Description
1. Background
N-butanol, an important chemical raw material, is expected to become one of the new generations of biofuels. At
present, the domestic industrial synthesis of N-butanol mainly adopts the low-pressure carbonyl synthesis method,
from the production process, propylene, CO, H2, and carbonyl are the main production materials. However, this
production method relies on non-renewable petroleum products as essential raw materials, which is not friendly to
the environment. Therefore, it is necessary to design an environmentally friendly production method for N-butanol
to meet the demand of environmental protection. At present, biosynthesis of N-butanol is an important research
field of butanol factory production. It has been found that N-butanol can be synthesized naturally in Clostridium,
but the tolerance of Clostridium bacteria is not good enough for large-scale production. Recently, Lactobacillus
Brevis with better N-butanol tolerance has been isolated by researchers. This project will build an
N-butanol-producing bacterium to meet the needs of industrial production and lay a foundation for subsequent
improvement.
2. Experiment Design
a) The metabolic pathway of N-butanol synthesis in Lactobacillus Brevis ATCC367.
Genes introduced into L. brevis are shown in red (Figure 1). The thl gene encodes
mercaptan, hbd encodes β -hydroxybutyrate CoA dehydrogenase, crt encodes 3-hydroxybutyrate CoA dehydrase, and ter
encodes trans-enol CoA reductase. Only when the four enzymes are expressed together can the engineering strain
achieve the metabolic process from glucose to N-butanol.
Figure 1. The metabolic pathway of N-butanol synthesis in Lactobacillus Brevis ATCC367.
General Experiment Procedure
First, the N-butanol fermentation-related genes, thlA, hbd, crt, and ter genes were amplified by PCR from the
Lactobacillus Brevis ATCC824 genomic DNA, and then amplicons were extracted.
Next, we extracted plasmids pIB184-vector from E. coli DH5α, ligated the target gene and the vector into complete plasmids with homologous recombinant enzymes, and transferred the recombinant plasmids into Streptococcus Brevis by electroporation.
Then, erythromycin was used to select whether the plasmid was inserted into the competent bacteria Streptococcus Brevis ATCC367 and screened, and the screened bacteria were coated on MRS solid medium dish. If there are positive colonies, we cultivate them in the liquid medium and incubate them in the anaerobic chamber.
Finally, when our bacteria grew, we measured the growth curve and detected the yield of N-Butanol (Figure 2).
Next, we extracted plasmids pIB184-vector from E. coli DH5α, ligated the target gene and the vector into complete plasmids with homologous recombinant enzymes, and transferred the recombinant plasmids into Streptococcus Brevis by electroporation.
Then, erythromycin was used to select whether the plasmid was inserted into the competent bacteria Streptococcus Brevis ATCC367 and screened, and the screened bacteria were coated on MRS solid medium dish. If there are positive colonies, we cultivate them in the liquid medium and incubate them in the anaerobic chamber.
Finally, when our bacteria grew, we measured the growth curve and detected the yield of N-Butanol (Figure 2).
Figure 2. General experiment procedure
3. Expected Result
1. Successfully construct thlA, hbd, crt, and ter genes into double-enzyme-digested vector pIB184.
2. Transform the recombinant plasmid into Streptococcus Brevis ATCC367 and identify the positive colony.
3. Detect the yield of N-Butanol and measure the growth curve of the engineered strain.
The study shows that Streptococcus Brevis has a high tolerance of N-butanol and is likely to be a candidate for producing high concentrations of N-butanol.
2. Transform the recombinant plasmid into Streptococcus Brevis ATCC367 and identify the positive colony.
3. Detect the yield of N-Butanol and measure the growth curve of the engineered strain.
The study shows that Streptococcus Brevis has a high tolerance of N-butanol and is likely to be a candidate for producing high concentrations of N-butanol.
4. Reference
1. Qi Li, Meixian Wu, Zhiqiang Wen, Yuan Jiang, Xin Wang, Yawei Zhao, Jinle Liu, Junjie Yang, Yu Jiang, Sheng Yang, Optimization of n-butanol synthesis in Lactobacillus brevis via the functional expression of thl, hbd, crt and ter, Journal of Industrial Microbiology and Biotechnology, Volume 47, Issue 12, 1 December 2020, Pages 1099–1108, https://doi.org/10.1007/s10295-020-02331-2
2. Li, J., Zhao, J. B., Zhao, M., Yang, Y. L., Jiang, W. H., & Yang, S. (2010). Screening and characterization of butanol-tolerant micro-organisms. Letters in applied microbiology, 50(4), 373–379. https://doi.org/10.1111/j.1472-765X.2010.02808.x
3. Berezina, O. V., Zakharova, N. V., Brandt, A., Yarotsky, S. V., Schwarz, W. H., & Zverlov, V. V. (2010). Reconstructing the clostridial n-butanol metabolic pathway in Lactobacillus brevis. Applied microbiology and biotechnology, 87(2), 635–646. https://doi.org/10.1007/s00253-010-2480-z
4. 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 Biot 77:1305–1316. https://doi.org/10.1007/s00253-007-1257-5
5. Mitchell WJ (1998) Physiology of carbohydrate to solvent conversion by Clostridia. In: Poole RK (ed) Advances in Microbial Physiology, vol 39. pp 31–130
6. Bowles LK, Ellefson WL (1985) Effects of butanol on Clostridium-acetobutylicum. Appl Environ Microb 50:1165–1170
7. 张云贤, 张华西, 余维新, 李杰灵, & 谭平华. (2015). 正丁醇的合成进展简述. 2015 中国化工学会学术年会.
2. Li, J., Zhao, J. B., Zhao, M., Yang, Y. L., Jiang, W. H., & Yang, S. (2010). Screening and characterization of butanol-tolerant micro-organisms. Letters in applied microbiology, 50(4), 373–379. https://doi.org/10.1111/j.1472-765X.2010.02808.x
3. Berezina, O. V., Zakharova, N. V., Brandt, A., Yarotsky, S. V., Schwarz, W. H., & Zverlov, V. V. (2010). Reconstructing the clostridial n-butanol metabolic pathway in Lactobacillus brevis. Applied microbiology and biotechnology, 87(2), 635–646. https://doi.org/10.1007/s00253-010-2480-z
4. 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 Biot 77:1305–1316. https://doi.org/10.1007/s00253-007-1257-5
5. Mitchell WJ (1998) Physiology of carbohydrate to solvent conversion by Clostridia. In: Poole RK (ed) Advances in Microbial Physiology, vol 39. pp 31–130
6. Bowles LK, Ellefson WL (1985) Effects of butanol on Clostridium-acetobutylicum. Appl Environ Microb 50:1165–1170
7. 张云贤, 张华西, 余维新, 李杰灵, & 谭平华. (2015). 正丁醇的合成进展简述. 2015 中国化工学会学术年会.