3. HA Fermentation Process in Engineered Host Microorganisms
As previously mentioned, industrial and scientific research is looking for optimized HA production methods with controlled parameters, high yields, low production costs, a high MW, and a high purity of the final product. HA bacterial production through metabolic engineering represents a promising strategy. Over the last years, several microorganisms have been engineered in order to produce HA. Different fermentation methods and culture media have been tested. Not only natural microbial producers of HA have been engineered in order to optimize the characteristics of the final product, but non-natural producers such as GRAS microorganisms (i.e., E. coli, B. subtilis, and B. megaterium) have been as well.
First of all, several researchers attempted to maximize HA production by tuning the microorganism culture conditions rather than engineering them. Parameters such as the initial pH of the broth, temperature, time of incubation, and agitation speed were evaluated as important parameters that influenced the yield of HA. Among several bacteria,
S. equi spp.
zooepidemicus was the most investigated. Güngör et al. found that the growing conditions that allowed them to improve the HA final yield (12 g/L) were pH 7.8, an incubation temperature of 33 °C, an incubation time of 24 h, and an agitation speed of about 187 rpm
[52]. In another recent study, UV-induced mutagenesis on
S. zooepidemicus allowed the researchers to further increase the HA yield, which reached about 4.2 g/L after 36 h of fermentation
[53]. In this case, the optimal fermentation was obtained by incubating it at 37 °C and pH 7.4 in a fermentation broth that contained glucose as the carbon source, phosphate as the essential nutrient, peptone 250 as the nitrogen source, and dibasic potassium phosphate as the phosphate source
[53]. Then, to enhance the efficiency of the hyaluronic acid production, a semi-continuous fermentation process that consisted of two-stage 3–L bioreactors was developed, and recombinant hyaluronidase
SzHYal (300 U/L) was added into the second stage bioreactor to reduce the broth viscosity. In this way, an HA yield of 30 g/L was successfully attained
[53]. Another strategy showed that as an alternative to glucose, molasses could be used as a carbon source in the fermentation medium of
S. zooepidemicus [54]. Indeed, the presence of this alternative and cheaper sugar allowed an increase in the yield of the HA up to 0.2 g/L compared to standard conditions (43 mg/L)
[54]. In addition to the pH, the incubation temperature, agitation speed, soil composition, and size of inoculum are key parameters of the fermentation process that affect the yield of the final product. Some researchers found that a streptococcal inoculum size of 10% had the greatest effect on the fermentation process and also that an agitation speed of 300 rpm could increase the HA yield
[55]. This may have occurred because at this rate of speed, a greater subdivision of air bubbles could occur with a consequent larger surface area for the gas–liquid mass transfer, which therefore led to a reduction in the thickness of the gas and liquid films responsible for the resistance to the mass transport
[55]. In this way, it is easy to understand that several parameters must be optimized in order to enhance the microbial production of HA starting from natural producer microorganisms.
With the aim of reducing extracted product endotoxins, recombinant non-natural microbial producers were designed, developed, and investigated in depth
[40]. A variety of organisms including
B. subtilis,
B. megaterium, and
E. coli have all been genetically engineered in order to produce HA
[56][57][58]. Genetically modified
E. coli strains have several potential advantages when compared to
Streptococcus strains and therefore offer a promising alternative
[40]. Additionally, compared to
Streptococcus strains, several
E. coli strains are non-pathogenic
[43], and they can be grown at a high cell density using cheap and simple media. As a result, the fermentative process of
E. coli could be improved through metabolic engineering
[59]. For these reasons, many studies have been carried out with recombinant
E. coli. In particular, a recent study compared the HA production in different
E. coli strains (pLysY/lq, Rosetta2, Rosetta2 (DE3) pLysS, and Rosetta-gami B (DE3) pLysS) and
B. megaterium (MS941) cells
[57]. Firstly, the researchers used different fermentation conditions for
E. coli cells. Initially, they engineered the bacterial cells by transforming them with different plasmids. The first introduction of the
hasA gene of
S. equi spp.
zooepidemicus in
E. coli Rosetta2 cells allowed them to obtain a yield of 8.8 mg/L
[57]. Then, the introduction of a second set of genes, which were
hasA,
hasB and
hasC genes in
E. coli pLysY/lq cells, allowed them to reach a yield of 208.3 mg/L (by adding MgCl
2, K
2HPO
4, and sorbitol to the fermentation broth)
[57]. Nasser et al. also attempted to obtain HA from Rosetta2 (DE3) pLysS, with a successful yields of 346.7 mg/L in Super Optimal broth with Catabolite repression (SOC) medium and 500 mg/L in terrific broth (TB) medium
[57]. Lastly, they analyzed the ability of Rosetta-gami B (DE3) pLysS to produce HA by transforming it with
hasA (hyaluronic acid synthase gene),
hasB (UDP-glucose dehydrogenase gene),
hasC (UDP-glucose pyrophosphorylase gene),
hasD (acetyltransferase gene),
hasE (phosphoglucoisomerase gene) genes; in this way, they obtained 585 mg/L of HA
[57].
In this case, it is also possible to note that the composition of the fermentation medium and the bacterial strain affect the HA yield. In a study by Nasser et al., it emerged that
B. megaterium was able to produce a higher amount of HA rather than
E. coli. Indeed, by expressing only the
hasA gene along with the addition of xylose, MgCl
2, K
2HPO
4 and sorbitol to Luria-Bertani (LB) fermentation broth, they were able to obtain 50 mg/L of HA. By expressing
hasA,
hasB, and
hasC with the addition of sucrose to LB fermentation broth, the yield of HA was about 2116.7 mg/L. In TB broth instead, the yield was decreased (2016 mg/L). At this point, they modified the fermentation medium, and they observed that in A5 + sodium 2-hydroxy-3-(morpholin-4-yl)propane-1-sulfonate (MOPSO) medium (containing yeast extract and a mineral medium based on MOPSO buffer), the amount of HA was about 1990 mg/L. As a final step, they introduced
hasA,
hasB,
hasC,
hasD, and
hasE genes in
B. megaterium cells. In LB medium with sucrose, the HA yield was about 2350 mg/L; in A5+MOPSO medium, the HA amount was about 2436 mg/L
[57].
Another study used
E. coli Rosetta (DE3) as the host to obtain the production of HA by optimizing the fermentation medium and inserting the
hasA and
hasE genes
[57]. In this case, the fermentation broth contained glucose as the carbon source, MgSO
4 as the Mg
2+ donor, and KH
2PO
4 and K
2HPO
4 as the phosphate sources
[57]. This study further demonstrated the effect of fermentation temperature on HA yield, since the maximum HA biosynthesis was observed when fermentation was run at less than 37 °C. Finally, it was observed that the gene expression inductor concentration also could affect the HA yield. The highest HA concentration was obtained when the cell growth was at the lowest; that is, when the concentration of the gene expression inductor was the highest. Indeed, in these conditions, the cellular growth decreased and the HA concentration increased
[57].
As previously mentioned, bacilli also can be used for HA production since they are safe, can be easily genetically tractable, and are able to grow in simple culture media
[60]. In particular, in a recent study
B. subtilis 3NA was engineered by overexpressing the
hasA gene of
S. zooepidemicus and its other endogenous genes involved in the HA biosynthetic pathway
[58]. The fermentation medium contained glycerol as the carbon and energy source, H
3PO
4 as the phosphate source, NH
4OH as the nitrogen source, MgSO
4 as the Mg
2+ donor and sulfur source, and finally KOH to buffer the pH
[58]. Hence, the obtained HA yield was comparable with the streptococcal one
[58].
Thus, all mentioned works allowed us to understand that the strain, exogenous genes, and fermentation conditions (i.e., soil composition, temperature, pH, concentration of the gene expression inductor, and agitation speed) are fundamental parameters that affect HA yield. The optimization of these parameters is fundamental for obtaining the highest HA amount from bacterial fermentation.
4. HA as a Powerful Bioactive Molecule
HA has been used for both medical and commercial applications thanks to its biocompatibility, biodegradability, and non-immunogenicity. Therefore, several HA-based products have been developed and are currently available on the market. HA showed its major potential for aesthetic applications but also was revealed to be promising in other biomedical fields. This is due to the fact that HA was revealed to be a versatile biomaterial with tunable properties that allowed the development of different kinds of devices (i.e., injectable formulations, gels, nanoparticles, sponges, and hydrogels) according to the injured tissue’s specific requirements. The main applications of HA as a powerful bioactive molecule are summarized in Figure 3.
Figure 3. HA: a powerful bioactive molecule. Schematical representation of the most important HA applications in the health-related sector.