Verena Siewers

Chalmers University of Technology

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Jens Nielsen

Chalmers University of Technology

127

Florian David

Chalmers University of Technology

Yun Chen

Zhejiang Chinese Medical University

Paulo Gonçalves Teixeira

Chalmers University of Technology

Shuobo Shi

Beijing University of Chemical Technology

Tyler W. Doughty

Chalmers University of Technology

Xiaowei Li

Novo Nordisk Foundation

Yongjin J. Zhou

Dalian Institute of Chemical Physics

Anastasia Krivoruchko

North-Caucasus Federal University

Martin K. M. Engqvist

Chalmers University of Technology

Cooperative Institutions

Chalmers University of Technology

124

Technical University of Denmark

Novo Nordisk Foundation

University of Gothenburg

Chinese Academy of Sciences

Centre National de la Recherche Scientifique

Foundation Center

University of Münster

Beijing University of Chemical Technology

University of Technology

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Production of 10-methyl branched fatty acids in yeast

Biotechnology for Biofuels (2021)

Hannah G. Blitzblau Andrew L. Consiglio Paulo Gonçalves Teixeira Donald V. Crabtree Shuyan Chen

Abstract Background Despite the environmental value of biobased lubricants, they account for less than 2% of global lubricant use due to poor thermo-oxidative stability arising from the presence of unsaturated double bonds. Methyl branched fatty acids (BFAs), particularly those with branching near the acyl-chain mid-point, are a high-performance alternative to existing vegetable oils because of their low melting temperature and full saturation. Results We cloned and characterized two pathways to produce 10-methyl BFAs isolated from actinomycetes and γ-proteobacteria. In the two-step bfa pathway of actinomycetes, BfaB methylates Δ9 unsaturated fatty acids to form 10-methylene BFAs, and subsequently, BfaA reduces the double bond to produce a fully saturated 10-methyl branched fatty acid. A BfaA-B fusion enzyme increased the conversion efficiency of 10-methyl BFAs. The ten-methyl palmitate production (tmp) pathway of γ-proteobacteria produces a 10-methylene intermediate, but the TmpA putative reductase was not active in E. coli or yeast. Comparison of BfaB and TmpB activities revealed a range of substrate specificities from C14-C20 fatty acids unsaturated at the Δ9, Δ10 or Δ11 position. We demonstrated efficient production of 10-methylene and 10-methyl BFAs in S. cerevisiae by secretion of free fatty acids and in Y. lipolytica as triacylglycerides, which accumulated to levels more than 35% of total cellular fatty acids. Conclusions We report here the characterization of a set of enzymes that can produce position-specific methylene and methyl branched fatty acids. Yeast expression of bfa enzymes can provide a platform for the large-scale production of branched fatty acids suitable for industrial and consumer applications.

Methylene

Unsaturated fatty acid

10.1186/s13068-020-01863-0

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Citations (18)

Metabolic engineering of Saccharomyces cerevisiae for overproduction of triacylglycerols

Metabolic Engineering Communications (2018)

Raphaël Ferreira Paulo Gonçalves Teixeira Michael Gossing Florian David Verena Siewers

Triacylglycerols (TAGs) are valuable versatile compounds that can be used as metabolites for nutrition and health, as well as feedstocks for biofuel production. Although Saccharomyces cerevisiae is the favored microbial cell factory for industrial production of biochemicals, it does not produce large amounts of lipids and TAGs comprise only ~1% of its cell dry weight. Here, we engineered S. cerevisiae to reorient its metabolism for overproduction of TAGs, by regulating lipid droplet associated-proteins involved in TAG synthesis and hydrolysis. We implemented a push-and-pull strategy by overexpressing genes encoding a deregulated acetyl-CoA carboxylase, ACC1S659A/S1157A(ACC1**), as well as the last two steps of TAG formation: phosphatidic phosphatase (PAH1) and diacylglycerol acyltransferase (DGA1), ultimately leading to 129 mg∙gCDW-1 of TAGs. Disruption of TAG lipase genes TGL3, TGL4, TGL5 and sterol acyltransferase gene ARE1 increased the TAG content to 218 mg∙gCDW-1. Further disruption of the beta-oxidation by deletion of POX1, as well as glycerol-3-phosphate utilization through deletion of GUT2, did not affect TAGs levels. Finally, disruption of the peroxisomal fatty acyl-CoA transporter PXA1 led to accumulation of 254 mg∙gCDW-1. The TAG levels achieved here are the highest titer reported in S. cerevisiae, reaching 27.4% of the maximum theoretical yield in minimal medium with 2% glucose. This work shows the potential of using an industrially established and robust yeast species for high level lipid production.

Overproduction

Acyltransferases

Metabolic Engineering

Phosphatidate

10.1016/j.meteno.2018.01.002

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Dynamic regulation of fatty acid pools for improved production of fatty alcohols in Saccharomyces cerevisiae

Microbial Cell Factories (2017)

Paulo Gonçalves Teixeira Raphaël Ferreira Yongjin J. Zhou Verena Siewers Jens Nielsen

In vivo production of fatty acid-derived chemicals in Saccharomyces cerevisiae requires strategies to increase the intracellular supply of either acyl-CoA or free fatty acids (FFAs), since their cytosolic concentrations are quite low in a natural state for this organism. Deletion of the fatty acyl-CoA synthetase genes FAA1 and FAA4 is an effective and straightforward way to disable re-activation of fatty acids and drastically increase FFA levels. However, this strategy causes FFA over-accumulation and consequential release to the extracellular medium, which results in a significant loss of precursors that compromises the process yield. In the present study, we aimed for dynamic expression of the fatty acyl-CoA synthetase gene FAA1 to regulate FFA and acyl-CoA pools in order to improve fatty alcohol production yields.We analyzed the metabolite dynamics of a faa1Δ faa4Δ strain constitutively expressing a carboxylic acid reductase from Mycobacterium marinum (MmCAR) and an endogenous alcohol dehydrogenase (Adh5) for in vivo production of fatty alcohols from FFAs. We observed production of fatty acids and fatty alcohols with different rates leading to high levels of FFAs not being converted to the final product. To address the issue, we expressed the MmCAR + Adh5 pathway together with a fatty acyl-CoA reductase from Marinobacter aquaeolei to enable fatty alcohol production simultaneously from FFA and acyl-CoA, respectively. Then, we expressed FAA1 under the control of different promoters in order to balance FFA and acyl-CoA interconversion rates and to achieve optimal levels for conversion to fatty alcohols. Expressing FAA1 under control of the HXT1 promoter led to an increased accumulation of fatty alcohols per OD600 up to 41% while FFA levels were decreased by 63% compared with the control strain.Fine-tuning and dynamic regulation of key metabolic steps can be used to improve cell factories when the rates of downstream reactions are limiting. This avoids loss of precursors to the extracellular medium or to competing reactions, hereby potentially improving the process yield. The study also provides knowledge of a key point of fatty acid regulation and homeostasis, which can be used for future design of cells factories for fatty acid-derived chemicals.

Fatty alcohol

Fatty Acid Metabolism

Fatty acid synthesis

Acyl-CoA

10.1186/s12934-017-0663-3

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Engineering Yeast to Improve Heterologous Abscisic Acid Production

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Maximilian Otto Michael Gossing Florian David Verena Siewers

Abstract Abscisic acid (ABA) is a high-value product with agricultural, medical and nutritional applications. We previously constructed an ABA cell factory by expressing the ABA metabolic pathway from Botrytis cinerea in the biotechnological workhorse Saccharomyces cerevisiae . In this study, we aimed to improve ABA production and explored various rational engineering targets mostly focusing on increasing the activity of two rate-limiting cytochrome P450 monooxygenases of the ABA pathway, BcABA1 and BcABA2. We evaluated the effects of overexpression and knock-down of cell membrane transporters, expression of heterologous cytochrome b5, overexpression of a rate-limiting heme biosynthesis gene and overexpression or knock-out of genes involved in ER membrane homeostasis. One of the genes involved in ER membrane homeostasis, PAH1 , was identified as the most promising engineering target. Knock-out of PAH1 improved ABA titers, but also caused a sever growth defect. By replacing the PAH1 promoter with a weak minimal promoter, it was possible to mediate the growth defect while still improving ABA production. In this report we were able to improve the ABA cell factory and furthermore provide valuable insights for future studies aiming to engineer cytochrome P450 monooxygenases. One-sentence summary In this study we explored various strategies to improve heterologous abscisic acid production in Saccharomyces cerevisiae and identified fine-tuning of the PAH1 gene as a promising engineering strategy.

Heterologous expression

Heterologous

Metabolic Engineering

10.1101/2023.06.07.544016

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Citations (1)

Implementation of communication‐mediating domains for non‐ribosomal peptide production in Saccharomyces cerevisiae

Biotechnology and Bioengineering (2010)

Verena Siewers Rita Santo San-Bento Jens Nielsen

Abstract Saccharomyces cerevisiae has in several cases been proven to be a suitable host for the production of natural products and was recently exploited for the production of non‐ribosomal peptides. Synthesis of non‐ribosomal peptides (NRPs) is mediated by NRP synthetases (NRPSs), modular enzymes, which are often organized in enzyme complexes. In these complexes, partner NRPSs interact via communication‐mediating domains (COM domains). In order to test whether functional interaction between separate NRPS modules is possible in yeast we constructed a yeast strain expressing two modules with compatible COM domains from two plasmids. Successful production as well as secretion of the expected dipeptide was detected. This opens the possibility of using yeast as a eukaryotic platform for fast assessment of new module combinations for the development of novel NRP compounds. Biotechnol. Bioeng. 2010;106: 841–844. © 2010 Wiley Periodicals, Inc.

10.1002/bit.22739

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The Influence of Microgravity on Invasive Growth inSaccharomyces cerevisiae

Astrobiology (2011)

Sebastiaan E. Van Mulders Catherine Stassen Luk Daenen Bart Devreese Verena Siewers

This study investigates the effects of microgravity on colony growth and the morphological transition from single cells to short invasive filaments in the model eukaryotic organism Saccharomyces cerevisiae. Two-dimensional spreading of the yeast colonies grown on semi-solid agar medium was reduced under microgravity in the Σ1278b laboratory strain but not in the CMBSESA1 industrial strain. This was supported by the Σ1278b proteome map under microgravity conditions, which revealed upregulation of proteins linked to anaerobic conditions. The Σ1278b strain showed a reduced invasive growth in the center of the yeast colony. Bud scar distribution was slightly affected, with a switch toward more random budding. Together, microgravity conditions disturb spatially programmed budding patterns and generate strain-dependent growth differences in yeast colonies on semi-solid medium. Key Words: Microgravity—Saccharomyces cerevisiae—Adhesion—Invasive growth—Budding—Proteomics. Astrobiology 11, 45–55.

Budding yeast

Strain (injury)

Budding

Proteome

10.1089/ast.2010.0518

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Citations (35)

Yeast perfume factory: metabolic engineering of Saccharomyces cerevisiae for plant isoprenoid biosynthesis

S. Gionata Christoph Knuf P. Siavash Y. Chen Jérôme Maury

Metabolic Engineering

Factory (object-oriented programming)

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Functional analysis of the cytochrome P450 monooxygenase Gene bcbot1 of Botrytis cinerea indicates. That botrydial is a strain-specific virulence factor

Verena Siewers Muriel Viaud Daniel Jiménez-Teja Isidro G. Collado Christian Schulze Gronover

Strain (injury)

Virulence factor

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Citations (7)

A Single Chromosome Strain ofS. cerevisiaeExhibits Diminished Ethanol Metabolism and Tolerance

bioRxiv (Cold Spring Harbor Laboratory) (2020)

Tyler W. Doughty Rosemary Yu Lucy Fang-I Chao Zhongjun Qin Verena Siewers

Abstract This study characterized the growth, metabolism, and transcriptional profile of a S. cerevisiae strain with a single large chromosome that was constructed via successive chromosomal fusions. The single chromosome strain exhibited a longer lag phase, increased doubling time, and lower final biomass concentration compared with a wildtype strain when grown on YPD. These phenotypes were amplified when ethanol was added to the medium or used as the sole carbon source. RNAseq analysis showed diminished induction of genes involved in diauxic shift, ethanol metabolism, fatty-acid ß-oxidation, and methylglyoxal catabolism during growth on ethanol compared to the reference strain. Enzyme-constrained metabolic modeling predicted that decreased flux through these poorly induced enzymes results in diminished ATP formation and decreased biomass accumulation observed. Together, these observations suggest that switch-like control of carbon source dependent gene expression in S. cerevisiae requires genome arrangement into multiple chromosomes.

Catabolism

Strain (injury)

10.1101/2020.08.22.256727

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Functional expression and characterization of five wax ester synthases in Saccharomyces cerevisiae and their utility for biodiesel production

Biotechnology for Biofuels (2012)

Shuobo Shi Juan Octavio Valle‐Rodríguez Sakda Khoomrung Verena Siewers Jens Nielsen

Wax ester synthases (WSs) can synthesize wax esters from alcohols and fatty acyl coenzyme A thioesters. The knowledge of the preferred substrates for each WS allows the use of yeast cells for the production of wax esters that are high-value materials and can be used in a variety of industrial applications. The products of WSs include fatty acid ethyl esters, which can be directly used as biodiesel. Here, heterologous WSs derived from five different organisms were successfully expressed and evaluated for their substrate preference in Saccharomyces cerevisiae. We investigated the potential of the different WSs for biodiesel (that is, fatty acid ethyl esters) production in S. cerevisiae. All investigated WSs, from Acinetobacter baylyi ADP1, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6 and Psychrobacter arcticus 273-4, have different substrate specificities, but they can all lead to the formation of biodiesel. The best biodiesel producing strain was found to be the one expressing WS from M. hydrocarbonoclasticus DSM 8798 that resulted in a biodiesel titer of 6.3 mg/L. To further enhance biodiesel production, acetyl coenzyme A carboxylase was up-regulated, which resulted in a 30% increase in biodiesel production. Five WSs from different species were functionally expressed and their substrate preference characterized in S. cerevisiae, thus constructing cell factories for the production of specific kinds of wax ester. WS from M. hydrocarbonoclasticus showed the highest preference for ethanol compared to the other WSs, and could permit the engineered S. cerevisiae to produce biodiesel.

Wax ester

10.1186/preaccept-1932279820621895

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Citations (88)