Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes

Lee, Won Heong; Seo, Seung Beom; Bae, Yi-Hyun; Nan, Hong; Jin, Yong Su; Seo, Jin Ho

doi:10.1007/s00449-012-0736-y

Cited by 90 publications

(38 citation statements)

References 25 publications

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“…Despite these genetic changes, engineered yeast still displays diauxic sugar consumption; glucose is preferentially consumed first, followed by xylose. In addition to ethanol, isobutanol [4–7], butanol [8], and fatty acid [9–12] biofuels have been generated from pure glucose and xylose by engineered S. cerevisiae , but not from sugars derived from lignocellulosic biomass.…”

Section: Introductionmentioning

confidence: 99%

Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose

Tran

Breuer

Narasimhan

et al. 2017

Biotechnol Biofuels

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BackgroundAcetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain an sn-3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers. Acetyl-TAGs can be synthesized by EaDAcT, a diacylglycerol acetyltransferase enzyme originally isolated from Euonymus alatus (Burning Bush). The heterologous expression of EaDAcT in different organisms, including Saccharomyces cerevisiae, resulted in the accumulation of acetyl-TAGs in storage lipids. Microbial conversion of lignocellulose into acetyl-TAGs could allow biorefinery production of versatile molecules for biofuel and bioproducts.ResultsIn order to produce acetyl-TAGs from abundant lignocellulose feedstocks, we expressed EaDAcT in S. cerevisiae previously engineered to utilize xylose as a carbon source. The resulting strains were capable of producing acetyl-TAGs when grown on different media. The highest levels of acetyl-TAG production were observed with growth on synthetic lab media containing glucose or xylose. Importantly, acetyl-TAGs were also synthesized by this strain in ammonia fiber expansion (AFEX)-pretreated corn stover hydrolysate (ACSH) at higher volumetric titers than previously published strains. The deletion of the four endogenous enzymes known to contribute to lcTAG production increased the proportion of acetyl-TAGs in the total storage lipids beyond that in existing strains, which will make purification of these useful lipids easier. Surprisingly, the strains containing the four deletions were still capable of synthesizing lcTAG, suggesting that the particular strain used in this study possesses additional undetermined diacylglycerol acyltransferase activity. Additionally, the carbon source used for growth influenced the accumulation of these residual lcTAGs, with higher levels in strains cultured on xylose containing media.ConclusionOur results demonstrate that S. cerevisiae can be metabolically engineered to produce acetyl-TAGs when grown on different carbon sources, including hydrolysate derived from lignocellulose. Deletion of four endogenous acyltransferases enabled a higher purity of acetyl-TAGs to be achieved, but lcTAGs were still synthesized. Longer incubation times also decreased the levels of acetyl-TAGs produced. Therefore, additional work is needed to further manipulate acetyl-TAG production in this strain of S. cerevisiae, including the identification of other TAG biosynthetic and lipolytic enzymes and a better understanding of the regulation of the synthesis and degradation of storage lipids.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0751-y) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose

Tran

Breuer

Narasimhan

et al. 2017

Biotechnol Biofuels

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“…The highest higher alcohols production from our experiment is higher than previous reports. For example, the highest isobutanol 151 mg/L can be obtained using S. cerevisiae as expression host viaby overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes404142. The highest isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol from C. crenatum without improvement in cell properties and harbouring mutant genes only reach to 1264.63 mg/L, 1026.61 mg/L, 748.35 mg/L43.…”

Section: Resultsmentioning

confidence: 99%

Metabolic engineering of Corynebacterium crenatium for enhancing production of higher alcohols

Lin

Wang

2016

Sci Rep

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Biosynthesis approaches for the production of higher alcohols as a source of alternative fossil fuels have garnered increasing interest recently. However, there is little information available in the literature about using undirected whole-cell mutagenesis (UWCM) in vivo to improve higher alcohols production. In this study, for the first time, we approached this question from two aspects: first preferentially improving the capacity of expression host, and subsequently optimizing metabolic pathways using multiple genetic mutations to shift metabolic flux toward the biosynthetic pathway of target products to convert intermediate 2-keto acid compounds into diversified C4~C5 higher alcohols using UWCM in vivo, with the aim of improving the production. The results demonstrated the production of higher alcohols including isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol from glucose and duckweed under simultaneous saccharification and fermentation (SSF) scheme were higher based on the two aspects compared with only the use of wild-type stain as expression host. These findings showed that the improvement via UWCM in vivo in the two aspects for expression host and metabolic flux can facilitate the increase of higher alcohols production before using gene editing technology. Our work demonstrates that a multi-faceted approach for the engineering of novel synthetic pathways in microorganisms for improving biofuel production is feasible.

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“…The three named isomers of butanol have different physical and chemical characteristics but all of them are considered as high value components either as chemicals or fuel. Various biological routes have been suggested for each of the three butanol isomers, such as amino acid pathways redirection for 1-butanol and isobutanol [2]–[4] and exploitation of the 1-butanol synthesis pathway in clostridium species [5]–[7]. 2-Butanol production can theoretically be introduced in yeast by a two-step conversion as found e.g.…”

Section: Introductionmentioning

confidence: 99%

2-Butanol and Butanone Production in Saccharomyces cerevisiae through Combination of a B12 Dependent Dehydratase and a Secondary Alcohol Dehydrogenase Using a TEV-Based Expression System

2014

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2-Butanol and its chemical precursor butanone (methyl ethyl ketone – MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuterii), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions.

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Isobutanol production in engineered Saccharomyces cerevisiae by overexpression of 2-ketoisovalerate decarboxylase and valine biosynthetic enzymes

Cited by 90 publications

References 25 publications

Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose

Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose

Metabolic engineering of Corynebacterium crenatium for enhancing production of higher alcohols

2-Butanol and Butanone Production in Saccharomyces cerevisiae through Combination of a B12 Dependent Dehydratase and a Secondary Alcohol Dehydrogenase Using a TEV-Based Expression System

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