Identification of a gene from Streptomyces rimosus M527 negatively affecting rimocidin biosynthesis and morphological differentiation.

The polyene macrolide rimocidin, produced by Streptomyces rimosus M527, was found to be highly effective against a broad range of fungal plant pathogens. Current understanding of the regulatory mechanism of rimocidin biosynthesis and morphological differentiation in S. rimosus M527 is limited. NsdA is considered a negative regulator involved in morphological differentiation and biosynthesis of secondary metabolites in some Streptomyces species. In this study, nsdAsr was cloned from S. rimosus M527. The role of nsdAsr in rimocidin biosynthesis and morphological differentiation was investigated by gene deletion, complementation, and over-expression. A ΔnsdAsr mutant was obtained using CRISPR/Cas9. The mutant produced more rimocidin (46%) and accelerated morphological differentiation than the wild-type strain. Over-expression of nsdAsr led to a decrease in rimocidin production and impairment of morphological differentiation. Quantitative RT-PCR analysis revealed that transcription of rim genes responsible for rimocidin biosynthesis was upregulated in the ΔnsdAsr mutant but downregulated in the nsdAsr over-expression strain. Similar effects have been described for Streptomyces coelicolor M145 and the industrial toyocamycin-producing strain Streptomyces diastatochromogenes 1628. KEY POINTS: • A negative regulator for sporulation and rimocidin production was identified. • The CRISPR/Cas9 system was used for gene deletion in S. rimosus M527.

Effects of addition of elicitors on rimocidin biosynthesis in Streptomyces rimosus M527.

The polyene macrolide rimocidin, produced by Streptomyces rimosus M527, is highly effective against a broad range of fungal plant pathogens, but at low yields. Elicitation is an effective method of stimulating the yield of bioactive secondary metabolites. In this study, the biomass and filtrate of a culture broth of Escherichia coli JM109, Bacillus subtilis WB600, Saccharomyces cerevisiae, and Fusarium oxysporum f. sp. cucumerinum were employed as elicitors to promote rimocidin production in S. rimosus M527. Adding culture broth and biomass of S. cerevisiae (A3) and F. oxysporum f. sp. cucumerinum (B4) resulted in an increase of rimocidin production by 51.2% and 68.3% respectively compared with the production under normal conditions in 5-l fermentor. In addition, quantitative RT-PCR analysis revealed that the transcriptions of ten genes (rimA to rimK) located in the gene cluster involved in rimocidin biosynthesis in A3 or B4 elicitation experimental group were all higher than those of a control group. Using a ß-glucuronidase (GUS) reporter system, GUS enzyme activity assay, and Western blot analysis, we discovered that elicitation of A3 or B4 increased protein synthesis in S. rimosus M527. These results demonstrate that the addition of elicitors is a useful approach to improve rimocidin production.Key Points • An effective strategy for enhancing rimocidin production in S. rimosus M527 is demonstrated. • Overproduction of rimocidin is a result of higher expressed structural genes followed by an increase in protein synthesis.

ToyA, a positive pathway-specific regulator for toyocamycin biosynthesis in Streptomyces diastatochromogenes 1628.

The nucleoside antibiotic toyocamycin (TM), which was produced by Streptomyces diastatochromogenes 1628, was found to be highly efficient against a broad range of plant pathogenic fungi. Despite its importance, little is known about the regulation TM biosynthesis. In this study, toyA, located in the TM biosynthetic gene cluster, was identified as a regulatory gene encoding a large ATP-binding regulator of the LuxR family (LAL-family). The role of toyA in TM biosynthesis in S. diastatochromogenes 1628 was investigated by gene deletion, complementation, and over-expression. Gene disruption of toyA resulted in almost loss of TM production. TM production in complemented strain was restored to the level comparable to that in the wild-type strain S. diastatochromogenes 1628. Over-expression of toyA separately controlled by promoter SPL57, SPL21, and permE* in wild-type strain S. diastatochromogenes 1628 led to a 2-fold, 1-fold, and 80% increase in TM production compared with wild-type strain S. diastatochromogenes 1628, respectively. Quantitative RT-PCR analysis revealed that the transcriptional level of toy structural genes was downregulated in the ΔtoyA mutant but restored in complemented strain and further upregulated in the toyA over-expression strain. The detection results from GFP reporter system in Escherichia coli and GUS reporter system and GUS activities in S. albus J1074 and S. diastatochromogenes 1628 showed that ToyA activated the expression of toyB and toyE operon directly and activated the expression of other toy structural genes indirectly. These results indicate that ToyA is essential for TM biosynthesis controlling the expression of structural genes.

Sequential improvement of rimocidin production in Streptomyces rimosus M527 by introduction of cumulative drug-resistance mutations.

Rimocidin is a polyene macrolide that exhibits a strong inhibitory activity against a broad range of plant-pathogenic fungi. In this study, fermentation optimization and ribosome engineering technology were employed to enhance rimocidin production in Streptomyces rimosus M527. After the optimization of fermentation, rimocidin production in S. rimosus M527 increased from 0.11 ± 0.01 to 0.23 ± 0.02 g/L during shake-flask experiments and reached 0.41 ± 0.05 g/L using 5-L fermentor. Fermentation optimization was followed by the generation of mutants of S. rimosus M527 through treatment of the strain with different concentrations of gentamycin (Gen) or rifamycin. One Genr mutant named S. rimosus M527-G37 and one Rifr mutant named S. rimosus M527-R5 showed increased rimocidin production. Double-resistant (Genr and Rifr) mutants were selected using S. rimosus M527-G37 and S. rimosus M527-R5, and subsequently tested. One mutant, S. rimosus M527-GR7, which was derived from M527-G37, achieved the greatest cumulative improvement in rimocidin production. In the 5-L fermentor, the maximum rimocidin production achieved by S. rimosus M527-GR7 was 25.36% and 62.89% greater than those achieved by S. rimosus M527-G37 and the wild-type strain S. rimosus M527, respectively. Moreover, in the mutants S. rimosus M527-G37 and S. rimosus M527-GR7 the transcriptional levels of ten genes (rimAsr to rimKsr) located in the gene cluster involved in rimocidin biosynthesis were all higher than those in the parental strain M527 to varying degrees. In addition, after expression of the single rimocidin biosynthetic genes in S. rimosus M527 a few recombinants showed an increase in rimocidin production. Expression of rimE led to the highest production.

Development and optimization of an intergeneric conjugation system and analysis of promoter activity in Streptomyces rimosus M527.

An efficient genetic transformation system and suitable promoters are essential prerequisites for gene expression studies and genetic engineering in streptomycetes. In this study, firstly, a genetic transformation system based on intergeneric conjugation was developed in Streptomyces rimosus M527, a bacterial strain which exhibits strong antagonistic activity against a broad range of plant-pathogenic fungi. Some experimental parameters involved in this procedure were optimized, including the conjugative media, ratio of donor to recipient, heat shock temperature, and incubation time of mixed culture. Under the optimal conditions, a maximal conjugation frequency of 3.05×10-5 per recipient was obtained. Subsequently, based on the above developed and optimized transformation system, the synthetic promoters SPL-21 and SPL-57, a native promoter potrB, and a constitutive promoter permE* commonly used for gene expression in streptomycetes were selected and their activity was analyzed using gusA as a reporter gene in S. rimosus M527. Among the four tested promoters, SPL-21 exhibited the strongest expression activity and gave rise to a 2.2-fold increase in ß-glucuronidase (GUS) activity compared with the control promoter permE*. Promoter SPL-57 showed activity comparable to that of permE*. Promoter potrB, which showed the lowest activity, showed a 50% decrease in GUS activity compared with the control permE*. The transformation system developed in this study and the tested promotors provide a basis for the further modification of S. rimosus M527.