Nutritional Requirements of Acetogen Clostridium sp. AWRP and Engineering for Vitamin B6 Prototrophy

Abstract

Acetogenic bacteria have been attracting attention because they can utilize carbon dioxide, the major cause of the greenhouse effect, as a carbon source. Clostridium sp. AWRP is a novel acetogen that exhibits high gas consumption and ethanol production. One of the unresolved issues with this bacterium was its nutritional requirements, which are indispensable for formulating a chemically defined growth medium. In this study, we determined that the cryptic auxotrophic behavior of Clostridium sp. AWRP is due to its lack of ability to utilize pyridoxine routinely supplemented to the culture medium. Replacement of pyridoxine with other forms of vitamin B6, pyridoxamine and pyridoxal-5-phosphate, supported the growth in a chemically defined medium. Additional essential nutrients were identified as panthothenate and biotin. To address vitamin B6 auxotrophy, we attempted to express the pdxST gene cluster either by introducing an overexpression plasmid or by integrating it into the genome. Engineered strains could be successfully grown without vitamin B6 under both heterotrophic and autotrophic conditions. The chemically defined minimal medium developed here will be useful for physiological and metabolic studies of Clostridium sp. AWRP.