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A cell-free system for production of 2,3-butanediol is robust to growth-toxic compounds.

ABSTRACT: The need for sustainable, low-cost production of bioenergy and commodity chemicals is increasing. Unfortunately, the engineering potential of whole-cell catalysts to address this need can be hampered by cellular toxicity. When such bottlenecks limit the commercial feasibility of whole-cell fermentation, cell-free, or in vitro, based approaches may offer an alternative. Here, we assess the impact of three classes of growth toxic compounds on crude extract-based, cell-free chemical conversions. As a model system, we test a metabolic pathway for conversion of glucose to 2,3-butanediol (2,3-BDO) in lysates of Escherichia coli. First, we characterized 2,3-BDO production with different classes of antibiotics and found, as expected, that the system is uninhibited by compounds that prevent cell growth by means of cell wall replication and DNA, RNA, and protein synthesis. Second, we considered the impact of polar solvent addition (e.g., methanol, n-butanol). We observed that volumetric productivities (g/L/h) were slowed with increasing hydrophobicity of added alcohols. Finally, we investigated the effects of using pretreated biomass hydrolysate as a feed stock, observing a 25% reduction in 2,3-BDO production as a result of coumaroyl and feruloyl amides. Overall, we find the cell-free system to be robust to working concentrations of antibiotics and other compounds that are toxic to cell growth, but do not denature or inhibit relevant enzymes.

SUBMITTER: Kay JE

PROVIDER: S-EPMC6951449 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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A cell-free system for production of 2,3-butanediol is robust to growth-toxic compounds.

Kay Jennifer E JE Jewett Michael C MC

Metabolic engineering communications 20191120

The need for sustainable, low-cost production of bioenergy and commodity chemicals is increasing. Unfortunately, the engineering potential of whole-cell catalysts to address this need can be hampered by cellular toxicity. When such bottlenecks limit the commercial feasibility of whole-cell fermentation, cell-free, or <i>in vitro</i>, based approaches may offer an alternative. Here, we assess the impact of three classes of growth toxic compounds on crude extract-based, cell-free chemical conversi ...[more]

PMID: 31934547

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Project description:BackgroundPreviously, a safe strain, Bacillus amyloliquefaciens B10-127 was identified as an excellent candidate for industrial-scale microbial fermentation of 2,3-butanediol (2,3-BD). However, B. amyloliquefaciens fermentation yields large quantities of acetoin, lactate and succinate as by-products, and the 2,3-BD yield remains prohibitively low for commercial production.Methodology/principal findingsIn the 2,3-butanediol metabolic pathway, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of 3-phosphate glyceraldehyde to 1,3-bisphosphoglycerate, with concomitant reduction of NAD(+) to NADH. In the same pathway, 2,3-BD dehydrogenase (BDH) catalyzes the conversion of acetoin to 2,3-BD with concomitant oxidation of NADH to NAD(+). In this study, to improve 2,3-BD production, we first over-produced NAD(+)-dependent GAPDH and NADH-dependent BDH in B. amyloliquefaciens. Excess GAPDH reduced the fermentation time, increased the 2,3-BD yield by 12.7%, and decreased the acetoin titer by 44.3%. However, the process also enhanced lactate and succinate production. Excess BDH increased the 2,3-BD yield by 16.6% while decreasing acetoin, lactate and succinate production, but prolonged the fermentation time. When BDH and GAPDH were co-overproduced in B. amyloliquefaciens, the fermentation time was reduced. Furthermore, in the NADH-dependent pathways, the molar yield of 2,3-BD was increased by 22.7%, while those of acetoin, lactate and succinate were reduced by 80.8%, 33.3% and 39.5%, relative to the parent strain. In fed-batch fermentations, the 2,3-BD concentration was maximized at 132.9 g/l after 45 h, with a productivity of 2.95 g/l·h.Conclusions/significanceCo-overexpression of bdh and gapA genes proved an effective method for enhancing 2,3-BD production and inhibiting the accumulation of unwanted by-products (acetoin, lactate and succinate). To our knowledge, we have attained the highest 2,3-BD fermentation yield thus far reported for safe microorganisms.

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A cell-free system for production of 2,3-butanediol is robust to growth-toxic compounds.

Publications

A cell-free system for production of 2,3-butanediol is robust to growth-toxic compounds.

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