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Engineering central pathways for industrial-level (3R)-acetoin biosynthesis in Corynebacterium glutamicum.

ABSTRACT: BACKGROUND:Acetoin, especially the optically pure (3S)- or (3R)-enantiomer, is a high-value-added bio-based platform chemical and important potential pharmaceutical intermediate. Over the past decades, intense efforts have been devoted to the production of acetoin through green biotechniques. However, efficient and economical methods for the production of optically pure acetoin enantiomers are rarely reported. Previously, we systematically engineered the GRAS microorganism Corynebacterium glutamicum to efficiently produce (3R)-acetoin from glucose. Nevertheless, its yield and average productivity were still unsatisfactory for industrial bioprocesses. RESULTS:In this study, cellular carbon fluxes in the acetoin producer CGR6 were further redirected toward acetoin synthesis using several metabolic engineering strategies, including blocking anaplerotic pathways, attenuating key genes of the TCA cycle and integrating additional copies of the alsSD operon into the genome. Among them, the combination of attenuation of citrate synthase and inactivation of phosphoenolpyruvate carboxylase showed a significant synergistic effect on acetoin production. Finally, the optimal engineered strain CGS11 produced a titer of 102.45 g/L acetoin with a yield of 0.419 g/g glucose at a rate of 1.86 g/L/h in a 5 L fermenter. The optical purity of the resulting (3R)-acetoin surpassed 95%. CONCLUSION:To the best of our knowledge, this is the highest titer of highly enantiomerically enriched (3R)-acetoin, together with a competitive product yield and productivity, achieved in a simple, green processes without expensive additives or substrates. This process therefore opens the possibility to achieve easy, efficient, economical and environmentally-friendly production of (3R)-acetoin via microbial fermentation in the near future.

SUBMITTER: Lu L

PROVIDER: S-EPMC7216327 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Engineering central pathways for industrial-level (3R)-acetoin biosynthesis in Corynebacterium glutamicum.

Lu Lingxue L Mao Yufeng Y Kou Mengyun M Cui Zhenzhen Z Jin Biao B Chang Zhishuai Z Wang Zhiwen Z Ma Hongwu H Chen Tao T

Microbial cell factories 20200512 1

<h4>Background</h4>Acetoin, especially the optically pure (3S)- or (3R)-enantiomer, is a high-value-added bio-based platform chemical and important potential pharmaceutical intermediate. Over the past decades, intense efforts have been devoted to the production of acetoin through green biotechniques. However, efficient and economical methods for the production of optically pure acetoin enantiomers are rarely reported. Previously, we systematically engineered the GRAS microorganism Corynebacteriu ...[more]

PMID: 32398078

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Project description:BackgroundLignocellulose provides the only practical carbohydrates feedstock for sustainable bioproduction of hydrocarbons as future alternative of fossil fuels. Production of hydrocarbons from lignocellulose is achieved by a biorefinery process chain including pretreatment to breakdown the crystalline structure for cellulase-catalyzed hydrolysis, detoxification of inhibitory compounds generated during pretreatment, enzymatic hydrolysis to fermentable monosaccharide sugars, and fermentation to hydrocarbon products. The major barriers on fermentative production of hydrocarbons from lignocellulose include two aspects: one is the inherent stress of pretreatment-derived inhibitors on microbial cells, the other is the toxicity of hydrocarbons to cell membranes. The microbial cell factory should be tolerant to both inhibitor stress and hydrocarbons toxicity.ResultsCorynebacterium glutamicum was selected as the starting strain of hydrocarbons synthesis since it is well adapted to lignocellulose hydrolysate environment. The dual hydrocarbon synthesis pathways were constructed in an industrial C. glutamicum S9114 strain. The first pathway was the regular one in microalgae composed of fatty acyl-acyl carrier protein (fatty acyl-ACP) reductase (AAR) and aldehyde deformylating oxygenase (ADO) with fatty acyl-ACP as precursor. The second pathway was the direct decarboxylation of free fatty acid by fatty acid decarboxylase (OleT) using the rich fatty acids from the disruption of the transcriptional regulator fasR gene. The transmembrane transportation of hydrocarbon products was avoided by secretively expressing the fatty acid decarboxylase (OleT) to the extracellular space. The hydrocarbons generation from glucose reached 29.2 mg/L, in which the direct decarboxylation pathway contributed more than 70% of the total hydrocarbons generation, and the AAR-ADO pathway contributed the rest 30%.ConclusionThe dual hydrocarbon synthesis pathways (OleT and AAR-ADO pathways) were constructed in the inhibitors tolerant C. glutamicum S9114 strain for hydrocarbon production using lignocellulose feedstock as the starting feedstock. When corn stover was used for hydrocarbons production after dry acid pretreatment and biodetoxification, the hydrocarbons generation reached 16.0 mg/L. This study provided a new strategy for hydrocarbons synthesis using microbial cell factory suitable for lignocellulose feedstock.

Project description:BackgroundCorynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin.ResultsHere, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 ε-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum ΔcrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum ΔcrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 ± 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum ΔcrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 ± 0.3 mg/g CDW were obtained.ConclusionC. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. glutamicum may serve as a potential host for carotenoid production.

Dataset Information

Engineering central pathways for industrial-level (3R)-acetoin biosynthesis in Corynebacterium glutamicum.

Publications

Engineering central pathways for industrial-level (3R)-acetoin biosynthesis in Corynebacterium glutamicum.

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