Clostridium acetobutylicum is one of the promising organisms for biofuel industry. Metabolic redesigning studies for production of butanol in C. acetobutylicum have been carried out. For this study, metabolic pathways of C. acetobutylicum including acid synthesis pathway, acid reassimilation pathway and solvent synthesis pathway have been redesigned to support butanol production.
At first, metabolic redesigning of C. acetobutylicum was carried out for enhanced butanol production.
Various genes involved in the dissimilation and reassimilation pathway of acetic and butyric acids in C. acetobutylicum were engineered to evaluate their impact on solventogenesis by measuring the fermentation products as well as by metabolic flux balance analysis. Disruption of pta gene encoding phosphotransacetylase on the acetate dissimilation pathway was particularly outstanding in enhancing butanol production. The pta-deficient C. acetobutylicum EKW was able to produce 18.5 g/L butanol as compared to 13.0 g/l butanol production in parent strain. In the next strategy, both the pta and buk genes were simultaneously disrupted to produce C. acetobutylicum BEKW strain capable of producing a maximum of 16.8 g/L butanol. Although, complete removal of acetic and butyric acid production could not be achieved in BEKW, phase transition from acidogenesis to solventogenesis was triggered earlier in BEKW as compared to both the parent and EKW strains. Phase transition was observed in just 6.3 h in BEKW as compared to more than 9.0 h in the other two strains. During the study on acid reassimilation pathway, it was found that acetate production pathway is not a reversible reaction in C. acetobutylicum. It was also observed that butanol could be produced from surplus acetyl-CoA via butyryl-CoA with butyrate dissimilation and reassmilation in EKW strain.
Secondly, a possible way to improve the economic efficacy of acetone-butanol-ethanol fermentation is to increase the butanol selectivity by eliminating the production of other by-products, such as acetone. For selective butanol production C. acetobutylicum M5(pIMP1E1ESL) was constructed by adhE1-groESL co-expression of C. acetobutylicum M5, a derivative strain of C. acetobutylicum ATCC 824, which does not produce solvents due to the lack of megaplasmid pSOL1. It was constructed pIMP1E1ESL cloned aad gene encoding aldehyde/alcohol dehydrogenase (AAD) and groESL gene encoding for groES and groEL chaperone protein. The batch fermentation studies showed that co-expression of aad and groESL restored 8.5 g/L butanol and 1.8 g/L ethanol formation in mutant M5 and suppressed production of acetone absolutely.
Thirdly, Clostridium acetobutylicum M5(pIMP1E1AB) was constructed by adhE1-ctfAB complementation of C. acetobutylicum M5. The gene products of adhE1-ctfAB catalyze the formation of acetoacetate and ethanol/butanol with acid re-assimilation in solventogenesis. Effects of the adhE1-ctfAB complementation of M5 were studied by batch fermentations under various pH and glucose concentrations, and by flux balance analysis using a genome-scale metabolic model for this organism. The metabolically engineered M5(pIMP1E1AB) strain was able to produce 11.1 g/L (154mM) butanol with 0.6 g/L (9.9mM) acetone at pH 5.5, resulting in a butanol selectivity (a molar ratio of butanol to total solvents) of 0.84, which is much higher than that (0.57 at pH 5.0 or 0.61 at pH 5.5) of the wild-type strain ATCC 824. Unlike C. acetobutylicum ATCC 824, higher level of acetate accumulation was observed during fermentation of M5 strain complemented with adhE1 and/or ctfAB. A plausible reason for this phenomenon is that cellular metabolism was shifted towards acetate production in order to compensate reduced ATP production during the largely growth-associated butanol formation by the M5(pIMP1E1AB) strain.
Finally, the acetoacetate decarboxylase gene(adc) in C. acetobutylicum ATCC824 was disrupted for highly selective butanol production. The butanol ratio increased from 66% to 88%, with acetone production reduced to approximately 0.50 g/L in the adc-disrupted mutant (AdKW).Also AdEKW disrupted both the pta and adc genes and AdBEKW strain, the triple knockout mutant, disrupted the pta, buk and adc genes simultaneously were constructed for the production of highly selective butanol. Although, complete removal of acetic and butyric acid production could not be achieved, acetic and butyric acid production ratio was lowed and acetone production was suppressed simultaneously. Butanol ratios of AdEKW and AdBEKW were 82% and 88%, respectively.