Genetic Engineering Of Pseudomonas Chlororaphis GP72 For The Enhanced Production Of 2-Hydroxyphenazine

Pseudomonas chlororaphis GP72 is a root-colonizing biocontrol strain isolated from the green pepper rhizosphere,which synthesizes three antifungal phenazine compounds,phenazine-1-carboxylic acid?PCA?,2-hydroxy-phenazine-1-carboxylic acid?2-OH-PCA?and 2-hydroxyphenazine?2-OH-PHZ?.Both PCA and 2-OH-PHZ could be used as antifungal pesticides.PCA is an effective antifungal pesticide and was registered as“Shenqinmycin”in2011 by the Ministry of Agriculture of China as a biologically synthesized fungicide,which is based on its effectiveness against specific phytopathogens and minimal toxicity toward the environment.According to PCA,2-OH-PHZ shows stronger fungistatic and bacteriostatic activity to some pathogens,such as Gaeumannomyces.graminis var.tritici which cause the take-all disease of wheat.The take-all disease is one of the most important root diseases of wheat worldwide,and no resistant cultivars or effective chemical fungicides are available,it is important to develop biological agents to control the disease.2-OH-PHZ is a potential antifungal pesticide,which has a broad application prospect.Although it may be practical to synthesize phenazines by chemical methods,the yield is low,and toxic byproducts such as lead oxide,aniline,o-phenylenediamine,and azobenzoate are produced.Therefore,the biocatalytic synthesis of 2-OH-PHZ using recombinant microorganisms provides an attractive alternative.Until now,2-OH-PHZ has been produced primarily by P.chlororaphis.P.chlororaphis GP72is a 2-OH-PHZ producing strain,although the production by the wild type is only 4.5 mg/L,it is possible to improve the 2-OH-PHZ production largely for its industrial production by genetic engineering and regulation.Different strategies were used to improve the yield of 2-OH-PHZ:knocking out the negative regulatory genes,enhancing the shikimate pathway,deleting the competing pathways of 2-OH-PHZ synthesis based on chorismate,and improving the activity of PhzO which catalyzes the conversion of PCA to 2-OH-PHZ,although the last two strategies did not give us satisfactory results.Firstly,disruption of four negative regulatory genes was used to enhance 2-OH-PHZ production.In P.chlororaphis GP72,the biosynthesis of phenazines has been traced to the shikimate pathway.This pathway begins with DAHP-synthase-mediated condensation of the central carbon metabolism intermediates erythrose 4-phosphate?E4P?and phosphoenol pyruvate?PEP?into 3-deoxy-D-arabinoheptulosonate7-phosphate?DAHP?.When PEP and E4P accumulate,it may channel the metabolic flow to biosynthetic routes of shikimate and chorismate,which are key metabolic precursors of phenazines.According to previous reports,in E.coli strains,increasing the availability of direct precursors PEP was used to enhance the shikimate pathway.PEP is a key intermediate of the central metabolism,it serves as a precursor in several biosynthetic pathways.Pyruvate synthesis from PEP catalyzed by pyruvate kinase?coded by pyk?is one of the main PEP consuming reactions.Therefore weakening or blocking the conversion of PEP to pyruvate is a key strategy to enhance PEP availability.In E.coli,two pyruvate kinases were detected,which were coded by pykA and pykF respectively.In contrast to the disruption of both pyk genes,a single inactivation of either the pykA or pykF genes may increase the quantity of PEP available for DAHP synthesis.This would not impede synthesis of pyruvate or the flux of pyruvate to acetyl-CoA.Similar to E.coli,P.chlororaphis GP72 also contains two pyruvate kinases,which were coded by pykA and pykF.In order to increase the availability of the PEP,the gene pykF of GP72 was selected to knock out.And after the analysis of HPLC,the 2-OH-PHZ production increased from 4.5 mg/L to 35 mg/L.In our previous research,a negative regulator RpeA?coded by rpeA?was insertionally mutated to construct strain GP72AN,which resulted in a fivefold increase of 2-OH-PHZ.In this work,we also knocked out rpeA from the GP72?pyk genome to construct GP72ND-1 with the non-scar deletion strategy.Similar to the result of insertional mutagenesis,the 2-OH-PHZ production of GP72ND-1 had a 5.8-fold increase and reached 239.8 mg/L.RpeA belongs to the RpeA/RpeB two-component signal transduction system?TCST?.Other than RpeA/RpeB,GacS/GacA is the best known TCST system in Pseudomonas.and is a master regulator of secondary metabolism,essential to phenazine production in P.chlororaphis and other Pseudomonas species.Studies have shown that GacA positively controls the expression of RsmX,RsmY,RsmZ,and other small non-coding RNAs which titrate the translational repressors RsmA and RsmE.The contribution made by RsmA and RsmE to phenazine production was assessed in P.chlororaphis30-84.Results indicated that RsmE is involved in the negative regulation of phenazines but RsmA is not.In order to enhance the yield of 2-OH-PHZ,the gene rsmE was knocked out of the strain GP72ND-1 genome to construct GP72ND-2,which resulted in the production of 2-OH-PHZ increasing from 239.8 mg/L to 273.5 mg/L.According to a previous report,in Pseudomonas protegens,the Lon protease can impair the stability of GacA protein and reduce the expression of Gac/Rsm signal transduction pathway.A lon gene knockout mutant has been previously shown to increase the production of antibiotics in P.protegens Pf-5.Importantly,the Gac/Rsm system is conserved in many Gram-negative bacteria,and it activates secondary metabolite production,including production of phenazines in P.chlororaphis 30-84.In order to gain more 2-OH-PHZ,the gene lon was also knocked out from GP72ND-2 to construct GP72ND-3.After fermentation,the yield of 2-OH-PHZ increased from 273.5 mg/L to 300.5mg/L.In this study,four negative regulatory genes?pykF,rpeA,rsmE and lon?were firstly knocked out of the strain GP72 genome stepwise.The yield of 2-OH-PHZ improved more than60 folds and increased from 4.5 mg/L to about 300 mg/L.Secondly,we tried to improve the 2-OH-PHZ production of GP72 by enhanceing the shikimate pathway.According to previous research,several successful strategies have been used to enhance the shikimate pathway in E.coli strains,for example,by increasing the availability of direct precursors PEP and E4P,improving DAHP synthesis in the shikimate pathway,enhancing the metablic flow through the biosynthetic pathway by impeding allosteric and transcriptional regulation,and identifying and interfering with rate-limiting enzymatic reactions.Disruption of pyruvate kinases is not the only way which could increase the availability of the direct precursors of shikimate pathway.Several other strategies have been reported.On the one hand,high PEP availability was achieved by improving the recycling of PYR to PEP by overexpression of gene ppsA?code PEP synthetase?.On the other hand,high E4P availability could be achieved by the overexpression of gene coding for a transketolase?tktA?.So ppsA and tktA in GP72 were selected for overexpression to enhance shikimate pathway.Further increases in carbon flux through the shikimate pathway were realized by removing the allosteric and transcriptional regions and by relieving impeding enzymatic reactions.The reactions catalyzed by DHQ dehydratase?coded by aroD?,quinate/shikimate dehydrogenase?coded by aroE?,dehydroquinic acid?DHQ?synthase?encoded by aroB?and DHAP synthetase?coded by aroG in E.coli?have been reported as limiting steps in the shikimate pathway.Therefore,four key genes of GP72?phzC,aroB,aroD,aroE which code DAHP synthase,DHQ synthase,DHQ dehydratase and SA dehydrogenase respectively?were also selected to enhance the shikimate pathway of GP72.Thus six key genes?ppsA,tktA,phzC,aroB,aroD and aroE?selected from different pathways were overexpressed to enhance the skimate pathway.According to previous report of Juminaga et al,genes located near the promoter usually show much higher levels of induction than those distant from the promoter.The reverse arrangement of genes in the operon was found to benefit from the formation of the desired product through higher concentrations of protein in the latter part of the pathway.Therefore,we followed this method,in which the gene order in the fusion plasmid was aroE-aroD-aroB-phzC-tktA-ppsA,and the reverse order of the expression pathway.When the plasmid pBbB5K-aroE-aroD-aroB-phzC-tktA-ppsA was overexpressed in GP72ND-3,the 2-OH-PHZ production increased to450.4 mg/L?which was near 100-fold of that in the wide type GP72?and phenazine derivatives production also increased to 1520 mg/L.Thirdly,we tried to enhancing the 2-OH-PHZ production by deleting the competing pathways of 2-OH-PHZ synthesis based on chorismate,and improving the activity of PhzO which catalyzes the conversion of PCA to 2-OH-PHZ.P.chlororaphis GP72 synthesizes phenazines with the enzymes encoded by the phz cluster using chorismate as a precursor.GP72 contains at least four additional chorismate-utilizing pathways for the synthesis of folate,tryptophan,co-enzyme Q,tyrosine and phenylalanine.The phenazine biosynthetic pathway must compete with these pathways for chorismate.In order to enhance the production of phenazines,we tried to block these chorismate-utilizing pathways.Thus,pabB/pabC encoding para-aminobenzoate synthase,trpE encoding anthranilate synthase,and pheA encoding chorismate mutase/prephenate dehydratase were all deleted from GP72.The attempt was not successful because of the bad growth of these mutants.According to previous research carried out by our group,in P.chlororaphis GP72,the enzyme PhzO catalyzed the conversion of PCA to 2-OH-PCA.And the 2-OH-PCA was then spontaneously decarboxylated to 2-OH-PHZ.Differing from other P.chlororaphis strains,PCA could not be completely converted to 2-OH-PHZ by PhzO in the GP72 strain because of the high production of PCA.Previous studies have shown the production of 2-OH-PCA in P.chlororaphis GP72AN to be relatively low,only 10%-20%of PCA.In this study,we analyzed the codon usage of phzO.Many rare codons were found to exist in this gene.To improve the expression of phzO,rare codons of the phzO were optimized,yielding phzO^op,the codon-optimized variant of phzO.The phzO^op was introduced into the GP72 and replaced the phzO gene successfully,generating corresponding strains.However,according to the fermentation result,optimization of the phzO gene did not improve the efficiency of PCA to 2-OH-PHZ conversion.This result suggested that the rare codon usage of phzO may not be the reason why PCA cannot be converted to 2-OH-PHZ completely.In this work,four regulatory genes pykF,rpeA,rsmE and lon were firstly knocked out of the strain GP72 genome step by step.Then,ppsA,tktA,phzC,aroB,aroD and aroE six genes were constructed into one BglBrick vector-pBbB5K and overexpressed to enhance the production of phenazine derivatives.A strain producing 450.4 mg/L of 2-OH-PHZ and 1520 mg/L of phenazine derivatives was obtained.This study laid a good foundation for the future industrial production and agricultural application of biopesticide phenazines.