Comparative Genomic Analysis of a High n-Butanol Tolerant and Producing Mutant Strain of Clostridium

n-Butanol is an important industrial chemical and also has been proposed as an alternative biofuel superior to ethanol. Biological production of n-butanol is promising because it can use renewable biomass as feedstock and is environmentally friendly. Unfortunately, n-butanol is more toxic than ethanol, which leads to the low final n-butanol titer and consequently high recovery cost. To improve n-butanol tolerance and final titer, a parent Clostridium strain commonly used in acetone-butanol-ethanol (ABE) fermentation was evolutionarily engineered to finally derive a mutant strain that produced 21 g/L n-butanol in free cell fermentation and 28 g/L in immobilized cell fermentation. Besides dramatically improved n-butanol tolerance and production, compared with the parental strain, the mutant also showed improved autolysis resistance, robust adjustment of membrane fatty acid composition, more complete substrate utilization, and remarkable proteomic change. To further investigate the molecular basis for the acquired traits of the mutant, the complete genomes of the parent and mutant strains were sequenced via the Illumina Hiseq2000 and comparative genomic analysis was conducted. SNP and short indel analyses revealed many variations which might have contributed to the characteristics of the hyper-n-butanol tolerant and producing mutant strain. The results can be used in further strain improvement via inverse metabolic engineering and evolutionary engineering.