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]
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]
| 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.
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]
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]
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]
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]
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]
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]