Hyaluronic acid (HA) or hyaluronan is a linear repeat of glucuronic acid and N-acetylglucosamine found in various animal parts, i.e., rooster combs, vitreous humour in eyes, umbilical cords, skin, and cartilage . HAs are high molecular mass molecules, usually more than a million Daltons, with viscoelastic properties that can maintain elasticity and moisture, reduce inflammation, and lubricate the movement of various body parts. It has been used for various biomedical applications; for example, as a diagnostic marker for cancer, rheumatoid arthritis and liver pathologies, reducing inflammation of the wound, and in drug delivery [2, 3]. HAs are also applied in certain ophthalmological and ontological surgeries, cosmetic regeneration, and soft tissue reconstruction as biocompatible and non-immunogenic materials . Moreover, low molecular mass HAs are involved in wound healing, angiogenesis, cell differentiation, tumour cell migration and apoptosis [5, 6].
Some bacteria, including Streptococcus sp. and Pasteurella multocida, produce HAs as part of their capsule and slime . P. multocida is a gram-negative bacterium that causes various diseases in livestock, including avian cholera, respiratory diseases, septicaemia, and atrophic rhinitis . This bacterium produces a capsule consisting of different polysaccharide compositions according to their capsular serotypes, including hyaluronic acid in serotype A, heparin in serotype D, and chondroitin sulfate in serotype F [8–10]. The capsule of type B contains arabinose, mannose, and galactose, while the content of capsular type E remains unclear . P. multocida serotype A produces a capsule containing HA similar to Streptococcus sp., algae, viruses and vertebrates [12, 13]. HA biosynthesis in these organisms involves nine genes, which encode glucokinase (glck), glucose-6-phosphate isomerase (pgi), phosphoglucomutase (pgm), UTP-glucose-1-phosphate uridyltransferase (galU), UDP-glucose 6-dehydrogenase (hyaC), L-glutamine:D-fructose-6-phosphate aminotransferase (glmS), phosphoglucosamine mutase (glmM), bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase (glmU), and hyaluronan synthase (hyaD) [12, 14].
HA is usually synthesized by two distinct pathways that synthesize HA precursors; these pathways begin with the phosphorylation of glucose by glucokinase to produce two precursors (UDP-glucuronic acid and N-acetylglucosamine). In the first reaction, phosphoglucomutase (Pgm) converts glucose-6-phosphate to glucose-1-phosphate, after which the phosphate group from UTP is transferred to glucose-1-phosphate via UTP-glucose-1-phosphate uridyltransferase (GalU) to produce UDP-glucose. UDP-glucose is oxidized by UDP-glucose 6-dehydrogenase (HyaC), deriving the first HA precursor, UDP-glucuronic acid. In the second pathway, glucose-6-phosphate is converted to fructose-6-phosphate by glucose-6-phosphate isomerase (Pgi) and then changed to fructose-6-phosphate by adding an amino group from a glutamine residue via L-glutamine:D-fructose-6-phosphate aminotransferase (GlmS) to produce glucosamine-6-phosphate, which is later modified by phosphoglucosamine mutase (GlmM) to yield glucosamine-1-phosphate. Glucosamine-1-phosphate is acetylated and phosphorylated by bifunctional N-acetylglucosamine-1-phosphate uridyltransferase/glucosamine-1-phosphate acetyltransferase (GlmU) to the second precursor, UDP-N-acetylglucosamine. Finally, hyaluronan synthase (HyaD) combines and polymerizes the two precursors, generating the HA polymer [15, 16]. Recent reviews by Peng et al. (2019) showed that the capsule biosynthesis genes in cap loci varied among different capsular types, particularly between types A, D, and F and type B. Comparative analysis of the HA biosynthetic genes from various organisms showed that the hyaluronan synthase from P. multocida was the only class II enzyme, while other organisms produced the class I hyaluronan synthase . Class II hyaluronan synthases are different from class I hyaluronan synthases in terms of structural topology and biosynthesis mechanism [18, 19]. In this step, the HA synthesized by class II hyaluronan synthases is connected to the cytoplasmic membrane and transported through the membrane directly to the extracellular matrix during chain elongation and not sulfated or chemically modified after biosynthesis [17, 20, 21].
Previous research improved HA production by genetical modification of the has operon in Streptococcus sp. However, production has a risk of pathogenic bacterial contamination . HA synthesis has been studied in many other microorganisms (Bacillus sp., Lactococcus sp., and Escherichia. coli) for the production of high-quality and safe-to-use HAs [16, 19]. Industrial-scale HA production was developed with recombinant E. coli and Bacillus sp. strains harbouring the hyaD genes from Streptococcus sp. and P. multocida [23, 24]. The recombinant B. subtilis strain produced HasA from Streptococcus equisimilis together with the co-expression of TuaD from B. subtilis. This recombinant bacterium produced a high level of HAs and had been used industrially . Another study optimized conditions for HA production in recombinant E. coli based on the expression of the hyaD gene of P. multocida ATCC 15742 (capsular type A:3) and the hyaC gene of E. coli strain K5 . The level of HA production in the recombinant bacterium increased (2.7 to 3.7 g/L (37%)) after varying the amount of supplemented oxygen and glucosamine. The synthesis also increased by up to 70% when fosfomycin was added to inhibit cell wall synthesis . Co-expression of the hyaC and hyaD genes from P. multocida gave the highest level of HAs (5.4 g/L) and stability compared to the co-expression of the hyaC gene from E. coli and hyaD from S. pyogenes . Although variations in the capsule biosynthesis genes of P. multocida have been studied, HA biosynthetic genes have not been explored and could be useful for further improving HA production. Therefore, this study aimed to compare the HA biosynthesis genes in P. multocida using sequence, pattern and structural analyses. The HA genes were compared across different capsular types of P. multocida and to their orthologues in other bacteria and humans. We proposed that variations observed within these genes could be useful targets for genetic engineering-based improvement of HA production.