Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols

Sheng, Jiayuan; Stevens, Joseph R.; Feng, Xueyang

doi:10.1038/srep26884

Cited by 72 publications

(40 citation statements)

References 41 publications

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“…The findings about the ER subcellular localization of MmFar1p (mouse fatty acid reductase) is relevant to engineering pathway compartmentalization as a way to optimize bioproduction or tailor product distribution (Avalos et al, 2013;Sheng et al, 2016;. Our observation that overexpression of a fatty acid desaturase led to improvements in fatty acid products in S. cerevisiae as in Y. lipolytica (Qiao et al, 2015) points to commonalities in fatty pathway engineering in both organisms.…”

Section: Discussionmentioning

confidence: 83%

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

d’Espaux

Ghosh

Runguphan

et al. 2017

Metabolic Engineering

105

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A B S T R A C TFatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing. We compared four heterologous FARs, finding highest activity and endoplasmic reticulum localization from a Mus musculus FAR. After screening an additional twenty-one single-gene edits, we identified increasing FAR expression; deleting competing reactions encoded by DGA1, HFD1, and ADH6; overexpressing a mutant acetyl-CoA carboxylase; limiting NADPH and carbon usage by the glutamate dehydrogenase encoded by GDH1; and overexpressing the Δ9-desaturase encoded by OLE1 as successful strategies to improve titer. Our final strain produced 1.2 g/L fatty alcohols in shake flasks, and 6.0 g/L in fed-batch fermentation, corresponding to~20% of the maximum theoretical yield from glucose, the highest titers and yields reported to date in S. cerevisiae. We further demonstrate high-level production from lignocellulosic feedstocks derived from ionic-liquid treated switchgrass and sorghum, reaching 0.7 g/L in shake flasks. Altogether, our work represents progress towards efficient and renewable microbial production of fatty acidderived products.

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Section: Discussionmentioning

confidence: 83%

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

d’Espaux

Ghosh

Runguphan

et al. 2017

Metabolic Engineering

105

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“…These alkyl hydroxycinnamates were not detected in cultures of a control strain expressing 4CL5 alone and fed with hydroxycinnamates and 1-dodecanol (data not shown). The microbial synthesis of alkyl hydroxycinnamates has never been reported and engineering strategies used for the production of fatty alcohols could be leveraged for their de novo synthesis [54, 55]. Moreover, using a strain that co-express FjTAL with 4CL5 and AtHHT3, we achieved the production of dodecyl p -coumarate by supplying only 1-dodecanol to the culture medium (Additional file 1: Fig.…”

Section: Resultsmentioning

confidence: 99%

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast

et al. 2016

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BackgroundBAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals.ResultsFor the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters. Similarly, we achieved for the first time the microbial production of polyamine hydroxycinnamate amides; monolignol, malate and fatty alcohol hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate.ConclusionWe demonstrate in this study the effectiveness of expressing members of the plant BAHD acyltransferase family in yeast for the synthesis of numerous valuable hydroxycinnamate and benzoate conjugates.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0593-5) contains supplementary material, which is available to authorized users.

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“…Many of the reports of engineered compartmentalization take advantage of the endogenous 4 cofactor supply or substrate pool in a particular organelle. For example, the mitochondria has been used for its supplies of acetyl-CoA 19,20 and α-ketoisovalerate 21 , the peroxisome for acyl-CoA 22,23 , farnesyl diphosphate 24 , and acetyl-CoA 18 , and the vacuole for halide ions and Sadenosylmethionine 25 . Organelles have also been harnessed for the reactions that they natively perform.…”

Section: Introductionmentioning

confidence: 99%

Repurposing the yeast peroxisome to compartmentalize a toxic enzyme enables improved (S)-reticuline production

Grewal¹,

Samson²,

Baker

et al. 2020

Preprint

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Eukaryotic cells compartmentalize metabolic pathways in organelles to achieve optimal reaction conditions and avoid crosstalk with other factors in the cytosol. Increasingly, engineers are researching ways in which synthetic compartmentalization could be used to address challenges in metabolic engineering. Here, we identified that norcoclaurine synthase (NCS), the enzyme which catalyzes the first committed reaction in benzylisoquinoline alkaloid (BIA) biosynthesis, is toxic when expressed cytosolically in Saccharomyces cerevisiae and, consequently, restricts (S)-reticuline production. We developed a compartmentalization strategy that alleviates NCS toxicity while promoting increased (S)-reticuline titer, achieved through efficient targeting of toxic NCS to the peroxisome while, crucially, taking advantage of the free flow of metabolite substrates and product across the peroxisome membrane. We identified that peroxisome protein capacity in S. cerevisiae becomes a limiting factor for further improvement of BIA production and demonstrate that expression of engineered transcription factors can mimic the oleate response for larger peroxisomes, further increasing BIA titer without the requirement for peroxisome induction with fatty acids. This work specifically addresses the challenges associated with toxic NCS expression and, more broadly, highlights the potential for engineering organelles with desired characteristics for metabolic engineering.

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Pathway Compartmentalization in Peroxisome of Saccharomyces cerevisiae to Produce Versatile Medium Chain Fatty Alcohols

Cited by 72 publications

References 41 publications

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

Engineering high-level production of fatty alcohols by Saccharomyces cerevisiae from lignocellulosic feedstocks

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast

Repurposing the yeast peroxisome to compartmentalize a toxic enzyme enables improved (S)-reticuline production

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