<i>Escherichia coli aceE</i> variants coding pyruvate dehydrogenase improve the generation of pyruvate‐derived acetoin

Moxley, W. Chris; Brown, R. E.; Eiteman, Mark A.

doi:10.1002/elsc.202200054

Cited by 4 publications

(2 citation statements)

References 61 publications

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“…The goal of this research was to examine pyruvate production from sucrose using E. coli W. Seven strains with differences in the aceE allele were first screened in shake flask studies: a wild-type aceE, a ΔaceE strain, and five containing mutations which have previously been shown to support pyruvate accumulation from glucose [25,27]. These mutated strains contain amino acid substitutions, which likely impact pyruvate dehydrogenase activity, although their effect on protein structure and activity has not been determined.…”

Section: Discussionmentioning

confidence: 99%

Pyruvic Acid Production from Sucrose by Escherichia coli Pyruvate Dehydrogenase Variants

Moxley

Eiteman

2023

Fermentation

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Sucrose is an abundant, cheap, and renewable carbohydrate which makes it an attractive feedstock for the biotechnological production of chemicals. Escherichia coli W, one of the few safe E. coli strains able to metabolize sucrose, was examined for the production of pyruvate. The repressor for the csc regulon was deleted in E. coli W strains expressing a variant E1 component of the pyruvate dehydrogenase complex, and these strains were screened in a shake flask culture for pyruvate formation from sucrose. The pyruvate accumulated at yields of 0.23–0.57 g pyruvate/g sucrose, and the conversion also was accompanied by the accumulation of some fructose and/or glucose. Selected strains were examined in 1.25 L controlled batch processes with 40 g/L sucrose to obtain time–course formation of pyruvate and monosaccharides. Pyruvate re-assimilation was observed in several strains, which demonstrates a difference in the metabolic capabilities of glucose- and sucrose-grown E. coli cultures. An engineered strain expressing AceE[H106M;E401A] generated 50.6 g/L pyruvate at an overall volumetric productivity of 1.6 g pyruvate/L·h and yield of 0.68 g pyruvate/g sucrose. The results demonstrate that pyruvate production from sucrose is feasible with comparable volumetric productivity and yield to glucose-based processes.

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Section: Discussionmentioning

confidence: 99%

Pyruvic Acid Production from Sucrose by Escherichia coli Pyruvate Dehydrogenase Variants

Moxley

Eiteman

2023

Fermentation

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“…knockdown or knockout of PDC reduces the flux toward the acetyl-CoA 56,57 . However, in one previous study, it was revealed that the knockout of the lpdA gene encoding another component of PDC could shift the carbon flux of glycolysis from the Embden-Meyerhof-Parnas (EMP) pathway to the Entner-Doudoroff (ED) pathway or Pentose Phosphate (PP) pathway, thereby increasing the intracellular level of NADPH 58 .…”

Section: Applying Tl-crispri To Optimize the Metabolic Fluxmentioning

confidence: 99%

Tunable translation-level CRISPR interference by dCas13 and engineered gRNA in bacteria

Kim,

Kim

et al. 2024

Nat Commun

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Although CRISPR-dCas13, the RNA-guided RNA-binding protein, was recently exploited as a translation-level gene expression modulator, it has still been difficult to precisely control the level due to the lack of detailed characterization. Here, we develop a synthetic tunable translation-level CRISPR interference (Tl-CRISPRi) system based on the engineered guide RNAs that enable precise and predictable down-regulation of mRNA translation. First, we optimize the Tl-CRISPRi system for specific and multiplexed repression of genes at the translation level. We also show that the Tl-CRISPRi system is more suitable for independently regulating each gene in a polycistronic operon than the transcription-level CRISPRi (Tx-CRISPRi) system. We further engineer the handle structure of guide RNA for tunable and predictable repression of various genes in Escherichia coli and Vibrio natriegens. This tunable Tl-CRISPRi system is applied to increase the production of 3-hydroxypropionic acid (3-HP) by 14.2-fold via redirecting the metabolic flux, indicating the usefulness of this system for the flux optimization in the microbial cell factories based on the RNA-targeting machinery.

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Citrate synthase variants improve yield of acetyl-CoA derived 3-hydroxybutyrate in Escherichia coli

Rajpurohit,

Eiteman

2024

Microb Cell Fact

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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.

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Escherichia coli aceE variants coding pyruvate dehydrogenase improve the generation of pyruvate‐derived acetoin

Cited by 4 publications

References 61 publications

Pyruvic Acid Production from Sucrose by Escherichia coli Pyruvate Dehydrogenase Variants

Pyruvic Acid Production from Sucrose by Escherichia coli Pyruvate Dehydrogenase Variants

Tunable translation-level CRISPR interference by dCas13 and engineered gRNA in bacteria

Citrate synthase variants improve yield of acetyl-CoA derived 3-hydroxybutyrate in Escherichia coli

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