Establishment Of The Hyaluronic Acid Synthesis Pathway In Lactococcus Lactis And Study On The Molecular Weight Regulating Mechanism Of Hyaluronic Acid Synthesized In Microbes

Hyaluronic acid(HA) is one kind of natural high molecular weight polysaccharide and plays structural,recognition and signaling roles in animals.HA has been used in medicines,cosmetics,drug delivery systems,vaccine aids,as well as in health foods. In industry,HA is produced using the gram-positive bacterium Streptococcus zooepidemicus. However,Streptococcus is a less-than-ideal source because of the potential to produce exotoxins,the difficulty in fermentation control or expensive medium.Therefore,it is meaningful and necessary to develop an alternative source of HA that avoids these pitfalls and enlarges the applications.The relative molecular weight(Mr) of HA is generally ranging from 10~4 to 10~7 Da in vertebrates and bacteria,and the biological roles of HA correlate with the length of the HA chain.HAs of different lengths often appear to have antagonistic or reverse effects in many cellular and tissue systems.The solution of HA with sizes greater than 10 kDa,which has outstanding moisture keeping ability,high viscoelasticity and lubricity,has been widely used in cosmetics and medicines.Recently,several groups have reported that short chain HA(～2 kDa-3.5 kDa) or HA oligosaccharides(～10-20 sugars in length) have interesting effects on cellular behavior.To extend the applications of HA and make better HA-containing biomedical products,it is necessary to obtain specially designed Mr or uniform size-defined HA.Vertebrates may be able to control the length of HA in vivo by differential expression of biosynthetic enzymes,but HA fermented by microbes is a mixture of a wide range of molecular masses.The in vitro study data support the theory that the relative ratio of catalysts to the precursor sugars may be a major factor in HA size control.Therefore, it is meaningful and necessary to investigate the regulation mechanism of HA size in bacteria. Lactoccus lactis,one of the most widely used Gram-positive lactic acid bacteria in food fermentations,is a generally regarded as safe(GRAS) strain and increasingly used in modern biotechnological applications.In virtue of great development of genetic engineering tools and the molecular characterization of this species in the last 25 years,a number of inducible expression promotors have been developed in L. lactis.Through these promotors,gene expression can be controlled by an inducer,a repressor or environmental factors,such as peptide,monosaccharide,or temperature. NICE(nisin controlled gene expression) system has become one of the most successful and widely used tools for regulating gene expression in Gram-positive bacteria.The lacF gene is commonly used as a food-grade selectable marker in the NICE system.In order to overcome the shortcomings of Streptococcus zooepidemicus as a HA production bacterium,we planned to construct some engineering GRAS recombinant strains based on Lactoecus lactis containing HA biosynthesis operon and lacF seletable marker.Another controllable expression system of L.lactis is based on the lacR promoter of the lac operon.The lacR promoter is a strong promoter that is virtually not subjected to catabolite repression and controlled by autoregulated LacR repressor.Both NICE system and the lacR promoter have been used to overexpress homologous or heterologous membrane-located proteins and regulate gene expression by quorum sensing-controlling in L.lactis.In this work,these characters of the lacR and the nisA promoters was used to regulate szHasA and szHasB expression,in investigating regulation mechanism of HA polymer size in recombinant L lactis. A series of enzymes are involved in the biosynthesis of HA in S.zooepidemicus, including hyaluronan synthase(HasA),UDP-glucose-6-dehydrogenase(HasB), UDP-glucose pyrophosphorylase(HasC) and glucosamine-1-phosphate N-acetyltransferase/UDP-N-acetylglucosamine pyrophosphorylase(HasD).HasA,the key enzyme in the production of HA,utilizes two sugar substrates(UDP-GlcA and UDP-GlcNAc) to synthesize HA.HasB,HasC and HasD are the enzymes in the synthesis pathway of the precursor sugars.UDP-glucose-6-dehydrogenase (UDP-GlcDH) and GlcN-1-P acetyl transferase/UDP-GlcNAc pyrophosphorylase (LACR) from L.lactis have the same catalyzing effects as HasB and HasD do. The szHasA,szHasB and szHasC were carried on pSH71-derived replicons and under the control of PnisA promoter in the expression plasmids pNZ8148-szHasA, pNZ8148-szHasB and pNZ8148-szHasC,respectively.The recombinant L.lactis strains with pNZ8148-szHasA,pNZ8148-szHasB and pNZ8148-szHasC were named as N9000-A,N9000-B and N9000-C.The HA biosynthesis operon of S. zooepidemicus was introduced into L.lactis NZ9000 and NZ3900 with the vectors of pNZ8148-szHasABC and pNZ8149-szHasABC,respectively,producing strain NFHA01 and NFHA03.Meanwhile,a recombinant HA biosynthesis operon was constructed and named as rHaop.In this artificial DNA fragment,the ORFs of szHasB and szHasC were displaced by the ORFs of ugd and LACR from L.lactis respectively.Then rHAop was introduced into L.lactis NZ3900 with the vector pNZ8149-rHAop,producing strain NFHA02.The concentrations of HA in the culture of these recombinant strains were analyzed and compared.About 0.22 g/L HA was detected in the NZ9000-A culture while HA cannot be detected in the control strain NZ9000,indicating that HA could be synthesized with the expression of HasA alone in L.lactis and the required precursors, UDP-GlcA and UDP-GIcNAc,could also be synthesized in the host strain.However, no HA was detected in the cultures of NZ9000-B and NZ9000-C.Furthermore,a final concentration of HA was about 0.6g/L in the NFHA01 culture,was about 170% higher than that of NZ9000-A,showing that HasA is essential for high heterologous HA production in L.lactis and improving the precursor sugar biosynthesis levels can obviously increase the production of HA.A final concentration of HA was about 0.49g/L in the NFHA03 culture,about 33%higher than that(about 0.38 g/L) in the culture of NFHA02..Therefore,it can be concluded that overexpression of the endogeneous enzymes,directing the steps in the synthesis pathway of the precursor sugars,is a more effective way to increase HA production than expressing exogenous enzymes in recombinant cells.HA fermented by microbes was a mixture of a wide range of molecular masses,but the biological roles of HA are correlated with the length of the polysaccharide chain, which is one of the limiting-factors to extend the applications of HA and make better HA-containing biomedical products.So,it has theoretical and practical value to obtain specially designed(?)w or uniform size-defined HA by industrial fermentation. The mechanism for size control of HA product has begun to be investigated recently. Jing et al.(2004) proved that HA polymers of a desired size could be obtained by controlling the reaction stoichiometry,such as molar ratio of precursors and acceptor molecules.The theory that the relative strength of the interaction between the catalyst and the precursor sugars may be a major factor in HA size control has been testified by Pummill.et al.(2003) in vitro.We considered that this theory could also be suitable in bacterium cells and should be investigated in vivo. To adjust the relative strength of the interaction between the catalyst and the precursor sugars,we obtained different ratios of HasA expression level to precursor sugar UDP-GlcA biosynthesis ability by regulating the szHasA and szHasB expression levels in a recombinant L.lactis.Two inducible expression promoters,nisA promoter and lacR promoter,were used in our work and gene expression could be controlled by nisin or lactose through these promoters.Thus,a dual-plasmid controlled expression system,which consisted of szHasA expression vector pNZ8148-szHasA and szHasB expression vector pNZ9531-szHasB,was constructed.The aim to regulate szHasA and szHasB expression levels in recombinant strain NFHA04 was accomplished by using this system.The ratio of szHasA to szHasB mRNA copies,the surface features of the ratio of catalytic ability of HA synthesis to the precursor sugar biosynthesis level in cells,was regulated strictly by changing the inducing concentration collocations with nisin and lactose.We analyzed the Mw and the concentration of HA under the different ratios, discovering that the theory that the relative strength of the interaction between the catalyst and the precursor sugar may be a major factor in HA size control was also suitable for in vivo HA synthesis of microbes,as we had expected.The average size of HA produced in high HasA/HasB mRNA ratio conditions was smaller than that in low ratio conditions.Altering the ratio of HA synthesizing ability to the concentration of precursor sugars might cause a change in HA size.The reason is probably that the distribution of precursor sugars among the HasA molecules is altered by changing HasA expression level and the precursor sugar biosynthesis ability.The distribution of precursor sugars to each HasA under the inducing condition of high concentration of nisin and low concentration of lactose would be relatively less than under the reverse inducing condition,so each HA polysaccharide chain gets less precursor sugars to lengthen itself catalyzed by HasA in the membrane.In the present study,the ratio of HasA expression level to the precursor sugars biosynthesis ability in bacteria, standing for the relative strength of the interaction between the catalyst and the precursor sugars,was proved to be an important facter for the size control of HA in bacteria.It has theoretical and practical value to determine the regulation mechanism of HA polymer size and this study puts forward a guide for establishing an efficacious way to control the size of HA in fermentation.