title: A Study on Enhanced Expression of 3-Hydroxypropionic Acid Pathway Genes and Impact on Its Production inLactobacillus reuteri.
abstract: 3-Hydroxypropionic acid (3-HP) is a novel antimicrobial agent against foodborne pathogens likeSalmonellaandStaphylococcusspecies.Lactobacillus reutericonverts glycerol into 3-HP using a coenzyme A-dependent pathway, which is encoded by propanediol utilization operon (pdu) subjected to catabolite repression. In a catabolite-repression-deregulatedL. reuteriRPRB3007, quantitative PCR revealed a 2.5-fold increase in the transcripts of the genespduP,pduWandpduLduring the mid-log phase of growth. The production of 3-HP was tested in resting cells in phosphate buffer and growing batch cultures in MRS broth of various glucose/glycerol ratios. Due to the upregulation of pathway genes, specific formation rate of 3-HP in the mutant strain was found to be enhanced from 0.167 to 0.257 g per g of cell dry mass per h. Furthermore, formation of 3-HP in resting cells was limited due to the substrate inhibition by reuterin at a concentration of (30±5) mM. In batch cultures, the formation of 3-HP was not observed during the logarithmic and stationary phases of growth of wild-type and mutant strains, which was confirmed by NMR spectroscopy. However, the cells collected in these phases were found to produce 3-HP after washing and converting them to resting cells. Lactate and acetate, the primary end products of glucose catabolism, might be the inhibiting elements for 3-HP formation in batch cultures. This was confirmed when lactate (25±5 mM) or acetate (20±5 mM) were added to biotransformation medium, which prevented the 3-HP formation. Moreover, the removal of sodium acetate and glucose (carbon source for lactic acid production) was found to restore 3-HP formation in the MRS broth in a similar manner to that of the phosphate buffer. Even though the genetic repression was circumvented by the up-regulation of pathway genes using a mutant strain, 3-HP formation was further limited by the substrate and catabolite inhibition.
authors.affiliation: Centre for Biotechnology, Anna University, Chennai 600 025, Tamil Nadu, India.
authors.forename: Gopal Ramakrishnan
authors.initials: GR
authors.lastname: Gopi
authors.affiliation: Centre for Biotechnology, Anna University, Chennai 600 025, Tamil Nadu, India.
authors.forename: Nehru
authors.initials: N
authors.lastname: Ganesh
authors.affiliation: Centre for Biotechnology, Anna University, Chennai 600 025, Tamil Nadu, India.
authors.forename: Suppuram
authors.initials: S
authors.lastname: Pandiaraj
authors.affiliation: Centre for Biotechnology, Anna University, Chennai 600 025, Tamil Nadu, India.
authors.forename: Balasubramaniyam
authors.initials: B
authors.lastname: Sowmiya
authors.affiliation: Centre for Biotechnology, Anna University, Chennai 600 025, Tamil Nadu, India.
authors.forename: Raman Gulab
authors.initials: RG
authors.lastname: Brajesh
authors.affiliation: Centre for Biotechnology, Anna University, Chennai 600 025, Tamil Nadu, India.
authors.forename: Subramanian
authors.initials: S
authors.lastname: Ramalingam
keywords: 3-hydroxypropionic acid; Lactobacillus reuteri; biotransformation; catabolite inhibition; catabolite repression