Overexpression of a non-native deoxyxylulose-dependent vitamin B6 pathway in Bacillus subtilis for the production of pyridoxine

Engineering Bacillus subtilis for the conversion of the antimetabolite 4-hydroxy-l-threonine to pyridoxine

“…However, growth of the vitamin B6 auxotrophic bacteria was only observed in the presence of an amino acid cocktail of Asp, Lys, Thr, Met and Ile (G1 AA). There is indeed experimental evidence that 4-HO-Thr inhibits biosynthesis of Asp-derived amino acids (see below; Commichau et al, 2014). Therefore, a B. subtilis mutant adapted to 4-HO-Thr could be advantageous for the production of PN from 4-HO-Thr.…”

“…In B. subtilis, amylase assays and growth experiments using the Synergy MX II multi-well plate reader (BioTek, USA) were performed as previously described . For the growth and PN production experiments, the media were supplemented with group 1 amino acids (G1 AA) containing Asp, Lys, Thr, Met and Ile, each at 0.5 g/l as described previously (Ludwig et al, 2002;Commichau et al, 2014).…”

“…1; Commichau et al, 2014). However, growth of the vitamin B6 auxotrophic bacteria was only observed in the presence of an amino acid cocktail of Asp, Lys, Thr, Met and Ile (G1 AA).…”

“…Possible drains for 4-hydroxy-threonine and 4-hydroxythreonine-phosphate 4-HO-Thr severely inhibits growth of E. coli and B. subtilis (Westley et al, 1971;Drewke et al, 1993;Commichau et al, 2014). Although the precise mechanism of 4-HO-Thr toxicity remains to be elucidated, it has been shown that 4-HO-Thr-P, the phosphorylated form of 4-HOThr (Fig.…”

High molecular weight DNA assembly in vivo for synthetic biology applications

“…[20,[51][52][53] B. subtilis is naturally competent and readily transformable with extracellular DNA. Extracellular DNA can be imported into the cytoplasm of B. subtilis cells in a single stranded form via B. subtilis transformation machinery.…”