Nawabi et al. 1

Journal Section: Biotechnology 1

Engineering E. coli for biodiesel production utilizing a bacterial fatty acid 2

methyltransferase 3

Running Title: Engineering E. coli for biodiesel production 4

5

Parwez Nawabia,b,1, Stefan Bauera, Nikos Kyrpidesa,b and Athanasios Lykidisa,b,1 6

7

aEnergy Bioscience Institute, University of California, Berkeley, CA 94720 8

bDepartment of Energy, Joint Genome Institute, Walnut Creek, CA 94598 9

10

1Corresponding Authors: 11

Athanasios Lykidis and Parwez Nawabi 12

DOE-Joint Genome Institute 13

2800 Mitchell Drive 14

Walnut Creek, CA 94598 15

Tel: 925-296-2570 16

Fax: 925-296-5850 17

Email: lykidis71@yahoo.com or pnawabi@lbl.gov 18

19

20

Abbreviations: FAME, fatty acid methyl ester; 3-OH-FAME, 3-hydroxy fatty acid methyl ester; 21

FFA, free fatty acid; 3-OH-FFA, 3-hydroxy free fatty acid; AdoMet, S-adenosylmethionine; 22

FAMT, fatty acid methyltransferase; FAT, fatty acyl-acyl carrier protein thioesterases.23

Formatted: Numbering: Continuous

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.05046-11 AEM Accepts, published online ahead of print on 16 September 2011

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ABSTRACT 24

The production of low cost biofuels in engineered microorganisms is of great interest due to the 25

continual increase in the world’s energy demands. Biodiesel is a renewable fuel that can 26

potentially be produced in microbes cost effectively. Fatty acid methyl esters (FAME) are a 27

common component of biodiesel and can be synthesized either from triacylglycerol or free fatty 28

acids (FFA). Here we report the identification of a novel bacterial fatty acid methyltransferase 29

(FAMT) which catalyzes the formation of FAMEs and 3-hydroxyl fatty acid methyl esters (3-30

OH-FAMEs) from the respective free acids and S-adenosylmethionine (AdoMet). FAMT 31

exhibits a higher specificity towards 3-hydroxy fatty acids (3-OH-FFAs), compared to FFAs, 32

synthesizing 3-hydroxy fatty acid methyl esters (3-OH-FAMEs) in vivo. We have also identified 33

bacterial members of the fatty acyl-ACP thioesterase (FAT) enzyme family with distinct acyl 34

chain specificities. These bacterial FATs exhibit increased specificity towards 3-hydroxy-acyl-35

ACP, generating 3-OH-FFAs which can subsequently be utilized by FAMTs to produce 3-OH-36

FAMEs. PhaG (3-hydroxyacyl ACP:CoA transacylase) constitutes an alternative route to 3-37

OH-FFA synthesis; coexpression of PhaG with FAMT led to the highest accumulation of 3-OH-38

FAMEs and FAMEs. The availability of AdoMet, the second substrate for FAMT, is an 39

important factor regulating the amount of methyl esters produced by bacterial cells. Our results 40

indicate that deletion of the global methionine regulator metJ and overexpression of methionine 41

adenosyltransferase results in increased methyl ester synthesis. 42

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INTRODUCTION 47

Biofuel research has recently focused on the synthesis of high energy density molecules 48

that would serve as gasoline or diesel substitutes. Molecules like butanol, isobutanol, fatty 49

alcohols, fatty acid ethyl esters, and long-chain hydrocarbons have high energy density, limited 50

water solubility and are compatible with existing infrastructure. The cellular pathways that have 51

recently attracted attention are the Clostridial pathway for isopropanol and butanol synthesis 52

(12), the amino acid pathway for the synthesis of higher alcohols (3) and the fatty acid pathway 53

for the production of fatty acids (16), fatty alcohols (29), fatty acid ethyl esters (15, 29) and long-54

chain hydrocarbons (Fig. 1) (5, 24, 31, 32). 55

Biodiesel is defined as fatty acid mono-alkyl esters and fatty acid methyl esters (FAMEs) 56

are the most common form. Currently FAMEs are synthesized predominantly via the 57

transesterification of triacylglycerols, coming mainly from plant oils. However, biodiesel 58

production from plant oils encounters various limitations, particularly the availability of oil-seed 59

supplies in suitable quantities and at competitive prices. Direct intracellular FAME synthesis in 60

bacteria is an attractive alternative to current methods of biodiesel production. It bypasses the 61

transesterification and subsequent purification steps, potentially increasing energy yields and 62

lowering production costs. Direct FAME synthesis could be achieved using the previously 63

described fatty acid O-methyltransferase (EC 2.1.1.15) (FAMT) enzyme. This enzymatic 64

activity which utilizes S-adenosylmethionine (AdoMet) to methylate the carboxyl group of FFA 65

has been described in Mycobacteria, however no genes have been cloned or enzymes purified 66

(Fig. 1) (1). 67

Engineering bacteria for FAME production via FAMT requires intracellular production 68

of both FFA and AdoMet. Bacterial fatty acid biosynthesis proceeds via a cytosolic multi-69

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enzyme system (38). Fatty acid synthesis in E. coli is tightly regulated at multiple points and is 70

coupled to membrane phospholipid biosynthesis by transcriptional and biochemical controls 71

(19). In bacteria there is no specific mechanism for terminating acyl chain elongation; however, 72

plants have a specific class of enzymes, fatty acyl-ACP thioesterases (FAT, EC 3.1.2.14), that 73

terminate acyl chain elongation by hydrolyzing the thioester bond of acyl-ACP thus releasing 74

FFAs. Expression of plant medium-chain FAT in an E. coli strain deficient in fatty acid 75

oxidation results in the accumulation of FFAs in the bacterial culture (10, 11, 14, 20, 23, 35, 37). 76

In addition to thioesterases, PhaG, a transferase involved in polyhydroxyalkanoate biosynthesis 77

has been reported to intercept the growing acyl chain during fatty acid biosynthesis (22). PhaG 78

is a 3-hydroxyacyl-ACP:Coenzyme A transferase (13) and catalyzes the transfer of 3-hydroxy-79

acyl groups from ACP to CoA. PhaG expression in E. coli leads to accumulation of 3-hydroxy 80

decanoate (40). AdoMet, the second substrate of FAMT, is synthesized by the action of 81

methionine adenosyltransferase (MAT), which catalyzes the reaction between methionine and 82

ATP (17, 18). AdoMet, in turn, regulates methionine levels by interacting with the global 83

methionine regulator MetJ (26, 36). MetJ is a repressor that controls the expression of the genes 84

involved in methionine biosynthesis (28, 30, 36) and increased levels of AdoMet down-regulates 85

the expression of methionine biosynthetic genes. 86

In this report we describe identification of a bacterial FAMT and the engineering of E. 87

coli to produce FAMEs and 3-OH-FAMEs by expressing FAMT and novel bacterial FATs that 88

exhibit distinct specificities. Production of FAMEs was further enhanced by increasing 89

intracellular AdoMet levels, by deleting metJ and overexpressing MAT. 90

91

MATERIALS AND METHODS 92 93

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Reagents. 3-OH-FFAs and 3-OH-FAMEs were purchased from Matreya. FFAs and FAMEs 94

were purchased from Sigma. BL21(DE3) and BL21(DE3)pLysS E. coli cells were purchased 95

from Novagen. Restriction enzymes were purchased from New England Biolabs. S-96

adenosylmethionine was purchased from Sigma-Aldrich and S-adenosyl-L-methionine [Methyl-97

3H] was purchased from MP Biomedicals. Gene synthesis was performed by Genscript Inc. The 98

full length rat MAT clone was purchased from Invitrogen. Genomic DNA for Clostridium 99

acetobutylicum ATCC824, Geobacter metallireducens, Mycobacterium marinum, and 100

Mycobacterium smegmatis was purchased from ATCC. Double expression pETDuet and 101

pCDFDuet vectors were purchased from Novagen. 102

Bacterial strains. The Keio collection ΔmetJ mutant (4) was purchased from the Yale E. coli 103

genetic stock center and the metJ mutation was transduced into BL21(DE3) E. coli strain using 104

P1 vir phage transduction. Kanamycin resistant colonies for ΔmetJ mutants were selected and 105

verified. Integration of the kanamycin cassette was determined by PCR, using the primers 106

metJfor 5′-CGGTAACGCCTGTACGGTAAACTATG and metJrev 5′-107

GTCCATGTATAAAAAGCGGTGGGTCGC which are external to the site of integration. A 108

PCR fragment of 1.6 kb, which was sequenced, confirmed integration of the kanamycin cassette 109

into the metJ site. All DNA sequencing was performed at the UC Berkeley sequencing facility. 110

Cloning. Rat MAT was PCR amplified from the full-length clone (clone ID 7368255, 111

Invitrogen) using primers ratMAT-Nde1F: 5′-112

GCACCATATGAATGGACCTGTGGATGGCTTGTGTGAC, ratMAT-Kpn1R: 5′-113

GCACGGTACC AAACACAAGCTTCTTGGGGACCTC and cloned into the NdeI- KpnI sites 114

of pETDuet vector generating an in frame S-tagged protein. 115

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The CAC_3591 gene was PCR amplified from C. acetobutylicum genomic DNA and inserted 116

into pCDFDuet vector within BamHI and PstI sites to generate pCDF-CaFAT; the primers used 117

were: cacFATfor: 5′-CGGGATCCGTCAAAGGTTGTTACTAAAAGAA and cacFATrev: 5′-118

GGCTGCAGTTATGATTTAATAAAATCAGTCTTTATTA. 119

Mmar_3356 and Msmeg_4347 were amplified from M. marinum and M. smegmatis genomic 120

DNA, respectively, and inserted into pETDuet vector within the EcoRI and HindIII sites to 121

generate pETDuet-MmFAMT and pETDuet-MsmegMT; the primers used were: MmFAMTfor, 122

5′-CCGGAATTCGCCACGGGAGATCAGGCTG; MmFAMTrev 5′-123

CCGAAGCTTTCAGGCGCGCTTGGCAAG; MsmegMTfor: 5′-124

CCGGAATTCGCCCAAATTCCGAGTGGC; and MsmegMTrev: 5′-125

CCGAAGCTTTCAGCCCGAGCGGCG. 126

FAT genes from C. phytofermentans, C. sporogenes, C. tetani, and M. marinum as well as the 127

Pseudomonas putida phaG and tesB from E. coli were synthesized by Genscript and cloned into 128

the BamHI-NotI sites of pCDFDuet to generate the respective expression vectors. Synthesized 129

sequences were codon optimized for E. coli expression. 130

Bacterial growth conditions. The strains used for the thioesterase expression studies were 131

grown in flasks with Luria Broth (LB), shaking at 37oC. For the FAMT expression studies, the 132

cells were grown in flasks with M9 minimal media supplemented with 2% glucose shaking at 133

37oC. The appropriate antibiotics were added to the cultures at the following concentrations: 50 134

mg/L ampicillin, 50 mg/L kanamycin, 50 mg/L spectinomycin, 34 mg/L chloramphenicol. 135

Transcription of heterologous genes was induced by the addition of 0.25 mM Isopropyl β-D-1-136

thiogalactopyranoside (IPTG) when the cells reached an OD600 of 0.4-0.6. To determine the 137

preferred substrate for FAMT, a fatty acid mixture composed of 400 μg of each of the 3-OH 138

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FFAs: C6:0, C8:0, C10:0, C12:0, C14:0, C16:0 and C18:0 was dried in a glass vial, resuspended 139

in ethanol. 250 μL of the ethanol-fatty acid mixture was added to 50 mL of culture in FAMT 140

expressing strains at the time of induction. The same experiment was also conducted with the 141

following FFAs: C10:0, C12:0, C14:0, C16:0, C16:1, and C18:1. The shaking cultures were 142

collected 20 h after induction and lipids were extracted from cell pellets and supernatants. The 143

extracted FFAs, FAMEs and 3-OH-FAMEs were separated and quantified as described below. 144

Lipid analysis. Bacterial cultures were harvested by centrifuging cultures for 20 min at 3220 g 145

to separate the pellet from the supernatant. The pellet was resuspended in PBS followed by the 146

extraction of both the pellet and supernatant lipids with chloroform:methanol (2:1). Prior to lipid 147

extraction, 50μg of the following internal standards, FFA C17:0, ME C17:0, 3-OH C17:0, and 3-148

OHME C17:0, were added to both the pellet and supernatant. The lower organic phase was 149

extracted, evaporated, and loaded onto thin layer chromatography (TLC) Silica Gel 60 plates 150

(250-μm thick). FFAs and FAMEs were separated using the solvent petroleum ether:diethyl 151

ether:acetic acid, 95:5:1, v/v/v (FFA Rf=0.15, FAME Rf=0.41). FFAs, 3-OH FFAs and 3-OH 152

FAMEs were separated using petroleum ether:diethyl ether:acetic acid, 50:50:1, v/v/v (3-OH 153

FFA Rf=0.15, 3-OH FAME Rf=0.35, and FFA Rf=0.55). The lipids on the TLC plate were 154

visualized by amido black staining (21) and spots corresponding to FFAs and 3-OH FFAs were 155

scraped into glass vials and derivatized with methanol:sulfuric acid (96:4) at 62oC. The 156

derivatized esters were extracted with hexane and analyzed by gas chromatography. Methyl 157

esters were scraped from the TLC plate and directly extracted with hexane for analysis by gas 158

chromatography. Gas chromatography was performed in a Clarus600 gas chromatogram (Perkin 159

Elmer) equipped with an Elite-5 (Perkin Elmer) column and a flame ionization detector for 160

effluent analysis. Hydrogen was used as the carrier gas at an initial flow rate of 2ml/min for 6 161

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min dropping to 1.5 ml/min for 8 min with a rate of 0.5 ml/min. The GC program was as 162

follows: initial temperature 125oC, ramped sequentially to 185oC at 12 oC /min, 215oC at 4 163

oC/min, and 275oC at 25oC/min. 164

Enzyme purification and FAMT assays. The plasmid encoding the FAMT gene was 165

transformed into BL21(DE3)pLysS E. coli and expression was initiated by the addition of 1mM 166

IPTG. The cells were grown overnight at 20oC, harvested by centrifugation, and protein 167

purification was performed using the Ni-NTA Spin Kit (Qiagen) according to the manufacturer 168

recommendations. The in vitro FAMT assay was modeled similar to the methyl jasmonate 169

synthase assays (25). FFAs or 3-OH –FFAs were dried in glass vials, resuspended in 100 mM 170

Tris-Hcl (pH 7.8), and solubilized by sonication. The second substrate, SAM, was added to the 171

reaction in both cold and radiolabeled [Methyl-3H] (0.55 μCi per reaction) forms. The substrates 172

were combined in an in vitro reaction also containing 300 mM KCl and purified FAMT in a final 173

volume of 100 μL. The reaction was incubated for 30 min at 37oC and stopped by the addition 174

of 100 μL hexane. After the addition of hexane, the reaction was vortexed and centrifuged; 175

[Methyl-3H] levels in the organic hexane phase was determined by a liquid scintillation. 176

Mass Spectrometry. Samples representing culture pellets or supernatants were derivatized with 177

N, O-bistrimethylsilyl-trifluoracetamide/1% TMSiCl (70°C for 30 min). Injection: 1 µL 1:10 178

split, injector temperature: 280°C, oven program: 3 min isocratic at 120°C then with 20°C/min to 179

320 °C, 3 min isocratic, carrier gas: helium 1 mL/min, mass spectrum was recorded from 50-600 180

m/z at a scan rate of 2.66 scans/s. Samples were derivatized with 0.2 mL DMDS and 10 µL 181

iodine (60 mg/mL in diethyl ether) at 70°C for 30 min. After cooling to room temperature, 0.2 182

mL iso-octane/iso-propanol (9+1, v/v) and 0.4 mL sodium thiosulfate (0.5 g/10 mL) were added. 183

After vigorous mixing, the mixture was centrifuged (3000 x g, 5 min) and the upper phase was 184

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derivatized with 100 µL N, O-bistrimethylsilyl-trifluoracetamide/1% TMClSi at 70°C for 30 185

min. Injection: 1 µL 1:10 split, injector temperature: 280 °C, oven program: 3 min isocratic at 186

120 °C then with 20°C/min to 325°C, 5 min isocratic, carrier gas: helium 1 mL/min, mass 187

spectrum was recorded from 50-600 m/z at a scan rate of 2.66 scans/s. GCMS was performed on 188

an Agilent 7890A gas chromatograph equipped with a Varian FactorFour VF5-ms (30 x 0.25 189

mm x 0.25 µm) capillary column coupled to an Agilent 5975C MSD. 190

191

RESULTS 192

Identification of bacterial FAMTs. The existence of FAMT enzyme activity has been reported 193

in Mycobacteria (1). Such a reaction would presumably involve the transfer of a methyl group 194

from AdoMet to the carboxyl group of a FFA, generating FAMEs. Similar reactions have been 195

described in plants such as jasmonate methyltransferase (25), benzoate and salicylate 196

methyltransferase (6), and gibberellin methyltransferase (34). These Arabidopsis enzymes form 197

a well-defined protein family, the SABATH methyltransferase family, (Pfam03492) and A. 198

thaliana contains 24 such genes. BLAST Searches of the Mycobacterial genomes using 199

Pfam03492 identified genes belonging to the carboxyl methyltransferase enzyme family. We 200

cloned and expressed, in E. coli, two genes, one from M. marinum (Mmar_3356), and another 201

from M. smegmatis (Msmeg_4347). These genes share 52% identity in their amino acid 202

sequences and their closest homolog in the A. thaliana genome is 25% identical (Fig. S1). To 203

test whether these genes exhibit FAMT activity, we expressed Mmar_3356 and Msmeg_4347 in 204

E. coli and provided either an extracellular mixture of fatty acids or 3-OH fatty acids (Fig. 2). 205

To ensure adequate AdoMet availability we performed this set of experiments in BL21 206

metJ::kan strains. Deletion of metJ prevents the feedback inhibition of methionine synthesis, 207

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thus allowing increased levels of methionine for the production of AdoMet (36). We also 208

utilized a double expression vector to co-express rat MAT, an enzyme previously shown to 209

significantly increase AdoMet levels in E. coli (2), with the Mycobacterial methyltransferases. 210

We provided free fatty acids or 3-OH free fatty acids with acyl chain lengths ranging from 10 to 211

18 carbons. In the cultures expressing the M. marinum gene, we detected the formation of 212

FAMEs and 3-OH-FAMEs, no FAMEs were detected in the absence of a methyltransferase (data 213

not shown). The total concentration of FAMEs in the culture was 1.87 μM (Fig. 2A). 214

Approximately 40% of the FAMEs were detected in the supernatant while the rest remained in 215

the cell pellet. When cells expressing Mmar_3356 were provided with 3-OH-FFAs, we also 216

detected the formation of 3-OH-FAMEs (Fig. 2B). However, we detected a much higher 217

concentration of 3-OH-FAMEs in the culture, 25.5 μM, compared to FAMEs. Approximately 218

94% of the 3-OH-FAMEs were detected in the supernatant. The two predominant methyl esters 219

formed were 3-OH-decanoic and 3-OH-dodecanoic acid methyl esters. These data strongly 220

suggest that the M. marinum gene encodes a functional FAMT with a preference towards C10:0 221

and C12:0 3-hydroxy fatty acids. 222

We performed similar experiments with E. coli cells expressing the M. smegmatis gene. 223

The cells were treated with the same mixture of either FFAs or 3-OH-FFAs as above. In these 224

cultures, we were unable to detect FAMEs, only 3-OH-FAMEs, albeit in significantly lower 225

quantities compared to the M. marinum gene (Fig. 2C). These observations suggest that 226

although Msmeg_4347 has some residual activity against 3-OH-FFAs its primary substrate may 227

be a different molecule. 228

In order to determine if Mmar_3356 is sufficient in methyl ester production, biochemical 229

assays were developed to determine if the purified protein exhibits in vitro FAMT activity and to 230

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determine its kinetic constants (Table 1). The catalytic efficiency of Mmar_3356 was highest 231

towards C10 and C12 3-OH-FFAs as substrates. The apparent Km for C10 3-OH-FFA was 99 232

μM and for AdoMet 80 μM (Fig. 3). The Km was slightly higher for 3-OH-C12 (129 μM, Table 233

1). Kinetic studies of 3-OH FAME production as a function of 3-OH C10 concentration at 234

different fixed concentrations of AdoMet yielded parallel lines indicative of a ping-pong reaction 235

mechanism (Fig. 3D) (7-9). These data correlate well with the in vivo observations and support 236

the conclusion that Mmar_3356 has a preference for the soluble C10:0 3-OH-FFA. 237

238

Identification of bacterial thioesterases with distinct acyl group specificities. The above 239

experiments utilized exogenous fatty acids to generate methyl esters. In situ generation of fatty 240

acids in E. coli is accomplished by expression of FATs. Based on the above results we sought to 241

identify FAT enzymes that would generate intracellular 3-OH FFAs. Many experiments have 242

been performed with plant FATs (11, 14, 20, 23, 35, 37) but none have reported the generation of 243

3-OH-FFAs. Therefore, we sought to identify novel FATs with the potential to synthesize the 244

desired 3-OH-FFAs. A computational survey of available genomic data identified a plethora of 245

bacterial genes belonging to the acyl-ACP thioesterase protein family (pfam01643). These genes 246

are widely distributed and their sequences are quite divergent even in organisms of the same 247

phylogenetic group (Table S1). The pair-wise identity of these genes was between 20-45% 248

indicating their significant sequence divergence. To determine whether these genes possess 249

thioesterase activity with distinct specificity compared to their plant counterparts we cloned and 250

expressed in E. coli, 6 bacterial genes (Table S1). We chose four Clostridial genes: CAC_3591 251

from C. acetobutylicum ATCC 824; CTC00119 from Clostridium tetani E88; CPHY_0251 from 252

Clostridium phytofermentans ISDg; CLOSPO00958 from Clostridium sporogenes ATCC 15579; 253

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and two genes, Mmar_0791 and Mmar_2977 from M. marinum. For comparative purposes we 254

also expressed the A. thaliana fatA gene. Figure 4 shows the specificity of the selected bacterial 255

thioesterases. Overexpression of the A. thaliana FATa in E. coli has previously been shown to 256

lead to the formation of C16:1 and C16:0 FFAs (11), we observed the same result (data not 257

shown). Expression of the bacterial FATs leads to overproduction of FFAs although differences 258

in FAT specificities are apparent. Contrary to A. thaliana FATa, the bacterial thioesterases have 259

broader substrate specificities leading to the accumulation of saturated and unsaturated FFAs 260

with chain lengths ranging between 10 and 18 carbons. The FFAs with shorter acyl chain length 261

(C8-C12) are found mainly in the supernatant whereas FFAs with longer acyl chains are 262

distributed among the cell pellet and the supernatant. Furthermore, expression of C. 263

acetobutylicum and C. phytofermentans thioesterases leads to significant production of 3-OH-264

FFAs with acyl chain length ranging mainly from C10 to C14. In addition to saturated 3-OH-265

FFAs, expression of CAC_3591 and CPHY_0251 results in the synthesis of mono-unsaturated 266

C12 and C14 3-OH-FFAs (Figure 4A and B, Fig. S2 and S3). The identity of these compounds 267

and the position of the double bond (omega-7) were verified by mass spectrometry (Fig. S2 and 268

S3). Unlike the bacterial thioesterases, expression of the plant thioesterase (AtFATa) does not 269

lead to formation of 3-OH-FFA. 3-OH-FFAs with acyl chain length up to 14 carbons were 270

found mainly in the supernatant whereas 3-OH FFAs with longer acyl chains were partitioned 271

among the cell pellet and the supernatant (Fig. 4). 272

273

In situ generation of FAMEs and 3-OH-FAMEs. To generate strains that will synthesize 274

FAMEs from endogenously produced FFAs we co-expressed MmFAMT with AtFATa or 275

CaFATa (CAC_3591) in shake-flask experiments under aerobic conditions. Coexpression of 276

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AtFATa and MmFAMT in the presence of rMAT resulted in the formation of FAMEs (Fig. 5A). 277

The total concentration of FAMEs in the culture was 10.2 μM (Fig. 5B). Approximately 48% of 278

the FAMEs were detected in the supernatant while the rest remained in the cell pellet (Fig. 5A). 279

The resulting FAME mixture consisted mainly of methyl palmitate and methyl palmitoleate with 280

smaller quantities of methyl myristate and methyl myristoleate, matching the profile of the FFAs 281

produced by the Arabidopsis thioesterase (Fig. 5A and B). These data indicate that MmFAMT is 282

able to generate FAMEs utilizing FFAs released endogenously by a plant thioesterase. Our 283

results from Figure 2, utilizing exogenous fatty acids, indicated that MmFAMT exhibits higher 284

specificity towards 3-OH fatty acids. Thus, we co-expressed CaFAT with MmFAMT in the 285

presence of rMAT, resulting in the synthesis of 3-OH-FAMEs in a final concentration of 33.25 286

μM (Fig. 5C). The predominant methyl ester was 3-hydroxy decanoic acid methyl ester (30.3 287

μM) with a minor percentage of 3-hydroxy dodecanoic acid methyl ester (2.95 μM) (Fig. 5C). 288

The majority of the methyl esters (95%) were found in the supernatant indicating an efficient 289

mechanism for the secretion of 3-OH-FAMEs. 290

The total quantity of 3-hydroxydecanoic acid produced by CaFAT makes up less than 291

33% of the total FFAs and 3-OH-FFAs (Fig. 4A). Thus, in order to maximize 3-OH-FAME 292

production, a more efficient enzyme is desirable for 3-OH FFA production. An alternative 293

pathway for synthesizing in situ 3-OH FFAs involves the coexpression of the (R)-3-294

hydroxydecanoyl-ACP:CoA transacylase gene (phaG) from Pseudomonas putida and 295

thioesterase II (tesB) from E. coli (39, 40). Previous work has shown that coexpression of phaG 296

and tesB, results in significantly higher 3-hydroxydecanoic acid production compared to the 297

thioesterases we tested (39, 40). When we co-expressed phaG and tesB, in addition to 3-OH 298

C10:0, the predominant 3-OH-FFA detected were 3-OH C14:1, 3-OH C14:0, 3-OH C16:1, and 299

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3-OH C16:0 FFAs (Fig. 6A). Coexpression of phaG and tesB with FAMT resulted in 300

accumulation of 3-OH-FAMEs, at 24 and 48 h post-induction, reaching a concentration of 70.5 301

μM/L at 48 h (Fig. 7). The majority of these 3OH-methyl esters (~91%) consisted of 3-OH 302

C10:0. In addition to 3-OH-FAMEs, we also detected the production of FAMEs at 24 h 303

(~15μM) and at 48 h (~10μM) (Fig. 7). It is not clear why there is a decrease in FAME 304

accumulation from 24 to 48 h, there may be some loss due to the volatile nature of FAMEs or 305

turnover of FAMEs. All of the FAME detected outside the cell corresponded to C12:0. A 306

separate set of experiments, that included triclosan, a FabI inhibitor implicated in increasing 3-307

OH-C10:0 production (39, 40) in cells expressing phaG and tesB, led to a slight decrease in all of 308

the 3-OH-FFAs; no increase in 3-OH-C10:0 was observed (Fig. 6B). Addition of triclosan in 309

phaG, tesB, and FAMT expressing cells did not result in an increase in methyl ester production, 310

suggesting that triclosan does not significantly affect fatty acid production in our experimental 311

system. 312

In order to determine how FAME production affects cell growth, we analyzed the growth 313

rate of cultures transformed with plasmids encoding the genes for phaG, tesB, FAMT, and rMAT. 314

Compared to uninduced cultures, the induced cells grew at a significantly slower rate, however, 315

after about 30 h post-induction their growth had reached within 75% of uninduced cells (Fig. 316

6C). Analysis of methyl esters revealed undetectable levels until 20 h post-induction and 317

maximal accumulation was detected at 48 h post-induction (Fig. 7C). 318

319

Regulation of FAME and 3-OH FAME synthesis by methionine metabolism. An important 320

factor in FAME and 3-OH FAME biosynthesis via FAMT is the concentration of AdoMet. 321

Previous work reported that expression of rMAT in E. coli leads to increased intracellular levels 322

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of AdoMet (2). To test whether the expression of rMAT leads to increased FAME synthesis we 323

cloned and expressed rMAT. Coexpression of rMAT with CaFATa and MmFAMT in wild-type 324

BL21 E. coli did not result in any significant increase in 3-OH-FAME synthesis (Fig. 8). 325

MetJ has been described as a major regulator of methionine biosynthesis acting by sensing 326

AdoMet levels and repressing the transcription of the genes operating in the methionine 327

biosynthetic pathway. Experiments utilizing a metJ deletion mutant resulted in an increase of 3-328

OH-FAME synthesis when CaFAT and MmFAMT were co-expressed (Fig. 8). In addition, 329

expression of rMAT in metJ mutants resulted in a further increase of 3-OH-FAMEs. These 330

results indicate that intracellular AdoMet concentrations are important determinants of methyl 331

ester formation. 332

333

DISCUSSION 334

The fatty acid biosynthetic pathway has taken a central stage in the efforts to generate 335

advanced biofuels (Fig. 1). We report the identification and characterization of a novel 336

methyltransferase that methylates fatty acids and 3-OH fatty acids utilizing AdoMet as the 337

methyl donor. Our bioinformatic analysis identified several Mycobacterial genes as potential 338

FAMTs; we cloned and tested two genes from M. marinum and M. smegmatis. We have 339

demonstrated both in vivo and in vitro that the M. marinum gene is a FAMT and its expression in 340

E. coli leads to FAME and 3-OH-FAME accumulation. In contrast, we were unable to determine 341

any activity for the M. smegmatis gene although its overall identity to Mmar_3356 is relatively 342

high (52%), indicating that the two genes have distinct enzymatic activities. The M. smegmatis 343

gene may have an activity against a fatty acid that is absent from E. coli, or some other variation 344

of the fatty acid core structure. Mycobacteria synthesize a plethora of fatty acid structures, 345

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saturated and unsaturated meroacids with variable degrees of substitutions (33) and it is possible 346

that the M. smegmatis gene product methylates one of these structures. Alternatively, the M. 347

smegmatis gene may methylate a molecule unrelated to fatty acids. 348

The in vitro assays indicate that MmFAMT is active on soluble substrates C10:0, C12:0 349

and C14:0. The Km for 3-OH C10:0 and 3-OH C12:0 fatty acids are essentially identical 350

whereas the Km for 3-OH C14:0 is approximately three times lower. Based on these Km values, 351

we would still expect to produce significantly higher levels of 3-OH C12:0 and 3-OH C14:0 352

methyl esters in cells generating in situ fatty acids via thioesterases, particularly CAC3591 which 353

generates significant amounts of saturated and unsaturated 3-OH C14 (Fig. 4A). We attribute 354

these observations to substrate solubility. Most probably 3-OH tetradecanoic acid is located on 355

the membrane exhibiting limited solubility and, therefore, is inaccessible to MmFAMT which is 356

clearly a soluble enzyme. 357

The higher specificity of the MmFAMT towards 3-OH fatty acids prompted us to 358

investigate the existence of FATs that would be able to generate 3-OH fatty acids. Plant FATs 359

have received broad attention since they are able to deregulate E. coli fatty acid biosynthesis. 360

However, no publication has reported the generation of 3-OH FFAs from plant thioesterases. 361

Therefore, we decided to investigate whether the bacterial homologs of plant FATs had similar 362

activity. All six genes that we tested increase FFA levels indicating that they are active 363

thioesterases. However, only two genes (CAC_3591 and CPHY_0251) generated significant 364

quantities of 3-OH FFAs indicating that this activity is not shared by all thioesterases. Although 365

we did not perform direct in vitro assays we postulate that these FATs act on acyl-ACP rather 366

acyl-CoA because we obtained similar results when we used fadD mutants which lack acyl-CoA 367

synthetase activity (data not shown). 3-OH FFAs have been described in the literature as 368

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effective antifungal compounds secreted by Lactobacillus plantarum (27). This is a probable 369

physiological function of the two bacterial FATs that generate 3-OH-FFAs. However, the 370

physiological function of the bacterial thioesterases that do not release 3-OH-FFAs remains 371

unclear. 372

A combination of FATs and FAMTs leads to FAME and 3-OH FAME production (Fig. 373

5). Besides FATs, expression of PhaG constitutes an alternative route to 3-OH-FFA production. 374

Combined expression of the PhaG-TesB with FAMT leads to the highest concentrations of 375

FAMEs and 3-OH-FAMEs, (Fig. 7) indicating that this pathway maybe more efficient in 376

intercepting the growing acyl chain than the FATs studied above. 377

In addition, methyl ester production also depends on the intracellular AdoMet 378

concentrations. Our data suggest the expression of the MAT enzyme alone is not enough to 379

increase methyl ester production. A deregulation of the transcriptional network that controls 380

methionine and AdoMet biosynthesis by deleting the global regulator MetJ is necessary to 381

increase methyl ester synthesis. These results are consistent with the current model of 382

methionine and AdoMet biosynthesis which places MetJ as a central regulator that responds to 383

elevated AdoMet concentrations and downregulates the methionine biosynthesis pathway. 384

The work described in this report provides an alternative route for the synthesis of biofuel 385

molecules. Direct intracellular synthesis of FAMEs and 3-OHFAMEs in E. coli bypasses the 386

extraction and transmethylation steps currently necessary in biodiesel production. The 387

coexpression of PhaG, TesB, FAMT, and rMAT achieved the highest yield of biodiesel, 80.5 uM 388

(16 mg/L). The FAME yields we achieved is roughly 40 times less than maximal fatty acid ethyl 389

esters reported and ~19 times less than that of alkanes (24, 29). However, our results suggest 390

that FAME production can be improved. In PhaG-TesB expressing cells, FAMT is able to 391

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convert virtually all of the 3-OH C10:0 FFA (90%) into 3-OH C10:0 FAMEs (Fig. 6C). This 392

strongly suggests that the bottleneck in methyl ester production in E. coli is due to a lack of 393

efficient thioesterase that hydrolyzes 3-OH C10:0. The main concern arising from the current 394

work is the mismatch between the specificities of the FAMT and FATs we used. MmFAMT 395

prefers soluble 3-OH C10:0 and C12:0 fatty acids; however, these fatty acids represent only a 396

minor percentage of the fatty acid pool released by the thioesterases we identified. Further work 397

will focus on identifying or engineering FAMT and FAT enzymes with similar specificities to 398

explore the limits of direct intracellular FAME synthesis. Furthermore, our in vitro assays with 399

purified FAMT shows that C12:0 fatty acid is an efficient FAMT substrate; however, FAMT 400

expressing cultures did not produce significant titers of FAME. We hypothesize that this may be 401

due to FAMT being a soluble enzyme distributed within the cytoplasm while free fatty acids are 402

predominately on the membrane, thus fatty acids are unlikely to have significant interaction with 403

FAMT. Localizing FAMT to the cytoplasmic side of the inner membrane may lead to 404

significantly higher interaction with fatty acids, therefore, significantly higher levels of FAME 405

production. The pathway described here is a first step in the generation of FAMEs and with 406

further optimization may lead to the production of a cost efficient next generation biofuel. 407

408

ACKNOWLEDGEMENTS 409

We thank Maria Billini for her excellent technical assistance. This work was funded by a grant 410

from the Energy Bioscience Institute to A. L. and by the Office of Science of the U.S. 411

Department of Energy under Contract No. DE-AC02-05CH112. 412

413

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35. Voelker, T. A., and H. M. Davies. 1994. Alteration of the specificity and regulation of 511 fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl 512 carrier protein thioesterase. J Bacteriol 176:7320-7327. 513

36. Weissbach, H., and N. Brot. 1991. Regulation of methionine synthesis in Escherichia 514 coli. Mol Microbiol 5:1593-1597. 515

37. Yuan, L., T. A. Voelker, and D. J. Hawkins. 1995. Modification of the substrate 516 specificity of an acyl-acyl carrier protein thioesterase by protein engineering. Proc Natl 517 Acad Sci U S A 92:10639-10643. 518

38. Zhang, Y. M., and C. O. Rock. 2008. Membrane lipid homeostasis in bacteria. Nat Rev 519 Microbiol 6:222-233. 520

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40. Zheng, Z., M. J. Zhang, G. Zhang, and G. Q. Chen. 2004. Production of 3-524 hydroxydecanoic acid by recombinant Escherichia coli HB101 harboring phaG gene. 525 Antonie Van Leeuwenhoek 85:93-101. 526

527 528

FIGURE LEGENDS 529

FIG. 1. Overview of the lipid biosynthetic pathways leading to biofuel related structures. 530

Expression of fatty acyl thioesterases (FAT) leads to the production of free fatty acids (FFA) 531

which can be converted to (1) fatty acid methyl esters (FAME) by FAMT using S-532

Adenosylmethionine (AdoMet) as the methyl donor or (2) fatty acid ethyl esters (FAEE) by the 533

action of a wax synthase (WS). Acyl-ACP can also be converted to fatty alcohols by expressing 534

acyl-ACP reductase (ACR) which is then converted to alkanes by aldehyde decarbonylase. The 535

circled enzymes are overexpressed in our system to produce FAME. 536

537

FIG. 2. Expression of MmFAMT leads to the formation of FAMEs and 3-OH-FAMEs. BL21 538

ΔmetJ::kan E. coli cells were transformed with double expression vectors expressing 539

Mmar_3356 (A and B) or Msmeg_4347 (C) and rat MAT. An FFA (A) or 3-OH FFA (B, C) 540

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cocktail mixture was added exogenously in each culture during induction. Lipids from cell 541

pellets and supernatants were extracted 20 h after induction and FAMEs and 3-OH FAMEs were 542

quantified as described under “Materials and Methods”. Data are representative of experiments 543

done three independent times. 544

545

FIG. 3. Kinetic characterization of MmFAMT. Kinetic parameters for 3-OH-C10-FFA (A), 546

C10-FFA (B), AdoMet (C), were determined for MmFAMT. Experiments for A and B were 547

performed at 2mM AdoMet. Experiments of variable AdoMet concentration (C) were performed 548

at 800 mM 3-OH-C10-FFA. Experiments in panel D were performed at three different AdoMet 549

concentrations: 1, 2, and 3 mM with 3-OH-C10-FFA. Enzyme assays were performed as 550

described under “Materials and Methods”. Error bars represent the standard deviation of 551

experiments done in triplicate. 552

553

FIG. 4. FFA and 3-OH-FFA production by E. coli strains expressing bacterial thioesterases. A: 554

CAC_3591, C. acetobutylicum; B: Cphy_0251, C. phytofermentans; C: Clospo00958, C. 555

sporogenes; D: CTC_0119, C. tetani; E: Mmar_2977, M. marinum; F: Mmar_0791, M. 556

marinum. Bacterial FATs were expressed in E. coli and FFAs and 3-OH-FFAs were analyzed as 557

described under “Materials and Methods”. Data are representative of experiments done three 558

independent times. 559

560

FIG. 5. Coexpression of thioesterases with MmFAMT leads to methyl ester formation. BL21 561

ΔmetJ::kan cells expressing either AtFATa (A and B) or CaFAT (C), with MmFAMT and 562

rMAT. Cultures were collected 24 h post-induction, lipids were extracted and FAMEs and 3-563

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OH-FAMEs separated by TLC and quantified by GC as described under “Materials and 564

Methods”. (A) GC chromatograms from extracts of the cell pellet and supernatant of AtFATa 565

expressing cells illustrating generation of FAMEs. (B) Relative concentrations and distribution 566

of the different FAME types in the pellet (grey bars) and supernatant (black bars) from AtFATa 567

expressing cells. (C) GC chromatograms from extracts of the cell pellet and supernatant of 568

CaFAT expressing cells illustrating production of 3-OH-FAMEs. Data are representative of 569

three independent experiments with similar results. 570

571

FIG. 6. 3-OH-FFA profiles of phaG and tesB expressing cells with and without triclosan. BL21 572

ΔmetJ::kan E. coli were transformed with a Duet expression vector expressing PhaG and TesB. 573

(A) Without triclosan (B) with triclosan. Cells were collected 48 h after induction and 3-OH 574

FFAs were determined in the cell pellet and supernatant. (C) The distribution of 3-OH FFAs in 575

the supernatant of PhaG-TesB expressing cells without FAMT (gray bars) with FAMT (black 576

bars). Data are representative of three independent experiments with similar results. 577

578

FIG. 7. Expression of phaG and tesB with FAMT leads to the production of FAME and 3-OH-579

FAME. BL21 ΔmetJ::kan E. coli were transformed with Duet coexpression vectors expressing 580

PhaG, TesB, FAMT, and rMAT. Cells were collected 24 h and 48 h after induction and (A) 581

FAMEs and (B) 3-OH FAMEs were quantified from the cell pellet and supernatant as described 582

under “Materials and Methods”. (C) Growth curves of uninduced (triangles) and induced (filled 583

squares) BL21 ΔmetJ::kan E. coli transformed plasmids encoding PhaG, TesB, FAMT, and 584

rMAT. Aliquots from each culture were collected 4, 8, 20, 30, and 48 hours postinduction and 585

analyzed for growth and methyl ester production. For the induced culture, the numbers below 586

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each time-point represents methyl ester production in μM. ND represents not detectable. Error 587

bars represent the standard deviation of triplicate experiments. 588

589

FIG. 8. MAT overexpression in ΔmetJ cells increases 3-OH-FAME production. BL21 or BL21 590

ΔmetJ::kan E. coli were transformed with Duet coexpression vectors expressing FAMT, CaFAT 591

and rMAT. Cells were collected 24 h after induction and FAMEs and 3-OH FAMEs were 592

quantified from the cell pellet and supernatant. Error bars represent the standard deviation of 593

triplicate experiments. 594

595

596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623

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Table 1. FAMT kinetic parameters for various fatty acid substrates. 624 625

FFA KM (μmol) kcat (sec-1) kcat / KM (mol/L) -1sec-1

3-OH C8:0 196±15 358±17 1.82×106

3-OH C10:0 99±6 1600±78 16.1×106

3-OH C12:0 129±11 1346±102 10.4×106

C8:0 77±4 510±32 6.6×106

C10:0 46±2 489±26 10.6×106

C12:0 103±5 1165±63 11.3×106

626

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Pyruvate

Carbon Source

AdoMet

Methionine

MAT

FIGURE 1

Alkanes

Acetyl-CoA

Acyl-ACP FFA

FAT

Acyl-CoA FOHWS

FAEEWS

FAMEFatty Alcohols

FAMTACRAld Decarb

AdoMet

FIG. 1. Overview of the lipid biosynthetic pathways leading to biofuel related structures. Expression of fatty acyl thioesterases (FAT) leads to the production of free fatty acids (FFA) which can be converted to(1) fatty acid methyl esters (FAME) by FAMT using S-Adenosylmethionine (AdoMet) as the methyl donor or (2) fatty acid ethyl esters (FAEE) by the action of a wax synthase (WS). Acyl-ACP can also be converted to fatty alcohols by expressing acyl-ACP reductase (ACR) which is than converted to alkanes b ld h d d b l Th i l d d i d FAMEby aldehyde decarbonylase. The circled enzymes are overexpressed in our system to produce FAME.

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FIGURE 2

M) 400

500

600A

FA

ME

(n

M

100

0

300

200

400

20000B

3-O

H-F

AM

E (

nM

)

4000

12000

8000

16000

0

FA

ME

(n

M)

150

200

250

300C

3-O

H-F

10:0

12:0

16:0

18:0

14:0

50

0

100

Pellet

Supernatant

FIG. 2. Expression of MmFAMT leads to the formation of FAMES and 3-OH-FAMEs. BL21 ΔmetJ:kanE. coli cells were transformed with double expression vectors expressing Mmar_3356 (A and B) or Msmeg_4347 (C) and rat MAT. An FFA (A) or 3-OH FFA (B, C) cocktail mixture was added exogenously in each culture during induction. Lipids from cell pellets and supernatants were extracted 20 h after induction and FAMEs and 3-OH FAMEs were quantified as described under “Materials and Methods”. Data are representative of experiments done three independent times.

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FIGURE 3

4

min

) A1.2n

)

B

3

1

2

03-O

H C

10:0

ME

(μm

ol/

0.01 0.020

0.2

0.81

0.40.6

0

1/v

(μm

ol/m

in)-1

1/s (μM)-1

1.2

0.4

0

C10

:0 M

E (

μmo

l/min

0.8

0.040

2

0

1/v

(μm

ol/m

in)-1

1/s (μM)-1

6

4

0.08 0.12

0

3-OH C:10 (μM)

3

200 400 600 800 10000 1200C:10 (μM)

200 400 600 800 10000 12000

0.6

0.8

1 mM

/min

)-1D2.5

1.5

2

E (

μmo

l/min

)

6

)-1

C

0.001 0.002 0.003 0.004 0.0050

0.2

0.4

0

1/s (μM)-1

0.006

3 mM2 mM

1/v

(μm

ol/

200 400 600 800 1000 1200

1

0

SAM (μM)

0.5

3-O

H C

10:0

ME

0

0.02 0.04 0.06 0.08 0.10

2

4

0

1/s (μM)-11/v

(μm

ol/m

in)

FIG. 3. Kinetic characterization of MmFAMT. Kinetic parameters for 3-OH-C10-FFA (A), C10-FFA (B), AdoMet (C), were determined for MmFAMT. Experiments for A and B were performed at 2mM AdoMet. Experiments of variable AdoMet concentration (C) were performed at 800 mM 3-OH-C10-FFA. Experiments in panel D were performed at three different AdoMet concentrations: 1, 2, and 3 mM with 3-OH-C10-FFA. Enzyme assays were performed as described under “Materials and Methods”. Error bars represent the standard deviation of experiments done in triplicaterepresent the standard deviation of experiments done in triplicate.

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100

200

300

3-O

HFA

/ O

D

40

80

120

M F

A /

OD

A FIGURE 4

80

40

60

A /

OD

B 10:0

12:1

12:0

14:1

14:0

16:0

100

nM 3

0

16:1

10:0

12:1

12:0

14:1

14:0

18:0

16:0

nM

0

HFA

/ O

D

400

600

10:0

12:1

12:0

14:1

14:0

18:0

16:1

16:0

18:1

20

40

nM F

A

0

6

A /

OD

nM 3

-OH

200

0

10:0

12:1

12:0

14:1

14:0

16:0

16:1

30

D

C

2

4nM

3-O

HFA

0

D250

60OD

10:0

12:1

12:0

14:1

14:0

16:0

16:1

12:1

12:0

14:1

14:0

18:0

16:1

16:0

18:1

10:0

10

20

nM F

A /

OD

0

10:0

12:1

12:0

14:1

14:0

18:0

16:1

16:0

18:1

50

100

150

nM F

A /

OD

0

200

250

20

40

60

nM 3

-OH

FA /

O

0

E 10:0

12:1

12:0

14:1

14:0

16:0

16:1

:0 :1 :0 :1 :0 :0:1 :0 :1

E

40

80

nM F

A /

OD

0

120

160

20

40

60

nM 3

-OH

FA /

OD

0

0 :1 0 1 0 0:1

10 12 12 14 14 1816 16 18

10

20

30

nM 3

-OH

FA /

OD

0

F

20

40

nM F

A /

OD

0

60

80

10:

12:

12:

14:

14:

16:

16:

Pellet

Supernatant

10:0

12:1

12:0

14:1

14:0

18:0

16:1

16:0

18:1

00

FIG. 4. FFA and 3-OH-FFA production by E. coli strains expressing bacterial thioesterases. A: CAC_3591, C. acetobutylicum; B: Cphy_0251, C. phytofermentans; C: Clospo00958, C. sporogenes; D: CTC_0119, C. tetani; E: Mmar_2977, M. marinum; F: Mmar_0791, M. marinum. Bacterial FATs were expressed in E. coli and FFAs and 3-OH-FFAs were analyzed as described under “Materials and Methods”. Data are representative of experiments done three independent times.

10:0

12:1

12:0

14:1

14:0

16:0

16:1

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FIGURE 5

A

900

1000

900

1000

SupernatantPellet

400

600

700

800

500mV

14:1

14:0

16:1

16:0

17:0

, st

d

400

600

700

800

500mV

14:1

14:0

16:1

16:0

17:0

, st

d

0 1 2 3 4 65 127 8 109 11

0

200

300

400

100

0 1 2 3 4 65 127 8 109 11

0

200

300

400

100

AM

E (

nM

)

1500

2000

2500

3000

Pellet

Supernatant

B

0 1 2 3 4 65 127 8 109 11Time (min)

0 1 2 3 4 65 127 8 109 11

FA

500

0

1000

16:1

12:0

16:0

18:0

14:0

14:1

900

1000

Pellet

C

900

1000

d

Supernatant

300

400

600

700

800

500mV

17:0

, st

dm

V

300

400

600

700

800

500

17:0

, st

d

12:0

10:0

Time (min)0 1 2 3 4 65 127 8 109 11

0

200

100

13 0 1 2 3 4 65 127 8 109 11

0

200

100

13

FIG. 5. Coexpression of thioesterases with MmFAMT leads to methyl ester formation. BL21 ΔmetJ:kan cellsFIG. 5. Coexpression of thioesterases with MmFAMT leads to methyl ester formation. BL21 ΔmetJ:kan cells expressing either AtFATa (A and B) or CaFAT (C), with MmFAMT and rMAT. Cultures were collected 24 h post-induction, lipids were extracted and FAMEs and 3-OH-FAMEs separated by TLC and quantified by GC as described under “Materials and Methods”. (A) GC chromatograms from extracts of the cell pellet and supernatant of AtFATa expressing cells illustrating generation of FAMEs. (B) Relative concentrations and distribution of the different FAME types in the pellet (grey bars) and supernatant (black bars) from AtFATa expressing cells. (C) GC chromatograms from extracts of the cell pellet and supernatant of CaFAT expressing cells illustrating production of 3-OH-FAMEs. Data are representative of three independent experiments with similar results.

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FIGURE 6μM

) 250- Triclosan300A

3-O

H-F

FA

(μ

50

0

150

100

200

4:1

0:0

4:0

8:1

2:0

2:1

6:1

6:0

141 14 111 1 1Pellet

Supernatant

H-F

FA

(μM

)

150

100

200

250+ Triclosan

B

3-O

H

50

0

100

14:1

10:0

14:0

18:1

12:0

12:1

16:1

16:0

M)

80

100C

3-O

H-F

FA

(μM

20

0

60

40

80

4:1

0:0

4:0

2:0

2:1

6:1

6:0

1410 141212 16 16

PhaG-TesB Supernatant

PhaG-TesB-FAMT Supernatant

FIG. 6. 3-OH-FFA profiles of phaG and tesB expressing cells with and without triclosan. BL21 ΔmetJ::kan E. coliwere transformed with a Duet expression vector expressing PhaG and TesB. (A) Without triclosan (B) with triclosan. Cells were collected 48 h after induction and 3-OH FFAs were determined in the cell pellet and supernatant. (C) The distribution of 3-OH FFAs in the supernatant of PhaG-TesB expressing cells without FAMT (gray bars) with FAMT (black bars). Data are representative of three independent experiments with similar results.

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FIGURE 7D 8

10

16OD

20

4

5

A B C

Uninduced

FA

ME

μM

/OD

24 h

2

0

4

6

48 h

4

0

8

12

16

3-O

H-F

AM

E μ

M/

40200

OD

60

0

1

0

2

3

4

10 5030

Induced

ND ND

80.5

27

44.6

Pellet

Supernatant

FIG. 7. Expression of phaG and tesB with FAMT leads to the production of FAME and 3-OH-FAME. BL21 ΔmetJ::kan E. coli were transformed with Duet coexpression vectors expressing PhaG, TesB, FAMT, and rMAT.

24 h 48 h 24 h 48 h 40200Time (h)

10 5030

Cells were collected 24 h and 48 h after induction and (A) FAMEs and (B) 3-OH FAMEs were quantified from the cell pellet and supernatant as described under “Materials and Methods”. (C) Growth curves of uninduced (triangles) and induced (filled squares) BL21 ΔmetJ::kan E. coli transformed plasmids encoding PhaG, TesB, FAMT, and rMAT. Aliquots from each culture were collected 4, 8, 20, 30, and 48 hours postinduction and analyzed for growth and methyl ester production. For the induced culture, the numbers below each time-point represents methyl ester production in μM. ND represents not detectable. Error bars represent the standard deviation of triplicate experiments.

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FIGURE 8

Pellet

Supernatant3-OHFAME

Pellet

SupernatantFAME

3

1

2

μM/O

D

3

0

+ +++

+ +++FAMT

CaFAT

MAT - +-+BL21 BL21ΔmetJ::Kan

FIG. 8. MAT overexpression in ΔmetJ cells increases 3-OH-FAME production. BL21 or BL21 ΔmetJ::kan E. coliwere transformed with Duet coexpression vectors expressing FAMT, CaFAT and rMAT. Cells were collected 24 h after induction and FAMEs and 3-OH FAMEs were quantified from the cell pellet and supernatant. Error bars represent the standard deviation of triplicate experiments.

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