Abstract
BackgroundStreptomyces chattanoogensis L10 is the industrial producer of natamycin and has been proved a highly efficient host for diverse natural products. It has an enormous potential to be developed as a versatile cell factory for production of heterologous secondary metabolites. Here we developed a genome-reduced industrial Streptomyces chassis by rational ‘design-build-test’ pipeline.ResultsTo identify candidate large non-essential genomic regions accurately and design large deletion rationally, we performed genome analyses of S. chattanoogensis L10 by multiple computational approaches, optimized Cre/loxP recombination system for high-efficient large deletion and constructed a series of universal suicide plasmids for rapid loxP or loxP mutant sites inserting into genome. Subsequently, two genome-streamlined mutants, designated S. chattanoogensis L320 and L321, were rationally constructed by depletion of 1.3 Mb and 0.7 Mb non-essential genomic regions, respectively. Furthermore, several biological performances like growth cycle, secondary metabolite profile, hyphae morphological engineering, intracellular energy (ATP) and reducing power (NADPH/NADP+) levels, transformation efficiency, genetic stability, productivity of heterologous proteins and secondary metabolite were systematically evaluated. Finally, our results revealed that L321 could serve as an efficient chassis for the production of polyketides.ConclusionsHere we developed the combined strategy of multiple computational approaches and site-specific recombination system to rationally construct genome-reduced Streptomyces hosts with high efficiency. Moreover, a genome-reduced industrial Streptomyces chassisS. chattanoogensis L321 was rationally constructed by the strategy, and the chassis exhibited several emergent and excellent performances for heterologous expression of secondary metabolite. The strategy could be widely applied in other Streptomyces to generate miscellaneous and versatile chassis with minimized genome. These chassis can not only serve as cell factories for high-efficient production of valuable polyketides, but also will provide great support for the upgrade of microbial pharmaceutical industry and drug discovery.
Highlights
Streptomyces chattanoogensis L10 is the industrial producer of natamycin and has been proved a highly efficient host for diverse natural products
Many studies had suggested that mobile genetic elements (MGEs), genomic islands (GIs) and biosynthesis gene clusters (BGCs) were dispensable
The results indicated that there were about 34 biosynthesis gene clusters (BGCs), 20 genomic islands (GIs) and 105 insertion sequence (IS) elements, and most of them were located at the two sub-telomeric regions
Summary
Streptomyces chattanoogensis L10 is the industrial producer of natamycin and has been proved a highly efficient host for diverse natural products. A tremendous number of secondary metabolites produced by Streptomyces were utilized as lead compounds in medicine such as clinically important anticancer, antibiotic, anti-inflammatory, antiviral, antiparasitic, antioxidant and anti-malaria drugs [1,2,3,4]. Genome analysis revealed that about 20–40 biosynthesis gene clusters were distributed in each Streptomyces Most of these gene clusters were cryptic under standard laboratory conditions. Lots of Streptomyces were quite difficult to cultivate or even uncultivable under laboratory conditions, grown slowly, lacked efficient genetic manipulation, possessed complicated regulatory networks or produced a large number of endogenous by-products which impeded the progress of drug discovery [6, 7]
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