Metabolic engineering of Thermoanaerobacterium strains AK17 and HG-8 for production of 1,2-propanediol

Abstract

The aim of this study is to genetically engineer the metabolic pathways of two thermophilic bacteria, Thermoanaerobacterium thermosaccharolyticum HG-8 and Thermoanaerobacterium AK17, for 1,2-propanediol production. Not many bacteria produce 1,2-propanediol naturally, but HG-8 is one of the best known natural 1,2-propanediol producer. For HG-8 the idea was to use homologous recombination to knock out genes that contribute to production of fermentation byproducts. Ethanol and lactate are produced in HG-8 in significant amounts. Lactate dehydrogenase catalyzes lactate formation and aldehyde-alcohol dehydrogenase catalyzes ethanol formation. By knocking out the ldh gene that codes for lactate dehydrogenase and the adhE gene that codes for aldehyde-alcohol dehydrogenase it might be possible to increase 1,2-propanediol production in the bacteria. The ldh transformation was successful and lactate production of the engineered strain did decrease. There was, however, no increase in 1,2-propanediol production. The adhE transformation was partly successful as it resulted in single crossover, expressing both the adhE gene but also the antibiotic resistance marker. Promising results were that this engineered strain produced more 1,2-propanediol than the wild type.AK17 cannot produce 1,2-propanediol naturally. The idea was to engineer the strain by cloning from HG-8 the genes that catalyze the missing steps of 1,2-propanediol production. The genes of interest were the genes that encode methylglyoxal synthase (MGS), methylglyoxal reductase (MGR) and glycerol dehydrogenase (GDH). This transformation was unsuccessful. Possibly because the plasmids have mutated or there is something in the transformed genes, or expression of them in AK17, that is having lethal effect on the bacteria.