Date Published: February 15, 2016
Publisher: Springer Berlin Heidelberg
Author(s): Jun Hui Sze, Jeremy C. Brownlie, Christopher A. Love.
Hyaluronic acid (HA) is a polysaccharide found in the extracellular matrix of vertebrate epithelial, neural and connective tissues. Due to the high moisture retention, biocompatibility and viscoelasticity properties of this polymer, HA has become an important component of major pharmaceutical, biomedical and cosmetic products with high commercial value worldwide. Currently, large scale production of HA involves extraction from animal tissues as well as the use of bacterial expression systems in Streptococci. However, due to concerns over safety, alternative sources of HA have been pursued which include the use of endotoxin-free microorganisms such as Bacilli and Escherichia coli. In this review, we explore current knowledge of biosynthetic enzymes that produce HA, how these systems have been used commercially to produce HA and how the challenges of producing HA cheaply and safely are being addressed.
Hyaluronic acid (HA) is a linear glycosaminoglycan polymer commonly found in the extracellular matrix of vertebrate epithelial, neural and connective tissues. It is also involved in many signalling pathways including those involved in embryonic development, wound healing, inflammation and cancer (Stern et al. 2006). HA is also a component of the extracellular capsule formed by some microorganisms, such as Streptococcus, that serves not only for adherence and protection, but also as a molecular mimicry to evade host’s immune system during its infection process (Wessels et al. 1991).
Hyaluronic acid (HA), also known as hyaluronan, is a linear glycosaminoglycan (GAG) composed of repeating disaccharides of β4-glucuronic acid (GlcUA)-β3-N-acetylglucosamine (GlcNAc) (Fig. 1). HA was first isolated and identified from the vitreous body of bovine’s eye (Meyer and Palmer 1934). Eventually this polysaccharide was found to be ubiquitously distributed in many parts of vertebrate tissues (brain, umbilical cord, synovial fluid in between joints, skin, rooster comb, neural tissues and epithelium) with differing concentrations and molecular weights (Fraser et al. 1997).Fig. 1Structure of a hyaluronic acid monomer. HA consists of glucuronic acid and N-acetylglucosamine that can be repeated up to 10,000 times or more (Cowman and Matsuoka 2005)
In general, for any bacterium to synthesise HA capsule, the HAS gene must be present as it is needed to polymerize UDP-sugar precursors into HA. The hasA was first isolated from S. pyogenes from Group A Streptococcus (GAS) and it was shown to have the capability to direct HA capsule biosynthesis in acapsular mutants as well as heterologous bacteria such as E. coli and Enterococcus faecalis (DeAngelis et al. 1993). Since then, other HA synthase (HAS) related genes have been identified in other organisms including Group C Streptococcus, algae, viruses and vertebrates. Analysis of the primary sequence and predicted structural topologies have shown HAS enzymes share many common features and have been divided into two categories, (based on similarities and differences in amino acid motifs, topology and mode of action) designated as Class I and Class II HAS (Table 1).Table 1Characteristics and features of Class I and Class II HA synthasesFeatureClass IClass IISource organismsStreptococci, amphibian, mammal, yeast, virusPasteurella multocidaAbility to be expressed as soluble active proteinNo (must be associated with cell membrane)Yes (amino acid residues 1–703)Number of GT2 module12HA chain growthAt reducing end—Streptococcus, humans and miceAt non-reducing end—Xenopus laevis and algal virusAt non-reducing endTopology of the proteinMultiple membrane associated domains throughout the whole proteinTwo catalytically independent domains, A1 and A2 attached to membrane via C-terminal regionIntrinsic polymerizationProcessiveNon-processiveSize (amino acids)417–588972Primer for initiation of HA synthesis?NoYesInvolvement of other molecules/proteins during HA translocationYes—involves lipid molecules (membrane bilayer)Yes—may involve capsular polysaccharide transport machinery (more studies needed to confirm)
HA’s essential functions in the human eye, synovial fluid of joints and in the epidermal layers, has led to considerable interest in developing new methods to successfully synthesise and deliver HA. A recent market analysis report predicted that as a consequence of an aging population and an increase in osteoarthritis the global market for HA visco-supplementation in humans alone was estimated to be more than $2.5 billion by 2017 (MRG.Net 2013).
Currently, industrial production of HA is based on either HA extraction from animal tissues or via large-scale bacterial fermentation with genetically modified strains. These processes are widely used and have been able to generate HA products with molecular weights above 1 MDa (as half-life of the molecule will increase and persist longer while maintaining its physiological function), which is desirable for biomedical and cosmetic use (Liu et al. 2011).
Given the widespread cosmetic and medical applications of HA, commercial interest in the rapid and safe production of HA remains strong. Cell-free and/or non-pathogenic bacterial expression systems are considered to be safer alternatives to existing production systems, though as discussed previously, are limited in their capacity to produce large amounts of HA. While genes encoding for Class I or Class II HAS enzymes cloned from diverse bacteria have successfully synthesised HA in novel non-pathogenic host bacteria such as E. coli, these enzymes have only ever been expressed independently (Mao et al. 2009; Yu and Stephanopoulos 2008). As both Class I and II HAS enzymes extend the growing HA polymer in unique ways, in theory these could act synergistically to produce HA. Such experiments should be possible in E. coli or other tractable bacterial expression systems.