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Engineering Carboxylic Acid Reductase for Selective Synthesis of Medium-Chain Fatty Alcohols in Yeast

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Engineering Carboxylic Acid Reductase for Selective Synthesis of Medium-Chain Fatty Alcohols in Yeast Engineering carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols in yeast Yating Hua,b,1, Zhiwei Zhua,b,c,1, David Gradischnigd, Margit Winklerd,e, Jens Nielsena,b,f,g,2, and Verena Siewersa,b aDepartment of Biology and Biological Engineering, Chalmers University of Technology, SE-41296 Gothenburg, Sweden; bNovo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE-41296 Gothenburg, Sweden; cSchool of Bioengineering, Dalian University of Technology, 116024 Dalian, China; dInstitute of Molecular Biotechnology, Graz University of Technology, A-8010 Graz, Austria; eAustrian Centre of Industrial Biotechnology, A-8010 Graz, Austria; fNovo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, DK-2800 Lyngby, Denmark; and gBioInnovation Institute, DK-2200 Copenhagen N, Denmark Contributed by Jens Nielsen, July 29, 2020 (sent for review May 26, 2020; reviewed by Hal S. Alper and Bernhard Hauer) Medium-chain fatty alcohols (MCFOHs, C6 to C12) are potential usually more abundant than the activated acyl-CoA and acyl- substitutes for fossil fuels, such as diesel and jet fuels, and have ACP in yeast cells (13). Moreover, thioesterases hydrolyzing wide applications in various manufacturing processes. While today acyl-CoA or acyl-ACP, thus releasing free FAs, can be used not MCFOHs are mainly sourced from petrochemicals or plant oils, mi- only for controlling the chain length of synthesized FAs but also crobial biosynthesis represents a scalable, reliable, and sustainable for increasing the FA synthesis flux, which is restricted by feed- alternative. Here, we aim to establish a Saccharomyces cerevisiae back inhibition mediated by acyl-CoA and acyl-ACP (14). platform capable of selectively producing MCFOHs. This was en- Therefore, fatty alcohol formation based on free FAs as sub- abled by tailoring the properties of a bacterial carboxylic acid re- strates through CAR enzymes possesses great advantages. Pre- ductase from Mycobacterium marinum (MmCAR). Extensive protein vious studies on engineering the FA synthesis pathway through engineering, including directed evolution, structure-guided semira- augmenting the precursor flux and improving FA synthetase tional design, and rational design, was implemented. MmCAR vari- (FAS) activity in yeast have generated microbial cells producing ants with enhanced activity were identified using a growth-coupled substantial amounts of medium-chain FAs (MCFAs) (Fig. 1A), high-throughput screening assay relying on the detoxification of which could be utilized for the synthesis of MCFOHs (15, 16). In the enzyme’s substrate, medium-chain fatty acids (MCFAs). Detailed our previous study (17), an engineered MCFA producing FAS characterization demonstrated that both the specificity and catalytic and MmCAR were expressed in yeast to generate aldehyde activity of MmCAR was successfully improved and a yeast strain precursors for alkane synthesis. Substantial quantities of harboring the best MmCAR variant generated 2.8-fold more MCFOHs produced by the engineered strain ZW540 (SI Ap- MCFOHs than the strain expressing the unmodified enzyme. pendix, Fig. S1) showed that endogenous alcohol dehydrogenases Through deletion of the native MCFA exporter gene TPO1,MCFOH and aldehyde reductases were able to support a strong down- production was further improved, resulting in a titer of 252 mg/L for stream flux toward MCFOH formation. However, a promiscuous the final strain, which represents a significant improvement in CAR enzyme reducing C6–C18 FAs is not ideal for MCFOH MCFOH production in minimal medium by S. cerevisiae. production, as it deprives the cell of C16–C18 long-chain FAs (LCFAs) that are essential for cell growth, and also invests carboxylic acid reductase | protein engineering | high-throughput NADPH and ATP in the formation of long-chain by-products screening | medium-chain fatty alcohols | Saccharomyces cerevisiae (18, 19). It is therefore of great interest to narrow the substrate spectrum of the enzyme or increase its activity on MCFAs to oncerns about climate change drive the scientific community Cto explore alternative ways to produce renewable biofuels Significance (1). With straight hydrocarbon chains ranging from C6 to C12, medium-chain fatty alcohols (MCFOHs) are designated as The yeast Saccharomyces cerevisiae has been utilized exten- higher alcohols in contrast to lower alcohols, such as butanol, sively as a cell factory to produce numerous products. The de ethanol, and methanol. MCFOHs are considered to be more novo synthesis of medium-chain fatty alcohols (MCFOHs) suitable to substitute the traditional diesel and jet fuels due to through conversion of free fatty acids by carboxylic acid re- their higher energy density, higher cetane number, and lower ductase (CAR) has been studied in yeast. However, the broad vapor pressure (2, 3). MCFOHs are also widely used in surfac- substrate specificity of CAR is one of the biggest challenges to tants, cosmetics, and plasticizers (4). Currently, fatty alcohols are improve MCFOH production. Here, different protein engineer- synthesized from petrochemical sources or produced from fatty ing strategies were employed to modify the CAR enzyme to- acids (FAs) extracted from oil seeds (5). A major obstacle with ward a more specific catalytic activity on shorter chain fatty sourcing from oil seeds is the competition with food production acids. For this, we created a high-throughput screening assay by using arable land and deforestation (6), and there is therefore relying on the detoxification of the toxic substrate to select for interest in producing MCFOHs through microbial fermentation, efficient enzyme variants. This study represents an important allowing for the utilization of a wide range of feedstocks (7), step forward in the selective synthesis of MCFOHs in yeast. including biomass. In organisms, fatty alcohols are derived from FAs. The bio- Author contributions: Y.H., Z.Z., J.N., and V.S. designed research; Y.H. performed research; synthesis of fatty alcohols can proceed through either fatty al- Y.H., Z.Z., D.G., and M.W. analyzed data; and Y.H., Z.Z., M.W., J.N., and V.S. wrote dehyde intermediates via two steps of two-electron reductions or the paper. directly from fatty acyl-CoA/ACP via a four-electron reduction Reviewers: H.S.A., The University of Texas at Austin; and B.H., University of Stuttgart. (8). Fatty aldehydes can be generated from 1) activated fatty The authors declare no competing interest. acyl-CoA/ACP by fatty acyl-CoA/ACP reductase [ACR (9) or Published under the PNAS license. AAR (10)] or 2) free FAs by carboxylic acid reductase (CAR) 1Y.H. and Z.Z. contributed equally to this work. (11). MmCAR from Mycobacterium marinum has shown the 2To whom correspondence may be addressed. Email: [email protected]. highest efficiency toward formation of fatty aldehydes that can This article contains supporting information online at https://www.pnas.org/lookup/suppl/ subsequently be converted to fatty alcohols in a previous study doi:10.1073/pnas.2010521117/-/DCSupplemental. (12). CARs utilize free FAs as substrates (Fig. 1A), which are First published September 1, 2020. 22974–22983 | PNAS | September 15, 2020 | vol. 117 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.2010521117 Downloaded by guest on September 26, 2021 A ‘MDH3 Δp β-oxidation ox1 O O Malate Oxaloacetete CoA CoA S RS RtME AnACL FAS* +MmACL Acetyl-CoA Fatty acyl-CoA Glucose Pyruvate Citrate FAS* CTP1 C O Citrate Acetyl-CoA Δtpo1 ROH Mitochondria a. ntr MCFA Medium-chain I B Fatty acid MmCAR modification approaches (MCFA) 88 541 651 726 731 1174 MmCAR MmCAR* Δhfd1 PCP O domain NpgA Adenylation domain (A domain) Random approach R H (directed evolution) Fatty aldehyde Rational design (FDH) Reductase domain ALR/ADH (R domain) Semi-rational design Rational design ROH Medium-chain Full-length Fatty alcohol Random approach (directed evolution) (MCFOH) Fig. 1. Overview of genetic manipulations in yeast enabling selective production of MCFOHs. (A) Schematic illustration of the pathway engineering strategies in background strain ZWE243. AnACL, ATP-citrate lyase from Aspergillus nidulans; Ctp1, mitochondrial citrate transporter; FAS*, engineered en- dogenous fatty acid synthase for MCFAs production; Hfd1, aldehyde dehydrogenase; ′Mdh3, malate dehydrogenase without peroxisomal signal; MmACL, ATP-citrate lyase from Mus musculus; MmCAR, carboxylic acid reductase from Mycobacterium marinum; NpgA, phosphopantetheinyl transferase from As- pergillus nidulans; Pox1, fatty acyl-CoA oxidase; RtME, malic enzyme from Rhodosporidium toruloides; . The genes encoding ′Mdh3, RtME, MmACL, AnACL, MICROBIOLOGY and Ctp1 were overexpressed to increase the acetyl-CoA supply, and POX1 and HFD1 genes were deleted to avoid reverse reactions. R represents C3–C9. (B) MmCAR was modified using diverse approaches. A domain (residues 88 to 541); PCP domain (residues 651 to 728); R domain (residues 731 to 1,174). (C) The predicted MCFA transporter gene TPO1 was deleted to prevent the secretion of MCFAs. more specifically produce MCFOHs. Protein engineering of a evolution as a powerful tool enables the development of reaction CAR enzyme would offer the opportunity to change its substrate schemes and catalytic mechanisms not found in nature (29), and specificity in the desired direction. could be utilized to access the untapped function of the CAR The CAR enzyme consists of an adenylation domain (A do- enzyme using efficient high-throughput selection.
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