Comparative genomic and transcriptomic analysis guides to further enhance the biosynthesis of erythromycin by an overproducer

Omics approaches have been applied to understand the boosted productivity of natural products by industrial high-producing microorganisms. Here, with the updated genome sequence and transcriptomic profiles derived from high-throughput sequencing, we exploited comparative omics analysis to further enhance the biosynthesis of erythromycin in an industrial overproducer, Saccharopolyspora erythraea HL3168 E3. By comparing the genome of E3 with the wild type NRRL23338, we identified fragment deletions inside 56 coding sequences and 255 single nucleotide polymorphisms over the genome of E3. Substantial numbers of genomic variations were observed in genes responsible for pathways which were interconnected to the biosynthesis of erythromycin by supplying precursors/cofactors or by signal transduction. Through comparative transcriptomic analysis, L-glutamine/L-glutamate and 2-oxoglutarate were identified as reporter metabolites. Around the node of 2-oxoglutarate, genomic mutations were also observed. Furthermore, the transcriptomic data suggested that genes involved in the biosynthesis of erythromycin were significantly up-regulated constantly, whereas some genes in biosynthesis clusters of other secondary metabolites contained nonsense mutations and were expressed at extremely low levels. Based on the omics association analysis, readily available strategies were proposed to engineer E3 by simultaneously overexpressing sucB (coding for 2-oxoglutarate dehydrogenase E2 component) and sucA (coding for 2-oxoglutarate dehydrogenase E1 component), which increased the erythromycin titer by 71% compared to E3 in batch culture. This work provides more promising molecular targets to engineer for enhanced production of erythromycin by the overproducer.

Uncovering and Engineering a Mini-Regulatory Network of the TetR-Family Regulator SACE_0303 for Yield Improvement of Erythromycin in Saccharopolyspora erythraea

Erythromycins produced by Saccharopolyspora erythraea have broad-spectrum antibacterial activities. Recently, several TetR-family transcriptional regulators (TFRs) were identified to control erythromycin production by multiplex control modes; however, their regulatory network remains poorly understood. In this study, we report a novel TFR, SACE_0303, positively correlated with erythromycin production in Sac. erythraea. It directly represses its adjacent gene SACE_0304 encoding a MarR-family regulator and indirectly stimulates the erythromycin biosynthetic gene eryAI and resistance gene ermE. SACE_0304 negatively regulates erythromycin biosynthesis by directly inhibiting SACE_0303 as well as eryAI and indirectly repressing ermE. Then, the SACE_0303 binding site within the SACE_0303-SACE_0304 intergenic region was defined. Through genome scanning combined with in vivo and in vitro experiments, three additional SACE_0303 target genes (SACE_2467 encoding cation-transporting ATPase, SACE_3156 encoding a large transcriptional regulator, SACE_5222 encoding α-ketoglutarate permease) were identified and proved to negatively affect erythromycin production. Finally, by coupling CRISPRi-based repression of those three targets with SACE_0304 deletion and SACE_0303 overexpression, we performed stepwise engineering of the SACE_0303-mediated mini-regulatory network in a high-yield strain, resulting in enhanced erythromycin production by 67%. In conclusion, the present study uncovered the regulatory network of a novel TFR for control of erythromycin production and provides a multiplex tactic to facilitate the engineering of industrial actinomycetes for yield improvement of antibiotics.

The bacterial iron sensor IdeR recognizes its DNA targets by indirect readout

ABSTRACTIdeR is the main transcriptional regulator controlling iron homeostasis genes in Actinobacteria, including species from the Corynebacterium, Mycobacterium, and Streptomyces genera, as well as the erythromycin-producing bacterium Saccharopolyspora erythraea. Despite being a well-studied transcription factor since the identification of the Diphtheria toxin repressor DtxR three decades ago, the details of how IdeR proteins recognize their highly conserved 19-bp DNA target remain to be elucidated. The results of our structural and mutational studies support a model wherein IdeR uses an indirect readout mechanism, identifying its targets via the sequence-specific DNA backbone structure rather than through direct contacts with the DNA bases. Furthermore, we show that IdeR efficiently recognizes a shorter palindromic sequence corresponding to a half binding site as compared to the full 19-bp target previously reported, expanding the number of potential target genes controlled by IdeR proteins.

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

Bacteria of the genus Saccharopolyspora produce important polyketide antibiotics, including erythromycin A (Sac. erythraea) and spinosad (Sac. spinosa). We herein report the development of an industrial erythromycin-producing strain, Sac. erythraea HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other Saccharopolyspora species and related actinomycetes. To facilitate the integration of natural product BGCs and auxiliary genes beneficial for the production of natural products, the erythromycin polyketide synthase (ery) genes were replaced with two bacterial attB genomic integration sites associated with bacteriophages ϕC31 and ϕBT1. We also established a highly efficient conjugation protocol for the introduction of large bacterial artificial chromosome (BAC) clones into Sac. erythraea strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into Sac. erythraea. The large spinosad gene cluster from Sac. spinosa and the actinorhodin gene cluster from Streptomyces coelicolor were successfully expressed in the ery deletion mutant. Deletion of the endogenous giant polyketide synthase genes pkeA1-pkeA4, the product of which is not known, and the flaviolin gene cluster (rpp) from the bacterium increased the heterologous production of spinosad and actinorhodin. Furthermore, integration of pJTU6728 carrying additional beneficial genes dramatically improved the yield of actinorhodin in the engineered Sac. erythraea strains. Our study demonstrated that the engineered Sac. erythraea strains SLQ185, LJ161, and LJ162 are good hosts for the expression of heterologous antibiotics and should aid in expression-based genome-mining approaches for the discovery of new and cryptic antibiotics from Streptomyces and rare actinomycetes.

Identification and Characterization of a Novel N- and O-Glycosyltransferase from Saccharopolyspora erythraea

Glycosyltransferases are important enzymes which are often used as tools to generate novel natural products. In this study, we describe the identification and characterization of an inverting N- and O-glycosyltransferase from Saccharopolyspora erythraea NRRL2338. When feeding experiments with 1,4-diaminoanthraquinone in Saccharopolyspora erythraea were performed, the formation of new compounds (U3G and U3DG) was observed by HPLC-MS. Structure elucidation by NMR revealed that U3G consists of two compounds, N1-α-glucosyl-1,4-diaminoanthraquinone and N1-β-glucosyl-1,4-diaminoanthraquinone. Based on UV and MS data, U3DG is a N1,N4-diglucosyl-1,4-diaminoanthraquinone. In order to find the responsible glycosyltransferase, gene deletion experiments were performed and we identified the glycosyltransferase Sace_3599, which belongs to the CAZy family 1. When Streptomyces albus J1074, containing the dTDP-d-glucose synthase gene oleS and the plasmid pUWL-A-sace_3599, was used as host, U3 was converted to the same compounds. Protein production in Escherichia coli and purification of Sace_3599 was carried out. The enzyme showed glycosyl hydrolase activity and was able to produce mono- and di-N-glycosylated products in vitro. When UDP-α-d-glucose was used as a sugar donor, U3 was stereoselective converted to N1-β-glucosyl-1,4-diaminoanthraquinone and N1,N4-diglucosyl-1,4-diaminoanthraquinone. The use of 1,4-dihydroxyanthraquinone as a substrate in in vitro experiments also led to the formation of mono-glucosylated and di-glucosylated products, but in lower amounts. Overall, we identified and characterized a novel glycosyltransferase which shows glycohydrolase activity and the ability to glycosylate “drug like” structures forming N- and O-glycosidic bonds.