Applied Microbiology and Biotechnology

, Volume 103, Issue 17, pp 6989–7001 | Cite as

Enhancement of acetyl-CoA by acetate co-utilization in recombinant Lactococcus lactis cultures enables the production of high molecular weight hyaluronic acid

  • Kirubhakaran Puvendran
  • Guhan JayaramanEmail author
Biotechnological products and process engineering


The molecular weight of hyaluronic acid (HA) is a critical property which determines its usage in various biomedical applications. This study investigates the correlation between the availability of a critical cofactor, acetyl-CoA, the concentration of a limiting precursor, UDP-N-acetylglucosamine (UDP-GlcNAc), and the molecular weight of HA (MWHA) produced by recombinant Lactococcus lactis MKG6 cultures. This strain expressed three heterologous HA-pathway genes obtained from the has operon of Streptococcus zooepidemicus in an ldh-mutant host strain, L. lactis NZ9020. A flux balance analysis, performed using the L. lactis genome-scale metabolic network, showed a positive correlation of acetyl-CoA flux with the UDP-GlcNAc flux and the experimental data on HA productivity. To increase the intracellular levels of acetyl-CoA, acetate was supplemented as a pulse feed in anaerobic batch cultures. However, acetate is effectively utilized only in the presence of glucose and exhaustion of glucose resulted in decreasing the final MWHA (1.5 MDa). Co-supplementation of acetate resulted in enhancing the acetyl-CoA and UDP-GlcNAc levels as well as the MWHA to 2.5 MDa. This logic was extended to fed-batch cultures, designed with a pH-based feedback control of glucose feeding and pulse acetate supplementation. When the glucose feed concentration was optimally adjusted to prevent glucose exhaustion or accumulation, the acetate utilization was found to be high, resulting in significantly enhanced levels of acetyl-CoA and UDP-GlcNAc as well as a MWHA of 3.4 MDa, which was sustained at this value throughout the process. This study provides the possibility of commercially producing high MWHA using recombinant L. lactis strains.


Acetyl-CoA Fed-batch process Hyaluronic acid Molecular weight Lactococcus lactis 



The authors would like to acknowledge the support provided by the Ministry of Human Resources Development (Govt. of India), for fellowship to Kirubhakaran Puvendran. We would also like to acknowledge the Department of Chemistry, IIT Madras, for helping us with NMR spectroscopy and thermo-gravimetric analysis.


This work was financially supported by the Department of Biotechnology (Ministry of Science and Technology, Govt. of India) through Project No. BT/PR13815/BBE/117/61/2015.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_9987_MOESM1_ESM.pdf (766 kb)
ESM 1 (PDF 766 kb)


  1. Avidan O, Brandis A, Rogachev I, Pick U (2015) Enhanced acetyl-CoA production is associated with increased triglyceride accumulation in the green alga Chlorella desiccata. J Exp Bot 66:3725–3735. CrossRefGoogle Scholar
  2. Badle SS, Jayaraman G, Ramachandran KB (2014) Ratio of intracellular precursors concentration and their flux influences hyaluronic acid molecular weight in Streptococcus zooepidemicus and recombinant Lactococcus lactis. Bioresour Technol 163:222–227. CrossRefGoogle Scholar
  3. Badri A, Raman K, Jayaraman G, (2019) Uncovering Novel Pathways for Enhancing Hyaluronan Synthesis in Recombinant Lactococcus lactis: Genome-ScaleMetabolic Modeling and Experimental Validation. Processes 7 (6):343.
  4. Boynton ZL, Bennett GN, Rudolph FB (1994) Intracellular concentrations of coenzyme A and its derivatives from Clostridium acetobutylicum ATCC 824 and their roles in enzyme regulation. Appl Environ Microbiol 60:39–44. Google Scholar
  5. Chauhan AS, Badle SS, Ramachandran KB, Jayaraman G (2014) The P170 expression system enhances hyaluronan molecular weight and production in metabolically-engineered Lactococcus lactis. Biochem Eng J 90:73–78. CrossRefGoogle Scholar
  6. Chen Y, Daviet L, Schalk M, Siewers V, Nielsen J (2013) Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism. Metab Eng 15:48–54CrossRefGoogle Scholar
  7. Chen WY, Marcellin E, Steen JA, Nielsen LK (2014) The role of hyaluronic acid precursor concentrations in molecular weight control in Streptococcus zooepidemicus. Mol Biotechnol 56:147–156. CrossRefGoogle Scholar
  8. Cheng F, Gong Q, Yu H, Stephanopoulos G (2016) High-titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum. Biotechnol J 11:574–584. CrossRefGoogle Scholar
  9. Chong BF, Nielsen LK (2003) Aerobic cultivation of Streptococcus zooepidemicus and the role of NADH oxidase. Biochem Eng J 16:153–162. CrossRefGoogle Scholar
  10. Flahaut NAL, Wiersma A, Van De BB, Martens DE, Schaap PJ, Sijtsma L, Santos DVAM, De Vos WM (2013) Genome-scale metabolic model for Lactococcus lactis MG1363 and its application to the analysis of flavor formation. Appl Microbiol Biotechnol 97:8729–8739. CrossRefGoogle Scholar
  11. Gao H, Du G, Chen J (2006) Analysis of metabolic fluxes for hyaluronic acid (HA) production by Streptococcus zooepidemicus. World J Microbiol Biotechnol 22:399–408. CrossRefGoogle Scholar
  12. Hmar RV, Prasad SB, Jayaraman G, Ramachandran KB (2014) Chromosomal integration of hyaluronic acid synthesis (has) genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis. Biotechnol J 9:1554–1564. CrossRefGoogle Scholar
  13. Hols P, Ramos A, Hugenholtz J, Delcour J, De Vos WM, Santos H, Kleerebezem M (1999) Acetate utilization in Lactococcus lactis deficient in lactate dehydrogenase: a rescue pathway for maintaining redox balance. J Bacteriol 181:5521–5526Google Scholar
  14. Jagannath S, Ramachandran KB (2010) Influence of competing metabolic processes on the molecular weight of hyaluronic acid synthesized by Streptococcus zooepidemicus. Biochem Eng J 48:148–158. CrossRefGoogle Scholar
  15. Jeeva P, Shanmuga Doss S, Sundaram V, Jayaraman G (2019) Production of controlled molecular weight hyaluronic acid by glucostat strategy using recombinant Lactococcus lactis cultures. Appl Microbiol Biotechnol 103:4363–4375.
  16. Jeong E, Shim WY, Kim JH (2014) Metabolic engineering of Pichia pastoris for production of hyaluronic acid with high molecular weight. J Biotechnol 185:28–36. CrossRefGoogle Scholar
  17. Jin P, Kang Z, Yuan P, Du G, Chen J (2016) Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168. Metab Eng 35:21–30. CrossRefGoogle Scholar
  18. Kang DY, Kim W-S, Heo IS, Park YH, Lee S (2010) Extraction of hyaluronic acid (HA) from rooster comb and characterization using flow field-flow fractionation (FIFFF) coupled with multiangle light scattering (MALS). J Sep Sci 33:3530–3536. CrossRefGoogle Scholar
  19. 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. CrossRefGoogle Scholar
  20. Kogan G, Solte’s L, Stern R, Gemeiner P (2006) Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. Biotechnol Lett 29:17–25. CrossRefGoogle Scholar
  21. Lin H, Castro NM, Bennett GN, San KY (2006) Acetyl-CoA synthetase overexpression in Escherichia coli demonstrates more efficient acetate assimilation and lower acetate accumulation: a potential tool in metabolic engineering. Appl Microbiol Biotechnol 71:870–874. CrossRefGoogle Scholar
  22. Liu L, Liu Y, Li J, Du G, Chen J (2011) Microbial production of hyaluronic acid: current state, challenges, and perspectives. Microb Cell Fact 10:99–108. CrossRefGoogle Scholar
  23. Mao Z, Chen RR (2007) Recombinant synthesis of hyaluronan by Agrobacterium sp. Biotechnol Prog 23:1038–1042. Google Scholar
  24. Mierau I, Kleerebezem M (2005) 10 Years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol 68:705–717. CrossRefGoogle Scholar
  25. Nakajima K, Kitazume S, Angata T, Fujinawa R, Ohtsubo K, Miyoshi E, Taniguchi N (2010) Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC. Glycobiology 20:865–871. CrossRefGoogle Scholar
  26. Novak K, Flöckner L, Erian AM, Freitag P, Herwig C, Pflügl S (2018) Characterizing the effect of expression of an acetyl-CoA synthetase insensitive to acetylation on co-utilization of glucose and acetate in batch and continuous cultures of E. coli W. Microb Cell Fact 17:1–15. CrossRefGoogle Scholar
  27. Oueslati N, Leblanc P, Harscoat-Schiavo C, Rondags E, Meunier S, Kapel R, Marc I (2014) CTAB turbidimetric method for assaying hyaluronic acid in complex environments and under cross-linked form. Carbohydr Polym 112:102–108. CrossRefGoogle Scholar
  28. Papagianni M, Avramidis N, Filiousis G (2007) Glycolysis and the regulation of glucose transport in Lactococcus lactis spp. lactis in batch and fed-batch culture. Microb Cell Fact 6:1–13. CrossRefGoogle Scholar
  29. Prasad SB, Ramachandran KB, Jayaraman G (2012) Transcription analysis of hyaluronan biosynthesis genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactis. Appl Microbiol Biotechnol 94:1593–1607. CrossRefGoogle Scholar
  30. Puvendran K, Anupama K, Jayaraman G (2018) Real-time monitoring of hyaluronic acid fermentation by in situ transflectance spectroscopy. Appl Microbiol Biotechnol 102:2659–2669. CrossRefGoogle Scholar
  31. Rajendran V, Puvendran K, Guru BR, Jayaraman G (2016) Design of aqueous two-phase systems for purification of hyaluronic acid produced by metabolically engineered Lactococcus lactis. J Sep Sci 39:655–662. CrossRefGoogle Scholar
  32. Ren LJ, Huang H, Xiao AH, Lian M, Jin LJ, Ji XJ (2009) Enhanced docosahexaenoic acid production by reinforcing acetyl-CoA and NADPH supply in Schizochytrium sp. HX-308. Bioprocess Biosyst Eng 32:837–843. CrossRefGoogle Scholar
  33. Schellenberger J, Que R, Fleming RMT, Thiele I, Orth JD, Feist AM, Zielinski DC, Bordbar A, Lewis NE, Rahmanian S, Kang J, Hyduke DR, Palsson BØ (2011) Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0. Nat Protoc 6:1290–1307. CrossRefGoogle Scholar
  34. Shanmuga Doss S, Pravinbhai N, Jayaraman G (2017) Improving the accuracy of hyaluronic acid molecular weight estimation by conventional size exclusion chromatography. J Chromatogr B 1060:255–261. CrossRefGoogle Scholar
  35. Song AAL, In LLA, Lim SHE, Rahim RA (2017) A review on Lactococcus lactis: from food to factory. Microb Cell Fact 16:1–15. CrossRefGoogle Scholar
  36. Sze JH, Brownlie JC, Love CA (2016) Biotechnological production of hyaluronic acid: a mini review. 3 Biotech.
  37. Vadali RV, Bennett GN, San K-Y (2004a) Cofactor engineering of intracellular CoA/acetyl-CoA and its effect on metabolic flux redistribution in Escherichia coli. Metab Eng 6:133–139. CrossRefGoogle Scholar
  38. Vadali RV, Horton CE, Rudolph FB, Bennett GN, San KY (2004b) Production of isoamyl acetate in ackA-pta and/or ldh mutants of Escherichia coli with overexpression of yeast ATF2. Appl Microbiol Biotechnol 63:698–704. CrossRefGoogle Scholar
  39. Yu H, Stephanopoulos G (2008) Metabolic engineering of Escherichia coli for biosynthesis of hyaluronic acid. Metab Eng 10:24–32. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Biotechnology, Bhupat and Jyoti Mehta School of BiosciencesIndian Institute of Technology MadrasChennaiIndia
  2. 2.Bioprocess and Metabolic Engineering Laboratory, Department of Biotechnology, Bhupat and Jyoti Mehta School of BiosciencesIndian Institute of Technology MadrasChennaiIndia

Personalised recommendations