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Resistance to furan derivatives and phenolic compounds plays an important role in the use of lignocellulosic biomass for biological production of chemicals and fuels. This study confirmed that expression of short-chain dehydrogenase/reductase (SDR) from Clostridium beijerinckii NCIMB 8052 significantly improved the tolerance of C. tyrobutyricum to furfural due to the enhanced activity for furfural reduction. And on this basis, co-expression of SDR and heat shock chaperones GroESL could simultaneously enhance the tolerance of C. tyrobutyricum to furan derivatives and phenolic compounds, which were the main inhibitors presented in dilute-acid lignocellulosic hydrolysates. Consequently, the recombinant strain ATCC 25755/sdr+groESL exhibited good performance in butyric acid production with corncob acid hydrolysate as the substrate. Batch fermentation in bioreactor showed that the butyrate produced by ATCC 25755/sdr+groESL was 32.8 g/L, increased by 28.1% as compared with the wild-type strain. Meanwhile, the butyrate productivity increased from 0.19 g/L·h to 0.29 g/L·h., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Butyric acid fermentation by Clostridium couples with the synthesis of acetic acid. But the presence of acetic acid reduces butyric acid yield and increases separation and purification costs of butyric acid. Hence, enhancing the butyrate/acetate ratio is important for economical butyric acid production. This study indicated that enhancing the acetyl-CoA to butyrate flux by overexpression of both the butyryl-CoA/acetate CoA transferase (cat1) and crotonase (crt) genes in C. tyrobutyricum could significantly reduce acetic acid concentration. Fed-batch fermentation of ATCC 25755/cat1 + crt resulted in increased butyrate/acetate ratio of 15.76 g/g, which was 2.24-fold higher than that of the wild-type strain. Furthermore, in order to simultaneously increase the butyrate/acetate ratio, butyric acid concentration and productivity, the recombinant strain ATCC 25755/ppcc (co-expression of 6-phosphofructokinase (pfkA) gene, pyruvate kinase (pykA) gene, cat1, and crt) was constructed. Consequently, ATCC 25755/ppcc produced more butyric acid (46.8 vs. 35.0 g/L) with a higher productivity (0.83 vs. 0.49 g/L·h) and butyrate/acetate ratio (13.22 vs. 7.22 g/g) as compared with the wild-type strain in batch fermentation using high glucose concentration (120 g/L). This study demonstrates that enhancing the acetyl-CoA to butyrate flux is an effective way to reduce acetic acid production and increase butyrate/acetate ratio.

Lignocellulosic biomass is the most abundant and renewable substrate for biological fermentation, but the inhibitors present in the lignocellulosic hydrolysates could severely inhibit the cell growth and productivity of industrial strains. This study confirmed that overexpressing of native groESL in Clostridium tyrobutyricum could significantly improve its tolerance to lignocellulosic hydrolysate-derived inhibitors, especially for phenolic compounds. Consequently, ATCC 25755/groESL showed a better performance in butyric acid fermentation with hydrolysates of corn cob, corn straw, rice straw, wheat straw, soybean hull and soybean straw, respectively. When corn straw and rice straw hydrolysates, which showed strong toxicity to C. tyrobutyricum, were used as the substrates, 29.6 g/L and 30.1 g/L butyric acid were obtained in batch fermentation, increased by 26.5% and 19.4% as compared with the wild-type strain, respectively. And more importantly, the butyric acid productivity reached 0.31 g/L·h (vs. 0.20-0.21 g/L·h for the wild-type strain) due to the shortened lag phase., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The response of Clostridium tyrobutyricum to butyric acid stress involves various stress-related genes, and therefore overexpression of stress-related genes can improve butyric acid tolerance and yield. Class I heat shock proteins (HSPs) play an important role in the process of protecting bacteria from sudden changes of extracellular stress by assisting protein folding correctly. The results of quantitative real-time PCR indicated that the Class I HSGs grpE, dnaK, dnaJ, groEL, groES, and htpG were significantly upregulated under butyric acid stress, especially the dnaK and groE operons. Overexpression of groESL and htpG could significantly improve the tolerance of C. tyrobutyricum to butyric acid, while overexpression of dnaK and dnaJ showed negative effects on butyric acid tolerance. Acid production was also significantly promoted by increased GroESL expression levels; the final butyric acid and acetic acid concentrations were 28.2 and 38% higher for C. tyrobutyricum ATCC 25755/groESL than for the wild-type strain. In addition, when fed-batch fermentation was carried out using cell immobilization in a fibrous-bed bioreactor, the butyric acid yield produced by C. tyrobutyricum ATCC 25755/groESL reached 52.2 g/L, much higher than that for the control. The improved butyric acid yield is probably attributable to the high GroES and GroEL levels, which can stabilize the biosynthetic machinery of C. tyrobutyricum under extracellular butyric acid stress.