid system...... 184

Figure 5-2 Characterization of the dynamic range of the Y3H assay for tetracyclines ...... 195

Figure 5-3 The modularity of the yeast three hybrid assay enables both screening and selecting for biosynthesis of target tetracyclines ...... 197

Figure 5-4 Differentiation of a producer and a non-producer strain of a target molecule by the Y3H assay for tetracyclines, demonstrating the applicability of the Y3H assay to metabolic engineering.

...... 199

Figure 5-5 LacZ readout of strain harboring plasmid encoding for LexA-TetR (PBA-8) and strain without the plasmid (PBA-5)...... 204

Figure 5-6 standard curve for TAN-1612 quantification in cultures...... 205

List of Schemes

Scheme 2-1 Two-step enzymatic conversion of anhydrotetracycline to tetracycline ...... 46

Scheme 3-1 Bacterial monooxygenases and their hypothesized substrates and products ...... 78

Scheme 3-2 Two-step enzymatic conversion of (6-demethyl-)anhydrotetracycline to (6-demethyl-

)6-epitetracycline ...... 91

Scheme 4-1 Biosynthetic plan for TAN-1612 6α-hydroxylation ...... 135

Scheme 4-2 Bacterial monooxygenases and their hypothesized substrates and products ...... 136

Scheme 5-1 Synthesis of the chemical inducer of dimerization (CID) Min-Mtxa ...... 193

List of Tables

Table 1-1 FDA approved tetracycline derivatives and their differentiating functional groups ...... 6

Table 1-2 Rates of hydrolysis of β-D-glycopyranosides in 0.5 M aqueous sulfuric acid ...... 23

Table 2-1 Strains used in this study ...... 58

Table 2-2 Plasmids used in this study ...... 58

Table 2-3 Sequences used in this study ...... 59

Table 3-1 Potential hits in the screen of DacO1 expression optimization constructs and other bacterial hydroxylases ...... 85

Table 3-2 UV/Vis spectroscopy results for hydroxylase strains cell lysates incubated with anhydrotetracycline and glucose-6-phosphate ...... 94

Table 3-3 Assay conditions for anhydrotetracycline hydroxylation and reduction in cell lysates of strains expressing DacO1, its fusion proteins and other bacterial hydroxylases ...... 95

Table 3-4 Strains used in this study ...... 105

Table 3-5 Plasmids used in this study ...... 106

Table 3-6 Sequences used in this study ...... 107

Table 4-1 Fungal monooxygenases biomined for TAN-1612 6α-hydroxylation in S. cerevisiae

...... 146

Table 4-2 Residues chosen for mutagenesis in OxyS ...... 149

Table 4-3 Rank of PgaE positive control in screen for TAN-1612 hydroxylase ...... 152

Table 4-4 Strains used in this study ...... 163

Table 4-5 Plasmids used in this study ...... 164

Table 4-6 Plasmid Library A – Strain EH-5-217-1 ...... 165

Table 4-7 Plasmid Library B – Strain EH-5-217-3 ...... 165

Table 4-8 Plasmid Library C – Strain EH-5-217-1 and EH-5-217-3...... 165

Table 4-9 Plasmid Library D – Strains EH-5-217-2 and EH-217-4 ...... 166

Table 4-10 Sequences used in this study ...... 166

Table 4-11 Primers used for OxyS saturation mutagenesis in this study ...... 177

Table 5-1 Strains used in this study ...... 206

Table 5-2 Plasmids used in this study ...... 207

Table 5-3 Sequences of the various TetR classes in the within the pMW103 backbone...... 207

xii

List of Abbreviations

5-FOA 5-fluoroorotic acid

AD activation domain

DBD DNA binding domain

DCM dichloromethane

DHFR dihydrofolate reductase

DMF dimethylformamide

EtOAc ethyl acetate

FAD flavin adenine dinucleotide

G6P glucose-6-phosphate

HPLC high-pressure liquid chromatography

HRMS high resolution mass spectroscopy kb kilobase pair

LCMS liquid chromatography mass spectrometry

MeCN Acetonitrile

MeOH methanol

MIC minimal inhibitory concentration

MS-MS tandem mass spectrometry

NADPH dihydronicotinamide adenine dinucleotide phosphate

NMR nuclear magnetic resonance

ONPG o-nitrophenyl β-d-galactopyranoside

PAGE polyacrylamide gel electrophoresis

xiii

PR protein receptor

PKS polyketide synthase r.t. room temperature

SDS sodium dodecyl sulfate

TetR tetracycline repressor

TIC total ion count

TFA trifluoroacetic acid

Tris Tris(hydroxymethyl)aminomethane w.t. wild type

X-Gal 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside

xiv

Acknowledgments

I would like to thank my advisor Prof. Virginia Cornish for a wide cast mentorship, for providing a very fertile environment for sciencemaking and for the pushes to dare scientifically.

I would like to thank my thesis committee members, Prof. Scott Snyder, Dr. George Ellestad, Prof.

Ruben Gonzalez and Prof. Anne-Catrin Uhlemann as well as my advisory committee member

Prof. Tristan Lambert. The valuable discussions with you throughout the years have been tremendously helpful to shape this research direction. I consider myself extremely lucky to have benefited from your highly relevant expertise to this research and your will to mentor.

I have profited immensely in various ways from interacting with the highly talented and committed members, past and present, of the Cornish and neighboring labs. Specifically, I would like to thank

Arden Lee, Pedro Baldera-Aguayo and Hyunwook Lee for their large contribution to this research and for being, each in their distinctive style, a pleasure to work with. I would like to thank Dr.

Andrew Anzalone, Dr. Nili Ostrov, Dr. Casey Brown, Dr. Sonja Billerbeck, Dr. Zhixing Chen, Dr.

Caroline Patenode, Dr. Marie Harton, Dr. Bertrand Adanve, Dr. Miguel Jimenez, Dr. Andy (Yao

Zong) Ng, Dr. Mia Shandell, Sean Bannier, Dr. Anna Kaplan and Dr. Andreas Hartel for collaborations and/or mentorship and/or technical assistance.

I would like to thank Prof. Yi Tang for sharing his time and wide-ranging knowledge, as well as plasmids pYR291 and pYR342 and yeast strain BJ5464-NpgA, Prof. Alan R. Davidson for sharing pET21d plasmid encoding TetR(B), Prof. Frank Foss for sharing synthetic FO and Dr. Bertrand T.

Adanve for the synthesis of Methotrexate-α-OtBu.

I would like to thank my family for making the immense effort it takes to be close while an ocean apart. I would like to thank my friends in Israel and the US for their support and company. Finally,

I would like to thank Jess for the love and support throughout the years. It’s been quite a journey.

To my Savtas. For your perseverance.

xvi

1 An introduction to tetracycline biosynthesis using

Saccharomyces cerevisiae

1.1 Chapter outline

This chapter provides an introduction to the pursuit of tetracycline antibiotics using S. cerevisiae.

First, the significance of the production of new antibiotic analogs from a human health perspective is mentioned (Section 1.2). Then, the general historical context for developing tetracycline antibiotics is given (Section 1.3.1), the antibiotic promise in making tetracycline analogs is discussed (Section 1.3.2) and a structural overview of FDA-approved tetracyclines follows

(Section 1.3.3).

Three modes of synthesis of tetracycline antibiotic are then introduced in the historical order in which they appeared, biosynthesis and semisynthesis using the original microbial hosts (Section

1.4.1), total synthesis (Section 1.4.2) and heterologous biosynthesis and semisynthesis (Section

1.4.3). The desirability of making 6-position analogs of tetracyclines as well as key desired properties of such analogs is then exemplified by natural products (Section 1.5.1), totally synthetic

(Section 1.5.2) and semisynthetic derivatives (Section 1.5.3).

The target tetracycline analogs pursued in this study are then outlined and the considerations in choosing their desired properties are then discussed (Sections 1.6.1, 1.6.2 and 1.6.3) as well as the reasons for employing S. cerevisiae in their synthesis (Section 1.6.4). Finally, an overview of the research plan and its execution throughout this dissertation is given (Section 1.7).

1.2 An antibiotic crisis

Developing treatments for antibiotic resistant bacterial infections is among the most urgent public health challenges world-wide.(1) The US Center for Disease Control reports over 2 million illnesses and 20,000 deaths annually in the United States as a result of antibiotic resistance.(1)

Novel antibiotic drug candidates have traditionally been accessed through natural product

2 screening, total synthesis, semisynthesis and combinatorial chemistry. However, the number of new antibiotics approved by the FDA has dropped significantly from previous decades, from 29 approvals in the 1980s to 9 in the 2000s.(2, 3) To reverse this trend, transformative new technologies for discovery of new antibiotics are needed.

1.3 Tetracycline antibiotics

1.3.1 General historical context for tetracycline antibiotics and tetracycline-

resistance

Tetracyclines are one of the most important classes of antibiotics,(4) but like other classes of antibiotics, have fallen prey to resistance.(5) From their discovery in the 1940’s, tetracyclines were used as broad spectrum antibiotics, as well as for other types of disease, such as periodontitis.(6)

The tetracyclines inhibit bacterial protein synthesis by binding reversibly to the 30S ribosomal subunit and sterically hindering aminoacyl-tRNA binding to the ribosomal A-site.(7, 8) However, bacteria have evolved a growing number of resistance mechanisms – including efflux pumps,(9) ribosomal protection proteins,(10) rRNA mutations,(11) and enzymatic degradation.(12)

1.3.2 Tetracycline analogs – small structural changes can lead to major

pharmaceutical improvements

Small changes in the tetracycline structure can lead to major and distinct pharmaceutically essential improvements. Examples include tetracycline analogs differing from tetracycline in only up to three positions and showing improved pharmacokinetic properties, binding affinity to the ribosome, activity against resistant strains and non-antimicrobial properties.(13-16)

Thus, for example, a difference in the position of one hydroxy group between tetracycline and doxycycline improves the half-life, tissue penetration and matrix metalloproteinase (MMP)

3 inhibition properties of the latter (Figure 1-1 and Table 1-1).(13) Due to such advantages, doxycycline is not only one of the most commonly clinically indicated tetracycline antibiotics, it is the only clinically available MMP inhibitor as of 2010 and is indicated for the treatment of periodontitis.(14)

Minocycline differs from tetracycline in only three functional groups, a hydroxy, a methyl and an

N,N-dimethylamino (Figure 1-1 and Table 1-1). Impressively, these differences are enough to improve minocycline relative to tetracycline in its pharmacokinetic profile, radical oxygen species scavenging and other anti-inflammatory properties.(13, 14) While tetracycline is unable to cross the blood-brain barrier (BBB), minocycline is known for its neuroprotective properties that have been demonstrated pre-clinically and clinically in the treatment of various neurological disease.

Examples include ischemia, traumatic brain injury and neuropathic pain, Parkinson's disease,

Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, multiple sclerosis and spinal cord injury.(15)

Lastly, tigecycline, being identical to minocycline except for the 9-t-butyl-glycylamido group has a better binding affinity to the A-site of the ribosome and an improved antimicrobial activity against tetracycline-resistant strains (Figure 1-1 and Table 1-1).(16) On the other hand, these changes also led to tigecycline being orally unavailable and furthermore, tigecycline use is associated with an increased risk of death.(17)

1.3.3 A structural overview of the FDA-approved tetracyclines

As of 2018, all nine FDA approved tetracycline antibiotics were either bacterial natural products or semisynthetic analogs derived from bacterial natural products or their intermediates, and all nine are produced commercially partly or wholly by fermentation (Figure 1-1).(6, 18-20) Three of these nine tetracycline antibiotics, chlortetracycline, meclocycline and metacycline, were

4 discontinued.(20) In 2018, three additional tetracycline antibiotics have received their FDA approval, omadacycline, sarecycline and eravacycline.(20-22) Importantly, FDA approved tetracycline derivatives are identical in all positions but for the 5α-, 6α-, 6β-, 7- and 9-positions

(Table 1-1).

Figure 1-1 Structural comparison of FDA-approved tetracyclines The common name, a full name outlining the structural relationship between the different derivatives and the year of FDA approval, are mentioned below the structures.(6, 20, 23)

Table 1-1 FDA approved tetracycline derivatives and their differentiating functional groups Namea 5α 6α 6β 7 9 Chlortetracycline Me OH Cl Oxytetracycline OH Me OH Tetracycline Me OH Demeclocycline OH Cl Doxycycline OH Me Minocycline dimethylamino Meclocycline OH CH2 CH2 Cl Metacycline OH CH2 CH2 Tigecycline dimethylamino tBu-glycylamido Sarecycline (methoxy(methyl)amino) methyl Omadacycline dimethylamino neopentylaminomethyl Eravacycline F pyrrolidinoacetamido aFDA approved tetracycline derivatives are identical in all positions but the 5α-, 6α-, 6β-, 7- and 9-positions. Empty cells indicate implicit Hs.

The identity of the FDA approved tetracyclines at all positions but for 5α, 6α, 7, 8 and 9 could be explained as stemming from two major reasons. One is the antibacterial ineffectiveness of tetracycline analogs generated so far in positions 10, 11, 11a, 12, 12a, 1, 2, 3 and 4. The other is limited semisynthetic and total synthetic access to modifications in positions such as 4a and 6α that could potentially yield successful tetracycline antibiotics but are awaiting efficient synthetic methods to access them. Both of these topics will be expanded on below.

Given that positions 10, 12a, 1, 2 and 3, as well as the β-diketone of 11,11a and 12 of tetracyclines

(Figure 1-1) are considered to interact with the ribosome and thus to be directly responsible for tetracyclines antibiotic activity,(8, 24) it is not surprising that modifications in these positions do not yield effective antibiotics. The 2-position is somewhat of an exception as modification at this position can yield tetracycline antibiotic prodrugs that hydrolyze to the unmodified 2-carboxamido functional group (Section 1.4.1). Modifications at the 4-position have also not yielded antibiotically active tetracyclines even though this position is not considered to be important for

6 ribosome binding. The prevalent hypothesis to explain the requirement for the 4α-dimethylamino group in tetracycline antibiotics is the importance of maintaining an equilibrium between a zwitterionic and a nonionic (lipophilic) structures (Figure 1-2, Section 1.4.1).(25) This equilibrium is considered crucial for tetracycline antibiotic activity as the zwitterionic form is the form of tetracycline that binds to the ribosome,(8) and the nonionic form is the form that can cross the cytoplasmic membrane.(26) It is therefore clear that the promising positions for modifications that could yield tetracycline antibiotics are likely to be limited to positions 4a, 5, 5a, 6, 7, 8 and 9.

Figure 1-2 Comparison of the tetracycline lipophilic and zwitterionic forms Adapted from (26).

In order to explain which modifications in which positions are of promise to obtain tetracycline analogs with desired antibiotic activity but have not yet been accessed, the following two subsections of the introduction are given. The first (Section 1.4) introduces three different fronts of tetracycline synthesis to access tetracycline antibiotic analogs, biosynthesis and semisynthesis using the original microbial hosts (Section 1.4.1), total synthesis (Section 1.4.2) and heterologous biosynthesis and semisynthesis (Section 1.4.3). Specifically, Section 1.4 discusses which modifications were so far inaccessible by all existing methods. The following section introduces desirable tetracyclines that are inaccessible by biosynthesis and semisynthesis using the original microbical hosts and by total synthesis and could be conceived to made by heterologous biosynthesis (Section 1.5). Finally, the last two sections of the introduction describe the plan to

7 access these target antibiotics using S. cerevisiae (Section 1.6) and the overview of the experimental progress and future plans towards these analogs (Section 1.7).

1.4 Making tetracycline antibiotics

In this section an overview of the three main methods for the production of tetracycline antibiotics is given, biosynthesis and semisynthesis using the original microbial hosts (Section 1.4.1), total synthesis (Section 1.4.2) and heterologous biosynthesis and semisynthesis (Section 1.4.3). This overview emphasizes two main points. For one, it is shown here that synthetic advancements in the tetracycline field have time and again led to the development of FDA approved tetracyclines.

The second point emphasized in this overview is the inaccessibility of key tetracycline antibiotic analogs through both of the established methods of tetracycline analog production, biosynthesis and semisynthesis using the original microbial hosts (Section 1.4.1) and total synthesis (Section

1.4.2).

Some example for the first main point include the FDA approval of minocycline on 1971 following the mutgenesis studies that enabled the fermentation of 6-demethyltetracyclines (e.g. demeclocycline) and the followup synthetic studies that enabled aromatic substitutions in the 7- position. This synthetic achievement was in turn followed up with studies on aromatic substitions of minocycline in the 9-position that gave birth to the tigecycline that received its FDA approval on 2005 (Section 1.4.1). Similarly, a breakthrough in the younger field of tetracycline total synthesis, enabling unprecedented modifications in the tetracycline D-ring such as 7- fluorotetracyclines, gave birth to eravacycline that was FDA approved on 2018 (Section 1.4.2).

Some examples for the second main point include promising tetracycline analogs at the 6- and 4a- positions that are further discussed in Section 1.5. It remains to be seen whether unique

8 transformations enabled by the much younger field of heterologous biosynthesis and semisynthesis of tetracyclines would yield FDA approved antibiotics (Section 1.4.3).

1.4.1 Biosynthesis and semisynthesis of tetracyclines using the original

microbial hosts

Tetracycline biosynthesis and semisynthesis using the original microbial hosts products can yield modifications in positions 1, 2, 3, 4, 5, 5a, 6, 7, 9, 11, 11a, 12 and 12a.(25) The original microbial hosts referred to here include Streptomyces aureofaciens and Streptomyces rimosus, the original producers of chlortetracycline and oxytetracycline, as well as the S. aureofaciens mutants that produce tetracycline and demeclocycline.(27) Despite the limitations on the positions that can be modified and more importantly despite the limitations on the functional group that could be introduced in these positions, this strategy yielded all FDA-approved tetracyclines that are not natural products prior to 2018.(19)

Considering that positions 12a, 1, 2 and 3 and the β-diketone of 11,11a and 12 are known to be important for the interaction of tetracyclines with the ribosome,(8, 24, 25) it is not surprising that modifications in these positions have never led to a new tetracycline antibiotic drug. I will therefore provide a short overview for biosynthetic and semisynthetic modifications using the original host that led to tetracyclines modified in positions 4, 5, 5a, 6, 7, and 9. For a broader review of the semisynthesis of tetracyclines using the original microbial hosts the reader is directed to the excellent book chapter by Rogalski.(25) For more recent reviews in the field, covering tigecycline and related derivatives as well, the reader is referred to references (6, 22, 28-31).

Modifications at the 4-position have so far not presented tetracycline analogs with improved antibiotic activity and most commonly have worsened antibiotic outcomes in comparison to the unmodified counterparts. For example, 4-epitetracycline, 4-dedimethylaminotetracycline and 4-

9 hydroxy-4-dedimethylaminotetracycline all present much higher MIC values relative to tetracycline.(25) Increasing bulk on the 4-position in 6-demethyltetracycline derivatives leads to reduced antibiotic activity as well. 6-demethyltetracycline derivatives containing methylethylamino, methylpropylamino, diethylamino and methyl(2-hydroxyethyl)-amino at the

4α-position instead of the 4α-dimethylamino group of 6-demethyltetracycline show 75%, 50%,

25% and 12%, respectively, of the antibiotic activity of tetracycline against Staphylococcus aureus while 6-demethyltetracycline maintains 96% of the activity.(32)

The study the effect of reducing bulk on the 4-amino group was attempted as well by synthesizing

4-monoalkylamines, but only the synthesis of 4β-methylamines has succeeded. Considering that

4-epitetracycline has much reduced antibiotic activity it is not surprising that these derivatives were much less effective antibiotics than tetracycline as well. The one monoalkylamino successfully generated, 6-demethyl-4-dedimethylamino-4-methylaminotetracycline has shown only 7% of the antibiotic activity of tetracycline.(32)

While 6-methylene tetracycline has effective antibiotic activity (e.g., MIC value of 0.2 against tetracycline-sensitive S. aureus), its 4-trimethylammonium derivative 6-methylenetetracycline methiodide does not (e.g., MIC value of 12 against tetracycline-sensitive S. aureus).(23) While some of the difference could stem from the partial epimerization at the 4-position for the methiodide, the partial epimerization alone cannot explain the difference. The lack of the proton on the 4-dimethylammonium required to form the characteristic H-bond of the zwitterionic form

(Figure 1-2) is hypothesized to explain why 4-quaternary ammonium derivatives cannot form the zwitterionic tetracycline form,(33) and could be a reason for their higher MIC values. 4- epitetracyclines would be incapable of forming the zwitterionic form (Figure 1-2) as well and similar problems could arise with increasing bulk on the 4-amino group.

The 4a proton was unmodified by biosynthesis and semisynthesis of tetracycline derivatives using the original microbial hosts, as is to be expected given that C-H activation of the tertiary 4a-proton is challenging in a highly functionalized molecule such as tetracycline and its natural and semisynthetic analogs.(25, 34) A 4a(12a)-anhydrotetracycline was made and found to have less than 1% the activity of tetracycline.(25)

Modifications at the 5-position enabled by biosynthesis and semisynthesis using the original microbial hosts were focused on modifications of oxytetracycline and doxycycline (Figure 1-1), such as esterification, and oxidation. In general, these modifications have either yielded tetracycline derivatives with similar or worse antibiotic activity.(25) Modifications at the 5a- position were mostly limited to dehydration, isomerization and 5a-methyl installation but have not produced improvements on biological activity.

Modifications at the 6-position have yielded many of the currently FDA-approved tetracyclines

(Figure 1-1, Table 1-1). Some of the 6-position analogs made had the following functional groups in the α and β positions, respectively: Me,OH; Me,H; H,Me; Me,-; H,OH; H,H; CH2,-; RSCH2,H;

OH,-; OH, HOCH2; PhCH2,H; RSOCH2,H; CH3COCH2,H; HOCH2CH2CH2,H; PhCH2CH2,H;

F,H; H,F; F,-; F,Me; H, OAc; with R indicating and alkyl, phenyl or a derivatized alkyl or phenyl and a hyphen indicating an SP2 carbon at the 6-position.(25) Thus, it is clear that alkyl derivatizations of the 6α-position were explored, as well as some heteroatoms such as fluorine.

However, the scope was still limited and has not included 6-epi-6-demethytetracyclines, a point that will be revisited in section 1.6. This is to be expected considering that natural tetracyclines such as tetracycline, oxytetracycline and chlortetracycline give ready access to a 6β- hydroxytetracyclines. Notably, 6-demethyltetracyclines such as demeclocycline (Figure 1-1) were accessed by strain mutagenesis.(25)

Experimental success with aromatic substitutions enabled initially the semisynthetic derivatization of the 7-position generating minocycline that later received FDA approval (Figure 1-1).(6) 9- position substation followed and thus the road was paved for the newer generation of semisynthetic tetracyclines, tigecycline, sarecycline and omadacycline (Figure 1-1).(21, 22, 25, 28, 35, 36)

However, the scope of possible substitutions in the D-ring has been significantly expanded with the Myers convergent approach to tetracycline total synthesis, covered in Section 1.4.2.

Modifications at the 2-position have generated tetracycline antibiotics that were approved outside the United States. Rolitetracycline, lymecycline (N-lysinomethyltetracycline) and clomocycline are such examples. However, the modifications in general made for tetracycline prodrugs with improved water solubility that hydrolyze to tetracyclines unmodified in the 2-position, which is their active form.(25)

1.4.2 Total synthesis of tetracycline antibiotics

Early efforts at the total synthesis of tetracyclines include the first total synthesis of a biologically active tetracycline, 6-demethyl-6-deoxytetracycline by the Woodward laboratory in the 1960s, albeit as a racemic mixture.(37) Later synthetic efforts of interest to antibiotically active analog generation included generating heteroatoms at the 6-position in derivatives such as 6- thiatetracycline.(25) However, the latter was found to be acting as an antibiotic by an alternative mechanism that does not involve the ribosome and was labeled, along with anhydrotetracycline, anhydrochlortetracycline, chelocardin and 4-epianhydrochlortetracycline, as an atypical tetracycline. Importantly, adverse side effects precluded the latter group from being perused as antibiotic agents.(38)

While other total syntheses of tetracyclines appeared over the years, (39, 40) the Myers’ group convergent enantioselective total synthesis of tetracyclines,(41) was the first one to produce a

12 totally synthetic FDA approved tetracycline.(20, 42) This convergent approach to tetracycline total synthesis permitted tetracycline analog modifications previously impossible, particularly in the 7-

, 8- and 9-positions. These modifications enabled the synthesis of TP-271, a clinical candidate and the FDA approved eravacycline, both 7-dedimethylamino-7-fluoro-9-amidominocyclines (Figure

1-1).(18, 43, 44) Additional tetracycline analogs that became accessible by later developments in this total synthesis approach are 5-oxo analogs with an oxygen instead of the C5 carbon and 5a- quaternary carbon analogs.(45, 46) Importantly, the Myers convergent synthesis of tetracyclines does involve a fermentation process in its first step, the enantioselective dihydroxylation of benzoic acid.(41, 47) However, this synthesis is clearly distinct from the semisynthetic efforts towards tetracyclines that modify fully formed tetracycline fermentation products (Section 1.4.1).

On the other hand, the synthetic strategy for forming the AB-ring precursor and then coupling it with the D-ring precursor across the C-ring limits modifications to the AB-ring junction and C- ring.(41) Specifically, the Michael-Claisen cyclization that forms the ABCD tetracycline core from the AB- and D-ring precursors demands a D-ring precursor that can undergo anionization at the carbon precursor to the C6 carbon and then serve as a nucleophile for 1,4-addition reaction to an

α,β-unsaturated carbonyl (Figure 1-3-a). This demand limits the functional diversity that can be convergently generated at the 6α-position (Figure 1-1). 6α-substituents that were thus published in the Myers group include alkyl, aryl, heteroaryl, and proton substituents.(41, 42) Thus, some 6- deoxytetracyclines such as doxycycline and some 6-deoxy-6-demethyl tetracyclines such as eravacycline can be convergently synthesized using this total synthesis strategy.(41, 42, 44)

However, other simple tetracyclines, such as tetracycline itself, require an alternative cyclization strategy and further oxygenation following the cyclization, and thus while the overall yield for doxycycline was 8.3%, the overall yield for tetracycline was 1.1%.(41, 48)

Substitutions at the 4a-positions also appear limited by the convergent approach to tetracycline total synthesis, as epoxide formation and opening on this position are critical to the formation of the AB-ring intermediate (Figure 1-3-b).(41) Finally, it is also unclear at this point whether this method of tetracycline total synthesis can compete economically with fermentation, or with semisynthesis from a fermentation intermediate, for commercial process chemistry of a wide range of tetracyclines indicated for a wide range of infectious diseases.

Figure 1-3 Key steps in the Myers convergent total synthesis of tetracyclines The key step to form the (a) ABCD precursor and (b) AB-ring precursor in the total synthesis of doxycycline. Adapted from (41)

1.4.3 Heterologous biosynthesis and semisynthesis of tetracyclines

Heterologous biosynthesis of natural products and their analogs is a rapidly developing field and is importing important biosynthetic pathways into tractable hosts, such as Escherichia coli and

Saccharomyces cerevisiae. This field relies heavily on recent breakthrough in the fields of DNA synthesis, DNA sequencing, methods in genetic manipulation of model organisms and information

14 regarding natural product pathways in a variety of organisms.(49) Heterologous biosynthesis is tackling the production of medicinally important and structurally complex natural products such as taxol,(50) erythromycin,(51) and lovastatin.(52) Notably, heterologous biosynthesis offers a greener or more economically feasible approach to the production of known compounds.(53, 54)

While much of the effort in heterologous biosynthesis is still focused on the production of natural products themselves, an ultimate goal is to obtain their unnatural derivatives as well.(55)

Heterologous biosynthesis in the tetracycline field was spearheaded by the Tang laboratory who produced in Streptomyces lividans K4 by herelogous expression oxytetracycline (Figure 1-1), dactylocyclinone (Figure 1-4) and SF2575 through the expression of their respective biosynthetic pathways.(56) Analog production of the antitumor agent SF2575 was also shown in this study by the elimination of biosynthetic enzymes from the SF2575 pathway. As is the common case for the heterologous production of other glycosylated natural products,(57) the aglycone, dactylocyclinone and not the glycosylated natural products dactylocyclines has been produced.(56)

The power of genetic modifications to the biosynthetic pathway in yielding tetracycline derivatives was historically used in the original hosts. For example, in the production of tetracycline and demeclocycline by mutants of Streptomyces aureofaciens that naturally produces chlortetracycline.(58) Demeclocycline is an intermediate in the semisynthesis of the important drug minocycline and a tetracycline drug on its own right (Figure 1-1).(59) While impressive for having taken place before full deciphering of the tetracycline biosynthetic pathway and prior to the development of efficient tools for genomic engineering, these two analogs were obtained by elimination of function rather than by introduction of function, which is more challenging. The

15 genetic revolution that intervened and the use of genetically tractable heterologous organisms such as S. cerevisiae are a potential avenue towards such challenging transformations.(49)

Importantly the Tang group has also heterologously expressed in S. cerevisiae the biosynthetic pathway of the fungal anhydrotetracycline derivative, TAN-1612.(60) Fungal anhydrotetracyclines such as TAN-1612 and viridicatumtoxin serve as excellent scaffolds for generating tetracycline analogs in S. cerevisiae (Section 1.6.4 and Chapter 4). While not pursued thus far in tetracyclines, heterologous semisynthesis combining the power of heterologous biosynthesis with the often complimentary advantages of synthetic chemistry is exciting. Such combination has led to the production of artemisinin from artemisinic acid fermented in yeast,(61) and could prove especially beneficial for generating tetracycline analogs (Section 1.6.4).

In conclusion, for each of the fronts of tetracycline synthesis, biosynthesis and semisynthesis using the original microbial hosts, total synthesis and heterologous biosynthesis and semisynthesis, the emergence of new capabilities is the enabling tool for powerful new antibiotics. Thus, the development of semisynthetic capabilities in the 6-, 7- and 9-positions enabled the synthesis of novel more powerful tetracycline antibiotics such as doxycycline, minocycline and tigecycline

(Section 1.4.1). The expansion of the range of possible modifications at the 7- and 9-positions by total synthesis enabled the synthesis of 7-fluoro-9-amidotetracycline antibiotics such as eravacycline (Section 1.4.2). Importantly however, key tetracycline analogs such as 6-demethyl-

6-epitetracyclines and 4a-hydroxytetracyclines, as well as their derivatives remained inaccessible through the existing synthetic approaches (Sections 1.4.1 and 1.4.2) It is now to be tested whether the new efforts enabling the heterologous expression of tetracycline biosynthetic pathways will give rise to new tetracycline antibiotic drugs (Section 1.4.3). Section 1.5 describes exciting potential structures for such tetracyclines.

1.5 Tetracycline analogs in the 6-position

The 6-position of tetracycline has been particularly useful for generating tetracycline analogs.

Indeed, no less than five different functional group combinations in the 6-position exist for FDA- approved tetracyclines (Figure 1-1 and Table 1-1). As noted by Rogalski in 1985, “Modifications at the C6 atom have produced by far the greatest success in evolving highly active tetracyclines.”(25) Three different classes of 6-position tetracycline analogs will be outlined here as inspiration for future tetracycline antibiotics analogs in the 6-position, the natural product dactylocyclines (Section 1.5.1) the totally synthetic 6α-aryltetracyclines (Section 1.5.2) and the semisynthetic 6α-methylenemercaptantetracyclines (Section 1.5.3).

1.5.1 Dactylocyclines – natural product 6-epiglycotetracyclines

Dactylocyclines are tetracycline antibiotics isolated in the early 1990’s from Dactylosporangium sp. (ATCC 53693) in a screen by Bristol-Myers Squibb for antibiotics active against tetracycline- resistant strains.(62) Dactylocyclines are 4a-hydroxy-6-epi-6-glyco-7-chloro-8- methoxytetracyclines (Figure 1-4). While the 7-chloro substitution is known for existing tetracycline antibiotics on the market, the 4a, 6, and 8 substitutions are unique (Figure 1-1).

Dactylocyclines were found to be antibiotically active, although not of an exceptional potency, with MIC values of 3.1-6.3 μg/mL against gram-positive tetracycline-resistant strains of

Straphylococcus aureus, Staphylococcus epidermis and Streptococcus faecalis. However, dactylocyclines exhibited high MIC values (>100 μg/mL) against gram-negative tetracycline- resistant strains.(63) Importantly, the antibiotic activity of dactylocyclines against tetracycline- resistant strains is dependent on the glycoside group, with dactylocyclinone, the aglycone of dactylocycline, not presenting similar antibiotic properties.(63)

Despite their promising antibacterial activity outlook, two main disadvantages prevented dactylocyclines’ further development as antibiotics. For one, dactylocyclines are extremely acid labile. Specifically, the glycoside installed on the tertiary hydroxyl group of the 6-epitetracycline aglycone dactylocyclinone is acid labile in mildly acidic conditions.(56, 62) Secondly, the fields of synthetic biology and metabolic engineering were not yet ripe to generate dactylocycline analogs at the early 1990’s when dactylocyclines were discovered. Twenty years later, with higher maturity of these fields, the pathway towards the dactylocycline aglycone, dactylocyclinone, was successfully heterologously expressed by the Tang group.(56) Thus, today there is an outlook for generating dacylocycline analogs that build on the advantages of the natural dactylocyclines and overcome their disadvantages (Section 1.6).

Figure 1-4 Structures of the dactylocycline family of natural products Adapted from (62).

1.5.2 6-demethyl-6-deoxy-6α-aryltetracyclines – totally synthetic tetracyclines

6-demethyl-6-deoxy-6α-aryltetracyclines and 6-demethyl-6-deoxy-6α-heteroaryltetracyclines have been accessed through the Myers convergent total synthesis of tetracyclines and described in

2008.(42) Among the 6α-aryls synthesized are included, phenyl, 4-toluyl and 3,5-eifluorophenyl.

While these derivatives showed good MIC values of <1 μg/mL against gram-positive tetracycline-

18 resistant strains, they showed values of ≥8 μg/mL against gram-negative tetracycline-resistant strains.(42) However, despite promising MIC values, when one of these derivatives, 6-demethyl-

6-deoxy-6α-phenyltetracycline was tested in vivo in mice infected with tetracycline-sensitive gram-positive S. aureus, it was found to be much less effective than tetracycline, requiring a concentration at least 10 times higher to achieve the same rescuing ability.(42) A possible reason for the ineffectiveness of 6α-aryltetracyclines against gram-negative tetracycline-resistant bacteria in vitro and against tetracycline-sensitive infections in vivo could be their high expected lipophilicity, a topic explored in Section 1.5.3.

1.5.3 6-demethyl-6-deoxy-6α-methylenemercaptantetracyclines –

semisynthetic tetracyclines

6-demethyl-6-deoxy-6α-methylenemercaptantetracyclines are an example for the potential importance of the polarity of 6α-substituents on the antibacterial activity of 6α-substituted 6- demethyltetracycline analogs. 6-demethyl-6-deoxy-6α-methylenemercaptantetracyclines were synthesized from the semisynthetic 6-methylenetetracycline and 6-methyleneoxytetracycline

(metacycline, Figure 1-1).(23)

Mercaptan derivatives of 6-methylenetetracycline with lipophilic substituents such as Ph and Bn showed a much reduced activity relative to tetracycline against the gram-negative Klebsiella pneumoniae (Figure 1-5-a). However, in the case of the phenyl and benzyl derivatives of 6- methyleneoxytetracycline the reduction in activity was lower, presumably due to the mediating hydrophilicity of the 5α-hydroxy (Figure 1-5-b). Similarly, in the case of the more polar benzyl and phenyl sulfoxide derivatives of 6-methylenetetracycline the reduction in activity relative to tetracycline was lower as well (Figure 1-5-c). The smaller and more polar acetyl derivatives

19 showed better activity in both mercaptan derivatives of 6-methylenetetracycline and 6- methyleneoxytetracycline (Figure 1-5-a and Figure 1-5-b).(23)

Figure 1-5 6-demethyl-6-deoxy-6α-methylenemercaptantetracyclines and their antibiotic activities (a) 6-demethyl-6-deoxy-6α-methylenemercaptantetracyclines derived from 6- methylenetetracycline, (b) 6-demethyl-6-deoxy-6α-methylenemercaptanoxytetracyclines derived from 6-methyleneoxytetracycline (metacycline) and (c) 6-demethyl-6-deoxy-6α- methylenemercaptantetracycline oxides derived from 6-methylenetetracycline. The numeric value stated for each of the derivative is its normalized turbidimetric bioassay value (relative to tetracycline) against the gram-negative Klebsiella pneumoniae.(23, 64)

Based on these results it was hypothesized that bulky and lipophilic substituents on the 6-position reduce antibacterial activity, especially against gram-negative organisms. Importantly, however,

MIC values for the derivatives of Figure 1-5 did not follow the above mentioned trend and were generally high, with the acetyl derivatives of 6-methyleneoxytetracycline performing better than the others.(23) It is of interest to see whether this strategy of improving gram-negative efficacy by reducing lipophilicity of the 6α-substituent, namely the glycoside, could be effective in the case of

6-demethyl-6-epiglycotetracyclines (Section 1.6).

1.6 Design of the biosynthesis of 6-demethyl-6-

epiglycotetracyclines using S. cerevisiae

Given the promise in tetracycline analogs in the 6-position to make effective antibiotics (Section

1.5) and given the limitations in existing synthetic methods to access key analogs in this position

(Section 1.4), I decided to focus my efforts on 6-position analogs. Specifically, inspired by the natural products dactylocyclines (Section 1.5.1) I focused my efforts towards making acid stable broad spectrum antibiotic derivatives of dactylocyclines effective against tetracycline-resistant strains. The sections below outline the specific structural elements of the target tetracycline analogs. Namely, making 6-demethyl derivatives for acid stability (Section 1.6.1), aiming for broad spectrum activity using glycoside libraries (Section 1.6.2) and ensuring antibiotic efficacy using 6-demethyl-6-epitetracyclines as opposed to 6-demethyltetracyclines (Section 1.6.3).

Finally, I describe why S. cerevisiae was chosen as the heterologous host for making these target tetracycline analogs (Section 1.6.4).

1.6.1 Targeting 6-demethyl derivatives of 6-epiglycotetracyclines for acid

stability

To improve on the extreme acid lability of dactylocyclines, I propose making 6-demethyl-6- epiglycotetracyclines, instead of 6-epiglycotetracyclines such as dactylocyclines. Dactylocyclines show promising antibacterial activity against tetracycline-resistant strains. However, dactylocyclines’ extreme acid lability prevents their development as antibiotics (Section 1.5.1). In weak acidic conditions dactylocyclines are known to convert to their aglycone, dactylocyclinone and thereby lose their antibiotic activity against tetracycline-resistant strains.(62, 63)

The extreme acid lability of dactylocyclines is likely originating in the tertiary 6α-hydroxy to which the glycoside is bound in dactylocyclines (Figure 1-4). As Figure 1-6 shows, the acidic hydrolysis bond splitting mechanism of the glycosidic bond is hypothesized to shift based on the aglycone ability to form a stable carbonium ion. Thus, in aglycones that cannot form stable carbonium ion, the oxonium ion would form on the glycoside and in aglycones that can form a stable carbonium ion the carbonium ion would form on the aglycone. Examples for aglycones that can form a stable carbonium ion are aglycones that are bound to the glycoside through a tertiary or benzylic hydroxyl.(65)

As expected according to this hypothesis, the acid hydrolysis reaction rate is increased in the case of a tert-butyl aglycone that can form a stable tertiary carbocation relative to the case of a methyl aglycone (Table 1-2). However, the adamantyl carbocation is not similarly stabilized and nevertheless when adamantyl is the aglycone the reaction rate is higher than in the case of other substituents such as Me, Et and Pr. This incongruence led to the hypothesis that F-strain produced by bulky aglycones enhances the hydrolysis rate as well.(65) Thus, in the case of the aglycone dactylocyclinone, where the aglycone is both bulky tertiary and benzylic, it is indeed expected to undergo facile hydrolysis in low acid concentrations.

Figure 1-6 Two possible C-O bond cleavage pathways in glycoside hydrolysis Adapted from (65).

Table 1-2 Rates of hydrolysis of β-D-glycopyranosides in 0.5 M aqueous sulfuric acid Aglycone Methyl Ethyl isopropyl tButyl Benzyl Adamantyl

Hydrolysis ratea 1 1.1 1.9 555.8 1.1 48.6 ahydroylsis rate is normalized to a methyl aglycone. The rate was calculated at 60 oC at 0.5 M aqueous sulfuric acid or extrapolated or interpolated from data at different temperatures.(66, 67)

To produce 6-epiglycotetracycline analogs inspired by dactylocyclines but with a much increased acid stability, I therefore propose to produce 6-demethyl-6-epiglycotetracyclines rather than 6- epiglycotetracyclines. An example for such tetracycline derivatives would be 6- demethyldactylocyclines. However, additional modifications are likely to be required to lead to improved activity of 6-demethyl-6-epiglycotetracyclines against tetracycline-resistant gram- positive and especially gram-negative strains. Such modifications are the subject of Section 1.6.2.

1.6.2 Targeting glycoside libraries on 6-demethyl-6-epiglycotetracyclines for

broad spectrum activity

For improving broad spectrum antibiotic activity against tetracycline-resistant strains I propose installing a library of glycoside moieties on the 6-demethyl-6-epitetracycline aglycone. The extremely limited tools in synthetic biology around the time of discovery of dactylocyclines restricted exploring unnatural derivatives in this manner. However, throughout the intervening years much progress has been achieved in producing diversified glycosylated natural products by employing glycoside libraries for natural product aglycones.(68-70) Progress in this field is fostered by the natural glycoside promiscuity of many glycosyltransferase.(68)

It was recently shown that installing sterically accessible amines on antibiotics that are only effective against gram-positive strains can assist in transforming them into broad spectrum antibiotics.(71) Thus, installing a library of glycosamines on 6-demethyl-6-epitetracyclines could

23 be a promising avenue to test whether tetracyclines active against gram-positive strains alone could be transformed into broad-spectrum tetracyclines in this manner as well. Many glycosamines with a primary amine have been previously reported in the literature and could be employed in this glycodiversification effort.(69)

More specifically for tetracyclines, it was shown that reducing lipophilicity of 6α-substituents or overall lipophilicity of 6-demethyl-6-deoxy-6α-methylmarcaptantetracyclines can increase their gram-negative activity (Section 1.5.3). Importantly, dactylocyclines were noted for being unusually more lipophilic than commercially available tetracyclines.(62) Their lipophilicity could theoretically be stemming from any of the differentiating elements between dactylocyclines and commercially available tetracyclines (Figure 1-1). These include a 4a-hydroxy, a 6-epi configuration, a 7-chloro and an 8-methoxy (Section 1.5.1, Figure 1-4). Given that the 4a-hydroxy and the 8-methoxy are unlikely to make dactylocyclines more lipophilic and given the presence of the 7-chloro in commercially available tetracyclines (Table 1-1) it is likely the uniqueness in the

6-position that makes dactylocyclines more lipophilic. The 6-epi configuration of dactylocyclines is unlikely to contribute to their unusually high lipophilicity relative to commercially available tetracyclines because 6α-methyltetracyclines are generally known to be more lipophilic than 6β- methyltetracyclines (Section 1.6.3). Thus, the 6-glycoside is the likely main contributor to dactylocyclines lipophilicity, especially considering that the glycoside of dactylocyclines is rather lipophilic with a 2,6-dideoxy and a 4-methoxy, leaving it with only one or zero hydroxyls (Figure

1-4). It would therefore be of interest to test whether 6-demethyl-6-epiglycotetracyclines with a variety of less lipophilic glycosides have broad-spectrum activity against tetracycline-resistant strains.

1.6.3 Targeting the 6-epi forms of 6-demethylglycotetracyclines for antibiotic

efficacy

C6 monosubstituted tetracyclines are generally more antibiotically active when substituted at the

α-position than when substituted at the β-position (Figure 1-1).(25) For example, doxycycline (6- deoxyoxytetracycline) had lower MIC values than 6-epidoxycycline (6-epi-6- deoxyoxytetracycline) in 7 / 7 gram-positive and gram-negative strains tested in one set of experiments and its in vivo protective dose was lower as well.(23) Similar results, although less pronounced, were reported for 6-deoxytetracycline versus 6-epi-6-deoxytetracycline,(23) and 6α- fluoro-6-demethyl-6-deoxytetracycline was also shown to be more effective than its 6-epimer, 6β- fluoro-6-demethyl-6-deoxytetracycline.

A possible explanation to the advantage 6α-tetracycline analogs have over their 6β counterparts is that 6α-analogs more efficiently maintain the important balance between the zwitterionic and the lipophilic forms of tetracyclines (Section 1.3.3 and Section 1.4.1). This explanation is supported by the following known trend in lipophilicity, with lipophilicity levels ranking oxytetraycline < 6- epi-6-deoxyoxytetracycline < 6-deoxyoxytetracycline, and with 6-epi-6-deoxyoxytetracycline showing only a modest increase in lipophilicity relative to oxytetracycline. While the increase in lipophilcity between oxytetracycline and its two 6-deoxy derivatives is expected based on a loss of a hydrophilic hydroxy group, the increase in lipophilicity between the two deoxyoxytetracycline epimers is less immediately reasoned. The commonly proposed reasoning is that a 6β-functional group leads to steric clashes with the 4α-dimethylamino and thus destabilizes the lipophilic conformation of the tetracycline, making the tetracycline derivative less lipophilic as a whole. In contrast, an 6α-substiuent does not disrupt the important balance between the lipophilic and zwitterionic forms of tetracycline derivatives.(72)

While these difference in lipophilicity and antibiotic activity are noted for 6α- and 6β-methyl groups, they are all the more relevant when a much bulkier glycoside is installed on the 6-hydroxyl.

It is thus perhaps not surprising that dactylocyclines, the only known 6-glycotetrayclines natural products, are 6-epitetracyclines while other tetracycline natural products such as oxytetracycline and chlortetracyclines are not. Thus, 6-demethyl-6-epiglycotetracyclines are proposed in this study and not 6-demethyl-6-glycotetracyclines. This point is of special importance given the higher accessibility of 6β-hydroxytetracyclines over their 6α-hydroxytetracycline counterparts in all methods of tetracycline preparation (Section 1.4.1 and Section 1.4.2 and Chapter 3).

1.6.4 S. cerevisiae as the heterologous host of choice for 6-demethyl-6-

epiglycotetracycline biosynthesis

Given that 6-demethyl-6-epiglycotetracyclines were marked here as highly desirable tetracycline targets to synthesize and test as antibiotics (Sections 1.6.1, 1.6.2 and 1.6.3), the question arises what would be the appropriate synthetic tool. Section 1.4.1 mentions the limitations in accessing

6-demethyl-6-epiglycotetracyclines by semisynthesis from the mainstream tetracycline fermentation products, such as tetracycline, oxytetracycline, chlortetracycline and demeclocycline, that are all 6β-hydroxy. Similarly, Section 1.4.2 mentions the difficulty in accessing 6-demethyl-

6-epitetracyclines by total synthesis due to the inherent limitations in the synthetic approach that requires constructing the C-ring from AB- and D-ring precursors. Recent advances in metabolic engineering and synthetic biology render it especially exciting to test heterologous biosynthesis for generating 6-demethyl-6-epitetracyclines, the precursors to the desired 6-demethyl-6- epiglycotetracyclines. Importantly, the following glycodiversification step can take place either in vivo or by isolating 6-demethyl-6-epitetracyclines and appending activated glycosides on them using glycosyltransferases in vitro or through chemical synthesis (Figure 1-7).

Figure 1-7 Retrosynthetic analysis of 6-demethyl-6-epiglycotetracyclines

Saccharomyces cerevisiae is uniquely positioned to the synthesis of 6-demethyl-6- epiglycotetracyclines. First, as a highly genetically tractable model organism that is easy to culture and quick to reproduce, S. cerevisiae has been demonstrated a useful heterologous host for the production of a variety of natural products of importance to human health.(61, 73-75) The advantages, sometimes unexpected, of performing chemistry in S. cerevisiae are also exemplified in this study (e.g., Section 2.4.2).

Nevertheless, 6-demethyl-6-epiglycotetracyclines or 6-demethyl-6-epitetracyclines could also theoretically be accessed from the native host Dactylosporangium sp. (ATCC 53693) or from an alternative heterologous host, such as S. lividans K4 previously used for dactylocyclinone biosynthesis.(56, 63) However, the native host Dactylosporangium sp. (ATCC 53693) is reported to grow at an exceedingly slow rate and to be completely inaccessible for genetic modifications.(56) It is yet to be determined whether an alternative heterologous host such as S. lividans K4 has better or worse outcomes than S. cerevisiae for this biosynthesis goal.

Importantly, the Tang laboratory has reported the heterologous biosynthesis of the fungal anhydrotetracycline TAN-1612 in S. cerevisiae (Section 1.4.3) and TAN-1612 is well positioned to serve as a scaffold for the production of 6-demethyl-6-epiglycotetracyclines in S. cerevisiae.

TAN-1612 holds important structural similarities to anhydrodactylocyclinone, such as the 4a- hydroxy and the 8-methoxy functional groups, that can render it a good starting point for further derivatization. These substitutions are not observed in the anhydrotetracycline precursors of the

27 commonly fermented tetracyclines, such as chloranhydrotetracycline and anhydrotetracycline.

Also favorably to the target molecules outlined (Figure 1-7), TAN-1612 lacks a 6-methyl group

(Figure 1-8).

Figure 1-8 Structural Comparison between anhydrotetracycline, chloranhydrotetracycline, TAN-1612 and anhydrotdactylocyclinone In grey are marked potentially desirable functional groups TAN-1612 has relative to the commercially available anhydrotetracycline as a scaffold for 6-demethyl-6-epiglycotetracycline biosynthesis. The stereochemistry of TAN-1612 at positions 4a and 12a was not yet confirmed by X-ray crystallography. Empty cells indicate implicit Hs.

However, TAN-1612 is also lacking structural features that are important for antibiotic activity in tetracyclines in the A-ring such as the 4α-dimethylamino and the 2-carboxamido (Figure 1-8). The furnishing of a 2-carboxamido derivative of TAN-1612 or a related molecule in S. cerevisiae could be envisioned by heterologously expressing the relevant genes from the oxytetracycline biosynthetic pathway of S. rimosus. An alternative pathway to harvest the relevant genes is the biosynthetic pathway towards the fungal polyketide viridicatumtoxin, which is homologous to that of TAN-1612. This effort would likely require evolving the TAN-1612 biosynthetic enzymes to the unnatural substrate.(76, 77)

The Yeast three hybrid (Y3H) system for metabolic engineering of tetracycline analogs could be crucial to assay mutants of the TAN-1612 pathway enzymes for relaxed substrate specificity towards the new 2-carboxamido intermediates (Section 1.7.4 and Chapter 5). Likewise, hydroxylation and oxidation of the 4-position of TAN-1612 can be envisioned by OxyE and OxyL

28 from the oxytetracycline pathway (78) evolved for TAN-1612 specificity by employing the Y3H as well. These steps could be followed by reductive amination to furnish the 4α-dimethylamino or a related functional group on TAN-1612 or a related derivative. Finally, fermentation of 6- demethyl-6-epiglycotetracyclines or 6-demethyl-6-epitetracyclines in S. cerevisiae could readily open up possibilities in the longer term for commercial production of new tetracycline therapeutics.

1.7 Research progress and future goals in the biosynthesis of

6-demethyl-6-epiglycotetracyclines using S. cerevisiae

1.7.1 Chapter 2 – Using Saccharomyces cerevisiae for the final steps of

tetracycline biosynthesis

This chapter describes the use of Saccharomyces cerevisiae for the final steps of tetracycline biosynthesis, setting the stage for total biosynthesis of tetracycline in S. cerevisiae. Specifically, data presented in this chapter supports the conversion of anydrotetracycline to tetracycline or an epimer in S. cerevisiae by heterologous expression of the 6β-hydroxylase enzyme form the oxytetracycline pathway, OxyS. This result is surprising as it was expected that the expression of a reductase enzyme that uses a cofactor not native to yeast would be required as well. It was also expected that OxyS would lead to an oxytetracycline intermediate and not to tetracycline by performing two hydroxylation steps as reported previously and not one as shown in this study. The ability to perform the required steps for tetracycline biosynthesis from anhydrotetracycline supports the ongoing efforts to produce 6-demethyl-6-epitetracyclines from anhydrotetracyclines

(Figure 1-7, Chapter 3 and Chapter 4).

1.7.2 Chapter 3 – Towards the biosynthesis of 6-demethyl-6-epitetracyclines

using S. cerevisiae

This chapter describes the work towards 6-demethyl-6-epitetracyclines biosynthesis in S. cerevisiae. 6α-hydroxylation of a 6-demethylanhydrotetracycline by an enzyme such as DacO1 is required for the synthesis of 6-demethyl-6-epitetracyclines as opposed to 6β-hydroxylation by enzymes such as OxyS leading to 6-demethyltetracyclines. However, previous attempts using multiple heterologous hosts to heterologously express DacO1 without the rest of the dactylocyclinone pathway have not yielded stably expressed protein. The chapter describes the successful development of a stably expressing form of DacO1 in S. cerevisiae by employing rational design approaches for optimizing DacO1 expression. This chapter also describes the successful hydroxylation of anhydrotetracycline by another 6α-hydroxylase candidate, PgaE. The next step towards 6-demethyl-6-epitetracyclines is to test the stably expressed DacO1 and PgaE in larger scale for 6α-hydroxylation of anhydrotetracycline and/or anhydrodactylocyclinone and/or

6-demethylanhydrodactylocyclinone.

1.7.3 Chapter 4 – Towards the biosynthesis of 6-demethyl-6-epitracyclines

from TAN-1612 in Saccharomyces cerevisiae

This chapter describes the work towards 6-demethyl-6-epitracycline derivatives of the fungal anhydrotetracycline TAN-1612 in Saccharomyces cerevisiae. TAN-1612 is indicated as a unique anhydrotetracycline scaffold for the production of 6-demethyl-6-epitetracyclines (Section 1.6.4), the precursors to the target molecules 6-demethyl-6-epiglycotetracyclines (Figure 1-7). The progress and future steps towards the directed evolution and biomining of a TAN-1612 6α- hydroxylase are outlined and the isolation and characterization of a new major product in a strain coexpressing PgaE and the TAN-1612 pathway is described.

1.7.4 Chapter 5 – a yeast three hybrid system for the metabolic engineering of

tetracycline derivatives

The transformation of anhydrotetracyline to tetracycline could be readily detected by a UV/Vis based assay due to the blue-shift in the excitation and emission of the molecule (Chapters 2-4).

However, most transformations of tetracyclines and other molecules that do not involve such a major UV/Vis shift, such as the proposed glycosylation and A-ring modifications (Section 1.6.4) require the development of a general assay for their detection. This chapter describes the development of a yeast three hybrid (Y3H) assay as such a general, high-throughput, versatile and readily implemented approach for the detection of target molecule biosynthesis. The Y3H assay is presented here as a tool for the reactions in the next steps towards the heterologous biosynthesis of 6-demethyl-6-epiglycotetracyclines in S. cerevisiae and beyond.

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2 Using Saccharomyces cerevisiae for the final steps of

tetracycline biosynthesis

*The contents of this chapter will be published in:

E. Herbst, V. W. Cornish, et al “Using Saccharomyces cerevisiae for the final steps of tetracycline biosynthesis”, in preparation.

2.1 Chapter outline

This chapter describes the use of Saccharomyces cerevisiae for the final steps of tetracycline biosynthesis, setting the stage for total biosynthesis of tetracycline in S. cerevisiae. Key results described in this chapter are that OxyS performs in S. cerevisiae just one hydroxylation step as opposed to two performed in vitro, thus enabling the biosynthesis of tetracycline instead of oxytetracycline (Section 2.4.1 and 2.5.1). In addition, this chapter describes the reduction of the hydroxylation product of OxyS in yeast cell lysate, without the need for heterologous expression of a dedicated reduction enzyme, such as OxyR (Section 2.4.2). This result is important as OxyR uses a cofactor not native to yeast. Thus, circumventing the need to express OxyR also circumvents the need to heterologously express the biosynthetic pathway of the cofactor or to extragenously supply it or its equivalent (Sections 2.4.2 and 2.5.2). Finally, this chapter describes mass spectrometry and UV/Vis results supporting the production of 5a(11a)-dehydrotetracycline using

S. cerevsiae even though its further hydroxylation and/or degradation hampered previous analysis when produced by OxyS in vitro and in vivo (Sections 2.4.2 and 2.5.2).

2.2 Introduction

S. cerevisiae has proven itself a useful heterologous host for the production of a variety of natural products of importance to human health. A key example is the production of artemisinic acid, a key intermediate in the production of artemisinin,(1) with more recent examples being the production of hydrocodone, Δ9-tetrahydrocannabinolic acid (THCA) and penicillin.(2-4) One of the promises in natural product biosynthesis using S. cerevisiae is the access to unnatural analogs of natural products.(3) Tetracyclines are one of the most important classes of antibiotics and tetracycline analogs are needed to combat the resistance to current tetracycline antibiotics on the

42 market given the current and impending antibiotic crisis (Chapter 1). Thus, from an applied science perspective, research on heterologous biosynthesis of tetracyclines is important for its potential to lead to new and effective tetracycline antibiotics. From a pure science perspective, heterologous biosynthesis of is often highly instructive for the study of the biosynthetic steps that take place in the natural hosts, as is exemplified throughout this chapter.

Granted, Heterologous biosynthesis of oxytetracycline was already shown in Streptomyces lividans K4‐11. This is a more readily achievable feat given the existence in that host of a biosynthetic pathway to cofactor F420, a required cofactor in the biosynthesis of tetracyclines that is not native to S. cerevisiae, and the capacity of bacterial heterologous hosts to express whole gene clusters from other bacteria.(5) However, its eukaryotic family lineage is also a key advantage for S. cerevisiae as a heterologous host to bacterial natural products such as tetracycline.

Expression in S. cerevisiae allows orthogonal and complementary chemistry to be performed, giving access to analogs that are otherwise inaccessible in heterologous expression in bacterial hosts.(6) Examples for the rich, useful, and sometimes unexpected chemical possibilities of S. cerevisiae in the heterologous biosynthesis of bacterial natural products such as tetracycline are demonstrated in this chapter (2.4.2 and 2.5.2).

2.3 Materials and methods

See Chapter 5 for general methods.

2.3.1 General protocol for hydroxylation/reduction assay in cell lysate

Fresh patches of strains harboring the plasmid for the hydroxylation and/or reduction enzyme and control strains were inoculated in 5 mL selective media (U- or HU-) in 15 mL culture tubes

(Corning 352059) and placed in shaker overnight to OD600 2-3. Overnight cultures were used to inoculate 100 mL selective media (U- or HU-) cultures in 500 mL conical flasks with a starting OD

43 of 0.01-0.05. Cells were grown to final OD of 0.6-0.8 before pelleting in 2 50 mL tubes (Corning

352098) at 4 °C, 4,000 rpm for 20 min. Each pellet was redissolved in 0.5 mL H2O and the suspension was distributed into two presterilized 1.5 mL Eppendorf tubes and pelleted at 14,000 rpm for 10 min at 4 °C. Pellets were stored at -20 °C prior to further use.

Pellets were weighed and thawn on ice. A 99:1 mixture of Y-PER yeast protein extraction reagent

(ThermoFisher Scientific 78991) and HALT protease inhibitor cocktail (ThermoFisher Scientific

PI87786) was added in a ratio of 3 μL mixture per mg pellet and placed on orbital shaker for 20 min at r.t., followed by 10 min centrifugation at 14,000 rpm at 4 °C and the cell lysate was transferred to a new 1.5 mL Eppendorf tube, kept on ice and used within 1 h.

The cell lysate (80 μL) was added as the last component to a 4 mL vial (Chemglass CG-4900-01) containing 280 μL of 143 mM Tris (pH 7.45), 7.7 mM anhydrotetracycline HCl (AdipoGen CDX-

A0197-M500), 4.3 mM NADPH tetrasodium hydrate (Sigma-Aldrich N7505), 26.4 mM mercaptoehtanol and 14.285 mM glucose-6-phosphate (in experiments labeled +G6P, 0 mM glucose-6-phophsate in all other experiments) and 40 μL glucose (278 mM) for final concentrations of 100 mM Tris, 5.4 mM anhydrotetracycline HCl, 3 mM NADPH, 18.5 mM mercaptoethanol, 0 or 10 mM G6P as indicated and 27.8 mM glucose. A septum was placed on top of the vial through which a needle was inserted to allow air exchange and the reaction was left at r.t. overnight. After night, 1 mL of MeOH was added, the contents were mixed and the reaction was filtered through a PTFE 0.2 μm filter (Acrodisc 4423) prior to analysis by mass and UV/VIS spectrometry.

2.3.2 Protocol for hydroxylation/reduction assay in whole cells

Fresh patches of strains harboring the plasmid for the hydroxylation and/or reduction enzyme and control strains were inoculated in 5 mL selective media (U- or HU-) in 15 mL culture tubes

(Corning 352059) and placed in shaker overnight to OD600 2-3. Overnight cultures were used to inoculate 100 mL selective media (U- or HU-) cultures in 500 mL conical flasks with a starting OD of 0.08. Cells were grown to an OD of 1.3 before placing at 15 °C for an additional 10 h until a final OD of 1.65. Cells were then pelleted in 2 50 mL tubes (Corning 352098) at 10 °C, 3,500 rpm for 5 min. Each pellet was redissolved in 0.5 mL H2O and the suspension was distributed into a presterilized 1.5 mL Eppendorf tube and pelleted at 11,000 rpm for 3 min at 10 °C. Pellets were placed on ice and used within 1 h.

Pellets from 50 mL culture were redissolved in H2O (1,025 μL) and added as the last component to 15 mL culture tubes (Corning 352059) containing 1,100 μL of 8 mg/mL anhydrotetracycline

HCl, 125 μL glucose solution in H2O (40%) and 250 μL 1 M Tris buffer pH 7.45 and were placed in shaker at 350 rpm at 21 °C for 27 h. Cultures were then pelleted and the supernatant was diluted

10X into H2O before being used for UV/VIS spectral measurements.

2.4 Results

2.4.1 6β-hydroxylation of anhydrotetracycline in S. cerevisiae

The first step required to convert anhydrotetracyclines to tetracyclines is the 6-hydroxylation of the anhydrotetracyclines. This step was studied in the oxytetracycline biosynthetic pathway and it was shown that anhydrotetracycline 6-hydroxylation is catalyzed by OxyS. A similar step is hypothesized to be catalyzed on 7-chloroanhydrotetracycline by the homologous enzyme Cts8 of the chlortetracycline pathway.(7) As tetracycline is biosynthesized in 7-chlorotetracycline producers with reduced chlorination ability,(8) anhydrotetracycline hydroxylation is likely to be the first in the final sequence of steps in tetracycline biosynthesis.

To test the capacity of Saccharomyces cerevisiae to hydroxylate an anhydrotetracycline, the model hydroxylating enzyme OxyS was used together with its native substrate, anhydrotetracycline

(Scheme 2-1). This enzyme-substrate pair was chosen as OxyS was shown to functionally express in Escherichia coli by the Tang laboratory and anhydrotetracycline is commercially available.(7)

The expression plasmid pSP-G1 was chosen in order to facilitate the coexpression of a reductase enzyme, to append antibody tags to both hydroxylase and reductase and to constitutively express both enzymes.(9) OxyS was cloned into pSP-G1 under the transcriptional control of the strong constitutive promoter TEF1 with a FLAG antibody tag at its C-terminus. The resulting plasmid

(AL-1-101) was transformed into FY251 and BJ5464-NpgA to generate strains EH-3-98-6 and

EH-3-248-1, respectively.

Scheme 2-1 Two-step enzymatic conversion of anhydrotetracycline to tetracycline

The OxyS-catalyzed reaction to convert anhydrotetracycline to 5a(11a)-dehydrotetracycline in S. cerevisiae is supported by mass spectrometry. When a cell lysate expressing OxyS is added to anhydrotetracycline the molecular ion corresponding to 5a(11a)-dehydrotetracycline ([M+H]+) has much higher ion counts compared to the molecular ion corresponding to anhydrotetracycline

([M+H]+). However, the opposite is the case when a cell lysate not expressing OxyS is added to anhydrotetracycline (Figure 2-1).

Figure 2-1 Mass spectrometry analysis of anhydrotetracycline hydroxylation in cell lysate expressing OxyS Cell lysate of OxyS expressing strain EH-3-248-1 (+OxyS) or a no-hydroxylase control EH-3- 248-8 (-OxyS) was placed overnight in Tris buffer (100 mM, pH 7.45) containing

47 anhydrotetracycline (5.4 mM), glucose (27.8 mM), NADPH (3 mM) and mercaptoethanol (18.5 mM). After night, MeOH was added, the contents were mixed and the reaction was filtered prior to MS analysis.

Following this encouraging result with a cell lysate, the ability of OxyS to hydroxylate anhydrotetracycline was tested in whole cells as well. As for the cell lysate, the molecular ion corresponding to 5a(11a)-dehydrotetracycline had much higher ion counts in the +OxyS sample relative to the -OxyS sample (data not shown). Given that anhydrotetracycline absorbs and fluoresces in the visible range, it was logical to test whether the reaction could be monitored using a simple UV/Vis assay using a spectrophotometer. Indeed, reducing the -OxyS spectrum from the

+OxyS spectrum shows a reduction in the 440 nm absorption and 570 nm emission peaks corresponding to anhydrotetracycline and a formation of 380 nm absorption and 500 nm emission peaks (Figure 2-2).

The blue shift in absorption and emission is supportive for the formation of 5a(11a)- dehydrotetracycline from anhydrotetracycline as anhydrotetracycline’s conjugation in the CD- rings is expected to be significantly reduced by 6-hydroxylation (Scheme 2-1). Indeed, the corresponding red shift in the absorption spectrum for the opposite transformation, where chlortetracycline is converted to anhydrochlortetracycline was previously reported (λmax = 373 nm to 438 nm, respectively).(10)

Figure 2-2 UV/Vis spectroscopy analysis of the reaction of anhydrotetracycline in whole cells expressing OxyS The spectra shown are Δ spectra, that is, the absorption/emission values for the OxyS expressing cells EH-98-6 minus the absorption/emission values of the no hydroxylase control EH-3-80-3 are shown.

2.4.2 5a(11a)-dehydrotetracycline reduction in S. cerevisiae

The second step in the synthesis of tetracycline from anhydrotetracycline is to reduce the 5a(11a)

α,β-unsaturated double bond (Scheme 2-1). The reduction at the 5a(11a) bond is known to be key to tetracyclines’ antibiotic activity, with 7-chlorotetracycline being over 20X more potent than 7- chloro-5a(11a)-dehydrotetracycline against Staphylococcus aureus.(11) In order to execute the reduction step OxyR, the reductase of 5a(11a)-dehydrooxytetracycline,(7) was placed under the control of a PGK1 promoter in the pSP-G1-OxyS plasmid AL-1-101 to generate AL-1-119-A-C7.

To function properly, OxyR requires cofactor F420, a cofactor that is not native to S. cerevisiae.

Cofactor Fo is an intermediate in cofactor F420 biosynthesis and is known to successfully replace cofactor F420 in several F420 dependent enzymes with similar Km and Kcat values.(12-15) Given that

Fo synthases, employing known yeast metabolites to biosynthesize Fo were previously characterized, an attempt was made to heterologousy produce Fo in S. cerevisiae. Fo synthase from

Thermobifida fusca and Chlamydomonas reinhardtii, was placed under the control of promoter pADH1 to generate plasmids AL-1-183-C and AL-1-183-D, respectively.(16, 17) However, following transformations of the plasmids to S. cerevisiae no Fo biosynthesis was confirmed by

MS (data not shown).

In order to test whether OxyR could be functional in S. cerevisiae with Fo as a cofactor, synthetic

Fo,(18) a generous gift from Frank Foss, was exogenously added to a yeast strain expressing OxyS,

OxyR and an Fo reductase. Fo reductase was placed under the control of pGPD (pTDH3) on PRS413 to generate AL-215-D-C1, AL-255-C1 and AL-235-C5, encoding F420 reductase from Mycobacterium tuberculosis, Archeoglobus fulgidus and Streptomyces griseus, respectively.(19-22) The reaction mixture in Tris buffer (pH 7.45) also contained anhydrotetracycline, glucose, NADPH, and in the

50 case of Fo reductase from M. tuberculosis, glucose-6-phosphate for Fo reduction. It was found that the levels of the molecular ion peak corresponding to tetracycline, the reduction product of

5a(11a)-dehydrotetracycline, are increased when glucose-6-phosphate is added (Figure 2-3).

Contrary to the expectation that glucose-6-phosphate serves as a substrate for the Fo reductase, it was found that neither Fo, the Fo reductase or OxyR increased the levels of the molecular ion peak corresponding to tetracycline either in the presence or absence of glucose-6-phosphate (data not shown).

Figure 2-3 Mass spectrometry of anhydrotetracycline hydroxylation and reduction in cell lysate expressing OxyS in the presence of G6P Cell lysate of OxyS expressing strain EH-3-248-1 was placed overnight in Tris buffer (100 mM, pH 7.45) containing anhydrotetracycline (5.4 mM), glucose (27.8 mM), NADPH (3 mM), 10 mM glucose-6-phosphate (+G6P) or 0 mM glucose-6-phosphate (-G6P) and mercaptoethanol (18.5 mM).

To test whether a native reductase to S. cerevisiae is reducing 5a(11a)-dehydrotetracycline, the native old yellow enzyme (OYE) reductases, OYE2 and OYE3 from S. cerevisiae were expressed under the strong promoter pPGK1, along with their fungal homolog OYE1 from Saccharomyces carlsbergensis.(23-26) OYE proteins are known to reduce the olefinic bond in α,β-unsaturated carbonyls, such as the reduction of 2-cyclohexenone to cyclohexanone and are known to have a flexible substrate specificity.(23) However, coexpression of the OYE proteins together with OxyS did not enhance the reduction compared to the expression of OxyS alone (data not shown).

2.5 Discussion

2.5.1 6β-hydroxylation of anhydrotetracycline in S. cerevisiae

When OxyS and OxyR were used in vitro, oxytetracycline (5-hydroxytetracycline) and not tetracycline was formed as the major product, indicating that OxyS is catalyzing both hydroxylation steps at C5 and C6 prior to OxyR reduction of the 5a(11a) double bond (Scheme

2-1).(7) Interestingly however, when expressed in S. cerevisiae, both in cell lysate and whole cells,

OxyS is catalyzing just one hydroxylation step (Figure 2-1 and data not shown). It could be that the absence of the 5-hydroxyl is the reason for the lack of contribution of OxyR to reduction as

OxyR’s native substrate is the doubly hydroxylated 5a(11a)-dehydrooxytetracycline and not the singly hydroxylated 5a(11a)-dehydrotetracycline. Given that DacO4 and CtcR are known to hydroxylate 5a(11a)-dehydrotetracycline,(7) an attempt of the hydroxylase-reductase pairs OxyS-

DacO4 and OxyS-CtcR is warranted in S. cerevisiae where OxyS performs only a single hydroxylation step.

Furthermore, previously in the absence of OxyR the reaction product of OxyS rapidly degraded both in vitro and in vivo in ΔoxyR Streptomyces lividans.(7) However, in the S. cerevisiae

53 expression system both in whole cells and in cell lysate the hydroxylation product of OxyS was analyzable (Figure 2-1, Figure 2-2). This could be explained by the additional degradation pathways possible due to the presence of the 5-hydroxy. For example, anhydrooxytetracycline (5- hydroxyanhydrotetracycline) is known to undergo B-ring scission degradation that is not possible in anhydrotetracycline.(27)

The ability to observe the hydroxylated product of anhydrotetracycline could also be because in this study prior to mass spectrometry analysis MeOH added and the samples were filtered, while in the in vitro study of OxyS acidic organic extraction using 1% AcOH in EtOAc was employed.(7)

Anhydrooxytetracycline is known to undergo B-ring scission specifically in the presence of dilute acid.(27).

Importantly, it could be that some double hydroxylation product is formed in the reactions of the

+OxyS S. cerevisiae strain with anhydrotetracycline (Figures 2-1, 2-2 and 2-3) but degraded prior to analysis. However, even if that is the case, it is still notable that a significant proportion of a singly hydroxylated product is formed as well (Figure 2-1), in contrast to the previously reported

OxyS double hydroxylation in vitro and in S. lividans.(7)

The literature reported ability to isolate and analyze 7-chloro-5a(11a)-dehydrotetracycline could provide further support for the potential role of the second hydroxylation step in destabilizing the

5a(11a)-dehydro product. 7-chloro-5a(11a)-dehydrotetracycline, lacking the 5α-hydroxy present in 5a(11a)-dehydrooxytetracycline, is produced by mutant strain S-1308 of Streptomyces lividans and can be isolated and analyzed.(11) The corresponding S. lividans wild type strain normally produces 7-chlorotetracycline, which, like tetracycline is also lacking the additional 5-hydroxy group. However, the 7-chloro group could also be responsible for the stability of 7-chloro-5a(11a)- dehydrotetracycline, previously reported to be the only stable 5a(11a)-dehydrotetetracycline of the

54 ones that have been prepared.(28) Purification of the hydroxylation product of anhydrotetracycline and NMR characterization will enable verifying whether 5a(11a)-dehydrotetracycline is indeed produced.

2.5.2 5a(11a)-dehydrotetracycline reduction in S. cerevisiae

Recently, the biosynthetic pathway of cofactor F420 was fully characterized and F420 was successfully heterologously produced in E. coli by recombinant expression of four genes.(29) The substrates for the F420 pathway, lactate, tyrosine, and 5-amino-6-ribitylamino-2,4[1H,3H]- pyrimidinedione, are native to yeast.(30, 31) Thus, a future attempt to natively produce F420 in yeast to allow functional expression of cofactor F420-dependent enzymes such as OxyR is now more feasible. Alternatively, cofactor F420 or its precursors might become more readily available to be exogenously supplied to S. cerevisiae through heterologous production in another organism such as E. coli. Importantly however, a reduction that takes place in S. cerevisiae in the absence of OxyR and F420 (Figure 2-3) potentially circumvents the need to heterologously express F420 in

S. cerevisiae, a formidable challenge on its own right.

Another alternative route to tetracycline from 5a(11a)-dehydrotetracycline is chemical reduction.

However, the process is not necessarily stereoselective and can yield an additional stereoisomer to tetracycline, 5a-epitetracycline, of significantly lesser antibiotic activity (e.g. against S. aureus) than tetracycline.(11) Stereoselectivity was achieved however in the chemical reduction of the

5a(11a)-bond of 7-chloro-5a(11a)-dehydrotetracycline and of the 6-peroxidated derivative of

5a(11a)-dehydrotetracycline.(32-34) Larger scale culturing, isolation and NMR analysis of the reduction product of OxyS hydroxylation (Figure 2-3) will be needed to confirm whether the reduction takes place in the 5a(11a)-bond and if it does, whether it is stereoselective.

While the conversion percent for the hydroxylation of anhydrotetracycline by OxyS is high (Figure

2-1), the conversion percent of the reduction reaction is much lower (Figure 2-3), assuming equal ionization capacity of anhydrotetracycline, its hydroxylated intermediate and its hydroxylated and reduced product (Scheme 2-1). Thus, optimization of the reduction reaction is likely to be needed.

An obvious method for optimization of the reduction reaction would be to identify the native reductase enzyme in S. cerevisiae and overexpress it. In order to identify the endogenous reductase enzyme in S. cerevisiae a screen could be done using a plasmid collection of yeast genes and a screening method such as described in the next chapter.(35) An alternative assay would be an overlay of the yeast on bacteria given the much higher antibiotic activity of tetracycline over

5a(11a)-dehydrotetracycline.(11) Instead of a library of yeast genes, a library of yeast deletion mutants could be screened, using similar assays, to identify yeast mutants that cannot reduce the hydroxylation products of OxyS.(36, 37)

Importantly however, for these assays to function, the reduction of OxyS’s hydroxylation product needs to happen in whole cells and not only in cell lysates and whether and in which conditions this is the case is still unanswered. Thus, a cell lysate assay employing a more specific library of yeast reductases or ene-reductases or even ene-reductases of α,β-unsat carbonyls is preferable.(38-

40) An alternative method will be to screen additional OYE proteins for the reduction step and potentially perform directed evolution for improved substrate recognition and stereoselectivity in similar methods to those described in the next chapters.(41-50) Finally, OxyR homologs with F420 or an intermediate in its biosynthesis are a potential alternative as well (Section 2.5.1).

2.6 Conclusion

The data presented in this chapter supports that the use of OxyS as the sole heterologously expressed enzyme in S. cerevisiae allows both a single hydroxylation and reduction steps of

56 anhydrotetracycline to take place using S. cerevisiae in the presence of G6P (Figure 2-3). This result is unexpected for two reasons. One, OxyS is known to perform two hydroxylation steps on anhydrotetracycline in vitro and in vivo. Two, it was expected that the coexpression of the dedicated reductase enzyme and the supply / heterologous biosynthesis of its nonnative cofactor would be required for the reduction step. Work on isolation and NMR analysis of the hydroxylation and hydroxylation + reduction products of anhydrotetracycline and 6- demethylanhydrotetracycline to complement the results presented in this chapter is ongoing.

The work presented in this chapter paves the road for total biosynthesis of tetracyclines using S. cerevisiae by expressing the additional bacterial biosynthetic enzymes from the oxytetracycline or the chlortetracycline pathways.(51-54) In addition, the work presented in this chapter paves the road for producing key tetracycline analogs from bacterial and fungal anhydrotetracyclines such as anhydrotetracycline, anhydrodactylocyclinone and TAN-1612, the subject of the next chapters.

2.7 Appendix

Table 2-1 Strains used in this study Genotype Source/Reference FY251 MATa leu2-Δ1 trpΔ63 ura3-52 his3-Δ200 ATCC 96098 BJ5464- MATα ura3-52 his3-Δ200 leu2-Δ1 a / (55) NpgA trp1 pep4::HIS3 δ:: pADH2-npgA-tADH2 prb1Δ1.6R can1 GAL EH-3-80-3 FY251 pSP-G1 This Study EH-3-98-6 FY-251 AL-1-101-C10 This Study EH-3-142-A AL-1-141-A-C6 This Study EH-3-142-B AL-1-151-A-C4 This Study EH-3-142-C AL-1-151-B-C2 This Study ΕΗ-5-153-8 FY-251 AL-1-119-A-C7 This Study EH-3-204-5 FY-251 AL-1-119-A-C7, AL-215-D-C1 This Study EH-3-204-6 FY-251 AL-1-119-A-C7, AL-255-C1 This Study EH-3-204-7 FY-251 AL-1-119-A-C7, AL-235-C5 This Study EH-3-204-8 FY-251 AL-1-119-A-C7, pRS413-pGAL1 This Study EH-3-204-9 FY-251 AL-1-101-C10, pRS413-pGAL1 This Study EH-3-248-1 BJ5464-NpgA AL-1-101-C10 This Study EH-3-248-8 BJ5464-NpgA pSP-G1 This Study a Y. Tang, University of California, Los Angeles, Department of Chemical and Biomolecular Engineering & Department of Chemistry and Biochemistry, Department of Bioengineering

Table 2-2 Plasmids used in this study Description Source/Reference pSP-G1 pTEF1-FLAG-tADH1, pPGK1-MYC-tCYC1, 2 μ, Ura Addgene 64736 pRS413- Shuttle vector (empty), His marker, CEN ATCC 87326 pGAL1 AL-1-101- pTEF1-oxyS-tADH1 in pSP-G1 This Study C10 AL-1-119-A- pTEF1-oxyS-tADH1, pPGK1-oxyR-tCYC1 in pSP-G1 This Study C7 AL-1-141-A- pTEF1-oxyS-tADH1, pPGK1-OYE1-tCYC1 in pSP-G1 This Study C6 AL-1-151-A- pTEF1-oxyS-tADH1, pPGK1-OYE2-tCYC1 in pSP-G1 This Study C4 AL-1-151-B- pTEF1-oxyS-tADH1, pPGK1-OYE3-tCYC1 in pSP-G1 This Study C2 AL-1-183-C- Fo synthase Thermobifida fusca This Study C8

AL-1-183-D- Fo synthase Chlamydomonas reinhardtii This Study C7 AL-1-215-D- FGD1 from Mycobacterium tuberculosis This Study C1 AL-1-255-C1 F420-dependent NADP+ oxidoreductase from This Study Archeoglobus fulgidus AL-1-235-C5 NADPH-dependent F420 reductase from Streptomyces This Study griseus

Table 2-3 Sequences used in this study The sequence for the hydroxylase OxyS from Streptomyces rimosus (GenBank AAZ78342.1) is shown capitalized within the context of the pSP-G1 backbone containing pTEF1, the FLAG tag and tADH1 (partial, decapitalized). The sequence for the reductase OxyR from Streptomyces rimosus (GenBank: DQ143963.2) is shown capitalized within the context of the pSP-G1 backbone containing pPGK1, the myc tag and tCYC1 (partial, decapitalized).

Sequences for the three F420 reductases from M. tuberculosis, A. fulgidus and S. griseus are shown along with the pGPD (pTDH3) promoter capitalized within the context of the pRS413 backbone containing the tCYC1 terminator (partial, decapitalized). The NCBI/Genbank reference sequences used for the Fo reductases from M. tuberculosis, A. fulgidus and S. griseus are CP023708.1, NC_000917.1 and NC_010572.1 (6172267..6172977). Prior to codon optimization, leucine codon in M. tuberculosis F420 reductases in subsequence LTGAACAACACCCGGTTT was changed to methionine so that the total sequence matches the protein sequence used for M. tuberculosis F420 crystallization.(19) pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa oxyS- gttttaattacaagcggccgcATGAGGTACGATGTTGTTATAGCTGGTGCAGGAC tADH1-in- CCACCGGTTTGATGTTAGCATGTGAACTTCGGCTGGCGGGTGCCAGA pSP-G1- ACTTTGGTTTTAGAAAGATTAGCCGAGCCTGTTGACTTCTCGAAAGCT AL-1-101- CTAGGAGTCCACGCTCGCACTGTTGAACTATTAGATATGAGAGGCCT C10 and CGGTGAAGGATTCCAGGCTGAAGCACCAAAGTTAAGAGGTGGTAATT AL-1-119- TTGCCTCATTAGGCGTCCCCCTGGATTTCTCATCATTTGATACTAGAC A-C7 ACCCATATGCATTGTTTGTTCCACAAGTACGAACTGAAGAACTGCTA ACAGGTAGAGCTTTGGAGCTAGGGGCGGAGCTGCGTCGTGGTCATGC CGTGACCGCCTTGGAACAAGATGCTGATGGTGTTACTGTGAGCGTGA CAGGCCCTGAAGGCCCATACGAAGTAGAATGTGCTTATTTGGTGGGC TGCGACGGTGGAGGCAGTACGGTGAGGAAACTATTGGGCATAGATTT TCCAGGTCAAGACCCACATATGTTTGCTGTCATCGCAGACGCAAGAT TCAGAGAAGAGCTTCCTCACGGAGAAGGTATGGGTCCTATGCGTCCT

TATGGTGTCATGAGACATGACCTTCGTGCATGGTTCGCAGCATTTCCG CTAGAACCAGACGTCTACAGAGCAACAGTCGCCTTTTTCGATAGACC GTATGCTGACAGGAGGGCGCCGGTAACGGAGGAGGATGTCAGAGCC GCGTTAACAGAAGTTGCTGGATCTGACTTTGGAATGCATGATGTAAG ATGGTTATCTCGTTTGACCGATACAAGTAGACAAGCAGAAAGGTATA GAGATGGGAGAGTTCTTTTGGCTGGTGATGCATGCCATATTCATTTGC CCGCTGGTGGCCAGGGACTGAACTTAGGATTTCAAGATGCCGTTAAC TTGGGTTGGAAGCTTGGTGCTACCATTGCCGGGACCGCTCCACCAGA GTTATTGGATACGTATGAAGCTGAGAGAAGGCCGATAGCCGCCGGTG TTTTGAGAAATACAAGGGCTCAAGCTGTTCTAATTGATCCAGATCCTC GCTACGAGGGTTTAAGGGAATTAATGATTGAATTGTTGCACGTTCCT GAGACTAATAGATATTTAGCGGGGCTAATCTCCGCATTAGACGTTAG ATACCCAATGGCTGGGGAACATCCATTGCTGGGAAGAAGAGTACCCG ACTTACCTCTTGTCACCGAAGATGGAACAAGACAGTTGTCCACTTATT TCCATGCTGCACGTGGCGTATTATTAACGTTAGGTTGTGATCAACCAC TAGCAGATGAAGCCGCTGCTTGGAAAGATAGGGTTGATTTAGTTGCA GCTGAAGGTGTGGCGGACCCTGGTTCTGCAGTAGATGGCTTAACTGC TTTACTTGTAAGGCCTGATGGTTACATTTGTTGGACAGCTGCCCCAGA AACTGGTACTGATGGATTGACAGACGCGCTGCGTACTTGGTTCGGCC CACCTGCAATGgtatcgatggattacaaggatgacgacgataagatctgagctcttaattaacaattcttcg ccagaggtttggtcaagtctccaatcaaggttgtcggct FGD1-in- ccctcactaaagggaacaaaagctggagctcAGTTTATCATTATCAATACTGCCATTTC pRS413- AAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTA pGPD- GTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAA tCYC1-AL- ATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGT 1-215-D- GAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTT C1 AAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGT TTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTA CAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAAT GGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTG ACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTG CTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTC CCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAG GTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTA CTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTT TCGACGGATACTAGTAAAATGGCTGAGTTGAAACTTGGCTACAAGGC ATCAGCTGAACAGTTTGCCCCAAGGGAGTTAGTCGAACTAGCAGTCG CTGCCGAAGCTCACGGTATGGATTCCGCTACTGTTTCCGACCATTTCC AACCATGGAGACACCAAGGTGGCCATGCACCATTCTCACTCAGTTGG ATGACAGCTGTTGGAGAAAGAACTAATAGATTGTTATTGGGGACGTC GGTACTCACCCCGACGTTTAGATACAACCCTGCGGTAATAGCACAGG CCTTTGCTACAATGGGATGTCTATATCCAAACCGGGTGTTTTTAGGTG TTGGTACTGGAGAGGCCTTGAATGAAATCGCCACTGGTTATGAAGGT GCTTGGCCTGAGTTCAAAGAAAGGTTTGCGCGTCTGCGCGAAAGCGT GGGGCTGATGAGACAATTATGGTCTGGTGATAGGGTAGATTTTGATG GAGATTATTATAGATTAAAAGGCGCGTCTATATATGACGTTCCGGAT

GGTGGTGTCCCTGTATATATTGCCGCCGGAGGACCAGCAGTTGCTAA ATATGCTGGCCGAGCTGGTGACGGTTTCATATGCACTTCAGGCAAGG GTGAAGAACTTTACACAGAAAAGTTGATGCCCGCCGTCAGAGAAGG GGCGGCAGCCGCTGACAGGTCTGTTGATGGCATAGACAAAATGATTG AGATCAAGATTTCATACGACCCAGATCCCGAATTAGCAATGAATAAC ACAAGATTTTGGGCACCTCTTAGTTTGACCGCAGAACAAAAGCATAG CATCGATGATCCAATTGAAATGGAAAAAGCTGCTGATGCTTTACCCA TCGAGCAAATTGCAAAAAGATGGATTGTTGCAAGTGATCCTGATGAA GCAGTGGAGAAAGTAGGACAGTACGTGACCTGGGGTTTAAATCATCT AGTTTTCCACGCTCCTGGGCATGATCAAAGAAGATTCTTGGAATTGTT TCAATCTGACCTAGCGCCAAGACTTCGTCGTCTGGGTTAACTCGAGtca tgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaac ctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtac agacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcg gccggtacccaat FNO-A- ccctcactaaagggaacaaaagctggagctcAGTTTATCATTATCAATACTGCCATTTC fulgidus-in- AAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTA pRS413- GTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAA pGPD- ATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGT tCYC1- GAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTT AL-1-255- AAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGT C1 TTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTA CAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAAT GGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTG ACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTG CTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTC CCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAG GTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTA CTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTT TCGACGGATACTAGTAAAATGAGGGTAGCTTTACTTGGTGGTACAGG AAATCTTGGAAAAGGCCTTGCCCTACGACTGGCCACGCTGGGCCATG AAATCGTAGTCGGAAGCAGAAGAGAAGAAAAGGCAGAGGCTAAAGC AGCAGAATATAGGCGCATTGCAGGTGACGCTTCCATCACTGGTATGA AAAATGAAGATGCCGCTGAGGCATGCGACATTGCTGTCTTGACCATT CCTTGGGAACATGCTATTGACACTGCCAGAGATTTGAAGAATATTTT ACGTGAGAAAATTGTTGTATCACCATTAGTTCCAGTGTCAAGAGGTG CAAAGGGCTTCACCTACTCGTCCGAAAGATCAGCGGCCGAGATTGTG GCTGAAGTTCTGGAATCTGAAAAAGTTGTTTCTGCGTTGCACACAAT ACCTGCTGCAAGATTTGCCAACTTGGATGAAAAATTTGATTGGGATG TTCCTGTTTGTGGTGATGATGACGAAAGTAAGAAAGTCGTCATGTCTT TAATAAGTGAAATAGATGGGTTGAGGCCGTTAGATGCTGGTCCCCTC TCTAACTCCCGTTTAGTGGAGAGCCTAACTCCATTGATATTGAACATC ATGAGATTCAATGGAATGGGTGAACTAGGGATCAAGTTTTTATAACT CGAGtcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaagga gttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttctt

ttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaagg ctttaatttgcggccggtacccaat FNO-S- ccctcactaaagggaacaaaagctggagctcAGTTTATCATTATCAATACTGCCATTTC Griseus-in- AAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTA pRS413- GTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAA pGPD- ATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGT tCYC1- GAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTT AL-1-235- AAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGT C5 TTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTA CAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAAT GGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTG ACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTG CTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTC CCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAG GTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTA CTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTT TCGACGGATACTAGTAAAATGACTACTCAAGACAGTGGGTCTGCACC GAAGCCTCCCGCCAAAGATCCATGGGATTTGCCAGATGTCAGCGCAC TGTCTGTTGGTGTCCTAGGTGGTACAGGACCACAGGGCAGGGGATTA GCCTACAGATTGGCGCGTGCTGGTCAAAAAGTGACCTTGGGCTCAAG AGATGCTGGACGCGCTGCTGATGCAGCGGCAGAGCTGGGCCATGGTG TGGAAGGTACTGATAATGCAGAATGCGCAAGAAGATCCGACATCGTT ATTGTTGCTGTACCTTGGGACGGTCATGCTAAAACTTTGGAAAGTTTA AGAGAAGAGTTGGCTGGCAAGCTGGTTATCGACTGTGTTAACCCCTT GGGGTTTGATAAGAAAGGTGCTTATGCTTTAAAACCAGAGGAGGGCT CGGCCGCTGAACAAGCTGCGGCGCTTCTCCCTGATTCACGAGTAACA GCAGCTTTCCACCATTTATCTGCTGTGCTTTTACAAGATGAATCCATT GAAGAAATTGATACCGATGTTTTGGTCTTAGGTGAAGCAAGGGCAGA CACGGATATTGTCCAGGCACTAGCAGGGCGTATACCAGGTATGAGAG GAATATTTGCCGGTCGTCTAAGAGGTGCCCACCAAGTTGAATCACTT GTAGCCAATTTAATATCTGTAAACAGAAGGTATAAGGCTCATGCCGG ACTAAGGGCCACAGACGTGTAACTCGAGtcatgtaattagttatgtcacgcttacattcac gccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttat agttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatac tgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcggccggtacccaat pPGK1- tggaatggcgggaaagggtttagtaccacatgctatgatgcccactgtgatctccagagcaaagttcgttcgatcgt oxyR- actgttactctctctctttcaaacagaattgtccgaatcgtgtgacaacaacagcctgttctcacacactcttttcttctaa tCYC1 in ccaagggggtggtttagtttagtagaacctcgtgaaacttacatttacatatatataaacttgcataaattggtcaatgc pSP-G1 aagaaatacatatttggtcttttctaattcgtagtttttcaagttcttagatgctttctttttctcttttttacagatcatcaagga AL-1-119- agtaattatctactttttacaacaaatataaaacaaggatccATGCCATTCACTCAAAAGGAGAT A-C7 and CACGTATTTAAGGGCTCAAGGCTACGGCCGACTAGCCACCGTCGGTG AL-1-26-1- CTCACGGTGAACCTCACAACGTGCCGGTATCTTTTGAAATTGATGAA C1 GAAAGAGGTACCATTGAAATAACAGGAAGGGATATGGGACGTTCCA GAAAATTCAGAAATGTTGCCAAAAGTGACAGAGTGGCATTTGTTGTT GATGATGTTCCATGCAGAGACCCAGAAGTTGTCAGAGCTGTTGTAAT ACATGGTACTGCACAGGCGCTTCCCACAGGCGGAAGAGAAAGGCGT

CCTCATTGTGCTGACGAAATGATTAGAATTCATCCAAGAAGAATAGT AACATGGGGTATCGAAGGTGATTTGTCAACTGGTGTTCATGCAAGAG ATATTACTGCTGAAGATGGTGGTAGAAGGgtcgacatggaacagaagttgatttccga agaagacctcgagtaagcttggtaccgcggctagctaagatccgctctaaccgaaaaggaaggagttagacaacc tgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtaca gacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaagatccagctgcatt aatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgct gcgctcgg

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3 Towards the biosynthesis of 6-demethyl-6-

epitetracyclines using S. cerevisiae

*The contents of this chapter will be published in:

E. Herbst, V. W. Cornish, et al “Biosynthesis of 6-demethyl-6-epitetracyclines using

Saccharomyces cerevisiae”, in preparation.

3.1 Chapter outline

This chapter describes the work towards 6-demethyl-6-epitetracyclines biosynthesis using S. cerevisiae. 6α-hydroxylation by an enzyme such as DacO1 is required for the synthesis of 6- demethyl-6-epitetracyclines as opposed to 6β-hydroxylation by enzymes such as OxyS leading to

6-demethyltetracycline (Scheme 3-1 and Scheme 3-2). However, previous attempts in multiple heterologous hosts to heterologously express DacO1 without the rest of the dactylocyclinone pathway have not yielded stably expressed protein. Thus, the chapter first describes rational design approaches for optimizing DacO1 expression in S. cerevisiae (Section 3.4.1). The rationale behind the choices for bacterial hydroxylases screened for 6α-hydroxylase activity on anhydrotetracycline is explained (Section 3.4.2). Screening for 6-hydroxylase activity towards the biosynthesis of 6- demethyl-6-epitetracyclines required the development of a multititer plate assay for anhydrotetracycline biosynthesis (Section 3.4.3). This assay was put to the use in screening for error-prone mutants of DacO1 in an attempt to identify 6α-hydroxylases of anhydrotetracycline

(Section 3.4.4). Finally, the results of testing the rational design DacO1 expression optimization library as well as the other bacterial candidates for 6α-hydroxylation in S. cerevisiae are described

(Section 3.4.5). Key results in this chapter are the the development of a stably expressing form of

DacO1 in S. cerevisiae (Figure 3-8), the identification of PgaE as an anhydrotetracycline hydroxylase (Figure 3-9 and Figure 3-10), and the discovery that a larger proportion of PgaE hydroxylated intermediates undergo reduction in the presence of glucose-6-phosphate than is the case in OxyS (Figure 3-11). Finally, the next required steps towards 6-demethyl-6-epitetracyclines biosynthesis in S. cerevsiae are discussed (Section 3.5).

3.2 Introduction

As mentioned in Chapter 1, the synthesis of 6-demethyl-6-epiglycotetracyclines for testing is highly desirable due to their highly promising outlook as antibiotics. The first two steps in the synthesis of 6-demethyl-6-epiglycotetracyclines are the synthesis of 6-demethyl-6-epitetracyclines from anhydrotetracyclines. This chapter shows the progress towards developing an S. cerevisiae platform for 6-demethyl-6-epitetracyclines biosynthesis using bacterial hydroxylase enzymes as a starting point for directed evolution and rational design as well as anhydrotetracycline as a model substrate. DacO1, a bacterial flavin-dependent monooxygenase homologous to OxyS was hypothesized to perform a 6α-hydroxylation on its native substrate that leads to 6- epiglycotetracyclines in its native host, Dactylosporangium sp. SC 14051 (ATCC 53693).(1, 2)

Thus, DacO1, along with other bacterial hydroxylases, is used here as a template to evolve a 6α- hydroxylase in S. cerevisiae towards the biosynthesis of 6-demethyl-6-epiglycotetracyclines.

3.3 Materials and methods

See Chapter 5 for general methods. See Chapter 2 for general protocol for hydroxylation/reduction assay in cell lysate.

3.3.1 General protocol for high throughput assay of hydroxylation/reduction

in whole cells in microtiter plates

Fresh colonies of strains harboring a plasmid encoding a hydroxylase and/or reductase and control strains (EH-3-98-6 encoding OxyS as positive control and EH-3-80-3 with no hydroxylase as negative control) were inoculated in 200 μL selective media (U- or HU-) in 96-well plates (Corning

353072) and placed in shaker overnight. Overnight cultures were used to inoculate 1 mL selective media (U- or HU-) cultures in deep well plates (Corning P-2ML-SQ-C-S) with an average starting

OD of 0.01. The plate was covered with two layers of SealMate film (Excel Scientific, SM-KIT-

BS) and placed in shaker overnight. Cells were then pelleted at 4 °C at 4,000 rpm. Each pellet was redissolved in 0.3 mL containing a 150 μL solution of 5 mg / mL anhydrotetracycline HCl, 30 μL

1 M Tris buffer pH 7.45, 15 μL 40% glucose solution in H2O and 105 μL H2O for final concentrations of 2.5 mg/mL anhydrotetracycline HCl, 100 mM Tris pH 7.45 and 2% glucose. The plates were then covered with two layers of SealMate film (Excel Scientific, SM-KIT-BS) and placed in shaker overnight at 800 rpm. 2 μL of Overnight suspensions were diluted into 198 μL of

H2O in Corning 3603 plates before UV/VIS spectroscopic measurements. UV/VIS spectroscopic measurements taken were as follows: (i) absorption spectrum 350 nm – 500 nm, (ii) emission spectrum 450 nm – 550 nm (λexcitation = 400 nm), (iii) excitation spectrum 305 nm – 455 nm (λemission

= 500 nm). Spectral steps in the spectra were 25 nm.

3.3.2 General protocol for western blots

Fresh patches or colonies of strains harboring the plasmid for the hydroxylation and/or reduction enzyme and control strains were inoculated in 3 mL selective media (U-) in 15 mL culture tubes

(Corning 352059) and placed in shaker overnight. Overnight cultures were used to reinoculate 3 mL selective media (U-) in 15 mL culture tubes (Corning 352059) and placed in shaker overnight with a starting OD of 0.01-0.05 (all western Blots shown in this study except for western blots shown in Figure 3-1 which were pelleted and lysed immediately). Cells were grown to final OD of 0.6-0.8 before pelleting in 2 separate microcentrifuge tubes (1 mL culture each) at 4 °C at 14,000 rpm, removing the supernatant and freezing at -20 °C prior to further use.

100 μL of a 99:1 mixture of Y-PER yeast protein extraction reagent (ThermoFisher Scientific

78991) and HALT protease inhibitor cocktail (ThermoFisher Scientific PI87786) were added to each microcentrifuge tube. The tubes were placed on orbital shaker for 20 min at r.t., followed by

10 min centrifugation at 14,000 rpm at 4 °C and the cell lysate was transferred to a new 1.5 mL microcentrifuge tube and kept on ice. Following 5 min at 95 °C with SDS loading buffer,(3) an

SDS-page was run with a Kaleidoscope Prestained Standard (Bio-Rad 161-0324, Figure 3-1) or a

Precision Plus Protein Kaleidoscope (Bio-Rad 161-0375, all other western blots shown),(4) the gels were transferred to PVDF blots (Thermo Fisher Scientific IB24001) and western blots were performed according to manufacturer guidelines (Thermo Fisher R951-25 and Sigma-Aldrich

A8592 for myc-and FLAG-tag, respectively) with BSA used instead of milk and analyzed with

Thremo Scientific 34000 substrate kit for horseradish peroxidase.

3.4 Results

3.4.1 Optimizing DacO1 expression in S. cerevisiae – rational design

Following the successful hydroxylation of anhydrotetracycline in S. cerevisiae by OxyS described in Chapter 2, the 6α-hydroxylation of anhydrotetracycline in S. cerevisiae by DacO1 was attempted. DacO1 is 66% homologous to OxyS and is assumed to perform the 6α-hydroxylation on its native substrate, anhydrodactylocyclinone, as opposed to the 6β-hydroxylation that OxyS performs on anhydrotetracycline (Scheme 3-1).(1)

DacO1 heterologous expression, along with the rest of the Dactylocyclinone pathway was previously attempted in Streptomyces lividans K4‐114 and has led to the successful biosynthesis of dactylocyclinone. Assuming correct assignment for the role of DacO1 in the dactylocycline pathway, given its homology to OxyS, this result supports that DacO1 was successfully heterologously expressed in S. lividans.(2) However, previous attempts to obtain soluble forms of heterologously expressed DacO1 from Streptomyces and E. coli, when heterologously expressed without the rest of its pathway, were not successful.(1)

My initial attempts to hydroxylate anhydrotetracycline with DacO1 yielded either little or no hydroxylation when assayed by mass spectrometry (data not shown) but little hydroxylation was supported by UV/Vis spectroscopy (Figure 3-4). Therefore, the expression levels of DacO1 were examined by western blotting with FLAG-tag to determine whether a problem in expression leads to the low/no hydroxylation levels with DacO1. Another likely explanation for low/no hydroxylation is difference between anhydrotetracycline, the model substrate I used, and anhydrodactylocyclinone, the native substrate of DacO1 (Scheme 3-1). Indeed, DacO1 expression levels were found to be significantly lower than its counterpart OxyS (Figure 3-1-a).

Figure 3-1 Western Blot of DacO1 and OxyS Cultures of DacO1, DacO4 and OxyS, OxyR encoding strains EH-3-153-7 and EH-3-153-8, respectively were lysed with Y-PER and labeled with Monoclonal ANTI-FLAG HRP antibody. Two biological replicates were used for each strain indicated as colony 1 and 2 (C1 and C2), respectively. The expected sizes are 55.1, 55.9, 16.3 and 17.5 for DacO1, OxyS, DacO4 and OxyR, respectively.

I therefore attempted to optimize the functional expression of DacO1 by a number of approaches.

Namely, fusion to a stable protein, switching to a transcriptional control of an inducible promoter, expression in a strain background that has protease deletions, lower temperature culturing and

75 coexpression of pathway proteins. The testing of these expression optimization approaches is described in Section 3.4.5.

Specifically, I attempted N-terminal fusions proteins previously shown to improve expression of heterologous proteins in yeast, ubiquitin,(5-8) superoxide dismutase,(9-11) Gal1(8) and γ-

IFN.(12) I also attempted a C- and N-terminal fusion to DacO4, the 5a(11a) reductase enzyme of the dactylocycline pathway that I verified to be stably expressed in yeast (Figure 3-1-b).

Given the homology between OxyS and DacO1, the successful heterologous expression of OxyS and the potential importance of the N-terminus of the protein to its in vivo stability,(13) I attempted three fusion proteins composed of an N-terminal portion of OxyS and a C-terminal portion of

DacO1. These fusion proteins include the first 37, 87 and 191 amino acids of OxyS, followed by the last 461, 411 and 307 amino acids of DacO1, respectively. In order to determine where to fuse the proteins I considered the structure of OxyS as determined previously by X-ray crystallography,(1) and made fusions proteins in disordered regions of the structure around positions 37, 87 and 191. The homology between DacO1 and OxyS made it simple to determine what are the corresponding amino acids at the DacO1 protein, although there is no guarantee the disordered regions in OxyS are also disordered in DacO1.

For inducible promoters I tested pGAL1, induced by galactose, and pADH2, a late stage promoter induced in low glucose / high EtOH.(14) The alternative strain background I chose is BJ5464-

NpgA, as the BJ-5464 strain background was previously shown useful for heterologous protein production in yeast.(15, 16) For a lower temperature culturing I attempted 25 °C.(17, 18)

The activity of polyketide biosynthetic enzymes is known to be dependent on an oligomeric complexation of biosynthetic enzymes. This is claimed for instance in the case of JadF, JadG and

JadH of the Jadomycin biosynthetic pathway.(19) Similarly, in the case of an oxytetracycline

76 producing strain, deletion of the gene encoding for OtcC, which is 99% identical to OxyS used in this study, led to production of a polyketide with a shorter chain length. Expression of a triply mutated, catalytically disfunctional OtcC, restored production of a polyketide with the same chain length. Thus, a post-polyketdie synthase (PKS) polyketide biosynthetic enzyme could have a structural role in the oligomeric protein complex responsible for polyketide biosynthesis.(20)

Potential further support for the potential that coexpression of additional proteins from the dactylocycline pathway has for improving DacO1 expression could be found in the case of DacO1 itself. As mentioned above, expression of DacO1 together with the rest of the dactylocyclinone pathway in S. lividans led to the production of dactylocyclinone.(2) However, expression of

DacO1 in the absence of the rest of the dactylocycline pathway in E. coli, Streptomyces,(1) or S. cerevisiae (Figure 3-1) yielded no/low levels of soluble protein. In order to provide DacO1 with enzymes potentially required to form a functional oligomeric complex I attempted the coexpression of DacJ and DacM2. DacJ and DacM2 were chosen since they are the closest enzymes to DacO1 in the dactylocycline gene cluster and they are proposed to share with DacO1 the functional role of aglycone tailoring.(2)

3.4.2 Searching the bacterial hydroxylase space for 6α-hydroxylation of

anhydrotetracyclines

Given the goal to perform 6α-hydroxylation on anhydrotetracyclines it was clear that there is a need to go beyond the 6β-hydroxylase OxyS. Expected challenges with the expression of Daco1

(Section 3.4.1) as well as incongruencies between the DacO1 native substrate anhydrodactylocyclinone and anhydrotetracycline affirmed the need to test other hydroxylases as well (Scheme 3-1). I therefore looked for homologous enzymes catalyzing hydroxylation in polyketides that are structurally related to anhydrotetracycline.

Scheme 3-1 Bacterial monooxygenases and their hypothesized substrates and products Bacterial flavin-dependent monooxygenases OxyS, DacO1, SsfO1, CtcN and PgaE and their native substrates. Marked in red are differences between the native substrate and anhydrotetracycline.

SsfO1, the hypothesized flavin-dependent monooxygenases of the 6α-position in SF2575 pathway, with 54% homology to OxyS, was an obvious candidate (Scheme 3-1).(2) Flavin-dependent monooxygenases of angucyclines, seemed like another promising source for relevant monooxygenases, given the structural similarity between angucyclines and tetracyclines.

Specifically, enzymes that catalyze monooxygenation of position 12 of the angucyclines,

78 analogous to position 6 of the tetracyclines seemed of particular interest.(21) I chose to test PgaE

(56% homology to OxyS), catalyzing the hydroxylation of UWM6 (Scheme 3-1), in the biosynthesis of gaudimycin C since it was previously both analyzed by X-ray crystallography and used in chemoenzymatic synthesis.(22, 23) Finally, CtcN, with 69% homology to OxyS was chosen since it was shown to be necessary for the 6-hydroxylation step in the chlortetracycline pathway.(24) The testing of these bacterial hydroxylases is described in Section 3.4.5.

3.4.3 Developing a microtiter plate assay for anhydrotetracycline

hydroxylation

Given the need to screen for a 6α-hydroxylase of anhydrotetracyclines I developed a high throughput screen for hydroxylation of anhydrotetracyclines based on the ready detection of anhydrotetracycline hydroxylation by UV/Vis spectroscopy (Figure 2-2). Following colony picking onto microtiter plates and overnight culturing to even out the OD across the different wells the cells were reinoculated for a 2nd overnight culture, pelleted and resuspended in a Tris reaction buffer containing anhydrotetracycline and glucose. The suspension was then diluted in H2O for

UV/VIS measurements (Figure 3-2).

Figure 3-2 A simplified outline of the microtiter plate assay for anhydrotetracycline hydroxylation.

3.4.4 Evolving a 6α-hydroxylase of anhydrotetracyclines – DacO1 random

mutagenesis

Given the low/no hydroxylation activity of DacO1 on anhydrotetracycline, DacO1 optimization was attempted by directed evolution. An error-prone mutagenesis library from DacO1 encoding plasmid AL-1-31-C7 was made with an average of 3 mutations per 1 kb and transformed into

FY251. The library was then assayed for anhydrotetracycline hydroxylation by the microtiter plate assay for anhydrotetracycline hydroxylation (Figure 3-2) with strains expressing OxyS, DacO1

(w.t.) and empty plasmid (EH-3-98-6, EH-3-80-2 and EH-3-80-3, respectively) as controls. Over

500 colonies were screened in the assay using 96-well plates with two potential hits identified. A typical plate with no hits, showing only the OxyS expressing strain positive control above background fluorescence as well as a plate displaying a hit are shown in Figure 3-3.

Figure 3-3 DacO1 error-prone mutagenesis screen excerpt DacO1 error-prone mutagenesis screen using the microtiter plate assay for anhydrotetracycline hydroxylation. (a) a plate with only the OxyS positive control significantly above background fluorescence. (b) a plate with both the OxyS positive control and a DacO1 mutant hit with fluorescence significantly above background. Wells 60 and 72 contain the positive control OxyS encoding strain EH-3-98-6. λex = 400 nm.

A followup on the two potential DacO1 mutant hits in the screen showed that one of them has a slightly increased fluorescence in the Δexcitation spectrum relative to DacO1 (Figure 3-4). The

81 new excitation peak at 375 nm, in 25 nm resolution, is similar to that found for OxyS hydroxylation of anhydrotetractycline at 380 nm, in 10 nm resolution, but of a much lower intensity (Figure 2-

2). Considering that the increase in fluorescence is rather minor I decided to focus in the interim on alternatives such as rational design approaches to optimize hydroxylation by DacO1 and exploring DacO1 homologs (Sections 3.4.1 and 3.4.2, respectively). The testing of these candidates is described in Section 3.4.5.

Figure 3-4 ΔExcitation spectrum for DacO1 and DacO1 error-prone PCR mutant The spectrum shown is a Δ spectrum, that is, the values shown are the emission values for the hydroxylase expressing cells minus the emission values of the no hydroxylase control EH-3-80-3.

λemission = 500 nm. Each value is the average of six biological replicates and the error bars represent standard error.

3.4.5 Testing DacO1 expression optimization constructs and other bacterial

hydroxylases

I screened the strains generated for the DacO1 optimization attempts as well as their controls, EH-

5-98-1 through EH-5-98-19, EH-3-248-1 through EH-3-248-8, EH-3-80-2, EH-3-80-3, EH-3-98-

6 and EH-5-115-1 through EH-5-115-5 (Sections 3.4.1 and 3.4.2) using the microtiter plate

UV/Vis assay (Section 3.4.3). Each of the 35 strains was assayed in four biological replicates, as well as in two culturing temperatures, 30 °C and 25 °C, starting from the 2nd inoculation stage

(Figure 3-2). The protocol for the microtiter plate UV/Vis assay (Section 3.3.1) was slightly modified to accommodate the strains encoding DacO1 controlled by inducible promoters. Thus,

EH-5-98-7 and EH-5-98-8 encoding pADH2-dacO1 were inoculated in YPD in the 2nd inoculation and EH-5-98-4 and EH-5-98-5, encoding pGAL1-dacO1 were inoculated in U- Raffinose and supplemented in the next morning with 66.7 μL of 30% galactose in H2O for a total concentration of 2%. UV/Vis measurements were taken following one night and after three nights of incubation with anhydrotetracycline for the 30 °C plates and after one night only for the 25 °C plates.

Strains that showed a blue shift in their peak in the Δexcitation spectrum from that of negative control strain EH-3-80-2 harboring empty pSP-G1 were indicated as potential hits in the assay and are shown in Table 3-1. Specifically, strains listed in the table have shown in the Δexcitation spectrum a peak of 405 nm, 380 nm or 355 nm as opposed to the 430 nm peak of negative control

EH-3-80-3, associated with anhydrotetracycline. Results from three measurements are shown, two measurements after overnight incubation of the cells suspensions in the buffer containing anhydrotetracycline, one for each of the culturing temperatures (25 °C and 30 °C) and another final measurement for the 30 °C culturing condition after 3 nights of suspension in the buffer containing anhydrotetracycline. The rank was calculated per plate measured for one of the measurements according to the emission at the peak maximum, and should be interpreted only as a rough indicator as peak emissions at three different wavelengths are ranked on the same scale.

The number 1 or 2 after the dot indicates the plate number from which the measurement was taken.

Reassuringly, the positive control OxyS, whose native substrate is anhydrotetracycline is leading the rank in highest emission resulting from a blue-shifted excitation. It is followed by the various

DacO1 fusion proteins as well as by hydroxylase alternatives to OxyS and DacO1. Surprisingly, empty pSP-G1 and OxyR in the alternative background strain, BJ-5464-NpgA have also displayed a blue-shifted peak in the excitation spectrum, although they are the lowest ranking (Table 3-1).

Also surprisingly, EH-3-80-2 encoding unoptimized DacO1 has not displayed a blue shifted excitation peak in the reduction spectrum in contrast to previous experiments (Figure 3-4). Strains that exhibited a blue-shifted excitation peak at 25 °C and not in 30 °C are: DacO1-DacO4,

OxySDacO1-191, no-AB-tags and DacO1-JCAT-BJ. I proceeded to analyze by western blot the hydroxylase expression of the strains that displayed a blue-shifted excitation peak in all three measurements (Figure 3-5, Figure 3-6 and Figure 3-7).

Table 3-1 Potential hits in the screen of DacO1 expression optimization constructs and other bacterial hydroxylases Strain Strain description 30 °C 25 °C 30 °C Rank 30 °C number measurement measure measure measurement I I ment I ment II EH-3-98-6 OxyS Y Y Y 1.1 EH-3-98-6 OxyS Y Y Y 1.2 EH-5-98-9 Ubiquitin-γ-IFN- Y Y Y 2.1 DacO1 EH-3-248-2 OxyS-OxyR-BJ Y Y Y 2.2 EH-5-98-1 Ubiquitin-DacO1 Y Y Y 3.1 EH-3-248-1 OxyS BJ Y Y Y 3.2 EH-5-98-10 γ-IFN-DacO1 Y Y Y 4.1 EH-5-115-4 OxyS JCAT Y Y Y 4.2 EH-3-98-2 hSOD-DacO1 Y Y 5.1 EH-3-248-7 PgaE-JCAT-BJ Y Y Y 5.2 EH-5-98-3 Gal1-DacO1 Y Y Y 6.1 EH-3-248-5 CtcN-JCAT-BJ Y Y Y 6.2 EH-5-98-12 DacO4-DacO1 Y Y Y 7.1 EH-5-115-5 DacO1-JCAT-BJ Y Y Y 7.2 EH-5-98-14 OxyS-DacO1-87 Y Y Y 8.1 EH-3-248-6 SsfO1-JCAT-BJ Y Y Y 8.2 EH-5-98-13 OxyS-DacO1-37 Y Y Y 9.1 EH-3-248-4 DacO1-BJ Y Y Y 9.2 EH-3-248-3 OxyR Y 10.2 EH-3-248-8 pSP-G1-empty-BJ Y Y Y 11.2 EH-98-11 DacO1-DacO4 Y EH-5-98-15 OxyS-DacO1-191 Y EH-5-98-6 DacO1-no-AB- Y tags EH-5-98-16 DacO1-JCAT-BJ Y EH-5-98-5 pGAL-DacO1-426 Y Y

Figure 3-5 Western blot analysis of DacO1 fusion proteins and other bacterial hydroxylases in FY251 Strain cultures were lysed with Y-PER and labeled with monoclonal ANTI-FLAG HRP antibody. Two biological replicates were used for each strain indicated as colony 1 and 2 (C1 and C2), respectively. Strain EH-5-98-1, EH-5-98-3, EH-5-98-9, EH-5-98-10 and EH-5-98-13 encode DacO1 fusions with ubiquitin, Gal1, Ubiquitin-γ-IFN, γ-IFN, and OxyS (first 37 amino acids) respectively. In the latter only the last 461 amino acids of DacO1 are encoded, while in the other

DacO1 fusion proteins the complete DacO1 protein is encoded. Strains EH-3-80-2, EH-3-80-3 and EH-3-98-6 encode DacO1, no hydroxylase and OxyS, respectively. The size (kDa) indicated below the gels is the expected size of the protein based on the amino acid sequence.

Figure 3-6 Western blot analysis of bacterial hydroxylases in BJ-5464-NpgA Strain cultures were lysed with Y-PER and labeled with monoclonal ANTI-FLAG HRP antibody. Two biological replicates were used for each strain indicated as colony 1 and 2 (C1 and C2), respectively. The size (kDa) indicated below the gels is the expected size of the protein based on the amino acid sequence.

Figure 3-7 Western blot analysis of DacO1-DacO4 and DacO4-DacO1 fusion proteins Strain cultures were lysed with Y-PER and labeled with monoclonal ANTI-Myc HRP antibody. Two biological replicates were used for each strain indicated as colony 1 and 2 (C1 and C2), respectively. The size (kDa) indicated below the gels is the expected size of the protein based on the amino acid sequence.

For the fusion protein constructs in FY251, ubiquitin-DacO1-C1, Gal1-DacO1-C1 and Ubiquitin-

IFN-DacO1-C1 slight bands are observed in the expected sizes that are not observed in the DacO1-

C1 control (55.0, 86.5 and 72.0 kDa, respectively, Figure 3-5). Bands at the expected sizes were also observed for OxyS-DacO1-37, SsfO1, OxyS and DacO1-C2, with SsfO1 and OxyS bands looking more prominent (55.5, 56.2, 55.9 and 55.1 kDa, respectively, Figure 3-5). OxyS-DacO1-

37 and DacO1-C2 show a 50 < band < 75 kDa that is not observed in the empty pSP-G1 negative

88 control. In all bands, proteins of lower mass are noticeable, potentially corresponding to proteolysis products of the hydroxylases retaining the C-terminal FLAG-tag.

For the protein constructs in BJ-5464-NpgA, clearly less degradation products are observed

(Figure 3-6). OxyS, SsfO1 and PgaE show very prominent bands at the expected sizes (55.9, 56.2 and 53.8 kDa) for both colonies; CtcN-C1 shows a slighter band in the expected size (51.9 kDa) and DacO1-JCAT-C2 shows an even slighter band of the expected size (55.1 kDa). In addition, the codon optimization method (COOL vs JCAT) does not seem to significantly alter expression levels in the case of OxyS, or significantly remediate expression levels in the case of DacO1

(Compare strains EH-3-248-1 and EH-3-248-4 with strains EH-5-115-4 and EH-5-115-5, respectively, Figure 3-6). Finally, even though BJ-5464-NpgA expression apparently reduced protein degradation for other hydroxylases (e.g. OxyS and SsfO1) it does not seem to have promoted significantly expression levels for DacO1 (Figure 3-5 and Figure 3-6).

The DacO4-DacO1 fusion did not yield a stably expressed protein. While DacO4 under pPGK1 and labeled with myc-tag is clearly expressed when unfused to DacO1, the DacO4-DacO1 fusion is not observed in the gel when under pPGK1 and labeled with myc-tag, in either FY251 or

BJ5464-NpgA background strain (Figure 3-7).

Since less protein degradation was observed for OxyS and SsfO1 in BJ5464-NpgA relative to

FY251 (Compare strain EH-3-248-1 and EH-3-248-6 to strains EH-3-98-6 and EH-5-98-17,

Figure 3-6 and Figure 3-5, respectively) it was logical to test whether the expression of the DacO1

N-terminal fusion proteins would be enhanced in BJ5464-NpgA as well. Strains EH-5-163-1 through EH-5-163-7 were therefore cloned and their DacO1 fusion expression was tested by western blot (Figure 3-8).

Figure 3-8 Western blot analysis of DacO1 and its fusion proteins in BJ5464-NpgA Strain cultures were lysed with Y-PER and labeled with monoclonal ANTI-FLAG HRP antibody. Two biological replicates were used for each strain indicated as colony 1 and 2 (C1 and C2), respectively. Strains EH-5-163-1, through EH-5-163-7 encode DacO1 fusions with ubiquitin,

Gal1, Ubiquitin-γ-IFN, γ-IFN, DacO4, OxyS (first 37 amino acids) and OxyS (first 87 amino acids), respectively. In the last two strains only the last 461 and 411 amino acids of DacO1 are encoded, respectively, while the other DacO1 fusion proteins the complete DacO1 protein is encoded. In contrast to DacO1 and its fusion proteins, PgaE expressing strain in this assay has an FY251 background. The size (kDa) indicated below the gels is the expected size of the protein based on the amino acid sequence.

In BJ5464-NpgA background, Ubiquitin-DacO1 fusion expression levels seem similar to that of unfused DacO1; Gal1-DacO1, Ubiquitin-IFN-DacO1-C2 and IFN-DacO1-C2 fusions gave bands at the expected sizes of 86.5 and 72.0, respectively, with IFN-DacO1-C2 showing the most prominent band; The OxyS N-terminal fusions to C-terminal DacO1, OxyS-DacO1-37 and OxyS-

DacO1-87 both gave bands at the expected size with OxyS-DacO1-87-C1 showing the more prominent band (Figure 3-8). Based on these results, the colonies showing better DacO1 expression levels were chosen for analysis of anhydrotetracycline hydroxylation by mass spectrometry (Scheme 3-2).

Scheme 3-2 Two-step enzymatic conversion of (6-demethyl-)anhydrotetracycline to (6- demethyl-)6-epitetracycline

Cell lysates of strains expressing hydroxylases that showed stronger bands of the expected sizes in the Western Blots were incubated in a Tris buffer with anhydrotetracycline and glucose-6- phosphate. The overnight incubations were then assayed by mass and UV/Vis spectrometry

(Figure 3-9 and Table 3-2). The strains that show hydroxylation levels potentially above background, as indicated by 443.4 ion counts that are more than double those for the no

91 hydroxylase negative control, are those encoding OxyS, PgaE and Ubiquitin-γ-IFN-DacO1

(labeled as 1, 4 and 9, respectively in Figure 3-9). These three strains, along with three additional ones encoding γ-IFN-DacO1, OxySDac37 and OxySDac87, also show a maximal emission and/or excitation in the Δspectra that differs from that of the background strain EH-3-248-8 encoding empty pSP-G1 (Table 3-2). Therefore, these six strains along with EH-3-248-4 and EH-3-248-8 as controls were assayed again by mass spectrometry in a wider set of conditions prior to larger scale reactions, isolation and analysis by NMR.

Figure 3-9 Mass spectrometry analysis of anhydrotetracycline hydroxylation and reduction by cell lysates of strains expressing DacO1, its fusion proteins and other bacterial hydroxylases The analyzed strains shown are EH-3-248-1, EH-3-248-4, EH-3-248-6, EH-3-248-7, EH-3-248-8, EH-5-163-1-C1, EH-5-163-2-C1, EH-5-163-2-C2, EH-5-163-3-C2, EH-5-163-4-C2, EH-5-163- 6-C1, EH-5-163-7-C1 encoding hydroxylases OxyS, DacO1, SsfO1, PgaE, pSPG1 (no hydroxylase negative control), Ubiquitin-DacO1, Gal1-DacO1, Gal1-DacO1, Ubiquitin-γ-IFN- DacO1, γ-IFN-DacO1, OxyS-DacO1-37 and OxyS-DacO1-87, respectively. Cell lysates were placed overnight in Tris buffer (100 mM, pH 7.45) containing anhydrotetracycline (5.4 mM),

93 glucose (27.8 mM), NADPH (3 mM), glucose-6-phosphate (10 mM) and mercaptoethanol (18.5 mM).

Table 3-2 UV/Vis spectroscopy results for hydroxylase strains cell lysates incubated with anhydrotetracycline and glucose-6-phosphate Strain no. Hydroxylase Max emission Max excitation (λexcitation = 280 nm) (λemission = 500 nm) 1 EH-3-248-1 OxySb 490 390 2 EH-3-248-4 DacO1 590 350 3 EH-3-248-6 SsfO1 580 350 4 EH-3-248-7 PgaEb 580 370 5 EH-3-248-8 pSPG1a 580 350 6 EH-5-163-1-C1 UbiDac 570 350 7 EH-5-163-2-C1 GalDac 570 350 8 EH-5-163-2-C2 GalDac 570 350 9 EH-5-163-3-C2 UbiIFNDacb 690 350 10 EH-5-163-4-C2 IFNDacb 460 350 11 EH-5-163-6-C1 OxySDac37b 420 370 12 EH-5-163-7-C1 OxySDac87b 430 470 a The maximum emission and maximum absorption spectra of EH-3-248-8 encoding no hydroxylase (empty pSP-G1) are shown in italics. For all other strains, the maximum value shown are from the Δspectrum of the corresponding strain minus the spectrum of the EH-3-248-8 strain encoding no hydroxylase (empty pSP-G1). b Maximum emission and excitation values for the Δspectra that are more than 10 nm different than the corresponding values obtained for strain EH-3-248-8 encoding no hydroxylase (empty pSP-G1) are shown in bold.

In order to select strains and conditions for larger scale reactions, strains encoding hydroxylases that performed favorably according to the Western Blot, mass spectrometry and UV/Vis spectroscopy results were analyzed in a larger set of conditions using mass spectrometry.

Following cell lysis, lysates were incubated overnight in four different conditions (Table 3-3) and analyzed by mass spectrometry. The rationale behind trying different conditions was that given that the conversion rates for all non-OxyS hydroxylases were significantly lower than for OxyS

(Figure 3-9), the reaction should be optimized to allow enough product to be isolated for NMR

94 analysis. The strains that show hydroxylation levels potentially above background, as indicated by

443.4 ion counts that are more than double those for the no hydroxylase negative control encoding empty pSP-G1, are OxyS and PgaE, with OxyS-DacO1-37 as the closest runner up (Figure 3-10).

These four strains will be used for a larger scale lysate experiment followed by product isolation and analysis by NMR spectroscopy. Condition D, maximizing the m/z peak associated with tetracycline ([M + H]+) per amount of cell lysate used will be used to attempt the isolation of tetracycline or its 6-epimer and condition B maximizing the m/z peak associated with 5a(11a)- dehydrotetracycline ([M + H]+) will be used to attempt the isolation of the latter or its 6-epimer.

Table 3-3 Assay conditions for anhydrotetracycline hydroxylation and reduction in cell lysates of strains expressing DacO1, its fusion proteins and other bacterial hydroxylases Condition no.a Atc (mM) G6P (10 mM) Cell lysate dilution factor into reaction mix A 5.4 + 5X B 5.4 - 5X C 21.6 + 5X D 21.6 + 10X aCell lysates were placed overnight in Tris buffer (100 mM, pH 7.45) containing in addition to anhydrotetracycline and glucose-6-phosphate at the concentrations mentioned above, glucose (27.8 mM), NADPH (3 mM) and mercaptoethanol (18.5 mM).

Figure 3-10 Mass spectrometry analysis of anhydrotetracycline hydroxylation and reduction in cell lysates of strains expressing DacO1, its fusion proteins and other bacterial hydroxylases The analyzed strains shown EH-3-248-1, EH-3-248-4, EH-3-248-7, EH-3-248-8, EH-5-163-3-C2, EH-5-163-4-C2, EH-5-163-6-C1, EH-5-163-7-C1 are encoding hydroxylases OxyS, DacO1, PgaE, pSPG1 (no hydroxylase negative control), Ubiquitin-γ-IFN-DacO1, γ-IFN-DacO1, OxyS- DacO1-37 and OxyS-DacO1-87, respectively.

While for both OxyS and PgaE, in the presence of G6P there is an increase in 445 ion counts associated with the product of both hydroxylation and reduction reactions (Figure 2-3 and Figure

3-10), the PgaE samples are consistently associated with higher 445/443 ion count ratios relative

96 to the OxyS samples in the presence of G6P (Figure 3-11). This supports that in the case of PgaE hydroxylation a larger fraction of the hydroxylation product ([M-H]+ = 443) gets further reduced

([M-H]+ = 445).

Figure 3-11 Mass spectrometry analysis of anhydrotetracycline hydroxylation and reduction in cell lysates of strains expressing OxyS and PgaE The analyzed strains shown are EH-3-248-1 and EH-3-248-7 encoding hydroxylases OxyS and PgaE, respectively in condition A (Table 3-3). TIC is total ion count. 443 and 445 refer to ion counts of these m/z values.

3.5 Discussion

3.5.1 Optimizing DacO1 expression in S. cerevisiae – rational design

DacO1 is hypothesized to be the 6-hydroxylase enzyme in the dactylocycline pathway based on its homology to OxyS, the 6-hydroxylase of the oxytetracycline pathway. Based on this assignment, DacO1 is performing its 6-hydroxylase function when heterologously expressed together with the rest of the dactylocyclinone pathway in S. lividans.(2) Heterologous expression of DacO1 exclusively does not yield a stably expressed protein in either E. coli or Stryptomyces,(1) and as shown in this study neither does it stably heterolougsly expressed in S. cerevisiae (Figure

3-1). These results taken together with the importance of coexpression of other pathway proteins to the function of other polyketide enzymes,(19, 20) led to the hypothesis that coexpression of

97 pathway proteins might be beneficial for DacO1 stable expression in S. cerevisiae. For the particular proteins whose coexpression with DacO1 in S. cerevisiae was attempted, DacJ and

DacM2, this hypothesis was not confirmed (Table 3-1). However, it could be that other or additional proteins from the dactylocycline pathway are needed for stable heterologous expression of DacO1. In contrast, the strategy to improve DacO1 expression by N-terminal fusions was successful (Sections 3.4.5 and 3.5.5).

3.5.2 Searching the bacterial hydroxylase space for 6α-hydroxylation of

anhydrotetracyclines

The successful identification of PgaE as a hydroxylase of anhydrotetracycline is shown in Section

3.4.5 and discussed in Section 3.5.5. PgaE heterologous expression in S. cerevisiae thus exemplifies the power of natural enzyme diversity harvesting and testing in S. cerevisiae as a heterologous host.

3.5.3 Developing a microtiter plate assay for anhydrotetracycline

hydroxylation

The microtiter plate assay for hydroxylation of anhydrotetracyclines is functional, as confirmed by differentiation between the OxyS encoding strain and the negative control strain (Figure 3-3) and its throughput could be further increased. One approach to increase the assay throughput would be to employ an automatic liquid dispenser / colony picker as well as to reduce the volume of each well in the assay.(25, 26) In the current assay format only 2 μL of the 300 μL suspension are needed for the final UV/Vis analysis. An alternative approach to increase the assay throughput would be to grow colonies on agar plates supplemented with anhydrotetracycline and monitor the change in fluorescence by a fluorescence agar plate imager and thus circumventing the need for

98 microtiter plate handling. However, given that anhydrotetracycline is toxic to S. cerevisiae at elevated concentrations it remains to be determined whether an agar plate UV/Vis assay is practical.(27, 28) On the other hand, that very toxicity could prove useful to develop a growth- based assay, as tetracyclines eukaryotic toxicity is significantly lower than that of anhydrotetracyclines. Thus, conversion of anhydrotetracycline to tetracycline could lead to an increased growth rate in media containing anhydrotetracycline.(27, 28)

The stereochemical difference between the expected products of anhydrotetracycline hydroxylation by a 6β-hydroxylase such as OxyS and a 6α-hydroxylase such as DacO1, 5a(11a)- dehydrotetracycline and 5a(11a)-6-epidehydrotetracycline, respectively, might lead to a difference in the absorption/emission of the respective products. Thus, an optimized spectrophotometer measurement protocol for the microtiter plate assay for anhydrotetracycline hydroxylation might be desired. However, in both cases both absorption and emission are expected to be blue-shifted, a difference that the current protocol is capable of detecting. DacO1 hydroxylation is supported by

UV/Vis evidence of the current protocol (Figure 3-4) although it is still to be determined whether

5a(11a)-6-epidehydrotetracycline is indeed synthesized from the reaction of DacO1 with anhydrotetracycline (Section 3.5.5).

3.5.4 Evolving a 6α-hydroxylase of anhydrotetracyclines – DacO1 random

mutagenesis

Importantly, the library-based (Section 3.4.4 and Section 4.4.3) and rational design-based (Section

3.4.1) approaches for optimizing DacO1 for 6α-hydroxylation of anhydrotetracycline in S. cerevisiae are not mutually exclusive. The winner of the DacO1 error-prone mutagenesis screen and additional winners of future screens could be sequenced to identify the amino acids at which mutations occurred and these sites could be chosen for saturation mutagenesis. The resulting

99 library of size ~100 could be then sequenced by the microtiter plate assay for anhydrotetracycline hydroxylation with a followup on screen hits as shown (Figure 3-4). The sequence of the confirmed hits can then be fused in DacO1-fusions that were confirmed to be effective in enhancing DacO1 expression levels such as UbiIFN-DacO1, IFN-DacO1, OxyS-DacO1-37 and OxyS-DacO1-87

(Figure 3-8). Alternatively the expression optimized DacO1 fusion porteins could be used for EP-

PCR or saturation mutagenesis as per Section 3.4.4 and Section 4.4.3.

3.5.5 Testing DacO1 expression optimization constructs and other

hydroxylases

The attempt to fuse a stable protein or protein fragment at the N-terminus of DacO1, or a DacO1 fragment in the case of the OxyS fusions, to improve DacO1 expression levels was successful

(Figure 3-8). However, the more stably expressed DacO1 fusions are still poor hydroxylases of anhydrotetracycline at best compared to OxyS (Figure 3-9 and Figure 3-10). A very likely hypothesis to explain the poor hydroxylation activity is that the natural substrate of DacO1, anhydrodactylocyclinone, is different enough from anhydrotetracycline to impede efficient hydroxylation of the latter. In fact, the hydroxylase SsfO1, whose heterologous expression was confirmed to be effective in S. cerevisiae (Figure 3-5 and Figure 3-6) did not prove an effective hydroxylase of anhydrotetracycline at all (Figure 3-9). The native substrate of DacO1 differs from anhydrotetracycline by having 8-methoxy, 7-chloro and 4a-hydroxy instead of protons in anhydrotetracycline and it could be that these functional groups serve as recognition elements in the substrate (Scheme 3-1). The 8-methoxy and 7-chloro substituents can could further have an electronic contribution to the activation energy of the hydroxylation reaction.

With stably expressed DacO1 fusion proteins at hand such as ubiquitin-γ-IFN-DacO1, γ-IFN-

DacO1, OxyS-DacO1-37 and OxyS-DacO1-87 (Figure 3-8), a few avenues could now be

100 explored: (i) upscale current hydroxylation setup (Table 3-3 and Section 3.4.5) and attempt isolation of product and analysis by NMR to verify whether the desired 6α-hydroxylation occurred.

(ii) mutagenize stably expressed DacO1 fusion proteins by error-prone PCR (Section 3.4.4) or saturation mutagenesis (Section 4.4.3) or a combination thereof and then test for anhydrotetracycline or 6-demethylanhydrotetracycline(29) hydroxylation by the mutants using the microplate UV/Vis assay (Section 3.4.3); (iii) test DacO1 fusion proteins hydroxylation of the

DacO1 native substrate, anhydrodactylocyclinone (Scheme 3-1) or 6- demethyanhydrodactylocyclinone; and (iv) test TAN-1612 hydroxylation by coexpressing the

DacO1 fusion proteins and the TAN-1612 pathway (Chapter 4). Given the high biological variation between the different colonies expressing DacO1-fusion proteins (Figure 3-8), testing several biological replicates for each strain for these approaches might be crucial.

Specifically for (iii), Anhydrodactylocyclinone and 6-demethylanhydrodactylocyclinone could be obtained either by deleting the dacO1 and dacO4 or the dacO1, dacO4 and dacM2 genes from plasmid pDac, respectively, and transforming the resulting plasmid into a K4-114 strain harboring pDacT1O3E.(2) Importantly, isolating anhydrodactylocyclinone and/or 6- demethylanhydrodactylocyclinone using such plasmids would support the functional assignment of DacO1, DacO4 and DacM2. Alternatively the DacO1 native substrate could be pursued by acidic dehydration of dactylocyclinone,(29) the aglycone of dactylocycline A, which can be purified either from its native producer Dactylosporangium sp. SC 14051 (ATCC 53693) or from the heterologous producer K4/pDac-T1O3E.(2, 30, 31) A coculture of a (6-demethyl-

)anhydrodactylocyclinone producing strain and the strain encoding the DacO1 fusion protein is another potential alternative. Finally, it is also potentially possible to feed the strain encoding the

101

DacO1 fusion protein with crude (6-demethyl-)anhydrodactylocyclinone without prior purification.

PgaE, a bacterial monooxygenase tested as a potential 6α-hydroxylase candidate (Section 3.4.2) was found to hydroxylate anhydrotetracycline (Sections 3.4.5 and 3.5.2). Furthermore, in the presence of PgaE more reduced product was obtained relative to the hydroxylated product, when compared to OxyS (Figure 3-11). Possible reasons for increased reduction proportion of hydroxylated anhydrotetracycline in the PgaE sample (Figure 3-11) could be (i) a lower hydroxylation rate in the case of PgaE results in less overload on reduction capacities (ii) a different hydroxylation product between OxyS and PgaE, such as different stereochemistry that leads to a more ready reduction of the PgaE hydroxylation product, (iii) an involvement of OxyS and/or

PgaE in the reduction process either directly by catalyzing it or indirectly by facilitating the process through product binding. These reasons could be tested by isolating the hydroxylation product from two different cell lysates expressing OxyS or PgaE and not containing G6P and analyzing the hydroxylated product by NMR. Studying and comparing the spectra would support or reject reason (ii). The isolated hydroxylated product can then be added to one of three cell lysates in the presence of G6P, expressing OxyS or PgaE or no hydroxylase. This will eliminate hydroxylation levels influence as per reason (i) and will enable testing whether OxyS and/or PgaE are involved in the reduction as per reason (iii), although it is still perhaps possible that involvement of OxyS and/or PgaE in the reduction happens only immediately after the hydroxylation before product release.

3.6 Conclusion

This chapter describes the progress towards 6-demethyl-6-epitetracyclines biosynthesis in S. cerevisiae (Scheme 3-2). The biosynthesis of 6-demethyl-6-epitetracyclines as opposed to 6-

102 demethyltetracyclines requires a 6α-hydroxylase, such as DacO1, as opposed to a 6β-hydroxylase such as OxyS (Scheme 3-1). However, past attempts to stably heterologoulsy express DacO1 without the rest of its pathway have been unsuccessful.(1) With the rational design attempt to stabilize DacO1 expression by N-terminal fusion proteins proving successful (Figure 3-8), the next step is to test the stably expressed DacO1 fusion proteins in larger scale for 6α-hydroxylation of anhydrotetracycline and/or anhydrodactylocyclinone and/or 6-demethylanhydrodactylocyclinone

(Section 3.5.5). This will allow product isolation followed by NMR analysis. In addition, given the differences between DacO1’s native substrate and anhydrotetracycline (Scheme 3-1) and given that DacO1 fusion proteins are poor hydroxylases of anhydrotetracycline at best (Figure 3-9 and

Figure 3-10), the stably expressed DacO1 fusion proteins could be subjected to mutagenesis

(Section 3.4.4 and Section 4.4.3) to identify mutants that readily hydroxylate the desired anhydrotetracycline substrate.

In addition to the use of rational design to improve DacO1 expression, this chapter also describes harvesting natural diversity in bacterial hydroxylases towards the search for a 6α-hydroxylase

(Section 3.4.2). With PgaE, one of these bacterial hydroxylases, proving as a hydroxylase of anhydrotetracycline (Figure 3-9 and Figure 3-10) the next step is to analyze its product after large scale preparation and isolation and to verify whether it is performing 6α-hydroxylation or 6- hydroxylation at all on anhydrotetracycline. It also remains to be determined why a larger proportion of the hydroxylation product of anhydrotetracycline undergoes reduction when PgaE is used as the hydroxylase in comparison to when OxyS is used (Figure 3-11) and whether either of these hydroxylases participates in reduction as well. Participation of OxyS or PgaE in the reduction reaction could theoretically take place by direct catalysis of the reduction or by facilitating catalysis through product binding. These open questions could be better understood by adding the isolated

103 hydroxylation product of OxyS and of PgaE, each to three separate cell lysates of strains encoding

OxyS, PgaE and no hydroxylases.

Finally, using a variant of the microtiter plate assay for anhydrotetracycline hydroxylation (Figure

3-2 and Figure 3-3), the promising 6α-hydroxylation candidates identified in this chapter (Table

3-2) were screened for hydroxylation of the fungal anhydrotetracycline TAN-1612 in the next chapter.

104

3.7 Appendix

Table 3-4 Strains used in this study Genotype Source/Reference FY251 MATa leu2Δ1 trpΔ63 ura3-52 his3-200 ATCC 96098 BJ5464- MATα ura3-52 his3-Δ200 leu2-Δ1 a / (15) NpgA trp1 pep4::HIS3 δ:: pADH2-npgA-tADH2 prb1Δ1.6R can1 GAL EH-3-80-2 FY251 AL-1-31-C7 This Study EH-3-80-3 FY251 pSP-G1 Chapter 2 EH-3-98-6 FY-251 AL-1-101-C10 Chapter 2 ΕΗ-5-153-7 FY-251 AL-1-173-B-C4 This Study ΕΗ-5-153-8 FY-251 AL-1-119-A-C7 Chapter 2 EH-3-248-1 BJ5464-NpgA AL-1-101-C10 Chapter 2 EH-3-248-2 BJ5464-NpgA AL-1-119-A-C7 This Study EH-3-248-3 BJ5464-NpgA AL-1-26-1-C1 This Study EH-3-248-4 BJ5464-NpgA AL-1-31-C7 This Study EH-3-248-5 BJ5464-NpgA AL-1-234-B-C2 This Study EH-3-248-6 BJ5464-NpgA AL-1-239-B-C3 This Study EH-3-248-7 BJ5464-NpgA AL-1-234-C-C1 This Study EH-3-248-8 BJ5464-NpgA pSP-G1 Chapter 2 EH-3-270-9 FY-251 AL-2-8-D This Study EH-5-19-2 FY-251 AL-2-8-D This Study EH-5-98-1 FY251 EH-5-49-A This Study EH-5-98-2 FY251 EH-5-49-B This Study EH-5-98-3 FY251 EH-5-49-C This Study EH-5-98-4 FY251 EH-5-80-1 This Study EH-5-98-5 FY251 EH-5-80-2 This Study EH-5-98-6 FY251 EH-5-80-3 This Study EH-5-98-7 FY251 EH-5-80-4 This Study EH-5-98-8 FY251 EH-5-80-5 This Study EH-5-98-9 FY251 EH-5-80-6 This Study EH-5-98-10 FY251 EH-5-80-7 This Study EH-5-98-11 FY251 EH-5-80-8 This Study EH-5-98-12 FY251 EH-5-80-9 This Study EH-5-98-13 FY251 EH-5-80-10 This Study EH-5-98-14 FY251 EH-5-80-11 This Study EH-5-98-15 FY251 EH-5-80-12 This Study EH-5-98-16 FY251 AL-1-239-A This Study EH-5-98-17 FY251 AL-1-239-B This Study EH-5-98-18 FY251 EH-5-90-A This Study EH-5-98-19 FY251 EH-5-90-A This Study EH-5-115-1 FY251 EH-5-90-A EH-5-105-Α This Study EH-5-115-2 FY251 AL-1-31-C7 EH-5-105-A This Study 105

EH-5-115-3 FY251 pSP-G1 EH-5-105-A This Study EH-5-115-4 BJ5464-NpgA AL-1-226-A This Study EH-5-115-5 BJ5464-NpgA AL-1-239-A This Study EH-5-163-1 BJ5464-NpgA EH-5-49-A This Study EH-5-163-2 BJ5464-NpgA EH-5-49-C This Study EH-5-163-3 BJ5464-NpgA EH-5-80-6 This Study EH-5-163-4 BJ5464-NpgA EH-5-80-7 This Study EH-5-163-5 BJ5464-NpgA EH-5-80-9 This Study EH-5-163-6 BJ5464-NpgA EH-5-80-10 This Study EH-5-163-7 BJ5464-NpgA EH-5-80-11 This Study EH-5-163-8 FY251 AL-1-234-C-C1 This Study a Y. Tang, University of California, Los Angeles, Department of Chemical and Biomolecular Engineering & Department of Chemistry and Biochemistry, Department of Bioengineering

Table 3-5 Plasmids used in this study Description Source/Reference pSP-G1 pTEF1-FLAG-tADH1, pPGK1-MYC-tCYC1, 2 μ, Ura Addgene 64736 pRS413-pGAL1 Shuttle vector (empty), His marker, CEN ATCC 87326 pRS416- pGAL1 Shuttle vector (empty), Ura marker, CEN ATCC 87332 pRS426- pGAL1 Shuttle vector (empty), Ura marker, 2 μ ATCC 87361 AL-1-26-1-C1 pPGK1-oxyR-tCYC1 in pSP-G1 This Studya AL-1-31-C7 pTEF1-dacO1-tADH1 in pSP-G1 This Study AL-1-101-C10 pTEF1-oxyS-tADH1 in pSP-G1 Chapter 2 AL-1-119-A-C7 pTEF1-oxyS-tADH1, pPGK1-oxyR-tCYC1 in pSP-G1 Chapter 2 AL-1-173-B-C4 pTEF1-dacO1-tADH1, pPGK1-dacO4-tCYC1 in pSP- This Study G1 AL-1-226-A-C2 pTEF1-oxyS-tADH1 in pSP-G1 JCAT codon opt. This Study AL-1-234-B-C2 pTEF1-ctcN-tADH1 in pSP-G1 JCAT codon opt. This Study AL-1-234-C-C1 pTEF1-pgaE-tADH1 in pSP-G1 JCAT codon opt. This Study AL-1-239-A-C2 pTEF1-dacO1-tADH1 in pSP-G1 JCAT codon opt. This Study AL-1-239-B-C3 pTEF1-ssfO1-tADH1 in pSP-G1 JCAT codon opt. This Study AL-2-8-D Error-prone mutagenesis library of AL-1-31-C7 This Study EH-5-49-A pTEF1-UBI4-dacO1-tADH1 in pSP-G1 This Study EH-5-49-B pTEF1-SOD1-dacO1-tADH1 in pSP-G1 This Study EH-5-49-C pTEF1-GAL1-dacO1-tADH1 in pSP-G1 This Study EH-5-80-1 pGAL1-dacO1-tCYC1 in pRS416 This Study EH-5-80-2 pGAL1-dacO1-tCYC1 in pRS426 This Study EH-5-80-3 pTEF1-dacO1-tADH1 in pSP-G1 (no antibody tag) This Study EH-5-80-4 pADH2-dacO1-tCYC1 in pRS416 This Study EH-5-80-5 pADH2-dacO1-tCYC1 in pRS426 This Study EH-5-80-6 pTEF1-UBI4-IFNG-dacO1-tADH1 in pSP-G1b This Study EH-5-80-7 pTEF1-IFNG-dacO1-tADH1 in pSP-G1b This Study EH-5-80-8 pPGK1-dacO4-dacO1-tCYC1 in pSP-G1 This Study

106

EH-5-80-9 pPGK1-dacO1-dacO4-tCYC1 in pSP-G1 This Study EH-5-80-10 pTEF1-oxyS-dacO1-37-tADH1 in pSP-G1 This Study EH-5-80-11 pTEF1-oxyS-dacO1-87-tADH1 in pSP-G1 This Study EH-5-80-12 pTEF1-oxyS-dacO1-191-tADH1 in pSP-G1 This Study EH-5-90-A pTEF1-dacO1-tADH1, pPGK1-dacJ-tCYC1 in pSP-G1 This Study EH-5-105-Α pGPD-dacM2-tCYC1 in pRS413 This Study a See Chapter 2 for sequence. b the sequence of UBI4 and of IFNG used in the gene contrasts here is partial. See Table 3-6

Table 3-6 Sequences used in this study Sequences for the hydroxylases DacO1 (GenBank: AFU65892.1), PgaE (PDB: 2QA1_A), SsfO1 (GenBank: GQ409537.1), CtcN (GenBank: D38214.1) and the DacO1 fusion proteins with UBI4 (GenBank: AF134781.1), SOD1 (GenBank: KJ892183.1), GAL1 (NM_001178368.1), IFNG (GenBank: X62468.1) and OxyS (AAZ78342.1) are shown capitalized within the context of the pSP-G1 backbone containing pTEF1, the FLAG tag and tADH1 (partial, decapitalized). Sequences for DacO4 (GenBank: JX262387.1), the DacO1-DacO4 and DacO4-DacO1 fusions and DacJ (GenBank: JX262387.1) are shown capitalized within the context of the pSP-G1 backbone containing pPGK1, the myc tag and tCYC1 (partial, decapitalized). The sequence for DacM2 (GenBank: JX262387.1) is shown along with the pGPD (pTDH3) promoter capitalized within the context of the pRS413 backbone containing the tCYC1 terminator (partial, decapitalized). The sequences for DacO1 in pRS backbone under the control of pGAL1 or pADH2 is shown capitalized along with the promoter (decapitalized in the case of pGAL1 and capitalized in the case of pADH2) within the context of the pRS backbone containing the tCYC1 terminator (partial, decapitalized). pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa dacO1- gttttaattacaagcggccgcATGTTAGTAGCCGGTGGTGGACCAACAGGGTTAT tADH1-in- GGTTGGCTGGTGAACTAAGGTTGGCCGGGGTTAGACCTCTTGTTTTA pSP-G1- GAGCGTAGGACAGAACCCTTTCCACATAGCAAAGCACTGGGTATCCA AL-1-31- TCCAAGAACTATTGAAATGTTGGAGTTGAGAGGCCTTGCTGGTAGAT C7 and AL- TTTTAGATGGCGCCCCGAAGCTGCCGAAAGGTCACTTTGCATCGCTTC 1-173-B- CTGTGCCCTTAGACTATGGTGCAATGGATACTCGACATCCGTACGGT C4 GTCTACCGTAAACAAGTGGAAACGGAAGCTTTATTAGCCGGCCACGC AACCAGATTAGGAGTTCCAGTCCGCAGAGGTCATGAACTTGTAGGCC TAAGACAGCACGATGATGCTGTAGTCGGTACTGTTCAAGGTCCTCAA GGTAGATACGAAGTCAGAGCCGCTTATCTTGTTGGTTGTGATGGAGG

107

AGGTTCTACCACAAGACGCCTGGCTGGGATAGATTTCCCTGGACAAG ATCCTGGTGTTGCCGTTTTGATGGCTGACGCAAGATTCCGGGACCCAT TACCCTCTGATCCAGCTATGGGTCCATTCAGACGCTATGGAGTCCTGA GACCTGATTTAAGGGCATGGTTTAGTGCTATTCCTTTACCAGAAGAA CAGGGTTTATGCAGAGTTATGGTTTTTTGGTATGGACGACAATTTGAA GATAGACGTGCTCCAGTAACTGAGGATGAAATGAGAGCAGCACTTGT TGATCTAGCGGGAACTGACTTTGGTATGTACGATGTTCAATGGTTGA CCAGATTCACAGACGCTTCCAGGCAAGCTGCAAGGTATAGATCTGGA AGGGTTTTCTTAGCTGGAGATGCTGCTCATACTGTCTTCCCCACTGGT GGCCCAGGTTTGAATGCAGGTTTACAAGATGCCATGAATTTAGGTTG GAAGTTAGCTGGAGCTGTTCATGGCTGGGGTGGTGCTTTACTAGATT CATATCATGATGAGCGTTACCCTGTAGCTGAACAAGTTTTGCGTGAC ACAAGAACACAATGTCTATTGTTAGATCCAGACCCTCGCTTTGTTCCA TTAAGAGAGACGTTAGCAGAACTATTGAGACTAGAACCCGTAAACA GATATTTCACCGGACACAACACGGCTCTAGCAGTTAGATACGATTTG GGTGGTGCCCATCCTGCAACGGGAGGCAGAATGCCAGATGTCTTGGT CGCAACTGGTGCTGGTCAAAGAAGGTTTTCAGATTGTTGTGTTACCG GGAGAGGGGTTCTTTTAGTCACTTCTGGTGCAGATAGGGGGGAACTG TTACGAGGATGGAAAGATAGAGTGGATGTGGTAACAGGTACCGTGG CAGGACCTCTTGATGCAGCTGATCATTTAGTTAGACCTGATGGTTATG TTGCATGGGCCGGTGGAGCTGGACACGATGAACCTGGTTTAGAAACT GCTCTTAGATTATGGTTTGGTGAACCAGTTAGTgtatcgatggattacaaggatgac gacgataaAatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggct pPGK1- tggaatggcgggaaagggtttagtaccacatgctatgatgcccactgtgatctccagagcaaagttcgttcgatcgt dacO4- actgttactctctctctttcaaacagaattgtccgaatcgtgtgacaacaacagcctgttctcacacactcttttcttctaa tCYC1-AL- ccaagggggtggtttagtttagtagaacctcgtgaaacttacatttacatatatataaacttgcataaattggtcaatgc 1-173-B- aagaaatacatatttggtcttttctaattcgtagtttttcaagttcttagatgctttctttttctcttttttacagatcatcaagga C4 agtaattatctactttttacaacaaatataaaacaaggatccATGTCATTCACTGCCAAGGAAGT TCAATATTTGCGTTCTCAGCAATTAGGAAGATTAGCCACCGTTGGTGC TGATGGTACACCACATAATGTTCCAGTAGGCTACAGATACAACGCTG ACTTGGGGACCATTGACATCACAGGAAGGGACTTAAGAAGAAGTAG GAAATATAGAGATGTTCAGGCTGGCTCGCGGGTGGCATTTATTGTTG ATGATCTACCGAGCACAGCACCTATAGTCGCCCGCGGGCTGGAGGTG AGGGGAACAGCTGAGGCGCTTTCTGGTGAAGAAGATTTGATACGTGT ACGACCTGCAAGAATCGTCACTTGGGGTATTGAAGCAGATTGGCAAG CTGGTCCTACTGGTAGAACGGTAAGGCCCACCACTCCAAGATCCGCG ACGgtcgacatggaacagaagttgatttccgaagaagacctcgagtaagcttggtaccgcggctagctaagatc cgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaag aacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgag aaggttttgggacgctcgaagatccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattg ggcgctcttccgcttcctcgctcactgactcgctgcgctcgg pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa oxyS- gttttaattacaagcggccgcATGAGATACGACGTTGTTATCGCTGGTGCTGGTCC tADH1 in AACTGGTTTGATGTTGGCTTGTGAATTGAGATTGGCTGGTGCTAGAA pSP-G1 CTTTGGTTTTGGAAAGATTGGCTGAACCAGTTGACTTCTCTAAGGCTT TGGGTGTTCACGCTAGAACTGTTGAATTGTTGGACATGAGAGGTTTG

108

JCAT GGTGAAGGTTTCCAAGCTGAAGCTCCAAAGTTGAGAGGTGGTAACTT codon opt. CGCTTCTTTGGGTGTTCCATTGGACTTCTCTTCTTTCGACACTAGACA (AL-1-226- CCCATACGCTTTGTTCGTTCCACAAGTTAGAACTGAAGAATTGTTGAC A) TGGTAGAGCTTTGGAATTGGGTGCTGAATTGAGAAGAGGTCACGCTG TTACTGCTTTGGAACAAGACGCTGACGGTGTTACTGTTTCTGTTACTG GTCCAGAAGGTCCATACGAAGTTGAATGTGCTTACTTGGTTGGTTGT GACGGTGGTGGTTCTACTGTTAGAAAGTTGTTGGGTATCGACTTCCCA GGTCAAGACCCACACATGTTCGCTGTTATCGCTGACGCTAGATTCAG AGAAGAATTGCCACACGGTGAAGGTATGGGTCCAATGAGGCCATAC GGTGTTATGAGACACGACTTGAGAGCTTGGTTCGCTGCTTTCCCATTG GAACCAGACGTTTACAGAGCTACTGTTGCTTTCTTCGACAGACCATA CGCTGACAGAAGAGCGCCAGTTACTGAAGAAGATGTTAGAGCTGCTT TGACTGAAGTTGCTGGTTCTGACTTCGGTATGCACGACGTTAGATGGT TGTCTCGTTTGACTGACACTTCTCGTCAAGCTGAAAGATACAGAGAT GGTAGAGTTTTGTTGGCTGGTGACGCTTGTCACATCCACTTGCCAGCT GGTGGTCAAGGTTTGAACTTGGGTTTCCAAGACGCTGTTAACTTGGG TTGGAAGTTGGGTGCTACTATCGCTGGTACTGCTCCACCAGAATTGTT GGACACTTACGAAGCTGAAAGAAGGCCAATCGCTGCTGGTGTTTTGA GAAACACTAGAGCGCAAGCTGTTTTGATCGACCCAGACCCAAGATAC GAAGGTTTGAGAGAATTGATGATCGAATTGTTGCACGTTCCAGAAAC TAACAGATACTTGGCTGGTTTGATCTCTGCTTTGGACGTTAGATACCC AATGGCTGGTGAACACCCATTGTTGGGTAGAAGAGTTCCAGACTTGC CATTGGTTACTGAAGATGGTACTAGACAATTGTCTACTTACTTCCACG CTGCTAGAGGTGTTTTGTTGACTTTGGGTTGTGACCAACCATTGGCTG ACGAAGCTGCTGCTTGGAAGGACAGAGTTGACTTGGTTGCTGCTGAA GGTGTTGCTGACCCAGGTTCTGCTGTTGACGGATTAACTGCTTTGTTG GTTAGACCAGACGGTTACATCTGTTGGACTGCTGCTCCAGAAACTGG TACTGACGGTTTGACTGACGCTTTGAGAACTTGGTTCGGTCCACCTGC TATGgtatcgatggattacaaggatgacgacgataagatctgagctcttaattaacaattcttcgccagaggtttg gtcaagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa dacO1- gttttaattacaagcggccgcATGTTGGTTGCTGGTGGTGGTCCAACTGGTTTGTG tADH1 in GTTGGCTGGTGAATTGAGATTGGCTGGTGTTAGACCATTGGTTTTGGA pSP-G1 AAGAAGAACTGAACCATTCCCACACTCTAAGGCTTTGGGTATCCACC JCAT CAAGAACTATCGAAATGTTGGAATTGAGAGGTTTGGCTGGTAGATTC codon opt. ( TTGGACGGTGCTCCAAAGTTGCCAAAGGGTCACTTCGCTTCTTTGCCA AL-1-239- GTTCCATTGGACTACGGTGCTATGGACACTAGACACCCATACGGTGT A) TTACAGAAAGCAAGTTGAAACTGAAGCTTTGTTGGCTGGTCACGCTA CTAGATTGGGTGTTCCAGTTAGAAGAGGTCACGAATTGGTTGGTTTG AGACAACACGACGACGCTGTTGTTGGTACTGTTCAAGGTCCACAAGG TAGATACGAAGTTAGAGCTGCTTACTTGGTTGGTTGTGACGGTGGTG GTTCTACTACTAGAAGATTGGCTGGTATCGACTTCCCAGGTCAAGAC CCAGGTGTTGCTGTTTTGATGGCTGACGCTAGATTCAGAGATCCATTG CCATCTGACCCAGCTATGGGTCCATTCAGAAGATACGGTGTTTTAAG ACCAGACTTGAGAGCTTGGTTCTCTGCTATCCCATTGCCAGAAGAAC AAGGTTTGTGTAGAGTTATGGTTTTCTGGTACGGTAGACAATTCGAA

109

GATAGAAGAGCGCCAGTTACTGAAGATGAAATGAGAGCTGCTTTGGT TGACTTGGCTGGTACTGACTTCGGTATGTACGACGTTCAATGGTTGAC TAGATTCACTGACGCTTCTCGTCAAGCTGCTAGATACAGATCGGGTA GAGTTTTCTTGGCTGGTGACGCTGCTCACACTGTTTTCCCAACTGGTG GTCCAGGTTTGAACGCTGGTTTGCAAGACGCTATGAACTTGGGTTGG AAGTTGGCTGGTGCTGTTCACGGTTGGGGTGGTGCTTTGTTGGACTCT TACCACGACGAAAGATACCCAGTTGCTGAACAAGTTTTGAGAGACAC TAGAACTCAATGTTTGTTGTTGGACCCAGACCCAAGATTCGTTCCATT GAGAGAAACTTTGGCTGAATTGTTGAGATTGGAACCAGTTAACAGAT ACTTCACTGGTCACAACACTGCTTTGGCTGTTAGATACGACTTGGGTG GTGCTCACCCAGCTACTGGTGGTAGAATGCCAGACGTTTTGGTTGCT ACTGGTGCTGGTCAAAGAAGATTCTCTGACTGTTGTGTTACTGGTAG AGGTGTTTTGTTGGTTACTTCTGGTGCTGACAGAGGTGAATTGTTGAG AGGTTGGAAGGACAGAGTTGACGTTGTTACTGGTACTGTTGCTGGTC CATTGGACGCTGCTGACCACTTGGTTAGACCAGACGGTTACGTTGCTT GGGCTGGTGGTGCTGGTCACGACGAACCAGGTTTGGAAACTGCTTTG AGATTGTGGTTCGGTGAACCAGTTTCTgtatcgatggattacaaggatgacgacgataag atctgagctcttaattaacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa ctcN- gttttaattacaagcggccgcATGGTTGTTGCTGGTGCTGGTCCAACTGGTTTGAT tADH1 in GTTGGCTTGTGAATTGGCTTTGGGTGGTGCTAGAGCTGTTGTTGTTGA pSP-G1 AAGAAGAAGAGAACCAGAAAAGCACTCTAAGGCTATGGGTATGCAA JCAT GGTAGAACTGTTGAATTGTTGGAATTGAGAGGTTTGTTGGACAGATT codon opt. CAAGGAAGGTGCTGGTGTTTTGCAAGGTGGTAACTTCGCTTCTTTGG (AL-1-234- GTGTTCCAATGAGATTCGAAGAATTTGACACTCAACCAACTCAATAC B-C2) GTTTTGTTGGTTCCACAATTGAGAACTGAAGAATTGTTGGCTGAAAG AGCTAGAGAATTGGGTGTTAGAATCGTTAGAGGTTCTGGTGTTACTG GTTTCGCTCAAGACGCTGACGGTGTTACTGTTGAAACTGACACTGGTT TGTTGAGAGCTAGATACTTGGTTGGTTGTGACGGTGGTTCTACTGTTA GAAAGGCTGCTGGTATCGGTTTCACTGGTCAAGACCCACACATGTAC GCTTTGATCGGTGACATGAGATTCTCTGGTGACTTGCCAAGAGGTGA AGGTTTGGGTCCAATGAGGCCAGTTGGTTTGGTTTACAGAGCTACTG TTGCTTGGTTCGACAGACCATTCGCTGACAGAAGAGCGCCAGTTACT GAAGAAGAAATGAGAGCTGCTTTGGTTGAACACACTGGTTCTGACCA CGGTATGCACGACGTTACTTGGTTGTCTCGTTTGACTGACGTTTCTCG TTTGGCTGACTCTTACAGATTGGGTAGAGTTTTGTTGGCTGGTGACGC TGCTCACATCCACTTGCCAGCTGGTGGTCAAGGTTTGAACTTGGGTTT CCAAGACGCTGTTAACTTGGGTTGGAAGTTGGCTGCTGTTGTTAGAG GTCACGGTACTGAAGAATTGTTGGACTCTTACGGTAGAGAAAGAAGG CCAATCGCTGACGGTGTTGTTAGAAACACTAGAACTCAAGCTGTTTT GATCGACCCAGACCCAAGATACGAAGCTCTCAGACAAACTTTGCCAC ACTTGATGGCTTTGCCAGACACTAACAGACACATGGCTGGTATGTTG TCTGGTTTCGACGTTGCTTACGGTGGTGGTGACCACCCATTGGTTGGT AGAAGAATGCCAGACGCTGAATTGATCACTGCTGACGGTCCAAGAA GAATCTCTGACTGTTTCGCTGGTGCTAGAGGTTTGTTGTTGTTGCCAG AACAAGGTCCAACTGCTTCTCCATTGGCTGCTTGGGCTGACAGAGTT

110

GACACTTTGACTGTTAAGTCTGGTGGTCCAGACCCAGACACTGCTCA CTTGGTTAGACCAGACGGTTACGTTGCTTGGGCTGGTGAACCAGCTA GAACTGAAGAATTGCACCACGCTGCTACTACTTGGTTCGGTGCTGCT GCTgtatcgatggattacaaggatgacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggt caagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa pgaE- gttttaattacaagcggccgcATGGACGCTGCTGTTATCGTTGTTGGTGCTGGTCC tADH1 in AGCTGGTATGATGTTGGCTGGTGAATTGAGATTGGCTGGTGTTGAAG pSP-G1 TTGTTGTTTTGGAAAGATTGGTTGAAAGAACTGGTGAATCTCGTGGTT JCAT TGGGTTTCACTGCTAGAACTATGGAAGTTTTCGACCAAAGAGGTATC codon opt. TTGCCAAGATTCGGTGAAGTTGAAACTTCTACTCAAGGTCACTTCGGT (AL-1-234- GGTTTGCCAATCGACTTCGGTGTTTTGGAAGGTGCTTGGCAAGCTGCT C-C1) AAGACTGTTCCACAATCTGTTACTGAAACTCACTTGGAACAATGGGC TACTGGTTTGGGTGCTGACATCAGAAGAGGTCACGAAGTTTTGTCTTT GACTGACGACGGTGCTGGTGTTACTGTTGAAGTTAGAGGTCCAGAAG GTAAGCACACTTTGAGAGCTGCTTACTTGGTTGGTTGTGACGGTGGT AGATCGTCTGTTAGAAAGGCTGCTGGTTTCGACTTCCCAGGTACTGCT GCTACTATGGAAATGTACTTGGCTGACATCAAGGGTGTTGAATTGCA ACCAAGAATGATCGGTGAAACTTTGCCAGGTGGTATGGTTATGGTTG GTCCATTGCCAGGTGGTATCACTAGAATCATCGTTTGTGAAAGAGGT ACTCCACCACAAAGAAGAGAAACTCCACCATCTTGGCACGAAGTTGC TGACGCTTGGAAGAGATTGACTGGTGACGACATCGCTCACGCTGAAC CAGTTTGGGTTTCTGCTTTCGGTAACGCTACTAGACAAGTTACTGAAT ACAGAAGAGGTAGAGTTATCTTGGCTGGTGACTCTGCTCACATCCAC TTGCCAGCTGGTGGTCAAGGTATGAACACTTCTATCCAAGACGCTGT TAACTTGGGTTGGAAGTTGGGTGCTGTTGTTAACGGTACTGCTACTGA AGAATTGTTGGACTCTTACCACTCTGAAAGACACGCTGTTGGTAAGA GATTGTTGATGAACACTCAAGCTCAAGGTTTGTTGTTCTTGTCTGGTC CAGAAGTTCAACCATTGAGAGATGTTTTGACTGAATTGATCCAATAC GGTGAAGTTGCTAGACACTTGGCTGGTATGGTTTCTGGTTTGGAAATC ACTTACGACGTTGGTACTGGTTCTCACCCATTGTTGGGTAAGAGAAT GCCAGCTTTGGAATTGACTACTGCTACTAGAGAAACATCTTCTACTG AATTATTACACACTGCTAGAGGTGTTTTGTTGGACTTGGCTGACAACC CAAGATTGAGAGCTAGAGCTGCTGCTTGGTCTGACAGAGTTGACATC GTTACTGCTGTTCCAGGTGAAGTTTCTGCTACTTCTGGTTTGAGAGAC ACTACTGCTGTTTTGATAAGACCAGACGGTCACGTTGCTTGGGCTGCT CCAGGTTCTCACCACGACTTGCCAATGGCTTTGGAAAGATGGTTCGG TGCTCCATTGACTGGTgtatcgatggattacaaggatgacgacgataagatctgagctcttaattaac aattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa ssfO1- gttttaattacaagcggccgcATGGAACCAGAAGTTGTTGTTGCTGGTGCTGGTCC tADH1 in AGTTGGTTTGATGTTGGCTTGTGAATTGAGAAGGCAAGGTGTTGGTG pSP-G1 TTGTTTTGGTTGAAAGAACTACTGAACCAGCTAACGACAACAGAGCG JCAT CAAGCTCTCCACGGTAGAACTATCCCAGTTTTGGACAGAAGAGGTTT codon opt. GTTGCCAAGATTCAGAGCTGCTGAAAGAGAATTGAACGGTGGTGGTT CTGGTGGTACTGAATCTCACAGAAAAAGACCATTGCCAAGAGGTCAC

111

(AL-1-239- TTCGCTGGTATCACTGGTTTATTACCATCTGCTCCAGACCCAGACCCA B-C3) GACGTTCCACCAGTTGTTTTCGTTCCACAATGGGTTACTACTAGATTG TTGACTGAACACGCTGCTGAATTGGGTGTTGTTTTGCACAGAGGTTCT GAAATCACTGCTGTTGAACAAGACGCTGACGGTGTTACTGTTGCTGT TTCTGGTGGTACTGCTCCAGCTAGATTGAGAGCTAGATACTTGATAG GTTGTGACGGTGCTAGATCGGTTGTTAGAAGATCGGCTGGTATCGAC TTCGCTGGTACTGGTGCTACTTTGTCTACTTTGGCTGCTGAAGTTCAC TTGGACGACCCAGCTGACTTGCCAGTTGGTTGGCACAGAACTCAACA CGGTTGTACTGTTATCTCTCCACACCCAGAAGGTGGTCACTCTCGTGT TGTTGTTATCGACTTCTCTGGTCCAGACGCTGACAGAGAAGCTCCAGT TACTTTGACTGAATTGGAAGAAAGAATCGCTGGTGTTTTGGGTAGAA AGGTTGCTATGTCTGACTTGAAGAACGCTCAAAGATACGGTGACGCT GCTAGATTGGCTGACAGATACAGAGTTGGTAGAGTTTTCTTGGCTGG TGACGCTGCTCACATCCACTACCCAGTTGGTGGTCAAGGTTTGAACA TCGGTTTGCAAGACGCTGTTAACTTGGCTTGGAAGTTGGCTGCTGACT TGAGAGGTTGGGCTCCAGACGGTTTGTTGGACACTTACCACACTGAA AGATACCCAGTTGCTGACTCTGTTTTGACTCACACTAGAGCGCAAGTT GCTTTGTTGGCTCCAGGTCCAAGAATCGACGCTTTGAGAACTTTGTTC ACTGACTTGATCGGTTTGGCTGACGTTTCTCGTTACTTGTCTGAAAAG ATGTCTGCTGCTGACGTTAGATACGACATGGGTGAAGCTGAACCACA CCCATTGACTGGTAGATTCGCTCCATCTTTGAGATTGGCTACTGAACA AGGTGAAAGAAGATTGGCTGACTTGTTGGCTGGTGGTAGACCATTGT TGTTGGACTTGTCTGGTAGAGCTGCTACTGCTGCTGTTGCTGCTAGAT GGGCTGACAGAGTTGACGTTCACGCTGCTACTTGTCCAGCTGAACCA GCTTTGGGTTCTTTGTTGATCAGACCAGACTCTTACGTTGCTTGGGCT TGTGCTCCAGGTGACGCTCCAGACCAAGTTGAAAGAGGTTTGGAAGC TGCTTTGAGAAGATGGTTCGGTGCTCCAGACCACCAAgtatcgatggattacaa ggatgacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtc ggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa UBI4- gttttaattacaagcggccgccattATGCAGATCTTCGTCAAGACGTTAACCGGTAA dacO1- AACCATAACTCTAGAAGTTGAATCTTCCGATACCATCGACAACGTTA tADH1 in AGTCGAAAATTCAAGACAAGGAAGGCATTCCACCTGATCAACAAAG pSP-G1 ATTGATCTTTGCCGGTAAGCAGCTCGAGGACGGTAGAACGCTGTCTG (EH-5-49- ATTACAACATTCAGAAGGAGTCGACCTTACATCTTGTCTTAAGACTA A) AGAGGTGGTATGTTAGTAGCCGGTGGTGGACCAACAGGGTTATGGTT GGCTGGTGAACTAAGGTTGGCCGGGGTTAGACCTCTTGTTTTAGAGC GTAGGACAGAACCCTTTCCACATAGCAAAGCACTGGGTATCCATCCA AGAACTATTGAAATGTTGGAGTTGAGAGGCCTTGCTGGTAGATTTTT AGATGGCGCCCCGAAGCTGCCGAAAGGTCACTTTGCATCGCTTCCTG TGCCCTTAGACTATGGTGCAATGGATACTCGACATCCGTACGGTGTCT ACCGTAAACAAGTGGAAACGGAAGCTTTATTAGCCGGCCACGCAACC AGATTAGGAGTTCCAGTCCGCAGAGGTCATGAACTTGTAGGCCTAAG ACAGCACGATGATGCTGTAGTCGGTACTGTTCAAGGTCCTCAAGGTA GATACGAAGTCAGAGCCGCTTATCTTGTTGGTTGTGATGGAGGAGGT TCTACCACAAGACGCCTGGCTGGGATAGATTTCCCTGGACAAGATCC

112

TGGTGTTGCCGTTTTGATGGCTGACGCAAGATTCCGGGACCCATTACC CTCTGATCCAGCTATGGGTCCATTCAGACGCTATGGAGTCCTGAGAC CTGATTTAAGGGCATGGTTTAGTGCTATTCCTTTACCAGAAGAACAG GGTTTATGCAGAGTTATGGTTTTTTGGTATGGACGACAATTTGAAGAT AGACGTGCTCCAGTAACTGAGGATGAAATGAGAGCAGCACTTGTTGA TCTAGCGGGAACTGACTTTGGTATGTACGATGTTCAATGGTTGACCA GATTCACAGACGCTTCCAGGCAAGCTGCAAGGTATAGATCTGGAAGG GTTTTCTTAGCTGGAGATGCTGCTCATACTGTCTTCCCCACTGGTGGC CCAGGTTTGAATGCAGGTTTACAAGATGCCATGAATTTAGGTTGGAA GTTAGCTGGAGCTGTTCATGGCTGGGGTGGTGCTTTACTAGATTCATA TCATGATGAGCGTTACCCTGTAGCTGAACAAGTTTTGCGTGACACAA GAACACAATGTCTATTGTTAGATCCAGACCCTCGCTTTGTTCCATTAA GAGAGACGTTAGCAGAACTATTGAGACTAGAACCCGTAAACAGATA TTTCACCGGACACAACACGGCTCTAGCAGTTAGATACGATTTGGGTG GTGCCCATCCTGCAACGGGAGGCAGAATGCCAGATGTCTTGGTCGCA ACTGGTGCTGGTCAAAGAAGGTTTTCAGATTGTTGTGTTACCGGGAG AGGGGTTCTTTTAGTCACTTCTGGTGCAGATAGGGGGGAACTGTTAC GAGGATGGAAAGATAGAGTGGATGTGGTAACAGGTACCGTGGCAGG ACCTCTTGATGCAGCTGATCATTTAGTTAGACCTGATGGTTATGTTGC ATGGGCCGGTGGAGCTGGACACGATGAACCTGGTTTAGAAACTGCTC TTAGATTATGGTTTGGTGAACCAGTTAGTgtatcgatggattacaaggatgacgacgat aagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa SOD1- gttttaattacaagcggccgccattATGGCGACGAAGGCCGTGTGCGTGCTGAAGGG dacO1- CGACGGCCCAGTGCAGGGCATCATCAATTTCGAGCAGAAGGAAAGT tADH1 in AATGGACCAGTGAAGGTGTGGGGAAGCATTAAAGGACTGACTGAAG pSP-G1 GCCTGCATGGATTCCATGTTCATGAGTTTGGAGATAATACAGCAGGC (EH-5-49- TGTACCAGTGCAGGTCCTCACTTTAATCCTCTATCCAGAAAACACGGT B) GGGCCAAAGGATGAAGAGAGGCATGTTGGAGACTTGGGCAATGTGA CTGCTGACAAAGATGGTGTGGCCGATGTGTCTATTGAAGATTCTGTG ATCTCACTCTCAGGAGACCATTGCATCATTGGCCGCACACTGGTGGT CCATGAAAAAGCAGATGACTTGGGCAAAGGTGGAAATGAAGAAAGT ACAAAGACAGGAAACGCTGGAAGTCGTTTGGCTTGTGGTGTAATTGG GATGTTAGTAGCCGGTGGTGGACCAACAGGGTTATGGTTGGCTGGTG AACTAAGGTTGGCCGGGGTTAGACCTCTTGTTTTAGAGCGTAGGACA GAACCCTTTCCACATAGCAAAGCACTGGGTATCCATCCAAGAACTAT TGAAATGTTGGAGTTGAGAGGCCTTGCTGGTAGATTTTTAGATGGCG CCCCGAAGCTGCCGAAAGGTCACTTTGCATCGCTTCCTGTGCCCTTAG ACTATGGTGCAATGGATACTCGACATCCGTACGGTGTCTACCGTAAA CAAGTGGAAACGGAAGCTTTATTAGCCGGCCACGCAACCAGATTAGG AGTTCCAGTCCGCAGAGGTCATGAACTTGTAGGCCTAAGACAGCACG ATGATGCTGTAGTCGGTACTGTTCAAGGTCCTCAAGGTAGATACGAA GTCAGAGCCGCTTATCTTGTTGGTTGTGATGGAGGAGGTTCTACCAC AAGACGCCTGGCTGGGATAGATTTCCCTGGACAAGATCCTGGTGTTG CCGTTTTGATGGCTGACGCAAGATTCCGGGACCCATTACCCTCTGATC CAGCTATGGGTCCATTCAGACGCTATGGAGTCCTGAGACCTGATTTA

113

AGGGCATGGTTTAGTGCTATTCCTTTACCAGAAGAACAGGGTTTATG CAGAGTTATGGTTTTTTGGTATGGACGACAATTTGAAGATAGACGTG CTCCAGTAACTGAGGATGAAATGAGAGCAGCACTTGTTGATCTAGCG GGAACTGACTTTGGTATGTACGATGTTCAATGGTTGACCAGATTCAC AGACGCTTCCAGGCAAGCTGCAAGGTATAGATCTGGAAGGGTTTTCT TAGCTGGAGATGCTGCTCATACTGTCTTCCCCACTGGTGGCCCAGGTT TGAATGCAGGTTTACAAGATGCCATGAATTTAGGTTGGAAGTTAGCT GGAGCTGTTCATGGCTGGGGTGGTGCTTTACTAGATTCATATCATGAT GAGCGTTACCCTGTAGCTGAACAAGTTTTGCGTGACACAAGAACACA ATGTCTATTGTTAGATCCAGACCCTCGCTTTGTTCCATTAAGAGAGAC GTTAGCAGAACTATTGAGACTAGAACCCGTAAACAGATATTTCACCG GACACAACACGGCTCTAGCAGTTAGATACGATTTGGGTGGTGCCCAT CCTGCAACGGGAGGCAGAATGCCAGATGTCTTGGTCGCAACTGGTGC TGGTCAAAGAAGGTTTTCAGATTGTTGTGTTACCGGGAGAGGGGTTC TTTTAGTCACTTCTGGTGCAGATAGGGGGGAACTGTTACGAGGATGG AAAGATAGAGTGGATGTGGTAACAGGTACCGTGGCAGGACCTCTTGA TGCAGCTGATCATTTAGTTAGACCTGATGGTTATGTTGCATGGGCCGG TGGAGCTGGACACGATGAACCTGGTTTAGAAACTGCTCTTAGATTAT GGTTTGGTGAACCAGTTAGTgtatcgatggattacaaggatgacgacgataagatctgagctctt aattaacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa GAL1- gttttaattacaagcggccgcATGACTAAATCTCATTCAGAAGAAGTGATTGTACC dacO1- TGAGTTCAATTCTAGCGCAAAGGAATTACCAAGACCATTGGCCGAAA tADH1 in AGTGCCCGAGCATAATTAAGAAATTTATAAGCGCTTATGATGCTAAA pSP-G1 CCGGATTTTGTTGCTAGATCGCCTGGTAGAGTCAATCTAATTGGTGAA (EH-5-49- CATATTGATTATTGTGACTTCTCGGTTTTACCTTTAGCTATTGATTTTG C) ATATGCTTTGCGCCGTCAAAGTTTTGAACGAGAAAAATCCATCCATT ACCTTAATAAATGCTGATCCCAAATTTGCTCAAAGGAAGTTCGATTT GCCGTTGGACGGTTCTTATGTCACAATTGATCCTTCTGTGTCGGACTG GTCTAATTACTTTAAATGTGGTCTCCATGTTGCTCACTCTTTTCTAAA GAAACTTGCACCGGAAAGGTTTGCCAGTGCTCCTCTGGCCGGGCTGC AAGTCTTCTGTGAGGGTGATGTACCAACTGGCAGTGGATTGTCTTCTT CGGCCGCATTCATTTGTGCCGTTGCTTTAGCTGTTGTTAAAGCGAATA TGGGCCCTGGTTATCATATGTCCAAGCAAAATTTAATGCGTATTACG GTCGTTGCAGAACATTATGTTGGTGTTAACAATGGCGGTATGGATCA GGCTGCCTCTGTTTGCGGTGAGGAAGATCATGCTCTATACGTTGAGTT CAAACCGCAGTTGAAGGCTACTCCGTTTAAATTTCCGCAATTAAAAA ACCATGAAATTAGCTTTGTTATTGCGAACACCCTTGTTGTATCTAACA AGTTTGAAACCGCCCCAACCAACTATAATTTAAGAGTGGTAGAAGTC ACTACAGaattccggATGTTAGTAGCCGGTGGTGGACCAACAGGGTTATG GTTGGCTGGTGAACTAAGGTTGGCCGGGGTTAGACCTCTTGTTTTAG AGCGTAGGACAGAACCCTTTCCACATAGCAAAGCACTGGGTATCCAT CCAAGAACTATTGAAATGTTGGAGTTGAGAGGCCTTGCTGGTAGATT TTTAGATGGCGCCCCGAAGCTGCCGAAAGGTCACTTTGCATCGCTTC CTGTGCCCTTAGACTATGGTGCAATGGATACTCGACATCCGTACGGT GTCTACCGTAAACAAGTGGAAACGGAAGCTTTATTAGCCGGCCACGC

114

AACCAGATTAGGAGTTCCAGTCCGCAGAGGTCATGAACTTGTAGGCC TAAGACAGCACGATGATGCTGTAGTCGGTACTGTTCAAGGTCCTCAA GGTAGATACGAAGTCAGAGCCGCTTATCTTGTTGGTTGTGATGGAGG AGGTTCTACCACAAGACGCCTGGCTGGGATAGATTTCCCTGGACAAG ATCCTGGTGTTGCCGTTTTGATGGCTGACGCAAGATTCCGGGACCCAT TACCCTCTGATCCAGCTATGGGTCCATTCAGACGCTATGGAGTCCTGA GACCTGATTTAAGGGCATGGTTTAGTGCTATTCCTTTACCAGAAGAA CAGGGTTTATGCAGAGTTATGGTTTTTTGGTATGGACGACAATTTGAA GATAGACGTGCTCCAGTAACTGAGGATGAAATGAGAGCAGCACTTGT TGATCTAGCGGGAACTGACTTTGGTATGTACGATGTTCAATGGTTGA CCAGATTCACAGACGCTTCCAGGCAAGCTGCAAGGTATAGATCTGGA AGGGTTTTCTTAGCTGGAGATGCTGCTCATACTGTCTTCCCCACTGGT GGCCCAGGTTTGAATGCAGGTTTACAAGATGCCATGAATTTAGGTTG GAAGTTAGCTGGAGCTGTTCATGGCTGGGGTGGTGCTTTACTAGATT CATATCATGATGAGCGTTACCCTGTAGCTGAACAAGTTTTGCGTGAC ACAAGAACACAATGTCTATTGTTAGATCCAGACCCTCGCTTTGTTCCA TTAAGAGAGACGTTAGCAGAACTATTGAGACTAGAACCCGTAAACA GATATTTCACCGGACACAACACGGCTCTAGCAGTTAGATACGATTTG GGTGGTGCCCATCCTGCAACGGGAGGCAGAATGCCAGATGTCTTGGT CGCAACTGGTGCTGGTCAAAGAAGGTTTTCAGATTGTTGTGTTACCG GGAGAGGGGTTCTTTTAGTCACTTCTGGTGCAGATAGGGGGGAACTG TTACGAGGATGGAAAGATAGAGTGGATGTGGTAACAGGTACCGTGG CAGGACCTCTTGATGCAGCTGATCATTTAGTTAGACCTGATGGTTATG TTGCATGGGCCGGTGGAGCTGGACACGATGAACCTGGTTTAGAAACT GCTCTTAGATTATGGTTTGGTGAACCAGTTAGTgtatcgatggattacaaggatgac gacgataagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggct pGAL1- ccctcactaaagggaacaaaagctggagctctagtacggattagaagccgccgagcgggtgacagccctccgaa dacO1- ggaagactctcctccgtgcgtcctcgtcttcaccggtcgcgttcctgaaacgcagatgtgcctcgcgccgcactgct tCYC1 in ccgaacaataaagattctacaatactagcttttatggttatgaagaggaaaaattggcagtaacctggccccacaaac pRS416 cttcaaatgaacgaatcaaattaacaaccataggatgataatgcgattagttttttagccttatttctggggtaattaatc and agcgaagcgatgatttttgatctattaacagatatataaatgcaaaaactgcataaccactttaactaatactttcaacat pGAL1- tttcggtttgtattacttcttattcaaatgtaataaaagtatcaacaaaaaattgttaatatacctctatactttaacgtcaag dacO1- gagaaaaaaccccggattctagaactagtATGTTAGTAGCCGGTGGTGGACCAACAGG tCYC1 in GTTATGGTTGGCTGGTGAACTAAGGTTGGCCGGGGTTAGACCTCTTG pRS426 TTTTAGAGCGTAGGACAGAACCCTTTCCACATAGCAAAGCACTGGGT (EH-5-80-1 ATCCATCCAAGAACTATTGAAATGTTGGAGTTGAGAGGCCTTGCTGG and EH-5- TAGATTTTTAGATGGCGCCCCGAAGCTGCCGAAAGGTCACTTTGCAT 80-2) CGCTTCCTGTGCCCTTAGACTATGGTGCAATGGATACTCGACATCCGT ACGGTGTCTACCGTAAACAAGTGGAAACGGAAGCTTTATTAGCCGGC CACGCAACCAGATTAGGAGTTCCAGTCCGCAGAGGTCATGAACTTGT AGGCCTAAGACAGCACGATGATGCTGTAGTCGGTACTGTTCAAGGTC CTCAAGGTAGATACGAAGTCAGAGCCGCTTATCTTGTTGGTTGTGAT GGAGGAGGTTCTACCACAAGACGCCTGGCTGGGATAGATTTCCCTGG ACAAGATCCTGGTGTTGCCGTTTTGATGGCTGACGCAAGATTCCGGG ACCCATTACCCTCTGATCCAGCTATGGGTCCATTCAGACGCTATGGA GTCCTGAGACCTGATTTAAGGGCATGGTTTAGTGCTATTCCTTTACCA

115

GAAGAACAGGGTTTATGCAGAGTTATGGTTTTTTGGTATGGACGACA ATTTGAAGATAGACGTGCTCCAGTAACTGAGGATGAAATGAGAGCA GCACTTGTTGATCTAGCGGGAACTGACTTTGGTATGTACGATGTTCAA TGGTTGACCAGATTCACAGACGCTTCCAGGCAAGCTGCAAGGTATAG ATCTGGAAGGGTTTTCTTAGCTGGAGATGCTGCTCATACTGTCTTCCC CACTGGTGGCCCAGGTTTGAATGCAGGTTTACAAGATGCCATGAATT TAGGTTGGAAGTTAGCTGGAGCTGTTCATGGCTGGGGTGGTGCTTTA CTAGATTCATATCATGATGAGCGTTACCCTGTAGCTGAACAAGTTTTG CGTGACACAAGAACACAATGTCTATTGTTAGATCCAGACCCTCGCTT TGTTCCATTAAGAGAGACGTTAGCAGAACTATTGAGACTAGAACCCG TAAACAGATATTTCACCGGACACAACACGGCTCTAGCAGTTAGATAC GATTTGGGTGGTGCCCATCCTGCAACGGGAGGCAGAATGCCAGATGT CTTGGTCGCAACTGGTGCTGGTCAAAGAAGGTTTTCAGATTGTTGTGT TACCGGGAGAGGGGTTCTTTTAGTCACTTCTGGTGCAGATAGGGGGG AACTGTTACGAGGATGGAAAGATAGAGTGGATGTGGTAACAGGTAC CGTGGCAGGACCTCTTGATGCAGCTGATCATTTAGTTAGACCTGATG GTTATGTTGCATGGGCCGGTGGAGCTGGACACGATGAACCTGGTTTA GAAACTGCTCTTAGATTATGGTTTGGTGAACCAGTTAGTgtatcgatggattac aaggatgacgacgataagatctgaaagcttatcgataccgtcgacctcgagtcatgtaattagttatgtcacgcttac attcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatt tttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgcatgtaaca ttatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttgcggccggtacccaat pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa dacO1- gttttaattacaagcggccgCATGTTAGTAGCCGGTGGTGGACCAACAGGGTTAT tADH1 in GGTTGGCTGGTGAACTAAGGTTGGCCGGGGTTAGACCTCTTGTTTTA pSP-G1 (no GAGCGTAGGACAGAACCCTTTCCACATAGCAAAGCACTGGGTATCCA antibody TCCAAGAACTATTGAAATGTTGGAGTTGAGAGGCCTTGCTGGTAGAT tag) (EH-5- TTTTAGATGGCGCCCCGAAGCTGCCGAAAGGTCACTTTGCATCGCTTC 80-3) CTGTGCCCTTAGACTATGGTGCAATGGATACTCGACATCCGTACGGT GTCTACCGTAAACAAGTGGAAACGGAAGCTTTATTAGCCGGCCACGC AACCAGATTAGGAGTTCCAGTCCGCAGAGGTCATGAACTTGTAGGCC TAAGACAGCACGATGATGCTGTAGTCGGTACTGTTCAAGGTCCTCAA GGTAGATACGAAGTCAGAGCCGCTTATCTTGTTGGTTGTGATGGAGG AGGTTCTACCACAAGACGCCTGGCTGGGATAGATTTCCCTGGACAAG ATCCTGGTGTTGCCGTTTTGATGGCTGACGCAAGATTCCGGGACCCAT TACCCTCTGATCCAGCTATGGGTCCATTCAGACGCTATGGAGTCCTGA GACCTGATTTAAGGGCATGGTTTAGTGCTATTCCTTTACCAGAAGAA CAGGGTTTATGCAGAGTTATGGTTTTTTGGTATGGACGACAATTTGAA GATAGACGTGCTCCAGTAACTGAGGATGAAATGAGAGCAGCACTTGT TGATCTAGCGGGAACTGACTTTGGTATGTACGATGTTCAATGGTTGA CCAGATTCACAGACGCTTCCAGGCAAGCTGCAAGGTATAGATCTGGA AGGGTTTTCTTAGCTGGAGATGCTGCTCATACTGTCTTCCCCACTGGT GGCCCAGGTTTGAATGCAGGTTTACAAGATGCCATGAATTTAGGTTG GAAGTTAGCTGGAGCTGTTCATGGCTGGGGTGGTGCTTTACTAGATT CATATCATGATGAGCGTTACCCTGTAGCTGAACAAGTTTTGCGTGAC ACAAGAACACAATGTCTATTGTTAGATCCAGACCCTCGCTTTGTTCCA

116

TTAAGAGAGACGTTAGCAGAACTATTGAGACTAGAACCCGTAAACA GATATTTCACCGGACACAACACGGCTCTAGCAGTTAGATACGATTTG GGTGGTGCCCATCCTGCAACGGGAGGCAGAATGCCAGATGTCTTGGT CGCAACTGGTGCTGGTCAAAGAAGGTTTTCAGATTGTTGTGTTACCG GGAGAGGGGTTCTTTTAGTCACTTCTGGTGCAGATAGGGGGGAACTG TTACGAGGATGGAAAGATAGAGTGGATGTGGTAACAGGTACCGTGG CAGGACCTCTTGATGCAGCTGATCATTTAGTTAGACCTGATGGTTATG TTGCATGGGCCGGTGGAGCTGGACACGATGAACCTGGTTTAGAAACT GCTCTTAGATTATGGTTTGGTGAACCAGTTAGTTGAgagctcttaattaacaattct tcgccagaggtttggtcaagtctccaatcaaggttgtcggcttgtctaccttgccagaaatttacgaaaagatggaaa agggtcaaatcgttggtagatacgttgttgacacttctaaataagcgaatttcttatgatttatgatttttattattaaataa gttataaaaaaaataagtgtatacaaattttaaagtgactcttaggttttaaaacgaaaattcttattcttgagtaactcttt cctgtaggtcaggttgctttctcaggtatagcatgaggtcgctccaattcagctggcgtaatagcgaaga pADH2- ccctcactaaagggaacaaaagctggagctcGCAAAACGTAGGGGCAAACAAACGGA dacO1- AAAATCGTTTCTCAAATTTTCTGATGCCAAGAACTCTAACCAGTCTTA tCYC1 in TCTAAAAATTGCCTTATGATCCGTCTCTCCGGTTACAGCCTGTGTAAC pRS416 TGATTAATCCTGCCTTTCTAATCACCATTCTAATGTTTTAATTAAGGG and ATTTTGTCTTCATTAACGGCTTTCGCTCATAAAAATGTTATGACGTTT pADH2- TGCCCGCAGGCGGGAAACCATCCACTTCACGAGACTGATCTCCTCTG dacO1- CCGGAACACCGGGCATCTCCAACTTATAAGTTGGAGAAATAAGAGA tCYC1 in ATTTCAGATTGAGAGAATGAAAAAAAAAAAAAAAAAAAAGGCAGAG pRS426 GAGAGCATAGAAATGGGGTTCACTTTTTGGTAAAGCTATAGCATGCC (EH-5-80-4 TATCACATATAAATAGAGTGCCAGTAGCGACTTTTTTCACACTCGAA and EH-5- ATACTCTTACTACTGCTCTCTTGTTGTTTTTATCACTTCTTGTTTCTTCT 80-4) TGGTAAATAGAATATCAAGCTACAAAAAGCATACAATCAACTATCAA CTATTAACTATATCGTAATACCATATGGCTAtctagaactagtATGTTAGTAG CCGGTGGTGGACCAACAGGGTTATGGTTGGCTGGTGAACTAAGGTTG GCCGGGGTTAGACCTCTTGTTTTAGAGCGTAGGACAGAACCCTTTCC ACATAGCAAAGCACTGGGTATCCATCCAAGAACTATTGAAATGTTGG AGTTGAGAGGCCTTGCTGGTAGATTTTTAGATGGCGCCCCGAAGCTG CCGAAAGGTCACTTTGCATCGCTTCCTGTGCCCTTAGACTATGGTGCA ATGGATACTCGACATCCGTACGGTGTCTACCGTAAACAAGTGGAAAC GGAAGCTTTATTAGCCGGCCACGCAACCAGATTAGGAGTTCCAGTCC GCAGAGGTCATGAACTTGTAGGCCTAAGACAGCACGATGATGCTGTA GTCGGTACTGTTCAAGGTCCTCAAGGTAGATACGAAGTCAGAGCCGC TTATCTTGTTGGTTGTGATGGAGGAGGTTCTACCACAAGACGCCTGG CTGGGATAGATTTCCCTGGACAAGATCCTGGTGTTGCCGTTTTGATGG CTGACGCAAGATTCCGGGACCCATTACCCTCTGATCCAGCTATGGGT CCATTCAGACGCTATGGAGTCCTGAGACCTGATTTAAGGGCATGGTT TAGTGCTATTCCTTTACCAGAAGAACAGGGTTTATGCAGAGTTATGG TTTTTTGGTATGGACGACAATTTGAAGATAGACGTGCTCCAGTAACT GAGGATGAAATGAGAGCAGCACTTGTTGATCTAGCGGGAACTGACTT TGGTATGTACGATGTTCAATGGTTGACCAGATTCACAGACGCTTCCA GGCAAGCTGCAAGGTATAGATCTGGAAGGGTTTTCTTAGCTGGAGAT GCTGCTCATACTGTCTTCCCCACTGGTGGCCCAGGTTTGAATGCAGGT TTACAAGATGCCATGAATTTAGGTTGGAAGTTAGCTGGAGCTGTTCA

117

TGGCTGGGGTGGTGCTTTACTAGATTCATATCATGATGAGCGTTACCC TGTAGCTGAACAAGTTTTGCGTGACACAAGAACACAATGTCTATTGT TAGATCCAGACCCTCGCTTTGTTCCATTAAGAGAGACGTTAGCAGAA CTATTGAGACTAGAACCCGTAAACAGATATTTCACCGGACACAACAC GGCTCTAGCAGTTAGATACGATTTGGGTGGTGCCCATCCTGCAACGG GAGGCAGAATGCCAGATGTCTTGGTCGCAACTGGTGCTGGTCAAAGA AGGTTTTCAGATTGTTGTGTTACCGGGAGAGGGGTTCTTTTAGTCACT TCTGGTGCAGATAGGGGGGAACTGTTACGAGGATGGAAAGATAGAG TGGATGTGGTAACAGGTACCGTGGCAGGACCTCTTGATGCAGCTGAT CATTTAGTTAGACCTGATGGTTATGTTGCATGGGCCGGTGGAGCTGG ACACGATGAACCTGGTTTAGAAACTGCTCTTAGATTATGGTTTGGTG AACCAGTTAGTgtatcgatggattacaaggatgacgacgataagatctgaaagcttatcgataccgtcga cctcgagtcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaagga gttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttctt ttttttctgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaagg ctttaatttgcggccggtacccaat pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa UBI4- gttttaattacaagcggccgccattATGCAGATCTTCGTCAAGACGTTAACCGGTAA IFNG- AACCATAACTCTAGAAGTTGAATCTTCCGATACCATCGACAACGTTA dacO1- AGTCGAAAATTCAAGACAAGGAAGGCATTCCACCTGATCAACAAAG tADH1 in ATTGATCTTTGCCGGTAAGCAGCTCGAGGACGGTAGAACGCTGTCTG pSP-G1 ATTACAACATTCAGAAGGAGTCGACCTTACATCTTGTCTTAAGACTA (EH-5-80- AGAGGTGGTATGCAAGACCCATATGTAAAAGAAGCAGAAAACCTTA 6) AGAAATATTTTAATGCAGGTCATTCAGATGTAGCGGATAATGGAACT CTTTTCTTAGGCATTTTGAAGAATTGGAAAGAGGAGAGTGACAGAAA AATAATGCAGAGCCAAATTGTCTCCTTTTACTTCAAACTTTTTAAAAA CTTTAAAGATGACCAGAGCATCCAAAAGAGTGTGGAGACCATCAAG GAAGACATGAATGTCAAGTTTTTCAATAGCAACAAAAAGAAACGAG ATGACTTCGAAAAGCTGACTAATTATTCGGTAACTGACTTGAATGTC CAACGCAAAGCAATACATGAACTCATCCAAGTGATGGCTGAACTGTC GCCAGCAGCTAAAACAGGGAAGCGAAAAAGGAGTCAGATGCTGTTT CGAGGTCGAAGAGCATCCCAGATGTTAGTAGCCGGTGGTGGACCAAC AGGGTTATGGTTGGCTGGTGAACTAAGGTTGGCCGGGGTTAGACCTC TTGTTTTAGAGCGTAGGACAGAACCCTTTCCACATAGCAAAGCACTG GGTATCCATCCAAGAACTATTGAAATGTTGGAGTTGAGAGGCCTTGC TGGTAGATTTTTAGATGGCGCCCCGAAGCTGCCGAAAGGTCACTTTG CATCGCTTCCTGTGCCCTTAGACTATGGTGCAATGGATACTCGACATC CGTACGGTGTCTACCGTAAACAAGTGGAAACGGAAGCTTTATTAGCC GGCCACGCAACCAGATTAGGAGTTCCAGTCCGCAGAGGTCATGAACT TGTAGGCCTAAGACAGCACGATGATGCTGTAGTCGGTACTGTTCAAG GTCCTCAAGGTAGATACGAAGTCAGAGCCGCTTATCTTGTTGGTTGT GATGGAGGAGGTTCTACCACAAGACGCCTGGCTGGGATAGATTTCCC TGGACAAGATCCTGGTGTTGCCGTTTTGATGGCTGACGCAAGATTCC GGGACCCATTACCCTCTGATCCAGCTATGGGTCCATTCAGACGCTAT GGAGTCCTGAGACCTGATTTAAGGGCATGGTTTAGTGCTATTCCTTTA CCAGAAGAACAGGGTTTATGCAGAGTTATGGTTTTTTGGTATGGACG

118

ACAATTTGAAGATAGACGTGCTCCAGTAACTGAGGATGAAATGAGA GCAGCACTTGTTGATCTAGCGGGAACTGACTTTGGTATGTACGATGTT CAATGGTTGACCAGATTCACAGACGCTTCCAGGCAAGCTGCAAGGTA TAGATCTGGAAGGGTTTTCTTAGCTGGAGATGCTGCTCATACTGTCTT CCCCACTGGTGGCCCAGGTTTGAATGCAGGTTTACAAGATGCCATGA ATTTAGGTTGGAAGTTAGCTGGAGCTGTTCATGGCTGGGGTGGTGCT TTACTAGATTCATATCATGATGAGCGTTACCCTGTAGCTGAACAAGTT TTGCGTGACACAAGAACACAATGTCTATTGTTAGATCCAGACCCTCG CTTTGTTCCATTAAGAGAGACGTTAGCAGAACTATTGAGACTAGAAC CCGTAAACAGATATTTCACCGGACACAACACGGCTCTAGCAGTTAGA TACGATTTGGGTGGTGCCCATCCTGCAACGGGAGGCAGAATGCCAGA TGTCTTGGTCGCAACTGGTGCTGGTCAAAGAAGGTTTTCAGATTGTTG TGTTACCGGGAGAGGGGTTCTTTTAGTCACTTCTGGTGCAGATAGGG GGGAACTGTTACGAGGATGGAAAGATAGAGTGGATGTGGTAACAGG TACCGTGGCAGGACCTCTTGATGCAGCTGATCATTTAGTTAGACCTG ATGGTTATGTTGCATGGGCCGGTGGAGCTGGACACGATGAACCTGGT TTAGAAACTGCTCTTAGATTATGGTTTGGTGAACCAGTTAGTgtatcgatgg attacaaggatgacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctccaatcaa ggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa IFNG- gttttaattacaagcggccgccattATGCAAGACCCATATGTAAAAGAAGCAGAAAA dacO1- CCTTAAGAAATATTTTAATGCAGGTCATTCAGATGTAGCGGATAATG tADH1 in GAACTCTTTTCTTAGGCATTTTGAAGAATTGGAAAGAGGAGAGTGAC pSP-G1 AGAAAAATAATGCAGAGCCAAATTGTCTCCTTTTACTTCAAACTTTTT (EH-5-80- AAAAACTTTAAAGATGACCAGAGCATCCAAAAGAGTGTGGAGACCA 7) TCAAGGAAGACATGAATGTCAAGTTTTTCAATAGCAACAAAAAGAA ACGAGATGACTTCGAAAAGCTGACTAATTATTCGGTAACTGACTTGA ATGTCCAACGCAAAGCAATACATGAACTCATCCAAGTGATGGCTGAA CTGTCGCCAGCAGCTAAAACAGGGAAGCGAAAAAGGAGTCAGATGC TGTTTCGAGGTCGAAGAGCATCCCAGATGTTAGTAGCCGGTGGTGGA CCAACAGGGTTATGGTTGGCTGGTGAACTAAGGTTGGCCGGGGTTAG ACCTCTTGTTTTAGAGCGTAGGACAGAACCCTTTCCACATAGCAAAG CACTGGGTATCCATCCAAGAACTATTGAAATGTTGGAGTTGAGAGGC CTTGCTGGTAGATTTTTAGATGGCGCCCCGAAGCTGCCGAAAGGTCA CTTTGCATCGCTTCCTGTGCCCTTAGACTATGGTGCAATGGATACTCG ACATCCGTACGGTGTCTACCGTAAACAAGTGGAAACGGAAGCTTTAT TAGCCGGCCACGCAACCAGATTAGGAGTTCCAGTCCGCAGAGGTCAT GAACTTGTAGGCCTAAGACAGCACGATGATGCTGTAGTCGGTACTGT TCAAGGTCCTCAAGGTAGATACGAAGTCAGAGCCGCTTATCTTGTTG GTTGTGATGGAGGAGGTTCTACCACAAGACGCCTGGCTGGGATAGAT TTCCCTGGACAAGATCCTGGTGTTGCCGTTTTGATGGCTGACGCAAG ATTCCGGGACCCATTACCCTCTGATCCAGCTATGGGTCCATTCAGACG CTATGGAGTCCTGAGACCTGATTTAAGGGCATGGTTTAGTGCTATTCC TTTACCAGAAGAACAGGGTTTATGCAGAGTTATGGTTTTTTGGTATGG ACGACAATTTGAAGATAGACGTGCTCCAGTAACTGAGGATGAAATGA GAGCAGCACTTGTTGATCTAGCGGGAACTGACTTTGGTATGTACGAT

119

GTTCAATGGTTGACCAGATTCACAGACGCTTCCAGGCAAGCTGCAAG GTATAGATCTGGAAGGGTTTTCTTAGCTGGAGATGCTGCTCATACTGT CTTCCCCACTGGTGGCCCAGGTTTGAATGCAGGTTTACAAGATGCCA TGAATTTAGGTTGGAAGTTAGCTGGAGCTGTTCATGGCTGGGGTGGT GCTTTACTAGATTCATATCATGATGAGCGTTACCCTGTAGCTGAACAA GTTTTGCGTGACACAAGAACACAATGTCTATTGTTAGATCCAGACCC TCGCTTTGTTCCATTAAGAGAGACGTTAGCAGAACTATTGAGACTAG AACCCGTAAACAGATATTTCACCGGACACAACACGGCTCTAGCAGTT AGATACGATTTGGGTGGTGCCCATCCTGCAACGGGAGGCAGAATGCC AGATGTCTTGGTCGCAACTGGTGCTGGTCAAAGAAGGTTTTCAGATT GTTGTGTTACCGGGAGAGGGGTTCTTTTAGTCACTTCTGGTGCAGATA GGGGGGAACTGTTACGAGGATGGAAAGATAGAGTGGATGTGGTAAC AGGTACCGTGGCAGGACCTCTTGATGCAGCTGATCATTTAGTTAGAC CTGATGGTTATGTTGCATGGGCCGGTGGAGCTGGACACGATGAACCT GGTTTAGAAACTGCTCTTAGATTATGGTTTGGTGAACCAGTTAGTgtatc gatggattacaaggatgacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctcca atcaaggttgtcggct pPGK1- tggaatggcgggaaagggtttagtaccacatgctatgatgcccactgtgatctccagagcaaagttcgttcgatcgt dacO4- actgttactctctctctttcaaacagaattgtccgaatcgtgtgacaacaacagcctgttctcacacactcttttcttctaa dacO1- ccaagggggtggtttagtttagtagaacctcgtgaaacttacatttacatatatataaacttgcataaattggtcaatgc tCYC1 in aagaaatacatatttggtcttttctaattcgtagtttttcaagttcttagatgctttctttttctcttttttacagatcatcaagga pSP-G1 agtaattatctactttttacaacaaatataaaacaaggatccATGTCATTCACTGCCAAGGAAGT (EH-5-80- TCAATATTTGCGTTCTCAGCAATTAGGAAGATTAGCCACCGTTGGTGC 8) TGATGGTACACCACATAATGTTCCAGTAGGCTACAGATACAACGCTG ACTTGGGGACCATTGACATCACAGGAAGGGACTTAAGAAGAAGTAG GAAATATAGAGATGTTCAGGCTGGCTCGCGGGTGGCATTTATTGTTG ATGATCTACCGAGCACAGCACCTATAGTCGCCCGCGGGCTGGAGGTG AGGGGAACAGCTGAGGCGCTTTCTGGTGAAGAAGATTTGATACGTGT ACGACCTGCAAGAATCGTCACTTGGGGTATTGAAGCAGATTGGCAAG CTGGTCCTACTGGTAGAACGGTAAGGCCCACCACTCCAAGATCCGCG ACGATGTTAGTAGCCGGTGGTGGACCAACAGGGTTATGGTTGGCTGG TGAACTAAGGTTGGCCGGGGTTAGACCTCTTGTTTTAGAGCGTAGGA CAGAACCCTTTCCACATAGCAAAGCACTGGGTATCCATCCAAGAACT ATTGAAATGTTGGAGTTGAGAGGCCTTGCTGGTAGATTTTTAGATGG CGCCCCGAAGCTGCCGAAAGGTCACTTTGCATCGCTTCCTGTGCCCTT AGACTATGGTGCAATGGATACTCGACATCCGTACGGTGTCTACCGTA AACAAGTGGAAACGGAAGCTTTATTAGCCGGCCACGCAACCAGATTA GGAGTTCCAGTCCGCAGAGGTCATGAACTTGTAGGCCTAAGACAGCA CGATGATGCTGTAGTCGGTACTGTTCAAGGTCCTCAAGGTAGATACG AAGTCAGAGCCGCTTATCTTGTTGGTTGTGATGGAGGAGGTTCTACC ACAAGACGCCTGGCTGGGATAGATTTCCCTGGACAAGATCCTGGTGT TGCCGTTTTGATGGCTGACGCAAGATTCCGGGACCCATTACCCTCTGA TCCAGCTATGGGTCCATTCAGACGCTATGGAGTCCTGAGACCTGATTT AAGGGCATGGTTTAGTGCTATTCCTTTACCAGAAGAACAGGGTTTAT GCAGAGTTATGGTTTTTTGGTATGGACGACAATTTGAAGATAGACGT GCTCCAGTAACTGAGGATGAAATGAGAGCAGCACTTGTTGATCTAGC

120

GGGAACTGACTTTGGTATGTACGATGTTCAATGGTTGACCAGATTCA CAGACGCTTCCAGGCAAGCTGCAAGGTATAGATCTGGAAGGGTTTTC TTAGCTGGAGATGCTGCTCATACTGTCTTCCCCACTGGTGGCCCAGGT TTGAATGCAGGTTTACAAGATGCCATGAATTTAGGTTGGAAGTTAGC TGGAGCTGTTCATGGCTGGGGTGGTGCTTTACTAGATTCATATCATGA TGAGCGTTACCCTGTAGCTGAACAAGTTTTGCGTGACACAAGAACAC AATGTCTATTGTTAGATCCAGACCCTCGCTTTGTTCCATTAAGAGAGA CGTTAGCAGAACTATTGAGACTAGAACCCGTAAACAGATATTTCACC GGACACAACACGGCTCTAGCAGTTAGATACGATTTGGGTGGTGCCCA TCCTGCAACGGGAGGCAGAATGCCAGATGTCTTGGTCGCAACTGGTG CTGGTCAAAGAAGGTTTTCAGATTGTTGTGTTACCGGGAGAGGGGTT CTTTTAGTCACTTCTGGTGCAGATAGGGGGGAACTGTTACGAGGATG GAAAGATAGAGTGGATGTGGTAACAGGTACCGTGGCAGGACCTCTTG ATGCAGCTGATCATTTAGTTAGACCTGATGGTTATGTTGCATGGGCCG GTGGAGCTGGACACGATGAACCTGGTTTAGAAACTGCTCTTAGATTA TGGTTTGGTGAACCAGTTAGTgtcgacatggaacagaagttgatttccgaagaagacctcgagt aagcttggtaccgcggctagctaagatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtcc ctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtacgc atgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaagatccagctgcattaatgaatcggcca acgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcgg pPGK1- tggaatggcgggaaagggtttagtaccacatgctatgatgcccactgtgatctccagagcaaagttcgttcgatcgt dacO1- actgttactctctctctttcaaacagaattgtccgaatcgtgtgacaacaacagcctgttctcacacactcttttcttctaa dacO4- ccaagggggtggtttagtttagtagaacctcgtgaaacttacatttacatatatataaacttgcataaattggtcaatgc tCYC1 in aagaaatacatatttggtcttttctaattcgtagtttttcaagttcttagatgctttctttttctcttttttacagatcatcaagga pSP-G1 agtaattatctactttttacaacaaatataaaacaaggatccATGTTAGTAGCCGGTGGTGGACC (EH-5-80- AACAGGGTTATGGTTGGCTGGTGAACTAAGGTTGGCCGGGGTTAGAC 9) CTCTTGTTTTAGAGCGTAGGACAGAACCCTTTCCACATAGCAAAGCA CTGGGTATCCATCCAAGAACTATTGAAATGTTGGAGTTGAGAGGCCT TGCTGGTAGATTTTTAGATGGCGCCCCGAAGCTGCCGAAAGGTCACT TTGCATCGCTTCCTGTGCCCTTAGACTATGGTGCAATGGATACTCGAC ATCCGTACGGTGTCTACCGTAAACAAGTGGAAACGGAAGCTTTATTA GCCGGCCACGCAACCAGATTAGGAGTTCCAGTCCGCAGAGGTCATGA ACTTGTAGGCCTAAGACAGCACGATGATGCTGTAGTCGGTACTGTTC AAGGTCCTCAAGGTAGATACGAAGTCAGAGCCGCTTATCTTGTTGGT TGTGATGGAGGAGGTTCTACCACAAGACGCCTGGCTGGGATAGATTT CCCTGGACAAGATCCTGGTGTTGCCGTTTTGATGGCTGACGCAAGAT TCCGGGACCCATTACCCTCTGATCCAGCTATGGGTCCATTCAGACGCT ATGGAGTCCTGAGACCTGATTTAAGGGCATGGTTTAGTGCTATTCCTT TACCAGAAGAACAGGGTTTATGCAGAGTTATGGTTTTTTGGTATGGA CGACAATTTGAAGATAGACGTGCTCCAGTAACTGAGGATGAAATGAG AGCAGCACTTGTTGATCTAGCGGGAACTGACTTTGGTATGTACGATG TTCAATGGTTGACCAGATTCACAGACGCTTCCAGGCAAGCTGCAAGG TATAGATCTGGAAGGGTTTTCTTAGCTGGAGATGCTGCTCATACTGTC TTCCCCACTGGTGGCCCAGGTTTGAATGCAGGTTTACAAGATGCCAT GAATTTAGGTTGGAAGTTAGCTGGAGCTGTTCATGGCTGGGGTGGTG CTTTACTAGATTCATATCATGATGAGCGTTACCCTGTAGCTGAACAAG

121

TTTTGCGTGACACAAGAACACAATGTCTATTGTTAGATCCAGACCCTC GCTTTGTTCCATTAAGAGAGACGTTAGCAGAACTATTGAGACTAGAA CCCGTAAACAGATATTTCACCGGACACAACACGGCTCTAGCAGTTAG ATACGATTTGGGTGGTGCCCATCCTGCAACGGGAGGCAGAATGCCAG ATGTCTTGGTCGCAACTGGTGCTGGTCAAAGAAGGTTTTCAGATTGTT GTGTTACCGGGAGAGGGGTTCTTTTAGTCACTTCTGGTGCAGATAGG GGGGAACTGTTACGAGGATGGAAAGATAGAGTGGATGTGGTAACAG GTACCGTGGCAGGACCTCTTGATGCAGCTGATCATTTAGTTAGACCT GATGGTTATGTTGCATGGGCCGGTGGAGCTGGACACGATGAACCTGG TTTAGAAACTGCTCTTAGATTATGGTTTGGTGAACCAGTTAGTATGTC ATTCACTGCCAAGGAAGTTCAATATTTGCGTTCTCAGCAATTAGGAA GATTAGCCACCGTTGGTGCTGATGGTACACCACATAATGTTCCAGTA GGCTACAGATACAACGCTGACTTGGGGACCATTGACATCACAGGAAG GGACTTAAGAAGAAGTAGGAAATATAGAGATGTTCAGGCTGGCTCG CGGGTGGCATTTATTGTTGATGATCTACCGAGCACAGCACCTATAGT CGCCCGCGGGCTGGAGGTGAGGGGAACAGCTGAGGCGCTTTCTGGTG AAGAAGATTTGATACGTGTACGACCTGCAAGAATCGTCACTTGGGGT ATTGAAGCAGATTGGCAAGCTGGTCCTACTGGTAGAACGGTAAGGCC CACCACTCCAAGATCCGCGACGgtcgacatggaacagaagttgatttccgaagaagacctcg agtaagcttggtaccgcggctagctaagatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggt ccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacagacgcgtgtac gcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaagatccagctgcattaatgaatcggc caacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcgg pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa oxyS- gttttaattacaagcggccgcATGAGGTACGATGTTGTTATAGCTGGTGCAGGAC dacO1-37- CCACCGGTTTGATGTTAGCATGTGAACTTCGGCTGGCGGGTGCCAGA tADH1 in ACTTTGGTTTTAGAAAGATTAGCCGAGCCTTTTCCACATAGCAAAGC pSP-G1 ACTGGGTATCCATCCAAGAACTATTGAAATGTTGGAGTTGAGAGGCC (EH-5-80- TTGCTGGTAGATTTTTAGATGGCGCCCCGAAGCTGCCGAAAGGTCAC 10) TTTGCATCGCTTCCTGTGCCCTTAGACTATGGTGCAATGGATACTCGA CATCCGTACGGTGTCTACCGTAAACAAGTGGAAACGGAAGCTTTATT AGCCGGCCACGCAACCAGATTAGGAGTTCCAGTCCGCAGAGGTCATG AACTTGTAGGCCTAAGACAGCACGATGATGCTGTAGTCGGTACTGTT CAAGGTCCTCAAGGTAGATACGAAGTCAGAGCCGCTTATCTTGTTGG TTGTGATGGAGGAGGTTCTACCACAAGACGCCTGGCTGGGATAGATT TCCCTGGACAAGATCCTGGTGTTGCCGTTTTGATGGCTGACGCAAGA TTCCGGGACCCATTACCCTCTGATCCAGCTATGGGTCCATTCAGACGC TATGGAGTCCTGAGACCTGATTTAAGGGCATGGTTTAGTGCTATTCCT TTACCAGAAGAACAGGGTTTATGCAGAGTTATGGTTTTTTGGTATGG ACGACAATTTGAAGATAGACGTGCTCCAGTAACTGAGGATGAAATGA GAGCAGCACTTGTTGATCTAGCGGGAACTGACTTTGGTATGTACGAT GTTCAATGGTTGACCAGATTCACAGACGCTTCCAGGCAAGCTGCAAG GTATAGATCTGGAAGGGTTTTCTTAGCTGGAGATGCTGCTCATACTGT CTTCCCCACTGGTGGCCCAGGTTTGAATGCAGGTTTACAAGATGCCA TGAATTTAGGTTGGAAGTTAGCTGGAGCTGTTCATGGCTGGGGTGGT GCTTTACTAGATTCATATCATGATGAGCGTTACCCTGTAGCTGAACAA

122

GTTTTGCGTGACACAAGAACACAATGTCTATTGTTAGATCCAGACCC TCGCTTTGTTCCATTAAGAGAGACGTTAGCAGAACTATTGAGACTAG AACCCGTAAACAGATATTTCACCGGACACAACACGGCTCTAGCAGTT AGATACGATTTGGGTGGTGCCCATCCTGCAACGGGAGGCAGAATGCC AGATGTCTTGGTCGCAACTGGTGCTGGTCAAAGAAGGTTTTCAGATT GTTGTGTTACCGGGAGAGGGGTTCTTTTAGTCACTTCTGGTGCAGATA GGGGGGAACTGTTACGAGGATGGAAAGATAGAGTGGATGTGGTAAC AGGTACCGTGGCAGGACCTCTTGATGCAGCTGATCATTTAGTTAGAC CTGATGGTTATGTTGCATGGGCCGGTGGAGCTGGACACGATGAACCT GGTTTAGAAACTGCTCTTAGATTATGGTTTGGTGAACCAGTTAGTgtatc gatggattacaaggatgacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctcca atcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa oxyS- gttttaattacaagcggccgcATGAGGTACGATGTTGTTATAGCTGGTGCAGGAC dacO1-87- CCACCGGTTTGATGTTAGCATGTGAACTTCGGCTGGCGGGTGCCAGA tADH1 in ACTTTGGTTTTAGAAAGATTAGCCGAGCCTGTTGACTTCTCGAAAGCT pSP-G1 CTAGGAGTCCACGCTCGCACTGTTGAACTATTAGATATGAGAGGCCT (EH-5-80- CGGTGAAGGATTCCAGGCTGAAGCACCAAAGTTAAGAGGTGGTAATT 11) TTGCCTCATTAGGCGTCCCCCTGGATTTCTCATCATTTGATACTCGAC ATCCGTACGGTGTCTACCGTAAACAAGTGGAAACGGAAGCTTTATTA GCCGGCCACGCAACCAGATTAGGAGTTCCAGTCCGCAGAGGTCATGA ACTTGTAGGCCTAAGACAGCACGATGATGCTGTAGTCGGTACTGTTC AAGGTCCTCAAGGTAGATACGAAGTCAGAGCCGCTTATCTTGTTGGT TGTGATGGAGGAGGTTCTACCACAAGACGCCTGGCTGGGATAGATTT CCCTGGACAAGATCCTGGTGTTGCCGTTTTGATGGCTGACGCAAGAT TCCGGGACCCATTACCCTCTGATCCAGCTATGGGTCCATTCAGACGCT ATGGAGTCCTGAGACCTGATTTAAGGGCATGGTTTAGTGCTATTCCTT TACCAGAAGAACAGGGTTTATGCAGAGTTATGGTTTTTTGGTATGGA CGACAATTTGAAGATAGACGTGCTCCAGTAACTGAGGATGAAATGAG AGCAGCACTTGTTGATCTAGCGGGAACTGACTTTGGTATGTACGATG TTCAATGGTTGACCAGATTCACAGACGCTTCCAGGCAAGCTGCAAGG TATAGATCTGGAAGGGTTTTCTTAGCTGGAGATGCTGCTCATACTGTC TTCCCCACTGGTGGCCCAGGTTTGAATGCAGGTTTACAAGATGCCAT GAATTTAGGTTGGAAGTTAGCTGGAGCTGTTCATGGCTGGGGTGGTG CTTTACTAGATTCATATCATGATGAGCGTTACCCTGTAGCTGAACAAG TTTTGCGTGACACAAGAACACAATGTCTATTGTTAGATCCAGACCCTC GCTTTGTTCCATTAAGAGAGACGTTAGCAGAACTATTGAGACTAGAA CCCGTAAACAGATATTTCACCGGACACAACACGGCTCTAGCAGTTAG ATACGATTTGGGTGGTGCCCATCCTGCAACGGGAGGCAGAATGCCAG ATGTCTTGGTCGCAACTGGTGCTGGTCAAAGAAGGTTTTCAGATTGTT GTGTTACCGGGAGAGGGGTTCTTTTAGTCACTTCTGGTGCAGATAGG GGGGAACTGTTACGAGGATGGAAAGATAGAGTGGATGTGGTAACAG GTACCGTGGCAGGACCTCTTGATGCAGCTGATCATTTAGTTAGACCT GATGGTTATGTTGCATGGGCCGGTGGAGCTGGACACGATGAACCTGG TTTAGAAACTGCTCTTAGATTATGGTTTGGTGAACCAGTTAGTgtatcgat

123

ggattacaaggatgacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctccaatc aaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa oxyS- gttttaattacaagcggccgcATGAGGTACGATGTTGTTATAGCTGGTGCAGGAC dacO1- CCACCGGTTTGATGTTAGCATGTGAACTTCGGCTGGCGGGTGCCAGA 191-tADH1 ACTTTGGTTTTAGAAAGATTAGCCGAGCCTGTTGACTTCTCGAAAGCT in pSP-G1 CTAGGAGTCCACGCTCGCACTGTTGAACTATTAGATATGAGAGGCCT (EH-5-80- CGGTGAAGGATTCCAGGCTGAAGCACCAAAGTTAAGAGGTGGTAATT 12) TTGCCTCATTAGGCGTCCCCCTGGATTTCTCATCATTTGATACTAGAC ACCCATATGCATTGTTTGTTCCACAAGTACGAACTGAAGAACTGCTA ACAGGTAGAGCTTTGGAGCTAGGGGCGGAGCTGCGTCGTGGTCATGC CGTGACCGCCTTGGAACAAGATGCTGATGGTGTTACTGTGAGCGTGA CAGGCCCTGAAGGCCCATACGAAGTAGAATGTGCTTATTTGGTGGGC TGCGACGGTGGAGGCAGTACGGTGAGGAAACTATTGGGCATAGATTT TCCAGGTCAAGACCCACATATGTTTGCTGTCATCGCAGACGCAAGAT TCAGAGAAGAGCTTCCTCACGATCCAGCTATGGGTCCATTCAGACGC TATGGAGTCCTGAGACCTGATTTAAGGGCATGGTTTAGTGCTATTCCT TTACCAGAAGAACAGGGTTTATGCAGAGTTATGGTTTTTTGGTATGG ACGACAATTTGAAGATAGACGTGCTCCAGTAACTGAGGATGAAATGA GAGCAGCACTTGTTGATCTAGCGGGAACTGACTTTGGTATGTACGAT GTTCAATGGTTGACCAGATTCACAGACGCTTCCAGGCAAGCTGCAAG GTATAGATCTGGAAGGGTTTTCTTAGCTGGAGATGCTGCTCATACTGT CTTCCCCACTGGTGGCCCAGGTTTGAATGCAGGTTTACAAGATGCCA TGAATTTAGGTTGGAAGTTAGCTGGAGCTGTTCATGGCTGGGGTGGT GCTTTACTAGATTCATATCATGATGAGCGTTACCCTGTAGCTGAACAA GTTTTGCGTGACACAAGAACACAATGTCTATTGTTAGATCCAGACCC TCGCTTTGTTCCATTAAGAGAGACGTTAGCAGAACTATTGAGACTAG AACCCGTAAACAGATATTTCACCGGACACAACACGGCTCTAGCAGTT AGATACGATTTGGGTGGTGCCCATCCTGCAACGGGAGGCAGAATGCC AGATGTCTTGGTCGCAACTGGTGCTGGTCAAAGAAGGTTTTCAGATT GTTGTGTTACCGGGAGAGGGGTTCTTTTAGTCACTTCTGGTGCAGATA GGGGGGAACTGTTACGAGGATGGAAAGATAGAGTGGATGTGGTAAC AGGTACCGTGGCAGGACCTCTTGATGCAGCTGATCATTTAGTTAGAC CTGATGGTTATGTTGCATGGGCCGGTGGAGCTGGACACGATGAACCT GGTTTAGAAACTGCTCTTAGATTATGGTTTGGTGAACCAGTTAGTgtatc gatggattacaaggatgacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctcca atcaaggttgtcggct pPGK1- tggaatggcgggaaagggtttagtaccacatgctatgatgcccactgtgatctccagagcaaagttcgttcgatcgt dacJ- actgttactctctctctttcaaacagaattgtccgaatcgtgtgacaacaacagcctgttctcacacactcttttcttctaa tCYC1 in ccaagggggtggtttagtttagtagaacctcgtgaaacttacatttacatatatataaacttgcataaattggtcaatgc pSP-G1 aagaaatacatatttggtcttttctaattcgtagtttttcaagttcttagatgctttctttttctcttttttacagatcatcaagga (EH-5-90- agtaattatctactttttacaacaaatataaaacaaggatccATGACCGCAGGACAGCTGCATCT A) TGACGTTCCGGAACCGCCTGGTTCATGGGTAGATGAAGCCACATTTA GGGCTGTTATGGGTGCTTTGCCATCCGGTGTTACAGTCGTGACGACCT TGGACCCAGATGGCAGACCAGTAGGCTTAACTTGTTCTGCGGCTTGT TCAGTTTCTCAAAGGCCTCCCTTATTACTTGTCTGCTTAAACGATAGA

124

AGCAGGGTACTAGATGCAATACTCAGAAGAGGTCAGTTCATAGTAAA TGTCCTACGCGACCGTAGAGAAGCAACATCGGCAGTCTTTGCCAGTA GAGCCGAAGATAGATTCGCAGCTGTGCCTTGGCAACCTGGGAGGCGA ACTGGTGTCCCTTGGTTGTTAGCAGACACGGTTGCTCATGCAGAATGT GAGGTGGCAGGTACTTTGCATGCTGGCGATCACGTTATCGTTGTTGG AGGTATTGTTGCGGGTGATGCGCACCCCGACCGTGTGGGGCCATTAA TGTATTGGAGACAAAGATACGGTGGATGGCCAGTAACAGATGATGCT AGAGAAATTGCATTGACTCTAGCTGCCGAGGGAgtcgacatggaacagaagttga tttccgaagaagacctcgagtaagcttggtaccgcggctagctaagatccgctctaaccgaaaaggaaggagttag acaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttc tgtacagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaagatccagc tgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgac tcgctgcgctcgg pGPD- ccctcactaaagggaacaaaagctggagctcAGTTTATCATTATCAATACTGCCATTTC dacM2- AAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTA tCYC1 in GTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAA pRS413 ATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGT (EH-5-105- GAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTT Α) AAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGT TTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTA CAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAAT GGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTG ACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTG CTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTC CCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAG GTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTA CTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTT TCGACGGATACTAGTAAAATGACTGATGTATCCGCCCATACTACAAC CGATGGGGCGGTTGCAAGGGCTCGCGCTGTGGTCAGACTAAATACTG CATATTTTCGGGCTAAGGTACTTCAAAGTGCTGTCGAACTAGGTGTTT TTGATCTTTTAGATGGAGGCCCACAACCTGCCGCTGACATTTGCGGA AAGTTAGGTATTAGACATAGACTCGCAGTGGACTATTTGGACGCATT GACGGGTTTAGGGTTGCTTGAAAGGGATGGTGAACGTTACAGAAACT CGCCAACAGCGGCTGAGTTCTTGGTCCAAGATCGACCCGCCTATTTG GGTGGAACCGCGAGGCAACATGCCAAATTACATTATCACGCTTGGGG TAAACTGGCCGATGCAATGAGAGAAGGCCGTGCTCAGAGCGCCGTG GCAGCTCAAGGTGCAAAAGCCTATGTAAATTACTATGAAGATTTGGG CCAGGCCAGGAGGGTCATGACACATATGGATGCTCATAACGGTTTCA CAGCGGATGAAATCGCTAGACAATTGGACTGGAGAGGATACGCCAC AGTAACAGATGTGGGCGGCGCTCGTGGGAATGTTGCAGCTCGAATTG TTCAAGCAGTGCCTCATTTGAGAGCAAATGTAGTTGACCTACCCGCA TTGCGACCTTTATTCGATGAGCTGATGGCCCAACTAGGAACTGCTGG TCAGGTTGAGTTTCACGGTGGTGACTTCTTTGCGGACCCGATACCAG AGTCTGACGTTATCGTTGTGGGCCACGTCCTGGCGGATTGGCCACAG CCAAGACGTGTGGAATTACTTAGAAGATGTCACGCAGCATTAAGACC TGGTGGAACGGTAGTTGTTTACGATGTTATGGTTGATGATGATAGAG

125

ATGACGTCGAAGCACTCCTACAAAGATTAAACTCTGCAATGATAAGG GACGATATGGGTGCTTACGCTGTTACTGAATGTGCTGGATATTTAAG AGAAGCTGGATTTACCGTAGAAAGGGCCCTGCGTACGGATACCATAA CTCGCGATCATTTTGTTATTGGTAGAAAAGCTGCATCATAACTCGAGt catgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagaca acctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgt acagacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacgctcgaaggctttaatttg cggccggtacccaat

126

3.8 References

1. Wang P, Bashiri G, Gao X, Sawaya MR, Tang Y. Uncovering the Enzymes that Catalyze the Final Steps in Oxytetracycline Biosynthesis. J Am Chem Soc. 2013;135(19):7138-7141. doi: 10.1021/ja403516u.

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9. Cousens LS, Shuster JR, Gallegos C, Ku L, Stempien MM, Urdea MS, Sanchez-Pescador R, Taylor A, Tekamp-Olson P. High level expression of proinsulin in the yeast, Saccharomyces cerevisiae. Gene. 1987;61(3):265-275. doi: 10.1016/0378-1119(87)90190-9.

10. Steimer KS, Higgins KW, Powers MA, Stephans JC, Gyenes A, George-Nascimento C, Luciw PA, Barr PJ, Hallewell RA, Sanchez-Pescador R. Recombinant polypeptide from the

127 endonuclease region of the acquired immune deficiency syndrome retrovirus polymerase (pol) gene detects serum antibodies in most infected individuals. J Virol. 1986;58(1):9-16.

11. Barr P, Steimer K, Sabin E, Parkes D, Georgenascimento C, Stephens J, Powers M, Gyenes A, Vannest G, Miller E. Antigenicity and immunogenicity of domains of the human immunodeficiency virus (HIV) envelope polypeptide expressed in the yeast Saccharomyces cerevisiae. Vaccine. 1987;5(2):90-101. doi: 10.1016/0264-410x(87)90053-3.

12. Barr PJ, Inselburg J, Green KM, Kansopon J, Hahm BK, Gibson HL, Lee-Ng CT, Bzik DJ, Li W-b, Bathurst IC. Immunogenicity of recombinant Plasmodium falciparum SERA proteins in rodents. Mol Biochem Parasitol. 1991;45(1):159-170. doi: 10.1016/0166-6851(91)90038-8.

13. Bachmair A, Finley D, Varshavsky A. In vivo half-life of a protein is a function of its amino-terminal residue. Science. 1986;234(4773):179-186. doi: 10.1126/science.3018930.

14. Peng B, Williams TC, Henry M, Nielsen LK, Vickers CE. Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: a comparison of yeast promoter activities. Microbial Cell Factories. 2015;14(1). doi: 10.1186/s12934-015-0278-5.

15. Ma SM, Li JWH, Choi JW, Zhou H, Lee KKM, Moorthie VA, Xie X, Kealey JT, Da Silva NA, Vederas JC, Tang Y. Complete Reconstitution of a Highly Reducing Iterative Polyketide Synthase. Science. 2009;326(5952):589-592. doi: 10.1126/science.1175602.

16. Jones EW. Tackling the protease problem in Saccharomyces cerevisiae. Methods Enzymol. 1991;194:428-453. doi: 10.1016/0076-6879(91)94034-A.

17. Romanos MA, Scorer CA, Clare JJ. Foreign gene expression in yeast: a review. Yeast. 1992;8(6):423-488. doi: 10.1002/yea.320080602.

18. Tøttrup HV, Carlsen S. A process for the production of human proinsulin inSaccharomyces cerevisiae. Biotechnol Bioeng. 1990;35(4):339-348. doi: 10.1002/bit.260350403.

19. Rix U, Wang C, Chen Y, Lipata FM, Remsing Rix LL, Greenwell LM, Vining LC, Yang K, Rohr J. The Oxidative Ring Cleavage in Jadomycin Biosynthesis: A Multistep Oxygenation Cascade in a Biosynthetic Black Box. ChemBioChem. 2005;6(5):838-845. doi: 10.1002/cbic.200400395.

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20. Perić-Concha N, Borovička B, Long PF, Hranueli D, Waterman PG, Hunter IS. Ablation of theotcC Gene Encoding a Post-polyketide Hydroxylase from the Oxytetracyline Biosynthetic Pathway inStreptomyces rimosusResults in Novel Polyketides with Altered Chain Length. J Biol Chem. 2005;280(45):37455-37460. doi: 10.1074/jbc.M503191200.

21. Kharel MK, Pahari P, Shepherd MD, Tibrewal N, Nybo SE, Shaaban KA, Rohr J. Angucyclines: Biosynthesis, mode-of-action, new natural products, and synthesis. Nat Prod Rep. 2012;29(2):264-325. doi: 10.1039/c1np00068c.

22. Koskiniemi H, Metsä-Ketelä M, Dobritzsch D, Kallio P, Korhonen H, Mäntsälä P, Schneider G, Niemi J. Crystal Structures of Two Aromatic Hydroxylases Involved in the Early Tailoring Steps of Angucycline Biosynthesis. J Mol Biol. 2007;372(3):633-648. doi: 10.1016/j.jmb.2007.06.087.

23. Kallio P, Liu Z, Mäntsälä P, Niemi J, Metsä-Ketelä M. Sequential Action of Two Flavoenzymes, PgaE and PgaM, in Angucycline Biosynthesis: Chemoenzymatic Synthesis of Gaudimycin C. Chem Biol. 2008;15(2):157-166. doi: 10.1016/j.chembiol.2007.12.011.

24. Dairi T, Nakano T, Aisaka K, Katsumata R, Hasegawa M. Cloning and Nucleotide Sequence of the Gene Responsible for Chlorination of Tetracycline. Biosci, Biotechnol, Biochem. 2014;59(6):1099-1106. doi: 10.1271/bbb.59.1099.

25. Kong F, Yuan L, Zheng YF, Chen W. Automatic Liquid Handling for Life Science. Journal of Laboratory Automation. 2012;17(3):169-185. doi: 10.1177/2211068211435302.

26. Dörr M, Fibinger MPC, Last D, Schmidt S, Santos-Aberturas J, Böttcher D, Hummel A, Vickers C, Voss M, Bornscheuer UT. Fully automatized high-throughput enzyme library screening using a robotic platform. Biotechnol Bioeng. 2016;113(7):1421-1432. doi: 10.1002/bit.25925.

27. Oliva B, Gordon G, McNicholas P, Ellestad G, Chopra I. Evidence that tetracycline analogs whose primary target is not the bacterial ribosome cause lysis of Escherichia coli. Antimicrob Agents Chemother. 1992;36(5):913-919. doi: 10.1128/aac.36.5.913.

28. Gossen M, Bujard H. Anhydrotetracycline, a novel effector for tetracycline controlled gene expression systems in eukaryotic cells. Nucleic Acids Res. 1993;21(18):4411-4412. doi: 10.1093/nar/21.18.4411.

29. Asai M. Studies on Pillaromycin A. II. The Structure of Pillaromycinone. Chem Pharm Bull (Tokyo). 1970;18(9):1706-1712. doi: 10.1248/cpb.18.1706.

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30. Wells JS, O'Sullivan J, Aklonis C, Ax HA, Tymiak AA, Kirsch DR, Trejo WH, Principe P. Dactylocyclines, novel tetracycline derivatives produced by a Dactylosporangium sp. I. Taxonomy, production, isolation and biological activity. J Antibiot. 1992;45(12):1892-1898. doi: 10.7164/antibiotics.45.1892.

31. Tymiak AA, Aklonis C, Bolgar MS, Kahle AD, Kirsch DR, O'Sullivan J, Porubcan MA, Principe P, Trejo WH. Dactylocyclines: novel tetracycline glycosides active against tetracycline- resistant bacteria. J Org Chem. 1993;58(3):535-537. doi: 10.1021/jo00055a002.

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4 Towards the biosynthesis of 6-demethyl-6-

epitracyclines from TAN-1612 in Saccharomyces

cerevisiae

*The contents of this chapter will be published in:

E. Herbst, V. W. Cornish, et al “Biosynthesis of 6-demethyl-6-epitetracycline analogs of TAN-

1612 in Saccharomyces cerevisiae”, in preparation.

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4.1 Chapter outline

This chapter describes the work towards 6-demethyl-6-epitracycline derivatives of the fungal anhydrotetracycline TAN-1612 in Saccharomyces cerevisiae. Building on the previous chapters describing anhydrotetracycline hydroxylations, this chapter begins by exploring the product formed by coexpressing the TAN-1612 pathway and the bacterial hydroxylase PgaE (Section

4.4.1). The testing of fungal hydroxylase homologs to DacO1, the 6α-hydroxylase of anhydrodactylocyclinone is then described (Section 4.4.2) followed by the description of a method for bacterial monooxygenase saturation mutagenesis near the anhydrotetracycline binding site, with OxyS as an example (Section 4.4.3). Finally, the screening process of this natural diversity and generated diversity by mutagenesis is described (Section 4.4.4) with future directions for followup discussed. A key result described in this chapter is the isolation and characterization of a new major product in a strain coexpressing PgaE and the TAN-1612 pathway that differs from the TAN-1612 major product in the strain expressing the TAN-1612 pathway without PgaE

(Figure 4-2, Figure 4-3 and Figure 4-5). The followup plan on this result and on the screen for

TAN-1612 hydroxylases is outlined in the Discussion.

4.2 Introduction

TAN-1612, a fungal anhydrotetracycline derivative originally produced by Aspergillus niger, has been introduced to S. cerevisiae by the Tang laboratory and our laboratory has further improved its titers in synthetic media, thus allowing the heterologous production of TAN-1612 derivatives.(1) TAN-1612 differs from anhydrotetracycline in five functional groups, an A-ring methyl ketone instead of the A-ring amide, a 4α-proton instead of the 4α-dimethylamino, a 4a- hydroxy instead of the 4a-proton, a 6-proton instead of the 6-methyl and an 8-methoxy instead of

132 the 8-proton (Scheme 4-2). The stereochemistry of TAN-1612 at the 4a,12a positions has not yet been verified, although the stereochemistry of the product of the homologous fungal polyketide pathway, viridicatumtoxin, has been determined to be the same as anhydrotetracycline in these positions.(2, 3) As outlined in the introduction chapter, the synthesis of 6-demethyl-6- epiglycotetracyclines is highly desirable (Section 1.6). TAN-1612 presents a unique scaffold for the biosynthesizing these derivatives in S. cerevisiae due to its ready biosynthesis in this heterologous host and its unique functional groups among anhydrotetracycline (Section 1.6.4).

Much of the knowledge generated in the process of anhydrotetracycline hydroxylation by OxyS and PgaE, as well as DacO1 expression optimization, described in the previous chapters, is directly relevant to the hydroxylation of TAN-1612 in S. cerevisiae as outlined in this chapter.

4.3 Materials and methods

See Chapter 5 for general methods. See Chapter 3 for general protocol for high throughput assay of hydroxylation/reduction assay in whole cells in microtiter plates and a general protocol for western blots.

4.3.1 General protocol for high throughput assay of TAN-1612 hydroxylation

in whole cells in microtiter plates

Fresh colonies of strains harboring a plasmid encoding the TAN-1612 pathway and a hydroxylase and control strains (EH-5-212-2 encoding PgaE and the TAN-1612 pathway as positive control and EH-5-212-4 encoding the TAN-1612 pathway and no hydroxylase as a negative control) were inoculated in 200 μL selective media (UT -) in 96-well plates (Corning 353072) and placed in shaker overnight. Overnight cultures were used to inoculate 300 μL selective media (UT-) cultures in deep well plates (Corning P-2ML-SQ-C-S) with an average starting OD of 0.01. The plate was covered with two layers of SealMate film (Excel Scientific, SM-KIT-BS) and placed in shaker at 133

800 rpm for three nights. 200 μL H2O were then added and after resuspension 20 μL were diluted into 180 μL of H2O in Corning 3603 plates before UV/VIS spectroscopic measurements. UV/VIS spectroscopic measurements taken were as follows: (i) absorption spectrum 350 nm – 600 nm, (ii) emission spectrum 450 nm – 650 nm (λexcitation = 400 nm), (iii) excitation spectrum 300 nm – 450 nm (λemission = 500 nm), (iii) excitation spectrum 300 nm – 450 (λemission = 560 nm). Spectral steps in the spectra were 50 nm.

4.3.2 Protocol for selecting amino acids for mutagenesis in hypothesized

binding pockets of monooxygenases

The structure of aklavinone-11 hydroxylase with FAD and aklavinone (PDB ID 3IHG) was loaded on PyMOL. Residues that are within 5 Å from the substrate aklavinone were selected as follows:

PyMOL>hide everything, all

PyMOL>select contacts, (resn VAK and chain A) around 5

Selector: selection "contacts" defined with 88 atoms.

PyMOL>select contacts_res, byres contacts

Selector: selection "contacts_res" defined with 268 atoms.

PyMOL>show sticks, contacts_res

Setting: seq_view set to on.

PyMOL>show sticks, resn vak

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4.4 Results

4.4.1 Testing PgaE and other bacterial hydroxylases for TAN-1612

hydroxylation

Considering the mass spectrometry support for hydroxylation of anhydrotetracycline by OxyS and

PgaE (Chapter 3, Section 3.4.5) it was logical to test them for TAN-1612 hydroxyaltion (Scheme

4-1). SsfO1 was tested as well because of its efficient expression in S. cerevisiae and 6α- hydroxylation of its native substrate (Section 3.4.5 and Scheme 4-2).

Scheme 4-1 Biosynthetic plan for TAN-1612 6α-hydroxylation

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Scheme 4-2 Bacterial monooxygenases and their hypothesized substrates and products Bacterial flavin-dependent monooxygenases OxyS, DacO1, SsfO1, CtcN and PgaE and their native substrates. Marked in blue are differences between the native substrate and TAN-1612.

The strain expressing the TAN-1612 pathway without an additional hydroxylase has an excitation peak at 420 nm for 560 nm emission and an emission peak of 530 nm for 400 nm excitation (Figure

4-1). This strain was confirmed to produce TAN-1612 as the major compound of 400 nm absorption (Figure 4-5-a and Figure 4-2). The strains expressing OxyS and SsfO1 have similar excitation and emission peaks, although both are increased for the sample encoding OxyS.

Notably, the strain encoding PgaE has a significantly reduced emission in 560 nm when excited at

136

420 +/- 30 nm (Figure 4-1), indicating that TAN-1612 is either not produced or produced in much lower quantities than in the other strains.

Figure 4-1 UV/Vis analysis of hydroxylation attempt of TAN-1612 by bacterial hydroxylases

(a) Excitation spectrum (λemission = 560 nm) (b) emission spectrum (λexcitation = 400 nm) of strains EH-5-212-1, EH-5-212-2, EH-5-212-3 and EH-5-212-4 harboring a plasmid encoding the TAN- 1612 pathway as well as a plasmid encoding OxyS, PgaE, SsfO1 and no hydroxylase, respectively. Emission and excitation spectra were taken after diluting cultures that were incubated for 3 nights in UT- media (4 mL) in 15 mL culture tubes (Corning 352059). Each data point and error bar represents the average and standard error of three biological replicates, respectively.

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In order to test which compound or compounds are being produced instead of TAN-1612 in the strain expressing the TAN-1612 pathway and PgaE, both that strain and the control strain expressing no hydroxylase were cultured in 500 mL scale to allow purification and further analysis by mass spectrometry and NMR. Following three nights of culturing each culture was extracted twice with EtOAc and the combined organic extract was washed with H2O and dried with Na2SO4 with the organic solvent removed under reduced pressure. The extract was then purified by semiprepative HPLC prior to NMR and mass spectrometry analysis.

As the absorption chromatogram of the semipreparative HPLC separation shows, the main product with regards to both 400 nm absorption and 254 nm absorption is modified in the +PgaE sample relative to the -PgaE control. While the main product in the -PgaE control elutes after 37 minutes, the main product in the +PgaE sample elutes after 34 minutes (Figure 4-2). Interestingly the -PgaE sample has a minor product eluting after 34 minutes, which could be the same product as the main product of the +PgaE sample, or a different one.

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Figure 4-2 UV/Vis chromatogram of the HPLC separation of extracts from +/- PgaE strains encoding the TAN-1612 pathway. Strains EH-5-212-2 and EH-5-212-4 harboring a plasmid encoding the TAN-1612 pathway as well as a plasmid encoding PgaE and no hydroxylase, respectively are indicated as +PgaE and -PgaE, respectively. Both strains were cultured in 500 mL scale for three nights. Prior to HPLC separation the culture was extracted twice with EtOAc and the combined organic extract was washed with

H2O and dried with Na2SO4, with the organic solvent removed under reduced pressure. Absorption chromatograms are shown for 254 and 400 nm for an HPLC separation method of 10:90 to 90:10

MeCN in 99.9% H2O/0.1% TFA over 1 h.

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The main product of each HPLC separation was isolated and analyzed by NMR and mass spectrometry. The mass spectrometry analysis of the -PgaE compound clearly supports that TAN-

+ + 1612 was isolated (MS (ES+): m/z calc’d for C21H19O9 , 415.1029; found 415.1026 [M + H]

(Figure 4-3-a) and the NMR spectrum matches the published TAN-1612 spectrum (Figure 4-5- a).(1) For the +PgaE sample, the mass spectrum shows four major peaks for an elution peak with absorbance at both 254 nm and 400 nm: 593.1218, 298.0743, 279.0966 and 278.0483 (Figure 4-3- b). The NMR spectrum of the sample does not show the methoxy and methyl ketone protons of shifts 3.89 and 2.58 ppm as well as the two aromatic protons of chemical shifts 7.05 and 6.45 ppm and does show additional 7 aromatic protons in their stead (Figure 4-5-b). The possible identity of the newly isolated major compound of the +PgaE +TAN-1612 pathway sample is discussed in

Section 4.5.1.

140

141

Figure 4-3 Mass spectrometry analysis of isolate from +/-PgaE strain encoding the TAN- 1612 pathway. Liquid chromatography mass spectrometry analyses for isolates from strains EH-5-212-2 and EH- 5-212-4 harboring a plasmid encoding the TAN-1612 pathway as well as a plasmid encoding PgaE and no hydroxylase, respectively are shown in (b) and (a), respectively. Both strains were cultured in 500 mL scale for three nights. Prior to HPLC separation the culture was extracted twice with

EtOAc and the combined organic extract was washed with H2O and dried with Na2SO4, with the organic solvent removed under reduced pressure. The LCMS separation method is 5:95 to 95:5

MeCN in 99.9% H2O/0.1% formic acid over 2 min.

Figure 4-4 MSMS analysis of isolate from +PgaE strain encoding the TAN-1612 pathway. Liquid chromatography MSMS analyses for isolate from strain EH-5-212-2 harboring a plasmid encoding the TAN-1612 pathway as well as a plasmid encoding PgaE with ion selection at mass

593. The LCMSMS separation method is 5:95 to 95:5 MeCN in 99.9% H2O/0.1% formic acid over 2 min.

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Figure 4-5 1H-NMR spectra of isolates from +/-PgaE strains encoding the TAN-1612 pathway.

143

1H-NMR spectra of isolates from strains EH-5-212-2 and EH-5-212-4 harboring a plasmid encoding the TAN-1612 pathway as well as a plasmid encoding PgaE and no hydroxylase, are shown in (b) and (a), respectively. Both strains were cultured in 500 mL scale for three nights. Prior to HPLC separation the culture was extracted twice with EtOAc and the combined organic extract was washed with H2O and dried with Na2SO4, with the organic solvent removed under reduced pressure. NMR spectra shown are in MeOD-d4 for the major product of each strain according to HPLC chromatogram (Figure 4-2). 1H NMR (for (b), δ>4 ppm, 500 MHz, MeOD- d4): δ 7.61 (dd, J = 8.0, 1.0 ,1H), 7.47 (d, J = 8.6, 2H), 7.22 (dd, J = 8.0, 1.1, 1H), 6.88 (dd, J = 8.6, 2H), 6.85 (dd, J = 8.0, 1H), 6.72 (s, 1H).

Figure 4-6 COSY NMR spectrum of isolate from +PgaE strain encoding the TAN-1612 pathway. Strain EH-5-212-2 harboring a plasmid encoding the TAN-1612 pathway as well as a plasmid encoding PgaE. Both strains were cultured in 500 mL scale for three nights. Prior to HPLC separation the culture was extracted twice with EtOAc and the combined organic extract was

144 washed with H2O and dried with Na2SO4, with the organic solvent was removed under reduced pressure. NMR spectra shown is in MeOD-d4 for the major product according to the HPLC chromatogram (Figure 4-2).

4.4.2 Searching the fungal hydroxylase space for 6α-hydroxylation of TAN-

1612

Given the expression challenges with the bacterial monooxygenase DacO1 (Section 3.4.1) it was logical to test fungal monooxygenases as well. Fungal monooxygenases were chosen based on homology to DacO1 and/or based on the presence of anhydrotetracycline monooxygenase in their

CDS product name. Table 4-1 shows the first twelve such enzymes attempted. Western blot analysis revealed that expression challenges could be prevalent with fungal hydroxylases as well, with only 25% of the fungal hydroxylases assayed displaying a band at the expected size (Figure

4-7). The strains that did not indicate any protein expression at the expected size encoded fungal monooxygenases of entries 1, 2, 4, 5, 6, and 11 from Table 4-1. The two strains that did exhibit protein expression at the expected size encoded fungal hydroxylases entries 8 and 12 from Table

4-1 (Figure 4-7). For the small sample tested, the two codon optimized fungal monooxygenases did not present a band of the expected size in S. cerevisiae. Two of the six non-codon optimized did presented such a band. The two strains that exhibited a protein of the expected size had a higher identity %, positives % and query cover with both DacO1 and OxyS than the six strains that did not exhibit a band of the expected size but not necessarily to a statistically significant degree

(Figure 4-7, Table 4-1 and data not shown).

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Table 4-1 Fungal monooxygenases biomined for TAN-1612 6α-hydroxylation in S. cerevisiae Entry Gene product Source organism GenBank Codon Identity Identity label entry optimi (%) / (%) / zed positives positives (%) / (%) / cover cover with with DacO1a OxySb 1 Anhydrotetracy Valsa mali strain CM00310 Y 30/44/266 25/39/388 cline 03-8 4.1 monooxygenas e 2 Anhydrotetracy Penicillium MNBE01 N 32/43/533 35/46/541 cline subrubescens 000128.1 monooxygenas strain CBS 132785 e 3 Anhydrotetracy Cercospora NC_0367 N 30/41/510 34/45/537 cline beticola 72.1 monooxygenas NC_036772 e 4 related to Phialocephala FJOG010 N 31/46/370 30/43/547 tetracenomycin subalpina strain 00008.1 polyketide UAMH 11012 synthesis hydroxylase tcmG 5 Anhydrotetracy Golovinomyces MCBR01 N 28/43/398 30/40/564 cline cichoracearum 007648.1 monooxygenas e 6 Anhydrotetracy Valsa mali var. KN71467 N 23/37/345 24/38/405 cline pyri strain 1.1 monooxygenas SXYL134 e 7 tetracycline 6- Aureobasidium KL584983 N 26/40/416 25/39/455 hydroxylase pullulans var. .1 protein pullulans 8 pentachlorophe Hypoxylon sp. KZ111246 N 34/45/520 38/49/531 nol 4- EC38 .1 monooxygenas e 9 probable 2- Phialocephala FJOG010 N 32/46/505 39/51/506 polyprenyl-6- subalpina strain 00031.1 methoxyphenol UAMH 11012

146

hydroxylase and related FAD- dependent oxidoreductase s 10 2-polyprenyl-6- Aspergillus oryzae AMCJ010 N 32/47/501 34/50/503 methoxyphenol 100-8 00140.1 hydroxylase 11 pentachlorophe Colletotrichum KB72597 Y 34/44/531 34/47/531 nol 4- orbiculare MAFF 6.1 monooxygenas 240422 e 12 unnamed Aspergillus oryzae AP007159 N 32/45/521 34/47/529 protein product; RIB40 .1 2-polyprenyl-6- methoxyphenol hydroxylase and related FAD- dependent oxidoreductase s" aDacO1 [Dactylosporangium sp. SC14051] GenBank: AFU65892.1 b OxyS [Streptomyces rimosus] GenBank AAZ78342.1

147

Figure 4-7 Western blot analysis of fungal hydroxylase expression in BJ5464-NpgA Strain cultures were lysed with Y-PER and labeled with Monoclonal ANTI-FLAG HRP antibody. The size (kDa) indicated below the gels is the expected size of the protein based on the amino acid sequence. Strains EH-5-227-1 through EH-5-227-9 encode Fungal monooxygenase entry 6, 5, 1, 4, 2, 2, 8, 11, 12, from Table 4-1, respectively.

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4.4.3 Evolving monooxygenases for 6α-hydroxylation of anhydrotetracyclines

– OxyS example

Considering the effective hydroxylation of anhydrotetracycline in yeast by OxyS (Chapter 2), its apparent lack of hydroxylation activity on TAN-1612 (Section 4.4.1) and an effective UV/Vis assay for anhydrotetracyclines hydroxylation (Sections 3.4.3-3.4.5), directed evolution of OxyS to accept TAN-1612 as a substrate seemed logical. In addition, OxyS structure was previously probed by X-ray crystallography, as well as the structure of a homologous protein, Aklavinone-11-

Hydroxylase, along with its native substrate, aklavinone (42% homology, PDB ID 4K2X and

3IHG, respectively).(4, 5) Thus, the substrate binding pocket of OxyS could be identified with some confidence and mutated accordingly.

In order to perform saturation mutagenesis on residues that are in proximity to the hypothesized substrate binding pocket of OxyS, residues of Aklavinone-11-Hydroxylase that are within 5 Å of aklavinone were listed (Section 4.3.2). Then the homologous residues in OxyS were noted as well

(Table 4-2). Some of these homologous residues in OxyS indeed sit in proximity to a cavity that is a continuation of the cavity in which FAD is situated in the OxyS crystal structure (Figure 4-8).

Table 4-2 Residues chosen for mutagenesis in OxyS Residue Residue context in Residue in Residue context Distance number 3IHG OxyS in OxyS from VAK in 3IHG (Å) 1 R45 PYPRAAG K42 FSKALGV 4-5 2 A47 PRAGQN A43 SKALGVH <4 3 G48 AAGQNPR G45 KALGVHAR 4-5 4 I72 ADDIRG - - <4 5 F79 QGDFVIR - - <4 6 I81 QGDFVIR - - 4-5 7 M101 DDMVAA - - 4-5 8 W113 PAGWAM L95 PYALFV 4-5 9 M115 PAGWAMLSQD F96 PYAL-FVPQV <4 10 M201 LTHMVGV M176 --HMFAV <4 11 W221 GTTGWYYL W211 DLRAWFAAFP <4

149

12 Y223 TGWYYLH F212 DLRAWFAAFPL <4 13 T232 PEFKGTFGPTDRP T225X ATVA <4 14 P235 TFGPTDRP F228 TVAFFDRP 4-5 15 F245 RHTLFVEYDPDEG V240X RRAPVTEED <4 16 W287 VDIQGWEMAARIA - - <4 17 M289 WEMAARIA - - 4-5 18 P314 VTPPTGGMSG P295X IHLPAGGQGL <4 19 T315 VTPPTGGMSG A296X IHLPAGGQGL <4 20 G316 VTPPTGGMSG G297X IHLPAGGQGL <4 21 G317 VTPPTGGMSG G298X IHLPAGGQGL <4 22 R373 AIYAQRMAP P357 LIDPDPRYE 4-5 23 M374 AIYAQRMAP R358 LIDPDPRYE 4-5 24 Y387 SVGYPET V372 LLHVPET 4-5

150

Figure 4-8 PyMOL illustrations of amino acids in proximity to the anhydrotetracycline substrate in aklavinone-11-Hydroxylase and in OxyS PyMOL illustration of (a) alkavinone and FAD (stick representation, carbons colored white) surrounded by amino acids of aklavinone-11-Hydroxylase within 5 Å of alkavinone (stick representation, carbons colored green, PDB ID 3IHG) and (b) FAD (carbons colored white) surrounded by OxyS (surface representation, carbons colored green, PDB ID 4K2X) and its amino

151 acids that are homologous to those of aklavinone-11-Hydroxylase amino acids that are within 5 Å of alkavinone in the structure shown in (a).

4.4.4 Library screening for TAN-1612 hydroxylation in S. cerevisiae

Following the assembly of the libraries of fungal monooxygenases (Section 4.4.2) and OxyS saturation mutagenesis (Section 4.4.3) the libraries were screened for TAN-1612 hydroxylation using a variant of the microtiter plate assay for anhydotetracycline hydroxylation (Section 3.4.3 and 4.3.1). As negative control a strain encoding the TAN-1612 pathway without an additional hydroxylase was used. As positive control a strain encoding the TAN-1612 pathway and PgaE was used, as that strain was already shown to produce an alternative major product instead of TAN-

1612 (Figure 4-2).

Given that the positive control strain expressing PgaE showed a significantly reduced emission at

560 nm upon 400 and 450 nm excitation, colonies were plotted according to their absorbance at these wavelengths and normalized to OD6oo: (400 + 450 nm) / 600 nm. When colonies of each 96- well plate was ranked according to this absorption criteria the PgaE positive control was ranked on average 83.6 (out of 96) with a standard deviation of 10.0 (Table 4-3 and Figure 4-9). A low rank of PgaE is expected in the likely scenario that most strains screened do not encode an efficient

TAN-1612 hydroxylase. Wells 84 and 96 containing no cells consistently ranked the lowest in the measurement of (400 + 450)/600 nm (Figure 4-9).

Table 4-3 Rank of PgaE positive control in screen for TAN-1612 hydroxylase Plate Strain Absorbance C1 Absorbance C2 (400 + 450)/600 (400 + 450)/600 a EH-5-217-2 89 59 b EH-5-217-2 91 92 c EH-5-217-2 94 68 d EH-5-217-4 88 79 e EH-5-217-4 78 85 f EH-5-217-4 93 87

152 g EH-5-217-1 and 81 87 EH-217-3 Average (all) 83.6 Standard 10.0 deviation (all)

Figure 4-9 Library screening for TAN-1612 hydroxylation in S. cerevisiae Y axes shows the sum of absorption at 400 and 450 nm divided by absorption at 600 nm while the X axes show the colony number. Plates a, b and c are screens of strain library EH-5-217-2. Plates d, e and f are screens of strain library EH-5-217-4. Both strain libraries EH-5-217-2 and EH-5- 217-4 encode the same OxyS saturation mutagenesis library and differ in the background TAN- 1612 producing strain. Plate g contains strain libraries EH-5-217-1 and EH-5-217-3 encoding bacterial and fungal hydroxylases, including selected DacO1 fusion proteins. Strains EH-5-212-1,

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EH-5-212-2 and EH-5-212-4 encoding the TAN-1612 pathway along with OxyS, PgaE and no hydroxylase, are labeled in blue, red and white, respectively.

4.5 Discussion

4.5.1 Testing PgaE and other bacterial hydroxylases for TAN-1612

hydroxylation

Despite the structural dissimilarities between UWM6, the native susbstrate of PgaE and TAN-

1612 (Scheme 4-2) it is PgaE and not OxyS or SsfO1 whose coexpression with the TAN-1612 pathway has led to the production of a different major product of 400 and 254 nm absorption instead of TAN-1612. Both the chromatograms for the HPLC separation of the +PgaE and -PgaE samples, as well as the excitation and emission spectra support that PgaE is converting TAN-1612 or an intermediate in its biosynthesis (Figure 4-2, Figure 4-1). It would therefore be of interest to test other enzymes involved in the 12-position hydroxylation of UWM6, such as CabE, LanE,

UrdE and GilOI.(6, 7)

The protons of chemical shifts 7.47 and 6.88 ppm with coupling constant of 8.6 Hz are likely two pairs of equivalent aromatic protons at ortho to each other and they are shown to interact in the

COSY spectrum as well (Figure 4-5 and Figure 4-6); the protons of chemical shifts 7.61, 7.22 and

6.85 ppm are likely three adjacent protons in one aromatic ring that has three other substituents, with the protons of chemical shifts 7.22 and 7.61 ppm likely meta to each other. Interactions between the protons of chemical shifts 7.61 and 6.85 as well as 7.22 and 6.85 ppm are also readily observed in the COSY spectrum and the coupling constants, characteristic of ortho interactions, are 8.0 Hz each (Figure 4-5 and Figure 4-6). The singlet at 6.72 ppm is likely a single proton in an aromatic ring, possibly the only remaining proton in the D-ring of the substituted TAN-1612 or its

154 intermediate (Figure 4-5). The absence of the methoxy shift in the new major product at 3.89 ppm

(Figure 4-5) can indicate that PgaE derivatization occurs prior to AdaD methylation, which is assumed to be the last step in TAN-1612 biosynthesis.(1) It could be that following hydroxylation by PgaE and a potential further derivatization by a yeast endogenous molecule, substrate affinity of AdaD to the modified product is too low to allow methylation. Examples for the moieties that might be part of the newly formed major compound are shown in Figure 4-10. Importantly, not all moieties are necessarily part of the same molecule, as can be verified by larger scale fermentation followed by further purification and NMR analysis. The aromatic moiety of Figure 4-10-c could theoretically represent a doubly hydroxylated TAN-1612 or any of the TAN-1612 intermediates of Figure 4-11. However such an assignment is questionable, given the lack of the protons of chemical shift 3.89 and 2.58 ppm corresponding to the methoxy and methyl ketone at the NMR spectrum of the newly formed major compound (compare Figure 4-5-a and Figure 4-5-b).

Figure 4-10 Possible moieties in the product isolated from +PgaE strain coexpressing the TAN-1612 bioysnthetic pathway Either of the two aromatic moieties of (a) could be responsible for generating the protons of chemical shifts 7.61, 7.22 and 6.85 ppm, the aromatic moiety of (b) could be responsible for generating protons of chemical shifts 7.47 and 6.88 ppm and either of the aromatic moieties of (c) could be responsible for generating a proton of chemical shifts 6.72 ppm in the 1H-NMR spectrum of the product isolated from +PgaE strain coexpressing the TAN-1612 bioysnthetic pathway (Figure 4-5). The squiggly lines represent any non-proton substituent.

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Figure 4-11 Possible intermediates in the biosynthesis of TAN-1612 TAN-1612 (1) and its intermediates 2-10 that can occur in the case of exclusion or disfunction of AdaB, AdaC or AdaD, the three post PKS biosynthetic enzymes of the TAN-1612 biosynthetic pathway, or combinations thereof.(1)

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The mass spectrum of the isolated compound from the +PgaE culture has major peaks with m/z values of 593.1218, 298.0743, 279.0966 and 278.0483 (Figure 4-3). The 593.1218 ion has an elution peak after 1.60 and after 1.47 min, but an absorption peak at 254 nm and 400 +/- 60 nm is noted only slightly before the 1.60 min (Figure 4-3) and not slightly before the 1.47 peak (the ions are first detected at the diode array before mass spectrometry scanning). Assuming that the two

593.1218 ions could be related stereoisomers of the same exact mass, it is perhaps doubtful whether any of them corresponds to a derivative of the TAN-1612 intermediates shown in Figure

4-11. This is because at any stereoisomeric form such compounds would be expected to have absorption at 400 and/or 254.

The 298.0743 and 278.0483 ions are four protons and one oxygen different in their m/z values.

Possibly a molecule of H2O + oxidation / reduction apart. In the MSMS spectrum for the ions of m/z 593 both the ions 278.0452 and 296.0551 appear (Figure 4-4), the former being the mass of

H2O less than the latter and the latter being H2 away from 298.0743 in its mass. Also of note is that the 593.1218 ion could be a result of a dimer of the 296.0551 ion plus the mass of a proton.

Thus, the 593.1218, 298.0743, 296.0551 and 278.0483 ions could correspond to chemical formulas

+ + + + of C32H21N2O10 , C16H12NO5 , C16H10NO5 , C16H8NO4 as they differ in 0.0022, 0.0028, 0.0008 and 0.0030 u, respectively, from the expected masses of these ions. These could be ions of some xanthurenic acid derivatives that PgaE might or might not have a contribution to their biosynthesis

(Figure 4-12). While the derivatives of xanthurenic acid shown in Figure 4-12 are not known in yeast, xanthurenic acid is a known yeast metabolite.(8) This proposal of structure or any other structure that involves only yeast metabolites and their derivatives could be explaining the mass and NMR spectra of the isolated major compound in the +PgaE sample (Figure 4-3, Figure 4-4,

Figure 4-5 and Figure 4-6). However, a major compound that does not involve a derivative of

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TAN-1612 or its intermediate still does not directly explain how the expression of PgaE leads to the reduction in the TAN-1612 absorbance and fluorescence (Figure 4-1 and Figure 4-2).

Figure 4-12 xanthurenic acid and its derivatives that could correspond to experimental mass and NMR spectra The xanthurenic acid derivatives presented in this figure have [M + H]+ values of 593.1196, 298.0715 and 296.0559 that are 0.0022, 0.0028 and 0.0008 amu from the experimental values detected 593.1218, 298.0743 and 296.0551 (Figure 4-3 and Figure 4-4). In addition, the protons of a molecule such as the one shown with chemical formula C16H11NO5 can correspond to the 6 proton types identified in the NMR spectrum (Figure 4-5 and Figure 4-6) and identified to potentially belong to the moieties described in Figure 4-10.

In order to determine the structure of the newly formed major compound with regards to 254 nm and 400 nm absorption in the +PgaE sample coexpressing the TAN-1612 pathway, the reaction would be repeated in larger scale to isolate enough of the newly formed compound for HSBC and

HMBC proton coupled carbon NMR spectra. Furthermore, the minor compound eluting at 34 min for the -PgaE sample will be isolated and analyzed as well to identify whether it is identical or at all related to the product isolated from the +PgaE sample that elutes at 34 min (Figure 4-2). This

158 characterization can assist in understanding the role PgaE has on the biosynthesis of the newly formed major compound with respect to 400 nm and 254 nm absorption in the +PgaE sample

(Figure 4-2). Another source of information would be the culturing and isolation of another control strain encoding PgaE but not encoding the TAN-1612 pathway and examining whether such strain displays a similar major peak in the HPLC chromatogram of 254 and 400 nm absorption as the strain that encodes the TAN-1612 pathway.

4.5.2 Searching the fungal hydroxylase space for 6α-hydroxylation of TAN-

1612

Importantly, the library generation approaches used to generate 6α-hydroxylases of anhydrotetracycline (Subsections 3.4.2 and 3.4.4) could be used for generating TAN-1612 hydroxylases (Subsections 4.4.2 and 4.4.3) and vice versa. The expression analysis of fungal monooxygenases showed that while branching towards fungal monooxygenases might be a useful strategy to find 6α-hydroxylases and specifically 6α-hydroxylases of TAN-1612, the fungal monooxygenases do not necessarily present an advantage over their bacterial counterparts as far as expression challenges in S. cerevisiae are concerned (Figure 4-7 and Chapter 3).

4.5.3 Evolving monooxygenases for 6α-hydroxylation of anhydrotetracyclines

Amino acids in OxyS were chosen for mutagenesis based on homology to amino acids in the proximity of the substrate in the crystal structure of RdmE and this approach could be implemented in other monooxygenases, such as PgaE or expression optimized DacO1 (Chapter 3). Importantly, implementing this approach does not require an existing crystal structure of the monooxygenase of interest. Also, as seen in the case of OxyS, at least some of the amino acids chosen for mutagenesis according to this strategy actually sit in proximity to the anhydrotetracycline cavity of OxyS (Figure 4-8). However, in the case of OxyS, an approach that uses the published crystal

159 structure of OxyS to choose additional amino acids for mutagenesis or to avoid mutating some of the amino acids that are not in proximity to the cavity might have been preferred. Further mutagenesis is being performed to explore the effect of additional mutations of OxyS in positions that are homologous to amino acids in RdmE structure that are up to 8 Å from aklavinone in 3IHG.

Finally, similar followup mutagenesis studies on stably expressed DacO1 fussion proteins, as well as PgaE will be directly relevant for testing 6α-hydroxylation of TAN-1612. Specifically for PgaE, much structural information is available to guide the amino acid choice for saturation mutagenesis from its crystal structure and from biochemical analyses.(6, 9-11)

4.5.4 Library screening for TAN-1612 hydroxylation in S. cerevisiae

In the screen for TAN-1612 hydroxylation, a variant of the microtiter plate assay for anhydrotetracycline hydroxylation described in Chapter 3 was used. Chapter 3 also discusses non- microtiter plate avenues for the anhydrotetracycline hydroxylation (Section 3.5.3) and these would be especially interesting to examine in the context of TAN-1612 hydroxylation, as TAN-1612 does not need to be exogenously supplied to the cells.

Similarly to the followup on the +PgaE +TAN-1612 encoding strain (Sections 4.4.1 and 4.5.1), strains with a lower (400 + 450)/600 nm absorption than the PgaE positive control (Figure 4-9) will be assayed for TAN-1612 hydroxylation by UV/Vis and mass spectrometry. If data supports production of an alternative major product to TAN-1612 these strains will be cultured in larger scale to allow purification and analysis of the product by NMR. If data does not support TAN-

1612 hydroxylation in any of the assayed strains, strain selection for further assay will be attempted based on other criteria such as 550 + 600 – 450 nm emission upon 400 nm excitation or 400 + 450

– 350 nm excitation for 560 nm emission. These criteria are based on the reduced emission and excitation peaks observed for the PgaE encoding positive control strain (Figure 4-1).

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4.6 Conclusion

This chapter describes the progress towards TAN-1612 hydroxylation in S. cerevisiae, the first step in the synthesis of 6-demethyl-6-epitetracycline from TAN-1612 in S. cerevisiae. This chapter shows that a strain expressing both PgaE and the TAN-1612 pathway produces a major compound with regards to 400 nm and 254 nm absorption that differs from TAN-1612 (Sections 4.4.1 and

4.5.1, Figure 4-1, Figure 4-2, Figure 4-3 and Figure 4-5). Future work towards TAN-1612 hydroxylation in S. cerevisiae should include a larger scale culturing of the +PgaE +TAN-1612 pathway strain to enable followup NMR experiments. In addition, the minor compound eluting at

34 min for the -PgaE sample (Figure 4-2) will be isolated and analyzed to verify whether it is identical or at all related to the product isolated from the +PgaE sample that elutes at 34 min.

Another control strain encoding PgaE but not encoding the TAN-1612 pathway will be cultured and examined to discover whether it displays a similar major peak in the HPLC chromatogram to that of the +PgaE +TAN-1612 pathway strain.

This chapter also describes a hydroxylase harvesting and mutagenesis strategy as well as a UV/Vis assay as a platform for identifying TAN-1612 hydroxylation. This platform was applied to the screening for TAN-1612 hydroxylation (Section 4.4.4) by OxyS mutants (Section 4.4.3), DacO1 fusion proteins (Sections 3.4.1 and 3.4.5), and other hydroxylases from bacterial (Sections 3.4.2 and 3.4.5) and fungal sources (Section 4.4.2). Followup culturing and spectroscopic analysis will be done on strains coexpressing the TAN-1612 pathway and one of the above mentioned hydroyxlases, identified as potentially hydroxylating TAN-1612 in the UV/Vis assay (Figure 4-9).

In addition to the followup on PgaE and on the other hydroxylases described in this chapter, other enzymes involved in the 12-position hydroxylation of UWM6, such as CabE, LanE, UrdE and

GilOI should also be tested for TAN-1612 hydroxylation.(6, 7) With a mutagenesis strategy that

161 does not require a crystal structure that could be used for DacO1 (Figure 4-8) and with the additional benefit of the PgaE crystal structure,(9) followup mutagenesis studies such as shown on

OxyS (Section 4.4.3) and on DacO1 (Section 3.4.4) on stably expressed DacO1 fusion proteins

(Section 3.4.5), as well as on PgaE could prove beneficial for 6α-hydroxylation of TAN-1612.

Identification and overexpression of a reductase responsible for 5a(11a)-dehydrotetracycline reduction in S. cerevisiae (Section 2.4.2 and 2.5.2) will be potentially useful for the reduction of the hydroxylation product of TAN-1612 as would be other efforts in the reduction of 5a(11a)- dehydrotetracycline in S. cerevisiae (Section 3.4.5 and 3.5.5). Finally, the anhydrotetracycline hydroxylations described in this chapter and in Chapters 2 and 3 relies heavily on the UV/Vis assay developed for the hydroxylation of anhydrotetracyclines (Section 3.4.3, 3.4.5, and 4.4.4). More broadly, high-throughput assays are of very high importance to small molecule production in microorganisms because of the inability to predict in advanced the successful strain modifications needed in the complex environment of the cell.(12) TAN-1612 and anhydrotetracycline hydroxylation belong to a special group of biochemical transformations that are easy to assay in high throughput because of the inherent change in the chromophore of the CD ring (Scheme 4-2).

However, the next steps for 6-demtehyl-6-epiglycotetracyclines from TAN-1612 will require a general, readily implemented, high-throughput assay for tetracycline biosynthesis in yeast. The development and characterization of such an assay is described in Chapter 5.

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4.7 Appendix

Table 4-4 Strains used in this study Strain Genotype Source/Reference BJ5464- MATα ura3-52 his3-Δ200 leu2-Δ1 a / (13) NpgA trp1 pep4::HIS3 δ:: pADH2-npgA-tADH2 prb1Δ1.6R can1 GAL PBA-482 BJ5464 ΔPEP4::pTDH3-Sb-NpgA-tENO2 P. A. Baldera- ΔPRB1::pTDH3-Sb-NpgA-tENO2 Aguayob PBA-1277 BJ5464 ΔPEP4::pHSP26-Sb-NpgA-tENO2 P. A. Baldera- ΔPRB1::pHSP26-Sb-NpgA-tENO2 Aguayob PBA-1333 PBA482 pPBA-L2BWG9 P. A. Baldera- Aguayob PBA-1337 PBA1277 pPBA-L6BWG9 P. A. Baldera- Aguayob EH-5-212-1 PBA-1337 AL-1-101-C10 This Study EH-5-212-2 PBA-1337 AL-234-C-C1 This Study EH-5-212-3 PBA-1337 AL-239-B This Study EH-5-212-4 PBA-1337 pSP-G1 (Addgene 64736) This Study EH-5-217-1 PBA-1337 Plasmid Libraries A + C This Studyc EH-5-217-2 PBA-1337 Plasmid Library D This Studyc EH-5-217-3 PBA-1333 Plasmid Libraries B + C This Studyc EH-5-217-4 PBA-1333 Plasmid Library D This Studyc EH-5-227-11 BJ5464-NpgA AL-2-72-B-C6 This Study EH-5-227-12 BJ5464-NpgA AL-2-72-D-C5 This Study EH-5-227-13 BJ5464-NpgA AL-2-72-F-C4 This Study EH-5-227-14 BJ5464-NpgA AL-2-79-J-C4 This Study EH-5-227-15 BJ5464-NpgA AL-2-79-K-C5 This Study EH-5-227-16 BJ5464-NpgA AL-2-79-L-C6 This Study EH-5-227-17 BJ5464-NpgA AL-2-86-D-C5 This Study EH-5-227-18 BJ5464-NpgA AL-2-86-I-C5 This Study EH-5-227-19 BJ5464-NpgA AL-2-86-A-C1 This Study a Y. Tang, University of California, Los Angeles, Department of Chemical and Biomolecular Engineering & Department of Chemistry and Biochemistry, Department of Bioengineering b Integrated Program in Cellular, Molecular and Biomedical Studies, Cornish Laboratory, Columbia University, New York, NY 10032, USA c Plasmid libraries compositions are described below

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Table 4-5 Plasmids used in this study Description Source/Reference pSP-G1 pTEF1-FLAG-tADH1, pPGK1-MYC-tCYC1, 2 μ, Ura Addgene 64736 AL-1-101-C10 pTEF1-oxyS-tADH1 in pSP-G1 Chapter 2 AL-1-234-C-C1 pTEF1-pgaE-tADH1 in pSP-G1 JCAT codon opt. Chapter 3 AL-1-239-B-C3 pTEF1-ssfO1-tADH1 in pSP-G1 JCAT codon opt. Chapter 3 AL-2-72-B-C6 pTEF1-Fungal-monooxygenase-6-tADH1 in pSP-G1c This Study AL-2-72-D-C5 pTEF1-Fungal-monooxygenase-5-tADH1 in pSP-G1c This Study AL-2-72-F-C4 pTEF1-Fungal-monooxygenase-1-tADH1 in pSP-G1c This Study AL-2-79-J-C4 pTEF1-Fungal-monooxygenase-4-tADH1 in pSP-G1c This Study AL-2-79-K-C5 pTEF1-Fungal-monooxygenase-2-tADH1 in pSP-G1c This Study AL-2-79-L-C6 pTEF1-Fungal-monooxygenase-2-tADH1 in pSP-G1c This Study AL-2-86-D-C5 pTEF1-Fungal-monooxygenase-8-tADH1 in pSP-G1c This Study AL-2-86-I-C5 pTEF1-Fungal-monooxygenase-11-tADH1 in pSP-G1c This Study AL-2-86-A-C1 pTEF1-Fungal-monooxygenase-12-tADH1 in pSP-G1c This Study EH-5-49-C pTEF1-GAL1-dacO1-tADH1 in pSP-G1 Chapter 3 EH-5-80-6 pTEF1-UBI4-IFNG-dacO1-tADH1 in pSP-G1 Chapter 3 EH-5-80-7 pTEF1-IFNG-dacO1-tADH1 in pSP-G1 Chapter 3 EH-5-80-10 pTEF1-oxyS-dacO1-37-tADH1 in pSP-G1 Chapter 3 EH-5-80-11 pTEF1-oxyS-dacO1-87-tADH1 in pSP-G1 Chapter 3 pAV124 Trp1, 2 μ acceptor vector for yeast golden gate Addgene 63190/(14) pPBA-AV124 Modified Trp1, 2 μ acceptor vector for yeast golden gate P. A. Baldera- Aguayoa pPBA- pHSP26-adaA-tPGK1_pPGK1-adaB-tADH1_pTEF1- P. A. Baldera- L2BWG9 adaC_tENO1_pTDH3-adaD_-tRPL15A on pPBA- Aguayoa AV124 pPBA- pHSP26-adaA-tPGK1_pHSP26-adaB-tADH1_pTEF1- P. A. Baldera- L6BWG9 adaC_-tENO1_pTDH3-adaD-tRPL15A on pPBA- Aguayoa AV124 a Integrated Program in Cellular, Molecular and Biomedical Studies, Cornish Laboratory, Columbia University, New York, NY 10032, USA b Fungal-monooxygenase-X refers to the fungal monooxygenase from Table 4-1 entry X. Thus, for example Fungal-monooxygenase-2 refers to the fungal monooxgenase from Table 4-1 entry 2.

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Plasmid libraries used for making strains EH-5-217-1 through EH-5-217-4 Table 4-6 Plasmid Library A – Strain EH-5-217-1 Library component Plasmid Plasmid common Colony # no. name 1 EH-5-49-C Gal1-DacO1 1 2 EH-5-80-6 9 - ubiIFN H 3 EH-5-80-7 9 - IFN H 4 EH-5-80-10 11-OxyS-DacO1-37 G 5 EH-5-80-11 11-OxyS-DacO1-87 H

Table 4-7 Plasmid Library B – Strain EH-5-217-3 Library component Plasmid Plasmid common Colony # no. name 1 EH-5-49-C Gal1-DacO1 1 2 EH-5-80-6 9 - ubiIFN H 3 EH-5-80-7 9 - IFN H 4 EH-5-80-10 11-OxyS-DacO1-37 G 5 EH-5-80-11 11-OxyS-DacO1-87 H 6 AL-234-C PgaE in pSPG1 1 7 AL-1-101 OxyS 10 8 AL-239-B SsfO1 3

Table 4-8 Plasmid Library C – Strain EH-5-217-1 and EH-5-217-3 no. Plasmid Plasmid common name Colony # 1 AL-2-72-B Atc Moase Valsa Mali Var Pyr C6 2 AL-2-72-D Atc Moase G Cichoaracearumi C5 3 AL-2-72-F Atc Moase Valsa Mali CM C4 4 AL-2-79-J Related to tetra hydroxylase P subalpina C4 5 AL-2-79-K Atc Moase P subrubescens C5 6 AL-2-79-L* Atc Moase P subrubescens C6 7 AL-2-86-D Pentachlorophenol-4-Moase Hypoxylon C5 8 AL-2-86-I Pentachlorophenol-4-Moase Colletotrichum orbiculare C5 9 AL-2-86-A 2-ployprenyl-6-methoxyphenol-hydroxylase Aspergillus C1 Oryzae RIB40 * AL-2-79-K and AL-2-86-L are the same plasmid but one of them does and the other does not encode a mutation in the sequence.

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Table 4-9 Plasmid Library D – Strains EH-5-217-2 and EH-217-4 no. Plasmid Plasmid common name 1 AL-2-57-A F96X OxyS Library 2 AL-2-57-B M176X OxyS Library 3 AL-2-57-C W211X OxyS Library 4 AL-2-57-D F212X OxyS Library 5 AL-2-57-E T225X OxyS Library 6 AL-2-57-F V240X OxyS Library 7 AL-2-57-G P295X OxyS Library 8 AL-2-57-H A296X OxyS Library 9 AL-2-83-A A43X OxyS Library 10 AL-2-83-B G297X OxyS Library 11 AL-2-83-C G298X OxyS Library

Table 4-10 Sequences used in this study Sequences for the hydroxylases fungal hydroxylases of Table 4-1 are shown capitalized within the context of the pSP-G1 backbone containing pTEF1, the FLAG tag and tADH1 (partial, decapitalized). pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa CM003104 gttttaattacaagcggccgcATGATGGGTGCAAAACACGTTATTAGAAGAGTGA .1-tADH1- CCAATTCGGAAAATGATACAGGATCAAGTTCGAAATCCACTACTTCA in-pSP-G1 TCTAATTCCGACAGGGACCATAATCCCTCTTCCTCTAATAGAGACAG (Entry 1 ATATCACAATGAAACATCACCTGCCAATATGATATTGATAAACGAGC Table 4-1, CACCTCCTACCACCTCCACAGAATTGGAGAGCGACACTCATATTAAC AL-2-72- GTACGTAGCGTTTCTCCATGTTTGGAAACTGGTTCCCTAAAAGCAGA F-C4) GCAAGCGCCTCAGAATGTCCAGAACCCACCACAGACACAGGACCAA GCTACTGAAGATACTGCCACCAACTCTCAACCGGCCTCAACGCAGTT CAGAGCTATCATTGTGGGAGGTGGCCCAAACGGTTTGTGTCTGGCAC ATGCGTTATACCTAGCTGGTATTGATTATGTTTTGCTAGAGCGCGGTG GTGAAATCATCAACCAATCTGGAGCCGGACTTGCTTTATGGCCTCATT CAGTTCGTATATTAGATCAATTGGGTCTGCTAGACGAAGCTAGGAAA CATTATTTCCCGATTAAGACAAAGCATAACCACCGCCCCGACGGGTC AGTCAGAGATGTTAACGATATGTTTGCCAAGGTAGAAGTTAATCACG GTCACCCCTGGATGCTGTTCCATCGAGTAACATTGCTAGAACTCTTGT GGGAAAATTTGCCTGATAAAGAGACTAGAGTAAAAGTCAACAAGAA GGTGGAAAGTGTAGTCTCCAACCATGATGGCGTCGTGGTTACATGTT CAGATGGAACATCTGAAGCAGGAAGTATAGTGATAGGTTGCGATGGT GTACACTCGACTGTTAGACAAGTCATGCATGACTTGAGAAGCGAGAA AAAGAAGAAAAGGCCAGGCCGAAAGCTCTCTCTGGGAAGGTCGCCG TCACAAGACAAGCCAATGAGAGCCCACTACTATGGGCTTATTGGTTG GATTCCGCTTCTTGATGGGTTACAACCTGCTGCTTGCTACGAAGTGAG AAGTGAGCCTAAAGGTAAAACCTTTACCATTTTAACGGGCGAAGATA CCGCTTACTTCATCGTGTATATTCATTTAGAAAAACCCACGAGAGAG

166

CGCAGCAGGCACACAGACGAGGATGCCGAAAGGTTGGCAGCCGCAT TGGCCGAGAACAAGATAACGAGGGAAATTACTTTTGGTGACCTATGG AGATCAAGGCGTTGGGGCAAAATGCTTGATTTTCAAGAAGGGTTCGT AGATAAATGGTATCATGAAAGAATCGTTTTAGTTGGCGATGCTGTCC ATAAGATGACTCCAAATGCGGGACTAGGCTTGAATGCTGGCTGGCAA GGTATCGCTGAATTAACGAACAGATTGCGGAGATTAGTTGTTGCAGA AGGACAAAGACCAGATGCAAGGTGCGTTGAAAAAGTTTTTCGAGGTT ACCAAGATAGTAGAAAGGGTATGGCAAAGAAAACTATGAGGTTTTCT TCTCTGTACACCCGTGTGGTAGCTAATCAGAGCTTACTGTATCGTTTT TGTGATAGAATGACACCAGCGGTTGGTGGGGATGTCGCATTATTAAA TACAATGGCTAGTCCTATTGTTAAAAAAGGTGTCACTTTCGACTTTGT AGCGGAACGTGATCATAAAGAAGGTAGAGTAAGATGGGTTCATCCA CAACATGTTCCAGCTGAAgtatcgatggattacaaggatgacgacgataagatctgagctcttaatt aacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa MNBE010 gttttaattacaagcggccgcATGACCAAGTCCAAGTTATACGACGTGATAATCG 00128.1- CGGGCGCCGGACCCGTCGGCCTCTTCCTCGCCCACGAACTCAGTCTC tADH1-in- CGCCAAATAGCAGTCCTAGTCCTAGAACGAACAACCCAACAAGACTC pSP-G1 GCCCTGGAAATCCGGACCTCTAGGCCTCCGAGGCCTAAACATCCAAT (Entry 2 CCATCGAAGCATTCTATCGCCGCGGTTTACTAGGCCAATTATTCAATC Table 4-1, TGGACGAAAGGCAAAAGTACCAACCGAGCAAGAAACCAGGTTTCCA AL-2-79- ATTCGGCGGCCACTTCGCCGGCATCATAATAAATGCCAATCAATTCG K-C5 or ATTTGCAACGCTGGAAGTACCGCCTCTCAGGTCCGGCGCTTGGTCCG AL-2-79- GGTCCAACGACGATGGATCAAGTGGAGAAGGTCCTAACGGCGCGGG L-C6) TGGAGAGTCTAGGCGTGAAGATTCTCCGCGGTCGATCTGTTAGCCGT ATCTCGCAGGATGAGGCAGGCGTTACAGTCGAAACAGACGATGACC ATTCCGAGTCATTTCGGGGCAAATGGCTCGTTGGCTGTGACGGTGGG CGCAGCGTTGTTCGAAAAGAAGCCGGCTTCGAATTCGTCGGTACAGA TGCCCAATTCACCGGCTACGCAACGCATGTCGAGATAGAAGGTCGCG AGAAGTTGAAGCCTGGTCTCAACGTTTCTGAAACGGGGATGTATATC AACGTAGTGCAGAATGGCGCTTTTCATCTCCTTGAATTTGACGGGGC GAACTTCGATCGCGCGGGTGATATCACGATCGATCACCTTCAGCGGG TCCTTGATCGGGCTATTGGTCGGACAGATGTGAAGATTACAAATGTG CATCTTGCTTCTTGTTATACCGATCGTTCAATGCAGGCGGCAAGTTAC CGGAGGAACCGCGTTCTTCTTGCCGGTGATGCAGCGCATATTCATTCT CCGCTCGGCGGGCAAGGACTGAATGTCGGACTAGGTGATGCGATGA ATTTGGGGTGGAAGCTTGCGGCTATAGTTCAGCGGGACCAAGGCCAG GGAGAGGATTCTGGGAATACGCATGATCTGACTCTTCTCGATACCTA CGAGAGCGAGAGACACCCCATTGCTGCGTCGGTTCTTGATTGGACTC GTGCGCAGGTCACGGCGCTGAGGCCGGATGCCTTTGGTCGGGCTACG CGCGCCCTGACGGAAGATCTGATCAAGACGGATGATGGGGCTAATCT GTTTATTGATCGGATCTGGGGACTTTCGCAGCGATATAAACTTGGCG AAGAGTCGAGGCCTACACATCCGCTTCTTGGGTCTAGTGCGCCTGAT TTCGAGTTTGGGAATGGGTCTAGGCTTGGCTCGAAGCTTGAGGAGGG ACGGGGGTTACTTCTTGATTTTGGGAACAGTAGTGAACTGGAATCAG TTGTTGATAGAAAATATTCTGGGAAGGTAGATTATCTTGCTCCAGAG

167

GCAAAGGAGAACTGTGGGCTGAGTGCCTTGTTGATTCGACCTGATGG AATCGTTGCATGGGTGGTGGAAGAAGATGCGCAGCACGATATTGATG CTTTGAAAGCTGCATTAGGGGATTGGTTCACTTTGgtatcgatggattacaaggat gacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa NC_03677 gttttaattacaagcggccgcATGACAACTAATCCAGAAGTCATCATCGTGGGCG 2.1- GCGGAGCAGTCGGCCTGGCCGCTGCATGCGAATTGGCCATCAAGAAT tADH1-in- GTCTCTGTTACGATCTTGGAGCGGGAATCTGGACCTGCAGCGCCATG pSP-G1 GAAGAACGAGAAGCTGGGCTTTCGCGAGCTGTTTGTGCCTGCCATCG (Entry 3 AGCACTTGTACCGCCGTGGCCTGCTGAACGACATGTTCCGCAATGAA Table 4-1) GAACGACCACAGACTATGCCGGAGAAGAAGGGTGGCTTTGAGTTTGC TGGTCATTTTGCTGGTATGATGATCAATGGAAACGATGTCAACTATTC ACACTGGGACGATACACACCTTCCCGGGCCTGCTTGTGTACCTGGAT CTTCGACACTGGGTCACATTGAGAATGTTCTGCGTGCACGTGCTGAG CAACTCAAAGTGCCTATTCTTACTGGCAAGAATGTGACTGGCATCGA AGACACAGACAGCGGCGTCAAGGTGTTCTGCGGCGAGGAAACATTC GAGGCTCAATATCTTGTCGGATGCGATGGTGGTCGGAGCACCATTCG CAAGAAAGCTGGGATTCCATTCATCGGCACAGAGCCCGAGTTCACTG GCTATGCTGTCCATTGCAAGCTCGACAACCCCTTTTTGCTCAAGCCCG GCTTCAATCGATGCCCAAATGGCGGACTCTACATCATGGCCGGCCCA CAGCACATCTATGCTGTAGATCACGACATCAGCTTTGACCGAGACCA AGAAGTAACAGCGGAACACTTTCAAAAGGTTCTGAGAAAGCTTTCCG GTACCAACGTGGTTGTTGAGCAGGTCGTTCTCGCATCCGCTTTCACCG ATCGCTGCAGACAAGCTGAGCAGTACCGCAAAGGCCGAGTCTTCCTT GCTGGCGATGCTGCCCACATCTACCCTCCATTCAGCGCCCAAGGACT CAACTGCGGTTACATCGATGCAGTGAATCTCGGTTGGAAGCTCGCTG CTTTGGTCAAGGGCTATGCTACTCCGGGCTTGATCGATACATACCAG CAAGAGCAACACCCAATGGCTGCGCGAGTACTTGATTGGGTGCGAGC ACAAATTGTTACCCTTCGACCTGACGCTCATGGCCGGGCTGTGGGCA ATGTTATGCGCGAGTTCCTCTACACCGAAGCTGGTGTGACATACTGC ATCGGTCGACAATGGGGTCTTGATGTGCGGTATGAGATCGGAGAGGG ACATAAGCTTGTCGGTCGCAGCGCTCTGGATTACGAGCTGGACGATG GCTCTCGACTGGGAACTAAGCTCGGCGCCGCGAACTTCACACTCGTG GCATTTGGTGACTGCAGTTCAGAGCTCACCAGCTCCGTCAACGAGCT TAACCCAGTGCTCGGATTCTGCAGAGCGAAGGGCGAGGAGAAGTCA GGCCTCCAGGGCGTCCTTGTACGTCCTGACGGGATCGTGTCGTGGGC ATCTGCAGATGCCCTCAACATCGAGACGATCAAGGCCAGCCTCGAAC GCTGGGTCGCGCTGCCAGCTGCTGTTGAGAAATTTGAATCTGTGTCTT ACGAGGCCAACCAGTTGTCCAACATTCAGACGCCATTGACGACTACT GTTCGATCGAATATCGGACGTGTATTTGGCAAGGATGGAGAGAAGTA CACTGGCCTTCCGACAGTGGTGACGGAAGTCCCGGCAACGGCTGATG CAgtatcgatggattacaaggatgacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggtc aagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa FJOG0100 gttttaattacaagcggccgcATGGGCGAGACCGATGTTCTGATAGTTGGTGGAG 0008.1- GGCCCACTGGATTGATGCTCGCCCTCGAGCTTACCAGCCAAGACATC

168 tADH1-in- CCATTCCGCATCATCGACTCAAGTCCTATTCGAAGCGACAAAAGCCG pSP-G1 GGCCTTGGTTCTCCATTCACGGACTCTGGAACTTCTCAATCGCCATGG (Entry 4 AATTGCTCAGGAGTTCGTTGACCGTGGAAATTTCAATTTGGCCGTGC Table 4-1, GCATCTTCGCCAACCAGAAGTTCGTGTTTGAGAACGATTTCAACATC AL-2-79-J- ATCGCTTTCAATGACACTATGTACCAGAACCCCTTGATTATCAGTCAG C4) GCAGAGATTGAATCGATCCTGGACGAAGTCTTGATGAAACATCAAGT GAAGGTGGAACGGCCCGTGACAGCGGAGAAGATATCACAAGATGAG ACTGGAGTTACTGCCGTACTACGCCACGAGGATGGAAGCGAAGAGTC ACTCCGTTGCAAATATGCTGTAGGCTGTGACGGCCCTCGCAGCATTG TACGAAATTCGGCAGGTCTACAATTCGAAGGAGCGGCGTACCCGCAG GATTTCATCCTCGCAGATGTGCACATGAAATGGGAGACTAGAGAATG CCTTCATATCTATCTTGGCGCCTCTGGGTTCATGATTGTCTTTCCGATG AAGGACAATGTCTGGCGTCTGATCTGTTCTCGCAGGGAAGCACTGGG CGCTGACGCTGAGCCAACTCTTGAGAATTTCCAGGAGATGTTGGACA AGCTGCTTCCTGAACCTGTTCAAATATTTGATCCAGTTTGGATCAGTC GATTTAAATTACACCATCGAATTGCAGACAATTACCGCGCTGGTCGA ATGCTCGTCGCGGGCGATGCAGCCCATGTCCATTCTCCTGCTGGTGG GCAAGGGATGAACACCGGTATGCACGATGCAGTCAATCTTGGCTGGA AGCTTGCAAGTGTCATTCGGGGCGAGAACGATGACTCTCTGTTGGAC TCGTACAATATCGAACGGCGTAGAGTTGGCCAGACTCTCCTGCAAGG CACAGATAGACTTTTCGAGTTTATGGCAACAACAAATCCTTTGTATCT CTTCATAAGGAACTACGTCATGCCTTTCATTATGCCCTGGGCCATGGC GATACCTGGCCGTCGAGCTCTTGCATATAGATTTGTATCCGAGTTGGG GATTCGATATCGCAAAAGTCCCATCGTTGGACAGGCAACGACTTGGA AAGGAACACTGAAAGGTGGAGATCGAGTACCCGACGGGAGACTCCT GAAGGGGAATATTGAGATCACTGTTCACTCGTTGCTTGGAGCACGCA AGCACAGTCTACTTCTGTTCTCGGGCGTGGACGGGGTCACCAGCACA GACGATCTGGAGAAAGCTCATGTGGAGTTCATCGAGGCCAGTGGTCG GTCGATCCCAGTATACACAATCACGAAATCATCCTCGGAGAACATAG AGATCATGGATCCGGAGGGGCTAGTGCATAGGCTGTTTGGCTTTACA GCATCCGGCTTTGTGTTAGTTCGACCCGATGGTCACATTGCGTTCATC GGGCCTCTCACGTCAATGGACGAGCTCAAGACGTGGATGGAGAGAgta tcgatggattacaaggatgacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctc caatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa MCBR010 gttttaattacaagcggccgcATGGATTGTGAAGTATTAATTATCGGTGCTGGGCC 07648.1- CACAGGACTTATGCTTGCCCTCGAACTTTCCCGCGAACAAATCCCCTT tADH1-in- CCGAATTCTCGATAGTCATTATTCTACTAAGACAGCCCATCAATCTCG pSP-G1 GTCTATAGTGGTTCACTCACGTTCCCTGGAGTTACTTGCCCGGCATGA (Entry 5 CTTAGCCGAAAGCTTCATCGCTAAAGGATTACTCATGGAAGAGATTC Table 4-1, GCTTCTTCTTGAAGCAAACCCTTAATGGGAAGAGTAGTTACCCTGGA AL-2-72- AGTCTCATGAATGATACAATTTATAAAAAACCACTTATCATTAGACA D-C5) ACCACTATCAGAGCAGCTTCTGGAAAAACGTCTTTGCGAGTATGGAG GCATTATTGAAAGGGGGGCAAGCGCTAAAAAAATGGTGATAGATGA AGATGGCGAGCGGGTGTCTGTTTTGGTACAGCATAGAATTCAGGAAG AAGAAGAGAAAGTAAGAGAGGAATTAATCAATTGTAAGTATGTTGT

169

AGGTTGTGATGGAGCTAACAGTATGGTACGAAAATTTGCGGGTATCG ACTTTCCGGGTACTTTATACCCTCAAGAGCTCATTCTCATTGATGCAC AGGTAGACTGGAACAATAAAATATGTCCACATGTTTTTATTGATAAG GAATTCGTGATCTTTATACCGCTAGACGAAACAGGTATCAACCGAAT CATCTGCCGGCGTCCTCCAAATGACACCAGCACTATCTCATCCAGAT CAAATCTGTTATCAGGATCATCGCCTTACCAAGAGCCTAGCCTATCC GAGTTTCATGATACAGTTATTTCTTCCGTTCCTGGTACGACTCGTATT CACAGCCCGATTTGGTCTTCCTCATTCCGTGTCTCACGTCGACTTGCT AGTACATATAGTGTGGGGCGGATCTTCCTTGCTGGTGACGCCGCTCA TGTCCATGCACCTATTGGCGGTCAAGGTATGAATACCGGTATTCAAG ATGCCGTAAACCTGGGTTGGAAACTTGCGCGTGTTATAAAATCAACT GCTCCGTCCTCTTTACTCAACTCATATAATGTAGAGCGATATAAAGTT GGTAGTGACACTGTTGAAAATACAGATCGCATGTTCAATATTTTAAT AACTACAAACCCTGTAAAAATCTGGTTACGAAACTTTCTTTTTCGCTG GGTTTTGCCATATTTCCTCAAACCAGATTTTGCAGACAGAAAAATTCG TTACATATCACAATTGTCAATTCGATATCGAAATAGTCCCGTTGTAGG CACGGCTTCTGTGTGGAAAGGAATTCTAAGAGGCGGAGATCGGGCAC CAGATGGATGGATGATGAATCCTGAAGGAGAAAATATCACTTTGCAC AGGCTTTTTAGGACATCAAATGATCATCTTTTCTTATTCAGCGGCCTA GACACTCTTGAATCGCGGATGGTCAATGATACTGTCCAGCAGCTAAA ACTCTGCAGCTCAGTTCTTGTGGTACACAAAATATATGACGGGAATT TTAAAGATAAAACAATTATCGATGGTTACATAGACCCAGGGGGTAAA GTTCACACCTTGTATCAGTTTATTGAGCCTGGTTACGTTCTTGTTCGA CCCGACGGATATATCTCATTTATAGGACCTATGACTTCACTAGATGA ACTGAAGAATTGGATGAATTCATATATGCAGGGAgtatcgatggattacaaggat gacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa KN714671. gttttaattacaagcggccgcATGGAGGCTAAGAAATCTTTCAAAGTTATCATTGC 1-tADH1- TGGTGGCAGTATCGTCGGCCTAACGCTGGCCAACGCGCTCGAGAAGG in-pSP-G1 CGGGTATTGATTTTCTTGTCCTCGAGAGGGGCGACATTGCTCCACAGC (Entry 6 TCGGCGCATCAGTCTCAATCTACTGCAATGCGTCCAGAGTCCTCGAC Table 4-1, CAGCTGGGAGTTTGGAAAGGCATTCACCAGAGCACAATCCCGCTGAC AL-2-72- TGATCGGCTATACTTCGATGAACATGGCCGCTTGTTTGAGGACAACA B-C6) AGATGATGAGGCTCATAGACGACAAGACTAAGCGGCCTATCACGTTC TTGAGAAGGGAGGCATATATCAGGGCTCTTTACGAAAACCTGAAGGA CAAGTCGAAGGTGAAGGGTCACTCTGGGCTGGTGTCCTTTGCCGAAG ATGATGAAGGCGTTTCAGTATTCACCAGTACCGGCGAGGAAATCAGG GGCAGCATTCTTGTGGGAGCAGACGGCATCCATAGTACCGTCCGGAA GCTCATGGCTGAGTCTGTCGCCAAGTCGGACCCTCAGAGGGCTAAGA ATCTCATCGAGGGCTTCACGGCAAACTACCGGACGATCTTCGGAACG TCCAAAAACCAGCGAAAAGACGACCCGAGCGTGCAGATGGTGGCAG ATAGTCTTTCCCATACCTCCTATTACCGTGGCGTCACGGGGGTCTGCG CCGTCGGCAACAGTGGGGTGATCTACTGGTTCCTCTTGGTGAAGGAG GACACGCCTTCGACAATGCCGAACTGCCCGCGCTACTCGGAGGCGGA TGCCGAAGAGACGCTTCGTAAATTCGGCCACCTTCATATGGGACCGG GATATACTTTCAATGACCTCTGGGAACTGAGGGAGAAAGGAGTCATG

170

GTACCGTTGCAGGAAGGCATCGTCGAAGGAAGCTGGAATAGTAGCG GGAGGGTTGTACTCATGGGCGACGCCGTGCACAAGTTCACTATAAGC GCGGGGCTGGGCGCCAATTTAGGCGTAGAAGGCGCTTGCCACCTGGT GAATGAGCTCCTCCCTGTCCTCAAAGAGACGGAAAACCCCGGAAAGC AAGAGATAAAGGATGTGCTCGACAGATACGAGGAGAAGCATCGTCC TCGAACTAAGATCTGTGCCGTATTGTCTAACTATTTGACCAAGTACGA GGCGATGGAGACCTGGTGGCTCAGACTTCTGAGGTTCATTATTCCTC GGATCCCGGACAGTTACAAGGCGAAGTCATTTGTGGACTTCATGTAT GGTGCTCCGATTCTCGATTTCCTGCCTCACCCGGGCAAGAGCTCTGCG gtatcgatggattacaaggatgacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggtcaagt ctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa KL584983. gttttaattacaagcggccgcATGTCGGTCTTACAATTCAATCTGAACGGCTTCGT 1-tADH1- GCCTGGCGACCCTCATGATCTCTACCGTGTCCACCAAGAAACCGCCG in-pSP-G1 AGATCGCAAACAATGTCCCGCAAGAAGTCGACGTCCTCATCATTGGC (Entry 7 TCTGGTCCAGCTGGTCTGGCTCTAGCAGCACAGCTATCTCTCTTTCCA Table 4-1) AGCATCACCACAAGAATCATTGAACAAAAGGCTGAACGACTAATCCT AGGCCAAGCCGATGGCCTCCAAGTCCGAAGCATCGAAATGTTCGAAG CATATGGCTTCAGCGAAAGAGTCCTCAAGGAAGCGTACTACATCAAC GAAACCACCTTCTGGAAGCCTGACGAGCAGCAACCGGACCGCATCGT GAGAAGTGGCAGGATTAAGGATACTGTAGACGATCTCTCCGAGTTCC CGCACGTCATCCTCAATCAGGCGAGAGTTCATGACTTCTTCCTTGATG TCATGCTCATGTCGCCGACAAGATTGCAACCGAGCTATTCACGCAAA CTCAAGCAGCTCAGCCTTCAACCAGCACAGGGCAACTCCTCGCACGC TACGTCCTCCGTGCAAGTTCAACTCGAGAGAACAGACTCCAGCCACG AGGGCGAAGTCGAGGATGTACGTGCTCGCTACGTCGTAGGCTGTGAT GGAGCCCGCAGTGCCGTCCGTCAAGCCCTCGGTCTCAAGCTGGAAGG CGACTCCGCAAACCAAGCTTGGGGTGTCATGGACGTACTTGCTGTAA CCGACTTCCCAGACTGGAGGACCAAGGTTGCAATCCACTCCGCGACA CAAGGCAGCATCTTGATCATCCCCAGAGAAGGAGGTTACCTTGTGCG CCTCTACATTGAACTCGACAAGCTCGACGCCAACGAACGTATCGCAA ACAAGAAGATTACCGCCGACAAGCTCATCGAGACCGCACAGCGCAT CTTCCATCCTTATGCTTTGGATGTCAAAGAAGTAGTCTGGCACTCTGT CTACGAGATTGGCCAGCGCCTGTGTACCAGATTCGACAACCTCACTC CGGAGACCGCTACAACAGAATCCCCAAACATCTTTATCGCAGGCGAC GCATGCCACACCCACAGCCCTAAAGCAGGTCAAGGCATGAATGTCAG CATGCAAGATGGCTACAATCTAGGCTGGAAGCTCGCCTCCGTGCTCA ATGGAATCTCTAACCCGGCCATCCTCCACACCTACTCCGCCGAACGT CAAAAAGTCGCTCAAGACCTCATCAACTTTGACCGAGAGATTGCTTC AATGTTTAGCGCCAGACCCAAGAGCCATGCCAAAGACACAGAGGGA GTCGACCCTGCTGAATTCCAAAAGTACTTTGTCAAGCAGGGCCTCTTC ATGGCTGGACTGGGCACAGCTTACTCGACTTCTGCTATCACCGCTGG ATCAGAGCATCAACACCTGGCCAAAGGTTTCCCCATCGGCATGAGAT TGCACTCTTCTCCAGTCATAAGATTGGCAGATGCAAAGCCGATCCAA CTAGGTCATGTGGTAAAAGCAGATGGAAGATGGAGAGTCTTCGTCTT CGCATCAGCCGAGGATCCCGCTTCGACCTCGTCAAGCTTCCACTTGG

171

CCTGCGAATCCCTGACCGAGCTTGTCAACAAGGTCAACCCTACTGGT GCTGATATCGACTCCGTCATCGATGTCAGAGGTATTCTGCAGCAGGG CCATGCTTCACTGAACATCAACCACATGCCTGCTATCGTGTGGCCGC AAAAGGGAAAGTATGGACTCAGAGATTACGAAAAGGTCTTCTGTGCT GAAGATGTGGATGGACATAGGGATATCTTTAATGCTAGGGATATCGA TCGCAGAGGCTGTATCGTGGTCGTACGACCTGACCAGTACGTCGCCA ACGTTCTGCCGTTGTGCGATCATGATGGTTTGTCAAGGTTCTTCGGAG GTTTCATGTTTGCAAGGGATgtatcgatggattacaaggatgacgacgataagatctgagctctt aattaacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa KZ111246. gttttaattacaagcggccgcATGACCAAGACTTACGATGTTATCGTTGCCGGCGC 1-tADH1- AGGCCCCATCGGCTTGTTTCTCGCCTGTGAGCTGAAGTTGGCGAACG in-pSP-G1 TTGACGTGCTAGTACTAGAGCGTGACACCAACCCCGAATCCCCCTGG (Entry 8 AAAAGCGAACCACTCGGCATACGAGGCTTGAATACGGTATCTGTCGA Table 4-1, GTCCTTCTATCGACGTGGGATGCTAGACAAGGTCACCTCCATGGAGC AL-2-86- GACCGAACTTCTTCAAGAAAGGCCCGGGGTTCCAATTCGGAGGCCAC D-C5) TTCGCCGGCATGCTACTGAATGCCAACAAGCTCGAAATGTCCCGTTA TAAGTACCGCCTCCAAGGCCCAGCATTGGTTCCCAACGGATCAAGTA TGGAACGTGTTGAGAAGGCCCTAACAGAGCGCGCTGAGAGCATTGG AGTAGATATTCTCAGGGGGAAGGGAGTCACGGAAGTCTCGCAGGAT GACAACGGCGTTACGGTGGAAGCAGGAGGCGAGACTTTCCATGCGA AATGGCTTGTCGGATGTGATGGCGGGCGGAGCACAGTTCGCAAGGCC GCTGGCTTCGACTTCGTCGGAACAAATCCTGAATGTACCGGTTACGC TTTTTTGGCCGATTTAGACCATCCCGAAAAGCTGAAGCCGGGGTTCC AACCCTCTGTAGGCGGCATGTACATCATAGGACATTGGGGTAACATG TACTTGCTAGACTTCGATGGTGGCGCTTTCGATCGTACGCAGGAGAT CACACCCGAGCACCTTAACGAAGTCATGGCCCGTGTGACAGGCACCG ACGTCAAGGTCACCAAGATTCATCAGGCATCCTCGTTTACCGACCGA TCTAAACAAGCGTCGACTTATCGAAAAGGCCGCGTTCTCTTAGCCGG CGACGCGGCGCACATCCATTCGCCTCTGGGAGCACAAGGATTGAATC TAGGGTTGGGAGATGCCGTGAACCTTGGCTGGAAATTGGCAGCGACA GTTCAGGCTTCTAAATCTGGAGAGGAACCAAAGGACCTCAGCCTACT CGATTCCTACGAGAAAGAGCGATATCCGATCGGGTCTTGGGTTCTTG AATGGACACGCGCTCAGATCTCGACGCTAAAGCCCGACATGTTCGGT ATCGCTCTTCGTAGGCTCATGAGCGATTTATTGGAGACGACTGATGG AACGAATTTATTCATCGATCGCATCTGGGGTCTGTCGCTGCGGTACG ATCTTGGCGACGAACACCCCGCCGTCGGCTGTAGCGCTCCCGATTTC GAATTCCACGACGGAACGAGGCTAGGTCCCAAGCTAGCGACGGGGC GAGGTCTATTGCTCGATTTCGGATCTAATGCAGAACTCAAGGATCTT GTCGGGAAATACGAGAGCAAGGTTGATTATCTTAGCACAGAGGCAA AGGACCAGCTGGGCCTGAAATCCTTGTTGATCCGCCCCGATGGCGTT GTGGCTTGGGTGGCTGAAGAGAAGCCTGATATGGACTCGGCGAAGG CCGCTCTGGAGAGATGGTTTGGGCTGGGTTCGAAAgtatcgatggattacaagga tgacgacgataagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggc t

172 pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa FJOG0100 gttttaattacaagcggccgcATGGAAAACCAGCTCTACGACGTCATCATTGTTGG 0031.1- CCGCGGTCCTGTTGGGCTATTTTTAGCATGCGAACTTCGCCTCACAGG tADH1-in- GCTCTCTGTCTTGGTTGTTGAGCGCCGCTCGAGCTCAGAGGGAGTGG pSP-G1 CGGAGACTCGCGCTTTTGTTGTCCATTGCCGCACACTCGAGATCCTCG (Entry 9 CATTCCGTGGTCTTCTTGGCAAGTTCCTCCAGGAGGGCACGAAGTCTC Table 4-1) CTTGGTGGCACTATGGCGTCTTGGACACCCGTTTGGATTTTAGCAGAT TTGGCCACGAAACGAAGCAGAATTTTGTCCTGTTAAGCCCACAGTTC AGAACTGAGGAGATTTTTCTGGAGCGTGCGAAGGAATTAGGCGTCGA CATTGTTCTGGGAATTCGCGTCGAGTCCGTGGAGCCTTCGACTACGTC TGTTACGATATATGGAACATCAAGCAGTCCAGATGGCGGCGCTAGAA GTTCGTTCAAGGCGAGAGGAAAGTATCTTGTCGGTGCGGATGGAGTG CGCAGTACCATCCGGAAGGCTACGGGCTTTGAATTCCCAGGTACGCC GGCAACACACACGGGTTTGACCGGCGAGGCAACATTGGGAGTGCCC ATGCCCTATCCCTACATTGCCAAAAACGAAGCAGGGCTTGTGATCGC CGTACAGCTGAATATTCCCAGCGGCCGGGCTCGCCTCAATGTTTTCAC CCCTGCTCGAGCTAATGTACCCGATTCGACCCCGGTAACGCTGGAAG ATTTCAACGAAGCATTACAAGAAGTCACTGGTGTTGACTACAAGCTC TCCAACCCTTGCATGCTTGGACGCTTCAACAACGAAGCCCGCTGCGT CGATACTTACCGGAAGGGACGCATCTTTCTCGCCGGTGATGCAGGCC ACCAGCACCTTCCAGCTGGCGGCCAAGGGCTCAACGTCGGTATTCAG GAAGCAACTAACCTTGGATGGAAACTTGGGGCCGTGATTCGCGGCTA TGCTCCGGACTCGCTATTGGATACTTATGAGACCGAAAGACTTCCAA TCGCACAAGGCGTCGTGCAGAGCACCACTGCTCAGTCTGTTCTCTTTT TTGCGCACAGCGGGCCGGAGTTGGCAATACGAAGTGTGGTGAACAC GTTGCTCAAAATACCAACAGCAAACCACGACATTGCTGTAGGAGTTA GTGGTTTCGGCGTCTCATACCCAAAGCTTCTTGACATGATACTTCCAG CTGGATGGGAGGCACTTCCAGAGGCGATCGCCGGGAAGCGAGCCCT AGATGTGAAGTTACGCGTCGGGAATGGCGAGGAAAAGCAGCTAAGC GACTATATGAGGGGAGGGAAGTGGGTTCAACTTCGGTTCCTTGACAA ACTCGCAGGAAGGCCGCCACTGCCGGCGTTCGAGGGGGCGACAGAG GTTGTGGATGTCGCTGAAGTTCTGGAGGGGAAAGGGAGCATGTACCG GGGCGCCTTGAGCGAACTGCTAATTCGCCCGGACGGTTATCTAGGGT TTGGAAAGCGCgtatcgatggattacaaggatgacgacgataagatctgagctcttaattaacaattcttcg ccagaggtttggtcaagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa AMCJ0100 gttttaattacaagcggccgcATGACCTACACTACTCATGACGTGGTGATTGTGGG 0140.1- AGCTGGTCCAGTTGGACTCTTCCTCGCTTGCGAACTACGCCTTGCCGG tADH1-in- TCTCTCCGTTCTGGTGGTCGAGAAACGAACAAATTCGGACGGCATGG pSP-G1 CGGAAACGCGTGCCTTTGTGATGCATGGTCGATCTTTGGAAATCTTCG (Entry 10 CCTCTCGCGGTCTGCTCGACTCTTTCGTTGAAGCGGGTCAAAAGACC Table 4-1) GACTGGTGGCATTACGGTGTTCTCGACACTCGTCTTGATTACAGTGTT TTCGGCCGTGAAACGGACCAGAACTACGTGTTACTCGTGCCTCAGTA CAAAACCGAGATGATCTTATTTCAACGCGCCGTGGATCTGGGTGCAG TGATTATCAAGGGTGTCCAGGTCGATTCAATACACGAATCCGGGCCT TATGTCGTTGCGAGGGGTTTCTACTGCAATAACAAACCTTTCATTGCC

173

AGCGGGAAATACCTCGTCGGTGCGGACGGTGTTCGCAGCACGATCCG CAAAATCGCCAACATCGAGTTCACTGGCAATCCACCTGTCAATACAG TCATGAGTGGCGAGGCTACACTAGGCACGGCCATGCCGAATCCCTAC ATTGTTCACAACGAGCACGGCCTAGTTATCGCCGCTGACCTGAGGGT CCCCAGCGGAAGAACCCGCCTAAATGTGTTCGCGAGTGATCGCGGCA CGGTTCCTGAGTCAGTTGAAGTGACTCTTGAGGAGATGAATCAGAGC TTGCAGAAAATAACCGGCGTTGACTACAAGCTTTCTAACCCTTGCAT GCTAAAGCGTTTCAGCAACGAACAACGGCTAGCGACAACGTATCGCC AAAATCGGATTTTCATCGTCGGAGATGCATGCCATAAACACCTCCCA GCCGGCGGCCAGGGCTTAAATGTCGGTCTCCAAGAAGCTCTGAATTT AGGATGGAAACTTGCCGCAGTCATCTCCAAGTCCGCCCCTGCTTCGTT ACTCGATACGTACGAAGAACGATGGCCAGTTGCCAAAGCTGTTGTAC AAAACACAACTTCACAGTCACTCTTATTTTTTGCCTCGTCTGGCCCAG AATGGGCGGTTAGGGAAGCGATCGACAAGCTTCTGCGTGTACCAGAG GCCAACAAGCGTTTGGCTAGGGAGATCAGCGGATTTTCTGTTGCCTA CCCCAAATCGCTGGATATGATACTTCCAGACGGATGGCGGGCCCTAC CAGAGAACATCCAGGGAAAGCGTGCATTGAATGTAAAGATGAGATT ACCAGATGGGATGGTAACCGAACTTCGTGATTACACCCAAGACGGAC GATGGATCCAGCTGCACCTTCCTGGAAAGCATATCATTGAACTCCGG CCTCCTCCGGCATTTGGTAACTGGACGACGGTAGTGGAAGTTGTCGA CATGCCAGATGAAGAAGAGAAGACGAGCTTGTATATGTGCGGAGTG AGAGAGATGCTGATCCGCCCGGATGGCTATTTGGCCTTTGGTCGAAT GGACGACgtatcgatggattacaaggatgacgacgataagatctgagctcttaattaacaattcttcgccaga ggtttggtcaagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa KB725976. gttttaattacaagcggccgcATGACTAATCAATATGATGTTGTAATTATTGGAGC 1-tADH1- TGGGCCAGTTGGATTATTATTGGCTTGTGAATTAGCTTTGGGAAAAA in-pSP-G1 CAAGCGTCATAGTTCTGGAAAGAGAAGCGTCGCCCGTATCTCCGTGG (Entry 11 AAAGAAGAACCGACTGGTATGAGAGGTTTGCACCTGCCTTCTAGTGA Table 4-1, AATTTTGTGGCGTAGGGGTTTGTTAGAAAAAATCTGGACATTGGAGG AL-2-86-I- GTAGACCACATGGCCCGACTAAGACTCCTGGATTTCAATTTGCAGGC C5) CATTTCGCCGGGATACCTCTTAACCTATCTCAGCTAGACTTAGATAGG TGGAAATATCGATTGAGGGGTCCATCATTAATGGGGGGTCCAATAAC AATTGCATTAATTGAGAGAGTGTTAGCAGAACGCGCTGAATCATTGG GAGTCGAAATTCATCGGGGTTGCGGGTTCGAGAGGATAGTCCAACAA TCCGCTGACGGCGTAACAATAGAAGCGGGTGAAGAAACCCAGACAT TTCATGCAAAATGGCTTATTGGTTGCGATGGCGGTCGTTCCCAAGTCC GTAAGGCTGCCGAAATCGACTTTCCTGGCACTGACGCGACCTTAACT GGCTACGTGATCCATTGTGATCTTGACCATCCTGAACGATTGTCACCT GGTTTCCAACCTACCAAACAGGGAATGTATATTTTTAGAAAGCCCGG CGCTCTCTACCTGATGGATTTCGACGGTGGTGCCGGCCAAACAAGAG AACCAAGCCAGCAGAGACTACAAGAAGTGATGGAGAGGGTTGTAGG GAGAACTGATATCAAGCTAGAGAAGGTTCACCTAGCAAGTGCGTTTA CGGACAGGGGCAAACAAGCCACAACGTATAGGAAGGGAAGAGTCCT CTTGGCTGGTGACGCAGCGCACATCCACCCACCACTTGGTGCACAAG GTATGAACGCCGGTTTGGGTGATGCCATGAATCTAGGATGGAAGTTA

174

GCTGCAACCGTTCGTGGAGAAGAGAAAGGAGTCCGGGCTTTCACCGT GTTGGATACCTATACTTCAGAGAGACATCCCGTTGGTCAATGGGTCTT AGAATGGAATTACGCACAAGTTGCTGCTCTAAAACCCGATGTAACAG GTTACGCCGTACAAAAATTAATGAGGGATTTGATTGCAACTGATGAC GGTACGAATTACTTCATAGATAATGTGTGGGGATTGTCTCAGCGCTA TGGTGACGGTGAAGGTGTTCATCCAGTAGTTGGAAGATCGGCCCCTG ATTTCACATTTAAAGATGGTAGTAGACTTGGGCCAAAATTAGAGGGT GGCAGAGGACTTTTTATAGATTTTGAAGATGTGGATCTGGAGAAAGC AGTTAAGCGATTCGAAAGAGTTGATTATCTCGGACAAAACGTGGAAG ATAGACGTGGAATCAGAGCTTTACTTATTAGACCAGATGGATTTGTT GCTTGGGCTGTAGAAGAGGGAGATGAACCAGACGCAGAGGGTTTAG AAACTGAATTAAAAAAGTGGTTTTCTTACgtatcgatggattacaaggatgacgacgat aagatctgagctcttaattaacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggct pTEF1- tcaagtttcagtttcatttttcttgttctattacaactttttttacttcttgctcattagaaagaaagcatagcaatctaatctaa AP007159. gttttaattacaagcggccgcATGGGCAAGACCTGGGACGTTATTATTGCTGGCG 1-tADH1- CTGGGCCGGTCGGTCTTTTCCTCGCCTGTGAACTGGCCATAGCTGGCG in-pSP-G1 TGTCGGTACTTGTACTGGAGCGAGAGATGCAGCCAGAGTCGCCTTGG (Entry 12 AAAGAGGGATTGTTTGGGCGTCGCGGTCTCTATACCCCGGCGGTCGA Table 4-1, GGCATTTTACCGCCGTGGCATCTTGAAGAGAATTTTCGGTGATGATG AL-2-86- AGCGTCCGACGCATTTGAAGAAGACTGAAGGCTTTCAATATGGAGGA A-C1) CACTTTGCGGGCTTGGTGCTTAATGCAAACAACATCGAGTTTTCACGC TGGCCATATCGCTTGCCAGGACCGTCCTTTCTTCCTGGTCCGACTTCT CTTGGACGTCTTGAGGCTGTGTTGTCGGAACGTGCAGAGACGCTGGG TGTCCAGATCCTTAGAGGTATGGAAGTCTCCCGGCTCGCTGATGAAG GTGAATCAGTCAAGGTATGGGCGGGCGACCAGTGGTTTACGGCTCAA TGGCTTGTCGGATGTGACGGTGGACGCAGCACAGTTCGGAAAACTGC TGGATTCGAGTTTGTCGGGACCGAAGCAGAATTCACTGGTTATATTG CGGTATGTGATCTCGACCGACCAGACTTATTGAAGTCAGGTTTCAAG CACACAAACTCGGGGATGTACATAGTTAGCGGACCTGGACAGTTGCA TGTTATCGATTTTGATAGGAGCTTTGATCGGTCACAAACTATCACGCG GGAACACTTCCAGGAAGTGCTACGGCGAGTGTCGGGCACAAACATC GTTGTCGAAGCGCTTCACCTAGCCTCCTCCTTCACCGATCGCTCGAAG CAAGCAACAGAATATCGTAGAGGACGCATCCTTCTGGCTGGTGACAG TGCCCACACACACTCTCCACTGGGCGCGCAAGGATTGAGTACAGGAA TTGGCGATGCGATGAATCTCGGCTGGAAGCTTGCGGCTACCGTCAAG GGGTTTGCATCTCCTGGTCTGCTAGACACCTACCACCAAGAGCGGCA TCCAGAGGCAGCTCGAGTGCTTGAGTGGACTCGCGCTCAGGTGGCGG CCTTACGCCCTGATCCATATGGCCAGGCTATTGCGAGTCTCATGCGG GACATGATCAATACCCAAGATGGGGCGACTTATCTCGCCGATCGCAT CTGGGGACTTTCAGTGCGCTATGGCCCGGGCGACGCGCATCCACTTG TCGGTTCTAGCGCTCCGGATTTTGAATTCGATGATGGAATGCGACTTG GAGCTAAGTTAGAGACAGGATCCTTCTTGGTGATTGACTTTGGGAGC AACAACCAGGTGGCAGAGCATGTGCAGTCCTTGCAGTCTCTGCAGTT CATGATTCAATACTGTGCATGTAGCGCAAAGGAGGAATTCGGGCTCA AGGGGTTACTCTTGCGACCTGATGGTGTGGTCGCATGGGTGTCGACG GAGGAGATAAATATAATACGACTGCATGTTGCATTGTCTCGTTGGAT

175

AAGTCTCCCTAGTTTCGAGGCTgtatcgatggattacaaggatgacgacgataagatctgagct cttaattaacaattcttcgccagaggtttggtcaagtctccaatcaaggttgtcggct

176

Table 4-11 Primers used for OxyS saturation mutagenesis in this study To create a saturation mutagenesis of the positions outlined in Table 4-2 the even numbered primers were used for PCR amplification with EH414 and the odd numbered primers were used for PCR amplification with EH745. The resulting amplicons were gel purified and used for Gibson Assembly with pSP-G1 (empty) digested with SpeI and NotI. EH414 GCTCATTAGAAAGAAAGCATAGC EH745 CTGGCGAAGAATTGTTAATTAAGAGC EH723 AGTGCGAGCGTGGACTCCTAGMNNTTTCGAGAAGTCAACAGGC A43X TC EH724 CTAGGAGTCCACGCTCGC A43X EH725 CAATGCATATGGGTGTCTAGTATCAA F96X EH726 CATCATTTGATACTAGACACCCATATGCATTGNNKGTTCCACAA F96X GTACGAACTGAAGAAC EH727 ATGTGGGTCTTGACCTGGAAAATC M176X EH728 AACTATTGGGCATAGATTTTCCAGGTCAAGACCCACATNNKTTT M176X GCTGTCATCGCAGACG EH729 TGCACGAAGGTCATGTCTCATG W211X EH730 TCATGAGACATGACCTTCGTGCANNKTTCGCAGCATTTCCGCTA W211X G EH731 CCATGCACGAAGGTCATGTCTC F212X EH732 GAGACATGACCTTCGTGCATGGNNKGCAGCATTTCCGCTAGAA F212X CC EH733 CAGCATACGGTCTATCGAAAAAGGCGACMNNTGCTCTGTAGAC T225X GTCTGGTTC EH734 GTCGCCTTTTTCGATAGACCG T225X EH735 CGGCGCCCTCCTGTCA V240X EH736 GTATGCTGACAGGAGGGCGCCGNNKACGGAGGAGGATGTCAG V240X AGC EH737 CTAAGTTCAGTCCCTGGCCACCAGCMNNCAAATGAATATGGCA P295X TGCATCACC EH738 GCTGGTGGCCAGGGACTG P295X EH739 ATCTTGAAATCCTAAGTTCAGTCCCTGGCCACCMNNGGGCAAA A296X TGAATATGGCATGCATC EH740 GGTGGCCAGGGACTGAACTTAG A296X EH741 TCCTAAGTTCAGTCCCTGGCCMNNAGCGGGCAAATGAATATGG G297X CA EH742 GGCCAGGGACTGAACTTAGGA G297X EH743 TTAACGGCATCTTGAAATCCTAAGTTCAGTCCCTGMNNACCAG G298X CGGGCAAATGAATATGG EH744 CAGGGACTGAACTTAGGATTTCAAG G298X EH812 GTGCGAGCGTGGACTCCTAGAGCMNNCGAGAAGTCAACAGGC K42X TCG EH813 GCTCTAGGAGTCCACGCTCGCACTG K42X

177

EH814 CAACAGTGCGAGCGTGGACMNNTAGAGCTTTCGAGAAGTCAA G45X CAG EH815 GTCCACGCTCGCACTGTTG G45X EH816 TGGGTGTCTAGTATCAAATGATGAG L95X EH817 TCTCATCATTTGATACTAGACACCCATATGCANNKTTTGTTCCA L95X CAAGTACGAACTGAAG EH818 GGCGACTGTTGCTCTGTAGA F228X EH819 TCTACAGAGCAACAGTCGCCNNKTTCGATAGACCGTATGCTGA F228X CA EH820 CATTAATTCCCTTAAACCCTCGTAGCGMNNATCTGGATCAATT P357X AGAACAGCTTGAG EH821 CGCTACGAGGGTTTAAGGGA P357X EH822 AGGATCTGGATCAATTAGAACAGCT R358X EH823 CTCAAGCTGTTCTAATTGATCCAGATCCTNNKTACGAGGGTTTA R358X AGGGAATTAATGATTG EH824 TAGCCCCGCTAAATATCTATTAGTCTCAGGMNNGTGCAACAAT V372X TCAATCATTAATTCCCT EH825 CCTGAGACTAATAGATATTTAGCGG V372X

178

4.8 References

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2. Chooi YH, Cacho R, Tang Y. Identification of the viridicatumtoxin and griseofulvin gene clusters from Penicillium aethiopicum. Chem Biol. 2010;17(5):483-494. doi: 10.1016/j.chembiol.2010.03.015.

3. Nicolaou KC, Hale CR, Nilewski C, Ioannidou HA, ElMarrouni A, Nilewski LG, Beabout K, Wang TT, Shamoo Y. Total synthesis of viridicatumtoxin B and analogues thereof: strategy evolution, structural revision, and biological evaluation. J Am Chem Soc. 2014;136(34):12137- 12160. doi: 10.1021/ja506472u.

4. Lindqvist Y, Koskiniemi H, Jansson A, Sandalova T, Schnell R, Liu Z, Mäntsälä P, Niemi J, Schneider G. Structural Basis for Substrate Recognition and Specificity in Aklavinone-11- Hydroxylase from Rhodomycin Biosynthesis. J Mol Biol. 2009;393(4):966-977. doi: 10.1016/j.jmb.2009.09.003.

5. Wang P, Bashiri G, Gao X, Sawaya MR, Tang Y. Uncovering the Enzymes that Catalyze the Final Steps in Oxytetracycline Biosynthesis. J Am Chem Soc. 2013;135(19):7138-7141. doi: 10.1021/ja403516u.

6. Patrikainen P, Kallio P, Fan K, Klika Karel D, Shaaban Khaled A, Mäntsälä P, Rohr J, Yang K, Niemi J, Metsä-Ketelä M. Tailoring Enzymes Involved in the Biosynthesis of Angucyclines Contain Latent Context-Dependent Catalytic Activities. Chem Biol. 2012;19(5):647-655. doi: 10.1016/j.chembiol.2012.04.010.

7. Kharel MK, Pahari P, Shepherd MD, Tibrewal N, Nybo SE, Shaaban KA, Rohr J. Angucyclines: Biosynthesis, mode-of-action, new natural products, and synthesis. Nat Prod Rep. 2012;29(2):264-325. doi: 10.1039/c1np00068c.

8. Kim J, Kim KH. Effects of minimal media vs. complex media on the metabolite profiles of Escherichia coli and Saccharomyces cerevisiae. Process Biochem. 2017;57:64-71. doi: 10.1016/j.procbio.2017.04.003.

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9. Koskiniemi H, Metsä-Ketelä M, Dobritzsch D, Kallio P, Korhonen H, Mäntsälä P, Schneider G, Niemi J. Crystal Structures of Two Aromatic Hydroxylases Involved in the Early Tailoring Steps of Angucycline Biosynthesis. J Mol Biol. 2007;372(3):633-648. doi: 10.1016/j.jmb.2007.06.087.

10. Kallio P, Liu Z, Mäntsälä P, Niemi J, Metsä-Ketelä M. Sequential Action of Two Flavoenzymes, PgaE and PgaM, in Angucycline Biosynthesis: Chemoenzymatic Synthesis of Gaudimycin C. Chem Biol. 2008;15(2):157-166. doi: 10.1016/j.chembiol.2007.12.011.

11. Kallio P, Patrikainen P, Belogurov GA, Mäntsälä P, Yang K, Niemi J, Metsä-Ketelä M. Tracing the Evolution of Angucyclinone Monooxygenases: Structural Determinants for C-12b Hydroxylation and Substrate Inhibition in PgaE. Biochemistry. 2013;52(26):4507-4516. doi: 10.1021/bi400381s.

12. Rogers JK, Taylor ND, Church GM. Biosensor-based engineering of biosynthetic pathways. Curr Opin Biotechnol. 2016;42:84-91. doi: 10.1016/j.copbio.2016.03.005.

13. Ma SM, Li JWH, Choi JW, Zhou H, Lee KKM, Moorthie VA, Xie X, Kealey JT, Da Silva NA, Vederas JC, Tang Y. Complete Reconstitution of a Highly Reducing Iterative Polyketide Synthase. Science. 2009;326(5952):589-592. doi: 10.1126/science.1175602.

14. Agmon N, Mitchell LA, Cai Y, Ikushima S, Chuang J, Zheng A, Choi W-J, Martin JA, Caravelli K, Stracquadanio G, Boeke JD. Yeast Golden Gate (yGG) for the Efficient Assembly of S. cerevisiae Transcription Units. ACS Synthetic Biology. 2015;4(7):853-859. doi: 10.1021/sb500372z.

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5 A yeast three hybrid assay for metabolic engineering

of tetracycline derivatives

*The contents of this chapter were published in:

E. Herbst, P. A. Baldera-Aguayo, H. Lee and V. W. Cornish “A Yeast Three Hybrid Assay for

Metabolic Engineering of Tetracycline Derivatives” Biochemistry, 2018, 57, 4726-4734. https://pubs.acs.org/doi/abs/10.1021/acs.biochem.8b00419

181

5.1 Chapter outline

Metabolic engineering stands to transform the discovery and production of a wide range of chemicals, but metabolic engineering currently demands considerable resource investments that restrict commercial application. To facilitate the applicability of metabolic engineering, general high-throughput and readily-implemented technologies are needed to assay vast libraries of strains producing desirable chemicals. Towards this end we describe here the development of a yeast three hybrid (Y3H) assay as a general, high-throughput, versatile and readily-implemented approach for the detection of target molecule biosynthesis. Our system detects target molecule biosynthesis through a change in reporter gene transcription that results from the binding of the target molecule to a modular protein receptor. We demonstrate the use of the Y3H assay for detecting the biosynthesis of tetracyclines, a major class of antibiotics, based on the interaction between tetracyclines and the tetracycline repressor protein (TetR). Various tetracycline derivatives can be detected using our assay, whose versatility enables its use both as a screen and a selection to match the needs and instrumentation of a wide range of end-users. We demonstrate the applicability of the Y3H assay to metabolic engineering by differentiating between producer and non-producer strains of the natural product anhydrotetracycline TAN-1612. The Y3H assay is superior to state- of-the-art HPLC-MS methods in throughput and limit of detection of tetracycline derivatives.

Finally, our establishment of the Y3H assay for detecting the biosynthesis of a tetracycline supports the generality of the Y3H assay for detecting the biosynthesis of many other target molecules.

182

5.2 Introduction

Metabolic engineering is a potentially greener and more economical approach than traditional organic synthesis for the production of a wide range of organic chemicals. As opposed to organic synthesis, metabolic engineering is capable of producing valuable chemicals from readily available and renewable carbon sources and significantly reducing the use of organic solvents and reagents.(1, 2) Traditional methods in DNA mutagenesis and increasingly, advances in DNA synthesis, DNA assembly and genome engineering are enabling high throughput strain construction in metabolic engineering.(3-8) High-throughput strain construction is necessary because not only the multi-gene biosynthetic pathway for the natural product, but also the underlying metabolism of the host must be optimized.(9) Assuming a biosynthetic pathway of 5 genes and 10 interacting host genes, to test just 5 variants for each of these genes results already in a library size exceeding 1010. However, the throughput of the most highly employed assaying methods such as LC-MS is only ~102-103 samples per day and is thus heavily limiting in assaying strains for successful metabolic engineering.(5, 9)

The currently employed methods for assaying metabolite production in metabolic engineering are either low-throughput and general or high-throughput but not general. LC-MS methods are general but low-throughput, enabling the screening of only about 102-103 samples per day.(9) Colorimetric, fluorometric and growth assays for molecules that are colored, fluorescent, essential for producer strain growth or could be easily converted to one of these, are high-throughput but not general.(10)

Attempts have been made to develop assays that are both high-throughput and general, but it is yet to be determined whether or not these assays could be widely implemented.(10, 11) Versatile, general, readily implemented high-throughput assays are required to transform metabolic

183 engineering from a field with a high potential to a field with an extensive real-world impact on the way chemicals are made.

We set out to adapt the Y3H assay, previously employed for various basic science applications by our laboratory and others,(12-18) to a general, readily implemented, high-throughput versatile assay for metabolic engineering. We wished to develop an assay that can be readily adapted to new target molecules, used as either a screen or a selection, and minimizes handling, to match varying needs and instrumentation of end-users.

Here, we demonstrate the application of the Y3H assay to detect the biosynthesis of tetracyclines, one of the major classes of antibiotics. We describe the engineering of the Y3H system to detect tetracyclines by synthesizing a minocycline-methotrexate chemical inducer of dimerization (CID) and cloning a LexA-TetR (tetracycline repressor) fusion protein.(14) Reporter gene expression responds to tetracycline in our system by competitive binding of tetracycline and the CID to the

LexA-TetR protein fusion (Figure 5-1). Our assay can report the presence of tetracyclines using various reporter gene outputs, enabling the use of the assay both as a screen and a selection to match the needs and instrumentation of a variety of end-users. We show the applicability of our assay to metabolic engineering through the differentiation between producer and non-producer strains of the natural product anhydrotetracycline, TAN-1612.

Figure 5-1 Detection of a target molecule biosynthesis by the yeast three hybrid system.

184

Low/no production of a target molecule is detected by high reporter gene expression (left). High production titers of the target molecule outcompete the chemical inducer of dimerization (CID) from the fusion protein receptor and are detected by lower gene expression (right). DBD = DNA binding domain; AD = activation domain; PR = protein receptor (e.g., TetR); DHFR = dihydrofolate reductase.

5.3 Materials and methods

General methods. Absorption and fluorescence spectra were recorded on Infinite-M200 fluorescent spectrometer. DNA sequences were purchased from IDT. Polymerases, restriction enzymes and Gibson Assembly mix were purchased from New England Biolabs. Sanger sequencing was performed by Genewiz. Yeast strains were grown at 30 °C and shaker settings were 200 rpm, unless otherwise indicated. Yeast transformations were done using the lithium acetate method.(19) Plasmids were cloned and amplified using Gibson Assembly and cloning strain C3040 (New England Biolabs).(20) Unless otherwise indicated, yeast strains were grown on synthetic minimal media lacking histidine and/or uracil and/or tryptophan and/or leucine, as indicated by the abbreviation HUTL-.(21) Yeast strain patches were obtained from glycerol stocks by streaking on an agar plate of synthetic medium lacking the appropriate amino acid markers, incubating at 30 °C for 3 days, patching single colonies onto a fresh agar plate and incubating at

30 °C overnight. Unless otherwise indicated, when 96 well plates were placed in the yeast shaker,

95% of the interface between the plate and its cover was wrapped with laboratory film (Parafilm,

Bemis PM999). All Y3H assays were performed with sterile components and three biological replicates. Protein homology was calculated by BLAST (https://blast.ncbi.nlm.nih.gov) using the standard sequence alignment parameters. DataExpress was used to analyze mass spectrometry

185 peaks. Protein visualization was performed using The PyMOL Molecular Graphics System,

Version 2.3 Schrödinger, LLC.

Codon optimization by COOL (http://cool.syncti.org/index.php) was used for all hydroxylases and reductases unless noted explicitly that JCAT or no optimization was used (http://www.jcat.de/).

Optimization parameters chosen were individual codon use, codon context GC content of 39.3% and S. cerevisiae organism. The following restriction sites were generally excluded: GAGACC,

GGTCTC, GGATCC, GAGCTC, CTCGAG, GAAGAC, GTCTTC, CGTCTC, GAGACG,

GAATTC, TTAATTAA, TCTAGA, ACTAGT, CCCGGG, CTGCAG, AAGCTT, GTCGAC,

ACGCGT, GGTACC, GCGGCCGC, AGATCT, GGCCGGCC, CCGCGG, GCTAGC,

CCATGG.

All chemical reactions were carried out under Argon atmosphere with anhydrous solvents unless otherwise indicated. Analytical high-pressure liquid chromatography (HPLC) was performed with a C-18 column, 2.6 μ, 250x4.6 mm, eluent given in parentheses. Unless stated otherwise, reactions were monitored using analytical HPLC with a separation gradient of 10% to 90% MeCN in 0.1%

(v/v) TFA aqueous solution over 20 minutes. Preparative HPLC was carried out with a C-18 5 μ column, 250x10 mm, eluent given in parentheses. NMR spectra were obtained using Bruker 400

MHz or 500 MHz instruments, as indicated. Unless stated otherwise, mass spectroscopy measurements were performed on Advion CMS mass spectrometer equipped with atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) sources. TAN-1612 quantification was performed using a phenyl-hexyl 1.7 μ 100x3 mm column, on a Waters SQD2 quadrupole mass spectrometer equipped with a UPC2 SFC inlet, a photodiode array (PDA) UV-

Vis detector, and a dual ESI/APCI probe. HRMS spectra were taken on a Waters ACQUITY UPLC

BEH C18 column (2.1 x 50 mm) at 30 °C with a flow rate of 0.8 mL/min. Unless stated otherwise,

186 all reagents, salts and solvents were purchased from commercial sources and used without further purification.

Synthesis of the Min-Mtx CID (1, Scheme 5-1). Step a. To 9-NH2-minocycline (100 mg, 0.2 mmol), Boc-11-aminoundecanoic acid (89 mg, 0.3 mmol), EDC (75 mg, 0.4 mmol) and HOBt (60 mg, 0.4) were added DMF (1 mL) and Et3N (139 μL, 1.0 mmol) and the reaction was mixed at r.t for 6 h. The reaction progress was monitored by RP-HPLC. Aqueous NaHCO3 (0.05 M)(22) was then added and the aqueous phase was extracted three times with CHCl3. The combined organic fraction was extracted with brine, the combined brine extracts were extracted twice with CHCl3, the combined organic fraction was dried with Na2SO4 and the solvent was removed under reduced pressure. The crude product was purified in two batches by preparative RP-HPLC (30-35% MeCN

1 in 99.9%:0.1% H2O:TFA, 45 min) to afford compound 3 (53.4 mg, 36%) as a yellow solid. H-

NMR (400 MHz, MeOD d4) δ 8.60 (s, 1H), 4.11 (s, 1H), 3.24 (dd, J = 15.4, 4.2, 1H), 3.14 (s, 6H),

3.08 (m, 3H), 3.02 (s, 6H), 2.50 (t, J = 7.4, 2H), 2.44 (t, J = 14.3, 1H), 2.24 (m, 1H), 1.72 (m, 1H),

1.62 (m, 1H), 1.43 (s, 9H), 1.42 (m, 4H) 1.38 (m, 2H), 1.32 (m, 10H). MS (APCI+): m/z calc. for

+ + - C39H58N5O10 : 756.42; found: 756.4 [M + H] ; MS (APCI-): m/z calc. for C39H56N5O10 : 754.40; found: 754.3 [M – H]-.

Steps b-d. To compound 3 (27.3 mg, 0.036 mmol) were added DCM (0.5 mL) and TFA (0.5 mL) and the reaction was mixed at r.t. for 12 min. The solvent was removed under reduced pressure.

PhMe was added and the solvent was again removed under reduced pressure. PhMe addition and solvent removal was repeated once more. Methotrexate-α-OtBu (27.5 mg, 0.054 mmol)(23, 24) and PyBOP (37.5 mg, 0.072 mmol) were added, followed by DIPEA (11.5 mg, 0.09 mmol) in

DMF (1 mL) and the reaction was mixed at r.t.. After 22 h EtOAc and aqueous NaHCO3 were added and the aqueous phase was extracted twice with EtOAc. The combined organic fraction was

187 extracted with brine and the brine extract was filtered. The solid residue was redissolved in MeOH and the solvent was removed under reduced pressure. The crude product was purified in two batches by preparative RP-HPLC (20-30% MeCN in 99.9%:0.1% H2O:TFA, 50 min). Following solvent removal under reduced pressure, TFA (2 mL) was added and the reaction was stirred at r.t. for 90 min. The reaction progress was monitored by RP-HPLC. The solvent was removed under reduced pressure to afford compound 1 (4.1 mg, 10%) as a yellow solid. 1H-NMR (500 MHz,

MeOD d4) δ 8.64 (s, 1H), 8.25 (s, 1H), 7.76 (d, J = 8.8, 2H), 6.86 (d, J = 8.8, 2H), 4.92 (s, 2H),

4.54 (dd, J = 8.8, 4.5, 1H), 4.08 (s, 1H), 3.27 (s, 3H), 3.11 (m, 2H), 3.00 (s, 6H), 2.83 (s, 3H), 2.80,

(s, 3H), 2.47 (m, 2H), 2.41-2.05 (m, 6H), 1.70-1.59 (m, 4H), 1.48-1.28 (m, 16H) MS (ESI+): m/z

2+ 2+ calc. for C54H71N13O12 : 1093.54; found: 546.9 [M + 2H] .

Y3H strain construction

LexA-TetR plasmids. Plasmids PBA-Gib5 and HL-249-1 through HL-249-6 encoding LexA-

TetR protein fusions for the various TetR classes were cloned by Gibson Assembly of the appropriate TetR sequence with pMW103 digested with restriction enzymes BamHI-HF and

EcoRI-HF (Table S3). All TetR sequences with the exception of TetR(B) were obtained as IDT gBlocks. TetR(B) sequence was amplified from pET21d-TetR, courtesy of A. Davidson.(25, 26)

LacZ assay strain construction. V506 was grown overnight in UT- media.(27) The culture was then diluted 104-105X with UT- and plated on UT- plates. Single colonies were streaked on HTU- and UT- plates and a streak growing on UT- but not on HTU-, indicating the loss of the pMW3(GSG)2rGR2 plasmid, was glycerol stocked as PBA5. This strain was further used for the transformation of the LexA-TetR plasmids to generate LacZ assay strains PBA8 and HL-260-1 through HL-260-7.

188

Growth assay strain construction. Yeast strain LW2635(28) was transformed with plasmid

PBA-Gib5 to generate strain PBA6. PBA6 was transformed with pMW2eDHFR(27) to generate the Y3H growth assay strain PBA14. mCherry reporter plasmid. Reporter plasmid HL-174-2 encoding mCherry under the transcriptional control of LexA operators was cloned by Gibson Assembly of an mCherry sequence amplified from yEpGAP-Cherry(29) and an 8LexA-min-pGal1 sequence amplified from pMW112(14) into pRS416 digested with SacI and XhoI.

Fluorescent protein assay strain construction. V506 was grown overnight in T- media. The culture was then diluted 104-105X with T- and plated on T- plates. Single colonies were streaked on TU-, HT- and T- plates and a streak growing on T- but not on UT- or on HT-, indicating the loss of the pMW106 and pMW3(GSG)2rGR2 plasmids, was glycerol stocked as PBA3. This strain was transformed with plasmid HL-174-2 to generate yeast strain HL-294-7. The latter was transformed with PBA-Gib5 to generate the Y3H fluorescent reporter assay strain HL-2-4-1.

TAN-1612 producer/non-producer strain construction. TAN-1612 producer and non-producer strains EH-3-54-4 and EH-3-54-2 were obtained by transforming plasmids pYR342 and pYR291 or pYR342 and pRS426, respectively, into yeast strain BJ5464-NpgA (Table 5-1 and Table

5-2).(30)

Protocol for fluorescent protein Y3H assay

Fresh patches of Y3H strain HL-2-4-1 were inoculated into HTU- media (1 mL) in 24-well plates

(flat, clear bottom, Corning #3526) and placed in shaker. After 24 hours the cells were pelleted by centrifugation at 3,250 rpm and resuspended in H2O (1 mL). Pelleting and resuspension were repeated once more. OD600 was measured by diluting 10X into H2O and the cells were diluted accordingly into H2O for OD600 = 1 to be used as a 10X solution. 20 μL cells of OD600 = 1 were

189 added to the assay plate as the last component in the assay plate (flat, clear bottom, black 96-well plate, Corning #3603). Each well of the assay plate contained 200 μL total liquid volume, 20 μL cells, 100 μL of 2X HTU- media of 4% raffinose and no glucose, 20 μL of 10X 20% galactose aqueous solution, 20 μL of 10X CID solution of 100 μM in 10% DMSO aqueous solution, 20 μL supernatant and 20 μL purified TAN-1612 in a 10% DMSO aqueous solution,(30) or a 10% DMSO aqueous solution. Starting conditions in the assay plate were OD600 = 0.1, 2% galactose, 2% raffinose, 0% glucose, 1X HTU-, 10 μM CID, 2% DMSO. The assay plate was placed in the 30 °C shaker overnight before measuring OD600 and 620 nm fluorescence (λex = 588 nm).(31)

TAN-1612 supernatants from flask cultures were obtained by inoculating TAN-1612 producing and non-producing strains, EH-3-54-4 and EH-3-54-2 respectively, in UT- media (5 mL) in 15 mL culture tubes (Corning 352059) and placing in shaker overnight; Overnight cultures were used to inoculate 100 mL YPD in 500 mL conical flasks with a starting OD600 of 0.08. After 68 h, 1 mL of culture was pelleted in 1.5 mL Eppendorf tubes at 14,000 rpm, sterile filtered and diluted 20X into H2O to be used as a 10X stock solution.

TAN-1612 supernatants from 96-well plate cultures were obtained by inoculating 12 and 84 colonies of TAN-1612 producing and non-producing strains, EH-3-54-4 and EH-3-54-2 respectively, in UT- media (0.5 mL) in a 96-well plate (Corning P-2ML-SQ-C-S). The plate was covered with two layers of SealMate film (Excel Scientific, SM-KIT-BS) and placed in a shaker overnight. Overnight cultures were used to inoculate 300 μL YPD in an identical setup with a starting OD600 of 0.1 and placed in 800 rpm shaker overnight. After 54 h, 150 μL of H2O were added to the cultures to prevent drying of cultures due to evaporation and two new layers of

Sealmate film were replaced. After 78 h, the cultures were pelleted at 3,250 rpm and the supernatant was diluted 20X into H2O to be used as a 10X stock solution.

190

Protocol for Y3H growth assay

Fresh patches of Y3H strain PBA14 were inoculated in HT- media (1 mL) and placed in shaker overnight. Cultures were then centrifuged at 3,000 rpm for 3 min and the pellets were resuspended with H2O (1 mL). Pelleting and resuspension was repeated twice more. OD600 was measured and the cells were diluted accordingly into H2O to OD600 = 1 to be used as a 10X stock solution. 20 μL cells of OD600 = 1 were added as the last component to the assay plate (flat, clear bottom, black

96-well plate, Corning #3603). Each well of the assay plate contained 200 μL total liquid volume,

20 μL cells, 100 μL of 2X HT- media with 4% raffinose, 4% galactose, 0% glucose, 0.4% 5-FOA,

20 μL of 0/250 μM 10X CID in 10% DMSO aqueous solution, 20 μL of doxycycline (0/50 μM in

10% EtOH aqueous solution) and 40 μL H2O. Starting conditions in the assay plate were OD600 =

0.1, 2% galactose, 2% raffinose, 0% glucose, 0.2% 5-FOA, 1X HT-, 25 μM CID, 1% DMSO, 1%

EtOH. The assay plate was placed in the 30 °C shaker for 5 days with daily measurements of

OD600.

Protocol for Y3H LacZ assay

Fresh patches of Y3H strains PBA-8 and HL-260-1 through HL-260-7 were inoculated into HTU- media (1 mL) in 24-well plates (flat, clear bottom, Corning #3526) and place at 30 °C shaker. After

24 hours the cells were pelleted by centrifugation at 3000 rpm for 5 min and resuspended in H2O

(1 mL). Pelleting and resuspension were repeated once more. OD600 was measured and the cells were diluted accordingly into H2O to OD600 = 1 to be used as a 10X solution. 20 μL cells of OD600

= 1 were added to the assay plate as the last component in the assay plate (clear v-bottom, clear

96-well plate, Corning #3894). Each well of the assay plate contained 200 μL total liquid volume,

20 μL cells, 100 μL of 2X HTU- media of 4% raffinose and no glucose, 20 μL of 10X 20% galactose aqueous solution, 20 μL of 10X CID solution in 10% DMSO aqueous solution or a 10%

191

DMSO aqueous solution, 20 μL of the tetracycline ligand in 10% EtOH aqueous solution and 20

μL water. Starting conditions in the assay plate are OD600 = 0.1, 2% galactose, 2% raffinose, 0% glucose, 1X HTU-, varying concentration CID, varying concentration of tetracycline ligand, 1%

DMSO and 1% EtOH. The assay plate was placed in the shaker overnight. OD measurements were then taken after 24 h and the cells were pelleted and resuspended in 100 μL Z-buffer (60 mM

Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 2 mM MgSO4, pH adjusted to 7, 2.7 mL/L β- mercaptoethanol added fresh the day of the assay). The cells were pelleted again, resuspended in

100 μL Y-PER buffer (Fisher Scientific, #78990) and lysed for 30 min at r.t. before the addition of o-Nitrophenyl β-D-galactopyranoside (ONPG) in Z-buffer (8.5 μL, 10 mg/mL). After 90 min a

Na2CO3 solution (1 M, 110 μL) was added to quench the reaction. After pelleting at 3,000 rpm,

175 μL of the supernatant was transferred into flat clear-bottom 96-well plates (Corning #353072) and OD420 was measured. The limit of detection was calculated as the concentration of molecule at which the signal-to-noise ratio of the assay was > 3:1.(32, 33)

5.4 Results

5.4.1 Y3H system design and CID design and synthesis

For a Y3H system detecting tetracyclines, we cloned a Y3H strain encoding LexA-TetR and

DHFR-B42 fusion proteins and synthesized a minocycline-methotrexate (Min-Mtx) CID (Figure

5-1). We chose to base our Y3H system on well-studied components. Namely, the LexA DNA binding domain (DBD) and B42 activation domain (AD),(34) respectively, introduced by Brent and coworkers,(14, 27, 35, 36) as well as the DHFR- methotrexate and TetR-tetracycline interactions. The latter two are highly characterized receptor-ligand interactions of picomolar affinity,(37) and the methotrexate-DFHR interaction has been used numerous times by our laboratory.(27, 35) We used LexA-TetR fusion proteins of TetR variants that were highly studied,

192 such as the variants from classes B and D,(37-39) as well TetR variants from the much less studied

TetR classes A,(40, 41) C,(42, 43) E,(44, 45) G,(46) and H.(47-50) TetR and LexA form stable homodimers, with each of the TetR monomers binding tetracyclines and LexA binding DNA as a dimer.(38, 51) Thus it is possible that the monomer ratio of TetR / LexA in the LexA-TetR fusion protein is unequal to 1, affecting accordingly the local concentrations of the Min-Mtx CID and

DHFR-B42 as well. In any case, increased levels of tetracyclines are expected to outcompete the

CID from the LexA-TetR protein fusion and lower reporter protein expression (Figure 5-1).

Our design and synthesis of the Min-Mtx CID is based on the wealth of structure activity relationship (SAR) data for the interaction of tetracyclines with TetR, as well as our laboratory’s previously reported CIDs.(27, 35) It is known from inspection of the high-resolution structure of

TetR bound to tetracycline derivatives as well as biochemical characterization that tetracyclines can be derivatized at the D ring without disrupting binding to TetR (Scheme 5-1).(37, 38, 52)

Moreover, the FDA-approved analog tigecycline and the clinical candidates eravacycline and TP-

271 are all 9-amidotetracyclines;(53, 54) thus, synthesis of a 9-amidotetracycline CID was logical.

Specifically, 9-aminominocycline was chosen as the starting material because of its high acid stability relative to tetracycline as well as its commercial availability.(55) The design and synthesis of the Mtx component of the Min-Mtx CID was based on our laboratory’s previously reported methotrexate CIDs (Scheme 5-1).(27, 35)

Scheme 5-1 Synthesis of the chemical inducer of dimerization (CID) Min-Mtxa

193

aReagents and conditions: (a) Boc-11-aminoundecanoic acid (1.5 equiv), EDC (2 equiv), HOBt (2 o o equiv), Et3N (5 equiv), DMF, 22 C, 6 h, 36%; (b) TFA:DCM (1:1), 22 C, 12 min; (c) Methotrexate-α-OtBu (1.5 equiv),(23, 24) PYBOP (2 equiv), DIPEA (2.5 equiv), DMF, 22 oC, 22 h; (d) TFA, 22 oC, 1.5 h, 10% over three steps. EDC = N-(3-Dimethylaminopropyl)-N′- ethylcarbodiimide hydrochloride, HOBt = 1-hydroxybenzotriazole, TFA = trifluoroacetic acid, PyBOP = (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, DIPEA = N- Ethyldiisopropylamine.

5.4.2 Initial characterization of the Y3H assay for tetracycline derivatives

With the Y3H strains and Min-Mtx CID at hand we verified the functionality of our Y3H system and determined the optimal CID concentration and TetR variant to further use in the assay. First, we confirmed that reporter gene output in our system is dependent upon the presence of the CID and determined the desired range of CID concentration to use in the assay (Figure 5-2a). We then tested candidates from each of the TetR classes as protein receptors and observed that the Y3H system is responsive to the CID with all known TetR classes except for the TetR(C) variant we used (Figure 5-2b). Given that TetR(B) and TetR(G) showed the best response to the CID and to maintain consistency with the highly studied TetR(B),(37-39) we focused our further experiments on TetR(B).

194

We show that our assay can differentiate sub-μM concentrations of various tetracyclines (Figure

5-2c), key for its applicability to differentiate between high and low producer strains. Our Y3H system differentiates doxycycline, tetracycline and 9-NH2-minocycline concentrations in the range of 2 – 200 nM, 0.02 – 2 μM and 0.2 – 20 μM, respectively. The limit of detection in our Y3H assay is ≤ 0.2 μM for doxycycline and ≤ 2 μM for tetracycline and 9-NH2-minocycline.(32)

Figure 5-2 Characterization of the dynamic range of the Y3H assay for tetracyclines Characterization of the dynamic range of the Y3H assay for tetracyclines in (a) varying concentrations of the Min-Mtx CID, (b) varying TetR classes and (c) varying target molecule structure and concentrations. LacZ was used as a reporter gene. Miller units were calculated as

195

(1000*OD420)/(well-volume(mL)*reaction-time(min)*OD600).(56) “+TetR” and “-TetR” are strains HL-260-5 and HL-260-7 encoding LexA-TetR(G) and LexA, respectively; Error bars represent the standard error of the mean (s.e.m) from three biological replicates. After confirming the functionality of the Y3H system for tetracyclines we proceeded to show that it can report the presence of tetracyclines with a modular output, key for enabling versatile use by various end-users. Using a URA3 reporter gene and growing the yeast in the presence of 5- fluorouracil (5-FOA) we show that yeast growth is doxycycline-dependent in the presence, but not in the absence of the Min-Mtx CID (Figure 5-3a). We additionally show that the Y3H assay for tetracyclines can be used as a colorimetric as well as a fluorometric assay, by employing a LacZ or and mCherry reporter gene, respectively (Figure 5-3b, Figure 5-3c). As expected, while reporter gene activity is dependent on CID and doxycycline in the Y3H strain, such dependence is not detected in a control strain excluding the eDFHR-AD fusion protein (Figure 5-5).

196

Figure 5-3 The modularity of the yeast three hybrid assay enables both screening and selecting for biosynthesis of target tetracyclines The modularity of the yeast three hybrid assay enables both screening and selecting for biosynthesis of target tetracyclines by enabling (a) a growth assay, (b) a colorimetric assay and a (c) fluorometric assay. (a) Yeast growth, (b) LacZ transcription and (c) mCherry transcription are dependent on doxycycline (Dox) in the presence, but not in the absence of the CID. CID, Dox concentrations = (a) 25 μM, 5 μM (b) 25 μM, 0.2 μM (c) 10 μM, 1 μM. Error bars represent the standard error of the mean (s.e.m) from three biological replicates. Miller units were calculated as

(1000*OD420)/(well-volume(mL)*reaction-time(min)*OD600).(56) mCherry (620 nm)/OD600 was calculated by dividing mCherry fluorescence (620 nm, λex = 588 nm) by OD600.

197

5.4.3 Applying the Y3H assay to the detection of target molecule biosynthesis

We demonstrate the applicability of the Y3H assay to metabolic engineering by differentiating between producer and non-producer strains of TAN-1612 (Figure 5-4a).(30) For the TAN-1612 producer strain we used S. cerevisiae strain EH-3-54-4, encoding all four genes of the TAN-1612 biosynthetic pathway as previously reported by Tang and coworkers and producing ~60 μM TAN-

1612 (Supplementary methods).(30) The non-producer control we used, S. cerevisiae strain EH-

3-54-2, does not encode the polyketide synthase AdaA and has no measurable production of TAN-

1612 (data not shown).

The Y3H assay for tetracyclines can differentiate between supernatants of producer and non- producer strains of TAN-1612 (Figure 5-4b). When purified TAN-1612 is spiked into non- producer supernatants, the resulting Y3H signal resembles the signal obtained with producer supernatant (Figure 5-4b). These results agree with our hypothesis that TAN-1612, present in the producer strain supernatant but not in the non-producer strain supernatant, outcompetes the CID from the LexA-TetR fusion protein.

The Y3H assay for tetracyclines also differentiates TAN-1612 producer colonies from a non- producing population cultured in a 96-well plate (Figure 5-4c). The 12 producer colonies gave an average mCherry fluorescence / OD600 signal of 132 with a standard deviation of 17 and the 84 non-producer colonies gave an average mCherry fluorescence / OD600 signal of 210 with a standard deviation of 16. Thus, assuming normal distribution there is less than 2.5% overlap in the Y3H signal from producer and non-producer colonies.

198

Figure 5-4 Differentiation of a producer and a non-producer strain of a target molecule by the Y3H assay for tetracyclines, demonstrating the applicability of the Y3H assay to metabolic engineering. (a) Workflow for screening colonies for production of a target molecule using the Y3H assay. (b) The Y3H assay for tetracyclines differentiates between the supernatants of TAN-1612 producer and TAN-1612 non-producer cultured in shake flasks. (c) The Y3H assay for tetracyclines differentiates the supernatants of TAN-1612 producer colonies from a non-producing population cultured in a 96-well plate. The non-producer strain does not encode AdaA, the polyketide synthase of the TAN-1612 biosynthetic pathway, while the producer strain encodes the complete

199 pathway (Table 5-1 and Table 5-2). The concentration of TAN-1612 in the Y3H assay well from the supernatant and from the purified sample is ~0.3 μM and 0.5 μM, respectively (Supplementary methods). mCherry (620 nm)/OD600 was calculated by dividing mCherry fluorescence (620 nm,

λex = 588 nm) by OD600. Error bars represent the standard error of the mean (s.e.m) from three biological replicates of the Y3H strain and two biological replicates of the TAN-1612 producer/non-producer.

5.5 Discussion

The Y3H assay for tetracyclines is advantageous compared to the state-of-the-art methods for tetracycline detection in terms of throughput, cost, instrumentation requirements and limit of detection. Being readily performed in 96-well microtiter plates, the Y3H assay throughput is >1000 samples / hour when measured by a standard laboratory plate reader (Infinite-M200). By comparison, the throughput of the previous state-of-the-art HPLC-based methods for tetracycline detection is <10 samples/hour.(33, 57) The use of a plate reader to measure the Y3H assay output is also advantageous in terms of cost to state-of-the-art chromatography-based methods such as

HPLC and even more so, LCMS. The price difference is both in the initial investment in instrumentation, as spectrophotometers are cheaper than LC instruments, and in the reagent costs of 96 well plates vs LC-grade solvents. In terms of limit of detection the Y3H assay has a limit of detection of ≤ 200 nM for doxycycline (Figure 5-2c), which is better than the state-of-the-art HPLC limit of detection of ≥ 4.5 μM for doxycycline.(33, 57) The low limit of detection our assay exhibits enables it to differentiate producer from non-producer strains even in low titers of initial strain development (Figure 5-4 and Section 5.7.1 5.7.2, ~60 μM). Differentiating between production levels in higher titer ballparks in later stages of producer strain optimization would simply require a higher dilution factor of supernatants into the Y3H strain media. Future generations of this Y3H

200 assay might include TetR mutants that are more specific and have a lower detection limit to tetracycline derivatives of interest.

The Y3H assay for metabolic engineering also compares well with our laboratory’s recently published FP assay,(58) because of two key differences in setup requirements and output. First, since the Y3H assay does not require the purification of the protein receptor, it is used much more readily to screen for the ultimate protein receptor for the assay. Thus, we show here the first comparison of all TetR classes for binding of a small molecule in an attempt to identify the best

TetR variant for differentiating between + and – CID (Figure 5-2b). Further optimization of a protein receptor for the assay of a specific target molecule is readily enabled in the Y3H assay simply by cloning the protein variants into the same plasmid backbone and transforming into yeast.

The versatility in choice of output in the Y3H assay is a key enabling factor for the advantages the

Y3H assay has over the FP assay since it makes the assay accessible to a wider range of users. The fluorescent output in the Y3H assay enables measurements with a standard laboratory plate reader, eliminating the requirement for anisotropy capacities (Figure 5-3c). The LacZ output could also be measured by a standard plate reader as well as by the naked eye (Figure 5-3b).

If the Y3H system and the metabolic pathway for the small molecule of interest are cloned into the same strain, colonies can be assayed for production even more directly, increasing throughput and minimizing handling. The Y3H assay can then enable screening colonies for production of the target molecule directly on an agar plate without the need for further handling of the colonies in microtiter plates. The agar plate screen could be performed with the naked eye or a simple camera by using X-Gal plates and the LacZ reporter or fluorescent camera/microscope by using the mCherry reporter. Finally, the Y3H assay throughput could be vastly increased by selecting for high producing strains en masse in flask or fermenter culturing using the growth assay or

201 fluorescence assisted cell sorting (FACS). Such en masse screening methods not only greatly enhance the throughput but also minimize the labor, instrumentation, cost and time, required to obtain results. The Y3H assay for tetracyclines can report the presence of a target molecule with any of the above mentioned outputs (Figure 5-3), enabling metabolic engineering applications for a range of end users with varying needs and instrumentation availabilities.

While a TetR-tetO-based assay could conceivably be developed for the metabolic engineering of tetracycline derivatives,(59) our TetR-based Y3H assay is advantageous for two main reasons. For one, the Y3H assay can potentially be applied to a greater variety of tetracycline derivatives as

TetR-binding to the tetracycline suffices and there is no demand for a TetR conformational change.

By comparison, signal transduction in the TetR-tetO system is dependent on coupling between tetracycline binding and a conformational change in TetR.(26, 38) It has been hypothesized that high-affinity binding may be a necessary but not sufficient property for TetR induction in the TetR-

TetO system.(60) In practice, while it was possible to detect binding to a tetracycline analog, 4- de(dimethylamino)-anhydrotetracycline, no induction was detected using a TetR-TetO-based assay.(37) Thus, the use of TetR in a Y3H system can facilitate the evolvement of TetR mutants with specific binding to tetracycline derivatives of interest in future generations of the assay.

Furthermore, the Y3H system for metabolic engineering can be readily modified to apply for many metabolites of interest beyond tetracyclines, as detailed below. In contrast, the TetR-TetO system is much more limited in the scope of molecules it could be applied to, namely transcriptional regulators.(61)

A Y3H system as described here for the metabolic engineering of tetracycline derivatives can be designed for any target molecule with a receptor. Given T, a desirable target, PR, a protein receptor for T, and T’, a readily derivatizable analog of T, two new components need to be generated for a

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Y3H assay to detect biosynthesis of T. The first is a Mtx-T’ CID and the second is a LexA-PR fusion protein (Figure 5-1). This approach is especially amenable for the development of assays for molecules of pharmacological interest. The reason is that such molecules often have a known protein target as well as an established derivatization chemistry and SAR that greatly support the design and synthesis of a CID. More broadly, building from the Cornish Laboratory’s early reports of chemical complementation,(17, 62, 63) this work advances the enabling technology of the Y3H system for the emerging field of synthetic biology for handling the diversity of engineering multi- gene pathways and assaying for chemistry not natural to the cell.

5.6 Conclusion

This chapter describes the development of a yeast three hybrid (Y3H) assay as a general, high- throughput, versatile and readily-implemented approach for the detection of target molecule biosynthesis. We demonstrate the use of the Y3H assay for detecting the biosynthesis of tetracyclines, a major class of antibiotics, based on the interaction between tetracyclines and the tetracycline repressor protein (TetR). We demonstrate the applicability of the Y3H assay to metabolic engineering by differentiating between producer and non-producer strains of the natural product anhydrotetracycline TAN-1612. The Y3H assay is superior to state-of-the-art HPLC-MS methods in throughput and limit of detection of tetracycline derivatives. Finally, our establishment of the Y3H assay for detecting the biosynthesis of an anhydrotetracycline supports the generality of the Y3H assay for detecting the biosynthesis of many other target molecules. Specifically, future versions of this assay to screen or select for further modifications of TAN-1612 that could not be assayed by the UV/Vis assay for hydroxylation of anhydrotetracycline used in the previous chapters are envisioned.

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5.7 Appendix

5.7.1 Supplementary figures

500 400 300 200

Miller Units Miller 100 0 + CID+ CID− CID − CID + CID+ CID− CID − CID +Dox -Dox +Dox -Dox +Dox -Dox +Dox -Dox +LexA-TetR -LexA-TetR

Figure 5-5 LacZ readout of strain harboring plasmid encoding for LexA-TetR (PBA-8) and strain without the plasmid (PBA-5). Only the strain with the LexA-TetR fusion protein responds to the CID. Addition of Dox lowers reporter gene expression only in the strain with LexA-TetR in the presence of the CID. Concentrations of CID and Dox are 25 μM and 200 nM, respectively. Miller units were calculated as (1000*OD420)/(well-volume(mL)*reaction-time(min)*OD600).(56) Error bars represent the standard error of the mean (s.e.m) from three biological replicates.

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14 12

410 nm) 410 10 - y = 0.016x + 8.1328 8 R² = 0.9933 6 4 2

Ln(absorption 390 Ln(absorption 0 0 50 100 150 200 250 300 TAN-1612 (μM)

Figure 5-6 standard curve for TAN-1612 quantification in cultures. A Purified TAN-1612 was analyzed at different concentrations on a separation gradient of 5% to

95% MeCN in supercritical CO2 over 5.5 minutes. Absorption between 390 nm to 410 nm was integrated at retention times of 0.87 – 1.50 min, which corresponds to TAN-1612 (MS (AI-): m/z - - - calc. for C21H17O9 : 413.09; found: 413.4 [M - H] ; MS (EI-): m/z calc. for C21H17O9 : 413.09; found: 413.4 [M - H]-).

5.7.2 Supplementary methods

Quantification of TAN-1612 concentration in cultures.

TAN-1612 cultures for quantification were obtained by inoculating two biological replicates of each of TAN-1612 producing and non-producing strains, EH-3-54-4 and EH-3-54-2 respectively, in UT- media (5 mL) in 15 mL culture tubes (Corning 352059) and placing in shaker overnight.

Overnight cultures were used to inoculate 100 mL YPD in 500 mL conical flasks with a starting

OD600 of 0.08. After 68 h, the 100 mL from the two biological replicates were combined and extracted with 200 mL of EtOAc in 8 x 50 mL falcon tubes (Corning 352098) by mixing at maximal speed in Orbital Shaker (Bellco Glass) at r.t. for 1.5 h and centrifuging at 3,300 rpm at r.t. for 15 min. The solvent of the combined organic phase was removed under reduced pressure

205 and the sample was redissolved in MeOH (1.5 mL) and filtered (Acrodisc® Syringe Filters, 13mm,

0.2 μm, Pall Laboratory, PTFE Membrane, 4422). The filtered sample was diluted 50X in MeOH and analyzed by Supercritical Fluid Chromatography / Mass spectrometry (SFC/MS) equipped with a photodiode array (PDA) UV-vis detector. Absorption between 390 nm to 410 nm was integrated at retention times of 0.87 – 1.50 min, which corresponds to TAN-1612 (MS (AI-): m/z

- - calc. for C21H17O9 : 413.09; found: 413.4 [M - H] . The TAN-1612 concentration in the extract was calculated to be 161 μM according to the following linear function: concentration=[ln(absorption 390-410 nm)-8.1328]/0.016 (Figure 5-6). The concentration of

TAN-1612 in the culture is therefore estimated to be ~60 μM, assuming a quantitive yield for the extraction process.

5.7.3 Supplementary tables

Table 5-1 Strains used in this study Strain Genotype Source/Reference FY250 MATα ura3-52 trp1 63 his3 200 leu2 1 gal L.P. Guarentea LW2635 MATa trp1Δ63 his3Δ200 8lexAop-Spo13-URA3 /(28) leu2Δ1 Gal+ BJ5464- MATα ura3-52 his3-Δ200 leu2-Δ1 Y. Tangb/(64) NpgA trp1 pep4::HIS3 δ:: pADH2-npgA-tADH2 prb1Δ1.6R can1 GAL V506 FY250 pMW106, pMW3(GSG)2rGR2, pMW2eDHFR /(27) PBA-5 FY250 pMW106, pMW2eDHFR This study HL-260-1 FY250 pMW106, pMW103TetR(A), pMW2eDHFR This study PBA-8 FY250 pMW106, pMW103TetR(B), pMW2eDHFR This study HL-260-2 FY250 pMW106, pMW103TetR(C), pMW2eDHFR This study HL-260-3 FY250 pMW106, pMW103TetR(D), pMW2eDHFR This study HL-260-4 FY250 pMW106, pMW103TetR(E), pMW2eDHFR This study HL-260-5 FY250 pMW106, pMW103TetR(G), pMW2eDHFR This study HL-260-6 FY250 pMW106, pMW103TetR(H), pMW2eDHFR This study HL-260-7 FY250 pMW106, pMW103(empty), pMW2eDHFR This study PBA-6 LW2635 PBA-Gib5 This study PBA-14 LW2635 PBA-Gib5, pMW2eDHFR This study PBA-3 FY250 pMW2eDHFR This study HL-294-7 FY250 pMW2eDHFR HL-174-2 This study HL-2-4-1 FY250 pMW2eDHFR pMW103TetR(B), HL-174-2 This study

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EH-3-54-4 BJ5464-NpgA pYR291, pYR342 This study EH-3-54-2 BJ5464-NpgA pRS426, pYR342 This study a L. P. Guarente, Massachusetts Institution of Technology, Biology Department b Y. Tang, University of California, Los Angeles, Department of Chemical and Biomolecular Engineering & Department of Chemistry and Biochemistry, Department of Bioengineering

Table 5-2 Plasmids used in this study Plasmid Description Source/Reference pMW2eDHFR pGal1-eDHFR-B42, trp marker, 2 μ /(27) pMW106 LacZ under control of 8 tandem LexA operators, Ura /(27, 65) marker pMW103 pADH1-LexA (empty), His marker, 2 μ R. Brenta/(65) pMW112 LacZ under control of 8 tandem LexA operators, Ura R. Brent/(14, 65) marker HL-249-1 pADH1-LexA-TetR(A)-tADH1 on pMW103 This study PBA-Gib5 pADH1-LexA-TetR(B)-tADH1 on pMW103 This study HL-249-2 pADH1-LexA-TetR(C)-tADH1 on pMW103 This study HL-249-3 pADH1-LexA-TetR(D)-tADH1 on pMW103 This study HL-249-4 pADH1-LexA-TetR(E)-tADH1 on pMW103 This study HL-249-5 pADH1-LexA-TetR(G)-tADH1 on pMW103 This study HL-249-6 pADH1-LexA-TetR(H)-tADH1 on pMW103 This study pRS416 Shuttle vector (empty), uracil marker, CEN ATCC87521 HL-174-2 8LexAOp-minpGal1-mCherry-tCyc1, Ura marker, CEN This study pYR291 AdaA expression in S. cerevisiae, uracil marker, 2 μ Y. Tangb/(30) pYR342 AdaB-D expression in S. cerevisiae, Tryp marker, 2 μ Y. Tang/(30) pRS426 Shuttle vector (empty), uracil marker, 2 μ ATCC77107 pET21d-TetR TetR(B) with C-term Histidine tag A. Davidsonc/(25) yEpGAP- mCherry, uracil marker, 2 μ Neta Deand/(29) Cherry a R. Brent, Fred Hutchinson Cancer Research Center, Division of Basic Sciences b Y. Tang, University of California, Los Angeles, Department of Chemical and Biomolecular Engineering & Department of Chemistry and Biochemistry, Department of Bioengineering c A. Davidson, Dept. of Molecular Genetics, Dept. of Biochemistry, Faculty of Medicine, University of Toronto d Neta Dean, Department of Biochemistry and Cell Biology, Stony Brook University

Table 5-3 Sequences of the various TetR classes in the within the pMW103 backbone.

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Sequences for the TetR classes are shown decapitalized within the context of LexA-DBD (partial, capitalized) in the 5’ end and the tADH1 terminator at the 3’ end (partial, capitalized). The NCBI and GenBank Reference Sequences used to order/clone DNA for the different TetR classes A, B, C, D, E, G and H are NC_006143.1, CP012140.1, NC_002056.1, CP000650.1, LKJQ01000048.1, CP003225.1 and AJ514834.1, respectively. TetR(A) – GCTGCGCGTCAGCGGGATGTCGATGAAAGATATCGGCATTATGGATG HL-249-1 GTGACTTGCTGGCAGTGCATAAAACTCAGGATGTACGTAACGGTCAG GTCGTTGTCGCACGTATTGATGACGAGGTTACCGTTAAGCGCCTGAA AAAACAGGGCAATAAAGTCGAACTGTTGCCAGAAAATAGCGAGTTT AAACCAATTGTCGTAGATCTTCGTCAGCAGAGCTTCACCATTGAAGG GCTGGCGGTTGGGGTTATTCGCAACGGCGACTGGCTGGAATTCatgacaa agttgcagccgaatacagtgatccgtgccgccctggacctgttgaacgaggtcggcgtagacggtctgacgacac gcaaactggcggaacggttgggggttcagcagccggcgctttactggcacttcaggaacaagcgggcgctgctc gacgcactggccgaagccatgctggcggagaatcatacgcattcggtgccgagagccgacgacgactggcgct catttctgatcgggaatgcccgcagcttcaggcaggcgctgctcgcctaccgcgatggcgcgcgcatccatgccg gcacgcgaccgggcgcaccgcagatggaaacggccgacgcgcagcttcgcttcctctgcgaggcgggtttttcg gccggggacgccgtcaatgcgctgatgacaatcagctacttcactgttggggccgtgcttgaggagcaggccgg cgacagcgatgccggcgagcgcggcggcaccgttgaacaggctccgctctcgccgctgttgcgggccgcgata gacgccttcgacgaagccggtccggacgcagcgttcgagcagggactcgcggtgattgtcgatggattggcgaa aaggaggctcgttgtcaggaacgttgaaggaccgagaaagggtgacgattagGATCCGTCGACCAT GGCGGCCGCTCGAGTCGACCTGCAGCCAAGCTAATTCCGGGCGAATT TCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAA GTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCT TATTCTTGAGTAACT TetR(B) – GCTGCGCGTCAGCGGGATGTCGATGAAAGATATCGGCATTATGGATG PBA-Gib5 GTGACTTGCTGGCAGTGCATAAAACTCAGGATGTACGTAACGGTCAG GTCGTTGTCGCACGTATTGATGACGAGGTTACCGTTAAGCGCCTGAA AAAACAGGGCAATAAAGTCGAACTGTTGCCAGAAAATAGCGAGTTT AAACCAATTGTCGTAGATCTTCGTCAGCAGAGCTTCACCATTGAAGG GCTGGCGGTTGGGGTTATTCGCAACGGCGACTGGCTGGAATTCatgtcta gattagataaaagtaaagtgattaacagcgcattagagctgcttaatgaggtcggaatcgaaggtttaacaacccgt aaactcgcccagaagctaggtgtagagcagcctacattgtattggcatgtaaaaaataagcgggctttgctcgacg ccttagccattgagatgttagataggcaccatactcacttttgccctttagaaggggaaagctggcaagattttttacgt aataacgctaaaagttttagatgtgctttactaagtcatcgcgatggagcaaaagtacatttaggtacacggcctaca gaaaaacagtatgaaactctcgaaaatcaattagcctttttatgccaacaaggtttttcactagagaatgcattatatgc actcagcgctgtggggcattttactttaggttgcgtattggaagatcaagagcatcaagtcgctaaagaagaaaggg aaacacctactactgatagtatgccgccattattacgacaagctatcgaattatttgatcaccaaggtgcagagccag ccttcttattcggccttgaattgatcatatgcggattagaaaaacaacttaaatgtgaaagtgggtcttagGATCC GTCGACCATGGCGGCCGCTCGAGTCGACCTGCAGCCAAGCTAATTCC GGGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAA AAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAA CGAAAATTCTTATTCTTGAGTAACT TetR(C) – GCTGCGCGTCAGCGGGATGTCGATGAAAGATATCGGCATTATGGATG HL-249-2 GTGACTTGCTGGCAGTGCATAAAACTCAGGATGTACGTAACGGTCAG

208

GTCGTTGTCGCACGTATTGATGACGAGGTTACCGTTAAGCGCCTGAA AAAACAGGGCAATAAAGTCGAACTGTTGCCAGAAAATAGCGAGTTT AAACCAATTGTCGTAGATCTTCGTCAGCAGAGCTTCACCATTGAAGG GCTGGCGGTTGGGGTTATTCGCAACGGCGACTGGCTGGAATTCatgaaca agctccaacgcgaggccgtgatccgaaccgcgctcgaactgcttaacgacgtgggcatggaaggtctaacgacg cgccgactggctgagcgcctcggggtgcaacagccagcgctctactggcatttcaagaacaagcgtgcgttgctc gacgcacttgccgaagccatgctgacgataaatcacacgcattcgacgccaagggatgacgacgactggcgttc gttcctgaagggcaatgcatgcagttttcgacgggcgttgctcgcttatcgcgatggcgcgcgtattcatgccggga cgcggccagccgcgccgcagatggaaaaagccgacgcgcagcttcgcttcctttgcgatgctggcttttcggcag gtgacgcgacctatgcgttgatggcaatcagctacttcaccgtcggcgctgttcttgagcagcaagctagcgaggc agacgccgaggagcggggcgaagatcagttgaccacctcagcgtctacgatgccggcgcgcctacagagcgc gatgaaaatcgtctacgaaggcggtccggacgcggcattcgagcgaggcctggctctcatcatcggcggtcttga aaaaatgaggctcactacgaacgacattgaggtgctgaagaatgttgacgaatagGATCCGTCGACCA TGGCGGCCGCTCGAGTCGACCTGCAGCCAAGCTAATTCCGGGCGAAT TTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATA AGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATT CTTATTCTTGAGTAACT TetR(D) – GCTGCGCGTCAGCGGGATGTCGATGAAAGATATCGGCATTATGGATG HL-249-3 GTGACTTGCTGGCAGTGCATAAAACTCAGGATGTACGTAACGGTCAG GTCGTTGTCGCACGTATTGATGACGAGGTTACCGTTAAGCGCCTGAA AAAACAGGGCAATAAAGTCGAACTGTTGCCAGAAAATAGCGAGTTT AAACCAATTGTCGTAGATCTTCGTCAGCAGAGCTTCACCATTGAAGG GCTGGCGGTTGGGGTTATTCGCAACGGCGACTGGCTGGAATTCatggcac ggctgaacagagaatcggttattgatgcggcactggaactgctgaatgagacagggattgacgggctgacgaccc gcaagctggcgcagaagctgggaatagaacagccgacactttactggcatgtgaaaaataaacgggcgttactgg atgcgctggcggtggagatcctggcgcgtcatcatgattattcactgcctgcggcgggggaatcctggcagtcattt ctgcgcaataatgcaatgagtttccgccgggcgctgctgcgttaccgtgacggggcaaaagtgcacctcggcacc cgccctgatgaaaaacagtatgatacggtggaaacccagttacgctttatgacagaaaacggcttttcactgcgcga cgggttatatgcgatttcagcggtcagtcattttacccttggtgccgtactggagcagcaggagcatactgccgccct gaccgaccgccctgcagcaccggacgaaaacctgccgccgctattgcgggaagcgctgcagattatggacagt gatgatggtgagcaggcctttctgcatggcctggagagcctgatccgggggtttgaggtgcagcttacggcactgt tgcaaatagtcggtggtgataaacttatcatccccttttgctagGATCCGTCGACCATGGCGGCC GCTCGAGTCGACCTGCAGCCAAGCTAATTCCGGGCGAATTTCTTATG ATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATA CAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTT GAGTAACT TetR(E) – GCTGCGCGTCAGCGGGATGTCGATGAAAGATATCGGCATTATGGATG HL-249-4 GTGACTTGCTGGCAGTGCATAAAACTCAGGATGTACGTAACGGTCAG GTCGTTGTCGCACGTATTGATGACGAGGTTACCGTTAAGCGCCTGAA AAAACAGGGCAATAAAGTCGAACTGTTGCCAGAAAATAGCGAGTTT AAACCAATTGTCGTAGATCTTCGTCAGCAGAGCTTCACCATTGAAGG GCTGGCGGTTGGGGTTATTCGCAACGGCGACTGGCTGGAATTCatggcac gactaagcttggacgacgtaatttcaatggcgctcaccctgctggacagcgaagggctagagggcttgactacgc gtaagctggcgcagtccctaaaaattgagcaaccgactctgtattggcacgtgcgcaacaagcagactcttatgaa catgctttcagaggcaatactggcgaagcatcacacccgttcagcaccgttaccgactgagagttggcagcagtttc tccaggaaaatgctctgagtttccgtaaagcattactggtccatcgtgatggagcccgattgcatatagggacctctc

209

ctacgcccccccagtttgaacaagcagaggcgcaactacgctgtctatgcgatgcagggttttcggtcgaggagg ctcttttcattctgcaatctatcagccattttacgttgggtgcagtattagaggagcaagcaacaaaccagatagaaaa taatcatgtgatagacgctgcaccaccattattacaagaggcatttaatattcaggcgagaacctctgctgaaatggc cttccatttcgggctgaaatcattaatatttggattttctgcacagttagatgaaaaaaagcatacacccattgaggatg gtaataaatagGATCCGTCGACCATGGCGGCCGCTCGAGTCGACCTGCAGC CAAGCTAATTCCGGGCGAATTTCTTATGATTTATGATTTTTATTATTA AATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCT TAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACT TetR(G) – GCTGCGCGTCAGCGGGATGTCGATGAAAGATATCGGCATTATGGATG HL-249-5 GTGACTTGCTGGCAGTGCATAAAACTCAGGATGTACGTAACGGTCAG GTCGTTGTCGCACGTATTGATGACGAGGTTACCGTTAAGCGCCTGAA AAAACAGGGCAATAAAGTCGAACTGTTGCCAGAAAATAGCGAGTTT AAACCAATTGTCGTAGATCTTCGTCAGCAGAGCTTCACCATTGAAGG GCTGGCGGTTGGGGTTATTCGCAACGGCGACTGGCTGGAATTCatgacca aactggacaagggcaccgtgatcgcggcggcgctagagctgttgaacgaggttggcatggacagcctgacgac gcggaagctcgctgaacgcctcaaggttcagcagcctgcgctttactggcatttccagaacaagcgagcgctgctt gatgcgctcgccgaggcgatgctggcggaacgccatacccgctcgctacccgaagagaatgaggactggcggg tgttcctgaaagagaatgccctgagcttcagaacggcgttgctctcttatcgggacggcgcgcgtatccatgccgg cactcgaccgacagaaccgaattttggcaccgccgagacgcaaatacgctttctctgcgcggagggcttttgtccg aagcgcgccgtttgggcgctccgggcggtcagtcactatgtggtcggttccgttctcgagcagcaggcatctgatg ccgatgagagagttccggacaggccagatgtgtccgagcaagcaccgtcgtccttcctgcacgatctgtttcacga gttggaaacagacggcatggatgctgcgttcaacttcggactcgacagcctcatcgctggtttcgagcggctgcgtt catctacaacagattagGATCCGTCGACCATGGCGGCCGCTCGAGTCGACCTGC AGCCAAGCTAATTCCGGGCGAATTTCTTATGATTTATGATTTTTATTA TTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGAC TCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACT TetR(H) – GCTGCGCGTCAGCGGGATGTCGATGAAAGATATCGGCATTATGGATG HL-249-6 GTGACTTGCTGGCAGTGCATAAAACTCAGGATGTACGTAACGGTCAG GTCGTTGTCGCACGTATTGATGACGAGGTTACCGTTAAGCGCCTGAA AAAACAGGGCAATAAAGTCGAACTGTTGCCAGAAAATAGCGAGTTT AAACCAATTGTCGTAGATCTTCGTCAGCAGAGCTTCACCATTGAAGG GCTGGCGGTTGGGGTTATTCGCAACGGCGACTGGCTGGAATTCatggcaa agctagataaagaacaagttattgatgatgcgttgattttacttaatgaagttggtattgaaggattaacaacgcgtaac gtggcgcaaaaaataggtgtggaacaacccacattgtattggcatgtaaaaaataaacgcgctttgttagatgcatta gcagaaactattttgcaaaagcaccatcatcatgttttgccattgccgaatgaaacatggcaggactttttgcgaaata acgcgaaaagcttccgccaagccttattaatgtatcgtgatggtggcaaaattcatgcgggaacacgcccctctgaa agtcaatttgagacatcagaacagcaactacagtttttgtgtgatgctgggtttagtctatctcaagccgtgtatgcatt aagctctattgcgcattttacattaggctccgtactggaaactcaagagcatcaagaaagccaaaaagagcgtgaaa aagtagagacggatactgttgcctatccgccattattaacccaagccgttgcaattatggatagtgataatggtgatg ctgcatttttgtttgtccttgatgtgatgatctctggacttgaaacagtattaaagagcgctaaatagGATCCGTC GACCATGGCGGCCGCTCGAGTCGACCTGCAGCCAAGCTAATTCCGGG CGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAA AAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGA AAATTCTTATTCTTGAGTAACT

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