Engineering Streptomyces diastatochromogenes 1628 to increase the production of toyocamycin

Abstract

Toyocamycin (TM) shows antifungal activity and can be used to treat plant diseases. TM is produced by Streptomyces diastatochromogenes 1628, but the production rate is always very low. Recently, the gene toyG was cloned; however, nothing was known about the influence of toyG on TM production. In this study, the rational combined co‐expression of toyG with frr encoding a ribosome recycling factor and vgb encoding Vitreoscilla hemoglobin was performed. Introduction of these genes in double‐ or triple‐combination into strain S. diastatochromogenes 1628 led to greatly increased TM production. Transcription analysis revealed that higher TM production in the constructed recombinant strains resulted from an increase in transcriptional levels of toyG gene. At the end, among all these different recombinant strains, the strain S. diastatochromogenes 1628‐VGF, harboring vgb, frr, and toyG placed under the control of PermE*, exhibited the largest increase in TM production. Notably, TM produced by S. diastatochromogenes 1628‐VGF reached the highest level of 319.2 mg/L at 72 h, while TM yield of the original strain 1628 was 147.1 mg/L at 84 h. The high‐yield, energy‐saving strain 1628‐VGF constructed in this study shall be beneficial for the industrial production of TM.

Cloning and Overexpression of the Toy Cluster for Titer Improvement of Toyocamycin in Streptomyces diastatochromogenes

“…To improve TM production further, we subsequently engineered the toy cluster to increase transcription of all toy genes. In our earlier study, the constitutive promoter permE ∗ , which is commonly used for gene expression in streptomycetes, was selected to increase the expression of favorable genes to improve TM production in S. diastatochromogenes 1628 ( Ma et al, 2014b ; Tao et al, 2016 ). Siegl et al (2013) developed a synthetic promoter library for actinomycetes based on the −10 and −35 consensus sequences of the constitutive and widely used ermEp1 promoter.…”

“…TM is produced by Streptomyces diastatochromogenes 1628, but the production level is always very low ( Ma et al, 2014a ). In this context, efforts to improve TM production have been made by the overexpression of structural genes ( Tao et al, 2016 ) or favorable genes ( Ma et al, 2014a ; Ma et al, 2014b ) and the pathway-specific regulatory gene toyA ( Xu et al, 2019 ) or ribosome engineering technology ( Ma et al, 2016 ). Expression of an extra copy of toyG and frr led to a 65.5% increase in TM production ( Tao et al, 2016 ).…”

“…In this context, efforts to improve TM production have been made by the overexpression of structural genes ( Tao et al, 2016 ) or favorable genes ( Ma et al, 2014a ; Ma et al, 2014b ) and the pathway-specific regulatory gene toyA ( Xu et al, 2019 ) or ribosome engineering technology ( Ma et al, 2016 ). Expression of an extra copy of toyG and frr led to a 65.5% increase in TM production ( Tao et al, 2016 ). In another study, expression of an extra copy of pathway-specific regulator toyA driven by different promoters led to up to twofold increase in TM production ( Xu et al, 2019 ).…”

Enhancement of toyocamycin production through increasing supply of precursor GTP in Streptomyces diastatochromogenes 1628

“…ToyA is controlling toyB-and toyE-expression by binding to both promoter regions [18]. It has also been shown that overexpression of toyF and toyG results in an improvement of TM production [14,16]. Thus, toyA, toyB, toyE, toyF and toyG are influencing TM production.…”

“…Streptomyces diastatochromogenes 1628 was shown to produce TM [15], but its limited TM yield is still a bottleneck for further industrialization and commercialization. To improve the synthesis level of TM, several methods from genetic modification to ribosome engineering have been applied to improve production [16][17][18]. In addition, it has been reported that TM is synthesized using GTP as the precursor by a multi-step reaction [19] (Figure S1).…”