Clostridium acetobutylicum is well known for its extraordinary ability to produce acetone, butanol and ethanol. The solvent producing Clostridia can use a wide variety of renewable biomass, and agricultural waste materials, to produce biobutanol, which makes the process environmental friendly. To explore the possibility of co-production of an additional high value product during biobutanol production, the C. acetobutylicum riboflavin operon ribGBAH was cloned under a strong clostridium promoter in an E. coli-Clostridium shuttle vector.  Constructs in which the gene start codon was changed to ATG (pJ-GR) or kept as the original clostridium start codon TTG (pJ-GN) were made. In E. coli, both plasmids complemented E. coli riboflavin mutants and secreted riboflavin in the medium during growth of the culture. In Clostridium acetobutylicum 824, riboflavin production is apparent directly on the transformation plate by the yellow color of the colonies. Furthermore, an extra copy of ribA, a dual function gene in the riboflavin operon, was placed on the above shuttle vectors to yield pJ-GRA and pJ-GNA. Riboflavin and butanol production differences were examined in the various recombinant strains.

Figure1. Picture shows a comparison of transformation plates between C. acetobutylicum 824 (pJ-GN), A, and control 824 (pJIR), B. C. acetobutylicum 824 (pJ-GN) secretes high amount of riboflavin into the agar plate to form a dark yellow color while 824 (pJIR) which express the control vector without a riboflavin expression cassette shows the normal light color.