Metabolic Engineering of Bacillus methanolicus MGA3 for Production of Acetoin
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Being a pure, inexpensive, non-food chemical that can be sustainably produced, methanol has attracted great interest as a carbon feedstock for microbial production of commodity/specialty chemicals. In this thesis, methanol-based production of acetoin was achieved by recombinant Bacillus methanolicus MGA3 strains. The alsSD operons from Bacillus licheniformis DSM13 and Bacillus subtilis 168 were cloned into expression vector pTH1mp and heterologously expressed in B. methanolicus MGA3, creating two acetoin-producing strains with different growth characteristics, MGA3 (pTH1mpLacO-alsSD_Bl(GTG)) and MGA3 (pTH1mpLacO-alsSD_Bs(GTG)), respectively. The potential of B. methanolicus MGA3 as an acetoin producer was evaluated by assessing acetoin tolerance and by functionally assaying the activity of recombinant enzymes produced. MGA3 (pTH1mpLacO-alsSD_Bl(GTG)) achieved the highest acetoin titer of 1.48 ± 0.24 g/L with yield of 0.23 ± 0.04g/L gacetoin/gmethanol, growing on methanol as sole carbon source. The relatively narrow engineering effort performed in this thesis highlights the potential of B. methanolicus MGA3 as a candidate for industrial production of acetoin. Parameters such as productivity, rate of methanol consumption, etc. are yet to be determined, which leaves room for future engineering strategies to further optimize yield. In addition, potential production of 2,3-butanediol, of which acetoin is the direct precursor, in B. methanolicus MGA3 was investigated.