Biotechnological production of hyaluronic acid: a mini review

Abstract

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.

Keywords: HA synthase; Hyaluronic acid; Microbial production; Streptococcus.

Fig. 1

Structure 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)

Fig. 2

Hyaluronic acid biosynthetic pathway in S. zooepidemicus. Glucose is first converted by hexokinase to form glucose-6-Phosphate which then enters one of the two distinct pathways to form UDP-glucuronic acid (pgm, hasC and hasB) or UDP-N-acetylglucosamine (hasE, glmS, glmM and hasD). These precursors are then bound together via the action of hyaluronic acid synthase or HAS (encoded by hasA in S. zooepidemicus) to form hyaluronic acid

Fig. 3

Schematic representation of HA biosynthesis in Class I and Class II Hyaluronic Acid Synthases (HAS). a Class I HAS are integral membrane proteins that catalyse the UDP-sugar addition to growing HA chain and may transport the hydrophilic HA polymer across the cell membrane of eukaryotes or Gram-positive bacteria. Lipid molecules (yellow circles) facilitate the HAS activity which allows it to direct HA translocation. b Class II HAS is a peripheral protein that also catalyses HA elongation. This enzyme is a hybrid of two glycosyltransferases that transfers GlcNAc-UDP and GlcUA-UDP at the non-reducing end of the HA chain. It was suggested that this protein may interact with other cell membrane proteins (capsular polysaccharide transport protein–purple block) in order to translocate HA across the cell membrane of Gram-negative bacteria (P. multocida). Blue and red dots represent GlcUA and GlcNAc, respectively. Orange triangle represents the UDP component of the sugar

Fig. 4

Position of non-reducing and reducing ends of a growing HA chain. Polymerisation of HA by Class I HAS occurs whereby the UDP group present at the reducing end of the HA polysaccharide is released to form a glycosidic bond between the growing chain and the new sugar-UDP (in this example GlcUA-UDP would be added, followed by GlcNAc-UDP). Class II HAS adds new sugar-UDPs to the non-reducing end of the HA polysaccharide. The HA growing chain remains attached to the same UDP group at the reducing end (throughout the whole polymerisation process) while subsequent new sugar-UDPs are being added to the non-reducing end. Yellow line represents the glycosidic bond formed between GlcUA and GlcNAc

Fig. 5

Overview of biosynthesis pathway for recombinant HA production in E. coli. In order to complete the HA biosynthetic pathway in the E. coli, expression of kfiD and pmHAS genes along with supplementation of glucosamine (with glucose) into the culture media are needed to allow the bacteria to produce HA (Mao et al. 2009)