Characterization of Growth in Escherichia coli Expressing Methylotrophic Genes

A pH-controlled bioreactor was used to determine batch system growth kinetics and rate of metabolite production in Escherichia coli expressing methylotrophic genes. Plasmids containing the genes for the methanol dehydrogenase (MDH) enzyme, for methanol oxidation, and for the ribulose monophosphate (RuMP) pathway, which consists of the 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) enzymes, for subsequent formaldehyde assimilation, were transformed into E. coli and the ability of these new strains to grow on methanol as a co-substrate with either glucose, xylose or yeast extract was assessed. The MDH gene was extracted from Geobacillus stearothermophilus while the RuMP genes originated from Bacillus methanolicus. An experiment utilizing this strain, inoculated into minimal media consisting of 1 g/L of yeast extract and 60 mM methanol, resulted in a significant methanol loss compared with evaporation rate. Repetitions of this experiment show a statistically significant improvement in biomass yield in the presence of methanol, with an average biomass yield of 0.28 g biomass per gram of yeast extract for the cultures containing both methanol and yeast extract, compared with an average yield of 0.22 grams biomass per gram of yeast extract for the cultures containing only yeast extract.  This result indicates incorporation of methanol into biomass and is evidence that the recombinant strain of E. coli is capable, with further recombination, of converting methanol into useful products.