Characterization of a Fatty Acyl-CoA Reductase from <i>Marinobacter aquaeolei</i> VT8: A Bacterial Enzyme Catalyzing the Reduction of Fatty Acyl-CoA to Fatty Alcohol

Willis, Robert M.; Wahlen, Bradley D.; Seefeldt, Lance C.; Barney, Brett M.

doi:10.1021/bi2008646

Cited by 107 publications

(138 citation statements)

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“…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%

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: 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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“…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 reductase enzymes acting on activated fatty acids or fatty aldehydes. M. aquaeolei VT8 contains at least two enzymes that have been found to produce fatty alcohols from several different substrates in vitro, including fatty aldehydes, fatty acyl-CoAs, and fatty acyl-acyl carrier proteins (ACPs) (4)(5)(6)(7). Additionally, both of the isolated enzymes from M. aquaeolei VT8 have significantly higher activities than those reported for other enzymes when tested in in vitro assays versus the enzyme isolated from Acinetobacter (4,(6)(7)(8).…”

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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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“…Heterologous expression of plant FARs in Escherichia coli, rapeseed (B. napus), and Saccharomyces cerevisiae led to a range of saturated and unsaturated alcohols of C14 to C26 chain length, depending on the host organism [100,114,115]. Besides plant FARs, microbial FAR genes [102,116,117] and FARs from vertebrates have also been isolated and functionally described [97,98].…”

Section: Far Propertiesmentioning

confidence: 99%

Biotechnological potential of insect fatty acid-modifying enzymes

Tupec

Buček

Valterová

2017

Zeitschrift Für Naturforschung C

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There are more than one million described insect species. This species richness is reflected in the diversity of insect metabolic processes. In particular, biosynthesis of secondary metabolites, such as defensive compounds and chemical signals, encompasses an extraordinarily wide range of chemicals that are generally unparalleled among natural products from other organisms. Insect genomes, transcriptomes and proteomes thus offer a valuable resource for discovery of novel enzymes with potential for biotechnological applications. Here, we focus on fatty acid (FA) metabolism-related enzymes, notably the fatty acyl desaturases and fatty acyl reductases involved in the biosynthesis of FA-derived pheromones. Research on insect pheromone-biosynthetic enzymes, which exhibit diverse enzymatic properties, has the potential to broaden the understanding of enzyme specificity determinants and contribute to engineering of enzymes with desired properties for biotechnological production of FA derivatives. Additionally, the application of such pheromone-biosynthetic enzymes represents an environmentally friendly and economic alternative to the chemical synthesis of pheromones that are used in insect pest management strategies.

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Characterization of a Fatty Acyl-CoA Reductase from Marinobacter aquaeolei VT8: A Bacterial Enzyme Catalyzing the Reduction of Fatty Acyl-CoA to Fatty Alcohol

Cited by 107 publications

References 31 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

Biotechnological potential of insect fatty acid-modifying enzymes

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