Transcriptional Response Of Rhodococcus Sp. R04 Cultured In Biphenyl And The Biotransformation Of Protocatechuate

With increasing of studies on the metabolic dynamic and mechanism of biodegradation to PCBs,the genome and transcriptome gradually become focus of PCBs metabolism. Research of genome and transcriptome not only can reveal molecular mechanism of the special biological process, but also analysis the metabolic characteristics of the whole cell. The widely application of transcriptomics, including detection of new genes, study of gene expression and regulation of microorganisms in different environments, and the analysis of metabolic pathways of special organic substances, are also highlighted. Transcriptomics, combined with the further development of sequencing technology and bioinformatics tools in the future, are likely to be comprehensively used in the study of biodegradation to PCBs.Investigating the transcriptional characteristics of Rhodococcus sp. R04 grown on different carbon source especially on the biphenyl, is to unearth the genes that participate in the transportation, metabolization and regulation of polychlorinated biphenyls (PCBs), and facilitate us to further understand the molecular mechanism of biodegradation of PCBs. Rhodococcus sp. R04, a PCBs degrader, was cultivated respectively on ethanol, glucose and biphenyl. The total RNA was extracted, and the cDNA was obtained by reverse transcription, subsequently, determined by high-throughput sequencing. The data obtained by sequencing were analysized to seek out the correlation between the PCBs metabolic network, gene transcription regulation and metabolic response. The gene expression of R04 grown on different medium was also detected by quantitative reverse transcription PCR with a subset of genes of interest. The sequencing results showed that 375 genes were upregulated during grown on biphenyl compared with on glucose,332 genes with on ethanol. Q-RT-PCR assays confirmed the sequencing results for selected genes. Those sequenced genes were classified into cellular component, molecular function and biological process, some of which were found to participate in multiple biological processes of biphenyl metabolite, such as transcriptional regulation, transport of biphenyl, antioxidant reaction and signal transduction. There are many genes encoding BphC and BphD isozymes in the biphenyl metabolic pathway in R04, but only bphC2 and bphDl were upregulated during growth on biphenyl. Sequence analysis suggested that benzoate was degraded via ortho cleavage pathway, meta cleavage pathway or protocatechuic acid pathway. Compared with grown on glucose and ethanol, it is significantly different in gene expression when cultured with biphenyl, which provide more valuable data for us to reveal the metabolic characteristics and metabolic regulation of PCBs.Protocatechuate is the precursor substance and active ingredient of variety of traditional Chinese medicine, and has important medicinalvalue. The domestic market demand for the protocatechuate is quite plenty, however, the classic chemical preparation for protocatechuate can damage the natural environment. In order to solve this problems, This study use bio-enzymatic preparations to product protocatechuate. We cloned the 4-hydroxybenzoate-3-monooxygenase gene into pET21a expression vector and then transformed the recombinant plasmid into expression host E.coli BL21(DE3) and achieved the biotransformation from p-HBA to protocatechuate. Protocatechuate production was improved by optimized experimental conditions, and the conversion of protocatechuate can reach 250 ～300μg per OD.