Biosynthesis of Long-Chain Ï-Hydroxy Fatty Acids by Engineered

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Biosynthesis of long-chain #-hydroxy fatty acids by engineered Saccharomyces cerevisiae Jingjing Liu, Chuanbo Zhang, and Wenyu Lu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00109 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 1, 2019

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Journal of Agricultural and Food Chemistry

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Biosynthesis of long-chain ω-hydroxy fatty acids by engineered Saccharomyces

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cerevisiae

3 4

Jingjing Liu a, Chuanbo Zhang a , Wenyu Lu a,b,c*

5

a

6

300072, PR China;

7

b

8

Education, Tianjin, 300072, PR China.

9

c

10

School of Chemical Engineering and Technology, Tianjin University, Tianjin

Key Laboratory of System Bioengineering (Tianjin University), Ministry of

SynBio Research Platform, Collaborative Innovation Center of Chemical Science

and Engineering (Tianjin), Tianjin, 300350, PR China

11 12

* Corresponding

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Tel.: +86-22-27892132; fax: +86-22-27400973.

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E-mail address: [email protected]

author: Wenyu Lu

15 16 17 18 19 20 21 22 23

ACS Paragon Plus Environment


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Abstract

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Long-chain hydroxy fatty acids (HFAs) are rare in nature but have many promising

26

industrial applications. In this study, we developed a biosynthesis method to produce

27

long-chain ω- hydroxy fatty acids. Through disruption of the acyl-CoA synthetases

28

FAA1 and FAA4 and the fatty acyl-CoA oxidase POX1, a Saccharomyces cerevisiae

29

strain was engineered to accumulate free fatty acids (FFAs). Subsequently, the

30

cytochrome P450 monooxygenase CYP52M1 from Starmerella bombicola was

31

introduced to convert FFAs to HFAs, leading to the production of C16 and C18 HFAs

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at the ω or ω-1 positions. Next, CYP52M1 was reconstituted with the homologous

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reductase S. bombicola CPR and the heterologous reductase Arabidopsis thaliana

34

cytochrome P450 reductase. The results showed that the CYP52M1-AtCPR1 system

35

significantly increased in FFA the hydroxylation. Moreover, a self-sufficient P450

36

enzyme system was constructed to achieve higher transformation efficiency. Finally,

37

fed-batch fermentation yielded as much as 347 ± 9.2 mg/L ω-HFAs.

38

Key words: Long-chain ω-hydroxy fatty acids, Saccharomyces cerevisiae,

39

Cytochrome P450, Cytochrome P450 reductase, Synthetic biology

40 41


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1. Introduction

43

Depletion of energy-related resources and increased levels of CO2 in the

44

atmosphere, leading to global climate change, have driven the development of

45

environmentally friendly processes, particularly those involving microbes, for the

46

production of fuels and commercial chemicals.1-3 In addition, microbial-based

47

processes

48

chemical selectivity, both of which facilitate their industrial development.4

49

Long-chain (C14–C18) hydroxy fatty acids (HFAs) are valuable chemicals that have a

50

carboxyl and a hydroxyl group, and they can be easily converted into other derivatives

51

that exhibit good reactivity, stability, and viscosity.5 Owing to their unique properties,

52

long-chain HFAs are widely used in various fields, such as food, chemical, and

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cosmetic industries, and serve as synthetic intermediates for pseudoceramides,

54

polyesters, and cyclic lactones.6 Recently, their pharmaceutical attributes, including

55

antibiotic, anti-inflammatory, and antidiabetic effects, have also been revealed.7, 8 In

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particular, the positions of the hydroxyl group in the fatty acid chain plays a vital role

57

in its physiological mechanisms and chemical applications. Hydroxylation of the

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hydrocarbon located close to the carboxyl group results in α- or β-hydroxylation,

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which affects human health.9 Omega HFAs (ω-HFAs) are fatty acids with a hydroxyl

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group at the terminal end. These multifunctional compounds have a broad range of

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applications, including use as adhesives, lubricants, and potential anticancer

62

agents,10,11 and serve as building materials for the synthesis of polymers, such as

63

bioplastics, with high resistance to water, low toxicity, good biocompatibility, and

have less safety concerns associated with them and also have and higher



64

excellent chemical versatility.12,13

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Given their broad commercial uses, substantial effort has been put into producing

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HFAs chemically or biologically. Direct synthesis of products with the desired

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specific hydroxylation is difficult to accomplish through chemical catalysis because of

68

the limitations of selective carbon atoms and harsh reaction conditions.14

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Alternatively, microorganisms such as yeast, 15 Escherichia coli, 16, 17 Bacillus18 allow

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producing HFAs through selective enzymatic hydroxylation. HFAs can be obtained

71

via two biocatalytic reactions: (a) hydration of the double bond(s) of an unsaturated

72

fatty acid by a hydratase, and (b) the oxidation of the fatty acid by an oxidase (e.g.

73

P450 monooxygenases). For example, gamma octalactone/decalactone can be

74

synthesized from octanoic or decanoic acid through gamma hydroxylation by

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Mortierella species.19 A large number of microorganisms producing hydratases

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(oleato and linoleato hydratases) are able to introduce a hydroxyl group at position 10

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or 13 of C18 unsaturated fatty acids.20 For example, Bacillus megaterium can

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hydroxylate oleic acid on carbon atoms 1, 2, and 3 to produce different hydroxy oleic

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acids.21 Candida tropicalis produces HFAs as by-products when cultured on alkanes

80

or fatty acids as the main carbon source.22 However, the engineering of fatty acid

81

biosynthesis with a self-sufficient P450 enzymatic complex in the same heterologous

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host is a new concept.

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Saccharomyces cerevisiae is a conventional and well-studied production host for

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which mature genetic tools for metabolic pathway manipulation are available. It has

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been successfully manipulated to produce numerous valuable platform chemicals and


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biofuels.23,

24

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medium, can utilize inexpensive renewable feedstock, and has a fast growth rate,

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facilitating its broad industrial utilization.25 Therefore, S. cerevisiae is an appropriate

89

candidate for de novo synthesis of HFAs from sugars. However, wild-type S.

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cerevisiae is incapable of accumulating free fatty acids (FFAs) and subsequently

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catalyzing them to HFAs because it possess a pathway for β-oxidation. Theoretically,

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to endow yeast with the ability to convert glucose to HFAs, it is necessary to

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overproduce FFAs by blocking FFA and preventing fatty acid degradation through

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β-oxidation, transform FFAs to HFAs via exogenous enzymatic hydroxylation, and

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integrate these two pathways into one metabolic route.

Furthermore, S. cerevisiae is easily cultured in chemically defined

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First, to accumulate high levels of FFAs in S. cerevisiae, it is essential to

97

interrupt FFA reactivation and prevent fatty acid degradation through β-oxidation.26, 27

98

In S. cerevisiae, fatty acids are mainly synthesized de novo as activated fatty acids

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(fatty acyl-CoAs) by cytosolic fatty acid synthase (FAS), which is composed of two

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subunits encoded by FAS1 and FAS2.28, 29 Subsequently, downstream modifications

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are necessary for the production and excretion of FFAs. S. cerevisiae has six known

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acyl-CoA synthetases encoded by FAA1, FAA2, FAA3, FAA4, FAT1, and FAT2,30 and

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FFAs will be rapidly re-activated by these fatty acyl-CoA synthetases to fatty

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acyl-CoAs, whose accumulation feedback hinders fatty acid biosynthesis and

105

secretion.31 Previous studies have shown that FFAs are secreted and accumulate after

106

deleting

107

Δfaa1Δfaa2Δfaa3Δfaa4 knockout strain did not excrete higher levels of FAA than a

acyl-CoA

synthetases,

encoded

by

FAA1


and

FAA4,

but

a


108

Δfaa1Δfaa4 strain.27 This is consistent with earlier research showing that the majority

109

of synthetase activity is attributed to the long-chain acyl-CoA synthetases FAA1 and

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FAA4.30 β-Oxidation is the catabolism of fatty acid molecules to generate acetyl-CoA

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and provide energy in the peroxisome, and is a major contributor to the loss of fatty

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acids.32, 33 POX1, encoding the fatty acyl-CoA oxidase ， is responsible for the first

113

step of this pathway. Li et al. removed POX1 and observed a 4-fold increase in FFA

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content when compared with that in the wild-type control.34

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After FFAs accumulate in the biocatalytic system, they will be persistently

116

converted to HFAs. The four types of enzymes involved in the biocatalysis of FAAs

117

to HFAs are cytochrome P450 monooxygenase, hydratase, hydroxylase, and

118

lipoxygenase; among these, cytochrome P450 monooxygenase is known to

119

hydroxylate FFAs to HFAs.21, 35 In eukaryote systems, class II P450 enzymes along

120

with their heme donor, cytochrome P450 reductase (CPR), are mainly involved in

121

catalyzing intricate reactions, such as regio- and stereo-specific hydroxylation of

122

various endogenous and exogenous compounds,36 and these reactions are known to be

123

rate-limiting steps.37 In general, terminal or subterminal HFAs are synthesized by

124

members of microbial CYPs, including CYP52 and CYP153, through hydroxylation

125

of fatty acids at specific positions. Several CYP52 genes have been identified in

126

Starmerella bombicola, and these genes encode isozymes with diverse substrate

127

specificities.38

128

In this study, we engineered S. cerevisiae to directly produce de novo ω-HFAs

129

(Fig. 1). We then evaluated the mechanisms of ω-HFAs generation in the engineered


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strains and analyzed the yield in fed-batch fermentation. The results showed that this

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method was promising for large-scale production of long-chain ω-HFAs.

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2. Materials and Methods

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2.1 Reagents, strains, and medium

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CYP52M1 (GenBank: EU552419), S. bombicola CPR (SbCPR; GenBank: EF050789),

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and Arabidopsis thaliana CPR1 (AtCPR1; GenBank: BT008426.1) were synthesized

137

with codon optimization by Jinsirui Biotechnology Co., Ltd. (Nanjing, China) and

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were cloned into plasmid pUC57. S. cerevisiae BY4741 was used as the parent strain

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for the construction of all engineered strains. The engineered yeast strains were grown

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in auxotrophic SD plates39 containing 20 g/L glucose, 6.7 g/L yeast nitrogen base, 2

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g/L amino acid mixture (without leucine, tryptophan, uracil, adenine, and histidine for

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auxotrophs as appropriate), and 20 g/L agar. Phanta Max Super-Fidelity DNA

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Polymerase was purchased from Vazyme Botech Co., Ltd. (Nanjing, China). Primers

144

were synthesized by GENEWIZ (Beijing, China). DNA gel mini purification and mini

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plasmid extraction kits were purchased from TIANGEN (Beijing, China).

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2.2 Strain construction

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The expression cassettes consisted of a promoter, structural gene, and terminator and

148

were constructed by fusion polymerase chain reaction (PCR). Next, 45-bp overlaps

149

were designed between the adjacent fragments such that they could be assembled into

150

S. cerevisiae genome. All promoters, terminators, and POX1, FAA1, FAA4 were

151

amplified from enomic DNA of S. cerevisiae BY4741. POX1A-HIS3-POX1B,



and

FAA4A-LEU2-FAA4B

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FAA1A-LEU2-FAA1B,

153

constructed by fusion PCR and were integrated into S. cerevisiae genome. The

154

PGK1p-CYP52M1-ADH1t,

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expression cassettes were constructed by fusion PCR and were integrated into the HO

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site of S. cerevisiae separately. Then a CYP52M1-AtCPR1 fusion gene was generated

157

using the widely used GSTSSGSG linker. All DNA fragments were transformed into

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yeast cells using the lithium acetate method.39All strains used in this study are listed

159

in Table 1. The expression cassettes and primers used for strain construction in this

160

study are shown in Fig. S1 and Table S1, respectively.

TDH3-SbCPR-TDH2t,

expression

and

cassettes

were

TDH3-AtCPR1-TDH2t

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2.3 Yeast strain cultivation and fermentation

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A single colony grown on SD plates was inoculated to 5-mL of yeast peptone

164

dextrose medium (YPD) and incubated overnight at 30°C with shaking at 220 rpm.

165

Then, aliquots were transferred to 250-mL shake flasks containing 30 mL of YPD at

166

an initial optical density at 600 nm (OD600) of 0.05 and cultivated at 30°C with

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shaking at 220 rpm for 4 days. Each sample was analyzed in triplicate.

168

The strain B06 was applied for batch and fed-batch fermentation in a 5-L

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bioreactor. A single colony obtained from the plate was inoculated into a 250-mL

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shake flask containing 30-mL of YPD and cultivated at 30°C with shaking at 220 rpm

171

overnight. For batch fermentation, the overnight culture was transferred to a 500-mL

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shake flask containing 100-mL of YPD with an initial OD600 0.05 and cultured at

173

30°C with shaking at 220 rpm for 18 h. The culture was then applied for seed culture,


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and a 10% (v/v) solution was inoculated into a 5-L bioreactor containing 2-L of YPD.

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Fermentation was carried out at 30°C with an initial pH of 5.5 and an air flow rate of

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2 L/min. Dissolved oxygen was maintained at approximately 35% by stirring. The pH

177

was controlled at 5.5 with NH3·H2O and 5 N H2SO4.40

178

During the fed-batch fermentation, glucose was used as the main carbon source

179

for cell growth and synthesis of target products. After 24 h, concentrated glucose

180

solution (500 g/L, 1000 mL) mixed with glutamate solution (10 g/L, 12 mL),

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microelement stock solution (12 mL/L, 10 mL), and vitamin stock solution (10 mL/L,

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12 mL)41 was intermittently fed into the bioreactor every 12 h to a glucose

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concentration 20 g/L.

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2.4 Extraction of FAAs and long-chain ω-HFAs

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The extraction process was conducted as described previously.42 Two milliliters of

187

culture broth was centrifuged at 10500×g for 5 min to collect the cell pellet and the

188

supernatant separately. Before extraction, C15:0-FFA (25 μg) or ω-OH-C15:0-FFA

189

(25 μg) was added as an internal standard. For the extraction of extracellular fatty

190

acids, 500 μL ethyl acetate was added, and the solution was oscillated for 10 min and

191

then centrifuged at 10500×g for 10 min. For intracellular fatty acids and HFAs, 500

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μL ethyl acetate was added to the cell pellet, oscillated for 10 min with glass beads,

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and then centrifuged at 10500×g for 10 min. The upper ethyl acetate layer was

194

recovered.

195



196

2.5 Gas chromatography-mass spectrometry (GC-MS) analysis and quantification of

197

FAAs and HFAs

198

The extract solution was evaporated with nitrogen gas and the extract was exposed to

199

2 mL of 2 N H2SO4 in methanol at 70°C for 1 h to generate fatty acid methyl esters

200

(FAMEs) and hydroxy fatty acid methyl esters (HFAMEs).43 For analysis of FFAs,

201

the FAMEs were extracted with hexane. For analysis of HFAs, the HFAMEs were

202

extracted with hexane, evaporated with nitrogen gas, and further converted into their

203

trimethylsilyl derivatives by incubation with an excess of N,O-bis (trimethylsilyl)

204

trifluoroacetamide at 70°C for 1 h.43 The solution was cooled down to room

205

temperature and filtered through a 0.22-μm membrane for GC-MS.

206

Briefly, a Bruker SCION 456 GC-FID was used for FAME quantification. The

207

injector and detector temperature were set at 300°C and 310°C, respectively. The

208

oven heating procedures were as previously described,44 and 1 μL sample was

209

injected at a split ratio of 10. Target product was quantified relatively by the peak area

210

normalized to the internal standard peak area. The structures of the HFAs were further

211

confirmed by GC-MS analysis. For this, 1 μL sample was analyzed at a split ratio of

212

10 on a Shimadzu GCMS-TQ8030 equipped with an Agilent Technologies HP-5MS

213

GC column (30 m × 0.250 mm × 0.25 μm) using helium as the carrier gas. The

214

injection temperature was set at 300°C, and the oven temperature program was as

215

follows: 100°C for 2 min, ramp rate of 40°C/min to 200°C and hold for 1 min, ramp

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rate of 4°C/min to 245°C and hold for 1 min, ramp rate of 30°C to 290°C and hold for

217

10 min.The ion source temperature was 300°C, and spectra were scanned from m/z


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30–600 with a solvent cutoff at 4 min. HFAs were identified by comparison with the

219

reference spectra from Wiley Registry of Mass Spectra. HFA production was

220

quantified by normalizing the peak area to the internal standard peak area.

221

3. Results and Discussion

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3.1 Construction of △Pox1△Faa1△Faa4 mutant S. cerevisiae and accumulation of

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long-chain FFAs

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To increase the FFA pool, it is necessary to consider that endogenous fatty acids can

225

be degraded by β-oxidation in peroxisomes,45 preventing FAAs accumulation. Thus,

226

we aimed to inactivate the β-oxidation pathway to improve fatty acid stability and

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availability in S. cerevisiae cell factories. β-Oxidation is executed by four enzymes:

228

acyl-CoA oxidase enol-CoA hydratase, 3-hydroxy acyl-CoA dehydrogenase, and

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3-oxoacyl-CoA thiolase. The first and rate-limiting step is catalyzed by acyl-CoA

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oxidase, encoded by POX1 in S. cerevisiae. This strategy results in functional

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disruption of the β-oxidation pathway, thereby preventing yeast cells from using fatty

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acids as a carbon source for cell growth and production.34 Thus, destruction of the

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β-oxidation pathway has become an effective strategy for the efficient accumulation

234

of fatty acids in the metabolic engineering of S. cerevisiae.

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Fatty acids are mainly synthesized de novo by a cytosolic FAS as activated fatty

236

acids (fatty acyl-CoAs) by condensation of acetyl-CoA and malonyl-CoA, and the

237

fatty acyl-CoAs are released as FFAs by thioesterase.29 Unfortunately, FFAs are

238

easily re-activated by fatty acyl-CoA synthetases to fatty acyl-CoAs, whose

239

accumulation feedback obstructs fatty acid biosynthesis and accumulation.28 To



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circumvent this, we aimed to knock out acyl-CoA synthetases. S. cerevisiae has six

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acyl-CoA synthetases, encoded by FAA1, FAA2, FAA3, FAA4, FAT1, and FAT2. The

242

peroxisomal FAT1 and FAT2 were found to be inessential for growth when fatty

243

acids were the carbon source.46 Further, FFAs are excreted from yeast cells into the

244

growth

245

Δfaa1Δfaa2Δfaa3Δfaa4 knockout strain did not show higher FAA levels than a

246

Δfaa1Δfaa4 strain.27 These results showed that the majority of synthetase activity was

247

centralized on the long-chain acyl-CoA synthetases FAA1 and FAA4. In a previous

248

study, yeast cells secreted 207 mg/L FFAs upon knockout of FAA1 and FAA4 and

249

overexpression of TesA, a native E. coli thioesterase.47 In another recent study, 520

250

mg/L FFAs were obtained by disruption of FAA1 and FAA4 and overexpression of a

251

truncated peroxisomal acyl-CoA thioesterase.48 Accordingly, we targeted these

252

synthetases to drive FFA overproduction in S. cerevisiae.

medium

upon

disruption

of

FAA1

and

FAA4;

however,

a

253

In this study, we performed a systematic analysis of the overproduction and

254

accumulation of FFAs by deleting the two main fatty acyl-CoA synthetases encoding

255

FAA1 and FAA4 genes to disrupt the re-activation process. Similarly, we disrupted

256

POX1, which encodes a fatty acyl-CoA oxidase, in order to interrupt fatty acid

257

degradation through β-oxidation. Generally, S. cerevisiae produces primarily C16 and

258

C18 FFAs. A wild-type strain produced only 3 mg/L FFAs.45 The cytochrome P450

259

enzyme CYP52M1 has been shown to preferentially oxidize C16-C18 saturated and

260

unsaturated FFAs preferentially. Therefore, we chose to only identify and measure the

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C16-C18 saturated and unsaturated FFAs. After cultivation for 4 days, processed


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samples were subjected to GC-MS analysis to confirm FFAs production. Four new

263

distinct peaks were detected (Fig. S2) and were further identified as hexadecanoic

264

acid (C16:1), hexadecenoic acid (C16:0), oleic acid (C18:1), and octadecanoic acid

265

(C18:0).49 The mass spectra of the four FFAs were shown in Figure S3, indicating that

266

the biosynthesis of C16-C18 FFAs in strain B03. The results showed that the total

267

yield of intra-cellular and extracellular C16-C18 FFAs was 336.1 mg/L in the

268

△Pox1△Faa1△Faa4 knockout strain B03 (Fig. 2).

269 270

3.2 Selection of CPRs for the CYP52M1 enzyme to oxidize FFAs

271

Cytochrome P450s and their heme donors, CPRs, carry out hydroxylation of

272

various endogenous and exogenous compounds. Cytochrome P450s are able to

273

catalyze complex reactions, such as the regio- and stereoselective oxidation of

274

inactivated hydrocarbon C-H bonds to the corresponding hydroxy (C–OH) products.50

275

The cytochrome P450 enzyme CYP52M1, from S. bombicola, oxidizes FFAs into

276

HFAs, and preferentially oxidizes C16 to C20 FFAs. Moreover, CYP52M1

277

hydroxylated fatty acids at their ω- and ω-1 positions.38 In addition, its

278

monooxygenase reactivity relies on the electron transfer compatibility of its redox

279

partner, CPR51, 52 Thus, it is essential to choose an appropriate functional CPR, which

280

will maximize the redox coupling efficiency of P450 enzymes and achieve optimal

281

CYP functional activity.

282

In this study, we chose two different CPR-encoding genes and co-expressed

283

these genes with CYP52M1 in strain B03, resulting in strains B04 (SbCPR-CYP52M1)



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284

and B05 (AtCPR1-CYP52M1). After cultivation for 4 days, the processed samples

285

were methylated and trimethylsilylated and analyzed by GC-MS. GC-MS analysis

286

revealed that four new compounds were generated. Compound 1 (Fig. 3A) showed a

287

mass spectrum with prominent ions at m/z 343 (M-15, loss of CH3), 311 (M-47, loss

288

of CH3 and CH3OH), and 117 [(CH3)3SiO+CHCH3] (Fig. 3B) and was identified as

289

ω-1-hydroxy palmitic acid.53 Compound 2 (Fig. 3A), displayed a mass spectrum with

290

prominent ions at m/z 343 (M-15, loss of CH3), 327 (M-31, loss of OCH3), 311

291

(M-47, loss of CH3 and CH3OH), and 103 [(CH3)3SiO+=CH2] (Fig. 3C) and was

292

identified as ω-hydroxy palmitic acid.49, 54 Compound 3 (Fig. 3A) exhibited a mass

293

spectrum with prominent ions at m/z 369 (M-15, loss of CH3), 337 (M-47, loss of CH3

294

and CH3OH), and 117 [(CH3)3SiO+CHCH3] (Fig. 3D) and was identified as

295

ω-1-hydroxy oleic acid.55 Compound 4 (Fig. 3A) displayed a mass spectrum with

296

prominent ions at m/z 369 (M-15, loss of CH3), 353 (M-31, loss of OCH3), 337

297

(M-47, loss of CH3 and CH3OH), and 103 [(CH3)3SiO+=CH2] (Fig. 3E), which was

298

identical to those of ω-hydroxy oleic acid54. For the first time, a robust long-chain

299

ω-HFAs producing S. cerevisiae was successfully established and highly specific

300

production of C16 and C18 ω-HFAs was achieved. GC analysis showed that B05

301

containing AtCPR1 from A. thaliana produced 83.2 mg/L long-chain ω-HFAs, which

302

was 2.2-fold higher than that in strain B04 containing SbCPR from S. bombicola, in

303

contrast to our expectations (Fig. 4A). Typically, a homologous CYP-CPR

304

reconstituted system is chosen because such a system will have high electron transfer

305

compatibility and coupling efficiency to enhance monooxygenase activity.55,


56

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306

However, in this study, the heterologous CYP-CPR reconstituted system

307

(AtCPR1-CYP52M1) showed a greater increase in the hydroxylation of FFAs,

308

supporting our hypothesis that CYP-CPR interaction plays a vital role in determining

309

the level of CYP catalytic efficiency. Therefore, we verified that the reactivity of

310

CYP52M1 between homologous and heterologous systems could be attributed to the

311

natural electron transfer compatibility and CYP-CPR interaction. Actually, The CPR

312

and P450s interaction efficiency was somewhat modulated dependent on the CPR. It

313

has been reported that CPR even influence the metabolite pattern of P450,58,

314

revealing the importance of the source of CPR in the functional activity of CYP

315

reconstituted systems in terms of long-chain ω-HFAs production.

316

3.3 Construction of a self-sufficient P450 enzyme system through fusion of CYP52M1

317

with AtCPR1 to improve long-chain ω-HFAs production

318

In the fermentation of strain B05, the precursor FFAs strongly accumulated and failed

319

to be transformed into ω-HFAs (data not shown), which could be explained by the

320

low catalytic efficiency of the CYP-CPR reconstituted system. Our previous studies

321

have shown that an artificial cytochrome P450-CPR fusion protein contributes to

322

improving the catalytic activity of fusion enzymes.60 Thus, we constructed an

323

artificial fusion protein containing CYP52M1 and AtCPR1 to verify the feasibility of

324

this method. The widely used linker GSTSSGSG was applied to construct the

325

CYP52M1-AtCPR1 fusion protein in strain B05, yielding strain B06.

59

326

As shown in Fig. 4B, the artificial CYP52M1-AtCPR1 fusion protein performed

327

better than co-expression of CYP52M1 and AtCPR1, with long-chain ω-HFAs



328

production of up to 148.7 mg/L in shake flask culture; this was a 1.8-fold increase

329

compared with that in strain B05. This result verified the feasibility of the method and

330

further emphasized the importance of the transformation efficiency of the cytochrome

331

P450 enzyme, which may be regarded as the rate-limiting step.

332 333

3.4 Batch and fed-batch fermentation of B06 in a 5-L bioreactor

334

The best long-chain ω-HFAs producing strain, B06, was used for batch and fed-batch

335

fermentation. During batch fermentation, cell growth, residual glucose concentration,

336

ethanol concentration, and ω-HFAs production were simultaneously monitored. As

337

shown in Fig. 5A, during the first 12 h, the cells grew very fast, glucose was

338

consumed quickly, and ω-HFAs synthesis began. Upon depletion of ethanol, ω-HFAs

339

began to accumulate gradually, and the highest yield obtained was 141.7 ± 3.5 mg/L,

340

with an OD600 of approximately 35.5.

341

Next, the strain B06 was applied for fed-batch fermentation. As shown in Fig. 5B,

342

the OD600 of B06 in fed-batch fermentation was approximately 2.8-fold higher than

343

that in batch fermentation. Furthermore, the ω-OHFA yield was significantly further

344

increased up to 347 ± 9.2 mg/L, indicating high-level long-chain ω-HFA production.

345

Taken together, we have constructed yeast cell factories for the production of FFAs

346

and long-chain ω-hydroxy fatty acids. These strains represent a beginning for the

347

establishment of yeast-based commercial bioprocesses for the production of

348

chemicals and advanced biofuels from renewable resources. Our metabolic

349

engineering strategies of pathway balancing not only facilitated the production of


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350

long-chain ω-hydroxy fatty acids but also provide valuable insights into construction

351

of yeast cell factories for production of other structures of HFAs.

352 353 354 355

Conflicts of interest The authors declare no conflicts of interest.

356

Acknowledgement

357

This work was supported by the National Natural Science Foundation of Chain (No.

358

21878220) and Major Research Plan of Tianjin (16YFXTSF00460).

359

Supporting Information

360

Table S1. Supplementary material Tables.

361

Figure S1. Supplementary material Figures.

362

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527 528 529 530 531 532 533 534

Figure Captions

535

Fig.1. Metabolic pathway from glucose to long-chain HFAs in S. Cerevisiae. FAS1

536

and FAS2, fatty acid synthase; TE, acyl-CoA thioesterase;

537

Fig.2. Production of C16-C18 FFAs by the engineered yeast strains B03 in shake

538

flasks. Data are the mean value of three independent experiments.

539

Fig.3. GC chromatogram and mass spectrum profile from S. cerevisiae B03 (control)

540

and B05 fermentations.

541

Fig.4. Production of long-chain HFAs by the engineered yeast strains B04-B06 in

542

shake flasks. Data are the mean value of three independent experiments.

543

Fig.5. Production of long chain HFAs by strain B06 in batch and fed-batch

544

cultivations in a 5-L bioreactor. (A) Batch fermentation of B06 in a 5-L bioreactor.

545

(B) Fed-batch fermentation of B06 in a 5-L bioreactor.

546 547 548



Page 26 of 32

549

Table 1 Strains involved in this study Name

Description

source

BY4741

MATa; leu2-3,112; met17; ura3-1; his3-11,15

Our lab

POX1 expression cassette in BY4741 was This B01 deleted

study This

B02

FAA1 expression cassette in B01 was deleted study This

B03

FAA4 expression cassette in B02 was deleted study CYP52M1 and SbCPR gene expression cassettes This

B04

were integrated into the HO site of B03 with study selection marker HIS3 CYP52M1

and

AtCPR1

gene

expression This

B05

cassettes were integrated into the HO site of B03 study with selection marker HIS3 CYP52M1-AtCPR1 fusion protein expression This

B06

cassettes were integrated into the HO site of B03 study with selection marker HIS3


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Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



Table of Contents


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Biosynthesis of Long-Chain Ï-Hydroxy Fatty Acids by Engineered

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