Analysis And Expression Of Key Genes Involved In The Guaiacol Biosynthesis By Alicyclobacillus

Alicyclobacillus spp.is a common spoilage bacterium in the juice industry.The main characteristic of juice spoilage caused by Alicyclobacillus spp.is that the bacterium can biosynthesise guaiacol in the juice,producing a "medicinal" and "preservative" taste,resulting in deterioration both in taste and flavour.In this study,we elucidate the main genes and proteins involved in guaiacol production,and identify the guaiacol biosynthetic gene cluster in A.acidoterrestriswith the most advanced method-transcriptomics and proteomics techniques.Besides,starting with the guaiacol synthesis gene cluster,the correlation between guaiacol production and guaiacol synthesis gene cluster and the relationship between gene expression in guaiacol biosynthesis gene cluster and guaiacol production wasexplored,separately by comparative genomics,and RT-q PCR method.We find that the genes in the synthetic gene cluster are closely related to the formation of guaiacol,thus r the molecular basis of guaiacol biosynthesis in Alicyclobacillus,and provide a theoretical basis for further understanding even solving the problem of juice corruption caused by Alicyclobacillus.The main research contents and results are as follows :(1)With RNA-seq-based transcriptomics and i TRAQ-based proteomics as the core tools,the differentially expressed genes and proteins of Alicyclobacillus alicyclicus under conditions of guaiacol production and non-guaiacol production were revealed.Furthermore,the genes related to guaiacol biosynthesis were mined by correlation analysis of transcriptomic and proteomic data.In the end,a total of 225 differentially expressed genes and 77 differentially expressed proteins were identified.Among them,the top three up-regulated genes in transcript levels arevdc B,vdc C and vdc Dgenes,whichencode vanilloic acid decarboxylase subunits and up-regulate transcript levlesby 626.47,185.01 and 52.81-fold respectivelyi,as well as the the protein expression levels by 2.12 and 2.64-fold respectively.It was predicted that the downstream gene of vdc BCD encoding pyridoxamine 5 ’-phosphate oxidase family protein was up-regulated by 47.35 times.The gene predicted to encode the Benzoic acid/H(+)cotransport Ben E family transporter located upstream of vdc BCD was up-regulated 2.69-fold.Hence,we conclude that the guaiacol biosynthesis gene cluster in Alicyclobacillusconsist of six genes,including one genepredicted to encode a transporter protein(gua A),one gene predicted to encode a Lys R family transcriptional regulator(gua B),three genes predicted to encode subunits of vanillic acid decarboxylase(vdc B,vdc C and vdc D)and one gene predicted to encode a pyridoxamine 5’-phosphate oxidase family protein(gua F).In addition,the expression levels of enzymes related to the degradation of aromatic compounds,such as catechol 2,3-plus dioxygenase,2-keto-4-pentenoate hydratase,2-hydroxymuconic acid-6-semialdehyde dehydrogenase and 3,4-dihydroxyphenylacetic acid-2,3-plus dioxygenase,as well as genes or proteins for transport proteins such as Fusarium acid resistance proteins,RND-type efflux pump family genes,and some ABC and MFS transporters The expression levels of differential genes related to the tricarboxylic acid cycle and ribosomal protein synthesis increased,while the expression of genes related to valine,leucine and isoleucine synthesis decreased.(2)The whole genome sequences of two Alicyclobacillus guaiacol-producing strains(A.dauci DSM 27800 and A.fastidiosus isolate KKP 3000)and one non-guaiacol-producing strain(A.fastidiosus standard strain DSM 17978)were determined by high-throughput sequencing.Then we analyse the whole genome sequence of a non-guaiacol producing strain(A.fastidiosus standard strain DSM 17978)by comparative genomics of the guaiacol biosynthesis gene cluster in Bacillus avium,and investigate the correlation between guaiacol production and the guaiacol synthesis gene cluster at the genome level.The genome sequencing results showed that the genome sizes of A.dauci DSM 28700,A.fastidiosus DSM17978 and A.fastidiosus KKP 3000 were 4,881,158,6,035,649 and 4,968,037 bp,respectively,with total number of genes are 5060,6343,5096 respectively and GC accounting for 50.90%,52.23% and 53.78% respectively.The COG classification indicated that the genes of all three strains were mainly concentrated on the functional group of substance metabolism and had strong substance metabolism ability.Comparative genomic analysis showed that:Alicyclobacillus have stable core structure and open pangenome.,and A.fastidiosus KKP 3000 and A.fastidiosus DSM 17978 were especially closely related.Besides,there were guaiacol biosynthetic gene clusters in A.fastidiosus KKP 3000 and A.suci VF-FSL-W10-0049,which were completely consistent with A.acidoterrestris ATCC 49025.Some genes in the synthetic gene cluster were found in A.herbarius DSM 13609,A.dauci DSM 28700 and A.acidiphilus NBRC 100859.Among the non-guaiacol-producing strains,only A.fastidiosus DSM 17978 had a defective version of the vdc operon.(3)Candidate internal reference genes were identified based on transcriptome analysis data of A.acidoterrestris under conditions of guaiacol production and non-guaiacol production.Furthermore,four algorithms,ge Norm,Norm Finder,Best Keeper and Re Finder were used to evaluate the stability of gene expression.The results showed that rha B and rpo B were the best reference genes.On this basis,RT-q PCR was used to determine the relative expression of each gene in the gene cluster during guaiacol production in A.acidoterrestris,and the relationship between the expression of each gene in the guaiacol biosynthesis gene cluster and guaiacol production was investigated.The results showed that the expression of six genes in the guaiacol biosynthesis gene cluster of A.acidoterrestris was closely related to guaiacol production.