Enhanced Butanol Production Obtained by Reinforcing the Direct Butanol-Forming Route in Clostridium acetobutylicum

Abstract

Butanol is an important industrial solvent and advanced biofuel that can be produced by biphasic fermentation by Clostridium acetobutylicum. It has been known that acetate and butyrate first formed during the acidogenic phase are reassimilated to form acetone-butanol-ethanol (cold channel). Butanol can also be formed directly from acetyl-coenzyme A (CoA) through butyryl-CoA (hot channel). However, little is known about the relative contributions of the two butanol-forming pathways. Here we report that the direct butanol-forming pathway is a better channel to optimize for butanol production through metabolic flux and mass balance analyses. Butanol production through the hot channel was maximized by simultaneous disruption of the pta and buk genes, encoding phosphotransacetylase and butyrate kinase, while the adhE1D485G gene, encoding a mutated aldehyde/alcohol dehydrogenase, was overexpressed. The ratio of butanol produced through the hot channel to that produced through the cold channel increased from 2.0 in the wild type to 18.8 in the engineered BEKW(pPthlAAD**) strain. By reinforcing the direct butanol-forming flux in C. acetobutylicum, 18.9 g/liter of butanol was produced, with a yield of 0.71 mol butanol/mol glucose by batch fermentation, levels which are 160% and 245% higher than those obtained with the wild type. By fed-batch culture of this engineered strain with in situ recovery, 585.3 g of butanol was produced from 1,861.9 g of glucose, with the yield of 0.76 mol butanol/mol glucose and productivity of 1.32 g/liter/h. Studies of two butanol-forming routes and their effects on butanol production in C. acetobutylicum described here will serve as a basis for further metabolic engineering of clostridia aimed toward developing a superior butanol producer.

Metabolic Engineering of Raoultella ornithinolytica BF60 for Production of 2,5-Furandicarboxylic Acid from 5-Hydroxymethylfurfural

“…Intron insertion into the chromosome involves a single-step transformation procedure with high efficiency. It is a promising approach to markerless mutagenesis, with consecutive introduction of several mutations into a single strain through curing of the donor plasmid (53,54).…”

Development of a Gene Knockout System Using Mobile Group II Introns (Targetron) and Genetic Disruption of Acid Production Pathways in Clostridium beijerinckii

“…The DNA target site is recognized primarily by base-pairing of easily modified intron RNA sequences, so the intron can be inserted into virtually any specific DNA target sites (8). Targetron gene knockout systems have been successfully employed in many Gram-positive and Gram-negative bacteria, including several clostridial species (4)(5)(6)(8)(9)(10)(11)(12).…”

“…Acid production in the acidogenic phase is significant and has been shown to play important roles in the switch to solventogenesis (13)(14)(15)(16). The inactivation of acetate and butyrate formation pathways in Clostridium acetobutylicum ATCC 824 has been shown to lead to modified product patterns and different cell physiology (4,12,(17)(18)(19).…”

Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052

“…SA-1 displays a metabolic carbon flow that is redirected mainly to the production of increased amounts of butanol and acetone. Redirecting the metabolic flow of carbon towards solvents to reach higher final butanol titres and yields has been shown previously by genetically modified C. acetobutylicum strains with disrupted genes responsible for butyric acid production (Harris et al, 2000;Jang et al, 2012b). Moreover, the overall butanol accumulation, ABE yields and solvent productivity of SA-1 are comparable to values previously reported for C. beijerinckii BA101, another butanol hyperproducing offspring of NCIMB 8052 (Formanek et al, 1997;Ezeji et al, 2007a, b;Qureshi & Blaschek, 2000;Lienhardt et al, 2002).…”