Chromosomal integration of hyaluronic acid synthesis (has) genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis

Abstract

Microbial production of hyaluronic acid (HA) is an attractive substitute for extraction of this biopolymer from animal tissues. Natural producers such as Streptococcus zooepidemicus are potential pathogens; therefore, production of HA by recombinant bacteria that are generally recognized as safe (GRAS) organisms is a viable alternative that is being extensively explored. However, plasmid-based expression systems for HA production by recombinant bacteria have the inherent disadvantage of reduced productivity because of plasmid instability. To overcome this problem, the HA synthesis genes (hasA-hasB and hasA-hasB-hasC) from has-operon of S. zooepidemicus were integrated into the chromosome of Lactococcus lactis by site-directed, double-homologous recombination developing strains VRJ2AB and VRJ3ABC. The chromosomal integration stabilized the genes and obviated the instability observed in plasmid-expressed recombinant strains. The genome-integrated strains produced higher molecular weight (3.5-4 million Dalton [MDa]) HA compared to the plasmid-expressed strains (2 MDa). High molecular weight HA was produced when the intracellular concentration of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-glucuronic acid (UDP-GlcUA) was almost equal and hasA to hasB ratio was low. This work suggests an optimal approach to obtain high molecular weight HA in recombinant strains.

High‐titer biosynthesis of hyaluronic acid by recombinant Corynebacterium glutamicum

“…Subsequently, we optimized the operon organization and found that the operon hasA-hasB was more suitable for HA synthesis in recombinant C. glutamicum. Similar results were obtained in two Gram-negative recombinant hosts, E. coli [14,16] and L. lactis [13]. In another Grampositive recombinant host, B. subtilis, operons hasA-hasB-hasC and hasA-hasB-hasD were both better than hasA-hasB [10].…”

“…Although restricted by pathogenicity, the wild type S. equi is still the most common producer of HA in the fermentation industry compared to recombinant B. subtilis , L. lactis and E. coli . C. glutamicum , a Gram‐positive microorganism widely used for producing diverse chemicals, particularly in the food industry such as acetohydroxy acid and amino acids, including L‐glutamate, L‐lysine, L‐ornithine, and L‐arginine , is not previously reported for use in HA production.…”

“…Although restricted by pathogenicity, the wild type S. equi is still the most common producer of HA in the fermentation industry compared to recombinant B. subtilis [10][11][12], L. lactis [13] and E. coli [14][15][16]. C. glutamicum, a…”

“…It was plagued by the low productivity and requirement for complex purification steps [6]. A microbial fermentation method was developed in Japan for accumulation of HA via Streptococcus in the 1980s [6][7][8][9], and a novel bio-route by fermentation of the engineered strains was proposed in recent years [10][11][12][13][14][15][16].…”

Editorial: Asian Congress on Biotechnology 2013

“…The work assumes significance in the sense that up‐conversion of infra‐red radiation will not only enhance algal cultivation, but would mitigate its heating effects also. The next article, by Prof. K. B. Ramachandran 's group [11], shows the successful integration of hyaluronic acid synthesis genes in the chromosome of Lactococcus lactis (a GRAS organism) for stable production of high molecular weight (3.5‐4.0 MDa) hyaluronic acid, which has high demand in several medical and cosmetic applications. The next research paper by Prof. Yoon‐Mo Koo and co‐workers [12] describes the application of ionic liquids as novel solvents for the synthesis of sugar fatty acid esters, which as bio‐surfactants are widely used in the food, pharmaceutical and cosmetic industries due to their non‐toxicity and high biodegradability.…”