Research on bioproduction of chemicals and fuels is of great interest due to the increasing demand for alternative, sustainable production processes. Clostridium pasteurianum represents an appealing microorganism for industrial bioproduction as it combines favourable properties regarding substrate utilization and product spectrum: C. pasteurianum is able to metabolize raw glycerol which accumulates as byproduct in biodiesel production. The main products of glycerol fermentation by C. pasteurianum are 1,3-propanediol and n-butanol, two compounds of high industrial value. The industrial potential of C. pasteurianum could not be fully exploited in the past, as molecular tools for the optimization of the cell´s me... More
Research on bioproduction of chemicals and fuels is of great interest due to the increasing demand for alternative, sustainable production processes. Clostridium pasteurianum represents an appealing microorganism for industrial bioproduction as it combines favourable properties regarding substrate utilization and product spectrum: C. pasteurianum is able to metabolize raw glycerol which accumulates as byproduct in biodiesel production. The main products of glycerol fermentation by C. pasteurianum are 1,3-propanediol and n-butanol, two compounds of high industrial value. The industrial potential of C. pasteurianum could not be fully exploited in the past, as molecular tools for the optimization of the cell´s metabolism were not available. Recently, a protocol was developed which enabled DNA transfer to C. pasteurianum for the first time. To date, methods for metabolic engineering of C. pasteurianum are poorly developed and inefficient. In this study, methods for metabolic engineering of C. pasteurianum were analyzed and optimized. Different DNA transfer methods were tested and compared regarding its applicability for transformation of C. pasteurianum. In contrast to conjugation, electroporation has proven to be more efficient for transformation of C. pasteurianum. A novel C. pasteurianum strain (R525) was isolated which exhibits 50-fold increased transformation efficiency. The genotype as well as the phenotype of the electrocompetent R525 strain were characterized and based on this strain, optimized strains were developed. By overexpression of pyruvate carboxylase, carbon flux of C. pasteurianum was shifted at the pyruvate-acetyl-CoA node, aiming to increase growth and product formation of C. pasteurianum on glycerol/ glucose cosubstrate. The created mutant reveals valuable new insights into the metabolism of C. pasteurianum, especially regarding its citrate cycle which is not fully elucidated so far. Several gene deletion methods were analyzed regarding their utility in C. pasteurianum. The CRISPR-Cas9 nickase system was successfully established for gene deletion in C. pasteurianum for the first time. By deletion of the glycerol dehydratase dhaB subunit, 1,3-propanediol production was shut down and butanol production was selectively enhanced. In addition, formation of the byproduct butyrate was eliminated in this strain. After optimization of fermentation conditions, butanol yields of 0.371 - 0.385 mol/mol substrate were reached with this strain, which are among the highest yields reported for C. pasteurianum to date. Glycerol dehydratase deleVI tion demonstrated for the first time that anaerobic cultivation of C. pasteurianum on glycerol without 1,3-propanediol formation is possible, revealing a new mechanism of redox regulation in C. pasteurianum. The results of this study contribute to the utilization of C. pasteurianum for prospective industrial biobutanol production in order to oppose conventional petrochemical processes a competitive sustainable alternative.
