Metabolic engineering of Clostridium tyrobutyricum for n‐butanol production through co‐utilization of glucose and xylose

Abstract

The glucose-mediated carbon catabolite repression (CCR) in Clostridium tyrobutyricum impedes efficient utilization of xylose present in lignocellulosic biomass hydrolysates. In order to relieve the CCR and enhance xylose utilization, three genes (xylT, xylA, and xylB) encoding a xylose proton-symporter, a xylose isomerase and a xylulokinase, respectively, from Clostridium acetobutylicum ATCC 824 were co-overexpressed with aldehyde/alcohol dehydrogenase (adhE2) in C. tyrobutyricum (Δack). Compared to the strain Ct(Δack)-pM2 expressing only adhE2, the mutant Ct(Δack)-pTBA had a higher xylose uptake rate and was able to simultaneously consume glucose and xylose at comparable rates for butanol production. Ct(Δack)-pTBA produced more butanol (12.0 vs. 3.2 g/L) with a higher butanol yield (0.12 vs. 0.07 g/g) and productivity (0.17 vs. 0.07 g/L · h) from both glucose and xylose, while Ct(Δack)-pM2 consumed little xylose in the fermentation. The results confirmed that the CCR in C. tyrobutyricum could be overcome through overexpressing xylT, xylA, and xylB. The mutant was also able to co-utilize glucose and xylose present in soybean hull hydrolysate (SHH) for butanol production, achieving a high butanol titer of 15.7 g/L, butanol yield of 0.24 g/g, and productivity of 0.29 g/L · h. This study demonstrated the potential application of Ct(Δack)-pTBA for industrial biobutanol production from lignocellulosic biomass.

Production of butyric acid from acid hydrolysate of corn husk in fermentation by Clostridium tyrobutyricum: kinetics and process economic analysis

“…Because of the requirement of extra energy in xylose transport, the net ATP yield from xylose was lower compared to that from glucose [ 13 , 14 ]. Although glucose metabolism can provide more ATP for cell growth and butyric acid production, glucose-mediated carbon catabolite repression (CCR) could inhibit the consumption of non-glucose sugars such as xylose also present in the lignocellulosic biomass hydrolysate [ 12 , 20 , 40 ]. Xylose utilization by C. tyrobutyricum was inhibited by glucose in free-cell fermentation, although the CCR could be relieved by engineering the cell to overexpress three genes in the xylose catabolism pathway [ 12 , 20 , 40 ].…”

“…Although glucose metabolism can provide more ATP for cell growth and butyric acid production, glucose-mediated carbon catabolite repression (CCR) could inhibit the consumption of non-glucose sugars such as xylose also present in the lignocellulosic biomass hydrolysate [ 12 , 20 , 40 ]. Xylose utilization by C. tyrobutyricum was inhibited by glucose in free-cell fermentation, although the CCR could be relieved by engineering the cell to overexpress three genes in the xylose catabolism pathway [ 12 , 20 , 40 ]. However, our results indicated that glucose mediated CCR would have little effect on butyric acid production from corn husk hydrolysate after cell adaptation in the FBB.…”

Metabolomics-driven approach to solving a CoA imbalance for improved 1-butanol production in Escherichia coli

“…1-Butanol titers from E. coli and Clostridium species are comparable. Recently reported engineered Clostridium species utilizing acetone-butanol-ethanol fermentation have been able to produce 19.1 g/L within 78 h (Xue et al, 2012), or 15.7 g/ L using co-utilization of glucose and xylose (Yu et al, 2015). The titer reported here is much higher compared to other non-native hosts such as in Saccharomyces cerevisiae (835 mg/L in 96 h) (Shi et al, 2016), cyanobacterium Synechococcus elongatus PCC 7942 (404 mg/L in 12 days) (Lan et al, 2013) and Lactobacillus brevis (300 mg/L in 60 h) (Berezina et al, 2010).…”

Unique genetic cassettes in a Thermoanaerobacterium contribute to simultaneous conversion of cellulose and monosugars into butanol

“…Thus, there is still a need to develop a consolidated bioprocessing technology for butanol production. Note that sustainable butanol production is also impeded by (i) product inhibition and carbon catabolite repression (CCR) ( 20 ) and (ii) complexities of downstream purification of butanol from other by-products such as acetone and ethanol ( 21 ). Hence, to achieve and simplify the consolidated bioprocessing, it is desirable to discover bacterial strains that can directly ferment cellulose and hemicellulose to butanol as the main product.…”