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

Teixeira, Paulo Gonçalves; Ferreira, Raphael; Zhou, Yongjin; Siewers, Verena; Nielsen, Jens

doi:10.1186/s12934-017-0663-3

Cited by 40 publications

(24 citation statements)

References 35 publications

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“…Based on these observations, one would assume that the phase of glucose consumption is particularly well suited for FA biosynthesis. However, yeast appears to have a greater capacity to produce FAs during growth on ethanol (Teixeira et al, 2017), which is supported by the results in this study (e.g. Figure 3A and 3B).…”

Section: Discussionsupporting

confidence: 88%

Investigation of putative regulatory acetylation sites in Fas2p ofSaccharomyces cerevisiae

Bergman

Wenning

Siewers

et al. 2018

Preprint

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Yeast metabolism is highly regulated, in part via coordinated reprogramming of metabolism on a transcriptional level, for example in response to environmental changes. Furthermore, regulation occurs on the protein level via posttranslational modifications directly affecting enzymatic activity -a mode of regulation that has the benefit of being very fast in response to environmental changes. One group of posttranslational modification that has been suggested to have a high impact on regulation of metabolism are acetylations. Around 4000 distinct protein acetylation sites have been found in Saccharomyces cerevisiae, many of which are located in central metabolic enzymes. However, reports on the verification of regulatory roles of specific acetylation sites on these metabolic enzymes have yet to emerge. This study investigates putative regulatory acetylation sites on Fas2p, which in concert with Fas1p is responsible for cytosolic fatty acid (FA) biosynthesis in S. cerevisiae. Fas2p stands out as one of the most highly acetylated proteins in yeast and is located at a branchpoint of acetyl-CoA metabolism. The amino acids (AAs) glutamine (Q) and arginine (R) were introduced to mimic a constitutively acetylated or non-acetylatable state at three separate lysine sites (K) (K83, K173 and K1551) confirmed to be acetylated in two independent studies, either separately or simultaneously. The results suggest that the residue replacement system in the specific case interferes with the enzymatic function of the fatty acid synthase (FAS), as QQQ and RRR triple mutants both reduce the amount of secreted free fatty acids (FFAs) in a faa1∆ faa4∆ yeast deletion mutant. The K173Q substitution significantly decreased C16 FA species at the expense of C18 FAs, while no such change could be observed for the corresponding K173R modification.3 KeywordsSaccharomyces cerevisiae, fatty acid synthase, regulation, protein acetylation, residue replacement system cytosolic/peroxisomal glyoxylate cycle. This pathway is essential for growth on C2 compounds (like acetate and ethanol), but also for degradation of FAs via β-oxidation, and produces succinate which can enter the TCA cycle .As a central metabolite, acetyl-CoA is an important building block in many anabolic reactions, but it also plays a role in the posttranslational modification of proteins by serving as a substrate for lysine acetyltransferases (KATs), which catalyze the transfer of acetyl groups to the epsilon-amino groups of lysines (Lys, K). In this way, fluctuating concentrations of acetyl-CoA, which are connected to the metabolic condition of the cell, are translated into dynamic protein acetylations that regulate a range of cellular functions. Enzymes that remove acetylations from proteins are called lysine deacetylases (KDACs). So far, seven KATs and nine KDACs, respectively, have been identified in yeast. KATs are classified based on their cellular localization, with being either of the nuclear type (A-type) or the cytoplasmic type (Btype), whereas KDACs are phylogenetically div...

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Section: Discussionsupporting

confidence: 88%

Investigation of putative regulatory acetylation sites in Fas2p ofSaccharomyces cerevisiae

Bergman

Wenning

Siewers

et al. 2018

Preprint

Self Cite

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“…Our work complements research in production of fatty acid-derived products in other microbial hosts in several ways (Liu et al, 2013;Teixeira et al, 2017). 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;.…”

Section: Discussionmentioning

confidence: 73%

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

d’Espaux

Ghosh

Runguphan

et al. 2017

Metabolic Engineering

104

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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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“…We also recommend the application of immobilized cells and a biofilm reactor for integrated production and recovery and for efficient use of multi-copy transformants. The de novo production of LCDCA can be further improved by generating a larger pool of free fatty acids [125], which have stronger stimulatory effect on the induction of the background ω-oxidation activity. This could be achieved by overexpressing the upstream and downstream targets of the fatty acid biosynthesis pathway.…”

Section: Discussionmentioning

confidence: 99%

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

2017

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This proof-of-concept study establishes Yarrowia lipolytica (Y. lipolytica) as a whole cell factory for the de novo production of long chain dicarboxylic acid (LCDCA-16 and 18) using glycerol as the sole source of carbon. Modification of the fatty acid metabolism pathway enabled creating a pool of fatty acids in a β-oxidation deficient strain. We then selectively upregulated the native fatty acid ω-oxidation pathway for the enhanced terminal oxidation of the endogenous fatty acid precursors. Nitrogen-limiting conditions and leucine supplementation were employed to induce fatty acid biosynthesis in an engineered Leu -modified strain. Our genetic engineering strategy allowed a minimum production of 330 mg/L LCDCAs in shake flask. Scale up to a 1-L bioreactor increased the titer to 3.49 g/L. Our engineered yeast also produced citric acid as a major by-product at a titer of 39.2 g/L. These results provide basis for developing Y. lipolytica as a safe biorefinery platform for the sustainable production of high-value LCDCAs from non-oily feedstock.

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

Cited by 40 publications

References 35 publications

Investigation of putative regulatory acetylation sites in Fas2p ofSaccharomyces cerevisiae

Investigation of putative regulatory acetylation sites in Fas2p ofSaccharomyces cerevisiae

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

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

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