Research On Systems Biology And Related Conversion Pathways In The Biosythesis Of L-Cysteine By Pseudomonas Sp. QR-101

Cysteine as a sulfydryl-containing amino acid, it has a wide range of uses inmedicine, food, feed, cosmetics and other industries. Cysteine is difficult tomicrobial fermentation production, has been using a hair hydrolysis method ofproduction. Pseudomonas sp. QR-101preserved in our laboratory can utilizeDL-ATC (DL-2-Amino-2-thiazoline-4-Carboxylic Acid) as substrate in the synthesisof L-cysteine. The Pseudomonas sp. QR-101cultivated under DL-ATC induced andun-induced conditions were compared in the aspects of gene transcription, proteinexpression and metabolite synthesis and degradation using ways of transcriptomics,proteomics and metabolomics. This will provide an approach for efficient productionof L-cysteine.Transcriptomic analysis found that there were90genes had Gene ExpressionDifference (DEGs, FDR≤0.001and|log2Ratio|≥1), of which55were up regulatedand35were down regulated. These genes involve in many aspects of the organismmetabolism including energy metabolism, sulfur metablism, L-cysteine synthesispathway and amino acid metabolism, et al. The log2Ratio of gene encoding ATChydrolase (atcB) increased17.4fold, it was influenced mostly and was also mostlyrelated to the L-cysteine synthesis from DL-ATC. Due to its up regulation, theaccumulation of L-cysteine was increased. Consequencely, genes involved in thecatabolism of L-cysteine were also up regulated. Besides, genes involved in sulfurmetabolism were down regulated while genes involved in energy metabolism wereup regulated.Two-dimensional gel electrophoresis was carried out to compare the proteinexpression profile of Pseudomonas sp. QR-101under DL-ATC induced andun-induced conditions. The protein spots showing distinctly different expressionpatterns were excised and carried out to mass spectrometric analysis. The proteinsinvolved in many aspects of the metabolism in bacterial, including transport system(flagellin, general amino acid ABC transporter periplasmic binding protein), signal transduction (putative serine protein kinase, PrkA), phosphate metabolism (PhoHfamily protein), amino acid metabolism (cysteine synthase/cystathionine beta-synthase family protein), reaction directly related to DL-ATC (ATC racemase, ATChydrolase), beta-lactamase and beta-lactamase domain. In these proteins, only generalamino acid ABC transporter periplasmic binding protein was down regulated and theother proteins were up regulated under DL-ATC induced condition. It can beconcluded that the whole metabolism network was changed due to the DL-ATCinduction.Metabolomic analysis was carried out to compare the metabolite accumulationprofiles of Pseudomonas sp. QR-101under DL-ATC induced and un-inducedconditions. UPLC/Q-TOF-MS was used to investigate the samples, principalcomponent analysis (PCA) was used to reveal the difference in the two samples and32potential biomarkers were recognized. These metabolites were involved in manymetabolic pathways including energy metabolism and amino acid metabolism. Itrevealed that the metabolism level of Pseudomonas sp. QR-101was increased underDL-ATC induced condition. The synthesis and accumulation of the other amino acidwere also influenced.LC-MS analysis revealed that this organism uses the N-pathway in theconversion of DL-ATC to L-cysteine. Genes encoding DL-ATC racemase (AtcA),L-ATC hydrolase (AtcB) and L-NCC amidohydrolase (AtcC), involved in thispathway, were cloned from Pseudomonas sp. QR-101and expressed in Escherichiacoli BL21via pET-28a(+). The three resulting enzymes were purified, their functionsidentified and their biochemical properties, including molecular mass, optimal pHand temperature, tolerance to ions, were described. In vitro catalysis experimentsusing these enzymes revealed that the bioconversion of L-cysteine from DL-ATCwas efficient in the presence of AtcA.In this study, using the means of systems biology, the enzymatic conversion ofL-cysteine process involves transcriptome, proteomics and metabolomics inPseudomonas sp. QR-101were clarified. This is the first report describingsimultaneous cloning and expression of atcA, atcB and atcC and characterization oftheir enzymes for L-cysteine production from DL-ATC via the N-pathway in Pseudomonas, enabling the complete N-pathway to be elucidated. The study hassignificant potential to guide the industrial production of L-cysteine using enzymaticconversion methods.