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Abstract Background The microbial chiral product (R)-3-hydroxybutyrate (3-HB) is a gateway to several industrial and medical compounds. Acetyl-CoA is the key precursor for 3-HB, and several native pathways compete with 3-HB production. The principal competing pathway in wild-type Escherichia coli for acetyl-CoA is mediated by citrate synthase (coded by gltA), which directs over 60% of the acetyl-CoA into the tricarboxylic acid cycle. Eliminating citrate synthase activity (deletion of gltA) prevents growth on glucose as the sole carbon source. In this study, an alternative approach is used to generate an increased yield of 3-HB: citrate synthase activity is reduced but not eliminated by targeted substitutions in the chromosomally expressed enzyme. Results Five E. coli GltA variants were examined for 3-HB production via heterologous overexpression of a thiolase (phaA) and NADPH-dependent acetoacetyl-CoA reductase (phaB) from Cupriavidus necator. In shake flask studies, four variants showed nearly 5-fold greater 3-HB yield compared to the wild-type, although pyruvate accumulated. Overexpression of either native thioesterases TesB or YciA eliminated pyruvate formation, but diverted acetyl-CoA towards acetate formation. Overexpression of pantothenate kinase similarly decreased pyruvate formation but did not improve 3-HB yield. Controlled batch studies at the 1.25 L scale demonstrated that the GltA[A267T] variant produced the greatest 3-HB titer of 4.9 g/L with a yield of 0.17 g/g. In a phosphate-starved repeated batch process, E. coli ldhA poxB pta-ackA gltA::gltA [A267T] generated 15.9 g/L 3-HB (effective concentration of 21.3 g/L with dilution) with yield of 0.16 g/g from glucose as the sole carbon source. Conclusions This study demonstrates that GltA variants offer a means to affect the generation of acetyl-CoA derived products. This approach should benefit a wide range of acetyl-CoA derived biochemical products in E. coli and other microbes. Enhancing substrate affinity of the introduced pathway genes like thiolase towards acetyl-CoA will likely further increase the flux towards 3-HB while reducing pyruvate and acetate accumulation.

Background: The microbial chiral product (R)-3-hydroxybutyrate (3-HB) is a gateway to several industrial and medical compounds. Acetyl-CoA is the key precursor for 3-HB, and several native pathways compete with 3-HB production. The principal competing pathway in wild-type Escherichia coli for acetyl-CoA is mediated by citrate synthase (coded by gltA), which directs over 60% of the acetyl-CoA into the tricarboxylic acid cycle. Eliminating citrate synthase activity (deletion of gltA) prevents growth on glucose as the sole carbon source. In this study, an alternative approach is used to generate an increased yield of 3-HB: citrate synthase activity is reduced but not eliminated by targeted substitutions in the chromosomally expressed enzyme. Results: Five E. coli GltA variants were examined for 3-HB production via heterologous overexpression of a thiolase (phaA) and NADPH-dependent acetoacetyl-CoA reductase (phaB) from Cupriavidus necator. In shake flask studies, four variants showed nearly 5-fold greater 3-HB yield compared to the wild-type, although pyruvate accumulated. Overexpression of either native thioesterases TesB or YciA eliminated pyruvate formation, but diverted acetyl-CoA towards acetate formation. Overexpression of pantothenate kinase similarly decreased pyruvate formation but did not improve 3-HB yield. Controlled batch studies at the 1.25 L scale demonstrated that the GltA[A267T] variant produced the greatest 3-HB titer of 4.9 g/L with a yield of 0.17 g/g. In a phosphate-starved repeated batch process, E. coli ldhA poxB pta-ackA gltA::gltA[A267T] generated 15.9 g/L 3-HB (effective concentration of 21.3 g/L with dilution) with yield of 0.16 g/g from glucose as the sole carbon source. Conclusions: This study demonstrates that GltA variants offer a means to affect the generation of acetyl-CoA derived products. This approach should benefit a wide range of acetyl-CoA derived biochemical products in E. coli and other microbes. Enhancing substrate affinity of the introduced pathway genes like thiolase towards acetyl-CoA will likely further increase the flux towards 3-HB while reducing pyruvate and acetate accumulation. [ABSTRACT FROM AUTHOR]

Biohydrogen production (BHP) can be achieved by direct or indirect biophotolysis, photo-fermentation and dark fermentation, whereof only the latter does not require the input of light energy. Our motivation to compile this review was to quantify and comprehensively report strains and process performance of dark fermentative BHP. This review summarizes the work done on pure and defined co-culture dark fermentative BHP since the year 1901. Qualitative growth characteristics and quantitative normalized results of H2 production for more than 2000 conditions are presented in a normalized and therefore comparable format to the scientific community. Statistically based evidence shows that thermophilic strains comprise high substrate conversion efficiency, but mesophilic strains achieve high volumetric productivity. Moreover, microbes of Thermoanaerobacterales (Family III) have to be preferred when aiming to achieve high substrate conversion efficiency in comparison to the families Clostridiaceae and Enterobacteriaceae. The limited number of results available on dark fermentative BHP from fed-batch cultivations indicates the yet underestimated potential of this bioprocessing application. A Design of Experiments strategy should be preferred for efficient bioprocess development and optimization of BHP aiming at improving medium, cultivation conditions and revealing inhibitory effects. This will enable comparing and optimizing strains and processes independent of initial conditions and scale. [ABSTRACT FROM AUTHOR]