Ergosterol Biosynthesis in Green Algae and its Phylogenetic Implications

by

Matthew B. Miller, B.S.

A Thesis

In

INTERDISCIPLINARY STUDIES

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of

MASTER OF SCIENCE

Approved

Committee:

Dr. W. David Nes Chair of the Committee

Dr. Robert W. Shaw

Mark Sheridan

Dean of the Graduate School

August, 2014

Copyright 2014, Matthew B. Miller

Texas Tech University, Matthew B. Miller, August 2014

ACKNOWLEDGMENTS

Enormous gratitude goes to my mentor, Dr. W. David Nes. His guidance, knowledge and advice have been appreciated well beyond this thesis. I would like to thank Dr. Bard for the gift of the KD7 and KD21 C. reinhardtii mutants. I would also like to thank our collaborators, Dr. William Snell and Dr. Qian Wang for growing, modifying and maintaining all of the cultures provided to us. Many thanks to my laboratory co- workers, especially Dr. Brad Haubrich, Crista Thomas, Alicia Howard, Dr. Garrett Mohr, and Presheet Patkar. Working with you all was a pleasure, and your willingness to share your knowledge was much appreciated.

To my mother and Father, I can’t imagine what the road would have been like if I didn’t have your unwavering support. Thank you for instilling in me a drive to succeed, and a stern hand when I needed it. Most of all, thank you for teaching me that if you want something, you had better be willing to work for it.

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TABLE OF CONTENTS

ACKNOWLEDGMENTS ...... iii ABSTRACT ...... v LIST OF TABLES ...... vii LIST OF FIGURES ...... viiii ABBREVIATIONS ...... ix CHAPTERS 1. ...... 1 1.1 Introduction ...... 1 1.2 Background ...... 1 1.3 Structure ...... 2 1.4 Sterol Nomenclature ...... 3 1.5 Fungal and Algal Sterols ...... 4 2. ISOLATION, IDENTIFICATION AND PURIFICATION OF STEROLS IN THE ALGA Chlamydomonas reinhardtii ...... 6 2.1 Materials and Methods ...... 6 2.2 Extraction (NSF) ...... 6 2.3 GC ...... 7 2.4 GC-MS ...... 8 2.5 RP-HPLC ...... 9 2.6 NMR ...... 10 3. RESULTS ...... 12 3.1 Sterols of Chlamydomonas reinhardtii ...... 14 3.2 Strengthening proposed the pathway ...... 16 3.3 Mutant studies ...... 17 3.4 25-Thialanosterol Inhibitor Study ...... 20 3.5 Isotopic labeling ...... 21 4. DISCUSSION ...... 24 4.1 Photosynthetic lineage...... 27 4.2 Evolutionary Divergence...... 28 BIBLIOGRAPHY ...... 30 APPENDIX

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A. MASS SPECTROMETER ANALYSIS ...... 33

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ABSTRACT

The green algae Chlamydomonas reinhardtii synthesizes the protosterol and converts it to (C24-methyl) and 7-dehydroporiferasterol (C24-ethyl) through a highly conserved sterol C24- methylation-C25-reduction (25(27)- olefin) pathway that is distinct from the well described acetate- to fungal and its conversion to ergosterol by the 24(28)-olefin pathway. 23 sterols were isolated and characterized by a combination of GC-MS and proton nuclear magnetic resonance spectroscopy analysis from a set of mutant, wild-type, and 25-thialanosterol- treated cells. The structure and stereochemistry of the final C24-alkyl sterol side chains possessed different combinations of 24-methyl/ethyl groups and 22(23)E and 25(27)- 2 double bond constructions. When incubated with [methyl- H3]methionine, cells incorporated three (into ergosterol) or five (into 7-dehydroporiferasterol) deuterium atoms into the newly biosynthesized 24-alkyl sterols, consistent only with a 25(27)- olefin pathway. Thus, our findings demonstrate that two separate isoprenoid-24-alkyl sterol pathways evolved in fungi and green algae, both of which converge to yield a common membrane insert ergosterol.

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LIST OF TABLES

3.1. Sterol composition of Chlamydomonas reinhardtii cells ...... 13 3.2. Diagnostic signals for sterols submitted for 1HNMR from Chlamydomonas reinhardtii ...... 18 4.1. Detected sterols with chromatographic and spectral properties from Chlamydomonas reinhardtii ...... 25

vi Texas Tech University, Matthew B. Miller, August 2014

LIST OF FIGURES

1.1. Some known functions of sterols among cells ...... 2

1.2. The common tetracyclic sterol frame with the 1,2-cyclopentanoperhydro- phenanthrene ring system ...... 3

1.3. A generic sterol structure showing the 4 domains ...... 3

1.4. The two most common naming systems used for sterols ...... 4

1.5. Proposed biosynthetic route to ergosterol in Chlamydomonas reinhardtii ...... 5

2.1. Dose Response of on GC...... 8

2.2. Dose Response curve of ergosterol on analytical HPLC ...... 10

3.1. Capillary GC trace of Chlamydomonas reinhardtii wild-type cells ...... 15

3.2. Mass spectra of ergosterol, 7-dehydroporiferasterol and cycloartenol ...... 16

3.3. Mass spectra and compound structures for cyclolaudenol and 24(28)-

methylenecycloartenol ...... 20

3.4. High end mass spectra of ergosterol and 7-dehydroporiferasterol incubated with

3 [methyl-2H ]-methionine...... 22

3.5. The sterol C24-alkylation/reduction pathways to alkylated products...... 23

4.1. Proposed sterol pathway from cycloartenol to end products in Chlamydomonas

reinhardtii ...... 26

A.1. Ergosta-5,7,22,25(27)-tetraenol ...... 33

A.2. Ergosta-5,7,22-trienol ...... 34

A.3. Ergosta-5,7,25(27)-trienol ...... 35

A.4. Ergosta-5,7-dienol ...... 36

A.5. Ergosta-7,25(27)-dienol ...... 37

A.6. Poriferasta-5,7,25(27)-trienol...... 38 vii Texas Tech University, Matthew B. Miller, August 2014

A.7. Poriferasta-5,7,22,25(27)-tetraenol ...... 39

A.8. Poriferasta-5,7,22-trienol ...... 40

A.9. Poriferasta-5,7-dienol ...... 41

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ABBREVIATIONS

c RRTc in HPLC amu Atomic Mass Unit eV Electron Volts

GC Gas Chromatograph

GC-MS Gas Chromatography-Mass Spectrometry

HP Hewlett Packard

KOH Potassium Hydroxide

MeOH Methanol

NMR Nuclear Magnetic Resonance

NSF Non-Saponafiable Fraction

RP-HPLC Reverse Phase High Performance Liquid Chromatography

RRTc Relative Retention Time to Cholesterol in GC

SBI Sterol Biosynthesis Inhibitor

SMT Sterol Methyl Transferase

TLC Thin Layer Chromatography

UHP Ultra High Purity

ix Texas Tech University, Matthew B. Miller, August 2014

CHAPTER 1

STEROLS

1.1 Introduction

There have been some conflicting reports in the literature regarding the biosynthetic route to ergosterol in green algae.1,4,5 This study intends to make a thorough analysis of the sterol pathway operating in the green alga Chlamydomonas reinhardtii. By using all of the current technologies afforded to us, i.e., NMR, HPLC, UV, GC, GC-MS, isotopic labeling, and inhibitor studies we were able to identify, characterize, and propose a biosynthetic pathway, for the 23 sterols isolated, and the phylogenetic implications of such a pathway.

1.2 Background

Sterols were first discovered by French chemists over 230 years ago. These pioneers noticed a crystalline component found in the gallstones of humans, this was later identified as cholesterol. Several years later it was noted by scientists working with bile stones that a similar phenomenon was observed and this substance was given the name

“Cholesterine” from the two Greek words “chole” and “stereos” meaning bile and solid respectively.1 After the substance was discovered to be an alcohol it was changed to cholesterol in countries speaking English. It is now recognized that sterols play several key roles throughout cells, such as membrane inserts and precursors to hormones and vitamins (Figure 1.1). Ergosterol, relevant to this study, was actually found to play significant roles in membranes before cholesterol. Our predecessors conducted studies with the technologies that they had available at the time, and were limited to melting

1 Texas Tech University, Matthew B. Miller, August 2014 point, crystallization, color, and optical characteristics in their attempts at natural product profiling and fractionation.1

Figure 1.1. Some known functions of sterols among cells. Adapted from 2.

1.3 Sterol Structure

Cholesterol and structurally similar compounds, “possessing the 1,2- cyclopentanoperhydrophenathrene ring system” account for the massive library of over

1000 natural products found in almost all living systems on earth (Figure 1.2).1 Sterols are synthesized de novo in most fungi, plants, and animals from complex biosynthetic pathways that can involve more than thirty enzymes.2 Cholesterol has 9 chiral centers and contains 4 distinct domains (Figure 1.3).1 The A domain contains the hydroxyl group at

C3 which is normally found in the  conformation. The B domain is comprised of the nucleus which can have varying degrees of unsaturation and methylation particularly at 2 Texas Tech University, Matthew B. Miller, August 2014

C4, and C14. Both of these characteristics can change the shape of the molecule and affect its chromatographic properties. The C domain is the stereocenter at C20 which is normally found in the right handed or R conformation. Lastly, the D domain which makes up the side chain can also have varying degrees of unsaturation and alkylation, particularly at C24.1

Figure 1.2. The common tetracyclic sterol frame with the 1,2-cyclopentanoperhydro- phenanthrene ring system. Adapted from 1.

Figure 1.3. A generic sterol structure showing the 4 domains. A shows the hydroxyl group at position C3. B is showing the sterol nucleus. C highlights the stereochemistry around C20. D is the sterol side chain. Adapted from 1.

1.4 Sterol Nomenclature

The official naming and numbering of sterols can be quite complicated. In the field of sterol research the conventional system, established in the book by

Fieser and Fieser, seems to be the more widely accepted and used system of numbering and naming.3 This is likely to assist in avoiding confusion of substitution and orientation particularly around C24, and C25 (Figure 1.4).2 This system accounts for the biosynthetic

3 Texas Tech University, Matthew B. Miller, August 2014 origin of C26 and C27, as well as allowing for , and  assignments to ethyl groups added to the nucleus (C4, C14) and side chain (C24).1

Figure 1.4. The two most common naming systems used for sterols. Adapted from 1. 1.5 Fungal and Algal Sterols

Prior to this study there was conflicting data on the sterol content of

Chlamydomonas reinhardtii. One such study postulated a pathway based on genomic sequencing rather than sterol isolation and identification.4 They proposed a route to ergosterol partially resembling that of the Saccharomyces cerevisiae. It has been well documented that fungi produce ergosterol from lanosterol.1 A key difference however, was that they proposed, unlike S. cerevisiae, that C. reinhardtii uses cycloartenol rather than lanosterol as its sterol precursor in the biosynthesis of ergosterol

(Figure 1.5).4 Another study worked with a mutated strain of C. reinhardtii.5 Here the researchers claim to have only been able to isolate end products and 24(28) sterols. It should be noted that in this study the researchers only did argentation (silver nitrate) thin- layer chromatography (TLC) for their chromatography step. Also, this group did not compare their findings to authentic standards. For these reasons, it is possible that they 4 Texas Tech University, Matthew B. Miller, August 2014 may have misidentified 25(27) sterols as 24(28) sterols. In stark contrast to this, a third group working with two of the same strains involved in our study, did identify several

25(27) sterols.5

Figure 1.5. Proposed biosynthetic route to ergosterol in Chlamydomonas reinhardtii comparted to that of the fungus S. cerevisiae. Adapted from 4.

5 Texas Tech University, Matthew B. Miller, August 2014

CHAPTER 2

ISOLATION, IDENTIFICATION AND PURIFICATION OF

STEROLS IN THE ALGA Chlamydomonas reinhardtii

2.1 Materials and Methods

GC capillary column (ZB-5) and both HPLC columns were purchased from

Phenomenex. All chemicals and solvents were purchased from Sigma, Fisher, or VWR, unless otherwise noted.

2.2 Extraction (NSF)

Samples were collected from our collaborator Dr. William J. Snell at UTSW

Medical School, (Dallas, TX) and extracted and characterized in the typical fashion as previously reported.6 Briefly, cell pellets were received on dry ice and promptly stored in a -80ºC freezer until ready for analysis. Cell pellets were weighed and then saponified with a volume ~10 times greater than the weight of the pellets in 10% aqueous methanolic potassium hydroxide (KOH) (10% w/v). Cells were saponified at ~78ºC for no less than 30 mins. After cooling to room temperature, cells were extracted three times with an equal volume of hexanes to obtain a non-saponafiable fraction (NSF). The

NSF was transferred to tared vials and then stripped of solvent by gentle heating under a stream of nitrogen gas. After drying the vials were then stored in our collection at -20ºC for further analysis.

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2.3 GC

Samples were taken from -20ºC storage and brought up in a volume of HPLC grade MeOH (methanol) based on the NSF weight to make the preparation 1mg/ml. It is important to standardize all samples by GC before injection onto the GC-MS and/or

HPLC. Because it was unknown exactly how much sterol was contained in the NSF the above is used as a starting point and then checked by GC. It is a standard operating procedure (SOP) in our lab to quantify nearly all sterols against cholesterol. This is done by taking a stock solution of cholesterol (1mg/ml) and shooting 1μl (1μg) of it onto the

GC. The area under the peak for our unknown sample (1μl) is divided by the peak area obtained for the standard of cholesterol. The result of this calculation is μgs of sample per

μl of solvent. By doing this it is possible to make much more accurate determinations

(especially in the μg range) of how much sterol is in a given sample. Since all injections were made manually into the GC, it is important for each laboratory member to perform a dose response to establish their consistency of injection volumes (Figure 2.1.). After quantification, if an adjustment in concentration needs to be made, the compound would then be stripped of solvent again, as previously described, and then brought up to a concentration of 1mg/ml in HPLC grade MeOH. After the sample has been brought up to

1mg/ml it is then filtered through a 0.2μm pore size filter, this is to remove any small particles or cell debris that may contaminate our samples and cause problems with our instrumentation. The sample would now be ready for the next step, which is either HPLC or GC-MS.

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50 45 40 35

30 25 20 (millions) 15 10 y = 4E+07x

Detector Integrated Area Area Integrated Detector 5 R² = 0.9987 0 0 0.2 0.4 0.6 0.8 1 1.2 Cholesterol (ug)

Figure 2.1. Cholesterol dose respone curve on GC. 2.4 GC-MS

The NSF (1ug total sterol injection) was routinely analyzed by GC-MS using a

HP 6890 gas chromatograph with a 30 m ZB-5 capillary column interfaced to a 5973 mass spectrometer at 70 eV; with a GC flow rate of UHP helium gas set to 1.2ml/min, injector port set to 250ºC, and the initial temperature was set to 170ºC, held for 1 min, and then ramped at 20ºC / min to a maximum temperature of 280ºC. Sterols were assigned an RRTc (relative retention time to cholesterol) by taking the sterol retention time and dividing it by the cholesterol standards retention time. Cholesterol had two retention times based on the capillary column that was installed into the GC-MS.

Cholesterol retention times were 13.8 min for the old column and 14.5 min for the new column.7 The difference in time can be attributed to the difference in column length.

Every time that the GC-MS ion source is cleaned ~10cm is clipped off of the injection side of the column. This is the portion of the column where the sample is injected and can get dirty very quickly, depending on the frequency of use. 8 Texas Tech University, Matthew B. Miller, August 2014

2.5 RP-HPLC

Samples were separated on an Agilent 1100 HPLC equipped with a photodiode array detector. This proved to be an invaluable tool for predicting the double bond character within a given sample by analyzing the UV spectra of a given peak. Some samples proved difficult to separate and multiple sterols were found to have eluted as one peak off of the HPLC. In order to separate these compounds, several injections were performed pooling this peak to collect more sample for further purification. The pooled fraction was stripped of solvent (as described) and then re-quantified (as described) for reinjection into the HPLC using another column with different chromatographic properties (Figure 2.1). All samples were run at a flow rate of 1ml/min. The solvents used were dependent on the column used. For the Luna column by Phenomenex 100% MeOH was used at a temperature of 20ºC. For the TOSOHAAS TSK gel column (ODS-120A)

65/35, v/v acetonitrile / isopropanol was used at 35ºC.

Samples were assigned an c value based on their retention times relative to that of cholesterol. For the Luna HPLC column cholesterol had a retention time of 16.5 min, and 26.8 min on the TSK gel. Though it was not an integral part of the present study, a dose response curve for sterols can be a helpful tool when quantifying sterol amounts on

HPLC (Figure 2.2).

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40 35

30 25 20 15 (thousands) 10 5 y = 1504.9x Detector Integrated Area Area Integrated Detector R² = 0.9995 0 0 5 10 15 20 25 30 Ergosterol (ug)

Figure 2.2. Dose Response of Ergosterol on Analytical HPLC. 2.6 NMR

NMR assignments were made by Dr. W. David Nes. Samples purified by HPLC were examined by proton nuclear magnetic resonance spectroscopy (1HNMR) to confirm structure and stereochemistry of the side-chain alkyl group at position C24. Samples were run in deuterochloroform solutions on a Varian Unity Inova 500MHz spectrometer with the chemical shifts referenced to chloroform resonating at 7.265 ppm and reported as  in ppm.

For reference see the natural products analysis flowchart (Figure 2.3.).

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Figure 2.3. Natural Products Analysis flow chart

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CHAPTER 3

RESULTS

When this project began, little was known about the sterol profile of

Chlamydomonas reinhardtii. It was reported that ergosterol, 7-dehydroporiferasterol, and another minor compound ergost-7-enol were synthesized by the alga.4,8-11 Tritiated -2,3-oxide assayed with a microsomal preparation was reported to go to only one product, cycloartenol.12 Unfortunately there was limited data for a proposed sterol biosynthetic route to ergosterol or 7-dehydroporiferasterol. Much of the data that could be found suggested a similar pathway to that of yeast, taking the 24(28)-olefin pathway.4

This study however suggests otherwise. After analyzing cells from C. reinhardtii, most of the compounds found were of the 25(27) side chain (Table 3.1).

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Table 3.1. Sterol composition of Chlamydomonas reinhardtii cells.

Sterola WTb KD7c KD21c 25-TLd KD7PYc 1 0.3 0.7 2.5 1.1 2 0.3 3 0.3 4 tre tr tr 0.9 5 tr tr tr tr tr 6 2.7 7 tr 8 2.6 14.9 8.1 21.4 9 1.6 10 3.5 3.0 0.5 11 0.5 6.4 10.6 12 21.3 13 0.9 4.6 14 50.7 36.6 15 70.4 16 37.2 27.5 1.5 17 18 15.5 19 0.5 20 8.8 21 22.6 21.5 22 19.4 12.7 23 1.7 2.9 24 35.0 26.8 a Structures of sterols are shown in Figure 4.1. b WT, wild-type cells. c Mutant cell lines. d 25TL, 25-thialanosterol salt treated cells e tr, trace amount of sterol at less than 0.3%: blank refers to no sterol detected in cells.

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3.1 Sterols of Chlamydomonas reinhardtii

In this study the wild type cells of C. reinhardtii were examined by GC-

MS and in total 11 sterols were detected (Figure 3.1). As previously reported, ergosterol, and 7-dehydroporiferasterol were detected as well as cycloartenol (Figure 3.2).5 These compounds matched the properties (RRTc, mass spectra, and UV spectra) for authentic standards. Significant amounts of the major compounds, ergosterol and 7- dehydroporiferasterol were obtained by HPLC at purity high enough to verify their identity by 1HNMR. The other compounds found in the GC trace were identified according to GC, RRTc’s and mass spectra compared to standards for cycloartenol,

4,14-dimethylergosta-8,25(27)-dienol, 4,14-dimethylergosta-8,24(28)-dienol, ergosta-7,25(27)-dienol, ergosta-8,25(27)-dienol, ergost-7-enol, poriferasta-7,25(27)- dienol, poriferasta-8,25(27)-dienol, and poriferas-7-enol. The detection of cycloartenol and C4-monomethyls was revealing, supporting that the alga evolved from photosynthetic ancestors. Another important finding was the detection of several 25(27)- sterols.7 The presence of these sterols supports that the biosynthesis of ergosterol in C. reinhardtii likely proceeds through a 25(27)-olefin pathway and not a 24(28)-olefin pathway as previously reported.4,7

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Figure 3.1. Gas-liquid chromatograph separation of sterols isolated from wild-type C. reinhardtii. Peaks in the total ion current (TIC) chromatograph correspond to peak 1, ergosta-5,7,22-trienol (ergosterol); peak 2, ergosta-8,25(27)-dienol; peak 3, ergosta- 7,25(27)-dienol; peak 4, ergost-7-enol; peak 5, 4 ,14 - dimethylergosta-8,25(27)-dienol; peak 6, 4 ,14 -dimethylergosta-8,24(28)- dienol; peak 7, porifersta-5,7,22-trienol (7- dehydroporifersterol); peak 8, porifersta-8,25(27)-dienol; peak 9, porifersta-7,25(27)- dienol; peak 10, poriferst-7-enol; and peak 11, cycloart-24(25)-enol (cycloartenol).

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Figure 3.2. From top to bottom. Mass spectra of ergosterol, 7-dehydroporiferasterol and cycloartenol. A-1, B-1, C-1 are the UV spectra for each compound.

3.2 Strengthening the proposed pathway

In order to generate a more complete sterol biosynthetic pathway for C. reinhardtii, strains that had been previously modified through genetic manipulation, and the wild-type strain were dosed with an inhibitor generated in this lab.6 These were then analyzed for their sterol composition. The mutant strains were chosen because it had been

16 Texas Tech University, Matthew B. Miller, August 2014 reported that they produced abnormal sterol profiles. The wild-type alga was also given an SBI (sterol biosynthesis inhibitor) in order to accumulate more sterol intermediates.

3.3 Mutant studies

The first strain analyzed was KD7 because it had been documented that this mutant synthesizes six different 25(27) sterols. Two of the sterols reported by previous researchers were found in the analysis of the wild-type cells, ergosta-7,25(27)-dienol, and poriferasta-7,25(27)-dienol.5 The other four sterols were found in the KD7 cells as well as two C4-methyl sterols, 4,14-dimethylergosta-8,25(27)-dienol, and

(Table 3.1). The detection of these 25(27) sterols strengthened the likelihood of a functioning 25(27)-olefin pathway to ergosterol.7

Another mutant strain, KD21, was analyzed for its sterol profile. This mutant had been previously reported to synthesize a C28-7-ene, and a C28-5,7-diene and their C29- ethyl homologs.5 In this study we were able to detect the four sterols previously reported as well as cycloartenol and obtusifoliol once again (Table 3.1).

With the use of analytical HPLC, and using two different columns (Luna, TSK gel), four sterols were isolated from KD7 and two from KD21. These six sterols were submitted for 1HNMR, and compared to reference materials from this lab to confirm their identity.13-15 These sterols were shown to contain one or more features of a 22E double bond, a C24-methyl or ethyl, and/or a 25(27) double bond in the side chain (Table 3.1).7

By using a combination of characterization tools, (MS, UV, 1HNMR) it was clear that both KD7 and KD21 sterols possessed either a 7 or 5,7 sterol nucleus.

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Table 3.2. Diagnostic signals for sterols submitted for 1HNMR from Chlamydomonas reinhardtii. Entry Sterol H-18 H-19 H-21 H-26 H-27 H-28 H-29 H-30 H-31 H-32 Olefinic protons 0.33 0.97 (s), 0.89 1.68 1.61 0.97 0.81 0.89 1 cycloart-24(25)-enol - - H24 - 5.11 (t) (s) 0.56 (d) (d) (s) (s) (s) (s) (d) 0.33 24-Methylcycloart- 0.96 (s), 0.859 1.64 4.67 1.00 0.81 0.97 0.90 2 - H27 - 4.67 (br d) 25(27)-enol (s) 0.55 (d) (br s) (br d) (d) (s) (s) (s) (d) 0.31 4.68 0.96 (s), 1.03 1.03 (s), 0.94 0.79 0.88 H28 - 4.68 (s), 3 cycloart-24(28)-enol 0.896 - (s) 0.53 (d) (d) 4.72 (s) (s) (d) 4.72 (br s) (d) (d) Ergosta-5,7,22- 0.63 0.95 1.04 0.82 0.84 0.92 9 - - - - 6, 7, 22, 23 trienol (s) (s) (d) (d) (d) (d) 0.596 0.924 0.837 0.75 0.77 0.92 H6- 5.56 (dd), 11 Ergosta-5,7-dienol - - - - (s) (s) (d) (d) (d) (d) H7 5.37 (dd) 4,14- 0.70 0.96 0.881 1.21 4.68 0.96 0.80 0.84 0.87 21 dimethylporiferasta- - H27 - 4.68 (br d) (s) (s) (d) (s) (br d) (d) (t) (d) (s) 8,25(27)-dienol Poriferasta-5,7- 0.60 0.92 0.84 0.79 0.81 0.92 H6 - 5.55 (dd), 24 - - - - dienol (s) (s) (d) (d) (d) (d) H7 - 5.39 (dd)

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The next strain analyzed was the KD7PY mutant strain generated by crossing the

KD7 mutant with wild type cells.7 This unique strain synthesized 13 sterols that were able to be detected, three of which were 4-methyl sterols that were undetected in the other strains. Cyclolaudenol and 24(28)-methylenecycloartenol were detected in minuscule amounts in the KD7PY strain as well as small amounts of 4,14- dimethylporiferasta-8,25(27)-dienol. The mass spectra of 24(28)-methylenecycloartenol and cyclolaudenol are nearly identical making identification by mass spectra alone nearly impossible (Figure 3.3). As previously reported in this lab, it was observed that these two structural isomers coelute on a GC packed column.16 The coelution of 25(27) and 24(28) sterols has also been noted by other researchers in this field.5 Using techniques developed in this lab from previous experiments, and with the aid of a capillary GC column, the separation of 25(27) and 24(28) sterols is possible. It has been noted that the 25(27) sterol will elute off of the column first followed by the 24(28) by a retention factor

(25(27)/24(28)) of roughly 0.99.14,15

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Figure 3.3. From top to bottom. The mass spectra and compound structures for cyclolaudenol and 24(28)-methylenecycloartenol. Adapted from 7. 3.4 25-Thialanosterol Inhibitor Study

Finally, C. reinhardtii was incubated with 25-thialanosterol salt. This compound was developed to inhibit the enzymological activity of C24-sterol methyltransferase.17

The 25-thialanosterol salt was incubated with C. reinhardtii cells at varying concentrations from 1-10 μM. Analysis of the cells incubated at 1μM yielded 10 sterols.

4,14-dimethylcholesta-8,24-dienol (31-norlanosterol), 14-methylcholesta-8,24- dienol (14-methylzymosterol), and cholesta-7,24-dienol, were all previously undetected

20 Texas Tech University, Matthew B. Miller, August 2014 sterols that happen to be un-alkylated at the C24 position. This indicated that the inhibitor was working effectively.17

3.5 Isotopic labeling

Ergosterol and 7-dehydroporiferasterol are the two most abundant sterols found in the wild type cells of C. reinhardtii. Feeding studies were performed with deuterated methionine to analyze how the deuterium atoms would be incorporated into the cells. Simultaneously, control cells were analyzed for their mass spectra of these compounds at the high mass end. Significant ions were detected at M+, M+(-methyl),

M+(-water) and M+(-methyl and water). For ergosterol these ions appeared at m/z 396,

381, 378, and 363 amu respectively. 7-Dehydroporiferasterol gave ions at m/z of 410,

395, 392, and 377 amu respectively. When the sterol profile was analyzed after the feeding of deuterated methionine it was found that ergosterol had increased in mass by three amu and 7-dehydroporiferasterol had increased by five amu. Ergosterol now showed significant ions at m/z 399, 396, 384, 381, 366, and 363, this mass spectra is consistent with the incorporation of three deuterium atoms into the side chain of the sterol.1 7-Dehydroporiferasterol showed significant ions at m/z 415, 413, 410, 400, 398, and 395, this is consistent with the incorporation of five deuterium atoms into the side chain of the sterol (Figure 3.4).1 These experiments corroborate findings in other green algae where 24-methyl/ethyl alkylations are attached thru a 25(27)-olefin pathway.18

These findings support the use of a 25(27)-olefin pathway in C. reinhartii and all but rule out the existence of a 24(28)-olefin pathway, where two and four deuterium atoms would

21 Texas Tech University, Matthew B. Miller, August 2014 have been incorporated into the side chain of the C24 alkylated methyl / ethyl sterol molecules respectively (Figure 3.5).7

Figure 3.4. From left to right, the high end mass spectra of ergosterol and 7- 2 dehydroporiferasterol incubated with [methyl- H3]-methionine. Adapted from 7.

22 Texas Tech University, Matthew B. Miller, August 2014

Figure 3.5. The sterol C24-alkylation reduction pathways to alkylated products (module 2 III). Deuteriums on C24 are from incubations with [methyl- H3] methionine. Labeling at 13C27 is shown by a dot. Adapted from 7.

23 Texas Tech University, Matthew B. Miller, August 2014

CHAPTER 4

DISCUSSION

In total, 23 sterol compounds were identified from Chlamydomonas reinhardtii including ergosterol and 7-dehydroporiferasterol, several of which possess a 25(27) double bond (Table 4.1). After compiling all of the compounds detected from the different algal strains, a plausible biosynthetic pathway to ergosterol was formed (Figure

4.1). The compounds that did not fit into the pathway were believed to be adventitious side-products.7 Using the current understanding of sterol biosynthesis which suggests that cycloartenol is an important intermediary compound, because of its 24(25) double bond it can be used as both the 24(28) precursor as well as the 25(27). This information allowed the following pathway to be suggested, cycloartenol  cyclolaudenol  ergosta-

8,25(27)-dienol  ergost-7-enol  ergosta-5,7,-dienol  ergosterol. By suggesting this biosynthetic pathway the typical 24(28) sterols that are characteristic of a 24(28)-olefin pathway are replaced by 25(27) sterols, like those that were found in the present studies.

This strengthens the argument that ergosterol biosynthesis in C. reinhardtii occurs through a 25(27)-olefin pathway.7

24 Texas Tech University, Matthew B. Miller, August 2014

Table 4.1. Detected sterols with chromatographic and spectral properties from Chlamydomonas reinhardtii.

Systematic Name Structurea GC (RRTc) UV (max) MW (M+) Cycloart-24(25)-enol 1 1.43 EA 426 24-Methyl cycloart-25(27)-enol 2 1.57 EA 440 24-Methyl cycloart-24(28)-enol 3 1.59 EA 440 4, 14-Dimethyl ergosta-8,25(27)-dienol 4 1.23 EA 426 4, 14-Dimethyl ergosta-8,24(28)-dienol 5 1.25 EA 426 Ergosta-8,25(27)-dienol 6 1.15 EA 398 4, 14-Dimethyl poriferasta-8,25(27)-dienol 7 1.40 EA 440 Ergosta-7,25(27)-dienol 8 1.17 EA 398 Poriferasta-8,25(27)-dienol 9 1.29 EA 412 Ergost-7-enol 10 1.23 EA 400 Poriferasta-7,25(27)-dienol 11 1.34 EA 412 Ergosta-5,7-dienol 12 1.20 282 398 Poriferast-7-enol 13 1.39 EA 414 Ergosta-5,7,22-trienol 14 1.10 282 396 Poriferasta-5,7-dienol 15 1.35 282 412 Poriferasta-5,7,22-dienol 16 1.28 282 410 Lanosta-8,24-dienolb 17 1.33 EA 426 4, 14-Dimethyl cholesta-8,24-dienol 18 1.15 EA 412 14-Methyl cholesta-8,24-dienol 19 1.07 EA 398 Cholesta-7,24-dienol 20 1.12 EA 384 Ergosta-5,7,25(27)-trienol 21 1.18 282 396 Ergosta-5,7,22,25(27)-tetraenol 22 1.12 282 394 Poriferasta-5,7,25(27)-trienol 23 1.33 282 410 Poriferasta-5,7,22,25(27)-tetraenol 24 1.26 282 408 a Structures of sterols are shown in Figure 4.1. b Lanosterol was not detected in the cells and is given for reference purposes only.

25 Texas Tech University, Matthew B. Miller, August 2014

Figure 4.1. Proposed sterol pathway from cycloartenol to end products in Chlamydomonas reinhardtii.

A similar pathway was constructed for 7-dehydroporiferasterol, the C29 end product found in C. reinhardtii. In this pathway a 24(28)-methylene intermediate is

26 Texas Tech University, Matthew B. Miller, August 2014 needed to serve as the substrate for the second alkylation at C24.7 In the present study two intermediates were detected with the required 24(28) double bonds. 24(28)- methylenecycloartenol, and obtusifoliol. The proposed pathway is as follows, cycloartenol  24(28)-methylenecycloartenol  obtusifoliol  4,14-dimethyl poriferasta-8,25(27)-dienol, the remaining sterols in the suggested pathway follow a

25(27)-olefin route to the end product 7-dehydroporiferasterol.7

4.1 Photosynthetic lineage

In studying reported evolution traits, data from multiple groups were gathered and it was concluded that a mevalonate-independent biosynthetic route to sterols is operating in Chlorophyta, diatoms and green algae.19-21 However, other groups of alga closely related to the Streptophyta exhibit characteristics of using the acetate-mevalonate pathway to sterols that align with land plants as well as the known animal and fungal systems.1,20,22 The present study shows that C. reinhardtii exhibits unique characteristics for the C24-alkylation-reduction reactions, solidifying that green algae produce ergosterol and 7-dehydroporiferasterol through the 25(27)-olefin pathway from cycloartenol. These finding suggest a photosynthetic lineage for C. reinhardtii, because typically organisms that use lanosterol as sterol intermediates are non-photosynthetic, and organism that use cycloartenol are photosynthetic.1 Evidence shows that brown and golden algae utilize the cycloartenol - 24(28)-olefin pathway in order to produce C24-ethyl/methyl sterols.23

Diatoms have been shown to use the cycloartenol - 24(28)-olefin pathway to synthesize

C24-methyl sterols.24 Lastly, in the unique choanflagellates and dinoflagellates their

27 Texas Tech University, Matthew B. Miller, August 2014 biosynthetic sterol pathway is reported to go from lanosterol to ergosterol or C30 thru the 24(28)-olefin pathway.25-27

4.2 Evolutionary Divergence

In order to bolster phyla-specific occurrences that are noted in module III, 24- sterol methyl transferase (SMT) was cloned from the ascomyetous fungus

Paracoccidiodes brasiliensis this organism was found to utilize both lanosterol and cycloartenol transforming them to one 24(28) product each.28,29 Though this fungus can use cycloartenol as a substrate for the SMT reaction, it is a dead-end product, and only the lanosterol product of SMT is further reacted to ergosterol. It is likely that the cycloartenol product cannot be further utilized because this fungus lacks the enzymes to break the cyclopropyl ring on the nucleus.1,23 Additional research is underway to explore if 24SMT from C. reinhardtii might be able to catalyze similar reactions with both lanosterol and cycloartenol. The results of these experiments should provide more insight into the alga’s ancestry.7

Though the data is inconclusive, there are subtle differences in de novo synthesis of how fungi make ergosterol relative to that of C. reinhardtii. Evidence supports most fungi starting with the acetate-mevalonate pathway to 3-IPP (isopentyl pyrophosphate) in module I. Green alga, like C. reinhardtii are suggested to use the mevalonate- independent methylerythritol 4-phosphate pathway to get to 3-IPP. In module II, fungi use the isoprenoid pathway to lanosterol, whereas green algae use the same pathway but make cycloartenol. Module III has fungi using the 24(28) pathway to reach ergosterol.

However alga, as reported here, use the 25(27) pathway to reach the same end product.7 28 Texas Tech University, Matthew B. Miller, August 2014

This highlights that there are significant differences in the routes taken by different organisms to synthesize ergosterol. It has been postulated that this is likely due to convergent evolution in the ancestors of these organisms while trying to find the best way to make sterol membrane inserts.7

One theory is that ancestral species of fungi may have lost the means to synthesize cycloartenol as a result of mutation.7 This could be due to “channel switching” in the cycloartenol synthase causing a change in product profile to now form lanosterol.1,7

Considering thermodynamic rational, lanosterol would be favored over cycloartenol in module II; whereas in module I green algae and fungi were both thought to operate under the acetate-mevalonate pathway. The occurrence of lanosterol synthases in a minority of algae and land plants and dinoflagellates operating a 25(27)-olefin pathway in order to

12,26,30-32 make C27 alkylated C30 sterols supports this theory.

It is also postulated that “channel switching” may at least partially explain the occurrence of multiple products from C24-methyl transferase. This event, which has phylogenetic ramifications, is likely due to evolution and could elucidate findings in higher plants and both fungi and algae that make sitosterol and ergosterol respectively.

Even if these enzymes are a product of mutations and functional divergence, the organization of the modules and the reactions contained by the modules to create the final sterols, still remains a mystery.33 It is hoped that as more information is gathered about the chemical and mechanistic nature of these enzymes through developing technologies such as X-ray crystallography, sequence alignments and other experiments, that our understanding of how the sterolome evolved will become more complete.

29 Texas Tech University, Matthew B. Miller, August 2014

BIBLIOGRAPHY

1. Nes, W. D. Biosynthesis of cholesterol and other sterols. Chem. Rev. 2011, 111, 6423-6451. 2. Kurzchalia, T. V. and Ward, S. Why do worms need cholesterol?. Nat. Cell Biol. 2003, 5(8), 684-688. 3. Feiser, L. F. and Feiser, M. Steroids. Reinhold, New York, 1959. 4. Brumfield, K. M., Moroney, J. V., Moore, T. S., Simms, A. and Donze, D. Functional characterization of the Chlamydomonas reinhardtii ERG3 ortholog, a gene involved in the biosynthesis of ergosterol. PLoS ONE. 2010. 5, e8659. 5. Bard, M., Wilson, K. J., and Thompson, R. M. Isolation of sterol mutants inChlamydomonas reinhardi: Chromatographic analyses. . 1978. 13(8), 533- 539. 6. Kanagasabai, R., Zhou, W., Liu, J., Nguyen, T. T. M., Veeramachaneni, et al. Disruption of ergosterol biosynthesis, growth, and the morphological transition in Candida albicans by sterol methyltransferase inhibitors containing sulfur at C-25 in the sterol side chain. Lipids. 2004. 39(8), 737-746. 7. Miller, M. B., Haubrich, B. A., Wang, Q., Snell, W. J., and Nes, W. D. Evolutionarily conserved Δ25(27)-olefin ergosterol biosynthesis pathway in the alga Chlamydomonas reinhardtii. J. Lipid Res. 2012. 53(8), 1636-1645. 8. Salimova, E., Boschetti, A., Eichenberger, W., and Lutova, L. Sterol mutants of Chlamydomonas reinhardtii Characterisation of three strains deficient in C24(28) reductase. Plant Physiol Biochem. 1999. 37(4), 241-249. 9. Gealt, M. A., Adler, J. H., and Nes, W. R. The sterols and fatty acids from purified flagella of Chlamydomonas reinhardi. Lipids. 1981. 16(2), 133-136. 10. Matthew, T., Zhou, W., Rupprecht, J., Lim, L., Thomas-Hall, et al. The metabolome of Chlamydomonas reinhardtii following induction of anaerobic H2 production by sulfur depletion. J. Biol. Chem. 2009. 284(35), 23415-23425. 11. Schwender, J., Gemünden, C., and Lichtenthaler, H. K. Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids. Planta. 2001. 212(3), 416-423. 12. Giner, J. L., Wünsche, L., Andersen, R. A., and Djerassi, C. Dinoflagellates cyclize squalene oxide to lanosterol. Biochem. Syst. Ecol. 1991. 19(2), 143-145. 13. Zhou, W., Cross, G. A., and Nes, W. D. Cholesterol import fails to prevent catalyst- based inhibition of ergosterol synthesis and cell proliferation of Trypanosoma brucei. J. Lipid Res. 2007. 48(3), 665-673. 14. Dennis, A. L., and Nes, W. D. Sterol methyl transferase. Evidence for successive C- methyl transfer reactions generating Δ24(28) -and Δ25(27)-olefins by a single plant enzyme. Tetrahedron Lett. 2002. 43(39), 7017-7021. 15. Zhou, W., Lepesheva, G. I., Waterman, M. R., and Nes, W. D. Mechanistic analysis of a multiple product sterol methyltransferase implicated in ergosterol biosynthesis in Trypanosoma brucei. J. Biol. Chem. 2006. 281(10), 6290-6296. 16. Nes, W. D., Norton, R. A., Crumley, F. G., Madigan, S. J., and Katz, E. R. Sterol phylogenesis and algal evolution. Proc. Natl. Acad. Sci. 1990. 87(19), 7565-7569.

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17. Kanagasabai, R., Zhou, W., Liu, J., Nguyen, T. T. M., Veeramachaneni, P. et al. Disruption of ergosterol biosynthesis, growth, and the morphological transition in Candida albicans by sterol methyltransferase inhibitors containing sulfur at C-25 in the sterol side chain. 2004. Lipids. 39(8), 737-746. 18. Goad, L. J., Lenton, J. R., Knapp, F. F., and Goodwin, T. W. side chain biosynthesis. 1974. Lipids, 9(8), 582-595. 19. Lombard, J., and Moreira, D. Origins and early evolution of the mevalonate pathway of isoprenoid biosynthesis in the three domains of life. Mol. Biol. Evol. 2011. 28(1), 87-99. 20. Lichtenthaler, H. K. Evolution of and isoprenoid biosynthesis in photosynthetic and non-photosynthetic organisms. Annu. Rev. Plant. Biol. 1999. 50, 47-65. 21. Cvejić, J. H., and Rohmer, M. CO2 as main carbon source for isoprenoid biosynthesis via the mevalonate-independent methylerythritol 4-phosphate route in the marine diatoms Phaeodactylum tricornutum and Nitzschia ovalis. Phytochemistry. 2000. 53(1), 21-28. 22. Nes, W. R. The biochemistry of plant sterols. Adv. Lipid Res. 1977. 15, 233-324 23. Goodwin, T. W. Biosynthesis of plant sterols and other triterpenoids. Biosynthesis of isoprenoid compounds. 1981. 1, 443-480. 24. Rubinstein, I., and Goad, L. J. Occurrence of (24S)-24-methylcholesta-5,22E-dien- 3β-ol in the diatom Phaeodactylum tricornutum. Phytochemistry. 1974. 13(2), 485- 487. 25. Giner, J. L., and Djerassi, C. Biosynthetic studies of marine lipids. 33. Biosynthesis of dinosterol, peridinosterol and gorgosterol: unusual patterns of bioalkylation in dinoflagellate sterols. J. Org. Chem. 1991. 56(7), 2357-2363. 26. Goad, J., and Akihisa, T. Analysis of Sterols. Blackie Academic and Professional, New York. 1997. 27. Kodner, R. B., Summons, R. E., Pearson, A., King, N., and Knoll, A. H. Sterols in a unicellular relative of the metazoans. Proc. Natl. Acad. Sci. 2008. 105(29), 9897- 9902. 28. Pereira, M., Song, Z., Santos-Silva, L. K., Richards, M. H., Nguyen, T. T. M., et al. Cloning, mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in biosynthesis. Biochim. Biophys. Acta. 2010. 1801(10), 1163-1174. 29. Nes, W. D., Koike, K., Jia, Z., Sakamoto, Y., Satou, T., et al. 9β, 19-Cyclosterol analysis by 1H and 13C NMR, crystallographic observations, and molecular mechanics calculations. J. Am. Chem. Soc. 1998. 120(24), 5970-5980. 30. Xue, Z., Duan, L., Liu, D., Guo, J., Ge, S., et al. Divergent evolution of oxidosqualene cyclases in plants. New Phytol. 2012. 193(4), 1022-1038. 31. Kolesnikova, M. D., Xiong, Q., Lodeiro, S., Hua, L., and Matsuda, S. Lanosterol biosynthesis in plants. Arch. Biochem. Biophys. 2006. 447(1), 87-95. 32. Ohyama, K., Suzuki, M., Kikuchi, J., Saito, K., and Muranaka, T. Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis. Proc. Natl. Acad. Sci. 2009. 106(3), 725-730.

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33. Neelakandan, A. K., Song, Z., Wang, J., Richards, M. H., Wu, X., et al. Cloning, functional expression and phylogenetic analysis of plant sterol 24C- methyltransferases involved in sitosterol biosynthesis. Phytochemistry. 2009. 70(17), 1982-1998.

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APPENDIX

Appendix A.1. Ergosta-5,7,22,25(27)-tetraenol.

33 Texas Tech University, Matthew B. Miller, August 2014

Appendix A.2. Ergosta-5,7,22-trienol.

34 Texas Tech University, Matthew B. Miller, August 2014

Appendix A.3. Ergosta-5,7,25(27)-trienol.

35 Texas Tech University, Matthew B. Miller, August 2014

Appendix A.4. Ergosta-5,7-dienol.

36 Texas Tech University, Matthew B. Miller, August 2014

Appendix A.5. Ergosta-7,25(27)-dienol.

37 Texas Tech University, Matthew B. Miller, August 2014

Appendix A.6. Poriferasta-5,7,25(27)-trienol.

38 Texas Tech University, Matthew B. Miller, August 2014

Appendix A.7. Poriferasta-5,7,22,25(27)-tetraenol.

39 Texas Tech University, Matthew B. Miller, August 2014

Appendix A.8. Poriferasta-5,7,22-trienol.

40 Texas Tech University, Matthew B. Miller, August 2014

Appendix A.9. Poriferasta-5,7-dienol.

41