Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Engineering carboxylic acid reductase for selective synthesis of medium-chain fatty alcohols in yeast.

ABSTRACT: Medium-chain fatty alcohols (MCFOHs, C6 to C12) are potential substitutes for fossil fuels, such as diesel and jet fuels, and have wide applications in various manufacturing processes. While today MCFOHs are mainly sourced from petrochemicals or plant oils, microbial biosynthesis represents a scalable, reliable, and sustainable alternative. Here, we aim to establish a Saccharomyces cerevisiae platform capable of selectively producing MCFOHs. This was enabled by tailoring the properties of a bacterial carboxylic acid reductase from Mycobacterium marinum (MmCAR). Extensive protein engineering, including directed evolution, structure-guided semirational design, and rational design, was implemented. MmCAR variants with enhanced activity were identified using a growth-coupled high-throughput screening assay relying on the detoxification of the enzyme's substrate, medium-chain fatty acids (MCFAs). Detailed characterization demonstrated that both the specificity and catalytic activity of MmCAR was successfully improved and a yeast strain harboring the best MmCAR variant generated 2.8-fold more MCFOHs than the strain expressing the unmodified enzyme. Through deletion of the native MCFA exporter gene TPO1, MCFOH production was further improved, resulting in a titer of 252 mg/L for the final strain, which represents a significant improvement in MCFOH production in minimal medium by S. cerevisiae.

SUBMITTER: Hu Y

PROVIDER: S-EPMC7502760 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Medium chain fatty acid (MCFAs) synthesis

Project description:Medium chain fatty acid (MCFAs) synthesis Metagenome

| PRJNA950190 | ENA

Anaerobic production of medium-chain fatty alcohols via a ?-reduction pathway.

Project description:In this report, we identify the relevant factors to increase production of medium chain n-alcohols through an expanded view of the reverse ?-oxidation pathway. We began by creating a base strain capable of producing medium chain n-alcohols from glucose using a redox-balanced and growth-coupled metabolic engineering strategy. By dividing the heterologous enzymes in the pathway into different modules, we were able to identify and evaluate homologs of each enzyme within the pathway and identify several capable of enhancing medium chain alcohol titers and/or selectivity. In general, the identity of the trans-2-enoyl-CoA reductase (TER) and the direct overexpression of the thiolase (FadA) and ?-hydroxy-acyl-CoA reductase (FadB) improved alcohol titer and the identity of the FadBA complex influenced the dominant chain length. Next, we linked the anaerobically induced VHb promoter from Vitreoscilla hemoglobin to each gene to remove the need for chemical inducers and ensure robust expression. The highest performing strain with the autoinduced reverse ?-oxidation pathway produced n-alcohols at titers of 1.8?g/L with an apparent molar yield of 0.2 on glucose consumed in rich medium (52% of theoretical yield).

| S-EPMC6072553 | biostudies-literature

Selective Extraction of Medium-Chain Carboxylic Acids by Electrodialysis and Phase Separation.

Project description:Carboxylic acids obtained via the microbial electrochemical conversion of waste gases containing carbon dioxide (i.e., microbial electrosynthesis) can be used in lieu of nonrenewable building-block chemicals in the manufacture of a variety of products. When targeting valuable medium-chain carboxylic acids such as caproic acid, electricity-driven fermentations can be limited by the accumulation of fermentation products in the culturing media, often resulting in low volumetric productivities and titers due to direct toxicity or inhibition of the biocatalyst. In this study, we tested the effectiveness of a simple electrodialysis system in upconcentrating carboxylic acids from a model solution mimicking the effluent of a microbial electrochemical system producing short- and medium-chain carboxylic acids. Under batch extraction conditions, the electrodialysis scheme enabled the recovery of 60% (mol mol-1) of the total carboxylic acids present in the model fermentation broth. The particular arrangement of conventional monopolar ion exchange membranes and hydraulic recirculation loops allowed the progressive acidification of the extraction solution, enabling phase separation of caproic acid as an immiscible oil with 76% purity.

| S-EPMC7992139 | biostudies-literature

Production of medium chain length fatty alcohols from glucose in Escherichia coli.

Project description:Metabolic engineering offers the opportunity to produce a wide range of commodity chemicals that are currently derived from petroleum or other non-renewable resources. Microbial synthesis of fatty alcohols is an attractive process because it can control the distribution of chain lengths and utilize low cost fermentation substrates. Specifically, primary alcohols with chain lengths of 12 to 14 carbons have many uses in the production of detergents, surfactants, and personal care products. The current challenge is to produce these compounds at titers and yields that would make them economically competitive. Here, we demonstrate a metabolic engineering strategy for producing fatty alcohols from glucose. To produce a high level of 1-dodecanol and 1-tetradecanol, an acyl-ACP thioesterase (BTE), an acyl-CoA ligase (FadD), and an acyl-CoA/aldehyde reductase (MAACR) were overexpressed in an engineered strain of Escherichia coli. Yields were improved by balancing expression levels of each gene, using a fed-batch cultivation strategy, and adding a solvent to the culture for extracting the product from cells. Using these strategies, a titer of over 1.6 g/L fatty alcohol with a yield of over 0.13 g fatty alcohol/g carbon source was achieved. These are the highest reported yield of fatty alcohols produced from glucose in E. coli.

| S-EPMC3866921 | biostudies-literature

Evolutionary engineering improves tolerance for medium-chain alcohols in <i>Saccharomyces cerevisiae</i>.

Project description:BackgroundYeast-based chemical production is an environmentally friendly alternative to petroleum-based production or processes that involve harsh chemicals. However, many potential alcohol biofuels, such as n-butanol, isobutanol and n-hexanol, are toxic to production organisms, lowering the efficiency and cost-effectiveness of these processes. We set out to improve the tolerance of Saccharomyces cerevisiae toward these alcohols.ResultsWe evolved the laboratory strain of S. cerevisiae BY4741 to be more tolerant toward n-hexanol and show that the mutations which confer tolerance occur in proteins of the translation initiation complex. We found that n-hexanol inhibits initiation of translation and evolved mutations in the α subunit of eIF2 and the γ subunit of its guanine exchange factor eIF2B rescue this inhibition. We further demonstrate that translation initiation is affected by other alcohols such as n-pentanol and n-heptanol, and that mutations in the eIF2 and eIF2B complexes greatly improve tolerance to these medium-chain alcohols.ConclusionsWe successfully generated S. cerevisiae strains that have improved tolerance toward medium-chain alcohols and have demonstrated that the causative mutations overcome inhibition of translation initiation by these alcohols.

| S-EPMC5880003 | biostudies-literature

Genetic engineering of medium-chain-length fatty acid synthesis in Dunaliella tertiolecta for improved biodiesel production.

Project description:Genetic engineering of microalgae to accumulate high levels of medium-chain-length fatty acids (MCFAs) represents an attractive strategy to improve the quality of microalgae-based biodiesel, but it has thus far been least successful. We demonstrate that one limitation is the availability of fatty acyl-acyl carrier protein (ACP) substrate pool for acyl-ACP thioesterase (TE). A combinational expression platform that involved plant lauric acid-biased TE (C12TE) and MCFA-specific ketoacyl-ACP synthase (KASIV) increased lauric acid (C12:0) and myristic acid (C14:0) accumulation by almost sevenfold and fourfold, respectively, compared with native strain. These findings suggest a platform for further investigation into the enlargement of MCFA acyl-ACP substrate pool as an approach to sustainably improve quality of microalgae-based biodiesel with regard to MCFA production.

| S-EPMC5705751 | biostudies-literature

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

Project description:Fatty alcohols are value-added chemicals and important components of a variety of industries, which have a >3 billion-dollar global market annually. Long chain fatty alcohols (>C12) are mainly used in surfactants, lubricants, detergents, pharmaceuticals and cosmetics while medium chain fatty alcohols (C6-C12) could be used as diesel-like biofuels. Microbial production of fatty alcohols from renewable feedstock stands as a promising strategy to enable sustainable supply of fatty alcohols. In this study, we report, for the first time, that medium chain fatty alcohols could be produced in yeast via targeted expression of a fatty acyl-CoA reductase (TaFAR) in the peroxisome of Saccharomyces cerevisiae. By tagging TaFAR enzyme with peroxisomal targeting signal peptides, the TaFAR could be compartmentalized into the matrix of the peroxisome to hijack the medium chain fatty acyl-CoA generated from the beta-oxidation pathway and convert them to versatile medium chain fatty alcohols (C10 &C12). The overexpression of genes encoding PEX7 and acetyl-CoA carboxylase further improved fatty alcohol production by 1.4-fold. After medium optimization in fed-batch fermentation using glucose as the sole carbon source, fatty alcohols were produced at 1.3 g/L, including 6.9% 1-decanol, 27.5% 1-dodecanol, 2.9% 1-tetradecanol and 62.7% 1-hexadecanol. This work revealed that peroxisome could be engineered as a compartmentalized organelle for producing fatty acid-derived chemicals in S. cerevisiae.

| S-EPMC4882508 | biostudies-literature

Increasing medium chain fatty acids production in Yarrowia lipolytica by metabolic engineering.

Project description:BackgroundOleaginous yeast Yarrowia lipolytica is an organism of choice for the development of biofuel and oleochemicals. It has become a chassis for metabolic engineering in order to produce targeted lipids. Understanding the function of key-enzymes involved in lipid metabolism is essential to design better routes for enhanced lipid production and for strains producing lipids of interest. Because medium chain fatty acids (MCFA) are valuable compounds for biokerosene production, we previously generated strains capable of producing MCFA up to 12% of total lipid content (Rigouin et al. in ACS Synth Biol 6:1870-1879, 2017). In order to improve accumulation and content of C14 fatty acid (FA), the elongation, degradation and accumulation of these MCFA in Yarrowia lipolytica were studied.ResultsWe brought evidence of the role of YALI0F0654 (YlELO1) protein in the elongation of exogenous or de novo synthesized C14 FA into C16 FA and C18 FA. YlELO1 deletion into a αFAS_I1220W expressing strain leads to the sole production of C14 FA. However, because this strain does not provide the FA essential for its growth, it requires being cultivated with essential fatty acids and C14 FA yield is limited. To promote MCFA accumulation in Y. lipolytica without compromising the growth, we overexpressed a plant diglyceride acyltransferase specific for MCFA and reached an accumulation of MCFA up to 45% of total lipid content.ConclusionWe characterized the role of YlELO1 in Y. lipolytica by proving its involvement in Medium chain fatty acids elongation. We showed that MCFA content can be increased in Yarrowia lipolytica by promoting their accumulation into a stable storage form (triacylglycerides) to limit their elongation and their degradation.

| S-EPMC6130074 | biostudies-literature

Fatty alcohols production by oleaginous yeast.

Project description:We have engineered Rhodosporidium toruloides to produce fatty alcohols by expressing a fatty acyl-CoA reductase from Marinobacter aquaeolei VT8. Production of fatty alcohols in flasks was achieved in different fermentation media at titers ranging from 0.2 to 2 g/L. In many of the conditions tested, more than 80 % of fatty alcohols were secreted into the cultivation broth. Through fed-batch fermentation in 7 L bioreactors, over 8 g/L of C(16)-C(18) fatty alcohols were produced using sucrose as the substrate. This is the highest titer ever reported on microbial production of fatty alcohols to date.

| S-EPMC4607723 | biostudies-literature

Metabolic Engineering for Enhanced Medium Chain Omega Hydroxy Fatty Acid Production in Escherichia coli.

Project description:Medium chain hydroxy fatty acids (HFAs) at ?-1, 2, or 3 positions (?-1/2/3) are rare in nature but are attractive due to their potential applications in industry. They can be metabolically engineered in Escherichia coli, however, the current yield is low. In this study, metabolic engineering with P450BM3 monooxygenase was applied to regulate both the chain length and sub-terminal position of HFA products in E. coli, leading to increased yield. Five acyl-acyl carrier protein thioesterases from plants and bacteria were first evaluated for regulating the chain length of fatty acids. Co-expression of the selected thioesterase gene CcFatB1 with a fatty acid metabolism regulator fadR and monooxygenase P450BM3 boosted the production of HFAs especially ?-3-OH-C14:1, in both the wild type and fadD deficient strain. Supplementing renewable glycerol to reduce the usage of glucose as a carbon source further increased the HFAs production to 144 mg/L, representing the highest titer of such HFAs obtained in E. coli under the comparable conditions. This study illustrated an improved metabolic strategy for medium chain ?-1/2/3 HFAs production in E. coli. In addition, the produced HFAs were mostly secreted into culture media, which eased its recovery.

| S-EPMC5808347 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data