Cell-Based Assay Approach For Glycosaminoglycan Polymerase High-Throughput Screening:Development And Applications

Glycosaminoglycans(GAGs)are a family of long linear heteropolysaccharides composed of repeating disaccharide units of hexuronic acid and either N-acetylglucosamine or N-acetylgalactosamine.They are also referred to as mucopolysaccharides.Based on the composition of monosaccharide residues,the type of glycosidic bond connection,and the distribution and number of sulfate groups,GAGs can be classified into six types:Hyaluronic acid(HA),chondroitin sulfate(CS),dermatan sulfate(DS),keratin sulfate(KS),heparan sulfate(HS),and heparin(HP).GAGs can interact with various biological signaling molecules such as growth factors,cytokines,and chemokines,making them important in a wide range of physiological processes.They also have extensive clinical applications as antitumor,anticoagulant,anti-inflammatory,and anti-aging agents.Glycosaminoglycan polymerases are a group of glycosyltransferases that catalyze the biosynthesis of GAGs.Among them,glycosaminoglycan polymerases are responsible for synthesizing various GAGs skeletons by transferring monosaccharides from monosaccharide donors to the end of the sugar chain.As an important tool in sugar engineering,glycosaminoglycan polymerases have been widely used in the in vitro enzymatic synthesis of heparosan,chondroitin,and hyaluronic acid,as well as in the construction of cell factories.However,the number of glycosaminoglycan polymerases that have been characterized for their catalytic properties is limited to only about 40 species.This,coupled with the strict substrate specificity of natural enzyme molecules,low catalytic activity,and heterologous expression levels,highlights the need to explore new sources of glycosaminoglycan polymerases elements.Therefore,it is crucial to develop methods for directed evolution and high-throughput screening of glycosaminoglycan polymerases activity,which can be applied to the exploration and modification of key enzymes involved in sugar chain synthesis.Protein directed evolution involves using artificially-driven evolution to modify the function and properties of proteins.By constructing a mutant protein library and combining it with high-throughput screening technology,new functional proteins can be obtained in a relatively short time.Two key factors in improving the success rate of directed evolution are developing "smarter" mutation libraries and more efficient activity screening methods.Screening for mutant proteins with an ideal phenotype is generally a random process.Increasing the abundance of mutant libraries can increase the probability of obtaining an ideal phenotype,but this also increases the difficulty and cost of screening.Therefore,developing high-throughput screening methods can greatly improve the success rate of directed evolution,and directly determine the efficiency of obtaining ideal phenotypes from large mutation libraries,reducing time and cost.However,the high-throughput analysis of glycosylation reactions during glycosaminoglycan polymerases is extremely challenging due to the lack of significant changes in fluorescence intensity or absorbance associated with glycosidic bond formation.This challenge seriously hinders the use of directed evolution to enhance the catalytic activity of related glycosyltransferase and broaden their substrate range.Therefore,developing high-throughput methods for analyzing glycosaminoglycan polymerases activity is of crucial application value.Based on the above back ground,this paper mainly includes:1.Establishment of a rapid assay method for chondroitin synthase activity based on Bacillus subtilis 168(B.subtilis 168)and its application in chondroitin synthase discovery.(1)Establishment of a rapid assay method for chondroitin synthase activity based on B.subtilis 168:Through a series of metabolic engineering modifications,our team has completed the construction of an engineered B.subtilis,and is named BS168SSAC.After inducing the expression of a dual-functional chondroitin synthase(KfoC),this strain produced an azido modified capsular polysaccharide chondroitin skeleton using N-(4-pentynyl)azidoacetyl-galactosamine(Ac4GalNAz).Due to the characteristic that azide groups are prone to click chemical reactions with alkynyl groups,covalent binding of alkynyl fluorescein to azide capsular polysaccharides carried by bacteria can be achieved.In this paper,based on the engineering strain,after a series of attempts,we successfully correlated the chondroitin synthase activity with the fluorescence intensity of the cell,and established a rapid analysis method for chondroitin synthase activity based on B.subtilis 168.(2)Optimization of a rapid assay method for chondroitin synthase activity based on B.subtilis 168:After exploring the optimal induction time and the concentration of an unnatural clickable labeled monosaccharide Ac4GalNAz during the screening process,this paper optimized the chondroitin skeleton synthesis pathway in host cells.Under above conditions,the fluorescence data analysis of azido chondroitin carrying BS168SSACD showed a more significant statistical difference compared to control cells.(3)Application of the rapid analysis method of chondroitin synthase activity based on B.subtilis 168 in chondroitin synthase discovery:A library of 10 genes with high similarity to known chondroitin synthase genes was constructed by using a phylogenetic tree.We then used the optimized rapid activity analysis method to efficiently screen the library.The fluorescence intensity of recombinant cells expressing various genes in the library was analyzed to identify six suspected chondroitin synthase encoding genes;subsequently,the six suspected genes were recombined and expressed using an E.coli expression system and purified to obtain soluble proteins.It was characterized that these recombinant enzyme molecules indeed possess chondroitin synthase activity.So far,we have established an efficient method for screening chondroitin synthase by using metabolic engineered B.subtilis 168 to label polysaccharides with unnatural groups by clicking chemical reactions,and screened six new members of the chondroitin synthase family.2.Establishment of a high throughput screening method for heparosan synthase activity based on Escherichia coli O10:K5(L):H4(E.coli K5)and its application in directed evolution of heparosan synthase.(1)Establishment of a high throughput screening method for heparosan synthase activity based on E.coli k5:Through a series of metabolic engineering modifications,we has completed the synthesis of an engineered E.coli K5,and is named K5SSAH.After inducing the expression of Pasteurella dual-functional heparosan synthase 2(PmHS2),this strain produced azido-modified capsular polysaccharides using exogenous N-(4-pentynyl)azidoacetyl-glucosamine(Ac4G1cNAz).Based on this engineering strain,we successfully correlated the activity of heparosan synthase with the fluorescence intensity of the strain through a series of attempts,and established a high throughput screening method for heparosan synthase activity based on E.coli K5.(2)Optimization of a high throughput screening method for heparosan synthase activity based on E.coli K5:We explored the optimal induction time and concentration of Ac4GlcNAz during the screening process to improve the fluorescence data difference between azidopolysaccharide carrying bacteria and control strains.(3)Application of high throughput screening method for heparosan synthase activity based on E.coli K5 in directed evolution of heparosan synthase:Using PmHS2 as the starting protein for directed evolution,through multiple sequence alignment,alanine scanning,and combining with the advanced structure of PmHS2,we established a heparosan synthase screening library(PmHS2 mutant sequences from 8000 different amino acid combinations).We used the above optimized conditions in combination with fluorescence activated cell sorting technology(FACS)to enrich cells with relatively high fluorescence intensity.Complete re screening by analyzing the fluorescence intensity of enriched recombinant cells using an enzyme-linked immunosorbent assay.Subsequently,the suspected gene was recombined and expressed using an E.coli BL21(DE3)expression system,and purified to obtain a soluble protein.Using substrates for glycosyltransferase reactions,characterization of these mutant enzyme molecules confirmed that their GlcNAc transferase activity was 4.48fold and 2.63-fold higher than that of wild type PmHS2,respectively.Subsequently,the corresponding unit point mutants in their combination mutants were designed and constructed to determine the contribution of a single site mutation to the improvement of PmHS2 activity.So far,using metabolic engineered E.coli K5,we have established a high-throughput screening method for heparosan synthase by clicking chemical reactions to label polysaccharides with unnatural groups.We have screened and obtained two active heparosan synthase mutants and identified key amino acid sites that affect the activity of PmHS2.The acquisition of the above highly active mutants demonstrates the potential of the screening method we have established to effectively apply to high-throughput screening of heparosan synthase.