RRC ID 35528
Author Ashok S, Mohan Raj S, Ko Y, Sankaranarayanan M, Zhou S, Kumar V, Park S.
Title Effect of puuC overexpression and nitrate addition on glycerol metabolism and anaerobic 3-hydroxypropionic acid production in recombinant Klebsiella pneumoniae ΔglpKΔdhaT.
Journal Metab Eng
Abstract 3-Hydroxypropionic acid (3-HP), an industrially important platform chemical, is used as a precursor during the production of many commercially important chemicals. Recently, recombinant strains of K. pneumoniae overexpressing an NAD(+)-dependent γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (PuuC) enzyme of K. pneumoniae DSM 2026 were shown to produce 3-HP from glycerol without the addition coenzyme B(12), which is expensive. However, 3-HP production in K. pneumoniae is accompanied with NADH generation, and this always results in large accumulation of 1,3-propanediol (1,3-PDO) and lactic acid. In this study, we investigated the potential use of nitrate as an electron acceptor both to regenerate NAD(+) and to prevent the formation of byproducts during anaerobic production of 3-HP from glycerol. Nitrate addition could improve NAD(+) regeneration, but decreased glycerol flux towards 3-HP production. To divert more glycerol towards 3-HP, a novel recombinant strain K. pneumoniae ΔglpKΔdhaT (puuC) was developed by disrupting the glpK gene, which encodes glycerol kinase, and the dhaT gene, which encodes 1,3-propanediol oxidoreductase. This strain showed improved cellular NAD(+) concentrations and a high carbon flux towards 3-HP production. Through anaerobic cultivation in the presence of nitrate, this recombinant strain produced more than 40±3mM 3-HP with more than 50% yield on glycerol in shake flasks and 250±10mM 3-HP with approximately 30% yield on glycerol in a fed-batch bioreactor.
Volume 15
Pages 10-24
Published 2013-1-1
DOI 10.1016/j.ymben.2012.09.004
PII S1096-7176(12)00099-7
PMID 23022570
MeSH Aldehyde Oxidoreductases / genetics Aldehyde Oxidoreductases / metabolism* Anaerobiosis / physiology Genetic Enhancement / methods* Glycerol / metabolism* Klebsiella pneumoniae / classification Klebsiella pneumoniae / physiology* Lactic Acid / analogs & derivatives* Lactic Acid / biosynthesis Nitrates / metabolism* Recombination, Genetic / genetics* Species Specificity Up-Regulation
IF 7.263
Times Cited 58
Prokaryotes E. coli NA