Fermentation of Sugar Mixtures Found in Lignocellulosic

FERMENTATION OF SUGAR MIXTURES FOUND IN LIGNOCELLULOSIC

HYDROLYSATE

USING SUBSTRATE-SELECTIVE ESCHERICHIA COLI

TIAN XIA

(Under the Direction of Mark.A. Eiteman)

ABSTRACT

Substrate-selective uptake is a novel approach for the simultaneous consumption of sugars found in biomass hydrolysates. Of the five strains studied, E. coli W showed generally the fastest growth on carbohydrates. The triple-knockout strain W glk , ptsG manZ (KD777) consumed 7 g/L L-arabinose or D-xylose within 5.5 h, and subsequently consumed glucose only slowly. The presence of glucose did affect the utilization of D- galactose by KD777. Similar results were obtained with W glk , ptsG manZ crr (KD915).

Two-sugar carbon-limited chemostats with KD777 at a growth rate of 0.25 h -1 showed that including glucose in the feed slowed xylose, arabinose, galactose consumption by

8%, 20%, and 45% respectively. However, glucose was not consumed in the presence of xylose.

INDEX WORDS: Fermentation, Lignocellulose, Substrate-selective, Glucose

FERMENTATION OF SUGAR MIXTURES FOUND IN LIGNOCELLULOSIC

HYDROLYSATE

USING SUBSTRATE-SELECTIVE ESCHERICHIA COLI

TIAN XIA

B.S., Zhejiang University of Technology, China, 2008

A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial

Fulfillment of the Requirements for the Degree

MASTER OF SCIENCE

ATHENS, GEORGIA

2011

Tian Xia

FERMENTATION OF SUGAR MIXTURES FOUND IN LIGNOCELLULOSIC

HYDROLYSATE

USING SUBSTRATE-SELECTIVE ESCHERICHIA COLI

TIAN XIA

Major Professor: Mark. A. Eiteman Committee: Sudhagar Mani Yajun Yan

Electronic Version Approved:

Maureen Grasso Dean of the Graduate School The University of Georgia May 2011

ACKNOWLEDGMENTS

The author acknowledges the Consortium for Biotechnology Research (CPBR),

NSF, and the Georgia Experiment Station. The author appreciates the academic and technical assistance from the major professor, the committee, and the lab colleagues.

TABLE OF CONTENTS

Page

ACKNOWLEDGMENTS ...... iv

LIST OF TABLES ...... vi

LIST OF FIGURES ...... vii

CHAPTER

1 INTRODUCTION ...... 1

Platform Organisms ...... 4

2 MATERIALS AND METHODS ...... 15

3 RESULTS AND DISCUSSION ...... 19

Results ...... 19

Discussion ...... 35

4 PROSPECTIVE WORK ...... 41

REFERENCES ...... 43

APPENDIX ...... 55

LIST OF TABLES

Page

Table 1.1: Useful biocatalyst traits for efficient fermentation of lignocelluloses...... 4

Table 2.1: Strains used in this study...... 15

Table 3.1: Specific growth rates (h -1) of E. coli strains on different carbon sources...... 19

Table 3.2: Maximum Specific growth rates (h -1) of E. coli strains on different carbon

sources...... 29

Table 3.3: Steady-state growth of E. coli KD777 on monosaccharides and mixtures...... 33

LIST OF FIGURES

Page

Figure 1.1: Composition of lignocelluloses ...... 3

Figure 1.2: Central metabolic routes of glucose in E.coli ...... 8

Figure 1.3: Metabolism of xylose to pyruvate ...... 10

Figure 1.4: Metabolism of L-arabinose ...... 10

Figure 1.5: Carbon catabolic repression mechanisms in Escherichia coli ...... 12

Figure 3.1: Growth and substrate consumption of E. coli KD777 on a mixture of xylose

and glucose...... 21

Figure 3.2: Growth and substrate consumption of E. coli KD777 on a mixture of

arabinose and glucose ...... 22

Figure 3.3: Growth and substrate consumption of E. coli KD777 on a mixture of

galactose and glucose ...... 23

Figure 3.4: Growth and substrate consumption of E. coli KD777 on a mixture of

galactose with glucose addition ...... 24

Figure 3.5: Growth and substrate consumption of E. coli KD915 on a mixture of xylose

and glucose...... 26

Figure 3.6: Growth and substrate consumption of E. coli KD915 on a mixture of

arabinose and glucose ...... 27

Figure 3.7: Growth and substrate consumption of E. coli KD915 on a mixture of

galactose and glucose ...... 28

vii

Figure 3.8: Growth and substrate consumption of E. coli KD777 on a mixture of xylose

and glucose with xylose addition ...... 30

Figure 3.9: Growth and substrate consumption of E. coli KD777 on a mixture of

arabinose and glucose with arabinose addition ...... 31

viii

CHAPTER 1

INTRODUCTION

Increasing petroleum costs, together with our increasing dependency on crude oil imports, have provided an opportunity for bio-based fuels and chemicals to become economically competitive. Currently, 65% of the oil consumed in the United States is imported. Roughly half of the total U.S. energy consumption was automotive fuel in 2005

(over 211 billion gallons). With the development of new technologies, supplementing or replacing current petroleum-based automotive fuels with sustainable resources is now feasible (Jarboe et al., 2007).

Lignocellulosic biomass is by far the most abundant source of renewable energy in the world. This natural, abundant and potentially cheap feedstock exists primarily as agricultural waste (e.g., wheat straw, corn stalks, soybean residues, sugar cane bagasse), industrial waste (e.g., pulp and paper industry), forestry residues, and municipal solid waste (Wiselogel et al., 1996). Totaling 10–50 billion tons of estimated annual production, this complex substance accounts for approximately 90% of the dry weight of plant material, and about 50% of the biomass in the world (Claasen et al., 1999).

Numerous environmental and social benefits would accrue by the replacement of petroleum-based automotive fuels with biochemicals such as ethanol derived from lignocellulosic materials (Olsson et al., 1996).

Lignocellulose is composed of three main fractions (Figure 1.1): cellulose (~45% of dry weight), hemicelluloses (~30% of dry weight), and lignin (~25% of dry weight)

(Wiselogel et al., 1996). Cellulose, the most abundant polymer on earth, is composed of thousands of molecules of anhydroglucose linked by β(1,4)-glycosidic bonds. The basic repeating unit is the disaccharide cellobiose. The secondary and tertiary conformation of cellulose, as well as its close association with lignin, hemicellulose, starch, protein and mineral elements, makes cellulose hydrolysis-resistant. Cellulose must be hydrolyzed chemically by dilute or concentrated acid, or enzymatically. Hemicellulose is a highly branched heteropolymer containing primarily sugar residues: hexoses (D-galactose, L- galactose, D-mannose, L-rhamnose, L-fucose), pentoses (D-xylose, L-arabinose), and uronic acids (D-glucuronic acid) (Zaldivar et al., 2001). Hemicellulose is more easily hydrolyzed than cellulose (Brigham et al., 1996). The composition of hemicellulose depends on the source of the raw material (Wiselogel et al., 1996). Xylose is the dominant pentose comprising the hemicellulose of most hardwood feedstocks. For instance, birch wood (roth) contains 89.3% xylose and 1% arabinose (Saha, 2003).

Softwood oat spelt contains over 70% xylose and 10% arabinose (Saha, 2003). In contrast, arabinose can constitute a significant fraction of the pentose derived from various agricultural residues and other herbaceous crops being considered for use as dedicated energy crops. For example, wheat contains 65.8% xylose and 33.5% arabinose, corn fiber contains 48-54% xylose and 33-35% arabinose (Saha, 2003). Arabinose represents 10–20% of the total pentoses in many herbaceous crops such as switchgrass

(Mohagheghi et al., 2002). Lignin is the most abundant aromatic (phenolic) polymer in nature, being the dehydration product of three monomeric alcohols (lignols), trans -p- coumaryl alcohol, trans -p-coniferyl alcohol, and trans -p-sinapyl alcohol, derived from p- cinnamic acid (Kirk et al., 1977).

Cellulose Cellulose (glu) 20-50%

Hemicellulose (xyl, ara, man, glu, gal) 20-40% Pectin (polygalacturonate) 2-20%

Other Lignin 8% (aromatics) 10-20%

Figure 1.1. Composition of lignocelluloses.

The biochemical conversion of lignocellulosic biomass into products by fungi and bacteria has been well developed. As described above, the utilization of lignocellulose demands hydrolysis before fermentation, and unlike corn processing, from which glucose is the only significant product, lignocellulosic substrates yield both pentoses and hexoses and other compounds. Therefore, the subsequent microbial conversion process is significantly more complex. The process requires not only an efficient microorganism able to ferment a variety of sugars (pentoses and hexoses) but also one which tolerates stress conditions (Zaldivar et al., 2001). Bacterial and yeast strains have been constructed using metabolic engineering to contain many desirable traits that are advantageous for ethanol production from lignocellulose-derived mixed sugars (Table 1.1). After several decades of modification, evaluation and modification, three main microbial platforms

have emerged from pilot studies (Zaldivar et al., 2001): Saccharomyces cerevisiae,

Zymomonas mobilis , and Escherichia coli . The development of a microbial biocatalyst that is capable of metabolizing all of the constitutive sugars would simplify the process and reduce the cost of biochemical production from lignocellulose.

Table 1.1. Useful biocatalyst traits for efficient fermentation of lignocellulose (modified from Picataggio and Zhang 1996). GRAS means Generally Regarded As Safe, as defined by the United States Food and Drug Administration (FDA) (Zaldivar et al., 2001).

Essential traits Desirable traits

Broad substrate utilization range Simultaneous sugar utilization

High ethanol yields and productivity Hemicellulose and cellulose hydrolysis

Minimal byproduct formation GRAS status

High ethanol tolerance Recyclable

Increased tolerance to inhibitors Minimal nutrient supplementation

Tolerance to process hardiness a Tolerance to low pH and high temperature aTransient adverse condition such as change in pH and temperature and/or increase in salt, sugar, or ethanol concentration.

Platform Organisms

The yeast Saccharomyces cerevisiae is the most widely microorganisms used for the production of ethanol. S. cerevisiae has a thick cytoderm, high sterol content, and high ethanol and lignocellulosic hydrolysate tolerance (Tang et al., 2006). S. cerevisiae can convert glucose to ethanol rapidly with few by-products at a low pH under anaerobic conditions, which minimizes contamination of bacteria and viruses. Since the genomic sequence of S. cerevisiae has been completed (Goffeau et al., 1996), this organism may

be readily manipulated, and its metabolic network analyzed using bioinformatic and molecular biology tools. A significant disadvantage in applying S. cerevisiae for lignocellulosic hydrolysates is that this organism cannot utilize any pentoses as a carbon source (Ho et al., 1998).

In order for S. cerevisiae to consume xylose, genes associated with xylose consumption and transport must be incorporated into this organism. Much effort has been made to create xylose-assimilating strains. For example, the xylose isomerase gene derived from the anaerobic fungus Piromyces has been introduced into S. cerevisiae

(Kuyper, 2005). Successful ethanol production from xylose has also been achieved using recombinant S. cerevisiae strains with heterologous xylose reductase (XR) and xylitol dehydrogenase (XDH) from P. stipitis coupled with the overexpression of S. cerevisiae xylulokinase (XK) (Ho et al., 1998). A recent study showed increased ethanol yield and decreased xylitol yield by moderate Sc XK overexpression with high activities of Ps XR and Ps XDH, with no inhibitory effect on either growth or xylose consumption from XK overexpression (Matsushika and Sawayama, 2008). Thus, only finely tuned overexpression of XK in S. cerevisiae leads to improved ethanol formation from xylose

(Matsushika et al., 2009). Although these recombinant yeasts can convert xylose to ethanol, xylose consumption rates remain insufficient for ethanol production from lignocellulosic materials (Ho et al., 1999). Therefore, several sugar transporters have also been investigated to improve xylose consumption. Changes in the expression of hexose transporters affect xylose uptake (Sedlak and Ho, 2004). Natural xylose-assimilating yeasts, such as Pichia and Candida species, have xylose transport systems, and these strains efficiently transport xylose inside cells (Kilian, et al., 1993). P. stipitis xylose

transporter Sut1 has the highest V MAX for xylose among the Sut transporters and among hexose transporters (Weierstall et al., 1999; Hamacher et al., 2002). By introducing the

SUT1 gene from P. stipitis into a xylose-assimilating S. cerevisiae strain, xylose uptake is improved (Akihiko Kondo et al., 2008).

Genetic manipulations are also required for S. cerevisiae to utilize the other common pentose arabinose. Although some yeasts will oxidize L-arabinose for growth and others will convert it to polyols, Candida auringiensis is one of the few yeasts that ferment L-arabinose to ethanol (Dien et al., 1996, Jeffries and Shi, 1999). Arabinose utilization in S. cerevisiae has been enabled by expressing bacterial (Becker and Boles,

2003) or fungal (Richard et al., 2003) arabinose pathways. For example, efficient growth and fermentation of arabinose was achieved by high-level expression of the bacterial arabinose pathway consisting of Bacillus subtilis L-arabinose isomerase ( araA ), E. coli

L-ribulokinase ( araB ) and E. coli L-ribulose-5-P 4-epimerase ( araD ), together with the endogenous pentose-transporting permease gene GAL2 (Becker and Boles, 2003). Genes of the pentose phosphate pathway (RPE1, RKI1, TKL1, and TAL1) were also overexpressed (Wisselink et al., 2009), followed by selection for improved arabinose growth by sequential transfer in arabinose medium (Becker and Boles, 2003).

A key problem in the co-fermentation of more than one sugar is carbon-catabolite repression (CCR). Glucose and related sugars repress the transcription of genes encoding enzymes required for the utilization of alternative carbon sources. Some genes are also repressed by other sugars such as galactose (Saier et al., 1996). In S. cerevisiae , for instance, the presence of glucose (or even fructose) is sensed by the cell and triggers an intracellular regulatory cascade that activates Mig1p, a DNA-binding protein (Ronne,

1995; Johnston, 1999). Mig1p binds to the promoters of alternative sugar-utilization genes, thus blocking the expression of genes encoding metabolism of alternative carbon sources (sucrose, galactose, maltose, etc.). Because of this regulatory scheme, when S. cerevisiae grows in a medium containing a mixture of glucose and another naturally fermentable sugar (sucrose, galactose or maltose) the metabolism is diauxic.

The Gram-negative bacterium Escherichia coli is being considered for ethanol production because this organism is able to consume all the sugars in lignocellulosic hydrolysate. E. coli and several enteric bacteria naturally possess a broad substrate- utilization range, metabolizing hexoses (glucose, mannose, galactose, fructose), pentoses

(xylose and arabinose), and uronic acids (galacturonic acid, glucuronic acid). As a mixed- acid producer, E. coli converts three carbon intermediates pyruvate and phosphoenolpyruvate (PEP) into a mixture of ethanol, lactate, acetate, succiante and formate (Clark, 1989; Zaldivar et al., 2001).

E. coli metabolizes glucose by the Embden-Meyerhof-Parnas pathway to produce numerous intermediary metabolites and energy (Figure 1.2). Through the EMP pathway, the six-carbon glucose is ultimately split into two three-carbon compounds. Among the metabolites of the central metabolism, PEP plays a key role in cell physiology. PEP is a phosphate donor in the PEP:carbohydrate phosphotransferase system (PTS) which transports and phosphorylates glucose, is a direct precursor of several amino acids, and is converted to pyruvate with the generation of ATP by the action of either of two pyruvate kinases (Postma et al., 1996; Valle et al., 1996). PEP can also be carboxylated to oxaloacetate.

glucose g a lP glucose g lk

glucose-6P p g i fructose-6P p fk A

fructose-1,6P 2 fb a A

dihydroxy-acetone-phosphate tp iA +

glyceraldehyde-3P gapA

glyceraldehyde-1,3P 2 pg k

3P-glycerate gpmA

2P-glycerate eno

PEP PEPC p y k A CO p y k F 2 lactate LDH

pyruvate H2 CO PYC PFL formate 2

PTA ACK acetyl CoA acetate

ACDH

ADH oxaloacetate ethanol CS Fig ure 1. 2. Central metabolic routes malate citrate of glucose in E. coli , including the AceB Embden-Meyerhof-Parnas pathway fumarate glyoxylate AceA isocitrate and key metabolites as well as the AceA genes and enzymes2 involved in their succinate transformation.

succinate (ext)

E. coli metabolizes pentoses such as D-xylose and L-arabinose through the pentose phosphate (PP) pathway. These aldopentoses are usually first converted, respectively, into D-xylulose-5-phosphate and D-ribose-5-phosphate (Sprenger, 1995).

For utilization of D-xylose, two divergently transcribed operons, xylAB and xylFGHR are required (Song and Park, 1997). The xylA and xylB genes respectively encode D-xylose isomerase and xylulose kinase (Sofia et al., 1994). The xylFGHR operon transcribed from the P F promoter and the weak P R promoter includes genes for the high-affinity transport system and a regulator protein XylR (Song and Park, 1997). XylR acts as an activator for both operons in the presence of xylose (Song and Park, 1997). Four operons in E. coli are involved in utilization of L-arabinose. The genes araBAD encode enzymes functioning in the catabolism of arabinose (Englesberg et al., 1965). The araFGH and araE genes encode proteins responsible for the high-affinity and low-affinity transport of arabinose

(Brown and Hogg, 1972; Clark and Hogg, 1981). The araC gene encodes a protein that regulates expression of the other arabinose operons and its own synthesis (Englesberg et al., 1965; Casadaban, 1976). Figure 1.3 and Figure 1.4 respectively summarize the pathways for the catabolism of D-xylose and L-arabinose.

Figure 1.3. Metabolism of xylose to pyruvate. Values for ATP correspond to the uptake and conversion of six molecules of xylose into 10 molecules each of ethanol and CO 2 (Tao et al., 2001).

Figure 1.4. Metabolism of L-arabinose (Sakakibara et al., 2009).

Glucose repression (i.e., "Carbon catabolite repression" or CCR) also affects pentose consumption in E. coli when the cells are exposed to multiple sugars. Like S. cerevisiae , E. coli tends first to metabolize from a mixture of carbon sources the one that affords the highest growth rate. The IIA Glc protein plays a central role in this process.

When glucose is present in the medium, this protein is not phosphorylated and in this state it binds to various non-PTS permeases, inhibiting uptake of non-PTS sugars (Figure

1.5). Non-phosphorylated IIA Glc also binds to the enzyme glycerol kinase (GK), inhibiting its activity (Novotny et al., 1985). In addition, non-phosphorylated IIB Glc binds the Mlc repressor protein, relieving repression from genes ptsHI, ptsG, mlc, manXYZ and malT (Plumbridge, 2002). When glucose is absent from the culture medium, IIAGlc and

IIB Glc exist primarily in the phosphorylated states. Under this condition, IIA Glc ~P binds to the enzyme adenylate cyclase (AC) activating its cAMP biosynthetic capacity. The cAMP concentration therefore increases in the cell, and cAMP binds to the cAMP receptor protein (CRP) to induce catabolite-repressed genes (Korner et al., 2003). The protein IIB Glc ~P loses its capacity to bind Mlc, so this protein binds to its target operator sequences, causing repression of genes involved in glucose uptake (Plumbridge, 2002).

Proteins EI and Hpr also have regulatory functions. In its non-phosphorylated state, Hpr activates glycogen phosphorylase (GP), whereas Hpr~P has a similar effect on BglG, a transcriptional activator of the bgl operon that encodes proteins involved in β-glucosidic sugars uptake and utilization (Seok et al., 2001). Non-phosphorylated EI binds to the chemotaxis protein CheA, inhibiting its autophosphorylation and thus causing smooth swimming (Lux et al., 1995). In addition, protein IIA Glc exerts negative control of expression for the gene encoding the σS subunit of RNA polymerase (Lux et al., 1995).

Figure 1.5. Carbon catabolic repression mechanisms in Escherichia coli (Gosset, 2005).

In summary, the PTS forms a complex regulatory network involved in coordinating cellular processes related to the cell's capacity to find, select, transport and metabolize a large number of carbon sources. Therefore, genetic alterations to PTS components can have wide-ranging effects on cell physiology. Historically, previous modification of the

PTS has focused on reducing or eliminating CCR, often with the goal of improving pentose consumption.

One method to improve pentose consumption in the presence of glucose is by a knockout of the ptsG gene encoding enzyme IICB Glc of the phosphotransferase system

(PTS) for carbohydrate transport ( Postma et al., 1993 ). A ptsG mutation in an E. coli ethanol production strain reduces the glucose-mediated repression of xylose consumption

(Nichols et al., 2001). A previously described ptsG::Tn5 mutant IT1168 metabolized

xylose and arabinose simultaneously with glucose, rather than using glucose preferentially (Kimata et al., 1997). This altered pattern of sugar use was also observed when the mutant was transformed with a plasmid carrying the genes for ethanol production and used to ferment a sugar mixture (Nichols et al., 2001). Though removal of the ptsG improves xylose consumption in the presence of glucose, 40% of the xylose remained when the glucose was depleted (Dien et al., 2002).

Several decades of research have focused on the development of a single organism that can consume xylose and glucose simultaneously instead of sequentially as a means to achieve high productivities for a product. However, even if an organism could consume the two sugars simultaneously, the sugar consumption rates would likely have a specific "ratio". That is, a single organism which consumes glucose and xylose in a single, fixed ratio (and which consumes glucose faster than xylose) would result in only one sugar (i.e., xylose) being utilized for a large portion of the process (Eiteman et al.,

2008). Essentially, a single microorganism is not able to adjust the rate of consumption of two substrates in order to match variable sugar concentrations which might be found in lignocellulosic biomass (Eiteman et al., 2008). For example, in batch culture with the E. coli ethanologenic strain K011 grown on a hemicellulose hydrolysate after 24 h only 11% of the xylose was consumed, while 80% of the glucose was consumed (Barbosa et al.,

1992). Similarly, genetically engineered S. cerevisiae containing genes to consume xylose still consumed less than 25% of the xylose by the time glucose was depleted

(Sedlak et al., 2003). Furthermore, a single-organism approach can be unstable over time: for example, a chemostat study demonstrated that the presence of both sugars caused a gradual increase in the by-product acetate, which ultimately led to a 20% decrease in

ethanol yield (Dumsday et al., 1999). Finally, the metabolic pathways to convert a hexose into a desired product at optimal yield and productivity might not correspond to the optimal metabolic pathways to convert a pentose into the same product. Preferably, a process converting xylose and glucose simultaneously into any product would make these pathways independent of one another, with glucose metabolism not influencing xylose metabolism and vice versa (Eiteman et al., 2008).

A new approach for the consumption of sugar mixtures is to use a "consortium" of the same species of microorganisms. This approach involves introducing into a mixed substrate stream several otherwise identical strains which each are able to consume only one particular substrate. Each strain will therefore effectively ignore other substrates while it carries out the one target conversion (Eiteman et al., 2008). An advantage of such

"substrate-selective uptake" is that the system can adapt to fluctuations in the feed stream; that is, cultures can grow in concert with a variable feed composition. In a study combining the xylose-selective (glucose deficient) strain E. coli ALS1073 with the glucose-selective (xylose deficient) strain E. coli ALS1074 glucose and xylose were simultaneously converted into lactate. By delaying the inoculation of one strain, the glucose and xylose consumption rates can be matched for optimal efficiency (Eiteman et al., 2009). Presumably additional metabolic engineering strategies could focus on improving the individual production strains independently. For example, the glucose- selective strain could be improved for the generation of a particular product without having to compromise on how those changes might impact the conversion of xylose to that product.

CHAPTER 2

MATERIALS AND METHODS

Strains

Escherichia coli strains were used in this study (Table 2.1). KD777 and KD915 were derived from W.

Table 2.1. Strains used in this study.

Strain Genotype Source

W Wild type ATCC 9637

B Wild type ATCC 11303

C Wild type ATCC 8739

MG1655 F-λ- rph -1 (wild type) CGSC 6300

KD777 W ptsG ::tet glk ::kan manZ ::cam

KD915 W ptsG ::FRT manZ ::cam glk ::FRT crr ::FRT

Growth Conditions

The basal medium used in all experiments contained (per L): 13.3 g KH2PO 4, 4.0 g (NH 4)2HPO 4, 1.2 g MgSO 4·7H 2O, 13.0 mg Zn(CH 3COO) 2·2H 2O, 1.5 mg CuCl 2·2H 2O,

15.0 mg MnCl 2·4H 2O, 2.5 mg CoCl 2·6H 2O, 3.0 mg H 3BO 3, 2.5 mg Na 2MoO 4·2H 2O, 100 mg Fe(III)citrate, 8.4 mg Na 2EDTA·2H 2O, 1.7 g citric acid, 4.5 mg thiamine·HCl, and 3-

7 g sugars (D-(+)-glucose, D-(+)-xylose, L-(+)-arabinose, D-(+)-galactose) as indicated in the results. Antibiotics appropriate for the particular strains were added at the following concentrations: 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, 36 mg/L kanamycin. The medium was adjusted to a pH of 7.0 with 20% NaOH.

Batch Cultures

For batch cultures, bacteria were first grown at 37°C with agitation of 250 rpm

(19 mm pitch) in 500 mL shake flasks containing 100 mL basal medium with 7 g/L of a single sugar. When the OD of this culture reached approximately 3, the flask contents were diluted with fresh basal medium so that 100 mL having an effective OD of 1.5 was used to inoculate the bioreactor containing 0.9 L basal medium with either 7 g/L of a single sugar or a sugar in the presence of glucose (both at a concentration of 7 g/L). Batch experiments were carried out in a 2.5 L bioreactor (Bioflo 2000, New Brunswick

Scientific Co., Edison, NJ) maintained at 37°C with air sparged at a flow rate of 1.0

L/min, an agitation of 500 rpm, and the pH controlled at 7.0 using 20% (w/v) NaOH.

A comparison of specific growth rates was completed by first growing cells at

37°C with agitation of 250 rpm (19 mm pitch) in 250 mL shake flasks containing 30 mL basal medium with 5 g/L of a single sugar. When the OD of this culture reached approximately 1, 5 mL was transferred to a second shake flask containing 45 mL basal

medium with 5 g/L of a single sugar from which optical density was measured at 0.5 –

1.0 h intervals to determine growth rates.

Chemostat

Steady-state cultures were carried out as chemostats in the same 2.5 L vessels used for batch cultures. After an initial batch phase, the medium was continuously supplied to achieve a dilution rate of 0.25 h -1. The volume was maintained at 1.0 L, and steady-state was attained after 5 residence times (20 h). Six carbon-limited cultures were completed with each carbon source present at 3 g/L: xylose, arabinose, galactose, and each of these sugars with glucose. A nitrogen-limited steady-state culture was conducted containing 5 g/L xylose, 3 g/L glucose and 0.50 g/L (NH 4)2HPO 4, with all other components unchanged. This medium composition resulted in a xylose C:N mass ratio of

22.6 (and total C:N mass ratio of 33.9). For these experiments, the pH was maintained at

7.0 using 20% NaOH, the temperature was maintained at 37°C, air was sparged at 0.5

L/min, and the agitation was 500 rpm to prevent oxygen limitation.

Analyses

For batch experiments, the OD measured at 600 nm (DU-650 spectrophotometer,

Beckman Instruments, San Jose, CA) was used to monitor cell growth, and this value was correlated to dry cell mass. For chemostat experiments, 20 mL of culture were used in triplicate to measure dry cell mass directly after oven drying 24 h at 60°C. An HPLC method was used to quantify organic compounds including glucose, xylose, arabinose, and galactose (Eiteman and Chastain, 1997). A Shimadzu HIC-6A ion-chromatography system with a 5 µL sample loop was used in this work. The UV detector was operated at

210 nm, and the flowrate of the eluent was 0.6 mL/min. A Coregel 64H (Interaction

Chromatography, San Jose, CA) ion-exclusion column (300×7.8 mm i.d.) of 10µm particle size was used in the analyses. The column is composed of sulfonated polystyrene divinyl benzene, providing an exchange capacity of 3.2 meq/g. The column was protected by a Coregel 64H guard column and was routinely stored in 1 mN H 2SO 4. The eluent was composed of sulfuric acid (Fisher Scientific, Fair Lawn, USA) and water deionized through a Modulab Analytical UF Polishing System to a conductivity of 18 M cm

(Continental Water Systems, Atlanta, Georgia, USA). Eluents having 7.0-16.0 mN H2SO 4 concentration were prepared daily and vacuum filtered through a 0.45 µm nylon filter

(Micron Separations, Westboro, USA). Standards from the stock solution and fermentation samples were diluted with deionized water if necessary and filtered through

0.45 µm nylon Acrodisc filters (Gelman Science, Ann Arbor, Michigan, USA) prior to injection. By measuring five identical samples of a single glucose solution, the relative error was obtained as 0.06%, which indicated the analytical variance of the HPLC.

CHAPTER 3

RESULTS AND DISCUSSION

Results

Growth rate comparison

Four different E. coli strains were used to compare their growth rates on each of the five monosaccharides found in lignocellulosic hydrolysates. Each of the growth rates was measured twice and the standard deviation was shown in the table. Generally K-12 derivative E. coli MG1655 showed lower growth rates on monosaccharides than strains

W, C and B (Table 3.1). E. coli W was selected as our platform organism.

Table 3.1. Specific growth rates (h -1) of E. coli strains on different carbon sources.

Substrate W s.d. B s.d. C s.d. MG1655 s.d.

D-Xylose 0.83 0.02 0.77 0.01 0.89 0.01 0.57 0.00

D-Glucose 0.98 0.02 0.95 0.03 0.95 0.01 0.72 0.00

L-Arabinose 0.93 0.02 0.86 0.00 0.92 0.01 0.66 0.01

D-Mannose 0.46 0.00 0.33 0.00 0.40 0.07 0.27 0.01

D-Galactose 0.64 0.02 0.78 0.02 0.71 0.05 0.33 0.01

Batch Growth using KD777 (original data were shown in appendix)

The development of a strain that is unable to consume glucose would be a valuable component of a consortium approach for simultaneous consumption of sugars found in lignocellulosic hydrolysates. KD777 has mutations in the ptsG , manZ and glk genes, a set of genes which has been described as preventing growth on glucose (Curtis and Epstein, 1975). In a controlled batch process KD777 consumed 7 g/L xylose completely in 5.5 h with a maximum specific growth rate of 0.81 h -1, essentially the same growth rate of 0.84 h -1 observed when the parental strain, E. coli W was grown in this medium. In the presence of both xylose and glucose, xylose was again completely consumed in about 5.5 h, during which time essentially no glucose was consumed (Figure

3.1). In the presence of glucose the maximum growth rate during xylose consumption was 0.78 h -1. After xylose was exhausted, glucose was slowly consumed during the subsequent 18 h, and the specific growth rate during glucose consumption was 0.03 h -1.

Analogous studies using KD777 were conducted in an arabinose medium. As the sole carbon source, 7 g/L arabinose was completely consumed in less than 5 h with a maximum specific growth rate of 0.84 h -1, slightly less than the growth rate of 0.94 h -1 observed for wild-type W. In a medium containing both arabinose and glucose, arabinose was consumed in 5 h at a mean growth rate of 0.81 h -1, and glucose was consumed slowly with a growth rate of 0.09 h -1 only after arabinose was depleted (Figure 3.2). Because the lag between arabinose utilization and glucose utilization was much greater than observed during growth on a xylose/glucose mixture, we replicated the arabinose/glucose batch experiment several times and also increased the concentration of the three antibiotics with essentially identical results.

10 6

4 OD 1

2 Xylose, Glucose (g/L) Xylose,

0.1 0 0 5 10 15 20 25 Time (h)

Figure 3.1. Growth and substrate consumption of E. coli KD777 on a mixture of xylose and glucose. OD: ; xylose: ; glucose: .

10 6

4 OD 1

2 Arabinose, Glucose (g/L) Arabinose,

0.1 0 0 10 20 30 40

Time (h)

Figure 3.2. Growth and substrate consumption of E. coli KD777 on a mixture of arabinose and glucose. OD: ; arabinose: ; glucose: .

Growth of KD777 on galactose and glucose mixtures was different from growth on the other two mixtures. In a medium containing galactose, this carbon source was consumed in less than 12 h with a mean specific growth rate of 0.42 h -1, significantly lower than the growth rate of 0.66 h -1 observed for E. coli W using galactose as the sole carbon source. In a medium containing both galactose and glucose, only 0.25 g/L galactose was consumed in the first 12.5 h. Then, the remaining galactose was consumed

over the next 9 h at a growth rate of 0.30 h -1. About 1.6 g/L glucose was consumed simultaneously with galactose from 17.5 h to 21.5 h, and the remaining glucose was consumed after galactose was depleted (Figure 3.3). In order to verify that glucose affected galactose consumption, another type of batch experiment was conducted. For this experiment, the culture was initiated on a medium containing galactose alone. When the OD reached about 1.0, approximately 6 g/L glucose was added. In this case, the initial

10 6

4 OD 1

2 Galactose, Glucose (g/L) Glucose Galactose,

0.1 0 0 5 10 15 20 25 30 Time (h)

Figure 3.3. Growth and substrate consumption of E. coli KD777 on a mixture of galactose and glucose. OD: ; galactose: ; glucose: . 7 g/L glucose and 7 g/L galactose initially.

growth rate of the cells on galactose alone was about 0.47 h -1, similar to the previous experiment with galactose as the sole carbon source. Although no lag phase was observed after the glucose was added, the growth rate on galactose shifted immediately to 0.26 h -1 and about one-third of the glucose was consumed in parallel with the galactose (Figure

3.4). Once galactose was depleted, the remaining glucose was consumed at a specific growth rate of 0.08 h -1.

-1 h µ = 0.08 10 -1 8 h 26 0. = µ 6 OD 1 -1 4 h 7 .4 0 = µ 2 Galactose, Glucose (g/L) Glucose Galactose,

0.1 0 0 5 10 15 20

Time (h)

Figure 3.4. Growth and substrate consumption of E. coli KD777 on a mixture of

galactose with glucose addition. OD: ; galactose: ; glucose: . 7 g/L galactose

initially with addition of 7 g/L glucose at 5 h.

Batch Growth using KD915

KD777 has a mutation in the ptsG gene encoding for the membrane-bound

Enzyme IIBC component of the glucose phosphotransferase uptake system. We were interested to determine whether a deletion in the Enzyme IIA component of the glucose uptake system in E. coli would further diminish glucose consumption in the presence of other monosaccharides. Therefore strain KD915 (W ptsG manZ glk crr ) was constructed and grown in a controlled bioreactor using various single carbohydrate and carbohydrate mixtures.

When KD915 was grown on xylose as the sole carbon source, this strain attained a specific growth rate of 0.65 h -1, about 20% lower than observed for KD777. In the presence of both xylose and glucose, xylose was consumed first at a growth rate of 0.62 h-1 (Figure 3.5). After xylose was exhausted, glucose was slowly consumed during the subsequent 18 h, and the specific growth rate during glucose consumption was 0.03 h -1.

10 6

4 OD 1

2 Xylose, Glucose (g/L) Xylose,

0.1 0 0 5 10 15 20 25 Time (h)

Figure 3.5. Growth and substrate consumption of E. coli KD915 on a mixture of xylose and glucose. OD: ; xylose: ; glucose: .

Similarly, arabinose as the sole carbon source was consumed by KD915 with a specific growth rate of 0.64 h-1, about 20% lower than KD777. In a medium containing both arabinose and glucose, arabinose was consumed at a mean growth rate of 0.64 h -1, and glucose was consumed slowly with a growth rate of 0.06 h -1 only after arabinose was depleted (Figure 3.6). A lag period between arabinose utilization and glucose utilization for KD915 was shorter than the lag period observed for KD777 (Figure 3.2).

10 8

OD 1 4

2 Arabinose, Glucose Glucose (g/L) Arabinose,

0.1 0 0 5 10 15 20 25 30 Time (h)

Figure 3.6. Growth and substrate consumption of E. coli KD915 on a mixture of arabinose and glucose. OD: ; arabinose: ; glucose: .

In a medium containing galactose, the sole carbon source was consumed by

KD915 in less than 8 h with a mean specific growth rate of 0.52 h-1. When both galactose and glucose were present in the medium, galactose was consumed in the first 10.25 h.

The majority of glucose was consumed simultaneously with galactose from 6 h to 10.25 h

(Figure 3.7). Growth rate on the mixture was 0.48 h -1.

8 10 6

4 OD 1

2 Galactose, Glucose (g/L) Glucose Galactose,

0.1 0 0 5 10 15 20 Time (h)

Figure 3.7. Growth and substrate consumption of E. coli KD915 on a mixture of galactose and glucose. OD: ; galactose: ; glucose: .

MG1655 and MG1655 crr Growth Rates

One unexpected result in comparing KD777 and KD915 was that the one additional knockout of the crr gene significantly reduced the growth rate of the microorganisms on xylose or arabinose. In order to determine whether a crr knockout alone affected growth on these pentoses, we constructed MG1655 crr and compared growth rates of this strain with wild type MG1655 in shake flask experiments. The maximum specific growth rate of MG1655 crr was 22% and 33% lower than the specific growth rate of MG1655, respectively, on xylose and arabinose (Table 3.2). Interestingly,

with galactose as the sole carbon source, the specific growth rate of MG1655 crr was

33% greater than the specific growth rate of MG1655.

Table 3.2. Maximum Specific growth rates (h -1) of E. coli strains on different carbon sources. (Each of the growth rates was measured twice.)

Strain Xylose s.d. Arabinose s.d. Galactose s.d.

MG1655 0.57 0.00 0.66 0.01 0.33 0.01

MC1655 crr 0.44 0.01 0.44 0.04 0.44 0.01

Batch Growth of KD777 with additional pentose

Results with batch growth of KD777 or KD915 in xylose-glucose or arabinose- glucose mixtures suggested that the onset of glucose consumption did not occur until the pentose was depleted. We sought to verify whether the presence of pentose effectively hindered glucose metabolism in KD777 by providing the culture with additional pentose after the initial quantity of pentose had been exhausted. For the case of a xylose-glucose mixture, we repeated the previous experiment, but added xylose to a concentration of about 7 g/L six hours after xylose was depleted, when the glucose concentration was approximately half its initial concentration (Figure 3.8). The additional xylose was consumed within 3 h, and during the initial part of that interval the glucose concentration remained unchanged. In fact, during the time when the xylose was just being depleted the glucose concentration increased from about 3 g/L to 4 g/L. After that additional dose of xylose had been exhausted, glucose consumption resumed and at a rate slower than the rate observed prior to the xylose addition.

10 8

OD 1 4

2 Xylose, Glucose (g/L) Xylose,

0.1 0 0 10 20 30 40

Time (h)

Figure 3.8. Growth and substrate consumption of E. coli KD777 on a mixture of xylose and glucose. Xylose was reintroduced into the medium after the initial xylose had been exhausted. OD: ; xylose: ; glucose: .

For the case of an arabinose-glucose mixture, approximately 7 g/L arabinose was added about 20 h after arabinose was depleted, when the glucose concentration had reached approximately half its initial concentration (Figure 3.9). This additional arabinose was consumed in two hours, and during that time the glucose concentration at first remained the same and then increased from about 3.5 g/L to 4.2 g/L. After that additional dose of arabinose had been exhausted, glucose consumption resumed at approximately the same rate observed prior to the arabinose addition.

10 8

OD 1 4

2 Arabinose, Glucose (g/L) Glucose Arabinose,

0.1 0 0 10 20 30 40 Time (h)

Figure 3.9. Growth and substrate consumption of E. coli KD777 on a mixture of arabinose and glucose. Arabinose was reintroduced into the medium after the initial arabinose had been exhausted. OD: ; arabinose: ; glucose: .

Carbon-Limited Steady-State Growth

In order to examine further the consumption of glucose supplied with other monosaccharides, we also conducted two-sugar carbon-limited chemostats using a growth rate of 0.25 h -1 for xylose, arabinose, galactose and their glucose mixtures.

Because these specific growth rates are far above the µMAX observed for growth on glucose alone by these strains but below the µMAX on arabinose, xylose or galactose, we expected that the microorganisms would only be able to consume a portion of the glucose

despite the system being carbon-limited . Because the presence of the crr knockout conferred insignificant additional reduction in glucose consumption, we used KD777 for these studies (Table 3.3).

Table 3.3. Steady-state growth of E. coli KD777 on monosaccharides and mixtures.

Nutrient Dilution Rate qS qG YCells/S Carbon Source(s) Limitation (h -1) (mmol/g·h) (mmol/g·h) (g/g)

Arabinose C 0.25 3.77 N/A 0.44

Arabinose + Glucose C 0.25 2.98 0.29 0.56

Xylose C 0.25 4.13 N/A 0.40

Xylose + Glucose C 0.25 3.80 0.43 0.44

Galactose C 0.25 3.67 N/A 0.45

Galactose + Glucose C 0.25 2.03 1.56 0.82

Xylose + Glucose N 0.25 4.94 -0.20† 0.34

qS: specific consumption rate of primary monosaccharide substrate (i.e., arabinose, xylose, or galactose) qG: specific glucose consumption rate

YCells/S: observed mass yield coefficient of biomass/primary monosaccharide

† Glucose was generated, and hence the consumption rate is negative.

For xylose metabolism, the presence of glucose slowed xylose consumption by

8%, and increased biomass yield (calculated on the basis of xylose) by 9%, indicating that essentially glucose replaced the xylose. For arabinose and galactose metabolism, the presence of glucose slowed the other monosaccharide uptake by 20% and 45%, respectively. In the mixture of xylose and glucose the ratio of monosaccharide consumption (X:G) was 8.8, and in a mixture of arabinose and glucose the ratio of

monosaccharide consumption (A:G) was 10.3, while in a mixture of galactose and glucose the ratio of monosaccharide consumption (Gal:G) was 1.3. Thus, given the dry mass of a single E. coli cell of 2.9 × 10 -13 g (Neidhardt et al., 1996), for each cell 200,000 xylose molecules were consumed per second to maintain a growth rate of 0.25 h -1, while in a mixture of xylose and glucose, 184,000 xylose molecules and 21,000 glucose molecules were consumed per second to maintain that growth rate. In comparison, for arabinose 183,000 molecules per second were consumed, while in an arabinose-glucose mixture, 145,000 arabinose molecules and 14,000 glucose molecules per second were consumed to maintain that growth rate. For galactose, 178,000 molecules per second were consumed, while in a galactose-glucose mixture, 98,000 galactose molecules and

76,000 glucose molecules per second were consumed to maintain that growth rate.

Nitrogen-Limited Steady-State Growth

Batch studies with KD777 using mixtures of xylose-glucose or arabinose-glucose suggested that glucose consumption did not occur until pentose was exhausted (Figures

3.1 and 3.2). Indeed, adding pentose back into the bioreactor after the initial quantity was exhausted not only curtailed glucose consumption, but this additional pentose appeared to lead to some glucose formation (Figures 3.8 and 3.9). In contrast, during the carbon- limited chemostats both pentose and glucose were consumed (Table 3.2). However, despite the continuous feeding of glucose and one of these two pentoses, the cells grew in the absence of pentose because the process was carbon-limited. We therefore sought to conduct a different steady-state process to determine whether xylose and glucose would be consumed simultaneously when xylose was present. To accomplish this condition, a nitrogen-limited chemostat with KD777 at a growth rate of 0.25 h -1 was conducted (Table

3.2). In this case, the specific xylose consumption was 4.94 mmol/g·h, an increase of 20% over the xylose consumption rate observed during the carbon-limited chemostat.

Interestingly, under nitrogen-limited growth the cells showed a specific glucose production rate of 0.20 mmol/g·h. The biomass yield (calculated on the basis of xylose) decreased by 15%. In this steady-state process, each cell consumed 240,000 xylose molecules and generated 9700 glucose molecules to maintain a growth rate of 0.25 h -1.

Discussion

E. coli KD777 and KD915 displayed the reverse of the typical diauxic growth.

That is, in a mixture of either arabinose or xylose and glucose, they metabolized the pentose first. The three genes ptsG , manZ and glk absent from these strains play important roles in glucose uptake (Curtis and Epstein, 1975). The ptsG gene encodes the

Enzyme IICB Glc of the glucose phosphotransferase system (Postma et al., 1993), and is quite specific for D-glucose (GPT). The manZ gene encodes the IID Man domain of the mannose PTS permease, which refers to mannose phosphotransferase (MPT) (Huber and

Erni, 1996) and is able to phosphorylate glucose and mannose, as well as their derivatives altered at the C2 position (Curtis and Epstein, 1975). The glk gene encodes glucokinase

(Curtis and Epstein, 1975), an enzyme which phosphorylates glucose in the cytoplasm using ATP in strains devoid of the PTS genes (Flores et al., 1996). Mutants lacking both

GPT and MPT grow very slowly on glucose, while E. coli lacking GPT, MPT and glucokinase previously were deemed unable to grow on glucose (Curtis and Epstein,

1975). Our study demonstrates that E. coli W with ptsG manZ glk or ptsG manZ glk crr mutations was indeed able to grow on glucose if the organism had grown first on another

monosaccharide. Furthermore, these three knockouts eliminated glucose-mediated repression of pentose consumption.

Although glucose consumption was largely curtailed until the pentose was exhausted, both KD777 and KD 915 were ultimately able to consume glucose. How did these organisms metabolize glucose in the absence of these key genes?

In order to metabolize glucose, the strains KD777 and KD915 first had to transport glucose. In fact, E. coli has several permeases that can translocate glucose, including the mannose-PTS system, ß-Methylgalactoside (Mgl) permease galactose transporter, the maltose affinity receptor LamB, as well as the galactose permease GalP

(Flores et al., 2005). In E. coli ptsH ptsI crr GalP replaces the transport functions of

IICB Glc (Flores et al., 2002). The Mgl system was initially identified as a high affinity galactose transport system (Ganesan and Rotman, 1965; Kalckar, 1971; Lengeler et al.,

1971). Mgl has a high affinity for both galactose and glucose via its periplasmic glucose/galactose binding protein, whose structure in the presence of ligands is well- defined and which has a K d of 0.2 µM for glucose (Vyas et al., 1991). The Mgl system contributes significantly to the growth and transport affinity for glucose at low extracellular glucose concentrations. Indeed under glucose limitation, Mgl can at least partially supplant the PTS for growth on glucose. The sugar-binding site of LamB has a weak but measurable affinity for glucose and other mono- and disaccharides (Benz et al.,

1987) which could facilitate the permeation of these substrates across the outer membrane. LamB functions as a broad specificity glycoporin under conditions of carbohydrate limitation (Death et al., 1993). Moreover, E. coli adapts to knockouts in phosphotransferase genes by elevating the expression of other genes responsible for

glucose and even secondary carbon sources uptake. For instance, the expression of galP, mglB and lamB increased as a result of ptsHI crr knockouts (Flores et al. 2005).

Similarly, transcript levels of galP, glk and pgi (the gene coding for phosphoglucose isomerase) in a ptsHI crr mutant called PB11 were respectively 13-, 2.2- and 6.6-fold higher than in the wild-type, and when glucose was the carbon source, PB11 upregulated rpoS , poxB, acs and ackA genes. As a result, PB11 showed improved capacity to consume acetate and glucose simultaneously (Flores et al. 2005). KD777 and KD915 likely retained the ability to transport glucose through any of these broad transport proteins.

In addition to transporting glucose across the cell membrane, however, strains

KD777 and KD915 also phosphorylated glucose in the absence of ptsG , manZ , glk and crr genes. Since glucose was blocked by the presence of xylose or arabinose, xylulokinase or L-ribulokinase may have phosphorylated glucose in the absence of their natural substrates, xylulose or ribulose. Xylulokinase and glucokinase are members of the hexokinase family of sugar kinases and share structural similarities (Bork et al., 1993).

Xylulokinase has activity with diverse substrates including xylitol, D-ribulose and D- arabitol (Di Luccio et al., 2007). Laboratory evolution of a strain of Pseudomonas putida expressing E. coli xylulokinase (and xylose isomerase) yielded a strain able to utilize L- arabinose as efficiently as D-xylose (Meijnen et al., 2008). L-ribulokinase, the key phosphorylating enzyme in the metabolism of L-arabinose, similarly shows activity toward several substrates and can phosphorylate all 2-ketopentoses (Lee et al., 2001).

Xylulukinase and L-ribulokinase might be responsible for glucose phosphorylation in the absence of glucose PTS system.

The comparison between KD777 and KD915 batch fermentations as well as the growth rates comparison experiments with MG1655 and MG1655 crr both led to the question why the crr gene knockout impacts pentose consumption. This phenomenon probably could be attributed to a low rate of cyclic AMP (cAMP) synthesis. In addition to their role in transport, some of the PTS proteins have also been implicated in the regulation of cell metabolism. The regulatory role of the PTS is illustrated by the pleiotropic nature of mutants defective in one or more components of the PTS. E. coli or

S. typhimurium mutant strains which lack enzyme I or HPr or both do not grow on a number of non-PTS sugars, including glycerol, melibiose, maltose, and lactose (Scholte and Postma, 1980). In E. coli the PTS is also involved in regulation of adenylate cyclase activity. The IIA Glc protein encoded by crr, which plays a central role in CCR, employs the phosphorylated states as glucose is absent from the culture medium. Upon binding of

IIA Glc ~P to the enzyme adenylate cyclase (AC), the cell synthesizes cAMP, which in turn binds to the cAMP receptor protein (CRP) and induces catabolite-repressed genes

(Korner et al., 2003). Indeed, the cAMP-CRP complex acts mainly as a transcriptional activator regulating more than a hundred genes and operons in E. coli (Busby and Kolb,

1996; Gosset et al., 2004). The absence of IIA Glc in KD915 would be expected to have reduced adenylate cyclase activity and cAMP concentration, and consequently repress the synthesis of the enzymes needed for the catabolism of the pentoses. Krin et al. (2002) demonstrated a crr – strain shows reduced cAMP levels because of the absence of IIA Glc

-mediated activation of the adenylate cyclase. When crr expression is regulated, a twofold reduction in the global amount of IIA Glc is sufficient to significantly decrease adenylate cyclase activity and the cAMP level (Krin et al., 2002). Similarly, S.

typhimurium crr mutants did not grow on several non-PTS compounds, including xylose, citrate, succinate, and malate, a deficiency which could be overcome by the addition of external cAMP (Scholte and Postma, 1980). Hence, in our study, the decreased growth rates of the crr knockouts on xylose and arabinose may be a result of relatively low cAMP level.

A remarkable result of these studies is that glucose is generated when xylose alone (or arabinose) was being consumed. Several enzymes might be responsible for this phenomenon. Glucose 6-P isomerase (phosphoglucose isomerase), which is located at the first juncture of the Embden-Meyerhof-Parnas (EMP) pathway, interconverts F-6-P and

G-6-P in the cytoplasm (Hua et al., 2003). Phosphoglucomutase catalyzes the reversible transformation of glucose 1-phosphate into glucose 6-phosphate via the enzyme-bound intermediate, glucose 1,6-bisphosphate (Ray and Peck, 1972), hence it can be used either to feed the pool of internal G-1-P from G-6-P or vice-versa. Glucose 1-phosphatase has broad substrate specificity for phosphorylated compounds but demonstrates its highest activity toward glucose-1-phosphate (Lee et al., 2003). Fructose 6-P is one product of the pentose phosphate pathway, and this compound could be converted to glucose 6- phosphate by glucose 6-P isomerase (encoded by pgi ), to glucose 1-P by phosphoglucomutase ( pgm ) and finally to glucose by glucose 1-phosphatase (agp ).

Alternatively, if xylulokinase does indeed phosphorylate glucose, it is possible that this same enzyme dephosphorylates one of these phosphoglucoses derived from fructose 6-P.

A previous study showed that yeast hexokinase was able to convert glucose-6-phosphate, mannose-6-phosphate and fructose-6-phosphate into glucose, mannose and fructose,

respectively (Viola et al., 1982). Once formed through any one of these mechanisms, glucose could not be metabolized because of the presence of xylose.

The mechanism regarding why the mere presence of glucose retarded D-galactose consumption is not clear. Previous study showed that in carbon-limited chemostat cultures, in which the steady-state substrate concentrations are very low, mixtures of

‘‘diauxic’’ carbon substrates are utilized simultaneously (Lendenmann and Egli, 1998).

When the bacterium was cultivated at dilution rates between 0.3 and 0.7 h-1 by supplying a 1:1 mixture of glucose and galactose, the two sugars were utilized simultaneously, and the steady-state concentration of a particular sugar during growth with mixtures of glucose and galactose was even proportional to its ratio in the medium feed (Lendenmann et al., 1996). Since the sugar concentrations are not high in our study and glucose and galactose might be competitively binding to permeases such as GalP, both of the two sugars were possibly transported into the cytoplasm slowly, so that glucose was competent to be consumed in galactose medium simultaneously.

CHAPTER 4

PROSPECTIVE WORK

The prospective work with respect to the aforementioned research would probably focus on the following aspects:

Figuring out the means that strains KD777 and KD915 phosphorylated glucose in the absence of ptsG, manZ, glk and crr genes. According to the accomplished experiments, additional genes encoding xylulokinase ( xylB ), ribulokinase ( araB ), and galactokinase ( galK ) could be knocked out so as to see whether glucose consumption is able to be further eliminated. Alternatively, it might help to incorporate plasmid with xylB, araB, or galK genes into the cells and overexpress xylulokinase, ribulokinase, or galactokinase in order to find out whether glucose phosphorylation would be improved in the glucose deficient strains. Furthermore, enzyme assay using purified xylulokinase, ribulokinase, and galactokinase could be conducted to investigate their activity on glucose phosphorylation. In addition, since different behaviors were observed among xylose, arabinose and galactose, we can study glucose consumption in the presence of different carbon sources, such as fructose, glycerol or non-PTS carbon source succinate.

Investigating the mechanism of glucose generation when xylose (or arabinose) alone was being consumed is also of great interest. One of the plans on the list is to repeat pentose addition batch experiment using D-[1-13C]-xylose. 13C-NMR isotopomer distribution analysis could be applied to estimate metabolic fluxes through biochemical reaction networks. We might be able to figure out whether glucose was generated from

pentose metabolism and to reveal the pathway and the mechanism of the glucose formation.

It’s worthwhile to try sugar mixture fermentation with multi-culture. Although glucose consumption hasn’t been excluded completely, the glucose deficient strains were able to utilize only pentose within a period of time. A set of E. coli knockouts could be substrate-selective by virtue of appropriate gene knockouts combinations. When growing in a sugar mixture, the consortium of strains will selectively consume arabinose, xylose and glucose. Furthermore, by conducting dual-phase sugar mixture fermentation, the three sugars will be effectively converted into various products such as succinate.

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APPENDIX

ARA 4/18/2009 Date A Reference KD777 Strain Park media with arabinose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with arabinose Inoculum Media After 8.0 hours growing in flask Time Inoculated 3.0 OD at transfer 46ml inoculum (OD600nm=3.0)+54 ml solution A Volume Inoculum 20% NaOH Base Antibiotics 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, and 36 mg/L kanamycin.

TIME OD ARABINOSE GLUCOSE PH ln(OD) (h) 600nm (g/L) (g/L) 0.0 0.19 7.42 0.00 7.00 -1.65 1.0 0.40 6.93 0.00 -0.91

2.0 0.87 6.27 0.00 -0.14

3.0 2.11 4.92 0.01 0.75

4.0 4.90 1.53 0.03 1.59

5.0 7.53 0.00 0.03 2.02

6.0 8.33 0.00 0.02 2.12

8.5 7.55 0.00 0.02 7.26 2.02 11.0 7.16 0.00 0.01 7.27 1.97 13.5 6.66 0.00 0.00 7.28 1.90 16.0 6.57 0.00 0.00 1.88

18.5 6.01 7.29 1.79

21.0 6.12 7.30 1.81

23.5 6.11 7.30 1.81

ARA-GLU-1

Date 4/18/2009

B Reference KD777 Strain Park media with arabinose and glucose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with arabinose Inoculum Media After 8.0 hours growing in flask Time Inoculated 3.0 OD at transfer 46ml inoculum (OD600nm=3.0)+54 ml solution A Volume Inoculum 20% NaOH Base Antibiotics 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, and 36 mg/L kanamycin.

TIME OD ARABINOSE GLUCOSE PH ln(OD) (h) 600nm (g/L) (g/L) 0.0 0.17 7.43 7.32 7.00 -1.76 1.0 0.33 6.94 7.30 -1.10

2.0 0.80 6.23 7.24 -0.22

3.0 1.92 5.05 7.32 0.65

4.0 4.36 2.14 7.29 1.47

5.0 7.65 0.00 7.22 2.04

6.0 8.40 0.00 7.33 2.13

8.5 8.08 0.00 7.02 7.20 2.09 11.0 7.84 0.00 6.92 7.19 2.06 13.5 7.82 0.00 6.63 7.19 2.06 16.0 8.22 0.00 6.56 2.11

18.5 8.11 0.00 6.17 7.18 2.09 21.0 9.11 0.00 5.64 7.16 2.21 23.5 9.98 0.00 4.64 7.12 2.30 26.0 12.52 0.00 2.50 7.00 2.53

ARA-GLU-2

Date 5/7/2009

C Reference KD777 Strain Park media with arabinose and glucose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 50/250ml shake flask 1000/2000ml fermenter Park media with arabinose Inoculum Media After 8.5 hours growing in flask Time Inoculated 3.23 OD at transfer Volume Inoculum 50ml inoculum (OD600nm=3.2265)+50 ml solution A 20% NaOH Base Antibiotics 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, and 36 mg/L kanamycin.

TIME OD ARABINOSE GLUCOSE PH ln(OD) (h) 600nm (g/L) (g/L)

0.0 0.22 6.59 6.88 7.00 -1.50 1.0 0.47 6.40 7.14 -0.76 2.0 1.08 5.70 7.13 0.08 3.0 2.68 4.13 7.24 6.93 0.99 4.0 7.10 0.23 7.04 6.62 1.96 5.0 8.06 0.00 7.21 6.67 2.09 6.0 8.12 0.00 7.01 6.71 2.09 9.0 7.33 0.00 6.87 6.73 1.99 11.0 7.24 0.00 6.56 1.98 13.5 6.80 0.00 6.98 1.92 16.0 7.11 0.00 6.39 1.96 18.5 6.98 0.00 6.41 1.94 21.0 7.26 0.00 5.83 1.98 23.5 7.92 0.00 5.48 6.72 2.07 26.0 9.46 0.00 3.50 6.68 2.25 28.5 12.38 0.00 0.36 6.58 2.52 31.0 12.62 0.00 0.06 6.38 2.54 33.5 12.34 0.00 0.04 6.41 2.51 36.0 12.22 0.00 0.03 6.41 2.50

ARA-GLU-3

Date 11/7/2009

M Reference KD777 Strain Park media with arabinose and glucose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 50/250ml shake flask 1000/2000ml fermenter Park media with arabinose Inoculum Media After 8.75 hours growing in flask Time Inoculated 2.19 OD at transfer Volume Inoculum 45ml inoculum (OD600nm=2.19)+55 ml solution A 20% NaOH 10 mL Base Antibiotics 10 mg/L chloramphenical,10 mg/L tetracycline, and 100 mg/L kanamycin.

TIME OD ARABINOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.0 0.14 6.61 6.63 -2.00

1.0 0.31 6.81 7.00 -1.18 2.0 0.69 6.11 6.63 -0.38 3.0 1.59 4.95 6.64 0.46 4.0 3.59 2.82 6.52 1.28 5.0 7.51 0.01 7.44 2.02 6.0 7.67 0.00 7.54 2.04 7.0 8.69 0.00 7.33 2.16 8.0 8.00 0.00 6.93 2.08 10.5 8.16 0.00 6.52 2.10 13.0 8.38 0.00 6.33 2.13 15.5 8.35 0.00 6.10 2.12 18.0 8.48 0.00 5.73 2.14 20.5 8.89 0.00 6.07 2.19 22.0 9.40 0.00 5.02 2.24 24.0 9.93 0.00 4.36 2.30

TIME OD ARABINOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 25.0 10.95 0.00 3.93 2.39 26.0 11.83 0.00 2.52 2.47 27.0 13.46 0.00 1.38 2.60 28.0 13.81 0.00 0.47 2.63 29.0 13.91 0.00 0.14 2.63 30.0 13.27 0.00 0.00 2.59 31.0 12.85 0.00 0.06 2.55

ARA-GLU-4

Date 11/7/2009

N Reference KD777 Strain Park media with arabinose and glucose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 50/250ml shake flask 1000/2000ml fermenter Park media with arabinose Inoculum Media After 8.75 hours growing in flask Time Inoculated 2.19 OD at transfer Volume Inoculum 45ml inoculum (OD600nm=2.19)+55 ml solution A 20% NaOH 10 mL Base Antibiotics 20 mg/L chloramphenical, 20 mg/L tetracycline, and 200 mg/L kanamycin.

TIME OD ARABINOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.0 0.13 6.76 6.81 -2.06

1.0 0.27 6.35 6.49 -1.31

2.0 0.49 6.01 6.37 -0.70

3.0 1.07 5.47 6.76 0.07

4.0 2.44 3.90 6.39 0.89

5.0 5.77 1.04 7.53 1.75

6.0 7.54 0.01 7.46 2.02

7.0 8.03 0.00 7.10 2.08

8.0 7.98 0.00 6.67 2.08

10.5 7.62 0.00 6.41 2.03

13.0 8.01 0.00 6.33 2.08

15.5 7.85 0.00 6.81 2.06

18.0 7.93 0.00 5.83 2.07

20.5 8.01 0.00 6.29 2.08

23.0 8.60 0.00 5.40 2.15

TIME OD ARABINOSE GLUCOSE ln(OD)

(h) 600nm (g/L) (g/L)

25.0 9.13 0.00 4.63 2.21

26.0 9.52 0.00 4.64 2.25

27.0 10.00 0.00 4.21 2.30

28.0 11.19 0.00 3.16 2.42

29.0 12.44 0.00 1.70 2.52

30.0 13.13 0.00 0.34 2.57 31.0 13.26 0.00 0.07 2.58 32.0 12.95 0.00 0.00 2.56

ARA-GLU-ARA

Date 8/17/2010

W Reference KD777 Strain Park media with arabinose and glucose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 50/250ml shake flask 1000/2000ml fermenter Park media with arabinose Inoculum Media After 9.25 hours growing in flask Time Inoculated 3.18 OD at transfer Volume Inoculum 50ml inoculum (OD600nm=3.18)+50 ml solution A 20% NaOH Base Antibiotics 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, and 36 mg/L kanamycin.

TIME OD ARABINOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.00 0.18 7.00 6.94 -1.72 1.00 0.39 6.44 6.54 -0.95 2.00 0.89 6.74 7.45 -0.12 3.00 2.14 5.06 6.96 0.76 4.00 4.53 1.41 6.46 1.51 5.50 7.12 0.00 6.85 1.96 6.75 7.16 0.00 6.54 1.97 8.75 7.31 0.00 6.76 1.99 11.75 7.15 0.00 6.48 1.97 14.75 7.37 0.00 7.00 2.00 16.75 7.98 0.00 6.82 2.08 17.75 7.62 0.00 6.63 2.03 18.75 8.07 0.00 6.59 2.09 20.75 8.11 0.00 6.36 2.09 22.75 8.27 0.00 5.49 2.11 24.75 9.51 0.00 5.60 2.25

TIME OD ARABINOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 26.75 9.98 0.00 4.69 2.30 27.75 10.38 7.51 3.92 2.34 29.25 13.39 2.67 3.68 2.59 30.25 14.31 0.22 4.33 2.66 31.25 14.29 0.00 4.31 2.66 33.25 13.95 0.00 3.84 2.64 35.25 13.80 0.00 3.2 2.62

GAL-1

Date 6/1/2009

D Reference KD777 Strain Park media with galactose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with galactose Inoculum Media After 17.5 hours growing in flask Time Inoculated 2.91 OD at transfer 45ml inoculum (OD600nm=2.91)+55 ml solution A Volume Inoculum 20% NaOH Base Antibiotics 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, and 36 mg/L kanamycin.

TIME OD GALACTOSE GLUCOSE PH ln(OD) (h) 600nm (g/L) (g/L)

0.0 0.15 7.29 0.01 7.00 -1.92 1.0 0.21 6.95 0.01 -1.55

2.0 0.33 7.10 0.01 -1.12

3.0 0.47 6.97 0.02 -0.76

4.0 0.66 6.86 0.02 -0.42

5.0 0.91 6.35 0.03 -0.09

6.0 1.29 6.32 0.04 0.26

8.0 2.76 4.95 0.07 1.01

10.0 5.99 0.05 0.09 1.79

12.0 7.01 0.02 0.01 7.16 1.95 14.0 6.71 0.03 0.01 7.17 1.90 16.0 6.50 0.03 0.01 1.87

GAL-2

Date 8/22/2009

K Reference KD777 Strain Park media with galactose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with galactose Inoculum Media After 17 hours growing in flask Time Inoculated 3.10 OD at transfer 46ml inoculum (OD600nm=3.10)+54 ml solution A Volume Inoculum 20% NaOH 7ml Base Antibiotics 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, and 36 mg/L kanamycin.

TIME OD GALACTOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.0 0.19 6.77 0.00 -1.66 1.5 0.38 6.26 0.01 -0.98 3.0 0.73 6.48 0.02 -0.32 4.0 1.15 6.27 0.02 0.14 5.0 1.86 5.25 0.03 0.62 6.0 2.96 4.26 0.05 1.08 7.0 5.10 2.45 0.06 1.63 8.0 7.47 0.04 0.03 2.01 9.0 7.44 0.00 0.00 2.01 10.0 7.38 0.00 0.00 2.00

GAL-GLU-1

Date 6/1/2009

E Reference KD777 Strain Park media with galactose and glucose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with galactose Inoculum Media After 17.5 hours growing in flask Time Inoculated 2.91 OD at transfer 45ml inoculum (OD600nm=2.91)+55 ml solution A Volume Inoculum 20% NaOH Base Antibiotics 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, and 36 mg/L kanamycin.

TIME OD GALACTOSE GLUCOSE PH ln(OD) (h) 600nm (g/L) (g/L)

0.0 0.14 7.45 7.29 7.00 -1.94 1.0 0.19 -1.64

2.0 0.27 7.18 7.01 -1.32

3.0 0.32 -1.15

4.0 0.37 6.96 7.25 -0.98

5.0 0.43 -0.85

6.0 0.48 7.07 7.20 -0.74

8.0 0.57 -0.57

10.0 0.71 6.68 7.26 -0.34

12.0 1.00 6.27 6.89 -0.01

14.0 1.55 5.89 7.30 0.44

16.0 2.91 4.57 7.01 1.07

21.0 12.03 0.99 1.31 7.12 2.49 22.0 12.59 0.00 0.45 7.13 2.53 23.0 12.07 0.00 0.16 2.49 24.0 11.53 0.00 0.15 7.15 2.45

GAL-GLU-2

Date 7/5/2009

F Reference KD777 Strain Park media with galactose and glucose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with galactose Inoculum Media After 12.5 hours growing in flask Time Inoculated 3.07 OD at transfer 50ml inoculum (OD600nm=3.07)+50 ml solution A Volume Inoculum 20% NaOH 11ml Base Antibiotics 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, and 36 mg/L kanamycin.

TIME OD GALACTOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.00 0.19 7.25 7.17 -1.66 1.00 0.21 7.15 7.10 -1.54 2.50 0.28 7.12 7.15 -1.29 4.00 0.32 7.04 7.13 -1.13 6.25 0.36 7.07 7.24 -1.02 8.00 0.36 7.02 7.23 -1.01 10.50 0.38 6.98 7.24 -0.96 12.50 0.45 7.00 7.33 -0.80 14.50 0.58 6.82 7.28 -0.55 16.50 1.00 6.46 7.27 0.00 17.50 1.43 6.11 7.26 0.36 18.50 2.26 5.16 7.03 0.82 19.50 3.64 3.90 6.94 1.29 20.50 6.24 1.61 6.37 1.83 21.00 7.71 0.30 5.77 2.04 21.50 9.25 0.00 5.64 2.22

TIME OD GALACTOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 22.00 9.78 0.00 4.63 2.28 22.50 10.26 0.00 3.76 2.33 22.75 10.93 0.00 3.16 2.39 23.00 11.91 0.00 2.72 2.48 23.25 11.91 0.01 2.05 2.48 23.50 12.39 0.01 1.56 2.52 23.75 13.24 0.02 1.28 2.58 24.00 13.41 0.02 0.97 2.60 24.50 13.46 0.03 0.56 2.60 25.00 13.28 0.03 0.24 2.59 25.50 13.18 0.04 0.19 2.58

GAL-GLU-3

Date 8/22/2009

L Reference KD777 Strain Park media with galactose and glucose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with galactose Inoculum Media After 17 hours growing in flask Time Inoculated 3.10 OD at transfer 46ml inoculum (OD600nm=3.10)+54 ml solution A Volume Inoculum 20% NaOH 11ml Base Antibiotics 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, and 36 mg/L kanamycin.

TIME OD GALACTOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.0 0.17 6.72 0.00 -1.80 1.5 0.35 6.16 0.01 -1.06 3.0 0.69 6.08 0.02 -0.37 4.0 1.09 6.14 0.02 0.08 5.0 1.41 5.21 5.45 0.34 6.0 1.68 4.72 5.35 0.52 8.0 2.76 4.49 5.68 1.01 9.0 3.59 4.26 6.07 1.28 10.0 4.78 2.96 5.44 1.56 11.0 6.48 1.54 5.39 1.87 12.0 8.42 0.00 4.69 2.13 13.0 9.61 0.00 4.30 2.26 14.0 10.26 0.01 3.11 2.33 15.0 11.19 0.02 2.01 2.42 16.0 12.14 0.03 0.83 2.50 17.0 12.63 0.05 0.19 2.54 18.0 12.38 0.06 0.00 2.52 19.0 12.16 0.05 0.00 2.50

Time(h) Event 4.0 Add 10ml of 700g/L glucose to the fermenter

XYL

Date 7/29/2009

G Reference KD777 Strain Park media with xylose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with xylose Inoculum Media After 9.5 hours growing in flask Time Inoculated 2.95 OD at transfer 44ml inoculum (OD600nm=2.95)+56 ml solution A Volume Inoculum 20% NaOH 8ml Base Antibiotics 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, and 36 mg/L kanamycin.

TIME OD XYLOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.0 0.16 7.62 0.01 -1.85 1.0 0.45 7.24 0.01 -0.81 2.0 0.95 6.29 0.01 -0.05 3.0 2.21 5.52 0.01 0.79 4.0 5.08 2.59 0.02 1.63 4.5 7.47 0.12 0.02 2.01 5.0 9.33 0.01 0.00 2.23 5.5 8.79 0.00 0.00 2.17 6.0 8.73 0.00 0.00 2.17

XYL-GLU

Date 7/29/2009

H Reference KD777 Strain Park media with xylose and glucose Media 37℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with xylose Inoculum Media After 9.5 hours growing in flask Time Inoculated 2.95 OD at transfer 44ml inoculum (OD600nm=2.95)+56 ml solution A Volume Inoculum 20% NaOH 12ml Base Antibiotics 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, and 36 mg/L kanamycin.

TIME OD XYLOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.00 0.16 6.37 6.20 -1.83 1.00 0.40 6.34 6.35 -0.92 2.00 0.85 6.24 6.63 -0.17 3.00 1.82 5.80 7.22 0.60 4.00 4.07 3.42 6.63 1.40 4.50 5.71 1.81 6.35 1.74 5.00 8.10 0.17 6.52 2.09 5.50 8.82 0.00 6.31 2.18 6.00 9.31 0.00 5.94 2.23 6.75 9.75 0.00 5.50 2.28 7.25 9.84 0.00 5.51 2.29 7.75 9.99 0.00 5.63 2.30 8.50 10.24 0.00 4.88 2.33 9.25 10.69 0.00 4.97 2.37 10.00 11.23 0.00 4.18 2.42 10.25 10.93 0.00 4.31 2.39

TIME OD XYLOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 10.75 11.16 0.00 4.20 2.41 11.50 11.54 0.00 3.80 2.45 12.50 12.28 0.00 2.79 2.51 12.75 11.99 0.00 2.99 2.48 13.50 12.22 0.00 2.45 2.50 14.50 12.77 0.02 2.49 2.55 15.50 13.02 0.00 1.91 2.57 16.50 12.91 0.03 1.66 2.56 17.50 13.64 0.03 1.46 2.61 18.50 13.85 0.03 1.07 2.63 19.50 14.48 0.03 0.83 2.67 20.50 14.54 0.04 0.62 2.68 21.50 14.56 0.04 0.38 2.68 22.50 14.45 0.04 0.34 2.67 23.50 14.21 0.04 0.32 2.65

XYL-GLU-XYL

Date 8/17/2010

X Reference KD777 Strain Park media with xylose and glucose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 50/250ml shake flask 1000/2000ml fermenter Park media with xylose Inoculum Media After 11 hours growing in flask Time Inoculated 3.08 OD at transfer Volume Inoculum 50ml inoculum (OD600nm=3.08)+50 ml solution A 20% NaOH Base Antibiotics 4.5 mg/L chloramphenical, 9.0 mg/L tetracycline, and 36 mg/L kanamycin.

TIME OD XYLOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.00 0.19 7.52 7.28 -1.68

1.25 0.47 6.93 6.93 -0.75

2.25 0.98 6.54 7.09 -0.02

3.75 3.01 4.41 7.14 1.10

4.25 4.53 2.86 6.97 1.51

5.00 6.88 0.15 6.98 1.93

6.00 7.77 0.00 6.96 2.05

8.25 8.78 0.00 5.64 2.17

10.00 9.24 0.00 4.82 2.22

12.00 10.51 6.15 2.82 2.35

13.00 11.27 5.86 2.86 2.42

14.00 12.96 2.45 3.05 2.56

15.00 13.60 0.00 3.92 2.61

16.00 14.27 0.00 3.72 2.66

17.00 13.72 0.00 3.49 2.62

19.00 12.82 0.00 3.12 2.55

TIME OD XYLOSE GLUCOSE ln(OD)

(h) 600nm (g/L) (g/L)

21.00 13.04 0.00 2.66 2.57

23.00 13.26 0.00 2.30 2.58

25.00 13.13 0.00 1.75 2.57

27.50 12.96 0.00 1.36 2.56

29.50 13.17 0.00 1.10 2.58 31.50 12.87 0.00 0.84 2.56 33.50 13.03 0.00 0.63 2.57

ARA

Date 5/5/2010

S Reference Strain KD915=ALS1034=W ptsG ::FRT glk ::FRT crr ::FRT manZ ::CAM Park media with arabinose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with arabinose Inoculum Media After 14 hours growing in flask Time Inoculated 3.17 OD at transfer 47ml inoculum (OD600nm=2.90)+53 ml solution A Volume Inoculum 20% NaOH 6 ml Base 4.5 mg/L chloramphenical Antibiotics

TIME OD ARABINOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.0 0.17 7.86 0.00 -1.79 1.0 0.34 7.71 0.03 -1.08 2.0 0.63 7.38 0.04 -0.47 3.0 1.16 6.88 0.06 0.15 4.0 2.28 5.64 0.09 0.82 5.0 4.30 2.65 0.11 1.46 6.0 8.19 0.00 0.12 2.10 7.0 8.98 0.00 0.09 2.20 8.0 8.90 0.00 0.07 2.19 9.0 8.84 0.00 0.07 2.18

ARA-GLU

Date 5/5/2010

T Reference Strain KD915=ALS1034=W ptsG ::FRT glk ::FRT crr ::FRT manZ ::CAM Park media with arabinose and glucose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with arabinose Inoculum Media After 14 hours growing in flask Time Inoculated 3.17 OD at transfer 47ml inoculum (OD600nm=2.90)+53 ml solution A Volume Inoculum 20% NaOH 10 ml Base 4.5 mg/L chloramphenical Antibiotics

TIME OD ARABINOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.00 0.17 7.88 7.82 -1.78 1.00 0.34 7.62 7.73 -1.08 2.00 0.61 7.37 7.78 -0.50 3.00 1.09 6.84 7.73 0.08 4.00 2.16 5.78 7.80 0.77 5.00 4.15 2.94 7.80 1.42 6.00 7.99 0.00 7.84 2.08 7.00 8.49 0.00 7.57 2.14 8.00 8.74 0.00 7.68 2.17 9.00 8.70 0.00 7.44 2.16 11.50 8.90 0.00 7.27 2.19 14.00 9.09 0.00 6.91 2.21 18.00 11.12 0.00 4.61 2.41 20.00 13.29 0.00 2.22 2.59 20.75 13.46 0.00 1.74 2.60 21.50 13.78 0.00 1.37 2.62 23.50 14.24 0.00 0.75 2.66 25.50 14.17 0.00 0.49 2.65 27.50 13.92 0.00 0.38 2.63

GAL

Date 7/1/2010

U Reference Strain KD915=ALS1034=W ptsG ::FRT glk ::FRT crr ::FRT manZ ::CAM Park media with galactose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with galactose Inoculum Media After 21 hours growing in flask Time Inoculated 3.13 OD at transfer 47ml inoculum (OD600nm=3.13)+53 ml solution A Volume Inoculum 20% NaOH 6 ml Base 4.5 mg/L chloramphenical Antibiotics

TIME OD GALACTOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.00 0.17 7.66 0.03 -1.77 2.00 0.64 7.15 0.04 -0.44 3.00 1.21 6.59 0.05 0.19 4.00 2.14 5.86 0.08 0.76 5.00 3.69 4.21 0.11 1.31 6.00 6.29 1.84 0.16 1.84 7.00 8.35 0.01 0.13 2.12 8.00 8.68 0.00 0.07 2.16 9.00 8.67 0.00 0.07 2.16

GAL-GLU

Date 7/1/2010

V Reference Strain KD915=ALS1034=W ptsG ::FRT glk ::FRT crr ::FRT manZ ::CAM Park media with galactose and glucose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with galactose Inoculum Media After 21 hours growing in flask Time Inoculated 3.13 OD at transfer 47ml inoculum (OD600nm=3.13)+53 ml solution A Volume Inoculum 20% NaOH 13 ml Base 4.5 mg/L chloramphenical Antibiotics

TIME OD GALACTOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.00 0.17 7.15 7.31 -1.78 2.00 0.38 7.09 7.34 -0.97 3.00 0.58 6.86 7.21 -0.54 4.00 0.92 6.58 7.11 -0.09 5.00 1.49 6.25 7.09 0.40 6.00 2.51 5.61 6.94 0.92 7.00 4.23 4.39 6.46 1.44 8.00 6.98 2.75 5.66 1.94 9.00 10.78 0.84 3.89 2.38 9.25 11.77 0.42 3.23 2.47 9.75 12.54 0.06 2.49 2.53 10.25 12.99 0.00 1.99 2.56 11.75 13.24 0.00 0.97 2.58 14.75 12.46 0.00 0.38 2.52 17.75 11.61 0.00 0.28 2.45

XYL

Date 4/22/2010

Q Reference Strain KD915=ALS1034=W ptsG ::FRT glk ::FRT crr ::FRT manZ ::CAM Park media with xylose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with xylose Inoculum Media After 20 hours growing in flask Time Inoculated 2.90 OD at transfer 46ml inoculum (OD600nm=2.90)+54 ml solution A Volume Inoculum 20% NaOH 10 ml Base 4.5 mg/L chloramphenical Antibiotics

TIME OD XYLOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.0 0.17 8.08 0.03 -1.78 1.0 0.35 7.86 0.03 -1.06 2.0 0.64 7.38 0.03 -0.44 3.0 1.29 6.72 0.04 0.25 4.0 2.49 5.29 0.04 0.91 5.0 5.07 2.61 0.05 1.62 6.0 8.04 0.04 0.00 2.08 7.0 8.59 0.00 0.03 2.15 8.0 8.36 0.00 0.03 2.12 9.0 8.13 0.00 0.03 2.10

XYL-GLU

Date 4/22/2010

R Reference Strain KD915=ALS1034=W ptsG ::FRT glk ::FRT crr ::FRT manZ ::CAM Park media with xylose and glucose Media 37 ℃ Temperature 7.0 PH 500rpm Agitation 1.0 L/min Air Flowrate Inoculum plate 100/500ml shake flask 1000/2000ml fermenter Park media with xylose Inoculum Media After 20 hours growing in flask Time Inoculated 2.90 OD at transfer 46ml inoculum (OD600nm=2.90)+54 ml solution A Volume Inoculum 20% NaOH 11 ml Base 4.5 mg/L chloramphenical Antibiotics

TIME OD XYLOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 0.0 0.17 7.81 7.65 -1.76 1.0 0.33 7.58 7.55 -1.10 2.0 0.62 7.35 7.60 -0.48 3.0 1.15 6.73 7.46 0.14 4.0 2.15 5.52 7.23 0.76 5.0 4.17 3.70 7.36 1.43 6.0 7.10 0.76 7.42 1.96 7.0 9.19 0.00 7.12 2.22 8.0 9.67 0.00 6.73 2.27 9.0 10.00 0.00 6.29 2.30 11.0 10.68 0.00 5.53 2.37 12.5 11.24 0.00 5.01 2.42 14.0 11.96 0.00 4.48 2.48 15.5 12.34 0.00 3.95 2.51 17.0 13.05 0.00 3.25 2.57

TIME OD XYLOSE GLUCOSE ln(OD) (h) 600nm (g/L) (g/L) 18.5 13.33 0.00 2.94 2.59 20.0 13.76 0.00 2.07 2.62 21.5 14.19 0.00 1.82 2.65 23.0 14.40 0.00 1.33 2.67 24.5 14.76 0.00 0.88 2.69 26.0 14.79 0.00 0.64 2.69 27.5 14.74 0.00 0.58 2.69