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Biosynthesis of Chondroitin in Engineered Corynebacterium glutamicum

  • Cheng, Fangyu (Department of Chemical Engineering, Tsinghua University) ;
  • Luozhong, Sijin (Department of Chemical Engineering, Tsinghua University) ;
  • Yu, Huimin (Department of Chemical Engineering, Tsinghua University) ;
  • Guo, Zhigang (Department of Chemical Engineering, Tsinghua University)
  • Received : 2018.10.31
  • Accepted : 2019.01.17
  • Published : 2019.03.28

Abstract

Chondroitin, the precursor of chondroitin sulfate, which is an important polysaccharide, has drawn significant attention due to its applications in many fields. In the present study, a heterologous biosynthesis pathway of chondroitin was designed in a GRAS (generally recognized as safe) strain C. glutamicum. CgkfoC and CgkfoA genes with host codon preference were synthesized and driven by promoter Ptac, which was confirmed as a strong promoter via GFPuv reporter assessment. In a lactate dehydrogenase (ldh) deficient host, intracellular chondroitin titer increased from 0.25 to 0.88 g/l compared with that in a wild-type host. Moreover, precursor enhancement via overexpressing precursor synthesizing gene ugdA further improved chondroitin titers to 1.09 g/l. Chondroitin production reached 1.91 g/l with the engineered strain C. glutamicum ${\Delta}L-CgCAU$ in a 5-L fed-batch fermentation with a single distribution $M_w$ of 186 kDa. This work provides an alternative, safe and novel means of producing chondroitin for industrial applications.

Keywords

References

  1. Vazquez JA, Rodriguezamado I, Montemayor MI, Fraguas J, Gonzalez MDP, Murado MA. 2013. Chondroitin sulfate, hyaluronic acid and chitin/chitosan production using marine waste sources: characteristics, applications and ecofriendly processes: a review. Mar. Drugs. 11: 747-774. https://doi.org/10.3390/md11030747
  2. Schiraldi C, Cimini D, De Rosa M. 2010. Production of chondroitin sulfate and chondroitin. Appl. Microbiol. Biotechnol. 87: 1209-1220. https://doi.org/10.1007/s00253-010-2677-1
  3. Roman E, Roberts I, Lidholt K, Kusche-Gullberg M. 2003. Overexpression of udp-glucose dehydrogenase in Escherichia coli results in decreased biosynthesis of k5 polysaccharide. Biochem. J. 374: 767-772. https://doi.org/10.1042/bj20030365
  4. Kwok JC, Warren P, Fawcett JW. 2012. Chondroitin sulfate: a key molecule in the brain matrix. Int. J. Biochem. Cell Biol. 44: 582-586. https://doi.org/10.1016/j.biocel.2012.01.004
  5. Lauder R. 2009. Chondroitin sulphate: a complex molecule with potential impacts on a wide range of biological systems. Complement. Ther. Med. 17: 56-62. https://doi.org/10.1016/j.ctim.2008.08.004
  6. Mikami T, Kitagawa H. 2013. Biosynthesis and function of chondroitin sulfate. BBA-Gen. Subjuects 1830: 4719-4733. https://doi.org/10.1016/j.bbagen.2013.06.006
  7. He W, Zhu Y, Shirke A, Sun X, Liu J, Gross RA, Koffas MAG, Linhardt RJ, Li M. 2017. Expression of chondroitin-4-O-sulfotransferase in Escherichia coli and Pichia pastoris. Appl. Microbiol. Biotechnol. 101: 6919-6928. https://doi.org/10.1007/s00253-017-8411-5
  8. Badri A, Williams A, Linhardt RJ, Koffas MAG. 2017. The road to animal-free glycosaminoglycan production: current efforts and bottlenecks. Curr. Opin. Biotechnol. 53: 85-92. https://doi.org/10.1016/j.copbio.2017.12.018
  9. Williams A, Linhardt RJ, Koffas MAG. 2018. Metabolic engineering of capsular polysaccharides. Emerg Top Life Sci. 2: 337-348. https://doi.org/10.1042/ETLS20180003
  10. Rodriguez ML, Jann B, Jann K. 2010. Structure and serological characteristics of the capsular k4 antigen of escherichia coli, O5:K4:H4, a fructose-containing polysaccharide with a chondroitin backbone. Eur. J. Biochem. 177: 117-124. https://doi.org/10.1111/j.1432-1033.1988.tb14351.x
  11. Bedini E, De Castro C, De Rosa M, Di Nola A, Iadonisi A, Restaino OF, et al. 2011. A microbiological-chemical strategy to produce chondroitin sulphate A, C. Angew. Chem. Int. Ed. Engl. 50: 6160-6163. https://doi.org/10.1002/anie.201101142
  12. Cimini D, Fantaccione S, Volpe F, De RM, Restaino OF, Aquino G. 2014. IS2-mediated overexpression of Kfoc in E. coli K4 increases chondroitin-like capsular polysaccharide production. Appl. Microbiol. Biotechnol. 98: 3955-3964. https://doi.org/10.1007/s00253-014-5506-0
  13. Zhang Q, Yao R, Chen X, Liu L, Xu S, Chen J. 2018. Enhancing fructosylated chondroitin production in Escherichia coli K4 by balancing the UDP-precursors. Metab. Eng. 47: 314-322. https://doi.org/10.1016/j.ymben.2018.04.006
  14. Wiles TJ, Kulesus RR, Mulvey MA. 2008. Origins and virulence mechanisms of uropathogenic Escherichia coli. Exp. Mol. Pathol. 85: 11-19. https://doi.org/10.1016/j.yexmp.2008.03.007
  15. He W, Fu L, Li G, Andrew JJ, Linhardt RJ, Koffas M. 2015. Production of chondroitin in metabolically engineered E. coli. Metab. Eng. 27: 92-100. https://doi.org/10.1016/j.ymben.2014.11.003
  16. Jin P, Zhang L, Yuan P, Kang Z, Du G, Chen J. 2016. Efficient biosynthesis of polysaccharides chondroitin and heparosan by metabolically engineered Bacillus subtilis. Carbohydr. Polym. 140: 424-432. https://doi.org/10.1016/j.carbpol.2015.12.065
  17. Zhou Z. Li Q, Huang H, Wang H, Wang Y, Du G, Chen J, Kang Z. 2018. A microbial-enzymatic strategy for producing chondroitin sulfate glycosaminoglycans. Biotechnol. Bioeng. 115: 1561-1570. https://doi.org/10.1002/bit.26577
  18. Guo Y, Han M, Yan W, Xu J, Zhang W. 2014. Generation of branched-chain amino acids resistant Corynebacterium glutamicum, acetohydroxy acid synthase by site-directed mutagenesis. Biotechnol. Bioprocess Eng. 19: 456-467. https://doi.org/10.1007/s12257-013-0843-x
  19. Neshat A, Mentz A, Ruckert C, Kalinowski J. 2014. Transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in Corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis. J. Biotechnol. 190: 55-63. https://doi.org/10.1016/j.jbiotec.2014.05.033
  20. Xu JZ, Wu ZH, Gao SJ, Zhang W. 2018. Rational modification of tricarboxylic acid cycle for improving llysine production in Corynebacterium glutamicum. Microb. Cell Fact. 17: 105. https://doi.org/10.1186/s12934-018-0958-z
  21. Z hang Y, Shang X, Lai S, Zhang Y, Hu Q, Chai X, et al. 2018. Reprogramming one-carbon metabolic pathways to decouple l-serine catabolism from cell growth in Corynebacterium glutamicum. Acs Synth. Biol. 101: 1-12.
  22. Cheng F, Gong Q, Yu H, Stephanopoulos G. 2016. High-titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum. Biotechnol. J. 11: 574-584. https://doi.org/10.1002/biot.201500404
  23. Cheng F, Luozhong S, Guo Z, Yu H, Stephanopoulos G. 2017. Enhanced biosynthesis of hyaluronic acid using engineered Corynebacterium glutamicum via metabolic pathway regulation. Biotechnol. J. 12: 1700268. https://doi.org/10.1002/biot.201700268
  24. Sambrook J, Fritsch EF, Maniatis T. 1988. Molecular cloning: A laboratory manual, 2nd edition. Cold Spring Harbor-Laboratory Press, Cold Spring Harbor, NY.
  25. Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A. 1994 Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145: 69-73. https://doi.org/10.1016/0378-1119(94)90324-7
  26. Bitter T, Muir HM. 1962. A modified uronic acid carbazole reaction. Anal. Biochem. 4: 330-334. https://doi.org/10.1016/0003-2697(62)90095-7
  27. Crameri A, Whitehorn EA, Tate E, Stemmer WP. 1996. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat. Biotechnol. 14: 315-319. https://doi.org/10.1038/nbt0396-315
  28. Shang X, Chai X, Lu X, Li Y, Zhang Y, Wang, G, et al. 2018. Native promoters of Corynebacterium glutamicum and its application in L-lysine production. Biotechnol. Lett. 40: 383-391. https://doi.org/10.1007/s10529-017-2479-y
  29. Liu Q, Ouyang S, Kim J, Chen G. 2007. The impact of PHB accumulation on L-glutamate production by recombinant Corynebacterium glutamicum. J. Biotechnol. 123: 273-279. https://doi.org/10.1016/j.jbiotec.2005.11.014
  30. Cimini D, Carlino E, Giovane A, Argenzio O, Dello Iacono I, De Rosa M, et al. 2016. Engineering a branch of the UDPprecursor biosynthesis pathway enhances the production of capsular polysaccharide in Escherichia coli O5:K4:H4. Biotechnol. J. 33: 1307-1315.
  31. Patek M, Holatko J, Busche T, Kalinowski J, Nesvera J. 2013. Corynebacterium glutamicum promoters: a practical approach. Microb. Biotechnol. 6: 103-117. https://doi.org/10.1111/1751-7915.12019
  32. Kaur M., Jayaraman G. 2016. Hyaluronan production and molecular weight is enhanced in pathway-engineered strains of lactate dehydrogenase-deficient Lactococcus lactis. Metab. Eng. Commun. 3: 15-23. https://doi.org/10.1016/j.meteno.2016.01.003
  33. Widner B, Behr R, Von Dollen S, Tang M, Heu T, Sloma A, et al. 2005. Hyaluronic acid p roduction in Bacillus subtilis. Appl. Environ. Microbiol. 71: 3747-3752. https://doi.org/10.1128/AEM.71.7.3747-3752.2005
  34. Westbrook AW, Ren X, Oh J, Mooyoung M, Chou CP. 2018. Metabolic engineering to enhance heterologous production of hyaluronic acid in Bacillus subtilis. Metab. Eng. 47: 401-413. https://doi.org/10.1016/j.ymben.2018.04.016
  35. Yang J, Cheng F, Yu H, Wang J, Guo Z, Stephanopoulos G. 2017. Key role of the carboxyl terminus of hyaluronan synthase in processive synthesis and size control of hyaluronic acid polymers. Biomacromolecules 18: 1064-1073. https://doi.org/10.1021/acs.biomac.6b01239

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