Genetic engineering of Clostridium acetobutylicum to enhance isopropanol-butanol-ethanol production with an integrated DNA-technology approach

Abstract

a b s t r a c tAcetone being a non-fuel solvent produced during the traditional acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum; reduces the overall fuel alcohol yield. However, the conversion of acetone into isopropanol has been recommended to improve the process economy. The present study aims to develop an engineered C. acetobutylicum DSM 792 strain to convert acetone into isopropanol by introducing the secondary alcohol dehydrogenase gene from Clostridium beijerinckii NRRL B593 using the allele-coupled exchange approach. Batch and continuous fermentation experiments were carried out with a modified strain C. acetobutylicum DSM 792-ADH to improve the isopropanol yield and titer. The growth and production behavior of the modified strain in stationary flask culture and controlled batch culture was studied. Almost 50% of acetone was converted into isopropanol with highest total solvent yield to be 0.39 g g À1 glucose. The modified strain also utilized sugar mixture and SO 2 eethanolewater spent liquor as a substrate to produce the solvents.

Synthetic Biology Tools for Genome and Transcriptome Engineering of Solventogenic Clostridium

“…• Complementing the mutant strain with a heterologous version of pyrE gene as a counter selective marker Tripathi et al, 2010;Heap et al, 2012;Ng et al, 2013;Bankar et al, 2015;Zhang N. et al, 2015;Croux et al, 2016;Ehsaan et al, 2016a; CRISPR/Cas • RNA-guided target specific DNA cleavage system…”

“…Similarly, successful expression of cellulosomal subunits in C. acetobutylicum has also been achieved using this method (Kovacs et al, 2013). Few other Clostridium species modified using ACE technology includes C. acetobutylicum, C. sporogenes, and C. difficile (Heap et al, 2012;Ng et al, 2013;Bankar et al, 2015;Ehsaan et al, 2016b;Minton et al, 2016;Willson et al, 2016).…”

Effective isopropanol–butanol (IB) fermentation with high butanol content using a newly isolated Clostridium sp. A1424

“…Butanol can also be produced through isopropanol–butanol–ethanol (IBE) fermentation with a small residual amount of acetone by wild-type strains [18–20] and engineered strains [1, 21, 22]. Isopropanol in the mixed alcohols can be utilized as a fuel additive [20].…”

“…However, there are only a few natural IBE producers reported to date, including Clostridium beijerinckii NRRL B-593 [19, 20] and Clostridium beijerinckii optinoii [18], and they show low production of butanol and alcohols even after process optimization [18, 20, 23]. Another approach is to genetically engineer ABE strains by expressing the isopropanol dehydrogenase from C. beijerinckii NRRL B-593 [1, 21, 22]. However, overexpression of isopropanol dehydrogenase alone caused an incomplete conversion of acetone to isopropanol and decrease of butanol production [1, 21, 22, 24].…”

“…Another approach is to genetically engineer ABE strains by expressing the isopropanol dehydrogenase from C. beijerinckii NRRL B-593 [1, 21, 22]. However, overexpression of isopropanol dehydrogenase alone caused an incomplete conversion of acetone to isopropanol and decrease of butanol production [1, 21, 22, 24]. Multiple genes had to be modified to restore butanol production [1, 24, 25].…”