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Metabolic engineering for the high-yield production of isoprenoid-based C? alcohols in E. coli.

ABSTRACT: Branched five carbon (C5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C5 alcohols that serve as potential biofuels. We first constructed a pathway for 3-methyl-3-buten-1-ol, where metabolite profiling identified NudB, a promiscuous phosphatase, as a likely pathway bottleneck. We achieved a 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB, which increased protein levels by 9-fold and reduced isopentenyl diphosphate (IPP) accumulation by 4-fold. To further optimize the pathway, we adjusted mevalonate kinase (MK) expression and investigated MK enzymes from alternative microbes such as Methanosarcina mazei. Next, we expressed a fusion protein of IPP isomerase and the phosphatase (Idi1~NudB) along with a reductase (NemA) to diversify production to 3-methyl-2-buten-1-ol and 3-methyl-1-butanol. Finally, we used an oleyl alcohol overlay to improve alcohol recovery, achieving final titers of 2.23?g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield), 150?mg/L of 3-methyl-2-buten-1-ol, and 300?mg/L of 3-methyl-1-butanol.

SUBMITTER: George KW

PROVIDER: S-EPMC4459108 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Metabolic engineering for the high-yield production of isoprenoid-based C₅ alcohols in E. coli.

George Kevin W KW Thompson Mitchell G MG Kang Aram A Baidoo Edward E Wang George G Chan Leanne Jade G LJ Adams Paul D PD Petzold Christopher J CJ Keasling Jay D JD Lee Taek Soon TS

Scientific reports 20150608

Branched five carbon (C5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C5 alcohols that serve as potential biofuels. We first constructed a pathway for 3-methyl-3-buten-1-ol, where metabolite profiling identified NudB, a promisc ...[more]

PMID: 26052683

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Project description:BackgroundCurrent petroleum-derived fuels such as gasoline (C5-C12) and diesel (C15-C22) are complex mixtures of hydrocarbons with different chain lengths and chemical structures. Isoprenoids are hydrocarbon-based compounds with different carbon chain lengths and diverse chemical structures, similar to petroleum. Thus, isoprenoid alcohols such as isopentenol (C5), geraniol (C10), and farnesol (C15) have been considered to be ideal biofuel candidates. NudB, a native phosphatase of Escherichia coli, is reported to dephosphorylate isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) into isopentenol. However, no attention has been paid to its promiscuous activity toward longer chain length (C10-C15) prenyl diphosphates.ResultsIn this study, the promiscuous activity of NudB toward geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) was applied for the production of isoprenoid alcohol mixtures, including isopentenol, geraniol, and farnesol, and their derivatives. E. coli was engineered to produce a mixture of C5 and C15 alcohols by overexpressing NudB (dihydroneopterin triphosphate diphosphohydrolase) and IspA (FPP synthase) along with a heterologous MVA pathway, which resulted in a total of up to 1652 mg/L mixture of C5 and C15 alcohols and their derivatives. The production was further increased to 2027 mg/L by overexpression of another endogenous phosphatase, AphA, in addition to NudB. Production of DMAPP- and FPP-derived alcohols and their derivatives was significantly increased with an increase in the gene dosage of idi, encoding IPP isomerase (IDI), indicating a potential modulation of the composition of the alcohols mixture according to the expression level of IDI. When IspA was replaced with its mutant IspA*, generating GPP in the production strain, a total of 1418 mg/L of the isoprenoid mixture was obtained containing C10 alcohols as a main component.ConclusionsThe promiscuous activity of NudB was newly identified and successfully used for production of isoprenoid-based alcohol mixtures, which are suitable as next-generation biofuels or commodity chemicals. This is the first successful report on high-titer production of an isoprenoid alcohol-based mixture. The engineering approaches can provide a valuable platform for production of other isoprenoid mixtures via a proportional modulation of IPP, DMAPP, GPP, and FPP syntheses.

Project description:BackgroundL(-)-carnitine production has been widely studied because of its beneficial properties on various diseases and dysfunctions. Enterobacteria possess a specific biotransformation pathway which can be used for the enantioselective production of L(-)-carnitine. Although bioprocesses catalyzed by enzymes or whole cells can overcome the lack of enantioselectivity of chemical methods, current processes for L(-)-carnitine production still have severe disadvantages, such as the low yields, side reactions and the need of high catalyst concentrations and anaerobic conditions for proper expression of the biotransformation pathway. Additionally, genetically engineered strains so far constructed for L(-)-carnitine production are based on plasmids and, therefore, suffer from segregational unstability.ResultsIn this work, a stable, high yielding strain for L(-)-carnitine production from low cost substrates was constructed. A metabolic engineering strategy was implemented in a multiple mutant for use in both growing and resting cells systems. The effect of mutations on gene expression and metabolism was analyzed to characterize the productivity constraints of the wild type and the overproducer strains. Precise deletion of genes which encode proteins of central and carnitine metabolisms were performed. Specifically, flux through the TCA cycle was increased by deletion of aceK (which encodes a bifunctional kinase/phosphatase which inhibits isocitrate dehydrogenase activity) and the synthesis of the by-product ?-butyrobetaine was prevented by deletion of caiA (which encodes a crotonobetainyl-CoA reductase). Both mutations led to improve the L(-)-carnitine production by 20 and 42%, respectively. Moreover, the highly regulated promoter of the cai operon was substituted by a constitutive artificial promoter increasing the biotransformation rate, even under aerobic conditions. Resting cells of the BW ?aceK ?caiA p37cai strain produced 59.6 mmol l(-1)?·?h(-1) of L(-)-carnitine, doubling the productivity of the wild type strain. In addition, almost total conversion was attained in less than two hours without concomitant production of the side product ?-butyrobetaine.ConclusionsL(-)-carnitine production has been enhanced by strain engineering. Metabolic engineering strategies herein implemented allowed obtaining a robust and high yielding E. coli strain. The new overproducer strain attained almost complete conversion of crotonobetaine into L(-)-carnitine with growing and resting cells, and even under aerobic conditions, overcoming the main environmental restriction to carnitine metabolism expression. So far, this is the best performing L(-)-carnitine production E. coli strain described.

Dataset Information

Metabolic engineering for the high-yield production of isoprenoid-based C? alcohols in E. coli.

Publications

Metabolic engineering for the high-yield production of isoprenoid-based C₅ alcohols in E. coli.

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