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Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives.

ABSTRACT: Background:Current 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. Results:In 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. Conclusions:The 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.

SUBMITTER: Zada B

PROVIDER: S-EPMC6058358 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives.

Zada Bakht B Wang Chonglong C Park Ji-Bin JB Jeong Seong-Hee SH Park Ju-Eon JE Singh Hawaibam Birla HB Kim Seon-Won SW

Biotechnology for biofuels 20180724

<h4>Background</h4>Current 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 co ...[more]

PMID: 30061932

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Project description:BACKGROUND: Riboflavin (vitamin B2), the precursor of the flavin cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), is used commercially as an animal feed supplement and food colorant. E. coli is a robust host for various genetic manipulations and has been employed for efficient production of biofuels, polymers, amino acids, and bulk chemicals. Thus, the aim of this study was to understand the metabolic capacity of E. coli for the riboflavin production by modification of central metabolism, riboflavin biosynthesis pathway and optimization of the fermentation conditions. RESULTS: The basic producer RF01S, in which the riboflavin biosynthesis genes ribABDEC from E. coli were overexpressed under the control of the inducible trc promoter, could accumulate 229.1 mg/L of riboflavin. Further engineering was performed by examining the impact of expression of zwf (encodes glucose 6-phosphate dehydrogenase) and gnd (encodes 6-phosphogluconate dehydrogenase) from Corynebacterium glutamicum and pgl (encodes 6-phosphogluconolactonase) from E. coli on riboflavin production. Deleting pgi (encodes glucose-6-phosphate isomerase) and genes of Entner-Doudoroff (ED) pathway successfully redirected the carbon flux into the oxidative pentose phosphate pathway, and overexpressing the acs (encodes acetyl-CoA synthetase) reduced the acetate accumulation. These modifications increased riboflavin production to 585.2 mg/L. By further modulating the expression of ribF (encodes riboflavin kinase) for reducing the conversion of riboflavin to FMN in RF05S, the final engineering strain RF05S-M40 could produce 1036.1 mg/L riboflavin in LB medium at 37°C. After optimizing the fermentation conditions, strain RF05S-M40 produced 2702.8 mg/L riboflavin in the optimized semi-defined medium, which was a value nearly 12-fold higher than that of RF01S, with a yield of 137.5 mg riboflavin/g glucose. CONCLUSIONS: The engineered strain RF05S-M40 has the highest yield among all reported riboflavin production strains in shake flask culture. This work collectively demonstrates that E. coli has a potential to be a microbial cell factory for riboflavin bioproduction.

Project description:BACKGROUND:The biosynthesis of high value-added compounds using metabolically engineered strains has received wide attention in recent years. Myo-inositol (inositol), an important compound in the pharmaceutics, cosmetics and food industries, is usually produced from phytate via a harsh set of chemical reactions. Recombinant Escherichia coli strains have been constructed by metabolic engineering strategies to produce inositol, but with a low yield. The proper distribution of carbon flux between cell growth and inositol production is a major challenge for constructing an efficient inositol-synthesis pathway in bacteria. Construction of metabolically engineered E. coli strains with high stoichiometric yield of inositol is desirable. RESULTS:In the present study, we designed an inositol-synthesis pathway from glucose with a theoretical stoichiometric yield of 1 mol inositol/mol glucose. Recombinant E. coli strains with high stoichiometric yield (>?0.7 mol inositol/mol glucose) were obtained. Inositol was successfully biosynthesized after introducing two crucial enzymes: inositol-3-phosphate synthase (IPS) from Trypanosoma brucei, and inositol monophosphatase (IMP) from E. coli. Based on starting strains E. coli BW25113 (wild-type) and SG104 (?ptsG::glk, ?galR::zglf, ?poxB::acs), a series of engineered strains for inositol production was constructed by deleting the key genes pgi, pfkA and pykF. Plasmid-based expression systems for IPS and IMP were optimized, and expression of the gene zwf was regulated to enhance the stoichiometric yield of inositol. The highest stoichiometric yield (0.96 mol inositol/mol glucose) was achieved from recombinant strain R15 (SG104, ?pgi, ?pgm, and RBSL5-zwf). Strain R04 (SG104 and ?pgi) reached high-density in a 1-L fermenter when using glucose and glycerol as a mixed carbon source. In scaled-up fed-batch bioconversion in situ using strain R04, 0.82 mol inositol/mol glucose was produced within 23 h, corresponding to a titer of 106.3 g/L (590.5 mM) inositol. CONCLUSIONS:The biosynthesis of inositol from glucose in recombinant E. coli was optimized by metabolic engineering strategies. The metabolically engineered E. coli strains represent a promising method for future inositol production. This study provides an essential reference to obtain a suitable distribution of carbon flux between glycolysis and inositol synthesis.

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.

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Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives.

Publications

Metabolic engineering of <i>Escherichia coli</i> for production of mixed isoprenoid alcohols and their derivatives.

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