Effect Of Expressing PHB Synthesis Genes PhbCAB Gene On Production Of Latate And Hyaluronic Acid By Streptococcus Zooepidemicus

Hyaluronic acid (HA) is a high molecuLar weight polysaccharide, whichis widely used in medicine and cosmetics. It has good ability of keeping waterand biocompatibility. These years, hyaluronic acid is industrially produced byfermentation of Streptococci. It has the advantage of low cost and goodquality. But in the fermentation process, large part of carbon source wastransformed to lactate and the productivity of HA is low. This study was tosolve these problems and make some improvements on HA production.Following results were achieved:hasB, which encodes the UDP-glucose dehydrogenase, was cloned fromthe genome of Steptococcus zooepidemicus (S. zooepidemicus). It wasexpressed in E.coli under T7 promoter and displayed the right activity. Theprotein sequence has 63.1% and 70.6% similarity to HasB of S. pyogene and Suberis, respectively. It had a special position in the phylogenetic tree based onUDP-glucose dehydrogenase gene sequence. These results means that thehasB of S. zooepidemicus has special characteristics, which is correlated withthe high HA synthesis ability of group C Streptococci.The PHB synthesis gene of Wautersia eutropha, phbCAB, wascoexpressed with the HA synthesis gene, hasABC, in E. coli. These twopathways could be coupled at the level of coenzyme. Although there was largeamount of PHB accumulation, no HA was synthesized. It means that E. coli isnot a suitable host for the production of HA.phbCAB gene was expressed in S. zooepidemicus harboring plasmidpEU308, an expression vector in Gram-positive bacteria. The activities ofPhbA and PhbB were, respectively, 3.13 U/mg and 1.23 U/mg. But no PhbCactivity was detected. And as a result, there was no PHB accumulation yet.NADPH was transformed to NADP in the reaction catalyzed by PhbB. Theexpression of phbAB provided an oxidation potential regeneration pathway. Inflask fermentation, the cells were in an oxygen-rich enviroment. There weretwo pathways for the bacteria to regenerate oxdation potential: one was thatNADH was oxidized to NAD by oxygen, the other was the lactate pathway.The first pathway could provide most part of oxidation potential. There wasno obvious influence on lactate formation. While in fermentor study, lactatepathway was almost the only way to regenerate oxidation potential, theintroduction of phbCAB gene reduced the lactate formation from 64 g/L to 41g/L. At the same time, the change of celluLar carbon flux led to about 1 g/Lincrease on HA production. These results indicated that the PHB synthesispathway could act as a global regulator to adjust the celluLaroxidation/reduction potential.