CHAIN Biotechnology Ltd.

Microbial formation of acetone, isopropanol, and butanol is largely restricted to bacteria belonging to the genus Clostridium. This ability has been industrially exploited over the last 100 years. The solvents are important feedstocks for the chemical and biofuel industry. However, biological synthesis suffers from high substrate costs and competition from chemical synthesis supported by the low price of crude oil. To render the biotechnological production economically viable again, improvements in microbial and fermentation performance are necessary. However, no comprehensive comparisons of respective species and strains used and their specific abilities exist today.

The genomes of a total 30 saccharolytic Clostridium strains, representative of the species Clostridium acetobutylicum, C. aurantibutyricum, C. beijerinckii, C. diolis, C. felsineum, C. pasteurianum, C. puniceum, C. roseum, C. saccharobutylicum, and C. saccharoperbutylacetonicum, have been determined; 10 of them completely, and compared to 14 published genomes of other solvent-forming clostridia. Two major groups could be differentiated and several misclassified species were detected.

Our findings represent a comprehensive study of phylogeny and taxonomy of clostridial solvent producers that highlights differences in energy conservation mechanisms and substrate utilization between strains, and allow for the first time a direct comparison of sequentially selected industrial strains at the genetic level. Detailed data mining is now possible, supporting the identification of new engineering targets for improved solvent production.

A review.The acetone-butanol-ethanol (ABE) fermentation was one of the largest biotechnol. operations ever performed, being beaten in size only by the ethanolic fermentationABE had its peak time between 1915 and 1950, but several plants were even operating until 2004.Meanwhile, the process might be revived, especially in China.Workhorses of the fermentation are solventogenic clostridia, namely,Clostridium acetobutylicum ,C. beijerinckii ,C. saccharobutylicum , andC. saccharoperbutylacetonicum .Their features in product formation, enzymes, regulation, genes and genomes, threats by phage infection, and newly developed sophisticated genetic tools for targeted strain improvement are summarized in this chapter.

Chirally pure (R)-1,3-butanediol ((R)-1,3-BDO) is a valuable intermediate for the production of fragrances, pheromones, insecticides and antibiotics. Biotechnological production results in superior enantiomeric excess over chemical production and is therefore the preferred production route. In this study (R)-1,3-BDO was produced in the industrially important whole cell biocatalyst Clostridium saccharoperbutylacetonicum through expression of the enantio-specific phaB gene from Cupriavidus necator. The heterologous pathway was optimised in three ways: at the transcriptional level choosing strongly expressed promoters and comparing plasmid borne with chromosomal gene expression, at the translational level by optimising the codon usage of the gene to fit the inherent codon adaptation index of C. saccharoperbutylacetonicum, and at the enzyme level by introducing point mutations which led to increased enzymatic activity. The resulting whole cell catalyst produced up to 20 mM (1.8 g/l) (R)-1,3-BDO in non-optimised batch fermentation which is a promising starting position for economical production of this chiral chemical.