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Engineering Escherichia coli for Glutarate Production as the C5 Platform Backbone.

ABSTRACT: Glutarate is a linear-chain dicarboxylic acid with wide applications in the production of polyesters and polyamides such as nylon-4,5 and nylon-5,5. Previous studies focused on the biological production of glutarate from lysine with low yields and titers. Here, we report on glutarate production by Escherichia coli using a five-step reverse adipate degradation pathway (RADP) identified in Thermobifida fusca By expressing the enzymes of RADP, the glutarate was detected by strain Bgl146 in shaken flasks. After fermentation optimization, the titer of glutarate by Bgl146 was increased to 4.7 ± 0.2 mM in shaken flasks. We further eliminated pathways for the major metabolites competing for carbon flux by CRISPR/Cas9 (?arcA, ?ldhA, ?atoB, and ?pflB). Moreover, the final strain Bgl4146 produced 36.5 ± 0.3 mM glutarate by fed-batch fermentation. These results constitute the highest glutarate titer reported in E. coliIMPORTANCE Glutarate is an important C5 linear-chain dicarboxylic acid, which is widely used in polyesters and polyamides such as nylon-4,5 and nylon-5,5 in the chemical industry. Glutarate is currently produced from the feedstocks derived from petroleum, specifically by oxidation of a mixture of cyclohexanone and cyclohexanol catalyzed by nitric acid. However, the chemical synthesis results in high pollution and dramatic greenhouse gas emission. Thus, the biological production of glutarate directly from the substrate is of great importance. Although there have been reports using Corynebacterium glutamicum to produce glutarate, it has serious limitations due to the limited lysine supply and long fermentation time. To solve this problem, a novel synthetic pathway was constructed in this study, and the highest glutarate titer was reported in Escherichia coli using a short fermentation time without lysine addition, making bio-based glutarate production much more feasible.

SUBMITTER: Zhao M

PROVIDER: S-EPMC6070760 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Engineering Escherichia coli for Glutarate Production as the C5 Platform Backbone.

Zhao Mei M Li Guohui G Deng Yu Y

Applied and environmental microbiology 20180801 16

Glutarate is a linear-chain dicarboxylic acid with wide applications in the production of polyesters and polyamides such as nylon-4,5 and nylon-5,5. Previous studies focused on the biological production of glutarate from lysine with low yields and titers. Here, we report on glutarate production by Escherichia coli using a five-step reverse adipate degradation pathway (RADP) identified in Thermobifida fusca By expressing the enzymes of RADP, the glutarate was detected by strain Bgl1 ...[more]

PMID: 29858204

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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:UNLABELLED:Engineering microbial hosts for the production of fungible fuels requires mitigation of limitations posed on the production capacity. One such limitation arises from the inherent toxicity of solvent-like biofuel compounds to production strains, such as Escherichia coli. Here we show the importance of host engineering for the production of short-chain alcohols by studying the overexpression of genes upregulated in response to exogenous isopentenol. Using systems biology data, we selected 40 genes that were upregulated following isopentenol exposure and subsequently overexpressed them in E. coli. Overexpression of several of these candidates improved tolerance to exogenously added isopentenol. Genes conferring isopentenol tolerance phenotypes belonged to diverse functional groups, such as oxidative stress response (soxS, fpr, and nrdH), general stress response (metR, yqhD, and gidB), heat shock-related response (ibpA), and transport (mdlB). To determine if these genes could also improve isopentenol production, we coexpressed the tolerance-enhancing genes individually with an isopentenol production pathway. Our data show that expression of 6 of the 8 candidates improved the production of isopentenol in E. coli, with the methionine biosynthesis regulator MetR improving the titer for isopentenol production by 55%. Additionally, expression of MdlB, an ABC transporter, facilitated a 12% improvement in isopentenol production. To our knowledge, MdlB is the first example of a transporter that can be used to improve production of a short-chain alcohol and provides a valuable new avenue for host engineering in biogasoline production. IMPORTANCE:The use of microbial host platforms for the production of bulk commodities, such as chemicals and fuels, is now a focus of many biotechnology efforts. Many of these compounds are inherently toxic to the host microbe, which in turn places a limit on production despite efforts to optimize the bioconversion pathways. In order to achieve economically viable production levels, it is also necessary to engineer production strains with improved tolerance to these compounds. We demonstrate that microbial tolerance engineering using transcriptomics data can also identify targets that improve production. Our results include an exporter and a methionine biosynthesis regulator that improve isopentenol production, providing a starting point to further engineer the host for biogasoline production.

Dataset Information

Engineering Escherichia coli for Glutarate Production as the C5 Platform Backbone.

Publications

Engineering Escherichia coli for Glutarate Production as the C<sub>5</sub> Platform Backbone.

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