Development of butanol-tolerant Bacillus subtilis strain GRSW2-B1 as a potential bioproduction host

Naoya Kataoka,Takahisa Tajima,Wanitcha Rachadech,Alisa S Vangnai,Junichi Kato

doi:10.1186/2191-0855-1-10

Abstract

As alternative microbial hosts for butanol production with organic-solvent tolerant trait are in high demands, a butanol-tolerant bacterium, Bacillus subtilis GRSW2-B1, was thus isolated. Its tolerance covered a range of organic solvents at high concentration (5%v/v), with remarkable tolerance in particular to butanol and alcohol groups. It was susceptible for butanol acclimatization, which resulted in significant tolerance improvement. It has versatility for application in a variety of fermentation process because it has superior tolerance when cells were exposed to butanol either as high-density, late-exponential grown cells (up to 5%v/v) or under growing conditions (up to 2.25%v/v). Genetic transformation procedure was optimized, yielding the highest efficiency at 5.17 × 103 colony forming unit (μg DNA)-1. Gene expression could be effectively driven by several promoters with different levels, where as the highest expression was observed with a xylose promoter. The constructed vector was stably maintained in the transformants, in the presence or absence of butanol stress. Adverse effect of efflux-mediated tetracycline resistance determinant (TetL) to bacterial organic-solvent tolerance property was unexpectedly observed and thus discussed. Overall results indicate that B. subtilis GRSW2-B1 has potential to be engineered and further established as a genetic host for bioproduction of butanol.

Highlights

Introduction nButanol is an important industrial chemical, widely used as a solvent, a stabilizer and feedstock for the production of polymers and plastics
Alternative hosts being engineered for butanol production are well-characterized, genetically-amenable microorganisms, such as Escherichia coli (Atsumi et al 2008,Inui et al 2008,; Nielsen et al 2009), Saccharomyces cerevisiae (Steen et al 2008), Clostridium ljungdahlii (Kopke et al 2010) and organic-solvent tolerant bacteria (OSTB), such as Pseudomonas putida S12 and Bacillus subtilis KS438 (Nielsen et al 2009)
Isolation of butanol-tolerant bacteria Most Gram negative OSTB have been isolated from soil samples, but a greater biodiversity of OSTB has been described in the marine environment because the relatively high salt concentration may induce multidrug efflux pump activity in bacteria, leading to their higher solvent tolerance (Sardessai and Bhosle 2002)

Summary

Introduction

Introduction nButanol (hereafter referred to as butanol) is an important industrial chemical, widely used as a solvent, a stabilizer and feedstock for the production of polymers and plastics. Alternative hosts being engineered for butanol production are well-characterized, genetically-amenable microorganisms, such as Escherichia coli (Atsumi et al 2008,Inui et al 2008,; Nielsen et al 2009), Saccharomyces cerevisiae (Steen et al 2008), Clostridium ljungdahlii (Kopke et al 2010) and organic-solvent tolerant bacteria (OSTB), such as Pseudomonas putida S12 and Bacillus subtilis KS438 (Nielsen et al 2009) They were capable of producing butanol, at relatively low yield, but the critical remaining problem was that they still severely suffer from butanol toxicity as their viability was significantly decreased at 0.75, 1.0, 1.25, 2.0%v/v butanol for P. putida, E. coli, B. subtilis, (Nielsen et al 2009), S. cerevisiae (Liu and Qureshi 2009) and Clostridia (Ezeji et al 2010,), respectively. To be suitable as a potential genetic engineered host for bioproduction of chemicals, other fundamental, but requisite, knowledge of the host regarding genetic competency, gene expression strength, etc. should be proven feasible

Objectives

Methods

Results

Conclusion