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

Theodore S. Groth,Eleftherios T. Papoutsakis,Keerthi P. Venkataramanan,Nicholas R. Sandoval

doi:10.1186/s13068-015-0408-7

Theodore S. Groth, Eleftherios T. Papoutsakis + Show 2 more

Open Access

https://doi.org/10.1186/s13068-015-0408-7

Copy DOI

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.

Highlights

Biodiesel production results in crude glycerol waste from the transesterification of fatty acids (10 % w/w)
Selection, and isolation of a mutant strain tolerant to crude glycerol Industrial butanol production from crude glycerol would require a strain that can readily grow on crude glycerol with a high growth rate and short lag phase and that can produce high butanol titers with good selectivity
The negative effect of crude glycerol on the growth of the wild type (WT) strain is apparent at 80 g/L (Fig. 1a), while no growth was observed at 150 g/L or higher

Summary

Introduction

Biodiesel 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. As glycerol is a more reduced carbon source than glucose, the theoretical yield for butanol production from glycerol is 17 % higher than from glucose on a carbon-mole basis. This reducing power is released in the initial step of glycerol degradation in the form of NADH. In a more recent study, a mutant Cpa strain was shown to produce up to 17.8 g/L butanol from pure glycerol at very high rates from high-density cultures, with minimal byproduct formation [9]

Methods

Results

Conclusion