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Type II polyketides (T2PKs) exhibit a wide range of structural diversity and potent pharmacological activities. However, the optimal chassis for the synthesis of T2PKs remains elusive, impeding the effective mining and production of these compounds. In this study, we identify Streptomyces aureofaciens J1-022, a high-yield producer of chlortetracycline, as a promising chassis for T2PKs synthesis. To mitigate precursor competition, we execute an in-frame deletion of two endogenous T2PKs gene clusters, resulting in a pigmented-faded host, designated Chassis2.0. Compared to conventional Streptomyces chassis, Chassis2.0 demonstrates enhanced efficiency in the production of oxytetracycline, achieving a 370% increase relative to commercial production strains. Additionally, the tri-ring type T2PKs, which includes actinorhodin and flavokermesic acid, are synthesized in Chassis2.0 with high efficiency. Furthermore, an unidentified biosynthetic gene cluster (BGC) associated with pentangular T2PKs is directly activated, leading to the production of a structurally distinct TLN-1. In conclusion, we successfully achieve the efficient synthesis of tri-ring type pigmented products, the overproduction of tetra-ring antibiotics, and the discovery of penta-ring type polyketides in Chassis2.0. These findings underscore the potential of Chassis2.0 as an optimal platform for the discovery and overproduction of T2PKs. Type II polyketides are natural pigments or antibiotics in daily products which are difficult to produce via traditional strategies. Here, the authors develop Streptomyces aureofaciens J1-022 as the chassis strain and engineering it for efficiently production of a diverse range of type II polyketides.

The shake flask cocultures of Aspergillus terreus and Streptomyces rimosus were investigated with regard to the production of mevinolinic acid (lovastatin), oxytetracycline, and other secondary metabolites (SMs). The aim of the study was to determine the effect of inoculum type (spore suspension or preculture) on the levels of SMs in the fermentation broth. Altogether, 17 SMs were detected, including 4 products with confirmed identities, 10 putatively annotated metabolites, and 3 unknown molecules. As observed over the course of qualitative and quantitative analyses, the selection of inoculum type markedly influenced the SM-related outcomes of cocultures. Depending on the coculture initiation procedure, replacing the preculture with spore inoculum positively affected the biosynthesis of oxytetracycline, butyrolactone I, (+)-geodin, as well as the molecules putatively identified as rimocidin, CE-108, and (+)-erdin. It was concluded that the comparative analyses of SM production in filamentous microbial cocultures and monocultures are dependent on the type of inoculum and thus the diversification of inocula is highly recommended in such studies. Furthermore, it was demonstrated that designing a coculture experiment that involves only a single type of inoculum may lead to the underestimation of biosynthetic repertoires of filamentous microorganisms.

The influence of the initial pH on the morphology and secondary metabolite production in cocultures and axenic cultures of Aspergillus terreus and Streptomyces rimosus was investigated. The detected secondary metabolites (6 of bacterial and 4 of fungal origin) were not found in the cultures initiated at pH values less than or equal to 4.0. The highest mean levels of oxytetracycline were recorded in S. rimosus axenic culture at pH 5.0. Initiating the axenic culture at pH 5.9 led to visibly lower product levels, yet the presence of A. terreus reduced the negative effect of non-optimal pH and led to higher oxytetracycline titer than in the corresponding S. rimosus axenic culture. The cocultivation initiated at pH 5.0 or 5.9 triggered the formation of oxidized rimocidin. The products of A. terreus were absent in the cocultures. At pH 4.0, the striking morphological differences between the coculture and the axenic cultures were recorded.

Heterologous expression of bacterial biosynthetic gene clusters is currently an indispensable tool for characterizing biosynthetic pathways. Development of an effective, general heterologous expression system that can be applied to bioprospecting from metagenomic DNA will enable the discovery of a wealth of new natural products. We have developed a new Escherichia coli-based heterologous expression system for polyketide biosynthetic gene clusters. We have demonstrated the over-expression of the alternative sigma factor σ(54) directly and positively regulates heterologous expression of the oxytetracycline biosynthetic gene cluster in E. coli. Bioinformatics analysis indicates that σ(54) promoters are present in nearly 70% of polyketide and non-ribosomal peptide biosynthetic pathways. We have demonstrated a new mechanism for heterologous expression of the oxytetracycline polyketide biosynthetic pathway, where high-level pleiotropic sigma factors from the heterologous host directly and positively regulate transcription of the non-native biosynthetic gene cluster. Our bioinformatics analysis is consistent with the hypothesis that heterologous expression mediated by the alternative sigma factor σ(54) may be a viable method for the production of additional polyketide products.

Streptomyces produce a broad spectrum of biologically active molecules such as oxytetracycline and rimocidin, which are widely used in human and animal treatments. microparticle-enhanced cultivation (MPEC) is one of the tools used for Streptomyces bioprocesses intensification by the control of mycelial morphology. In the present work, morphological changes of Streptomyces rimosus caused by the addition of 10 µm talc microparticles in MPEC were correlated with the biosynthetic activity of the microorganism. Comparing the runs with and without microparticles, major morphological changes were observed in MPEC, including the deformation of pellets, variation of their size, appearance of hyphae and clumps as well as the aggregation of mycelial objects. The presence of talc microparticles also influenced the levels of the studied secondary metabolites produced by S. rimosus. Comparing control and MPEC runs, the addition of talc microparticles increased the amounts of oxytetracycline (9-fold), 2-acetyl-2-decarboxamido-oxytetracycline (7-fold), milbemycin A3+4[O] (3-fold) and CE 108 (1.5-fold), while rimocidin (27-ethyl) and milbemycin β11+4[O] production was reduced. In summary, the addition of talc microparticles to S. rimosus cultivations led to the development of smaller morphological forms like hyphae and clumps as well as to the changes in the amounts of secondary metabolites.

Heterologous expression is an important strategy to activate biosynthetic gene clusters of secondary metabolites. Here, it is employed to activate and manipulate the oxytetracycline (OTC) gene cluster and to alter OTC fermentation process. To achieve these goals, a fast-growing heterologous host Streptomyces venezuelae WVR2006 was rationally selected among several potential hosts. It shows rapid and dispersed growth and intrinsic high resistance to OTC. By manipulating the expression of two cluster-situated regulators (CSR) OtcR and OtrR and precursor supply, the OTC production level was significantly increased in this heterologous host from 75 to 431 mg/l only in 48 h, a level comparable to the native producer Streptomyces rimosus M4018 in 8 days. This work shows that S. venezuelae WVR2006 is a promising chassis for the production of secondary metabolites, and the engineered heterologous OTC producer has the potential to completely alter the fermentation process of OTC production.

There is a critical need to develop novel antibiotics to combat antimicrobial resistance. Streptomyces species are very rich source of antibiotics, typically encoding 20–60 biosynthetic gene clusters (BGCs). However, under laboratory conditions, most are either silent or poorly expressed so that their products are only detectable at nanogram quantities, which hampers drug development efforts. To address this subject, we used comparative genome analysis of industrial Streptomyces rimosus strains producing high titers of a broad spectrum antibiotic oxytetracycline (OTC), developed during decades of industrial strain improvement. Interestingly, large-scale chromosomal deletions were observed. Based on this information, we carried out targeted genome deletions in the native strain S. rimosus ATCC 10970, and we show that a targeted deletion in the vicinity of the OTC BGC significantly induced expression of the OTC BGC, as well as some other silent BGCs, thus suggesting that this approach may be a useful way to identify new natural products.

Previous studies identified a MarR (multiple antibiotic resistance regulator) family transcription factor OtrR in the oxytetracycline biosynthetic gene cluster, which regulated the expression of an efflux pump OtrB. The genes otrB and otrR were divergent arranged and the inter-ORF (open reading frame) region between the two genes contained the promoter otrBp. In this study, we demonstrated that the reverse complementary sequence of otrBp contained the promoter of otrR, and its activity was also repressed by OtrR by sharing the same operator otrO within otrBp, and allosteric regulated by oxytetracycline. Our findings offered a solid base for the synthetic biological application of the bi-direction promoter in controlling two elements at the same time using only one signal molecule.

Background Oxytetracycline (OTC) is a broad-spectrum antibiotic commercially produced by Streptomyces rimosus . Despite its importance, little is known about the regulation of OTC biosynthesis, which hampered any effort to improve OTC production via engineering regulatory genes. Results A gene encoding a Streptomyces antibiotic regulatory protein (SARP) was discovered immediately adjacent to the otrB gene of oxy cluster in S. rimosus and designated otcR . Deletion and complementation of otcR abolished or restored OTC production, respectively, indicating that otcR encodes an essential activator of OTC biosynthesis. Then, the predicted consensus SARP-binding sequences were extracted from the promoter regions of oxy cluster. Transcriptional analysis in a heterologous GFP reporter system demonstrated that OtcR directly activated the transcription of five oxy promoters in E. coli , further mutational analysis of a SARP-binding sequence of oxyI promoter proved that OtcR directly interacted with the consensus repeats. Therefore, otcR was chosen as an engineering target, OTC production was significantly increased by overexpression of otcR as tandem copies each under the control of strong SF14 promoter. Conclusions A SARP activator, OtcR, was identified in oxy cluster of S. rimosus ; it was shown to directly activate five promoters from oxy cluster. Overexpression of otcR at an appropriate level dramatically increased OTC production by 6.49 times compared to the parental strain, thus demonstrating the great potential of manipulating OtcR to improve the yield of OTC production.

Increasing the self-resistance levels of Streptomyces is an effective strategy to improve the production of antibiotics.To increase the oxytetracycline（OTC） production in Streptomyces rimosus,we investigated the cooperative effect of three co-overexpressing OTC resistance genes：one gene encodes a ribosomal protection protein（otrA） and the other two express efflux proteins（otrB and otrC）.Results indicated that combinational overexpression of otrA,otrB,and otrC（MKABC） exerted a synergetic effect.OTC production increased by 179%in the recombinant strain compared with that of the wild-type strain M4018.The resistance level to OTC was increased by approximately two-fold relative to the parental strain,thereby indicating that applying the cooperative effect of self-resistance genes is useful to improve OTC production.Furthermore,the previously identified cluster-situated activator OtcR was overexpressed in MKABC in constructing the recombinant strain MKRABC;such strain can produce OTC of approximately7.49 g L~（（-1））,which represents an increase of 19%in comparison with that of the OtcR-overexpressing strain alone.Our work showed that the cooperative overexpression of self-resistance genes is a promising strategy to enhance the antibiotics production in Streptomyces.