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Expressing a Cytosolic Pyruvate Dehydrogenase Complex To Zhang et al. Microb Cell Fact (2020) 19:226 https://doi.org/10.1186/s12934-020-01493-z Microbial Cell Factories RESEARCH Open Access Expressing a cytosolic pyruvate dehydrogenase complex to increase free fatty acid production in Saccharomyces cerevisiae Yiming Zhang1†, Mo Su1†, Ning Qin1, Jens Nielsen1,2,3 and Zihe Liu1* Abstract Background: Saccharomyces cerevisiae is being exploited as a cell factory to produce fatty acids and their derivatives as biofuels. Previous studies found that both precursor supply and fatty acid metabolism deregulation are essential for enhanced fatty acid synthesis. A bacterial pyruvate dehydrogenase (PDH) complex expressed in the yeast cytosol was reported to enable production of cytosolic acetyl-CoA with lower energy cost and no toxic intermediate. Results: Overexpression of the PDH complex signifcantly increased cell growth, ethanol consumption and reduced glycerol accumulation. Furthermore, to optimize the redox imbalance in production of fatty acids from glucose, two endogenous NAD+-dependent glycerol-3-phosphate dehydrogenases were deleted, and a heterologous NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase was introduced. The best fatty acid producing strain PDH7 with engineering of precursor and co-factor metabolism could produce 840.5 mg/L free fatty acids (FFAs) in shake fask, which was 83.2% higher than the control strain YJZ08. Profle analysis of free fatty acid suggested the cyto- solic PDH complex mainly resulted in the increases of unsaturated fatty acids (C16:1 and C18:1). Conclusions: We demonstrated that cytosolic PDH pathway enabled more efcient acetyl-CoA provision with the lower ATP cost, and improved FFA production. Together with engineering of the redox factor rebalance, the cyto- solic PDH pathway could achieve high level of FFA production at similar levels of other best acetyl-CoA producing pathways. Keywords: The pyruvate dehydrogenase complex, Free fatty acids, Redox factor, Saccharomyces cerevisiae Background modifcation, and their levels are therefore tightly regu- Bioproduction of fatty acids and their derivatives serves lated to ensure in vivo homeostasis [1]. Intensive eforts as a sustainable route to alleviate the dilemma between have been carried out to improve the titer, rate and yield the growing demands of high-energy–density fuels and (TRY) of fatty acid-derived biofuels, focusing mainly on concerns on their environmental impacts. However, fatty the deregulation of fatty acid metabolism and redirection acids are involved in several essential cellular processes, of carbon fux [2–4]. such as membrane synthesis, energy storage, and protein Regarding deregulation of fatty acid metabolism, dele- tions of dominant acyl-CoA synthetases (FAA1, FAA4), *Correspondence: [email protected] and fatty acyl-CoA oxidase (POX1) catalyzing the frst †Yiming Zhang and Mo Su contributed equally to this work step in β-oxidation, could signifcantly accumulate free 1 Beijing Advanced Innovation Center for Soft Matter Science fatty acids (FFAs) [5–7]. Moreover, deletions of main and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, No.15 North Third Ring Road East, sterol ester formation genes (ARE1, ARE2), and phos- Chaoyang District, Beijing 100029, People’s Republic of China phatidate phosphatase genes (PAH1, DPP1, LPP1) could Full list of author information is available at the end of the article also deregulate fatty acid synthesis and increase the © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhang et al. Microb Cell Fact (2020) 19:226 Page 2 of 9 production of total fatty acids [8]. Combining deletion of Previous studies showed that high glycerol produc- several lipid regulation mechanisms resulted in a reduced tion was often observed in FFA production strains, indi- complexity of the fatty acid metabolic network, leading to cating a high NADH pressure [7]. Glycerol synthesis enhanced FFA accumulation [8, 9]. from dihydroxyacetone phosphate consists of two steps, Besides engineering lipid metabolic network, engineer- catalyzed by NAD+-dependent glycerol-3-phosphate ing of precursor supply and redox cofactor regeneration dehydrogenase (encoded by GPD1 and GPD2) and glyc- was also essential for carbon fux redirection towards erol-3-phosphate phosphatase (encoded by GPP1 and FFA production. Each elongation cycle in fatty acid syn- GPP2), respectively (Fig. 1). Glycerol accumulation is also thesis extends the backbone with a C2 unit and requires 2 an issue in bioethanol production and has therefore been molecules of NADPH as cofactor. Endogenous synthesis studied for years. Several studies found that glycerol for- of cytosolic acetyl-CoA, the main C2 metabolite, is often mation could be decreased with altered redox-cofactor limited and several heterologous enzymes have therefore specifcity [11, 19–22], and could be eliminated through been expressed to increase acetyl-CoA supply for FFA inactivation of both GPD1 and GPD2 with the expense synthesis, including NAD +-dependent alcohol dehydro- of a dramatic reduction in cell growth [23]. As alterna- genase (AdhE), NAD+-dependent acylating acetaldehyde tives, an NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (EutE) [10], ATP citrate lyases (ACLs) [6, dehydrogenase (GapN) was demonstrated to enable res- 7, 11–13], xylulose-5-phosphate phosphoketolase (XpkA) cuing the negative efects and improve ethanol yield [24, and phosphotransacetylase (Pta) [14]. Compared to these 25]. Moreover, since GapN irreversibly converts glycer- strategies, expression of cytosolic pyruvate dehydro- aldehyde-3-phosphate to 3-phosphoglycerate and pro- genase (PDH) is attractive because of its lower energy duces NADPH (Fig. 1), it was reported to increase the cost [15]. Te PDH complex consists of three catalytic synthesis of NADPH demanding bioproducts, including subunits, including pyruvate dehydrogenase (E1), dihy- 3-hydroxypropionic acid (3-HP) and polyhydroxybu- drolipoyl transacetylase (E2), and dihydrolipoyl dehy- tyrate (PHB) [26, 27]. Terefore, it could potentially elim- drogenase (E3), and requires four kinds of cofactors TPP, inate glycerol accumulation and increase FFA production lipoate, FAD and NAD+. Among three PDH complexes by interruption of glycerol synthesis and expression of that have been expressed in the cytosol of yeast, PDH GapN. from Enterococcus faecalis was demonstrated to enable Te goal of this study is to investigate the efect of complete replacement of the native pathway for cyto- expressing the cytosolic PDH complex on FFA produc- solic acetyl-CoA synthesis, and was less sensitive to high tion in yeast strains with deregulated fatty acid metabo- NADH/NAD+ ratios compared with other PDH com- lism and optimized cytosolic redox balance (Fig. 1). Te plexes [16–18]. However, one concern with PDH expres- strategies reported here may shed lights for production sion for FFA production is the redox imbalance between of other acetyl-CoA and NADPH demanding chemicals redox couples of NAD+/NADH and NADP+/NADPH. as well. Glucose AMP ATP Redox balance Faa1,4 Hfd1 G3P Fay acyl-CoA Fay acid Fay aldehyde + Gpd1, NADH NAD+ NADP+ NADP Gpd2 GapN Fas1,2 Pah1, NAD+ NADH NADPH NADPH Lpp1, Glycerol 3PG Malonyl-CoA Dpp1, ADP Are1 - HCO3 , Acc1 NAD+ NADH ATP cPDH Pox1 Triacylglycerol Precursor PyruvateAcetyl-CoA FFA supply PDH bypass β-oxidaon Sterol ester Synthesis Fig. 1 Schematic representation of engineering strategies for FFA production in S. cerevisiae. G3P glyceraldehyde-3-phosphate, 3PG 3-phospho-glycerate, cPDH cytosolic pyruvate dehydrogenase. A cytosolic PDH complex was evaluated in a faa1Δ faa4Δ pox1Δ hfd1Δ mutant YJZ08 for free fatty acid production, in combination with fatty acid metabolism engineering via pah1Δ lpp1Δ dpp1Δ are1Δ and redox cofactor engineering via GPD deletion and GapN expression Zhang et al. Microb Cell Fact (2020) 19:226 Page 3 of 9 Results and discussion 1.90 g/L glycerol with YJZ08 (Fig. 2a), suggesting that Expression of a bacterial PDH complex to increase cytosolic PDH did not cause an NADH burden in YJZ08. the acetyl‑CoA supply Teoretically, a combination of the PDH route with A FFA producing strain YJZ08 with four gene deletions the phosphoketolase (PK) pathway can result in a higher (faa1Δ faa4Δ pox1Δ hfd1Δ) was used as the background maximum FFA yield [15], since the PDH route could strain for cytosolic PDH evaluation [6]. Te three subu- produce acetyl-CoA with low energy cost and the PK nits of PDH complex, encoded by pdhA, pdhB,
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