Engineered Streptomyces lividans Strains for Optimal Identification and Expression of Cryptic Biosynthetic Gene Clusters

Abstract

Streptomyces lividans is a suitable host for the heterologous expression of biosynthetic gene clusters (BGCs) from actinomycetes to discover “cryptic” secondary metabolites. To improve the heterologous expression of BGCs, herein we optimized S. lividans strain SBT5 via the stepwise integration of three global regulatory genes and two codon-optimized multi-drug efflux pump genes and deletion of a negative regulatory gene, yielding four engineered strains. All optimization steps were observed to promote the heterologous production of polyketides, non-ribosomal peptides, and hybrid antibiotics. The production increments of these optimization steps were additional, so that the antibiotic yields were several times or even dozens of times higher than the parent strain SBT5 when the final optimized strain, S. lividans LJ1018, was used as the heterologous expression host. The heterologous production of these antibiotics in S. lividans LJ1018 and GX28 was also much higher than in the strains from which the BGCs were isolated. S. lividans LJ1018 and GX28 markedly promoted the heterologous production of secondary metabolites, without requiring manipulation of gene expression components such as promoters on individual gene clusters. Therefore, these strains are well-suited as heterologous expression hosts for secondary metabolic BGCs. In addition, we successfully conducted high-throughput library expression and functional screening (LEXAS) of one bacterial artificial chromosome library and two cosmid libraries of three Streptomyces genomes using S. lividans GX28 as the library-expression host. The LEXAS experiments identified clones carrying intact BGCs sufficient for the heterologous production of piericidin A1, murayaquinone, actinomycin D, and dehydrorabelomycin. Notably, due to lower antibiotic production, the piericidin A1 BGC had been overlooked in a previous LEXAS screening using S. lividans SBT5 as the expression host. These results demonstrate the feasibility and superiority of S. lividans GX28 as a host for high-throughput screening of genomic libraries to mine cryptic BGCs and bioactive compounds.

Engineering the Erythromycin-Producing Strain Saccharopolyspora erythraea HOE107 for the Heterologous Production of Polyketide Antibiotics

“…We have previously demonstrated that pJTU6728, an attP ϕ BT 1 -based integrative plasmid carrying the transcription factor gene nusG , the global regulator gene afsS cla , and the two drug-efflux pump genes mdfA co and lrmA co , increased the heterologous production of secondary metabolites in an S. lividans host ( Peng et al, 2018 ). To assess the effects of pJTU6728 on the heterologous production of actinorhodin in Sac.…”

“…spinosa NRRL18395. pJTU6728 is a derivative of the integrative vector pMS82 and carries the integration site attP and int loci of the Streptomyces temperate phage ϕBT1 ( Gregory et al, 2003 ) and contains the transcription factor gene nusG , global regulator genes afsRS cla , and the two drug efflux pump genes mdfA co and lrmA co ( Peng et al, 2018 ). pJTU6728 was used to improve the production of heterologous antibiotics in Sac.…”

“…Various techniques, for example, the transformation-associated recombination system ( Kouprina and Larionov, 2016 ), integrase-mediated recombination system ( Olorunniji et al, 2019 ), and bacterial artificial chromosome (BAC) system ( Sosio et al, 2000 ), have been adapted for cloning some large, intact BGCs. However, the current heterologous hosts of actinomycetes mostly belong to the Streptomyces genus, including Streptomyces coelicolor ( Gomez-Escribano and Bibb, 2011 ) ; Streptomyces lividans ( Xu et al, 2016 , 2020 ; Zhao et al, 2016 ; Gao et al, 2017 ; Chen et al, 2018 ; Peng et al, 2018 ) ; Streptomyces avermitilis ( Komatsu et al, 2013 ) ; and Streptomyces albus ( Chater and Wilde, 1976 ; Myronovskyi et al, 2018 ). Few heterologous hosts are derived from rare actinomycetes, mainly due to the lack of efficient genetic manipulation systems that would enable the substantial strain engineering required for removing internal competitive biosynthetic pathways, such as polyketide BGCs ( Pfeifer and Khosla, 2001 ).…”

Clavulanic Acid Production by Streptomyces clavuligerus: Insights from Systems Biology, Strain Engineering, and Downstream Processing

“…Mathematical estimations point out that Streptomycetes are able to produce around 100,000 antibiotics and only 3% of them have been discovered [ 15 ]. Similarly, the existence of cryptic biosynthetic gene clusters (BGCs) suggests a higher capacity of antibiotic production in those organisms [ 16 , 17 , 18 ].…”

Bioprospecting Through Cloning of Whole Natural Product Biosynthetic Gene Clusters

“…Atratumycin (Yang et al, 2019) Neoabyssomicin/abyssomicin (Tu et al, 2018) Avermectins (Deng et al, 2017) Murayaquinone (Peng et al, 2018) FAC libraries…”