Rational construction of genome-reduced and high-efficient industrial Streptomyces chassis based on multiple comparative genomic approaches

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 chassis S. 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.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1055-7) contains supplementary material, which is available to authorized users.

Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

“…The shared perspectives of synthetic and systems biology together brought into realization the concept of minimal genomes along with their proposed advantages across diverse fields of applications, including expression of heterologous proteins. A growing number of reports already highlight the advantages of genome-reduced microbial strains outperforming their wild-type counterparts in terms of productivity of target proteins [113] and desired biomolecules [114], which result from the elimination of unfavorable features for protein expression and rewired metabolic networks redirected to produce heterologous proteins. In this section, we attempt to provide a brief overview of the concept, design, and mechanism underlying synthesis of the minimal genome.…”

“…Due to relative ease of underlying procedures and cheaper costs, a larger number of top-down genome reduction projects have been published to date as compared with that of bottom-up synthesis (Table 2). Strains subjected to top-down genome-reductions to date include E. coli [123][124][125][126][127], Streptomyces strains [114,[128][129][130][131][132][133], B. subtilis [92,[134][135][136][137][138], L. lactis [139], Pseudomonas putida [140,141], Corynebacterium glutamicum [142], and a yeast strain [143]. This strategy enabled the removal of unnecessary proportions of genome at large scale without causing palpable defects in the target organisms (except in a few instances [135,144]) and sometimes yielded improved cellular performances in terms of growth rates, cell density, transformation efficiency, and protein productivity compared with their wild-type counterparts.…”

Synthetic Biology Tools for Novel Secondary Metabolite Discovery in Streptomyces

“…In vitro Sticky end ligation: pSBAC [113] Blunt end ligation: ICE [95] Gibson assembly: CATCH and MSGE [89,96] In vivo Recombination in native host: IR [114] Recombination in E. coli: LLHR [117] Recombination in yeast: TAR, DNA assembler, DiPac, and mCRISTAR [97,115,116,118] Transferring BGC vector to the expression host Conjugation pUWLcre [136] Protoplast transformation pSKC2 and pOJ446 [137] Target secondary metabolite production by expression of the BGC vector Integrative pSET152, pCAP01, and pESAC [118,138] Replicative pSKC2 and pUWL201 [139] expression vectors in previous studies were integrative vectors, which are more stable after serial generations [109]. Genetic stability is very important for the further fermentation process of secondary metabolites [121]. However, the copy number of the integrative SM-BGC vector is only one compared to multi-copy replicative vectors.…”

“…They include genomic islands (GI), IS elements, and endogenous CRISPR array regions that decrease genomic stability. For example, NGRs in S. avermitilis (1.48 Mb) and S. chattanoogenisis (0.7 Mb) were selected, based on comparative genomics of Streptomyces genomes, and deleted by λ-Red system or Cre/loxP recombination system [121,125]. As expected, deletion of NGRs increased the fitness level of the engineered strain relative to the wild-type strain with beneficial effects on morphology, ATP level, NADPH level, transformation efficiency, and genetic stability in S. chattanoogenisis [121].…”

“…Development of synthetic biology tools can also be exploited to construct the Streptomyces chassis for heterologous expression of novel SM-BGCs. So far, S. albus J1074 with deletion of 15 BGCs and S. chattanoogensis L10 with deletion of 0.7 Mb of a nonessential arm seem to be the best Streptomyces chassis for heterologous expression [121,122]. A further challenge might be the construction of the "superhost Streptomyces chassis" by removing all endogenous SM-BGCs, nonessential genes, and genomic regions and adding all precursor synthetic genes.…”