Improving the Clostridium acetobutylicum butanol fermentation by engineering the strain for co-production of riboflavin

Abstract

Solvent-producing clostridia are well known for their capacity to use a wide variety of renewable biomass and agricultural waste materials for biobutanol production. To investigate the possibility of co-production of a high value chemical during biobutanol production, the Clostridium acetobutylicum riboflavin operon ribGBAH was over-expressed in C. acetobutylicum on Escherichia coli-Clostridium shuttle vector pJIR750. Constructs that either maintained the original C. acetobutylicum translational start codon or modified the start codons of ribG and ribB from TTG to ATG were designed. Riboflavin was successfully produced in both E. coli and C. acetobutylicum using these plasmids, and riboflavin could accumulate up to 27 mg/l in Clostridium culture. Furthermore, the C. acetobutylicum purine pathway was modified by over-expression of the Clostridium purF gene, which encodes the enzyme PRPP amidotransferase. The function of the plasmid pJaF bearing C. acetobutylicum purF was verified by its ability to complement an E. coli purF mutation. However, co-production of riboflavin with biobutanol by use of the purF over-expression plasmid was not improved under the experimental conditions examined. Further rational mutation of the purF gene was conducted by replacement of amino acid codons D302 V and K325Q to make it similar to the feedback-resistant enzymes of other species. However, the co-expression of ribGBAH and purFC in C. acetobutylicum also did not improve riboflavin production. By buffering the culture pH, C. acetobutylicum ATCC 824(pJpGN) could accumulate more than 70 mg/l riboflavin while producing 190 mM butanol in static cultures. Riboflavin production was shown to exert no effect on solvent production at these levels.

Effect of iron limitation and fur gene inactivation on the transcriptional profile of the strict anaerobe Clostridium acetobutylicum

“…After . Moreover, Cai & Bennett (2011) showed that overproduction of RF did not influence the solvent production significantly, which is in agreement with our results.…”

“…3c). It has been reported recently that overexpression of the ribDBAH operon in C. acetobutylicum leads to accumulation of about 70 mg RF l 21 in culture (Cai & Bennett, 2011). Considering the industrial potential of this bacterium, it was tempting to quantify the amounts generated by the fur : : int strain.…”

Simultaneous Fermentation of Glucose and Xylose to Butanol by Clostridium sp. Strain BOH3

“…This corresponds to a yield of 0.25 g/g for butanol and 0.36 g/g for total solvents of acetone, butanol, and ethanol (ABE). Interestingly, riboflavin also appeared during the fermentation process and reached a concentration of 110.5 mg/liter, which is much higher than those found in previous studies using either wild-type (50 mg/liter) or gene-modified Clostridium strains (70 mg/liter) (3,8). In comparison, when glucose (60 g/liter) was used as the sole carbon source, the substrate consumption curves almost over- lapped with that of xylose ( Fig.…”

Analysis of redox responses during TNT transformation by Clostridium acetobutylicum ATCC 824 and mutants exhibiting altered metabolism

“…Escherichia coli cultures were grown aerobically at 37°C in Luria-Bertani medium, C. acetobutylicum was grown anaerobically at 37°C in buffered clostridium growth medium (CGM) in a Forma Scientific anaerobic chamber (Thermo Forma, Marietta, OH) as described previously (Cai and Bennett 2011;Zhao et al 2005). For E. coli recombinant strains, the medium was supplemented with ampicillin (100 μg/mL), chloramphenicol (35 μg/mL), kanamycin (50 μg/mL), or erythromycin (200 μg/mL) as appropriate.…”