Bioconversion Of Rebaudioside C And Its Catabolism Mechanism By Paenarthrobacter Ilicis CR5301

Steviol glycosides is a high sweetness,low calorie natural sweetener extracted from the leaves of Stevia rebaudiana Bertoni,a member of the Asteraceae（Compositae）family.It is an ideal substitute for sucrose and is known as the“third sugar source in the world”.The resource utilization of byproducts of steviol glycosides has always been a challenge for manufacturers.The main component of byproduct is Rebaudioside C（RC）,which is the third most abundant component in Stevia rebaudiana Bertoni.Due to its licorice and bitter aftertaste,its application is limited.Microbial conversion of RC to prepare other high-value derivatives with physiological activity is an effective way to broaden its application.At present,there are few microorganisms with RC conversion activity,and there are shortcomings such as low conversion efficiency and long time consumption.Meanwhile,there is no report on the key enzymes of microbial conversion of RC and the pathway and mechanism of RC catabolism.Therefore,in order to explore the feasibility of efficient conversion of RC by microorganisms to prepare Steviol（STE）,then to analyze the key genes（enzymes）,pathways and mechanisms of RC catabolism in microorganisms,this study systematically investigated the RC conversion,catabolism capabilities,furthermore the mechanism of the screened strain CR5301 by genome,transcriptome,target metabolite identification and genetic engineering techniques.The specific research content and results are as follows:（1）RC was selected as the only carbon source,a bacterial strain CR5301 that can efficiently convert RC was screened from stevia planting soil by plate separation and HPLC identification methods.The bacterial strain belongs to the species Paenarthrobacter ilicis and named P.ilicis CR5301 by taxonomic identification of cell morphology and 16S r RNA gene.P.ilicis CR5301 can convert all high purity RC to steviol,and can also convert other glycosides components in the byproducts of steviol glycosides to STE.Compared with previous studies,it was found that P.ilicis CR5301 was currently the best microorganism for microbial conversion RC and byproducts of steviol glycosides to STE.And it not only has good industrial application potential,but also is an ideal model for the study of RC catabolism mechanism.（2）The complete genome of P.ilicis CR5301 was sequenced by high-throughput sequencing method combining the second generation Illumina Hi Seq and the third generation Nanopole.The results showed that the complete chromosome genome of P.ilicis CR5301 was a closed circular DNA molecule without plasmid.The total length of the chromosome genome is 4748281 bp,the（G+C）%content is 62.92%,and it contains 4458 coding genes,including18 r RNA operon and 54 t RNAs.P.ilicis CR5301 is the first of P.ilicis species to have its complete genome determined and the largest known genome in the genus Paenarthrobacter.Gene function analysis showed that 379,163,301 and 174 carbohydrate active enzyme genes were annotated in COG,GO,KEGG and CAZy databases,respectively.After removing redundancy,the P.ilicis CR5301 genome contained 523 carbohydrate active enzyme genes.By comparing the molecular structure of RC and its metabolite steviol,18 glycoside hydrolase genes were found to be closely related to the hydrolysis and metabolism of RC.These results provide clear genetic background and potential key gene information for the study of RC catabolic mechanism in P.ilicis CR5301.（3）The whole genome expression profile and targeted metabolites of P.ilicis CR5301 were determined and analyzed under RC absence（control group）and presence（treatment group）conditions by high throughput transcriptional sequencing（RNA-seq）technology and targeted metabolite identification（HPLC,UPLC-Triple-TOF/MS）techniques.It was found that P.ilicis CR5301 in the treatment group could convert RC to three intermediate products（Duc A,Duc B,Duc A1）and one final product（STE）compared with the control group.There are two hydrolytic pathways for RC to STE:RC→Duc A→Duc A1→STE and RC→Duc B→Duc A1→STE.RNA-Seq sequencing results showed that 105 genes were significantly differentially expressed after RC addition,and 53 GO entries and 7 Pathway entries were significantly enriched,which were mainly involved in carbohydrate metabolism and transport functions.And3 glycoester hydrolase genes（0600,1935,and 1936）and 6 glycoside hydrolase genes（1932,1940,1941,1944,1947,and 3616）may play key roles in RC catabolism.Five metabolic pathways（map00040,map00051,map00500,map00910,and map02010）showed significant metabolic responses to RC addition.In addition,RT-q PCR verified the accuracy and reliability of RNA-Seq sequencing.Based on the above results,this study proposed a model of RC catabolism in strain CR5301,including the detailed process,complete pathway and cell localization of RC catabolism,and demonstrated that RC catabolism occurs within cells through identification of intracellular and extracellular enzyme activities.These results clearly clarified the key genes,complete pathways and metabolic mechanisms of RC catabolism in bacteria at the level of transcription and metabolism for the first time,providing a new theoretical basis and important experimental basis for the future research on microbial conversion and catabolism of RC.（4）The keyβ-glucosidase coding genes bgl X1944,bgl X1940 and bgl X1932 in P.ilicis CR5301 were cloned,expressed,purified and enzyme activity identified by genetic engineering,affinity chromatography and substrate RC conversion experiments.The genes bgl X1944,bgl X1940,and bgl X1932 were successfully heterologously expressed in E.coli BL21（DE3）,and the three purified recombinase Bgl X1944,Bgl X1940,and Bgl X1932 all had the hydrolytic active ofβ-1,3-glucosidic linkage at C13 andβ-glucosyl ester linkage at C19 of RC.But the products of hydrolysis of RC and catalytic efficiency by these enzymes were different,and only Bgl X1932 could hydrolyze RC to Duc B first,and then hydrolyze Duc B to Duc A1.Through the characterization of the enzymatic properties of Bgl X1932,it was found that the optimal p H of Bgl X1932 for hydrolyzing RC was 8.0,and it had strong tolerance in the range of p H 2.0-10.0.The optimal temperature is 25℃,and below 45℃,the enzyme activity remains above65.8%.The kinetic parameters K_m,K_cat and K_cat/K_m of Bgl X1932 on RC were 17.73 mmol/L,59.22×10³ s^-1 and 3.34×10³L/mmol·s,indicating that the Bgl X1932 has good affinity and catalytic efficiency for substrate RC.Finally,Duc B and Duc A1 were successfully prepared by Bgl X1932 under the optimal catalytic conditions.The purity of Duc B and Duc A1 was 92.65%and 91.13%respectively,which was the first report on the enzymatic preparation of Duc B and Duc A1.