In this paper, glucose respiratory quotient (RQ)-feedback control was developed for erythromycin production with a recombinant strain Saccharopolyspora erythraea ZL1004. RQ was confirmed to be an ...ideal online parameter for regulating glucose feed rate. Through feeding glucose to control RQ at 0.85 during 45-100 h and 0.95 during 100-185 h, erythromycin titer and erythromycin A concentration were reached 11.88 and 8.82 g l super(-1) in 50 l fermenter, which were increased by 8.3 and 6.1 % as compared to that with glucose pH-feedback control, respectively. When glucose RQ-feedback control was scaled up to 372-m super(3) fermenter, erythromycin titer and erythromycin A concentration at 155 h were reached 9.12 and 7.12 g l super(-1), respectively, which were 10.5 and 9.4 % higher than that with the original technology (glucose pH-feedback control). To the best of our knowledge, this is the first report on the successful application of glucose RQ-feedback control in erythromycin production, especially in 372-m super(3) fermenter.
Propanol had been widely used as a precursor for erythromycin synthesis in industrial production. However, the knowledge on the exact metabolic fate of propanol was still unclear. In the present ...study, the metabolic fate of propanol in industrial erythromycin-producing strain Saccharopolyspora erythraea E3 was explored via 13C labeling experiments. An unexpected pathway in which propanol was channeled into tricarboxylic acid cycle was uncovered, resulting in uneconomic catabolism of propanol. By deleting the sucC gene, which encodes succinyl-CoA synthetase that catalyse a reaction in the unexpected propanol utilization pathway, a novel strain E3-ΔsucC was constructed. The strain E3-ΔsucC showed a significant enhancement in erythromycin production in the chemically defined medium compared to E3 (786.61 vs 392.94 mg/L). Isotopically nonstationary 13C metabolic flux analysis were employed to characterize the metabolic differences between Saccharopolyspora erythraea E3 and E3-ΔsucC. The results showed that compared with the starting strain E3, the fluxes of pentose phosphate pathway in E3-△sucC increased by almost 200%. The flux of the metabolic reaction catalyzed by succinyl-CoA synthetase in E3-ΔsucC was almost zero, while the glyoxylate bypass flux significantly increased. These new insights into the precursor utilization of antibiotic biosynthesis by rational metabolic engineering in Saccharopolyspora erythraea provided the new vision in increasing industrial production of secondary metabolites.
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•13C labeling experiment revealed the metabolic fate of n-propanol.•A high erythromycin-producing industrial strain has been constructed by engineering the succinyl-CoA synthetase gene.•13C INST-MFA was applied to compare WT and engineered strain E3-ΔsucC.•This work demonstrates the example of “design-build-validate-guidance” metabolic engineering cycle.
Erythromycins (Ers) are clinically potent macrolide antibiotics in treating pathogenic bacterial infections. Microorganisms capable of producing Ers, represented by Saccharopolyspora erythraea, are ...mainly soil-dwelling actinomycetes. So far, Actinopolyspora erythraea YIM90600, a halophilic actinomycete isolated from Baicheng salt field, is the only known Er-producing extremophile. In this study, we have reported the draft genome sequence of Ac. erythraea YIM90600, genome mining of which has revealed a new Er biosynthetic gene cluster encoding several novel Er metabolites. This Er gene cluster shares high identity and similarity with the one of Sa. erythraea NRRL2338, except for two absent genes, eryBI and eryG. By correlating genotype and chemotype, the biosynthetic pathways of 3'-demethyl-erythromycin C, erythronolide H (EH) and erythronolide I have been proposed. The formation of EH is supposed to be sequentially biosynthesized via C-6/C-18 epoxidation and C-14 hydroxylation from 6-deoxyerythronolide B. Although an in vitro enzymatic activity assay has provided limited evidence for the involvement of the cytochrome P450 oxidase EryFAc (derived from Ac. erythraea YIM90600) in the catalysis of a two-step oxidation, resulting in an epoxy moiety, the attempt to construct an EH-producing Sa. erythraea mutant via gene complementation was not successful. Characterization of EryKAc (derived from Ac. erythraea YIM90600) in vitro has confirmed its unique role as a C-12 hydroxylase, rather than a C-14 hydroxylase of the erythronolide. Genomic characterization of the halophile Ac. erythraea YIM90600 will assist us to explore the great potential of extremophiles, and promote the understanding of EH formation, which will shed new insights into the biosynthesis of Er metabolites.
The microbial genome remains a huge treasure trove for the discovery of diverse natural products. Saccharopolyspora erythraea NRRL23338, the industry producer of erythromycin, has a dozen of ...biosynthetic gene clusters whose encoding products are unidentified. Heterologous expression of one of the polyketide clusters pks7 in Streptomyces albus B4 chassis resulted in the characterization of its function responsible for synthesizing both 6-methylsalicyclic acid and 6-ethylsalicyclic acid. Meanwhile, two new 6-ethylsalicyclic acid ester derivatives were isolated as shunt metabolites. Their structures were identified by comprehensive analysis of MS and NMR experiments. Putative functions of genes within the pks7 BGC were also discussed.
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Saccharopolyspora erythraea produces a large number of secondary metabolites with biological activities, including erythromycin. Elucidation of the mechanisms through which the production of these ...secondary metabolites is regulated may help to identify new strategies for improved biosynthesis of erythromycin. In this paper, we describe the systematic prediction and analysis of small non-coding RNAs (sRNAs) in S. erythraea, with the aim to elucidate sRNA-mediated regulation of secondary metabolite biosynthesis. In silico and deep-sequencing technologies were applied to predict sRNAs in S. erythraea. Six hundred and forty-seven potential sRNA loci were identified, of which 382 cis-encoded antisense RNA are complementary to protein-coding regions and 265 predicted transcripts are located in intergenic regions. Six candidate sRNAs (sernc292, sernc293, sernc350, sernc351, sernc361, and sernc389) belong to four gene clusters (tpc3, pke, pks6, and nrps5) that are involved in secondary metabolite biosynthesis. Deep-sequencing data showed that the expression of all sRNAs in the strain HL3168 E3 (E3) was higher than that in NRRL23338 (M), except for sernc292 and sernc361 expression. The relative expression of six sRNAs in strain M and E3 were validated by qRT-PCR at three different time points (24, 48, and 72 h). The results showed that, at each time point, the transcription levels of sernc293, sernc350, sernc351, and sernc389 were higher in E3 than in M, with the largest difference observed at 72 h, whereas no signals for sernc292 and sernc361 were detected. sernc293, sernc350, sernc351, and sernc389 probably regulate iron transport, terpene metabolism, geosmin synthesis, and polyketide biosynthesis, respectively. The major significance of this study is the successful prediction and identification of sRNAs in genomic regions close to the secondary metabolism-related genes in S. erythraea. A better understanding of the sRNA-target interaction would help to elucidate the complete range of functions of sRNAs in S. erythraea, including sRNA-mediated regulation of erythromycin biosynthesis.
c-di-AMP is an essential and widespread nucleotide second messenger in bacterial signaling. For most c-di-AMP synthesizing organisms, c-di-AMP homeostasis and the molecular mechanisms pertaining to ...its signal transduction are of great concern. Here we show that c-di-AMP binds the N-acetylglucosamine (GlcNAc)-sensing regulator DasR, indicating a direct link between c-di-AMP and GlcNAc signaling. Beyond its foundational role in cell-surface structure, GlcNAc is attractive as a major nutrient and messenger molecule regulating multiple cellular processes from bacteria to humans. We show that increased c-di-AMP levels allosterically activate DasR as a master repressor of GlcNAc utilization, causing the shutdown of the DasR-mediated GlcNAc signaling cascade and leading to a consistent enhancement in the developmental transition and antibiotic production in Saccharopolyspora erythraea. The expression of disA, encoding diadenylate cyclase, is directly repressed by the regulator DasR in response to GlcNAc signaling, thus forming a self-sustaining transcriptional feedback loop for c-di-AMP synthesis. These findings shed light on the allosteric regulation by c-di-AMP, which appears to play a prominent role in global signal integration and c-di-AMP homeostasis in bacteria and is likely widespread in streptomycetes that produce c-di-AMP.
The limited catalytic efficiency of cellulose-degrading enzymes restricts cellulose digestion. We investigated the transcriptional regulation of genes encoding key cellulose degrading enzymes, namely ...β-glucosidases, in the industrial actinobacterium Saccharopolyspora erythraea. We observed that the expression of most β-glucosidase-encoding genes was controlled by the availability of nitrogen and phosphate via their respective global regulators, namely GlnR and PhoP. Electrophoretic mobility shift assay demonstrated that GlnR and PhoP bound directly to the promoters of β-glucosidase-encoding genes. Deletion of glnR resulted in lower transcript levels and activity of β-glucosidases, leading to decreased bacterial growth on cellulose. Overexpression of glnR and phoP or nitrogen/phosphate starvation increased the transcript levels and total activity of β-glucosidases. Moreover, GlnR/PhoP-mediated cellobiose utilization was also observed in Streptomyces coelicolor A3(2). These findings provide insights into the regulatory roles played by GlnR and PhoP in coordinating nitrogen/phosphate metabolism and carbohydrate utilization, and indicate potential strategies for cellulose fermentation in the production of bio-based chemicals by actinobacteria.
The regulatory mechanisms underlying the uptake and utilization of multiple types of carbohydrates in actinomycetes remain poorly understood. In this study, we show that GlnR (central regulator of ...nitrogen metabolism) serves as a universal regulator of nitrogen metabolism and plays an important, previously unknown role in controlling the transport of non-phosphotransferase-system (PTS) carbon sources in actinomycetes. It was observed that GlnR can directly interact with the promoters of most (13 of 20) carbohydrate ATP-binding cassette (ABC) transporter loci and can activate the transcription of these genes in response to nitrogen availability in industrial, erythromycin-producingSaccharopolyspora erythraea. Deletion of theglnRgene resulted in severe growth retardation under the culture conditions used, with select ABC-transported carbohydrates (maltose, sorbitol, mannitol, cellobiose, trehalose, or mannose) used as the sole carbon source. Furthermore, we found that GlnR-mediated regulation of carbohydrate transport was highly conserved in actinomycetes. These results demonstrate that GlnR serves a role beyond nitrogen metabolism, mediating critical functions in carbon metabolism and crosstalk of nitrogen- and carbon-metabolism pathways in response to the nutritional states of cells. These findings provide insights into the molecular regulation of transport and metabolism of non-PTS carbohydrates and reveal potential applications for the cofermentation of biomass-derived sugars in the production of biofuels and bio-based chemicals.
Bacteria of the genus
produce important polyketide antibiotics, including erythromycin A (
) and spinosad (
). We herein report the development of an industrial erythromycin-producing strain,
.
...HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other
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 (
) genes were replaced with two bacterial
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
strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into
. The large spinosad gene cluster from
and the actinorhodin gene cluster from
were successfully expressed in the
deletion mutant. Deletion of the endogenous giant polyketide synthase genes
-
, the product of which is not known, and the flaviolin gene cluster (
) 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
strains. Our study demonstrated that the engineered
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
and rare actinomycetes.
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