Whole-genome sequence of an evolved Clostridium pasteurianum strain reveals Spo0A deficiency responsible for increased butanol production and superior growth

Abstract

BackgroundBiodiesel production results in crude glycerol waste from the transesterification of fatty acids (10 % w/w). The solventogenic Clostridium pasteurianum, an anaerobic Firmicute, can produce butanol from glycerol as the sole carbon source. Coupling butanol fermentation with biodiesel production can improve the overall economic viability of biofuels. However, crude glycerol contains growth-inhibiting byproducts which reduce feedstock consumption and solvent production.ResultsTo obtain a strain with improved characteristics, a random mutagenesis and directed evolution selection technique was used. A wild-type C. pasteurianum (ATCC 6013) culture was chemically mutagenized, and the resulting population underwent 10 days of selection in increasing concentrations of crude glycerol (80–150 g/L). The best-performing mutant (M150B) showed a 91 % increase in butanol production in 100 g/L crude glycerol compared to the wild-type strain, as well as increased growth rate, a higher final optical density, and less production of the side product PDO (1,3-propanediol). Wild-type and M150B strains were sequenced via Single Molecule Real-Time (SMRT) sequencing. Mutations introduced to the M150B genome were identified by sequence comparison to the wild-type and published closed sequences. A major mutation (a deletion) in the gene of the master transcriptional regulator of sporulation, Spo0A, was identified. A spo0A single gene knockout strain was constructed using a double--crossover genome-editing method. The Spo0A-deficient strain showed similar tolerance to crude glycerol as the evolved mutant strain M150B. Methylation patterns on genomic DNA identified by SMRT sequencing were used to transform plasmid DNA to overcome the native C. pasteurianum restriction endonuclease.ConclusionsSolvent production in the absence of Spo0A shows C. pasteurianum differs in solvent-production regulation compared to other solventogenic Clostridium. Growth-associated butanol production shows C. pasteurianum to be an attractive option for further engineering as it may prove a better candidate for butanol production through continuous fermentation.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0408-7) contains supplementary material, which is available to authorized users.

Disruption of the Reductive 1,3-Propanediol Pathway Triggers Production of 1,2-Propanediol for Sustained Glycerol Fermentation by Clostridium pasteurianum

“…Hence, inactivation of the upstream gene encoding glycerol dehydratase (dhaBCE) and deletion of the entire 1,3-PDO regulon represent superior strategies for developing a completely 1,3-PDO-deficient mutant of C. pasteurianum. 1,3-PDO titers could be further decreased through inactivation of the Spo0A transcriptional regulator, as recently demonstrated by Sandoval et al (16), resulting in enhanced glycerol utilization and decreased production of 1,3-PDO (Table 5). A number of promising C. pasteurianum strains have also been generated via random mutagenesis and directed evolution, such as the hyper n-butanol-producing strain described by Malaviya et al (12).…”

“…Given the organism's unique central metabolism, several studies have explored the glycerol-to-butanol fermentation carried out by C. pasteurianum (9)(10)(11). To enable metabolic engineering of C. pasteurianum, methodologies have recently been reported for electrotransformation (14), group-II-intron-mediated gene disruption (15), gene deletion and integration (16), antisense RNA (asRNA) gene knockdown (17), and clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) genome editing (18). We also sequenced the C. pasteurianum genome (19) and provided genomic analysis of the organism's central metabolism (20).…”

“…We also sequenced the C. pasteurianum genome (19) and provided genomic analysis of the organism's central metabolism (20). C. pasteurianum genome sequences have been reported by at least five additional groups, covering the type strain (ATCC 6013/DSM 525) (16,(21)(22)(23) and two nontype strains (BC1 and NRRL B-598) (24). A method for electroporation of a unique acetone-producing aerotolerant strain was also described recently (25), demonstrating widespread interest in C. pasteurianum as a biocatalyst for crude glycerol valorization.…”

“…We previously downregulated the primary C. pasteurianum hydrogenase-encoding gene (hydA) using asRNA and observed superior growth characteristics, as well as enhanced titers of the reduced end products n-butanol and ethanol (17). Concurrently, Sandoval et al (16) employed random mutagenesis and directed evolution to identify the Spo0A master transcriptional regulator as a key target for gene inactivation. Deletion of the corresponding spo0A gene yielded improved glycerol dissimilation and n-butanol yields, as well as decreased levels of 1,3-PDO.…”

Exponential Growth and Solvents-Production of <i>Clostridium acetobutylicum</i> ATCC 824 on TYA Media Containing Sucrose and Glucose as Different Sole Carbon Sources

“…The spores germinate once the environmental conditions become favourable for living and they also get revived if heat shocked (> 80 degree centigrade at 10 minutes) [3]. Sporulation is triggered by low pH, the presence of carbohydrates and at times a quorum sensing mechanism at high population densities [4,5]. The metabolic pathway of the Clostridium acetobutylicum is divided into two distinct phases: acidogenic phase and solventogenic phase [6].…”