The Mechanism Of High Production And Synthetic Process Enhancement Of Phenazine-1-carboxamide In Pseudomonas Chlororaphis

Pseudomonas chlororaphis HT66 is a root-colonizing biocontrol strain isolated from the rice rhizosphere,which exhibits wide-spectrum resistance to fungal phytopathogens because of its capability to produce PCN.PCN is converted from PCA via a transamidase reaction catalyzed by Phz H.Compared with PCA which is a powerful biocontrol agent（designated as’Shenqinmeisu’）,PCN showed higher antifungal activity against the tomato/Fusarium oxysporum system and has superior stability.Thus,PCN is a promising agricultural antibiotic.Nevertheless,the low yields production of PCN（425 mg/L）by P.chlororaphis wild-type strain HT66restricts its commercial objective.In our previous work,a high PCN-producing strain,P.chlororaphis P3,was obtained by subjecting strain HT66 to multiple rounds of chemical and physical mutagenesis and selection.The PCN production of mutant P3was 3.99 times that of HT66.Although many genes were identified to undergo point mutation（s）in the genome of P3,the physiological changes that resulted in the increased PCN yield in strain P3 are not clear.In this research,the isobaric tag for relative and absolute quantification（iTRAQ）technique was applied to study the expression level changes in the proteome between the wild-type strain HT66 and the mutant strain P3.The mechanism of high PCN production in mutant P3 was revealed.Then,various genetic engineering strategies were used to enhance PCN production in P3 based on the iTRAQ results and literatures.The main contents and findings were described as follows:An iTRAQ-based quantitative proteomic analysis was used to study the changes in the cellular proteome between the wild-type HT66 and mutant P3.A total of 452proteins were differentially expressed（fold change>1.5 or<0.7,P<0.05）,of which181 proteins were upregulated and 271 proteins were downregulated.Of the 452differentially expressed proteins,most were functionally mapped into PCN biosynthesis pathway or other related metabolisms.The upregulation of proteins,including Phz A/B,Phz D,Phz F,Phz G,and Phz H,involved in PCN biosynthesis was in agreement with the efficient production of PCN in P3.A number of proteins that function primarily in energy production,amino acid metabolism,and secondary metabolism played important roles in PCN biosynthesis.Notably,proteins involved in the uptake and conversion of phosphate,inorganic nitrogen sources,and iron improved the PCN production.Furthermore,the type VI secretion system may participate in the secretion or/and indirect biosynthetic regulation of PCN in P.chlororaphis.In addition,a large proportion of differentially expressed proteins were involved in cell motility,cell wall/membrane/envelope biogenesis,chemotaxis and signal transduction,indicative of a substantial overall phenotypic difference resulting from the chemical mutagenesis of the mutant P3 compared with the wild-type strain HT66.Many experiments were applied to confirm some hypotheses for iTRAQ results.First,to further assess the expression of the phz gene cluster at the translational level in strains HT66 and P3,a lac Z reporter plasmid named p BBR-phz′-′lac Z（translational fusion）was constructed to analyze theβ-galactosidase activity.We observed that theβ-galactosidase activity from the p BBR-phz′-′lac Z translational fusion plasmid in mutant P3 was significantly higher than that of the wild-type strain HT66,suggesting that the expression of the phz gene cluster was enhanced at the translational level in P3,which was in agreement with the iTRAQ result.Second,to detect the importance of Phz E protein in the PCN biosynthesis,we overexpressed the phz E gene in mutant P3,resulting in 18-21%increase of the PCN level in P3,which suggests that Phz E is crucial for PCN biosynthesis.Third,fermentation by addition of ions suggests that lower concentration of K₂HPO₄,addition of Fe Cl₃ and inorganic nitrogen in medium greatly improved PCN production in P.chlororaphis.Finally,deletion of gene clusters of T6SS in our strains indicates that T6SS may participate in the secretion and/or biosynthetic regulation of PCN and each locus of the T6SS probably plays an independent role.Further studies are required to investigate the complex relevance of HS1,HS2,and HS3 in the secretion and/or biosynthetic regulation of PCN.Various genetic engineering strategies by blocking or weakening competing pathways of PCN biosynthesis were used to improve PCN production in P3.The results indicated that blocking tryptophan biosynthesis pathway and weakening phenylalanine biosynthesis pathway greatly affected the growth of strain.However,blocking the PEP conversion pathway greatly improved PCN production.PCN production was 2589 mg/L.Based on analysis of theβ-galactosidase（Lac Z）activity,we found that an inhibitor region located in the 5’-UTR of phz gene cluster.The prediction of secondary structure of 5’-UTR m RNA showed that a hairpin structure was formed in the 5’-UTR（+22nt-+78nt）.The PCN production greatly improved when the sequence containing the hairpin structure was deleted.q RT-PCR experiment suggests that the hairpin structure affected PCN production by regulating the expression of phz genes.The integration of phz E into a specific-site of genome improved PCN production.Deletion of ofa,hcn and fit gene clusters had slightly influence for PCN biosynthesis,and the PCN production of mutant P3AI was 3673 mg/L,which is 2.1 times that of P3.The KB medium was modified according to iTRAQ results and used to cultivate mutant strain.The results showed that the lower concentration of K₂HPO₄in KB medium and addition of Fe Cl₃ and NH₄Cl in KB medium greatly improved the yields of PCN.The effect of Fe Cl₃on PCN production was more obvious.The mutant P3AI was fermented in KB medium containing lower concentration of K₂HPO₄and addition of Fe Cl₃ and NH₄Cl,and PCN production was 5731 mg/L.The result of q RT-PCR showed that the lower concentration of K₂HPO₄ and addition of Fe Cl₃ in KB medium enhanced the PCN production by increasing the expression of phz genes in P.chlororaphis,but the mechanism was not clear.In this paper,the basic mechanism of high-yield PCN of P3 strain was first explored by proteomics analysis.Some hypotheses were confirmed by various experiments.A series of mutant strains with high yield of PCN were obtained by genetic engineering methods.The PCN production of the mutant strain was further improved by fermenting using the modified KB medium.In addition,we discovered some potential factors and gene sites that may play important roles in the regulation of phenazines biosynthesis in P.chlororaphis.This study laid a theoretical and technical basis for the subsequent regulation and biosynthesis of phenazines in P.chlororaphis.