A Yeast-Based Biosensor for Screening of Short- and Medium-Chain Fatty Acid Production

Baumann, Leonie; Rajkumar, Arun S.; Morrissey, John P.; Boles, Eckhard; Oreb, Mislav

doi:10.1021/acssynbio.8b00309

Cited by 38 publications

(38 citation statements)

References 36 publications

(107 reference statements)

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“…However, it is possible that the cells intrinsically upregulate factors that drive the production of LCFA (e.g., Acc1). Therefore, adaptive laboratory evolution, as a possible approach to increase the OA tolerance, should be accompanied by high-throughput methods capable of measuring the intrinsic OA production, such as the recently developed biosensors (Baumann et al, 2018), to prevent the selection of adapted strains exhibiting an unfavorable FA product profile. Uncoupling biomass generation from production is another promising strategy to improve the production of toxic products (Yu et al, 2017), that could also be tested for OA biosynthesis in follow-up studies.…”

Section: Systematic Analysis Of Reactions Limiting Oa Productionmentioning

confidence: 99%

Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations

Wernig

Baumann

Boles

et al. 2021

Biotech & Bioengineering

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The eight-carbon fatty acid octanoic acid (OA) is an important platform chemical and precursor of many industrially relevant products. Its microbial biosynthesis is regarded as a promising alternative to current unsustainable production methods. In Saccharomyces cerevisiae, the production of OA had been previously achieved by rational engineering of the fatty acid synthase. For the supply of the precursor molecule acetyl-CoA and of the redox cofactor NADPH, the native pyruvate dehydrogenase bypass had been harnessed, or the cells had been additionally provided with a pathway involving a heterologous ATP-citrate lyase. Here, we redirected the flux of glucose towards the oxidative branch of the pentose phosphate pathway and overexpressed a heterologous phosphoketolase/phosphotransacetylase shunt to improve the supply of NADPH and acetyl-CoA in a strain background with abolished OA degradation. We show that these modifications lead to an increased yield of OA during the consumption of glucose by more than 60% compared to the parental strain. Furthermore, we investigated different genetic engineering targets to identify potential factors that limit the OA production in yeast. Toxicity assays performed with the engineered strains suggest that the inhibitory effects of OA on cell growth likely impose an upper limit to attainable OA yields. K E Y W O R D S acetyl-CoA, octanoic acid, phosphoketolase, phosphotransacetylase 1 | INTRODUCTION Fatty acids (FAs) with various chain lengths and their derivatives with different functional groups are important compounds in modern industry, which have numerous applications like fuels, cosmetics, pharmaceuticals, and food additives. Recently, engineering microbial FA production has attracted attention as an alternative to established methods such as petrochemistry or oil palm cultivation, which have lately been criticized for their environmental impact. In Saccharomyces cerevisiae, biosynthesis of FAs in the cytosol is catalyzed by the large multidomain fatty acid synthase (FAS) complex, which naturally generates long-chain fatty acid (LCFA, C14-C18) as building blocks of membranes or for storage lipids. The biosynthesis is initiated by cytosolic acetyl-CoA (AcCoA) and maturing FAs are elongated by AcCoA-derived malonyl-CoA until This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Section: Systematic Analysis Of Reactions Limiting Oa Productionmentioning

confidence: 99%

Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations

Wernig

Baumann

Boles

et al. 2021

Biotech & Bioengineering

Self Cite

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“…Among the screened promoters, we identified the previously reported short‐ and medium‐chain fatty acid promoter sensor pPDR12 . We also identified three further promoters—pTDH1, pPHO3, and pUBC13—that obviously upregulated mCherry expression upon exposure of cells to C6 or C12 fatty acid.…”

Section: Resultsmentioning

confidence: 99%

“…Further, a G‐protein‐coupled receptor from mammals has been used to detect even‐chain C8–C12 fatty acids in S. cerevisiae . Recently, the endogenous short‐ and medium‐chain fatty acid promoter sensor pPDR12 of S. cerevisiae has been reported and showed its highest sensitivity toward C6 . However, much more work needs to be done to study MCFA‐responsive promoters in S. cerevisiae .…”

Section: Introductionmentioning

confidence: 99%

Discovery and identification of medium‐chain fatty acid responsive promoters in Saccharomyces cerevisiae

Han

et al. 2020

Engineering in Life Sciences

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Medium-chain fatty acids (MCFAs) and their derivatives are important chemicals that can be used in lubricants, detergents, and cosmetics. MCFAs can be produced in several microbes, although production is not high. Dynamic regulation by synthetic biology is a good method of improving production of chemicals that avoids toxic intermediates, but chemical-responsive promoters are required. Several MCFA sensors or promoters have been reported in Saccharomyces cerevisiae. In this study, by using transcriptomic analysis of S. cerevisiae exposed to fatty acids with 6-, 12-, and 16-carbon chains, we identified 58 candidate genes that may be responsive to MCFAs. Using a fluorescence-based screening method, we identified MCFA-responsive promoters, four that upregulated gene expression, and three that downregulated gene expression. Dose-response analysis revealed that some of the promoters were sensitive to fatty acid concentrations as low as 0.02-0.06 mM. The MCFA-responsive promoters reported in this study could be used in dynamic regulation of fatty acids and fatty acid-derived products in S. cerevisiae. K E Y W O R D Sdose response, medium-chain fatty acid, promoter, Saccharomyces cerevisiae, transcriptome Abbreviation: MCFAs, medium-chain fatty acids.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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“…[ 114 ] Based on this phenomenon, substrate‐responsive natural transporter promoters were discovered and related transcription factors were designed, which have been utilized to develop biosensors for high‐throughput screening of high‐producing strains. [ 115 ] These elements were even found to have the ability of a feedback regulation in native transporter expression. [ 66,116 ] Therefore, these dynamic regulatory elements can be used to control heterologous transporters and pathways in scale‐up fermentations.…”

Section: Resultsmentioning

confidence: 99%

Transporter Engineering for Microbial Manufacturing

Zhu

Zhou

Wang

et al. 2020

Biotechnology Journal

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Microbes play an important role in biotransformation and biosynthesis of biofuels, natural products, and polymers. Therefore, microbial manufacturing has been widely used in medicine, industry, and agriculture. However, common strategies including enzyme engineering, pathway optimization, and host engineering are generally inadequate to obtain an efficient microbial production system. Transporter engineering provides an alternative strategy to promote the transmembrane transfer of substrates, intermediates, and final products in microbial cells and thus enhances production by alleviating feedback inhibition and cytotoxicity caused by final products. According to the current studies in transport engineering, native transporters usually have low expression and poor transportation ability, resulting in inefficient transport processes and microbial production. In this review, current approaches for transporter mining, characterization, and verification are comprehensively summarized. Practical approaches to enhance the transport system in engineered cells, such as balancing transporter overexpression and cell growth, and evolution of native transporters are discussed. Furthermore, the applications of transporter engineering in microbial manufacturing, including enhancement of substrate utilization, concentration of metabolic flux to the target pathway, and acceleration of efflux and recovery of products, demonstrate its outstanding advantages and promising prospects.

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A Yeast-Based Biosensor for Screening of Short- and Medium-Chain Fatty Acid Production

Cited by 38 publications

References 36 publications

Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations

Production of octanoic acid in Saccharomyces cerevisiae: Investigation of new precursor supply engineering strategies and intrinsic limitations

Discovery and identification of medium‐chain fatty acid responsive promoters in Saccharomyces cerevisiae

Transporter Engineering for Microbial Manufacturing

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