Dynamic engineering of 1-alkenes biosynthesis and secretion in yeast

Dec 28, 2017 - ... Yating Hu, Zhiwei Zhu, Verena Siewers, and Jens Nielsen. ACS Synth. Biol. , Just Accepted Manuscript. DOI: 10.1021/acssynbio.7b0033...
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Dynamic engineering of 1-alkenes biosynthesis and secretion in yeast Yongjin J. Zhou, Yating Hu, Zhiwei Zhu, Verena Siewers, and Jens Nielsen ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.7b00338 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Dynamic engineering of 1-alkenes biosynthesis and

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secretion in yeast

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Yongjin J. Zhou1,2*, Yating Hu2,3, Zhiwei Zhu2,3, Verena Siewers2,3,

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Jens Nielsen2,3,4*

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Division of Biotechnology, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, PR China Department of Biology and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE-41296 Gothenburg, Sweden Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE-41296 Gothenburg, Sweden Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark

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*Correspondence to: Jens Nielsen E-mail: [email protected]

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Yongjin J. Zhou E-mail: [email protected]

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Abstract

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Microbial production of fatty acid-derived hydrocarbons offers a great opportunity

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to sustainably supply biofuels and oleochemicals. One challenge is to achieve a high

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production rate. Besides, low efficiency in secretion will cause high separation costs

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and it is therefore desirable to have product secretion. Here, we engineered the

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budding yeast Saccharomyces cerevisiae, to produce and secrete 1-alkenes by

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manipulation of the fatty acid metabolism, enzyme selection, engineering the

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electron transfer system and expressing a transporter. Furthermore, we

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implemented a dynamic regulation strategy to control the expression of membrane

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enzyme and transporter, which improved 1-alkene production and cell growth by

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relieving the possible toxicity of overexpressed membrane proteins. With these

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efforts, the engineered yeast cell factory produced 35.3 mg/L 1-alkenes with more

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than 80% being secreted. This represents a 10-fold improvement compared with

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earlier reported hydrocarbon production by S. cerevisiae.

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Key words: Yeast cell factories; Dynamic regulation; Fatty acids; Hydrocarbons;

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Metabolic engineering

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Introduction

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Microbial utilization of biomass for production of biofuels represents a sustainable

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solution for provision of liquid transportation fuels with a reduced carbon footprint.1,

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2

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of surfactants and lubricants, and also have the potential to serve as “drop-in”

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compatible fuels due to their high energy density.

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The fatty acid biosynthesis pathway has attracted significant attention for

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production of oleo-chemicals and biofuels. Among these, 1-alkenes can be

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synthesized from activated fatty acids (fatty acyl-CoA/ACP) by polyketide synthases,3,

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4

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iron-dependent fatty acid decarboxylases.6, 7 All of these pathways were discovered

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in bacteria and some of these enzymes have been heterologously expressed in

Long-chain linear 1-alkenes (α-olefins) are widely used as feedstock for production

or from free fatty acids (FFAs) by H2O2-dependent P450 enzymes5 and

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Escherichia coli for 1-alkene production.8 It is also interesting to explore the potential

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of 1-alkene production in the industrially widely applied Saccharomyces cerevisiae,

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which has high robustness and tolerance towards harsh fermentation conditions as

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well as its already wide use for bioethanol production.9, 10 However, compared to E.

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coli, productivity and yield of fatty acid-derived hydrocarbons are much lower in

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engineered S. cerevisiae,11-17 as well as in the oleaginous yeast Yarrowia lipolytica.18,

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19

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had a titer of 100 mg/L, while an engineered S. cerevisiae only produced < 5 mg/L

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1-alkenes.13, 14, 17 This might be attributed to a more complex yeast metabolism, an

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unsuitable cellular environment for the heterologous enzymes and/or the lack of

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suitable cofactors. Furthermore, due to the hydrophobic nature of hydrocarbons

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they may tend to accumulate in the lipid bi-layer of the cell membrane and hence

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not be secreted to the extracellular medium, which is an obstacle for product

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separation.

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Here, we aim to engineer an ideal yeast cell factory for production and secretion of

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1-alkene by pathway screening, engineering of the electron transfer system, overall

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dynamic metabolic balancing and heterologous transporter expression (Figure 1).

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Figure 1. Schematic overview of metabolic pathways engineered for the production of 1-alkenes in S.

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cerevisiae. Multiple arrows indicate multiple steps and single arrows represent a single step. Fatty

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acid consumption pathways were eliminated by deleting fatty acyl-CoA synthetase encoding genes

With regard to 1-alkene production, an engineered E. coli with FFA accumulation

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FAA1 and FAA4, and fatty acyl-CoA oxidase encoding gene POX1. 1-Alkenes can be synthesised from

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free fatty acids catalysed by H2O2-dependent P450 enzyme OleT, nonheme iron oxidase UndA or

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desaturase-like enzyme UndB. The NADH-dependent putidaredoxin (Pdx)-putidaredoxin reductase

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(Pdr) system was introduced to handle the electron transfer involved in the UndB catalysed FFA

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decarboxylation. Pdxox and Pdxred represent the oxidative and reductive state of Pdx, respectively.

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FATP1, encoding long chain fatty acid transporter protein 1, was also expressed for exporting

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1-alkenes.

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Results and Discussion

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Enzyme selection for 1-alkene production

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Previous studies explored the P450 fatty acid decarboxylase OleT from

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Jeotgalicoccus sp. ATCC8456 for 1-alkene production in S. cerevisiae, which enabled

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the production of 0.2-3.7 mg/L 1-alkenes,13, 14 only about 5 % compared with an

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engineered E. coli strain.20 Speculating that this enzyme works poorly in yeast we

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searched for a more efficient pathway for 1-alkene production by using the fatty acid

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over-producing strain YJZ06,12 which carries triple deletions of the fatty acyl-CoA

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synthase genes FAA1/4 and the fatty acid oxidase gene POX1 (Figure 2A). Besides the

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H2O2-dependent P450 enzyme OleT, we also evaluated the recently identified

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non-heme iron oxidase UndA6 and two different variants of the desaturase-like

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enzyme UndB.7 All these fatty acid decarboxylase genes were expressed under

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control

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UASTEF-UASCIT-UASCLB-TDH3p)12 and cloned into the pYX212 plasmid backbone.

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Expression of OleT from Jeotgalicoccus enabled production of 0.92 mg/L of 1-alkenes,

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while expression of PpUndA from Pseudomonas putida F1 resulted in a lower

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1-alkene titer of 0.57 mg/L (Figure 2B). It was reported previously that more than 3

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mg/L of medium chain 1-alkenes were produced by S. cerevisiae expressing PpUndA

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under simple cultivation in vials.17 Here, the low 1-alkene production via expression

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of PpUndA was attributed to the fact that PpUndA shows poor activity toward long

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chain fatty acids (LCFAs)6 and the fatty acids of host strain YJZ06 are mainly LCFAs.12

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Interestingly, the desaturase-like UndB enzymes were much more efficient in terms

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of 1-alkene biosynthesis in S. cerevisiae (Figure 2B). Among the two enzymes

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evaluated, PmUndB from Pseudomonas mendocina enabled production of 2.90 mg/L

of

the

enhanced

strong

promoter

eTDH3p

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1-alkenes whereas expression of PfUndB from Pseudomonas fluorescens Pf-5

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resulted in a titer of 5.76 mg/L (Figure 2B). This is different from findings in E. coli,

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where PmUndB was found to be more efficient than PfUndB for 1-alkene

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production.7 This performance variation might be owing to the differences in protein

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expression level and/or in the cellular environment for enzyme activity.

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Figure 2: Engineering 1-alkene biosynthesis in S. cerevisiae by enzyme evaluation and fatty acid

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manipulation A) Schematic overview of the metabolic pathways used for production of 1-alkenes

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from FFAs via three different routes. B) Evaluation of enzymes for total 1-alkene production in the

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fatty acid overproducing strain YJZ06 that carries deletions of POX1, FAA1 and FAA4. C) The effect of

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manipulating the production of FFAs on total 1-alkene biosynthesis in a strain expressing PfUndB. All

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data represent the mean ± s.d. of three yeast clones.

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We then investigated the effect of FFA accumulation on 1-alkene production (Figure

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2C). The deletion of POX1 and/or FAA4 had a marginal effect on 1-alkene production,

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while FAA1 deletion improved it by 36%. Furthermore, the triple deletion of POX1

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and FAA1/4 led to a 2.8-fold higher 1-alkene titer (6.20 mg/L) compared with the

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wild-type background. The 1-alkene titers were well correlated with the free fatty

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acid levels (Figure 2C), which was also observed in previous studies using OleT for

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1-alkene production

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fatty acids were essential for 1-alkene production with using UndB.

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Cofactor engineering for enhanced 1-alkene biosynthesis

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1-Alkene biosynthesis from FFAs is an oxidation process involving an electron

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transfer process. Several studies suggested that the lack of an efficient reduction

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system could be one of the reasons for the low activities of fatty acid decarboxylases

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both in vitro21 and in vivo.13 We therefore compared three electron transfer systems:

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ferredoxin-ferredoxin reductase (Fd/Fnr) and flavodoxin-ferredoxin reductase

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(Fld/Fnr)

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putidaredoxin-putidaredoxin reductase from P. putida (Pdx/Pdr, encoded by camB

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and camA, respectively) that uses NADH as cofactor (Figure 3A). Expression of the

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ferredoxin- and flavodoxin-based electron transfer systems improved 1-alkene

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production by 42% and 58%, respectively, while the putidaredoxin reductase system

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increased 1-alkene production by 108% (Figure 3B). To facilitate electron

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channelling,22 we fused the Pdx/Pdr system to PfUndB with different linkers.

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However, this endeavour resulted in a much lower 1-alkene production (Figure S1),

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which might be owing to an impaired function of the membrane-bound enzyme

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PfUndB after protein fusion. It is worth mentioning that the use of NADH as electron

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donor by Pdx/Pdr may be responsible for the improved 1-alkene biosynthesis in S.

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cerevisiae as this yeast has a much higher level (>7-fold) of NADH than NADPH.23

from

E.

. These results suggested that high levels of precursor free

coli,

which

use

NADPH

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Figure 3: Comparison of different electron transfer systems for total 1-alkene production in a FFA

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overproducing strain expressing PfUndB. A) Schematic overview of the metabolic pathway including

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electron transfer systems from E. coli (Fd/Fnr, Fld/Fnr) and P. putida (Pdx/Pdr), respectively. B) Total

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1-alkene production in engineered strains. Data represent the mean ± s.d. of three yeast clones.

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Dynamic regulation of UndB to improve 1-alkene production with relieved

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metabolic burden

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UndB was proposed to be a membrane-bound desaturase-like enzyme by

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bioinformatics analysis (Figure 4A),7 and the enzyme may therefore also be involved

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in export of the 1-alkenes as we found that the majority of the 1-alkenes produced

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was secreted to the extracellular medium (Figure S2). Furthermore, fluorescence

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microscopy analysis of the PfUndB-GFP carrying strain AE17 showed that this

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enzyme predominantly localized to the membrane (Figure 4B). Initially, we used a

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strong promoter, eTDH3p, for high-level expression of PfUndB, but this resulted in

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reduced growth (strain AE24, Figure 4C), which might be due to eTDH3p-based

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transcription being strongly upregulated in the glucose phase.24

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High-level expression of a membrane protein might cause stress on cell growth.

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Furthermore, cells showed a much lower fatty acid accumulation during the glucose

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phase compared to the ethanol phase (Figure S4), and TDH3p-based transcription is

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therefore not synchronized with FFA accumulation. We therefore fine-tuned the

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expression of undB to balance cell growth and product formation. To reach this goal,

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we designed a dynamic control system to separate cell growth and 1-alkene

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production into two different phases by using the carbon source-dependent

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promoter of GAL7 (GAL7p), which is repressed at high glucose levels and activated at

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glucose depletion. Expression of PfundB under control of GAL7p, combined with the

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deletion of the transcription factor Gal80 (strain AE38), resulted in a 100% higher

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final titer of 1-alkenes compared to strain AE24 constitutively expressing PfUndB

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under control of eTDH3p (Figure 4C). Strain AE24 produced more 1-alkenes in the

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glucose phase compared to strain AE38. Both strains secreted the majority of the

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1-alkenes produced (>60%) and the GAL7p driven expression of PfundB improved the

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production of both extracellular and intracellular 1-alkenes (Figure S2). Furthermore,

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strain AE38 showed better growth in the glucose phase resulting in a higher final

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biomass yield and glucose consumption rate compared to strain AE24 (Figure 4C and

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Figure S3). Furthermore, there was a much higher 1-alkene production (Figure 4C)

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and lower specific FFA accumulation (Figure S4) in the cultures of strain AE38

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compared to those of AE24. These results suggested that dynamic control could

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relieve the toxicity of expressing the membrane protein PfUndB and improve

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1-alkene production by separating cell growth and 1-alkene production into two

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different phases, which could provide a feasible approach to improve the

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performance of cell catalysts with toxic pathways or enzymes.

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Figure 4: Dynamic control of membrane enzyme and transporter expression for 1-alkene production

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and secretion. A) Schematic view of 1-alkene biosynthesis and secretion. B) Fluorescence microscopy

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analysis of AE17 that carried an undB-gfp fusion gene. C) Dynamic control of UndB expression for

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enhanced total 1-alkene biosynthesis. Expression of UndB under control of GAL7p (strain AE38) was

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compared to expression under control of eTDH3p (strain AE24). D) Expression of the LCFA transporter

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FATP1 for improving 1-alkene secretion and production. Strain AE39 contains FATP1, while AE38 was

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used as control. E) Total 1-alkene profiles of AE38 and AE39 expressing FATP1 at 48 h and 70 h. All

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data represent the mean ± s.d. of three yeast clones.

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Expressing a transporter for improved 1-alkene production and secretion

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Downstream extraction and purification usually greatly raises the overall cost of

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production, and if the product is intracellular there is a need for cell disruption and

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extraction by using environmentally unfriendly solvents.25, 26 Engineering product

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secretion is therefore attractive for improving the process economy. Furthermore,

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this may also relieve product inhibition and toxicity. Previous studies successfully

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improved terpene production by using resistance-nodulation-cell division (RND)27 or

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ATP binding cassette (ABC)25, 28 transporters. Here, we alternatively explored the

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long chain fatty acid transporter protein 1 (FATP1) from Homo sapiens29 to improve

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1-alkene production, based on the similar hydrophobic property between LCFAs and

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1-alkenes. In order to alleviate the toxicity of membrane protein FATP1 expression in

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the glucose phase, we expressed FATP1 using HXT6p,30 which was expected to be

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highly expressed after glucose depletion when 1-alkene production is initiated.

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Genome integration of a FATP1 expression cassette (strain AE39) had a marginal

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effect on cell growth and fatty acid production (Figure S5), but significantly improved

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the extracellular and total 1-alkene production after 70 h of cultivation by 40% and

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37%, respectively, compared with the control strain AE38 (Figure 4D). There was

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little difference in the 1-alkene profiles between AE38 and AE39, but slightly more

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C17 1-alkenes were accumulated at 70 h compared with at 48 h (Figure 4E). The

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FATP1 expressing strain AE39 clearly showed the highest 1-alkene secretion resulting

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in an extracellular final titer of 29.3 mg/L corresponding to 83% of the total

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1-alkenes produced at 48 h. Although yeasts have been extensively explored for

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production of fatty acid derived hydrocarbons, the titers are still very low and most

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products accumulate intracellularly, which limits future industrial application. Here,

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our S. cerevisiae strain AE39 exhibited the highest reported production of 1-alkenes

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from free fatty acids in yeast, and this represents a 10-fold improvement compared

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to previously engineered S. cerevisiae cell factories11-15 (Table 1). Moreover, product

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secretion provides great benefits for industrial production as it will decrease the

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separation costs.

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In summary, we engineered a yeast cell factory for production and secretion of

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1-alkenes by cofactor engineering, transporter engineering and dynamic enzyme

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control, which enabled the highest reported titers of 1-alkenes by a eukaryotic cell

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factory.

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Table 1: Fatty acid-derived hydrocarbons in engineered eukaryotic cell factories Host

Product

Total (mg/L)

Extracellular (mg/L)

Ref.

S. cerevisiae Aspergillus carbonarius Y. lipolytica S. cerevisiae

Alkanes

0.1-3.2

0

11-13, 15

Alkanes

1.0

N.D.

31

N.D. 0

18

N. D

17

29.3

This study

S. cerevisiae S. cerevisiae 221

Alkanes 23.5 1-Alkenes 3.7 Medium chain 3.0 1-alkenes 1-Alkenes 35.3

N.D., not detected

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Methods

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Yeast strains, plasmids and reagents. The plasmids and strains used in this study are listed

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in the Tables S1 and S2, respectively. The primers (Table S3) were ordered from

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Sigma-Aldrich. Genes oleT, PpundA, PfundB and PmundB (Table S7) were codon-optimized

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for yeast expression and synthesized by Genscript or Invitrogen. PrimeStar DNA polymerase

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was purchased from TaKaRa. Zymoprep™ Yeast Plasmid Miniprep II was supplied by Zymo

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Research Corp. Restriction enzymes, DNA gel purification and plasmid extraction kits were

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purchased from Thermo Fisher Scientific. Analytical standards for quantification of 1-alkenes

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and fatty acid methyl esters (FAMEs) were supplied by Sigma-Aldrich.

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Strain cultivation. Yeast strains were normally cultivated in YPD media consisting of 10 g/L

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yeast extract (Merck Millipore), 20 g/L peptone (Difco), and 20 g/L glucose (Merck Millipore).

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Strains containing URA3 and/or HIS3 based plasmids/cassettes were selected on synthetic

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complete media without uracil or L-histidine (SC-URA, SC-HIS or SC-URA-HIS), which

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consisted of 6.7 g/L yeast nitrogen base (YNB) without amino acids (Formedium), 20 g/L

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glucose (Merck Millipore) and 0.77 g/L complete supplement mixture without corresponding

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nutrition (CSM-URA, CSM-HIS or CSM-HIS-URA, Formedium). The URA3 maker was removed

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and selected against on SC+5-FOA plates, which contained 6.7 g/L YNB, 0.77 g/L complete

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supplement mixture and 0.8 g/L 5-fluoroorotic acid. Strains containing the amdSYM32

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cassette were selected on SM+AC media (5 g/L (NH4)2SO4, 3 g/L KH2PO4, 0.5 g/L MgSO4∙7H2O,

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6.6 g/L K2SO4, 0.6 g/L acetamide, 20 g/L glucose, trace metal and vitamin solutions33

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supplemented with 40 mg/L histidine and/or 60 mg/L uracil if needed). Strains containing

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the kanMX cassette were selected on YPD plates containing 200 mg/L G418 (Formedium).

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Shake flask batch fermentations for production of alkenes, were carried out in minimal

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medium containing 5 g/L (NH4)2SO4, 14.4 g/L KH2PO4, 0.5 g/L MgSO4∙7H2O, 30 g/L glucose,

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trace metal and vitamin solutions33 supplemented with 40 mg/L histidine and/or 60 mg/L

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uracil if needed. Cultures were inoculated, from 24 h precultures, at an initial OD600 of 0.1

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using 15 mL minimal medium in 100 mL unbaffled shake flasks and cultivated at 200 rpm,

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30 °C for 72 h.

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Genetic engineering. All episomal vectors or genome-integrated pathways (Figure S6) were

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constructed by the modular pathway engineering (MOPE) strategy.34 Briefly, genes,

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promoters, and terminators were amplified from the yeast genome or the custom

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synthesized templates (Table S4). Then, gene expression modules, consisting of a promoter,

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a structural gene, a terminator, and the promoter of the next module for homologous

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recombination, were assembled by one-pot fusion PCR (Table S5).34 The modules were gel

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purified and transformed to the S. cerevisiae (Table S6). Deletion of GAL80 was performed

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by using an amdSYM gene as selection marker. The deletion cassettes were constructed by

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fusing 200-600 nucleotide homologous arms with the amdSYM expression module32 and the

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clones were selected on a SM+AC media. The genomic manipulations were verified with

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colony PCR by using the genomic DNA, which was prepared by a quick extraction method as

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previously described.35

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Product extraction and quantification. Intracellular 1-alkenes were extracted by a

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microwave-based method modified from our previous study.36 Cell pellets were collected

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from 5 mL cell cultures and subjected to freezer drying for 48 h. Freeze-dried cells were

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mixed with 4 mL of chloroform-methanol (2:1, v/v) in an extraction glass tube containing 0.5

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mg/L hexadecane as internal standard. The extraction tubes were vigorously vortexed and

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then place in the microwave reaction vessel (12 cm X 3 cm I.D., 0.5 cm thickness; Milestone

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Start D, Sorisole Bergamo, Italy) that contained 30 mL of Mili-Q water inside and then sealed

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with a TFM screw cap. The vessel was heated using a microwave digestion system equipped

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with a PRO-24 medium pressure high throughput rotor (Milestone). The temperature

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programing of microwave extraction was ramped to 60 °C (from room temperature, using

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800 W for 24 vessels) within 6 min and kept constant for 10 min. After the samples were

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cooled down to room temperature, 1 mL of NaCl (0.73% w/v) was added and then the

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samples were vortexed vigorously. Thereafter, the samples were centrifuged at 1000 x g for

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10 min allowing for phase separation and the organic phase was transferred into a new

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clean extraction tube. The extracted sample was then pre-concentrated by drying under

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vacuum, and re-suspended with 200 µL n-hexane.

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The extracellular 1-alkenes were extracted from the supernatant by using n-hexane. 2 mL

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supernatant were added to 2 mL hexane containing 1 mg/L hexadecane and the mixtures

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were shaken for 30 min by using a vortex mixer (1000 rpm). The samples were centrifuged at

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2000 x g for 10 min allowing for phase separation and the organic phase was transferred into

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a new clean extraction tube for analysis. The intracellular and extracellular extraction

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solvents were analysed by a GC-FID (Focus GC with a flame ionization detector (FID), Thermo

285

Fisher Scientific) equipped with a Zebron ZB-5MS GUARDIAN capillary column (30 m x 0.25

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mm x 0.25 µm, Phenomenex). The GC program for 1-alkenes was as follows: initial

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temperature of 50 °C, hold for 5 min; then ramp to 310 °C at a rate of 10 °C per min and hold

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for 6 min. The temperature of inlet and detection were kept at 250 °C and 300 °C,

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respectively. The flow rate of the carrier gas (nitrogen) was set to 1.0 mL per minute, and

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data were analyzed by using the Xcalibur software.

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Free fatty acids were simultaneously extracted and methylated by dichloromethane

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containing methyl iodide as methyl donor12, 37. Cell cultures from shake flask were mixed

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well and 100 µl cell cultures were diluted 2-fold with water, then 10 µl base catalyst 40%

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tetrabutylammonium was added immediately followed by addition of 200 µl

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dichloromethane containing 200 mM methyl iodide as methyl donor and 100 mg/L

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pentadecanoic acid as an internal standard. The mixtures were shaken for 30 min at 1400

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rpm by using a vortex mixer, and then centrifuged at 3500 g to promote phase separation.

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160 µl dichloromethane layer was transferred into a GC vial with glass insert, and

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evaporated 4 hours to dryness. The extracted methyl esters were re-suspended in 160 µl

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hexane and then analysed by GC-FID. The GC program was as follows: initial temperature of

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40 °C, hold for 2 min; ramp to 130 °C at a rate of 30 °C per minute, then raised to 280 °C at a

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rate of 10 °C per min and hold for 3 minutes. The temperature of inlet and detection were

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kept at 250 °C and 300 °C, respectively. Final quantification was performed using the

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Xcalibur software.

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Fluorescence microscopy analysis. For UndB localization analysis, the C-termini of the

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proteins were fused to a green fluorescent protein (GFP). The encoding genes were

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transformed into the yeast strain YJZ304. The cells were cultivated in SC-URA or minimal

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media for 48 h at 30 °C, 200 rpm. 3 µL of the cell cultures were dropped onto microscope

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slides and then viewed with a LEICA DMI4000B microscope (Leica Microsystems CMS

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GmbH).

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SUPPORTING INFORMATION

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The Supporting Information is available free of charge on the ACS Publications

313

website at DOI: xxx

314

Strains, plasmids, and gene sequences used in this study; and other supporting

315

figures described in the text (PDF)

316 317

AUTHOR INFORMATION

318 319 320 321 322 323 324 325

Corresponding Author Yongjin J. Zhou: Tel/Fax: +86 (0)411 84771060, E-mail: [email protected] Jens Nielsen: Tel: +46 (0)31 772 3804, Fax: +46 (0)31 772 3801, Email: [email protected] ORCID Yongjin J. Zhou: 0000-0002-2369-3079. Zhiwei Zhu: 0000-0002-2925-5758

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326 327 328

Jens Nielsen: 0000-0002-9955-6003

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Y.J.Z and J.N. conceived the study and designed the experiments. Y.J.Z performed all

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experiments and analyzed the data. Y.H. and Z.Z participated in the genetic

331

engineering and product quantification. V.S. assisted with data analysis and

332

interpretation. Y.J.Z and J.N. wrote the manuscript. All authors revised and approved

333

the manuscript.

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Notes

335

The authors declare no competing financial interest

Author Contributions

336 337 338

Acknowledgements

339

The authors acknowledge funding from the Novo Nordisk Foundation, the Knut and

340

Alice Wallenberg Foundation, FORMAS and Vetenskapsrådet (to J.N.), and research

341

grant from Dalian Institute of Chemicals Physics, CAS (Grant: DICP DMTO201701 to

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Y.J.Z.).

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References

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[8] Herman, N. A., and Zhang, W. (2016) Enzymes for fatty acid-based hydrocarbon biosynthesis, Curr. Opin. Chem. Biol. 35, 22-28. [9] Mattanovich, D., Sauer, M., and Gasser, B. (2014) Yeast biotechnology: teaching the old dog new tricks, Microb. Cell Fact. 13, 34. [10] Caspeta, L., Chen, Y., Ghiaci, P., Feizi, A., Buskov, S., Hallstrom, B. M., Petranovic, D., and Nielsen, J. (2014) Altered sterol composition renders yeast thermotolerant, Science 346, 75-78. [11] Buijs, N. A., Zhou, Y. J., Siewers, V., and Nielsen, J. (2015) Long-chain alkane production by the yeast Saccharomyces cerevisiae, Biotechnol. Bioeng. 112, 1275-1279. [12] Zhou, Y. J., Buijs, N. A., Zhu, Z., Qin, J., Siewers, V., and Nielsen, J. (2016) Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast cell factories, Nat. Commun. 7, 11709. [13] Zhou, Y. J., Buijs, N. A., Zhu, Z., Gomez, D. O., Boonsombuti, A., Siewers, V., and Nielsen, J. (2016) Harnessing yeast peroxisomes for biosynthesis of fatty-acid-derived biofuels and chemicals with relieved side-pathway competition, J. Am. Chem. Soc. 138, 15368-15377. [14] Chen, B., Lee, D. Y., and Chang, M. W. (2015) Combinatorial metabolic engineering of Saccharomyces cerevisiae for terminal alkene production, Metab. Eng. 31, 53-61. [15] Foo, J. L., Susanto, A. V., Keasling, J. D., Leong, S. S., and Chang, M. W. (2017) Whole-cell biocatalytic and de novo production of alkanes from free fatty acids in Saccharomyces cerevisiae, Biotechnol. Bioeng. 114, 232-237. [16] Kang, M. K., Zhou, Y. J., Buijs, N. A., and Nielsen, J. (2017) Functional screening of aldehyde decarbonylases for long-chain alkane production by Saccharomyces cerevisiae, Microb. Cell Fact. 16, 74. [17] Zhu, Z., Zhou, Y. J., Kang, M. K., Krivoruchko, A., Buijs, N. A., and Nielsen, J. (2017) Enabling the synthesis of medium chain alkanes and 1-alkenes in yeast, Metab. Eng. 44, 81-88. [18] Xu, P., Qiao, K. J., Ahn, W. S., and Stephanopoulos, G. (2016) Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals, Proc. Natl. Acad. Sci. U S A 113, 10848-10853. [19] Blazeck, J., Liu, L., Knight, R., and Alper, H. S. (2013) Heterologous production of pentane in the oleaginous yeast Yarrowia lipolytica, J. Biotechnol. 165, 184-194. [20] Liu, Y., Wang, C., Yan, J., Zhang, W., Guan, W., Lu, X., and Li, S. (2014) Hydrogen peroxide-independent production of alpha-alkenes by OleTJE P450 fatty acid decarboxylase, Biotechnol. Biofuels 7, 28. [21] Dennig, A., Kuhn, M., Tassoti, S., Thiessenhusen, A., Gilch, S., Bulter, T., Haas, T., Hall, M., and Faber, K. (2015) Oxidative decarboxylation of short-chain fatty acids to 1-alkenes, Angew Chem. Int. Ed. 54, 8819-8822. [22] Sibbesen, O., De Voss, J. J., and Montellano, P. R. (1996) Putidaredoxin reductase-putidaredoxin-cytochrome p450cam triple fusion protein. Construction of a self-sufficient Escherichia coli catalytic system, The Journal of biological chemistry 271, 22462-22469.

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acids in Escherichia coli; next generation biofuels with improved cold-flow properties, Metab. Eng. 26, 111-118.

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Figure for Table of Contents

Dynamic engineering

Suger Free fatty acid

1-Alkene

1-Alkene

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&]PµŒ í

%LRPDVV GHULYHG VXJDU

$FHW\O &R$ 1$'+

POX1

)DWW\ DF\O &R$ FAA1,4

)UHH IDWW\ DFLG 1$' 1$'+

$ONHQH

3GU

8QG%

$ONHQH

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1-Alkenes (mg/L)

B A Acetyl-CoA POX1

OleT

Fatty acyl-CoA

OleT

C Free fatty acid

UndB

1-Alkene

UndA PmUndB PfUndB

Strain AE1

FAA1,4

H2O2

7 6 5 4 3 2 1 0

O2 + 2H+

UndA

8 7 6 5 4 3 2 1 0 pox1Δ faa4Δ faa1Δ

AE3

AE2

1-Alkenes

AE16

1.2

FAAs

Strain

1.0 0.8 0.6

0.4 0.2 0.0

‒ ‒ ‒

+ ‒ ‒

AE11 AE12

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+ + ‒

+ ‒ +

AE14 AE15

+ + + AE16

FFAs (g/L)

Figure 2

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1-Alkenes (mg/L)

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Figure 3 Glucose

A

B

10

NADH

8

Acetyl-CoA

Free fatty acid NADPH Fnr

Fd/Fldred

NADP+

Fd/Fldox

UndB

Pdxred

NADH Pdr

Pdxox

NAD+

1-Alkenes (mg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

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6 4 2 0 Control

Strain

AE16

Fd/Fnr AE22

1-Alkene

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Fld/Fnr AE23

Pdx/Pnr AE21

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B

Free fatty acid

C

(Intra) 1-alkene

UndB

10 μm

1-Alkenes (mg/L)

A

1-Alkene-AE38 1-Alkene-AE24 OD600-AE38 OD600-AE24

30 25

16 14 12 10 8 6 4 2 0

20 15 10

5 0 0 10 20 30 40 50 60 70 80 Time (h)

(Extra) 1-alkene

D

E

Extracellular-AE39 Extracellular-AE38 Total-AE39 Total-AE38

40 35 30 25 20 15 10 5 0

OD600

Figure 4

1-Alkenes (mg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

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48 h

48 h 1-Tridecene 1,7-Pentadecadiene

70 h

70 h

1-Pentadecene 1,8-Heptadecadiene 1-Heptadecene

0

10

20

30 40 50 Time (h)

60

70

AE38

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AE39