Development of novel Bacillus subtilis strain for 3-hp overproduction

Stančik, Ivan Andreas (2016) Development of novel Bacillus subtilis strain for 3-hp overproduction. Diploma thesis, Faculty of Science > Department of Biology.

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Abstract

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.

Item Type: Thesis (Diploma thesis)
Keywords: Bacillus subtilis, metabolic engineering, lactate, glycerol, ldh knock-out
Supervisor: Mijakovic, Ivan
Co-supervisor: Franjević, Damjan
Date: 2016
Number of Pages: 35
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Grozdana Sirotic
Date Deposited: 21 Nov 2016 11:22
Last Modified: 21 Nov 2016 11:22
URI: http://digre.pmf.unizg.hr/id/eprint/5316

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