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

Abstract

Background: 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.

Results: 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).

Conclusions: 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.

Keywords: Comparative genomics; Markerless deletion; Pseudomonas chlororaphis; Reduced-genome; Secondary metabolite.

Fig. 1

An overview of the reduced genome of P. chlororaphis MDS22. Deletions of MD1 through MD22 were illustrated on the circular map of the GP72 genome. The green and purple circles represent the genomes of GP72 and the largest deletion mutant (MDS22), respectively

Fig. 2

Growth curves of parent strain and genome-reduced mutants. Growth trends of all 23 strains were divided into three groups. a Group 1: GP72(rpeA-) and MDS1–8. b Group 2: MDS9–18. c Group 3: MDS19–22. Strains were grown in KMB liquid medium adding 40 mg/L Km at 28 °C and 180 rpm. The values are presented as the means of three replicates, and the error bars indicate the standard deviation (SD)

Fig. 3

Phenotypic characterization of the parent strain GP72(rpeA-) and the genome-reduced mutants (MDS10 and MDS22). a Colony morphology assays were performed on 10 g/L tryptone broth plates (1% agar) containing Congo Red (40 μg/ml) and Coomassie Brilliant Blue (20 μg/ml). Strains were grown in LB liquid medium to late exponential phase, and then 10 μl were spotted onto plates and incubated at 28 °C for 6 days [49]. b Flagellar swimming assays were performed by using semi solid agarose plates. The swimming status was photographed after overnight incubation at 28 °C [51]. Three replicates were used for each sample and experiments repeated for twice

Fig. 4

Production of phenazines by the parent strain and genome-reduced mutants. Production of PCA, 2-OH-PCA, and 2-OH-PHZ by the parent strain GP72(rpeA-) and its mutants after 24 h of fermentation in KMB medium at 28 °C and 180 rpm. The phenazines were extracted and measured by HPLC at a wavelength of 254 nm. The bar indicates the group mean and the error bar indicates SD from triplicate experiments

Fig. 5

Metabolism of strains GP72(rpeA-), MDS10 and MDS22 on Biolog PM5 plates. Suspensions were added to each well of the PM5 microplates at a volume of 150 μl and incubated at 28 °C for 72 h [86]. Red and blue curves denote growth trends of GP72(rpeA-) and MDS10, respectively, while the black one shows information of MDS22, which indicates a significant defect in substance utilization. Wells A1 and A2 are negative control and positive control, respectively. The information of other 94 wells is listed in Additional file 6