Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases

Liu, Aiqiu; Tan, Xiaoming; Yao, Lun; Lü, Xuefeng

doi:10.1007/s00253-013-5027-2

Cited by 64 publications

(70 citation statements)

References 37 publications

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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: 74%

“…Besides our earlier report expressing a Mus musculus FAR in S. cerevisiae (Runguphan and Keasling, 2013), other labs have reported on heterologously expressing a FAR from Tyto alba (Feng et al, 2015) and two from Marinobacter hydrocarbonasticus (Liu et al, 2013;Wahlen et al, 2009;Willis et al, 2011). To determine the variant most effective for fatty alcohol production in S. cerevisiae, we chromosomally integrated the four FAR coding sequences (MmFAR1, TaFAR1, MaFAR2, and MaFAR7, codon-optimized sequences in Suppl.…”

Section: Pull: Comparing Fatty Acyl-coa Reductase Variantsmentioning

confidence: 99%

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

d’Espaux

Ghosh

Runguphan

et al. 2017

Metabolic Engineering

103

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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: 74%

Section: Pull: Comparing Fatty Acyl-coa Reductase Variantsmentioning

confidence: 99%

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

d’Espaux

Ghosh

Runguphan

et al. 2017

Metabolic Engineering

103

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“…As mentioned previously, additional activities toward fatty acyl-CoA and fatty acyl-ACP have been reported for the fatty aldehyde reductase from M. aquaeolei VT8 since the initial characterization of this enzyme (4,5). Additionally, others have recently demonstrated that the fatty aldehyde reductase (Maqu_2220, or NCBI accession number YP_959486) resulted in improved fatty alcohol yields when heterologously expressed in E. coli versus several other fatty acyl-CoA reductases (21).…”

Section: Resultsmentioning

confidence: 99%

Fatty Alcohols for Wax Esters in Marinobacter aquaeolei VT8: Two Optional Routes in the Wax Biosynthesis Pathway

Lenneman

Ohlert

Palani

et al. 2013

Appl Environ Microbiol

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The biosynthesis of wax esters in bacteria is accomplished by a unique pathway that combines a fatty alcohol and a fatty acyl coenzyme A substrate. Previous in vitro enzymatic studies indicated that two different enzymes could be involved in the synthesis of the required fatty alcohol in Marinobacter aquaeolei VT8. In this study, we demonstrate through a series of gene deletions and transcriptional analysis that either enzyme is capable of fulfilling the role of providing the fatty alcohol required for wax ester biosynthesis in vivo, but evolution has clearly selected one of these, a previously characterized fatty aldehyde reductase, as the preferred enzyme to perform this reaction under typical wax ester-accumulating conditions. These results complement previous in vitro studies and provide the first glimpse into the role of each enzyme in vivo in the native organism.T he global cycle of oil is of interest from the standpoints of both energy and the environment, as efforts by humankind to obtain this valuable resource can result in substantial releases of crude oil through incidents such as the Deepwater Horizon oil spill of 2010 in the Gulf of Mexico. We also note that crude oil from natural deposits is routinely released into aqueous environments, such as the oceans, by natural processes where geological reserves meet surface waters. These environments have allowed natural populations of organisms, such as marine bacteria, to evolve to utilize these supplies, rich in reduced carbon, for use as a biological fuel source. A primary focus related to oil-degrading marine bacteria is the oxidation of these oils to meet energy requirements of the living cell. Interestingly, for certain marine bacteria found to utilize and degrade oils, these bacteria are also capable of producing natural lipids that have economic values similar to those obtained from harvesting sperm whales prior to the late 20th century, even when the bacteria are grown on simple organic acids or carbohydrates. We selected the marine bacterium Marinobacter aquaeolei VT8, which was isolated from an oil well off the coast of Vietnam (1), as a model bacterial species to study metabolic processes in an oil-metabolizing and neutral lipid-accumulating bacteria. In addition to growing on long-chain hydrocarbons, M. aquaeolei VT8 also produces a natural hydrocarbon, the wax ester, when grown on simple citric acid cycle intermediates, such as succinate or citrate, as the sole carbon source (1-3), indicating that all of the precursors required for the biosynthesis of wax ester are indigenous to this strain.Biosynthesis of wax esters is accomplished by the combination of several different enzymes. The wax ester synthase/acyl-coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT) enzyme catalyzes the reaction of a fatty acyl-CoA substrate with a fatty alcohol (Fig. 1). While the fatty acyl-CoA utilized by the WS/ DGAT is proposed to come directly from the fatty acyl-CoA pool, the fatty alcohol is believed to be produced through the action of several reducta...

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“…While many thioesterases have been ex-plored for long-chain fatty acid production (14)(15)(16)(17), few studies have focused on those that prefer short-chain acyl-CoAs. Several broad-specificity acyl-CoA thioesterases, including E. coli TesB and Saccharomyces cerevisiae Pte1p, can be used for SCFA production but lack the specificity necessary for optimizing biosynthetic pathways (11,18).…”

mentioning

confidence: 99%

Functional Screening and In Vitro Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli

McMahon

Prather

2014

Appl Environ Microbiol

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b Short-chain fatty acid (SCFA) biosynthesis is pertinent to production of biofuels, industrial compounds, and pharmaceuticals from renewable resources. To expand on Escherichia coli SCFA products, we previously implemented a coenzyme A (CoA)-dependent pathway that condenses acetyl-CoA to a diverse group of short-chain fatty acyl-CoAs. To increase product titers and reduce premature pathway termination products, we conducted in vivo and in vitro analyses to understand and improve the specificity of the acyl-CoA thioesterase enzyme, which releases fatty acids from CoA. A total of 62 putative bacterial thioesterases, including 23 from the cow rumen microbiome, were inserted into a pathway that condenses acetyl-CoA to an acyl-CoA molecule derived from exogenously provided propionic or isobutyric acid. Functional screening revealed thioesterases that increase production of saturated (valerate), unsaturated (trans-2-pentenoate), and branched (4-methylvalerate) SCFAs compared to overexpression of E. coli thioesterase tesB or native expression of endogenous thioesterases. To determine if altered thioesterase acyl-CoA substrate specificity caused the increase in product titers, six of the most promising enzymes were analyzed in vitro. Biochemical assays revealed that the most productive thioesterases rely on promiscuous activity but have greater specificity for product-associated acyl-CoAs than for precursor acyl-CoAs. In this study, we introduce novel thioesterases with improved specificity for saturated, branched, and unsaturated short-chain acyl-CoAs, thereby expanding the diversity of potential fatty acid products while increasing titers of current products. The growing uncertainty associated with protein database annotations denotes this study as a model for isolating functional biochemical pathway enzymes in situations where experimental evidence of enzyme function is absent.

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Fatty alcohol production in engineered E. coli expressing Marinobacter fatty acyl-CoA reductases

Cited by 64 publications

References 37 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

Fatty Alcohols for Wax Esters in Marinobacter aquaeolei VT8: Two Optional Routes in the Wax Biosynthesis Pathway

Functional Screening and In Vitro Analysis Reveal Thioesterases with Enhanced Substrate Specificity Profiles That Improve Short-Chain Fatty Acid Production in Escherichia coli

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