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Model-guided metabolic rewiring to bypass pyruvate oxidation for pyruvate derivative synthesis by minimizing carbon loss.

ABSTRACT: Engineering microbial hosts to synthesize pyruvate derivatives depends on blocking pyruvate oxidation, thereby causing severe growth defects in aerobic glucose-based bioprocesses. To decouple pyruvate metabolism from cell growth to improve pyruvate availability, a genome-scale metabolic model combined with constraint-based flux balance analysis, geometric flux balance analysis, and flux variable analysis was used to identify genetic targets for strain design. Using translation elements from a ~3,000 cistronic library to modulate fxpK expression in a bicistronic cassette, a bifido shunt pathway was introduced to generate three molecules of non-pyruvate-derived acetyl-CoA from one molecule of glucose, bypassing pyruvate oxidation and carbon dioxide generation. The dynamic control of flux distribution by T7 RNAP-mediated synthetic small RNA decoupled pyruvate catabolism from cell growth. Adaptive laboratory evolution and multi-omics analysis revealed that a mutated isocitrate dehydrogenase functioned as a metabolic switch to activate the glyoxylate shunt as the only C4 anaplerotic pathway to generate malate from two molecules of acetyl-CoA input and bypass two decarboxylation reactions in the tricarboxylic acid cycle. A chassis strain for pyruvate derivative synthesis was constructed to reduce carbon loss by using the glyoxylate shunt as the only C4 anaplerotic pathway and the bifido shunt as a non-pyruvate-derived acetyl-CoA synthetic pathway and produced 22.46, 27.62, and 6.28 g/L of l-leucine, l-alanine, and l-valine by a controlled small RNA switch, respectively. Our study establishes a novel metabolic pattern of glucose-grown bacteria to minimize carbon loss under aerobic conditions and provides valuable insights into cell design for manufacturing pyruvate-derived products.IMPORTANCEBio-manufacturing from biomass-derived carbon sources using microbes as a cell factory provides an eco-friendly alternative to petrochemical-based processes. Pyruvate serves as a crucial building block for the biosynthesis of industrial chemicals; however, it is different to improve pyruvate availability in vivo due to the coupling of pyruvate-derived acetyl-CoA with microbial growth and energy metabolism via the oxidative tricarboxylic acid cycle. A genome-scale metabolic model combined with three algorithm analyses was used for strain design. Carbon metabolism was reprogrammed using two genetic control tools to fine-tune gene expression. Adaptive laboratory evolution and multi-omics analysis screened the growth-related regulatory targets beyond rational design. A novel metabolic pattern of glucose-grown bacteria is established to maintain growth fitness and minimize carbon loss under aerobic conditions for the synthesis of pyruvate-derived products. This study provides valuable insights into the design of a microbial cell factory for synthetic biology to produce industrial bio-products of interest.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC10949502 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Model-guided metabolic rewiring to bypass pyruvate oxidation for pyruvate derivative synthesis by minimizing carbon loss.

Zhang Yun Y Wang Xueliang X Odesanmi Christianah C Hu Qitiao Q Li Dandan D Tang Yuan Y Liu Zhe Z Mi Jie J Liu Shuwen S Wen Tingyi T

mSystems 20240205 3

Engineering microbial hosts to synthesize pyruvate derivatives depends on blocking pyruvate oxidation, thereby causing severe growth defects in aerobic glucose-based bioprocesses. To decouple pyruvate metabolism from cell growth to improve pyruvate availability, a genome-scale metabolic model combined with constraint-based flux balance analysis, geometric flux balance analysis, and flux variable analysis was used to identify genetic targets for strain design. Using translation elements from a ~3 ...[more]

PMID: 38315666

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Project description:Simple Summary The fermentative production of desired chemicals from renewable resources is one of the promising biosynthetic routes to replace the petrochemical-based process. Gamma-aminobutyric acid (GABA) can be synthesized from l-glutamic acid and used as a building block for the synthesis of butyrolactam and polyamide 4 (nylon 4). The genome-scale metabolic model can predict the growth ability and metabolic flux distribution by genetic disturbances, which provides a strategy to construct a microbial cell factory for GABA and butyrolactam biosynthesis. Here, we performed model-guided metabolic engineering of Corynebacterium glutamicum ATCC13032 for GABA and butyrolactam fermentation from glucose. The biosynthetic pathways of GABA and butyrolactam were constructed by overexpressing the heterologous genes using a bi-cistronic expression cassette. The genetic modifications of the metabolic network cooperatively forced the carbon flux toward GABA and butyrolactam synthesis. This study provides new insights into engineering industrial microorganisms to produce target chemicals from renewable carbon sources. Abstract Gamma-aminobutyric acid (GABA) can be used as a bioactive component in the pharmaceutical industry and a precursor for the synthesis of butyrolactam, which functions as a monomer for the synthesis of polyamide 4 (nylon 4) with improved thermal stability and high biodegradability. The bio-based fermentation production of chemicals using microbes as a cell factory provides an alternative to replace petrochemical-based processes. Here, we performed model-guided metabolic engineering of Corynebacterium glutamicum for GABA and butyrolactam fermentation. A GABA biosynthetic pathway was constructed using a bi-cistronic expression cassette containing mutant glutamate decarboxylase. An in silico simulation showed that the increase in the flux from acetyl-CoA to α-ketoglutarate and the decrease in the flux from α-ketoglutarate to succinate drove more flux toward GABA biosynthesis. The TCA cycle was reconstructed by increasing the expression of acn and icd genes and deleting the sucCD gene. Blocking GABA catabolism and rewiring the transport system of GABA further improved GABA production. An acetyl-CoA-dependent pathway for in vivo butyrolactam biosynthesis was constructed by overexpressing act-encoding ß-alanine CoA transferase. In fed-batch fermentation, the engineered strains produced 23.07 g/L of GABA with a yield of 0.52 mol/mol from glucose and 4.58 g/L of butyrolactam. The metabolic engineering strategies can be used for genetic modification of industrial strains to produce target chemicals from α-ketoglutarate as a precursor, and the engineered strains will be useful to synthesize the bio-based monomer of polyamide 4 from renewable resources.

Dataset Information

Model-guided metabolic rewiring to bypass pyruvate oxidation for pyruvate derivative synthesis by minimizing carbon loss.

Publications

Model-guided metabolic rewiring to bypass pyruvate oxidation for pyruvate derivative synthesis by minimizing carbon loss.

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