Expression And Regulation Of Phenazine Synthesis Cluster & Fermentation Optimization Of Phenazine-1-carboxylic Acid

Pseudomonas sp. strain M18, isolated as a new plant growth-promoting rhizobacteria around the melon roots in the suburb of Shanghai, would be promising for use as a powerful bio-control agent due to its secondary metabolite phenazine-1-carboxylic acid (PCA) which has a broad-spectrum antibacterial activity. It is well known that GacA/GacS two-component signal transduction system, quorum sensing system and environmental factors are involved in the synthesis of PCA. The objectives of this study were to: (1) confirm the regulation elements of phz cluster in M18 and the regulatory mechanisms of both GacA/GacS and QS system related to the synthesis of PCA at molecular level; (2) deal with the effects of environmental factors on PCA synthesis at cell level; (3) improve PCA yields with the helps of optimized medium, batch and fed-batch optimal control strategies which were attributed to the fermentation technology of strain M18G. The following are main results.Strain M18 have the similar genetic background to those of Pseudomonas aeruginosa and Pseudomonas fluorescens, so the conservative primers for strain M18 were designed according to the sequences of phz gene clusters from P. aeruginosa able to involve in the synthesis of PCA. Two phz gene clusters amplified from strain M18, named as phzA1B1C1D1E1F1G1(phz1)and phzA2B2C2D2E2F2G2(phz2), shared a 99% homology with that of P. aeruginosa PAO1. The flank sequences of two phz clusters in strain M18 were close to that of P. aeruginosa PAO1, but the non-coding region downstream of the phzA2-G2 in strain M18 contained a three-144bp-repeat sequence which did not exist in strain P. aeruginosa PAO1. It meant that some genetic differences in contributing to the formation of PCA existed between strains M18 and PAO1. Meanwhile, two mutants of strain M18P1 and M18P2 with two phz clusters knocked out were constructed. The phz2 cluster was dominant in the PCA biosynthesis of strain M18, although PCA synthesis were regulated by environmental factors.The mutant strain M18GP1 and M18GP2 with phz1 and phz2 clusters knocked-out based on gacA-inactivated strain M18G, respectively. Both RT-PCR and phz-lacZ fusions methods was used to analyze the expression features of two phz clusters and the regulation on them by global regulator GacA in strain M18. It was found to that GacA have a selective regulation model on two phz gene clusters. The phz1 expression was positive regulated, while phz2 expression was negative regulated by gacA at transcription level. Therefore, the regulatory activity of GacA in strain M18 on the whole transcription level did not favor PCA synthesis due to negatively regulation of phz2 clusters expression.5'race method was used to confirmed the transcription start sites at regulation regions of two phz clusters , while the regulation region of gene were systemic knocked out and phz-lacZ fusion with different length plasmids were constructed. There existed a powerful inhibitor regions located in the downstream 1 to 90 bp of phz1 transcription start site, which led to inhibit phz1expression at post-transcription level and nearly abolish its role on PCA synthesis. Gene gacA did not regulate the inhibitor at post-transcriptional level. Another inhibitor region located in the downstream 1 to 131 of phz2 transcription start site, which also led to inhibit phz2 expression at post-transcription level, but negatively regulated by GacA. Furthermore, regulation of LasR and RhlR from QS system also affected the expression of both phz gene clusters. Rhl promoted the expression of phz1 and phz2, while LasR only promoted the expression of phz1 but negatively regulated phz2 expression. Correspondingly, Rhl promoted the yield of PCA in strain M18,but LasR negatively supported the PCA synthesis. The differences of GacA/GacS and QS system in regulating phenazine synthesis between strain M18 and strain PAO1 might attribute to their evolution pressures, because the two strains were adapted themselves to different niches.The effects of environmental physical-chemical factor on PCA synthesis were investigated in strain M18G. It was seen that the regulator GacA/GacS in strain M18 was sensitive to high temperature, high oxygen and low pH or alkaline environments. However, the mutant M18G adapted easily stress conditions due to the knocked-out of its gacA. In addition, the simulation of different nutrients simulated plant rhizoshpere exudation showed the nutrition took up differently between the strain M18 and the mutant M18G. Glucose and organic nitrogen promoted the mutant M18 to synthesis more PCA than strain M18 did. It was clear that the mutant M18G had a better environment tolerance than that of strain M18, and showed high commercial potential for the production of PCA of M18G.Then the mutant M18G which gacA was inactivated used to produce the secondary metabolite PCA. Four key components from 12 fermentation nutrients with the help of stastic Plackett-Burman design (PB) and Response Surface Method (RSM) were determined in promoting PCA production. The analysis of quadratic equation showed the four key nutrients were soybean meal (33.4 g/L), glucose (12.7 g/L), soy peptone (10.9 g/L), and ethano (13.8 g/L). Soybean meal and ethanol played much more crucial roles in improving the production of PCA. The highest PCA production reached 1.89 g/L after the optimized fermentation process went to 60h, and the PCA yield was increased to 6 fold compared to the non-optimized fermentation process. 10L fermentor trials were to prove the advantages of the optimized fermentation process. Furthermore, the batch fermentation of PCA with mutant M18G was studied according to previous optimal medium. Controlled-agitation of 250rpm (aeration 660L/h) produced 1873 mg/L of PCA after 60 h fermentation. A kinetic model composing of the parameters including cell growth, product formation and substrate consumption, was constructed to describe the batch fermentation process. The fitting curves of three parameters by model were consistent with the experiment values. The control of agitation by different phases, especially relatively lower in latter fermentation phases, promoted PCA yield to 2190mg/L. The concentrations of original glucose and dissolved oxygen in medium affected PCA in batch fermentation of mutant M18G. The fixing of initial glucose concentration to 12-14 g/L and dissolved oxygen to 20% in latter fermentation period improved PCA yields. Feeding of Glucose to fermentor in two-phase modes favored PCA production to 2597 mg/L if total quantity of glucose feed controlled to 6.6g/L throughout the whole fermentation of mutant M18G. In this case, the synthesis rate of PCA (rp) was 36.1 mg/L·h. The production and synthesis rate of PCA increase of 31.7% and 9.39% in fed-batch fermentation over than that of batch fermentation.In conclusion, this present study explained how the two phz gene clusters of Pseudomonas strain M18 were expressed and regulated by GacA/GacS and QS systems plus environmental conditions to synthesis of PCA. Genetic modification of strain M18 to mutant M18G significantly promoted the production of PCA with 9-fold higher in coupling with the optimized liquid fermentation technology including RSM, batch fermentation and fed-batch strategies.This study gives a new way to apply strain M18 as a higher PCA production to biological control.