Erythromycin Production Research Articles

Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea.

Glycosylation represents an attractive target for protein engineering of novel antibiotics, because specific attachment of one or more deoxysugars is required for the bioactivity of many antibiotic and antitumour polyketides. However, proper assessment of the potential of these enzymes for such combinatorial biosynthesis requires both more precise information on the enzymology of the pathways and also improved Escherichia coli-actinomycete shuttle vectors. New replicative vectors have been constructed and used to express independently the dnmU gene of Streptomyces peucetius and the eryBVII gene of Saccharopolyspora erythraea in an eryBVII deletion mutant of Sac. erythraea. Production of erythromycin A was obtained in both cases, showing that both proteins serve analogous functions in the biosynthetic pathways to dTDP-L-daunosamine and dTDP-L-mycarose, respectively. Over-expression of both proteins was also obtained in S. lividans, paving the way for protein purification and in vitro monitoring of enzyme activity. In a further set of experiments, the putative desosaminyltransferase of Sac. erythraea, EryCIII, was expressed in the picromycin producer Streptomyces sp. 20032, which also synthesises dTDP-D-desosamine. The substrate 3-alpha-mycarosylerythronolide B used for hybrid biosynthesis was found to be glycosylated to produce erythromycin D only when recombinant EryCIII was present, directly confirming the enzymatic role of EryCIII. This convenient plasmid expression system can be readily adapted to study the directed evolution of recombinant glycosyltransferases.

Isocratic separation of erythromycin, related substances and degradation products by liquid chromatography on XTerra RP18

A simple, sensitive, selective and robust isocratic LC method is described for the analysis of erythromycin on XTerra RP18. The main component, erythromycin A, is separated from all known related substances and degradation products. Several unknown impurities are also separated. Acetonitrile-0.2 MK2HPO4pH7.0-water, (35∶5∶60, v/v) was used as a mobile phase at 1.0 mL min−1. UV detection was at 215 nm. The robustness of the method was evaluated by a full-factorial experimental design.

Novel octaketide macrolides related to 6-deoxyerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase

Background: The macrolide antibiotic erythromycin A, like other complex aliphatic polyketides, is synthesised by a bacterial modular polyketide synthase (PKS). Such PKSs, in contrast to other fatty acid and polyketide synthases which work iteratively, contain a separate set or module of enzyme activities for each successive cycle of polyketide chain extension, and the number and type of modules together determine the structure of the polyketide product. Thus, the six extension modules of the erythromycin PKS (DEBS) together catalyse the production of the specific heptaketide 6-deoxyerythronolide B.Results: A mutant strain of the erythromycin producer Saccharopolyspora erythraea, which accumulates the aglycone intermediate erythronolide B, was found unexpectedly to produce two novel octaketides, both 16-membered macrolides. These compounds were detectable in fermentation broths of wild-type S. erythraea, but not in a strain from which the DEBS genes had been specifically deleted. From their structures, both of these octaketides appear to be aberrant products of DEBS in which module 4 has ‘stuttered’, that is, has catalysed two successive cycles of chain extension.Conclusions: The isolation of novel DEBS-derived octaketides provides the first evidence that an extension module in a modular PKS has the potential to catalyse iterative rounds of chain elongation like other type I FAS and PKS systems. The factors governing the extent of such ‘stuttering’ remain to be determined.

Interspecies complementation in Saccharopolyspora erythraea : elucidation of the function of oleP1, oleG1 and oleG2 from the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus and generation of new erythromycin derivatives.

Two glycosyltransferase genes, oleG1 and oleG2, and a putative isomerase gene, oleP1, have previously been identified in the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus. In order to identify which of these two glycosyltransferases encodes the desosaminyltransferase and which the oleandrosyltransferase, interspecies complementation has been carried out, using two mutant strains of Saccharopolyspora erythraea, one strain carrying an internal deletion in the eryCIII (desosaminyltransferase) gene and the other an internal deletion in the eryBV (mycarosyltransferase) gene. Expression of the oleG1 gene in the eryCIII deletion mutant restored the production of erythromycin A (although at a low level), demonstrating that oleG1 encodes the desosaminyltransferase required for the biosynthesis of oleandomycin and indicating that, as in erythromycin biosynthesis, the neutral sugar is transferred before the aminosugar onto the macrocyclic ring. Significantly, when an intact oleG2 gene (presumed to encode the oleandrosyltransferase) was expressed in the eryBV deletion mutant, antibiotic activity was also restored and, in addition to erythromycin A, new bioactive compounds were produced with a good yield. The neutral sugar residue present in these compounds was identified as L-rhamnose attached at position C-3 of an erythronolide B or a 6-deoxyerythronolide B lactone ring, thus indicating a relaxed specificity of the oleandrosyltransferase, OleG2, for both the activated sugar and the macrolactone substrate. The oleP1 gene located immediately upstream of oleG1 was likewise introduced into an eryCII deletion mutant of Sac. erythraea, and production of erythromycin A was again restored, demonstrating that the function of OleP1 is identical to that of EryCII in the biosynthesis of dTDP-D-desosamine, which we have previously proposed to be a dTDP-4-keto-6-deoxy-D-glucose 3, 4-isomerase.

The Effect of Rapeseed Oil Uptake on the Production of Erythromycin and Triketide Lactone by Saccharopolyspora erythraea.

Saccharopolyspora erythraea was grown in an oil-based process medium at two different laboratory scales. The initial concentration of rapeseed oil in the medium was shown not to affect the growth, while addition of oil significantly increased erythromycin A production. Increasing the agitation speed at the 2 L scale increased the growth of the culture and the production of erythromycin A but had little effect on the level of oil remaining in the fermentation. Maximum oil utilization of 50% (w/w) was obtained in both 2 and 7 L cultures. Gas chromatography analysis of the process medium showed that there was no accumulation of fatty acids and glycerides during the fermentations. The specific oil utilization by the recombinant strain of S. erythraea was significantly lower compared to the wild-type strain grown at the same scale and initial oil concentration; the recombinant strain also produced lower concentrations of the novel polyketide, triketide lactone.

Kinetics and manipulation of hyphal breakage and its effect on antibiotic production

Erythromycin production in 1.5 l continuous cultures of Saccharopolyspora erythraea, was correlated with the mean mycelial fragment size, which was influenced by culture agitation (750–1 500 rev./min marine/disk turbine impellors). Kinetic parameters for hyphal breakage were determined, and revealed that more highly branched mycelial fragments (hyphal growth unit ∼14 μm, compared to ∼21 μm) were more resistant to breakage. Branching rate depended on growth rate, the more highly branched mycelium being observed at the higher dilution rate. The breakage rate was inversely related to the mycelial fragment diameter at all dilution rates. Manipulating the size distribution of mycelial fragment by using an ultrasonic filter improved specific antibiotic productivity by 33%.

Effect of shear on morphology and erythromycin production in Saccharopolyspora erythraea fermentations

A complex medium was used to investigate the effects of shear on the S. erythraea fermentation at 7-l scale. Maximum biomass was 11.1 ± 0.5 g l−1 at 1250 rpm (tip speed = 4.45 ms−1), whereas it was 12.7 ± 0.2 g l−1 at 350 rpm (tip speed = 1.07 ms−1). Specific erythromycin production was not stirrer speed dependent in the range of 350 to 1000 rpm and decreased by 10% at stirrer speed of 1250 rpm. Morphological measurements using image analysis showed that the major axis of the mycelia (both freely dispersed and clumps) decreased after the end of the rapid growth phase to a relatively constant value (equilibrium size) dependent on the stirrer speed. The mechanical properties of the cell wall were examined by disruption of fermentation broth in homogeniser and it was shown that mechanical strength of the cell wall increased in a large extent during deceleration phase.

Effect of shear on morphology and erythromycin production in

Effect of shear on morphology and erythromycin production in

Construction of new vectors for high-level expression in actinomycetes.

A new integrative vector (pCJR24) was constructed for use in the erythromycin producer Saccharopolyspora erythraea and in other actinomycetes. It includes the pathway-specific activator gene actII-ORF4 from the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor. The actI promoter and the associated ribosome binding site are located upstream of an NdeI site (5'-CATATG-3') which encompasses the actI start codon allowing protein(s) to be produced at high levels in response to nutritional signals if these signals are faithfully mediated by the ActII-ORF4 activator. Several polyketide synthase genes were cloned in pCJR24 and overexpressed in S. erythraea after integration of the vector into the chromosome by homologous recombination, indicating the possibility that the S. coelicolor promoter/activator functions appropriately in S. erythraea. pCJR24-mediated recombination was also used to place the entire gene set for the erythromycin-producing polyketide synthase under the control of the actI promoter. The resulting strain produced copious quantities of erythromycins and precursor macrolides when compared with wild-type S. erythraea. The use of this system provides the means for rational strain improvement of antibiotic-producing actinomycetes.

Influence of carbon and nitrogen source on synthesis of NADP+-isocitrate dehydrogenase, methylmalonyl-coenzyme A mutase, and methylmalonyl-coenzyme A decarboxylase inSaccharopolyspora erythraeaCA340

Methylmalonyl-coenzyme A (CoA) mutase, methylmalonyl-CoA decarboxylase and NADP+-isocitrate dehydrogenase activity profiles were determined in Saccharopolyspora erythraea CA340, an erythromycin producer, grown in glucose or ammonium chloride. Results show that methylmalonyl-CoA mutase and isocitrate dehydrogenase activities were associated with growth, while methylmalonyl-CoA decarboxylase maximal activity was observed at stationary phase. Isocitrate dehydrogenase synthesis was stimulated by glucose and repressed by ammonium; glucose affected negatively the synthesis of methylmalonyl-CoA mutase, whereas methylmalonyl-CoA decarboxylase production was not affected by carbon or nitrogen sources. These results suggested that the negative effect of glucose or ammonium on erythromycin production could be due, in part, to lower pools of succinyl-CoA and methylmalonyl-CoA.

A genetically engineered strain of Saccharopolyspora erythraea that produces 6,12-dideoxyerythromycin A as the major fermentation product.

The erythromycin producer, Saccharopolyspora erythraea ER720, was genetically engineered to produce 6,12-dideoxyerythromycin A, a novel erythromycin derivative, as the major macrolide in the fermentation broth. Inspection of the biosynthetic pathway for erythromycin would suggest that production of this compound could be achieved simply through the disruption of two genes, that encoding the erythromycin C-6 hydroxylase (eryF) and that encoding the erythromycin C-12 hydroxylase (eryK). The double mutant, however, was found to produce a mixture of 6,12-dideoxyerythromycin A and the precursor, 6-deoxyerythromycin D. Complete conversion to the desired product (to the limit of detection by TLC) was achieved by inserting an additional copy of the eryG gene, encoding the erythromycin 3"-O-methyltransferase and driven by the ermE* promoter, into the S. erythraea chromosome.

Reactor heterogeneity with saccharopolyspora erythraea airlift fermentations

Bioreactor heterogeneity has been studied in a multiconfigurable pilot-scale airlift reactor (0.25 m3) which created different degrees of heterogeneity. The impact of the two sparger configurations, i.e. in the draft tube or the annulus, in conjunction with a marine propeller fitted at the base of the downcomer, on the physiology of Saccharopolyspora erythraea was studied. Cellular growth, morphology, and productivity were compared between airlift and stirred tank reactors. Dissolved oxygen tension heterogeneity caused by differences in dissolved oxygen tension around the vessel did not affect growth, but the reduction of heterogeneity improved the specific erythromycin production rate and final specific production. Erythromycin production was shown to be proportional to the energy dissipation rate. The enhancement of bubble coalescence with increasing apparent viscosity led to the reduction of the sectional gas holdups and the improvement of liquid mixing. The extent of the changes with increasing apparent viscosity was dependent on the broth morphology, reactor configurations, and operating conditions. Copyright 1998 John Wiley & Sons, Inc.

High Performance Liquid Chromatographic Assay of Erythromycin Salts and Esters in Bulk and Pharmaceutical Dosage Forms

Recently, several liquid chromatographic methods for the assay of erythromycin base were developed and are being considered for the routine assay of erythromycin. In this study, we developed simple and rugged liquid chromatographic methods for the assay of erythromycin salts and esters, and used these methods for the assay of erythromycin estolate, erythromycin ethylsuccinate, erythromycin stearate, erythromycin gluceptate, and erythromycin lactobionate in bulk and pharmaceutical formulations. The HPLC methods proved to be simple, sensitive, versatile, and rugged. The developed methods provide an attractive alternative to current official microbiological methods. The HPLC assay results for all commercial erythromycin products tested comply with the USP Antibiotics-Microbial assay specification.

Targeted gene inactivation for the elucidation of deoxysugar biosynthesis in the erythromycin producer Saccharopolyspora erythraea.

The production of erythromycin A by Saccharopolyspora erythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-alpha-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV), ORF17 (eryCIV) and ORF7 (eryBII) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates.

The loading domain of the erythromycin polyketide synthase is not essential for erythromycin biosynthesis in Saccharopolyspora erythraea.

6-Deoxyerythronolide B synthase (DEBS) is a large multifunctional enzyme that catalyses the biosynthesis of the erythromycin polyketide aglycone. DEBS is organized into six modules, each containing the enzymic domains required for a single condensation of carboxylic acid residues which make up the growing polyketide chain. Module 1 is preceded by loading acyltransferase (AT-L) and acyl carrier protein (ACP-L) domains, hypothesized to initiate polyketide chain growth with a propionate-derived moiety. Using recombinant DNA technology several mutant strains of Saccharopolyspora erythraea were constructed that lack the initial AT-L domain or that lack both the AT-L and ACP-L domains. These strains were still able to produce erythromycin, although at much lower levels than that produced by the wild-type strain. In addition, the AT-L domain expressed as a monofunctional enzyme was able to complement the deletion of this domain from the PKS, resulting in increased levels of erythromycin production. These findings indicate that neither the initial AT-L nor the ACP-L domains are required to initiate erythromycin biosynthesis; however, without these domains the efficiency of erythromycin biosynthesis is decreased significantly. It is proposed that in these mutants the first step in erythromycin biosynthesis is the charging of KS1 with propionate directly from propionyl-CoA.

Physiological controls of erythromycin production by Saccharopolyspora erythraea are exerted at least in part at the level of transcription

Erythromycin production by Saccharopolyspora erythraea is confined to the idiophase, derepressed by low initial glucose or phosphate concentration, and repressed when ammonium was used as nitrogen source. The idiophase limitation and effects of low phosphate or ammonium correlated with amounts of ery mRNA revealed by Northern blotting, indicating that they act at least partly at the transcriptional level. © Rapid Science Ltd. 1998

Improved erythromycin production in a genetically engineered industrial strain of Saccharopolyspora erythraea.

An industrial erythromycin production strain of Saccharopolyspora erythraea spp. was used to demonstrate that careful genetic engineering can significantly improve productivity. The chromosomally integrated Vitreoscilla hemoglobin gene (vhb) was shown to enhance the final titer of erythromycin by some 70% compared to the original S. erythraea spp. Overall, specific erythromycin yields were about 2.5 g of erythromycin/g of total protein for S. erythraea::vhb but <1 for the S. erythraea spp. The maximum rates of biosynthesis were 57.5 mg of erythromycin/(L/h) and 24.3 mg/(L/h) for the recombinant strain S. erythraea::vhb and S. erythraea spp., respectively. Overall space-time yield was 100% higher for the S. erythraea::vhb fermentation (1.1 g of erythromycin/(L/day)) than for the S. erythraea spp. fermentation (0. 56 g of erythromycin/(L/day)). The genetic stability of the recombinant strain was high, and no selective pressure was needed throughout the cultivations. Expression of functional Vitreoscilla hemoglobin throughout the cultivations was verified by CO difference spectrum assays.

Use of reaction calorimetry to monitor and control microbial cultures producing industrially relevant secondary metabolites

In this article, bench-scale heat flux calorimetry is applied to monitor complex microbial systems producing secondary metabolites and having a rheologically complex behavior. With such systems, biological metabolic activity can only be measured accurately if calorimetry is improved by on-line correction for stirring power variations using torque measurement. First, a successful application to the production of the antibiotic erythromycin by Saccharopolyspora erythraea is presented. During a batch-culture study, it was shown that heat-flux calorimetry can indicate the two main phases of the process, the exact moment of any substrate depletion, and the nature of the depleted substrate. A fed-batch strategy was set up to optimize erythromycin production. Cultures controlled by calorimetry with pulsed addition of the N-source during the trophophase and of the C-source during the idiophase allowed a higher productivity to be reached. Second, an application of heat-flux calorimetry to the production of a bioinsecticide by the sporulating bacterium Bacillus sphaericus is presented. This project aims to understand, model and control the factors that effect growth, sporulation and insecticide production in a chemically defined medium. A batch-culture study has shown that calorimetry can be used to monitor the different phases of the process (growth, sporulation) and the different substrate depletions. The use of a control strategy to optimize production of the insecticidal protein is now under investigation.

Isolation of isoflavones from soy-based fermentations of the erythromycin-producing bacterium Saccharopolyspora erythraea.

A search for an abundant and economical source of isoflavones, particularly genistein, led to the discovery that the erythromycin-producing organism Saccharopolyspora erythraea also produces this promising new cancer-prevention agent. Erythromycin fermentation is a large-scale, soybean-based process used world-wide for the commercial production of this medically important antibiotic. Results from this study indicate that genistin (the glucoside form of genistein), which is added to the fermentation in the soybean media, was converted to genistein through the action of a beta-glucosidase produced by the organism. Genistein was co-extracted with erythromycin from the fermentation broth, then separated from erythromycin during the second step of the purification process for the production of erythromycin.

A physiological model for the control of erythromycin production in batch and cyclic fed batch culture.

Reported differences in antibiotic production dynamics resulting from altering the growth-limiting nutrient (growth-dissociated production in carbon-limited culture and apparent growth-associated production in nitrogen-limited culture) are due to the different effects on growth kinetics. The substrate affinity for nitrate is significantly lower than that for glucose, resulting in nitrogen limitation effectively occurring throughout the culture. Glucose limitation occurs later in the culture, coinciding with the induction of antibiotic production. Induction occurs at the start of nitrogen-limited culture so that production appears to be growth-associated. Evidence that this hypothesis is consistent with production kinetics in cyclic fed batch culture was also obtained.