Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production with high butyrate/acetate ratio
- 383 Downloads
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.
KeywordsMetabolic engineering Butyric acid Clostridium tyrobutyricum Butyrate/acetate ratio Butyryl-CoA/acetate CoA transferase Crotonase
This work was funded by the National Natural Science Foundation of China (21676098), the State Key Laboratory of Pulp and Paper Engineering (2017C03), the China Postdoctoral Science Foundation Funded Project (2017M612667), and the Fundamental Research Funds for the Central Universities (2017BQ084).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Inui M, Suda M, Kimura S, Yasuda K, Suzuki H, Toda H, Yamamoto S, Okino S, Suzuki N, Yukawa H (2008) Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli. Appl Microbiol Biotechnol 77(6):1305–1316. https://doi.org/10.1007/s00253-007-1257-5 CrossRefPubMedGoogle Scholar
- Jang YS, Lee JY, Lee J, Park JH, Im JA, Eom MH, Lee J, Lee SH, Song H, Cho JH (2012) Enhanced Butanol production obtained by reinforcing the direct Butanol-forming route in Clostridium acetobutylicum. mBio 3(5):e00314–e00312. https://doi.org/10.1128/mBio.00314-12 CrossRefPubMedPubMedCentralGoogle Scholar
- Jiang L, Wang JF, Liang SZ, Wang XN, Cen PL, Xu ZN (2010a) Production of butyric acid from glucose and xylose with immobilized cells of Clostridium tyrobutyricum in a fibrous-bed bioreactor. Appl Biochem Biotechnol 160(2):350–359. https://doi.org/10.1007/s12010-008-8305-1 CrossRefPubMedGoogle Scholar
- Jiang L, Cai J, Wang JF, Liang SZ, Xu ZN, Yang ST (2010b) Phosphoenolpyruvate-dependent phosphorylation of sucrose by Clostridium tyrobutyricum ZJU 8235: evidence for the phosphotransferase transport system. Bioresour Technol 101(1):304–309. https://doi.org/10.1016/j.biortech.2009.08.024 PubMedCrossRefGoogle Scholar
- Rephaeli A, Zhuk R, Nudelman A (2000) Prodrugs of butyric acid from bench to bedside: synthetic design, mechanisms of action, and clinical applications. Drug Develop Res 50(3-4):379–391. https://doi.org/10.1002/1098-2299(200007/08)50:3/4<379::AID-DDR20>3.0.CO;2-8Google Scholar
- Sillers R, Al-Hinai MA, Papoutsakis ET (2009) Aldehyde-alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentations. Biotechnol Bioeng 102(1):38–49. https://doi.org/10.1002/bit.22058 CrossRefPubMedGoogle Scholar
- Wang Y, Li XZ, Milne CB, Janssen H, Lin WY, Phan G, Hu HY, Jin YS, Price ND, Blaschek HP (2013) Development of a gene knockout system using mobile group II introns (Targetron) and genetic disruption of acid production pathways in Clostridium beijerinckii. Appl Environ Microbiol 79(19):5853–5863. https://doi.org/10.1128/aem.00971-13 CrossRefPubMedPubMedCentralGoogle Scholar
- Yu MR, Du YM, Jiang WY, Chang WL, Yang ST, Tang IC (2012) Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum. Appl Microbiol Biotechnol 93(2):881–889. https://doi.org/10.1007/s00253-011-3736-y CrossRefPubMedGoogle Scholar
- Zhao Y, Tomas CA, Rudolph FB, Papoutsakis ET, Bennett GN (2005) Intracellular Butyryl phosphate and acetyl phosphate concentrations in Clostridium acetobutylicum and their implications for solvent formation. Appl Environ Microbiol 71(1):530–537. https://doi.org/10.1128/AEM.71.1.530-537.2005 CrossRefPubMedPubMedCentralGoogle Scholar
- Zhou X, Lu XH, Li XH, Xin ZJ, Xie JR, Zhao MR, Wang L, Du WY, Liang JP (2014) Radiation induces acid tolerance of Clostridium tyrobutyricum and enhances bioproduction of butyric acid through a metabolic switch. Biotechnol Biofuels 7:22. https://doi.org/10.1186/1754-6834-7-22 CrossRefPubMedPubMedCentralGoogle Scholar