Developing genome-reduced Pseudomonas chlororaphis strains for the production of secondary metabolites

Xuemei Shen, Zheng Wang, Xianqing Huang, Hongbo Hu, Wei Wang, Xuehong Zhang

The current chassis organisms or various types of cell factories have considerable advantages and disadvantages. Therefore, it is necessary to develop various chassis for an efficient production of different bioproducts from renewable resources. In this context, synthetic biology offers unique potentialities to produce value-added products of interests. Microbial genome reduction and modification are important strategies for constructing cellular chassis and cell factories. Many genome-reduced strains from Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum and Streptomyces, have been widely used for the production of amino acids, organic acids, and some enzymes. Some Pseudomonas strains could serve as good candidates for ideal chassis cells since they grow fast and can produce many valuable metabolites with low nutritional requirements and strong environmental adaptability. Pseudomonas chlororaphis GP72 is a non-pathogenic plant growth-promoting rhizobacterium that possesses capacities of tolerating various environmental stresses and synthesizing many kinds of bioactive compounds with high yield. These include phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ), which exhibit strong bacteriostatic and antifungal activity toward some microbial pathogens. We depleted 685 kb (10.3% of the genomic sequence) from the chromosome of P. chlororaphis GP72(rpeA-) by a markerless deletion method, which included five secondary metabolic gene clusters and 17 strain-specific regions (525 non-essential genes). Then we characterized the 22 multiple-deletion series (MDS) strains. Growth characteristics, production of phenazines and morphologies were changed greatly in mutants with large-fragment deletions. Some of the genome-reduced P. chlororaphis mutants exhibited more productivity than the parental strain GP72(rpeA-). For example, strain MDS22 had 4.4 times higher production of 2-OH-PHZ (99.1 mg/L) than strain GP72(rpeA-), and the specific 2-OH-PHZ production rate (mmol/g/h) increased 11.5-fold. Also and MDS10 had the highest phenazine production (852.0 mg/L) among all the studied strains with a relatively high specific total phenazine production rate (0.0056 g/g/h). In conclusion, P. chlororaphis strains with reduced genome performed better in production of secondary metabolites than the parent strain. The newly developed mutants can be used for the further genetic manipulation to construct chassis cells with the less complex metabolic network, better regulation and more efficient productivity for diverse biotechnological applications.