Korean Journal of Agricultural Science (농업과학연구)

Institute of Agricultural Science, CNU (충남대학교 농업과학연구소)
권호 표지
DOI QR코드

DOI QR Code

Development of succinate producing Cellulomonas flavigena mutants with deleted succinate dehydrogenase gene

  • Lee, Heon-Hak (Department of Bio-Environmental Chemistry, Chungnam National University) ;
  • Jeon, Min-Ki (Department of Bio-Environmental Chemistry, Chungnam National University) ;
  • Yoon, Min-Ho (Department of Bio-Environmental Chemistry, Chungnam National University)
  • Received : 2017.01.16
  • Accepted : 2017.03.08
  • Published : 2017.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

This study was performed to produce succinic acid from biomass by developing mutants of Cellulomonas flavigena in which the succinate dehydrogenase gene (sdh) is deleted. For development of succinate producing mutants, the upstream and downstream regions of sdh gene from C. flavigena and antibiotic resistance gene (neo, bla) were inserted into pKC1139, and the recombinant plasmids were transformed into Escherichia coli ET12567/pUZ8002 which is a donor strain for conjugation. C. flavigena was conjugated with the transformed E. coli ET12567/pUZ8002 to induce the deletion of sdh in chromosome of this bacteria by double-crossover recombination. Two mutants (C. flavigena H-1 and H-2), in which sdh gene was deleted in the chromosome, were constructed and confirmed by PCR. To estimate the production of succinic acid by the two mutants when the culture broth was fermented with biomass such as CMC, xylan, locust gum, and rapeseed straw; the culture broth was analyzed by HPLC analysis. The succinic acid in the culture broth was not detected as a fermentation products of all biomass. One of the reasons for this may be the conversion of succinic acid to fumaric acid by sdh genes (Cfla_1014 - Cfla_1017 or Cfla_1916 - Cfla_1918) which remained in the chromosomal DNA of C. flavigena H-1 and H-2. The other reason could be the conversion of succinyl-CoA to other metabolites by enzymes related to the bypass pathway of TCA cycle.

Keywords

References

  1. Abt B, Foster B, Lapidus A, Clum A, Sun H, Pukall R, Lucas S, Del Rio TG, Nolan M, Tice H, Cheng JF, Pitluck S, Liolios K, Ivanova N, Mavromatis K, Ovchinnikova G, Pati A, Goodwin L, Chen A, Palaniappan K, Land M, Hauser L Chang YJ, Jeffries CD, Rohde M, Goker M, Woyke T, Bristow J, Eisen JA, Markowitz V, Hugenholtz P, Kyrpides NC, Klenk HP. 2010. Complete genome sequence of Cellulomonas flavigena type strain (134T). Standards in Genomic Sciences 3:15-25. https://doi.org/10.4056/sigs.1012662
  2. Bierman M, Logan R, O'brien K, Seno ET, Rao RN, Schoner BE. 1992. Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. Gene 116:43-49. https://doi.org/10.1016/0378-1119(92)90627-2
  3. Bretz K. 2015. Succinic acid production in fed-batch fermentation of Anaerobiospirillum succiniciproducens using glycerol as carbon source. Chemical Engineering & Technology 38:1659-1664. https://doi.org/10.1002/ceat.201500015
  4. Choi S, Song H, Lim SW, Kim TY, Ahn JH, Lee JW, Lee MH, Lee SY. 2016. Highly selective production of succinic acid by metabolically engineered Mannheimia succiniciproducens and its efficient purification. Biotechnology and Bioengineering 113: 2168-2177. https://doi.org/10.1002/bit.25988
  5. Christopherson MR, Suen G, Bramhacharya S, Jewell KA, Aylward FO, Mead D, Brumm PJ. 2013. The genome sequences of Cellulomonas fimi and "Cellvibrio gilvus" reveal the cellulolytic strategies of two facultative anaerobes, transfer of "Cellvibrio gilvus" to the genus Cellulomonas, and proposal of Cellulomonas gilvus sp. nov. PloS One 8:e53954. https://doi.org/10.1371/journal.pone.0053954
  6. Dubos RJ. 1928. The decomposition of cellulose by aerobic bacteria. Journal of bacteriology, 15: 223.
  7. Flett F, Mersinias V, Smith CP. 1997. High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA-restricting streptomycetes. FEMS Microbiology Letters 155:223-229. https://doi.org/10.1111/j.1574-6968.1997.tb13882.x
  8. Han JG, Oh SH, Jeong MH, Kim SS, Seo HB, Jeong KH, Jang YS, Kim IC, Lee HY. 2009. Two-step high temperature pretreatment process for bioethanol production from rape stems. KSBB Journal 24:489-494. [in Korean]
  9. Huang S, Millar AH. 2013. Succinate dehydrogenase: The complex roles of a simple enzyme. Current Opinion in Plant Biology 16:344-349. https://doi.org/10.1016/j.pbi.2013.02.007
  10. Jojima T, Noburyu R, Suda M, Okino S, Yukawa H, Inui M. 2016. Improving process yield in succinic acid production by cell recycling of recombinant Corynebacterium glutamicum. Fermentation 2:5. https://doi.org/10.3390/fermentation2010005
  11. Kang HW, Ryu YW. 2009. Inhibition of oligomycin biosynthesis by olmA5 gene knock-out in Streptomyces avermitilis. KSBB Journal 24:279-286. [in Korean]
  12. Kim MS, Yoon MH, Choi WY. 2007. Taxonomic studies on the cellulolytic bacterium Cellulomonas uda CS 1-1. CNU Journal of Agricultural Science 34:99-109. [in Korean]
  13. Lee HH, Jeon MK, Yoon MH. 2016. Pretreatment and enzymatic saccharification process of rapeseed straw for production of bioethanol. Korean Journal of Agricultural Science 43:641-649. [in Korean]
  14. Li N, Zhang B, Chen T, Wang Z, Tang YJ, Zhao X. 2013. Directed pathway evolution of the glyoxylate shunt in Escherichia coli for improved aerobic succinate production from glycerol. Journal of Industrial Microbiology & Biotechnology, 40:1461-1475. https://doi.org/10.1007/s10295-013-1342-y
  15. Lin H, Bennett GN, San KY. 2005a. Metabolic engineering of aerobic succinate production systems in Escherichia coli to improve process productivity and achieve the maximum theoretical succinate yield. Metabolic Engineering 7:116-127. https://doi.org/10.1016/j.ymben.2004.10.003
  16. Lin H, Bennett GN, San KY. 2005b. Fed-batch culture of a metabolically engineered Escherichia coli strain designed for high-level succinate production and yield under aerobic conditions. Biotechnology and Bioengineering 90:775-779. https://doi.org/10.1002/bit.20458
  17. Mazodier P, Petter R, Thompson C. 1989. Intergeneric conjugation between Escherichia coli and Streptomyces species. Journal of Bacteriology 171:3583-3585. https://doi.org/10.1128/jb.171.6.3583-3585.1989
  18. Nigam PS, Singh A. 2011. Production of liquid biofuels from renewable resources. Progress in Energy and Combustion Science 37:52-68. https://doi.org/10.1016/j.pecs.2010.01.003
  19. Paget MS, Chamberlin L, Atrih A, Foster SJ, Buttner MJ. 1999. Evidence that the extracytoplasmic function sigma factor $\zeta$E is required for normal cell wall structure in Streptomyces coelicolor A3 (2). Journal of Bacteriology 181:204-211.
  20. Pateraki C, Patsalou M, Vlysidis A, Kopsahelis N, Webb C, Koutinas AA, Koutinas M. 2016. Actinobacillus succinogenes: Advances on succinic acid production and prospects for development of integrated biorefineries. Biochemical Engineering Journal 112:285-303. https://doi.org/10.1016/j.bej.2016.04.005
  21. Rezaei MN, Aslankoohi E, Verstrepen KJ, Courtin CM. 2015. Contribution of the tricarboxylic acid (TCA) cycle and the glyoxylate shunt in Saccharomyces cerevisiae to succinic acid production during dough fermentation. International Journal of Food Microbiology 204:24-32. https://doi.org/10.1016/j.ijfoodmicro.2015.03.004
  22. Song H, Lee SY. 2006. Production of succinic acid by bacterial fermentation. Enzyme and Microbial Technology 39:352-361. https://doi.org/10.1016/j.enzmictec.2005.11.043
  23. Werpy T, Petersen G, Aden A, Bozell J, Holladay J, White J, Manheim A, Elliot D, Lasure L, Jones S, Gerber M, Ibsen K, Lumberg L, Kelly S. 2004. Top value added chemicals from biomass. Volume 1-Results of screening for potential candidates from sugars and synthesis gas (No. DOE/GO-102004-1992). Department of Energy Washington DC.
  24. Zeikus JG, Jain MK, Elankovan P. 1999. Biotechnology of succinic acid production and markets for derived industrial products. Applied Microbiology and Biotechnology 51:545-552. https://doi.org/10.1007/s002530051431