Development of novel Bacillus subtilis strain for 3-HP overproduction

Bacillus subtilis je Gram-pozitivna bakterija sposobna za rast na glicerolu kao izvoru ugljika i postizanjem velike biomase u industrijskim fermentorima. Nedavno, rekombinantni sojevi B. subtilis su preko ekspresije B12-ovisne glicerol dehidrataze s aldehidom dehidrogenazom konstruirani za dobivanje 3-hidroksipropionske kiseline (3-HP) iz glicerola. No, ti rekombinantni sojevi B. subtilis nisu proizveli 3-HP, dok je laktat bio glavni nusprodukt, vjerojatno kroz metilglioksalni ciklus. Naš cilj je bio konstruirati rekombinantni soj B. subtilis sa kodon-optimiziranim biosintentskim putem koji sadrži glicerol dehidratazu i njegove reaktivatore (DhaB123, GdrAB) zajedno s aldehid dehidrogenazom (PuuC) iz bakterije Klebsiella pneumoniae (soj BSsynK). Ovaj heterologni ciklus uveden u B. subtilis rezultira proizvodnjom laktata nakon 24 sata i tek onda stvaranjem 3-HP s najvišim doprinosom od 200 mg/L. Kako bi se otklonio laktat, na temelju računalne FBA simulacije, deletiran je gen za glicerol kinazi (glpK) u BSsynK soju, što rezultira sojem ΔglpKBSsynK, koji više ne može rast na glicerolu, pa je uzgajan na glukozi, a glicerol je dodan kasnije nakon dosegnute određene biomase. Ova strategija uspješno je preusmjerila metabolički tok ugljika prema produkciji 3-HP umjesto prema glikolizi, te je laktat u potpunosti uklonjen te 3-HP doprinos je dosegnuo 1 g/L nakon nakon 60 sati uzgoja bakterijske kulture.Bacillus subtilis, a Gram-positive model bacterium can grow on glycerol as the carbon source and reaching high biomass in fermenters. Recently, recombinant strains of B. subtilis overexpressing few coenzyme B12-dependent glycerol dehydratases with an aldehyde dehydrogenase were constructed to produce 3-hydroxypropionic acid from glycerol. These recombinant B. subtilis strains could not produce 3-HP while forming lactate as the main by-product, possibly through the methylglyoxal pathway. We aimed to show how the recombinant B. subtilis strains with the codon-optimized biosynthetic pathway with the glycerol dehydratase and its reactivators (DhaB123, gdrAB) and the aldehyde dehydrogenase (PuuC) from Klebsiella pneumoniae (strain BSsynK). This heterologous pathway when introduced into in B. subtilis resulted in lactate production up to 24 hours and 3-HP was formed with the highest titer of 200 mg/L. To eliminate lactate, based on in silico predictions, the glycerol kinase (glpK) was knocked out in the BSsynK strain which resulted in ΔglpKBSsynK strain which was able to grow on glycerol anymore, therefore it was cultivated on glucose, and glycerol was added after a suitable biomass. This strategy successfully directed the carbon flux towards 3-HP instead of glycolysis, lactate was removed and the 3-HP titer was improved up to 1 g/L after 60 hours