No abstract

Streptomyces are one of the most important industrial microorganisms for the production of proteins and small-molecule drugs. Previously reported flow cytometry-based screening methods can only screen spores or protoplasts released from mycelium, which do not represent the filamentous stationary phase Streptomyces used in industrial cultivation. Here we show a droplet-based microfluidic platform to facilitate more relevant, reliable and rapid screening of Streptomyces mycelium, and achieved an enrichment ratio of up to 334.2. Using this platform, we rapidly characterized a series of native and heterologous constitutive promoters in Streptomyces lividans 66 in droplets, and efficiently screened out a set of engineered promoter variants with desired strengths from two synthetic promoter libraries. We also successfully screened out several hyperproducers of cellulases from a random S. lividans 66 mutant library, which had 69.2–111.4% greater cellulase production than the wild type. Our method provides a fast, simple, and powerful solution for the industrial engineering and screening of Streptomyces in more industry-relevant conditions.

The polyether ionophore antibiotic monensin is produced by Streptomyces cinnamonensis and is used as a coccidiostat for chickens and growth-promoting agent for cattle. Monensin biosynthetic gene cluster has been cloned and partially characterized. The GntR-family transcription factor DasR regulates antibiotic production and morphological development in Streptomyces coelicolor and Saccharopolyspora erythraea. In this study, we identified and characterized the two-level regulatory cascade of DasR to monensin production in S. cinnamonensis. Forward and reverse genetics by overexpression and antisense RNA silence of dasR revealed that DasR positively controls monensin production under nutrient-rich condition. Electrophoresis mobility shift assay (EMSA) showed that DasR protein specifically binds to the promoter regions of both pathway-specific regulatory gene monRII and biosynthetic genes monAIX, monE and monT. Semi-quantitative RT-PCR further confirmed that DasR upregulates the transcriptional levels of these genes during monensin fermentation. Subsequently, co-overexpressed dasR with pathway-specific regulatory genes monRI, monRII or monH greatly improved monensin production.

Actinomycetes are versatile secondary metabolite producers with great application potential in industries. However, industrial strain engineering has long been limited by the inefficient and labor-consuming plate/flask-based screening process, resulting in an urgent need for product-driven high-throughput screening methods for actinomycetes. Here, we combine a whole-cell biosensor and microfluidic platform to establish the whole-cell biosensor and producer co-cultivation-based microfluidic platform for screening actinomycetes (WELCOME). In WELCOME, we develop an MphR-based Escherichia coli whole-cell biosensor sensitive to erythromycin and co-cultivate it with Saccharopolyspora erythraea in droplets for high-throughput screening. Using WELCOME, we successfully screen out six erythromycin hyper-producing S. erythraea strains starting from an already high-producing industrial strain within 3 months, and the best one represents a 50% improved yield. WELCOME completely circumvents a major problem of industrial actinomycetes, which is usually genetic-intractable, and this method will revolutionize the field of industrial actinomycete engineering.

Background Lincomycin, produced by Streptomyces lincolnensis, is a lincosamide antibiotic and widely used for the treatment of the infective diseases caused by Gram-positive bacteria. The mechanisms of lincomycin biosynthesis have been deeply explored in recent years. However, the regulatory effects of LmbU that is a transcriptional regulator in lincomycin biosynthetic (lmb) gene cluster have not been fully addressed. Results LmbU was used to search for homologous LmbU (LmbU-like) proteins in the genomes of actinobacteria, and the results showed that LmbU-like proteins are highly distributed regulators in the biosynthetic gene clusters (BGCs) of secondary metabolites or/and out of the BGCs in actinomycetes. The overexpression, inactivation and complementation of the lmbU gene indicated that LmbU positively controls lincomycin biosynthesis in S. lincolnensis. Comparative transcriptomic analysis further revealed that LmbU activates the 28 lmb genes at whole lmb cluster manner. Furthermore, LmbU represses the transcription of the non-lmb gene hpdA in the biosynthesis of l-tyrosine, the precursor of lincomycin. LmbU up-regulates nineteen non-lmb genes, which would be involved in multi-drug flux to self-resistance, nitrate and sugar transmembrane transport and utilization, and redox metabolisms. Conclusions LmbU is a significant pleiotropic transcriptional regulator in lincomycin biosynthesis by entirely activating the lmb cluster and regulating the non-lmb genes in Streptomyces lincolnensis. Our results first revealed the pleiotropic regulatory function of LmbU, and shed new light on the transcriptional effects of LmbU-like family proteins on antibiotic biosynthesis in actinomycetes.

CRISPR/Cas9-mediated base editors, based on cytidine deaminase or adenosine deaminase, are emerging genetic technologies that facilitate genomic manipulation in many organisms. Since base editing is free from DNA double-strand breaks (DSBs), it has certain advantages, such as a lower toxicity, compared to the traditional DSB-based genome engineering technologies. In terms of Streptomyces, a base editing method has been successfully applied in several model and non-model species, such as Streptomyces coelicolor and Streptomyces griseofuscus. In this study, we first proved that BE2 (rAPOBEC1-dCas9-UGI) and BE3 (rAPOBEC1-nCas9-UGI) were functional base editing tools in Streptomyces lividans 66, albeit with a much lower editing efficiency compared to that of S. coelicolor. Uracil generated in deamination is a key intermediate in the base editing process, and it can be hydrolyzed by uracil DNA glycosidase (UDG) involved in the intracellular base excision repair, resulting in a low base editing efficiency. By knocking out two endogenous UDGs (UDG1 and UDG2), we managed to improve the base editing efficiency by 3.4−67.4-fold among different loci. However, the inactivation of UDG is detrimental to the genome stability and future application of engineered strains. Therefore, we finally developed antisense RNA interference-enhanced CRISPR/Cas9 Base Editing method (asRNA-BE) to transiently disrupt the expression of uracil DNA glycosidases during base editing, leading to a 2.8−65.8-fold enhanced editing efficiency and better genome stability. Our results demonstrate that asRNA-BE is a much better editing tool for base editing in S. lividans 66 and might be beneficial for improving the base editing efficiency and genome stability in other Streptomyces strains.

Monensin, produced by Streptomyces cinnamonensis, is a polyether ionophore antibiotic widely used as a coccidiostat and a growth-promoting agent in agricultural industry. In this study, cyclic AMP receptor protein (Crp), the global transcription factor for regulation of monensin biosynthesis, was deciphered. The overexpression and antisense RNA silencing of crp revealed that Crp plays a positive role in monensin biosynthesis. RNA sequencing analysis indicated that Crp exhibited extensive regulatory effects on genes involved in both primary metabolic pathways and the monensin biosynthetic gene cluster (mon). The primary metabolic genes, including acs, pckA, accB, acdH, atoB, mutB, epi and ccr, which are pivotal in the biosynthesis of monensin precursors malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA, are transcriptionally upregulated by Crp. Furthermore, Crp upregulates the expression of most mon genes, including all PKS genes (monAI to monAVIII), tailoring genes (monBI-monBII-monCI, monD and monAX) and a pathway-specific regulatory gene (monRI). Enhanced precursor supply and the upregulated expression of mon cluser by Crp would allow the higher production of monensin in S. cinnamonensis. This study gives a more comprehensive understanding of the global regulator Crp and extends the knowledge of Crp regulatory mechanism in Streptomyces.

Erythromycin is a clinically important drug produced by the rare actinomycete Saccharopolyspora erythraea. In the wide-type erythromycin producer S. erythraea NRRL 23338, there is a lack of systematical method for promoter engineering as well as a well-characterized promoter panel for comprehensive metabolic engineering. Here we demonstrated a systematical promoter acquiring process including promoter characterization, engineering and high-throughput screening by the droplet-microfluidic based platform in S. erythraea NRRL 23338, and rapidly obtained a panel of promoters with 21.5-fold strength variation for expression fine-tuning in the native host. By comparative qRT-PCR of S. erythraea NRRL 23338 and a high-producing strain S0, potential limiting enzymes were identified and overexpressed individually using two screened synthetic promoters. As a result, erythromycin production in the native host was improved by as high as 137.24 folds by combinational gene overexpression. This work enriches the accessible regulatory elements in the important erythromycin-producing strain S. erythraea NRRL 23338, and also provides a rapid and systematic research paradigm of promoter engineering and expression fine-tuning in the similar filamentous actinomycete hosts.