Metabolic Engineering of Clostridium acetobutylicum ATCC 824 for Isopropanol-Butanol-Ethanol Fermentation

Abstract

Clostridium acetobutylicum naturally produces acetone as well as butanol and ethanol. Since acetone cannot be used as a biofuel, its production needs to be minimized or suppressed by cell or bioreactor engineering. Thus, there have been attempts to disrupt or inactivate the acetone formation pathway. Here we present another approach, namely, converting acetone to isopropanol by metabolic engineering. Since isopropanol can be used as a fuel additive, the mixture of isopropanol, butanol, and ethanol (IBE) produced by engineered C. acetobutylicum can be directly used as a biofuel. IBE production is achieved by the expression of a primary/secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B-593 (i.e., adh B-593 ) in C. acetobutylicum ATCC 824. To increase the total alcohol titer, a synthetic acetone operon (act operon; adc-ctfA-ctfB) was constructed and expressed to increase the flux toward isopropanol formation. When this engineering strategy was applied to the PJC4BK strain lacking in the buk gene (encoding butyrate kinase), a significantly higher titer and yield of IBE could be achieved. The resulting PJC4BK(pIPA3-Cm2) strain produced 20.4 g/liter of total alcohol. Fermentation could be prolonged by in situ removal of solvents by gas stripping, and 35.6 g/liter of the IBE mixture could be produced in 45 h.

Enhanced isopropanol–butanol–ethanol (IBE) production in immobilized column reactor using modified Clostridium acetobutylicum DSM792

“…The acetone produced during the traditional ABE fermentation was preferentially converted into isopropanol to improve the process economy [1,6]. Hence, C. acetobutylicum which is a commonly used microorganism for ABE fermentation was engineered to convert acetone into isopropanol by introducing the secondary alcohol dehydrogenase gene from C. beijerinckii NRRL B593 using allele-coupled exchange approach.…”

“…Hence, C. acetobutylicum which is a commonly used microorganism for ABE fermentation was engineered to convert acetone into isopropanol by introducing the secondary alcohol dehydrogenase gene from C. beijerinckii NRRL B593 using allele-coupled exchange approach. C. acetobutylicum does not possess a gene for secondary alcohol dehydrogenase (adh) which is required to produce 2-propanol (isopropanol) from acetone [6,27]. C. beijerinckii NRRL B593 contain a NADPH dependent primary/secondary alcohol (isopropanol) dehydrogenase (EC 1.1.1.1) to produce a mixture of isopropanol and n-butanol.…”

“…However, the co-production of acetone in the ABE process is considered as un-desirable because of its corrosiveness to rubber engine parts and its poor fuel properties [2][3][4][5][6]. Some natural Clostridium beijerinckii strains [7] produce isopropanol, instead of acetone, together with butanol and ethanol (IBE) to produce alcohol biofuel mixture which is considered as a 'green solvent'.…”

Advances in consolidated bioprocessing systems for bioethanol and butanol production from biomass: a comprehensive review

“…Co-immobilization of saccharolytic molds with ethanol producing yeast 4.08% (v/v), and a YE/s of 0.41 after 9 d Lee et al, 2012 . integrated haploid strains, each expressing either α-amylase or glucoamylase gene.…”

“…After fermentation optimization, the mixed culture of both strains showed an increased amylase activity by 10 folds, and enhanced ABE production rates by upto 5.4 and 6.5 folds from soluble starch and cassava starch, respectively, compared to those of the single culture of C. butylicum. Lee et al, (2012) used a co-immobilization system for direct ethanol production from sweet potato. In their work, the saccharification and fermentation conditions were optimized for co-immobilization of saccharolytic molds (A. oryzae and Monascus purpureus) with S. cerevisiae.…”