Several chromosomally expressed AceE variants were constructed in
- Award ID(s):
- 1413862
- PAR ID:
- 10055679
- Date Published:
- Journal Name:
- Applied and Environmental Microbiology
- Volume:
- 82
- Issue:
- 24
- ISSN:
- 0099-2240
- Page Range / eLocation ID:
- 7176 to 7184
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Escherichia coli ΔldhA ΔpoxB ΔppsA and compared using glucose as the sole carbon source. These variants were examined in shake flask cultures for growth rate, pyruvate accumulation, and acetoin production via heterologous expression of thebudA andbudB genes fromEnterobacter cloacae ssp. dissolvens . The best acetoin‐producing strains were subsequently studied in controlled batch culture at the one‐liter scale. PDH variant strains attained up to four‐fold greater acetoin than the strain expressing the wild‐type PDH. In a repeated batch process, the H106V PDH variant strain attained over 43 g/L of pyruvate‐derived products, acetoin (38.5 g/L) and 2R,3R‐butanediol (5.0 g/L), corresponding to an effective concentration of 59 g/L considering the dilution. The acetoin yield from glucose was 0.29 g/g with a volumetric productivity of 0.9 g/L·h (0.34 g/g and 1.0 g/L·h total products). The results demonstrate a new tool in pathway engineering, the modification of a key metabolic enzyme to improve the formation of a product via a kinetically slow, introduced pathway. Direct modification of the pathway enzyme offers an alternative to promoter engineering in cases where the promoter is involved in a complex regulatory network. -
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ABSTRACT The non‐renewable petrochemical phenol is used as a precursor to produce numerous fine and commodity chemicals, including various pharmaceuticals and phenolic resins. Microbial phenol biosynthesis has previously been established, stemming from endogenous tyrosine via tyrosine phenol lyase (TPL). TPL, however, suffers from feedback inhibition and equilibrium limitations, both of which contribute to reduced flux through the overall pathway. To address these limitations, two novel and non‐natural phenol biosynthesis pathways, both stemming instead from chorismate, were constructed and comparatively evaluated. The first proceeds to phenol in one heterologous step via the intermediate
p ‐hydroxybenzoic acid, while the second involves two heterologous steps and the associated intermediates isochorismate and salicylate. Maximum phenol titers achieved via these two alternative pathways reached as high as 377 ± 14 and 259 ± 31 mg/L in batch shake flask cultures, respectively. In contrast, under analogous conditions, phenol production via the established TPL‐dependent route reached 377 ± 23 mg/L, which approaches the maximum achievable output reported to date under batch conditions. Additional strain development and optimization of relevant culture conditions with respect to each individual pathway is ultimately expected to result in further improved phenol production. Biotechnol. Bioeng. 2016;113: 1745–1754. © 2016 Wiley Periodicals, Inc. -
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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 bygltA ), which directs over 60% of the acetyl-CoA into the tricarboxylic acid cycle. Eliminating citrate synthase activity (deletion ofgltA ) 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 ) fromCupriavidus 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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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/AEM.02178-16
