Reduction of Feedback Inhibition in Homoserine Kinase (ThrB) ofCorynebacterium glutamicumEnhancesl-Threonine Biosynthesis

ABSTRACTThe industrial production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been hindered by high cost and a complex control strategy caused by the addition of propionate. In this study, based on analysis of the PHBV biosynthesis process, we developed a PHBV biosynthetic pathway from a single unrelated carbon source via threonine biosynthesis inEscherichia coli. To accomplish this, we (i) overexpressed threonine deaminase, which is the key factor for providing propionyl-coenzyme A (propionyl-CoA), from different host bacteria, (ii) removed the feedback inhibition of threonine by mutating and overexpressing thethrABCoperon inE. coli, and (iii) knocked out the competitive pathways of catalytic conversion of propionyl-CoA to 3-hydroxyvaleryl-CoA. Finally, we constructed a series of strains and mutants which were able to produce the PHBV copolymer with differing monomer compositions in a modified M9 medium supplemented with 20 g/liter xylose. The largest 3-hydroxyvalerate fraction obtained in the copolymer was 17.5 mol%.

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Homoserine kinase from the phototrophic bacteriumRhodospirillum rubrumis not sensitive to feedback inhibition by l-threonine

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.1987.tb02522.x ◽

1987 ◽

Vol 48 (1-2) ◽

pp. 93-96

Author(s):

Britta Finkelnburg ◽

Jobst-Heinrich Klemme

Keyword(s):

Feedback Inhibition ◽

Homoserine Kinase

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Homoserine kinase from the phototrophic bacterium Rhodospirillum rubrum is not sensitive to feedback inhibition by ?-threonine

FEMS Microbiology Letters ◽

10.1016/0378-1097(87)90142-x ◽

1987 ◽

Vol 48 (1-2) ◽

pp. 93-96

Author(s):

B Finkelnburg

Keyword(s):

Rhodospirillum Rubrum ◽

Feedback Inhibition ◽

Phototrophic Bacterium ◽

Homoserine Kinase

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Directed Evolution of Methanococcus jannaschii Citramalate Synthase for Biosynthesis of 1-Propanol and 1-Butanol by Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.02046-08 ◽

2008 ◽

Vol 74 (24) ◽

pp. 7802-7808 ◽

Cited By ~ 178

Author(s):

Shota Atsumi ◽

James C. Liao

Keyword(s):

Escherichia Coli ◽

Directed Evolution ◽

Specific Activity ◽

Feedback Inhibition ◽

Evolutionary Strategy ◽

Keto Acid ◽

Methanococcus Jannaschii ◽

Acid Deficiency ◽

Acid Pathway ◽

Threonine Biosynthesis

ABSTRACT Biofuels synthesized from renewable resources are of increasing interest because of global energy and environmental problems. We have previously demonstrated production of higher alcohols from Escherichia coli using a 2-keto acid-based pathway. Here, we took advantage of the growth phenotype associated with 2-keto acid deficiency to construct a hyperproducer of 1-propanol and 1-butanol by evolving citramalate synthase (CimA) from Methanococcus jannaschii. This new pathway, which directly converts pyruvate to 2-ketobutyrate, bypasses threonine biosynthesis and represents the shortest keto acid-mediated pathway for producing 1-propanol and 1-butanol from glucose. Directed evolution of CimA enhanced the specific activity over a wide temperature range (30 to 70°C). The best CimA variant was found to be insensitive to feedback inhibition by isoleucine in addition to the improved activity. This CimA variant enabled 9- and 22-fold higher production levels of 1-propanol and 1-butanol, respectively, compared to the strain expressing the wild-type CimA. This work demonstrates (i) the first production of 1-propanol and 1-butanol using the citramalate pathway and (ii) the benefit of the 2-keto acid pathway that enables a growth-based evolutionary strategy to improve the production of non-growth-related products.

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Role of homoserine and threonine pathway intermediates as precursors for the biosynthesis of aminoethoxyvinylglycine in Streptomyces sp. NRRL 5331

Microbiology ◽

10.1099/mic.0.26851-0 ◽

2004 ◽

Vol 150 (5) ◽

pp. 1467-1474 ◽

Cited By ~ 6

Author(s):

Mónica Fernández ◽

Yolanda Cuadrado ◽

Jesús F. Aparicio ◽

Juan F. Martín

Keyword(s):

Gene Disruption ◽

Homoserine Dehydrogenase ◽

Streptomyces Sp ◽

Threonine Synthase ◽

Homoserine Kinase ◽

Negative Effect ◽

Branching Point ◽

Threonine Biosynthesis ◽

Null Mutants

The genes hom, thrB and thrC, encoding homoserine dehydrogenase, homoserine kinase (HK) and threonine synthase, respectively, involved in the last steps of threonine biosynthesis, have been studied in Streptomyces sp. NRRL 5331, the producer of the ethylene synthetase inhibitor aminoethoxyvinylglycine (AVG), in order to determine their role in the biosynthesis of AVG. Different null mutants were obtained by plasmid-mediated disruption of each of the three genes. thrC gene disruption had no effect on AVG production, while the disruption of thrB blocked HK activity and substantially reduced the yield of this metabolite, probably due to the accumulation of homoserine and/or methionine which have a negative effect on AVG biosynthesis. Disruption of hom (thrA) completely blocked AVG biosynthesis, indicating that homoserine lies at the branching point of the aspartic-acid-derived biosynthetic route that leads to AVG. The four carbon atoms of the vinylglycine moiety of AVG derive, therefore, from homoserine.

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