(670d) Metabolic Engineering of Thermobifida Fusca for One-Step Producing Fine Chemicals from Biomass

Our computational analysis and experimental results demonstrate that a cellulolytic actinobacterium Thermobifida fusca produces propionic acid. Secretion of propionic acid was observed experimentally using HPLC and mRNA transcription of key genes (Tfu_2971 and Tfu_1546) were measured using real-time PCR as an indicator of in vivo transcription activity. The conversion of propionate to propanoyl-CoA can be theoretically achieved by an acetyl-CoA synthetase (gene Tfu_1546) that is natively found in T. fusca. The remaining two reactions to convert propanoyl-CoA to propionaldehyde then to 1-propanol can be achieved using a single bifunctional butyraldehyde/alcohol dehydrogenase such as the adhE2 gene found in C. acetobutylicum. Based on the genetic modification platform, adhE2 gene was knock into the genome DNA of T. fusca to produce 1-propanol. The highest 1-propanol titer (0.48 g/L) was achieved for growth on switchgrass. These results represent the first demonstration of direct conversion of untreated lignocellulosic biomass to 1-propanol in an aerobic organism and illustrate the potential utility of T. fusca as an aerobic, cellulolytic bioprocess organism. Moreover, Thermobifida fusca mutant strain muS excretes butyric acid. In order to achieve a high yield of butyric acid, the effect of different carbon sources: mannose, xylose, lactose, cellobiose, glucose, sucrose and acetates, on butyric acid production was studied. The highest yield of butyric acid was 0.67 g/g-C (g-butyric acid/g-carbon input) on cellobiose. The best stir speed and aeration rate for butyric acid production were found to be 400 rpm and 2 vvm in a 5-L Fermentor. The maximum titer of 2.1 g/L butyric acid was achieved on 9.66 g/L cellulose. In order to test the production of butyric acid on lignocellulosic biomass, corn stover was used as the substrate, on which there was 2.37 g/L butyric acid produced under the optimized conditions. In addition, butyric acid synthesis pathway was identified involving five genes that catalyzed reactions from acetyl-CoA to butanoyl-CoA in T. fusca.