Substantial improvement of toyocamycin production in Streptomyces diastatochromogenes by cumulative drug-resistance mutations

Abstract

Toyocamycin is a member of the nucleoside antibiotic family and has been recognized as a promising fungicide for the control of plant diseases. However, low productivity of toyocamycin remains an important bottleneck in its industrial production. Therefore, dramatic improvements of strains for overproduction of toyocamycin are of great interest in applied microbiology research. In this study, we sequentially selected for mutations for multiple drug resistance to promote the overproduction of toyocamycin by Streptomyces diastatochromogenes 1628. The triple mutant strain, SD3145 (str str par), was obtained through sequential screenings. This strain showed an enhanced capacity to produce toyocamycin (1500 mg/L), 24-fold higher than the wild type in GYM liquid medium. This dramatic overproduction was attributed at least partially to the acquisition of an rsmG mutation and increased gene expression of toyA, which encodes a LuxR-family transcriptional regulator for toyocamycin biosynthesis. The expression of toyF and toyG, probably directly involved in toyocamycin biosynthesis, was also enhanced, contributing to toyocamycin overproduction. By addition of a small amount of scandium (ScCl3·6H2O), the mutant strain, SD3145, produced more toyocamycin (2664 mg/L) in TPM medium, which was the highest toyocamycin level produced in shake-flask fermentation by a streptomycete so far. We demonstrated that introduction of combined drug resistance mutations into S. diastatochromogenes 1628 resulted in an obvious increase in the toyocamycin production. The triple mutant strain, SD3145, generated in our study could be useful for improvement of industrial production of toyocamycin.

Substantial improvement of tetraene macrolide production in Streptomyces diastatochromogenes by cumulative drug resistance mutations

“…In our previous work, we demonstrated that the triple mutant SD3145 produced a higher yield (1959 mg/L) of toyocamycin, which was 11-fold greater than that produced by the wild-type 1628. In addition, this mutant grew slowly [17]. In general, drug resistance is obtained accompanied by cost of growth fitness.…”

“…Realtime quantitative PCR (qRT-PCR) was carried out using an Applied Biosystems StepOne-Plus Real-Time PCR System with FastStar Universal SYBR Green Master (Rox) (Roche) [17]. The gene primers used in qRT-PCR reactions are listed in S1 Table. For each gene, all PCR reactions were carried out in triplicate within a single plate, with rpoA used as the reference gene.…”

“…The introduction of drug-resistance by ribosome engineering often markedly changes the growth characteristic of mutants such as growth velocity in addition to antibiotic productivity [17,18]. The batch fermentation experiments were scaled up using a 5-L fermentor to further investigate the growth characteristic of wild-type strain 1628 and mutant strains (G1-99, G2-13, G3-13, G4-118 and G5-59) (Fig 1A-1F).…”

“…This ribosome engineering technology holds several advantages, i) the ability to screen for drug-resistance mutations through a simple selection that can be carried out on drug-containing plates even in the case of mutations with extremely low frequency (e.g., <10 −10 ) and ii) the ability to select for mutations in the absence of known genetic information [16]. Interestingly, the introduction of several drug resistance mutations has a cumulative effect on antibiotic production [17,18].…”

“…In our previous work, a strain S. diastatochromogenes (No.1628) was screened for biocontrol [17,19]. Further studies demonstrated that this strain can produce at least 7 tetraene macrolides on the basis of ultraviolet absorption characteristics.…”