Engineering:Microbial hyaluronic acid production

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Template:Cs1 config Microbial hyaluronic acid production refers to the process by which microorganisms, such as bacteria and yeast, are utilized in fermentation to synthesize hyaluronic acid (HA).[1] HA is used in a wide range of medical, cosmetic, and biological products because of its high moisture retention and viscoelasticity qualities.[2] HA had originally been extracted from rooster combs in limited quantities.[3] However, challenges such as low yields, high production costs, and ethical issues associated with animal-derived HA has driven the development of microbial production methods for HA.[4]

Although there are other methods for instance chemical synthesis and modification, chemoenzymatic synthesis, enzymatic synthesis; microbial fermentation has been preferred to produce HA because of economical advantages.[5]

Bacterial production

Some bacteria, such as Streptococcus, develop an extracellular capsule that contains HA. This capsule functions as a molecular mimic to elude the host's immune system during the infection process in addition to providing adherence and protection.[6] Streptococcus zooepidemicus was used for first commercially HA fermentation, and that is most used bacteria since provides high yields although it is a pathogen microorganism.[7]

Encoding of HA production is carried out by hasA, hasB, hasC, hasD and hasE genes in S. zooepidemicus.[8]

Genes and their functions HA production in S. zooepidemicus
Gene Enzyme Function Reference
hasA Hyaluronic acid synthase HA synthesis and

transport

[9]
hasB UDP-glucose dehydrogenase UDP-GlcA

biosynthesis

[10][11]
hasC UDP-glucose pyrophosphorylase UDP-GlcA

biosynthesis

[12]
hasD Acetyltransferase and

pyrophosphorylase (bifunctional)

UDP-GlcNAc

biosynthesis

[13]
hasE Phosphoglucoisomerase UDP-GlcNAc

biosynthesis

[13]

Genetically modified producers were developed such as Kluysveromyces  lactis,[14]  Lactococcus  lactis,[15] Bacillus  subtilis,[16] Escherichia  coli,[17]  and Corynebacterium glutamicum[18][19] because of S. zooepidemicus's pathogeny.

Biological process

Intracellular factors

Metabolism

Intermediates are used from  pathways  essential  to  support cell  growth,  such  as  the  production  of  organic  acids,  polysaccharides during the HA production.[20] HA is not an essential metabolite, and it competes other metabolites to attend the carbon flux in the cell.[4] Reduction potential of S. zooepidemicus may have a role in hyaluronic acid production, because 2 NAD+ are consumed during the synthesis of one monomer. Although NAD+ does not control HA synthesis when NADH oxidase over-expressed,[21] it has a big role in biomass formation.

Some studies showed that balanced intracellular concentration of precursors and their fluxes balanced provides higher molecular weight such as UDP-acetylglucosamine concentration.[22][23] Enzymes such as hyaluronidase,[24] β-glucuronidase[25] of S. zooepidemicus decrease yield of HA. HA concentration is increased by deletion of associated genes of these enzymes.[24][25]

On the other hand, some enzymes induce HA production such as sucrose-6-phosphatate hydrolase,[26] and hyaluronan synthase.[27] Using combined approaches with these two type enzymes is a good strategy for high yield HA production.[20]

Membrane

HA is produced around the cell, serving as a barrier against the host immune system by the bacteria. Only 8% of HA remains as attached the cell when cells arrived stationary phase. Biosurfactants such as sodium dodecyl sulfate (SDS) are used to gain this product.[28] Hyaluronan synthase, that is a membrane-binding enzyme, is one of the factors that reduces the production of HA. Hyaluronan synthase limits hyaluronic acid production by affecting cell morphology.[28]

Environmental factors

pH

Organic acids formed during HA production by S. zooepidemicus cause pH to decrease[20] Although HA production without pH control is cheaper, it prefers since provides high hyaluronic acid yields.[29][30]

Temperature

HA production is affected regarding to yield and molecular weight by temperature.[31] HA production increases while bacterial cells are growing above 37 °C. However, HA yield decreases while molecular weight is higher with fermentation under 32 °C.[30]

Aeration

Although S. zooepidemicus is an aerotolerant anaerobe, hyaluronic acid production is affected by oxygen because NADH/NAD+ balance of cells changes with oxygen amount. Controlling oxygen during the cultivation via agitation rate provides increase both HA yield and molecular weight.[32]

Culture Media Components

The carbon source is one of the media components that has effects on production of microbial HA.[20] Although the glucose[33][34] is most used one as a carbon source for the HA production; molasses,[35] sucrose,[36] and maltose[32] are used for microbial production.

HA production needs also many amino acids in the culture media therefore nitrogen source concentration has a key.[37]

See also

References

  1. ↑ "Microbial Hyaluronic Acid Production: A Review". Molecules 28 (5): 2084. February 2023. doi:10.3390/molecules28052084. PMID 36903332. 
  2. ↑ "Biotechnological production of hyaluronic acid: a mini review". 3 Biotech 6 (1): 67. June 2016. doi:10.1007/s13205-016-0379-9. PMID 28330137. 
  3. ↑ "Microbial production of hyaluronic acid: the case of an emergent technology in the bioeconomy" (in en). Biofuels, Bioproducts and Biorefining 15 (6): 1604–1610. 2021. doi:10.1002/bbb.2285. ISSN 1932-104X. 
  4. ↑ 4.0 4.1 "Microbial hyaluronic acid production". Applied Microbiology and Biotechnology 66 (4): 341–351. January 2005. doi:10.1007/s00253-004-1774-4. PMID 15599518. 
  5. ↑ "Prospective bacterial and fungal sources of hyaluronic acid: A review". Computational and Structural Biotechnology Journal 20: 6214–6236. 2022. doi:10.1016/j.csbj.2022.11.013. PMID 36420162. 
  6. ↑ "Hyaluronic acid capsule is a virulence factor for mucoid group A streptococci". Proceedings of the National Academy of Sciences of the United States of America 88 (19): 8317–8321. October 1991. doi:10.1073/pnas.88.19.8317. PMID 1656437. Bibcode: 1991PNAS...88.8317W. 
  7. ↑ "Comparative Economic Analysis Between Endogenous and Recombinant Production of Hyaluronic Acid". Frontiers in Bioengineering and Biotechnology 9. 2021-07-21. doi:10.3389/fbioe.2021.680278. PMID 34368093. 
  8. ↑ "Genetic and biochemical characterization of genes involved in hyaluronic acid synthesis in Streptococcus zooepidemicus". Applied Microbiology and Biotechnology 100 (8): 3611–3620. April 2016. doi:10.1007/s00253-016-7286-1. PMID 26758299. 
  9. ↑ "Hyaluronic acid synthesis operon (has) expression in group A streptococci". The Journal of Biological Chemistry 270 (31): 18452–18458. August 1995. doi:10.1074/jbc.270.31.18452. PMID 7629171. 
  10. ↑ "Molecular characterization of hasB from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP-glucose dehydrogenase activity". The Journal of Biological Chemistry 268 (10): 7118–7124. April 1993. doi:10.1016/S0021-9258(18)53153-7. PMID 8463246. 
  11. ↑ "A hyaluronan-based polysaccharide peptide generated by a genetically modified Streptococcus zooepidemicus". Carbohydrate Research 478: 25–32. May 2019. doi:10.1016/j.carres.2019.04.005. PMID 31042589. 
  12. ↑ "Molecular characterization of hasC from an operon required for hyaluronic acid synthesis in group A streptococci. Demonstration of UDP-glucose pyrophosphorylase activity". The Journal of Biological Chemistry 270 (48): 28676–28680. December 1995. doi:10.1074/jbc.270.48.28676. PMID 7499387. 
  13. ↑ 13.0 13.1 "Evolution of the hyaluronic acid synthesis (has) operon in Streptococcus zooepidemicus and other pathogenic streptococci". Journal of Molecular Evolution 67 (1): 13–22. July 2008. doi:10.1007/s00239-008-9117-1. PMID 18551332. Bibcode: 2008JMolE..67...13B. 
  14. ↑ "Heterologous Hyaluronic Acid Production in Kluyveromyces lactis". Microorganisms 7 (9): 294. August 2019. doi:10.3390/microorganisms7090294. PMID 31466214. 
  15. ↑ "Production of controlled molecular weight hyaluronic acid by glucostat strategy using recombinant Lactococcus lactis cultures". Applied Microbiology and Biotechnology 103 (11): 4363–4375. June 2019. doi:10.1007/s00253-019-09769-0. PMID 30968163. 
  16. ↑ "Application of hydrocarbon and perfluorocarbon oxygen vectors to enhance heterologous production of hyaluronic acid in engineered Bacillus subtilis". Biotechnology and Bioengineering 115 (5): 1239–1252. May 2018. doi:10.1002/bit.26551. PMID 29384194. 
  17. ↑ "Cloning and expression of hyaluronan synthase (hasA) in recombinant Escherichia coli BL21 and its hyaluronic acid production in shake flask culture" (in en). Malaysian Journal of Microbiology. 2019. doi:10.21161/mjm.190444. ISSN 2231-7538. http://mjm.usm.my/index.php?r=cms/entry/view&id=2423&slug=Cloning-and-expression-of-hyaluronan-synthase-%28-has-A%29-in-recombinant-Escherichia-coli-BL21-and-its-hyaluronic-acid-production-in-shake-flask-culture. 
  18. ↑ "Hyaluronic acid production with Corynebacterium glutamicum: effect of media composition on yield and molecular weight". Journal of Applied Microbiology 117 (3): 663–678. September 2014. doi:10.1111/jam.12553. PMID 24863652. 
  19. ↑ "Preparation, purification, and characterization of low-molecular-weight hyaluronic acid". Biotechnology Letters 43 (1): 133–142. January 2021. doi:10.1007/s10529-020-03035-4. PMID 33131008. 
  20. ↑ 20.0 20.1 20.2 20.3 "Microbial hyaluronic acid production in the 21 century: a roadmap toward high production, tailored molecular weight". Observatório de la Economía Latinoamericana 22 (3): e3913. 2024-03-27. doi:10.55905/oelv22n3-185. ISSN 1696-8352. https://ojs.observatoriolatinoamericano.com/ojs/index.php/olel/article/view/3913. 
  21. ↑ "Amplifying the cellular reduction potential of Streptococcus zooepidemicus". Journal of Biotechnology 100 (1): 33–41. January 2003. doi:10.1016/S0168-1656(02)00239-0. PMID 12413784. 
  22. ↑ "Ratio of intracellular precursors concentration and their flux influences hyaluronic acid molecular weight in Streptococcus zooepidemicus and recombinant Lactococcus lactis". Bioresource Technology 163: 222–227. July 2014. doi:10.1016/j.biortech.2014.04.027. PMID 24814248. Bibcode: 2014BiTec.163..222B. 
  23. ↑ "Influence of competing metabolic processes on the molecular weight of hyaluronic acid synthesized by Streptococcus zooepidemicus". Biochemical Engineering Journal 48 (2): 148–158. 2010. doi:10.1016/j.bej.2009.09.003. ISSN 1369-703X. 
  24. ↑ 24.0 24.1 "Improved Yield of High Molecular Weight Hyaluronic Acid Production in a Stable Strain of Streptococcus zooepidemicus via the Elimination of the Hyaluronidase-Encoding Gene". Molecular Biotechnology 59 (6): 192–199. June 2017. doi:10.1007/s12033-017-0005-z. PMID 28500482. 
  25. ↑ 25.0 25.1 "Increase in hyaluronic acid production by Streptococcus equi subsp. zooepidemicus strain deficient in beta-glucuronidase in laboratory conditions". Applied Microbiology and Biotechnology 71 (4): 415–422. July 2006. doi:10.1007/s00253-005-0173-9. PMID 16292534. 
  26. ↑ "Construction of efficient Streptococcus zooepidemicus strains for hyaluoronic acid production based on identification of key genes involved in sucrose metabolism". AMB Express 6 (1): 121. December 2016. doi:10.1186/s13568-016-0296-7. PMID 27896786. 
  27. ↑ "Enhanced hyluronic acid production in Streptococcus zooepidemicus by over expressing HasA and molecular weight control with Niscin and glucose". Biotechnology Reports 16: 65–70. December 2017. doi:10.1016/j.btre.2017.02.007. PMID 29296591. 
  28. ↑ 28.0 28.1 "Effect of oxygen and shear stress on molecular weight of hyaluronic acid". Journal of Microbiology and Biotechnology 18 (4): 718–724. April 2008. PMID 18467866. 
  29. ↑ "Microbial production of hyaluronic acid from agro-industrial by-products: Molasses and corn steep liquor" (in en). Biochemical Engineering Journal 117: 181–187. 2017. doi:10.1016/j.bej.2016.09.017. Bibcode: 2017BioEJ.117..181A. 
  30. ↑ 30.0 30.1 "Efficient production of high-molecular-weight hyaluronic acid with a two-stage fermentation". RSC Advances 8 (63): 36167–36171. October 2018. doi:10.1039/C8RA07349J. PMID 35558483. Bibcode: 2018RSCAd...836167L. 
  31. ↑ "Regulation of hyaluronic acid molecular weight and titer by temperature in engineered Bacillus subtilis". 3 Biotech 9 (6): 225. June 2019. doi:10.1007/s13205-019-1749-x. PMID 31139540. 
  32. ↑ 32.0 32.1 "Aerobic cultivation of Streptococcus zooepidemicus and the role of NADH oxidase". Biochemical Engineering Journal. Biopolymers 16 (2): 153–162. 2003. doi:10.1016/S1369-703X(03)00031-7. ISSN 1369-703X. Bibcode: 2003BioEJ..16..153F. 
  33. ↑ "Evaluation of magnetic nanoparticles influence on hyaluronic acid production from Streptococcus equi". Carbohydrate Polymers 192: 135–142. July 2018. doi:10.1016/j.carbpol.2018.03.037. PMID 29691005. 
  34. ↑ "Development of In Situ Product Recovery (ISPR) System Using Amberlite IRA67 for Enhanced Biosynthesis of Hyaluronic Acid by Streptococcus zooepidemicus". Life 13 (2): 558. February 2023. doi:10.3390/life13020558. PMID 36836914. Bibcode: 2023Life...13..558A. 
  35. ↑ "Hyaluronic acid production by utilizing agro-industrial waste cane molasses". 3 Biotech 12 (9): 208. September 2022. doi:10.1007/s13205-022-03265-5. PMID 35935546. 
  36. ↑ "Factorial design in fermentation medium development for hyaluronic acid production by Streptococcus zooepidemicus". Acta Scientiarum. Technology 42. 2019. doi:10.4025/actascitechnol.v42i1.42729. ISSN 1807-8664. 
  37. ↑ "Growth and amino acid requirements of hyaluronic-acid-producing Streptococcus zooepidemicus" (in en). Applied Microbiology and Biotechnology 47 (3): 309–312. 1997. doi:10.1007/s002530050932. ISSN 1432-0614. 



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