Precursor-directed production of erythromycin analogs by Saccharopolyspora erythraea.

Diketide N-acetylcysteamine (diketide NAC) thioester precursors were fed to 6-Deoxyerythronolide B synthase (DEBS) ketosynthase-1 inactivated (KS1 degree) Saccharopolyspora erythraea strains to produce 13-substituted erythromycin analogs. This direct feeding process potentially represents a simplified production process over the current analog production system. Titers of these analogs were observed to increase linearly with the diketide concentration up to a precursor-specific saturation level. However, the rate of product formation was lower and the rate of diketide consumption higher with S. erythraea than was previously observed with a recombinant strain of Streptomyces coelicolor. Several strategies were pursued to address the issue of these high diketide consumption rates: (1) elucidation of the locale of diketide degradation, (2) addition of beta-oxidation inhibitors to the cultures, and (3) addition of a sacrificial diketide enantiomer to occupy putative degradative enzymes. Additionally, repeated addition of diketide to an S. erythraea KS1 degrees culture indicated that the titer of these erythromycin analogs is also currently limited by a shorter production period than observed during erythromycin synthesis by the parent strain. These results indicate potential avenues for expanding the use of this precursor-directed system from the generation of limited quantities of erythromycin analogs to a large-scale production system for these compounds.     

Improved production of erythromycin by Saccharopolyspora erythraea by various plant oils

Various plant oils (50 g l^–1) increased the production of erythromycin by Saccharopolyspora erythraea. Maximum titer of erythromycin in media containing black cherry kernel, walnut, rapeseed, olive and cottonseed oils and control medium were 3.5, 2.8, 2.6, 2.1, 1.9, 0.7 g l^–1, respectively. Erythromycin production media containing rapeseed or cottonseed oil was growth-dependent but not in other media used.     

Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production

Background  

There is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.     

Cosynthesis of monofluoroacetate and 4-fluorothreonine by resting cells of blocked mutants of Streptomyces cattleya NRRL8057

Streptomyces cattleya NRRL 8057 produces monofluoroacetate and 4-fluorothreonine from inorganic fluoride. Mutants blocked in fluorometabolite production were prepared by chemical mutagenesis, and cosynthesis experiments with these blocked mutants were carried out by suspending cells of one blocked mutant in the supernatant broth of another blocked mutant. The harvest age of the cells, pH of the buffer, potassium fluoride concentration and glycerol supplementation were optimized for the monofluoroacetate production by a resting-cell suspension of S. cattleya. Successful cosynthesis with pairs of the mutants characterized four distinctive blocked sites in the order N-82, N-44, N-43 and N-47. Additional preparation of blocked mutants by UV irradiation and their cosynthesis assay confirmed that U-303, U-304, U-400 and U-500 were blocked in later steps than N-47. O’Hagan et al. recently proposed that fluoroacetaldehyde, the hypothetical precursor of monofluoroacetate and 4-fluorothreonine, derives from 5′-fluoro-5′-deoxyadenosine, the first fluorinated metabolite synthesized from S-adenosyl- -methionine and inorganic fluoride by the novel enzyme ‘fluorinase’. We were able to detect fluorinase activity in crude extracts of wild type and N-47 mutant strains, but not in the other mutant strains whose blocked steps flanked that of N-47.     

An assessment of seed quality on erythromycin production by recombinant Saccharopolyspora erythraea strain

An assessment of seed quality on erythromycin production by recombinant strain Saccharopolyspora erythraea ZL1004 was investigated in 15 l fermenter. Adding 10 g/l corn steep liquor and 30 g/l soybean flour in seed medium were beneficial to improve cell growth, and the maximal biomass reached 36% at 40 h. Enzyme activity in cell showed that the maximal protease and minimum amylase were appeared in this stage. Compared with the control in 50 l fermenter, the cell metabolism with inoculation of the optimized seed cultivation was obviously quicker, and physiological response such as oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) were also improved. The maximal erythromycin A production was 9160 U/ml at 215 h, which was increased by 21.63% with respect to the control. It was the first report to integrate cell growth characteristics and physiological response method to assess the seed quality for erythromycin production.     

Improvement of erythromycin production by Saccharopolyspora erythraea in molasses based medium through cultivation medium optimization

In the present work, erythromycin production was carried out in submerged culture using Saccharopolyspora erythraea. Different experiments were conducted to optimize the cultivation medium through the change of carbon and nitrogen sources to cheaper one in order to reduce the cost of medium and to utilize sugar cane molasses as one of major sugar industry by-products in Egypt. It was found that the addition of sugar cane molasses a sole carbon source at a concentration of 60 g/l accompanied by corn steep liquor (as organic N-source) in combination with ammonium sulphate (as inorganic N-source) gave the maximal erythromycin production. The antibiotic production in this medium reached about 600 mg/l which is about 33% higher than the value obtained in glucose based medium. On the other hand, the addition of n-propanol in concentration of 1% (v/v) increased the antibiotic production reaching about 720 mg/l after 144 h. Concluding, the new medium formulation based on cheap carbon source, sugar cane molasses, was a good alternative solution for the production of erythromycin economically.     

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^у at 1250 rpm (tip speed = 4.45 ms^у), whereas it was 12.7 - 0.2 g l^у at 350 rpm (tip speed = 1.07 ms^у). 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.     

Application of oxygen uptake rate and response surface methodology for erythromycin production by Saccharopolyspora erythraea

A process for efficient production of erythromycin by Saccharopolyspora erythraea using statistical designs and feeding strategy was developed. The critical nutrient components were selected in accordance with fractional factorial design and were further optimized via response surface methodology. Three significant components (ZnSO₄, citric acid threonine) were identified for the optimization study. The optimum levels of these significant variables were determined with Box–Behnken design, which were ZnSO₄ 0.039 g/l, citric acid 0.24 g/l and threonine 0.42 g/l, respectively. A novel feeding strategy based on oxygen uptake rate (OUR) measurement was developed successfully to increase the flux of erythromycin biosynthesis, in which the optimized nutrient components was fed in the 50 l stirred bioreactor when OUR began to decline at 46 h. The maximum erythromycin production reached 10,622 U/ml, which was 11.7% higher than the control in the same cultivation conditions. It was the first report to integrate physiological parameter OUR and statistical methods to optimize erythromycin production.     

The effect of culture conditions on the production of erythromycin by Saccharopolyspora erythraea in batch culture

Summary Saccharopolyspora erythraea growth is inhibited when grown at a low constant dissolved oxygen tension (DOT) of 10% air saturation. However, the specific erythromycin production is virtually identical to that of a culture where the DOT did not fall below 65%. In addition, at constant DOT (10%) a stirrer speed of 750 rpm in a 7 litre causes mechanical damage to the mycelia in comparison with result at 500 rpm.     

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production

Engineering of the methylmalonyl-CoA (mmCoA) metabolite node of the Saccharopolyspora erythraea wild-type strain through duplication of the mmCoA mutase (MCM) operon led to a 50% increase in erythromycin production in a high-performance oil-based fermentation medium. The MCM operon was carried on a 6.8kb DNA fragment in a plasmid which was inserted by homologous recombination into the S. erythraea chromosome. The fragment contained one uncharacterized gene, ORF1; three MCM related genes, mutA, mutB, meaB; and one gntR-family regulatory gene, mutR. Additional strains were constructed containing partial duplications of the MCM operon, as well as a knockout of ORF1. None of these strains showed any significant alteration in their erythromycin production profile. The combined results showed that increased erythromycin production only occurred in a strain containing a duplication of the entire MCM operon including mutR and a predicted stem-loop structure overlapping the 3' terminus of the mutR coding sequence.     

A new modular polyketide synthase in the erythromycin producer Saccharopolyspora erythraea

A previously unidentified set of genes encoding a modular polyketide synthase (PKS) has been sequenced in Saccharopolyspora erythraea, producer of the antibiotic erythromycin. This new PKS gene cluster (pke) contains four adjacent large open reading frames (ORFs) encoding eight extension modules, flanked by a number of other ORFs which can be plausibly assigned roles in polyketide biosynthesis. Disruption of the pke PKS genes gave S. erythraea mutant JC2::pSBKS6, whose growth characteristics and pattern of secondary metabolite production did not apparently differ from the parent strain under any of the growth conditions tested. However, the pke PKS loading module and individual pke acyltransferase domains were shown to be active when used in engineered hybrid PKSs, making it highly likely that under appropriate conditions these biosynthetic genes are indeed expressed and active, and synthesize a novel polyketide product.     

Fermentation optimization and industrialization of recombinant Saccharopolyspora erythraea strains for improved erythromycin a production

Improvement of Erythromycin A (Er-A) production and purity by metabolic engineering of the industrial erythromycin-producing strains Saccharopolyspora erythraea strians ZL1004 and ZL1007, in which the amounts of tailoring enzymes EryK (a P450 hydroxylase) and EryG (an S-adenosylmethionine-dependent O-methyltransferase) for biotransformation of Erythromycin D to Er-A were modulated, was performed in a 50 L fermentor. Addition of 15 g/L of corn steep liquor to the medium increased Er-A production; maximum Er-A production was 8,196 U/mL at 191 h, which was 81.8% higher than that of control (4,507 U/mL at 184 h). Er-B impurities were completely eliminated, whereas Er-C impurities were only 153 U/mL at 191 h. Analysis of intra- and extracellular metabolites and key enzyme activities in central carbon metabolism revealed that the pool of TCA cycle intermediates was enhanced by the addition of corn steep liquor and induced an increase in erythromycin biosynthesis. There were no significant differences between strains ZL1004 and ZL1007 regarding Er-A production and impurity accumulation. Compared to wild type strain, Er-A production was improved by 23.9% while Er-C was reduced by 83.9% and Er-B was completely eliminated. Furthermore, fermentation of recombinant strain ZL1004 was successfully scaled up from laboratory scale (50 L fermentor) to industrial scale (25 and 132 m³), with similar levels of Er-A production and purity obtained.     

Reconstruction of the Saccharopolyspora erythraea genome-scale model and its use for enhancing erythromycin production

Genome-scale metabolic reconstructions are routinely used for the analysis and design of metabolic engineering strategies for production of primary metabolites. The use of such reconstructions for metabolic engineering of antibiotic production is not common due to the lack of simple design algorithms in the absence of a cellular growth objective function. Here, we present the metabolic network reconstruction for the erythromycin producer Saccharopolyspora erythraea NRRL23338. The model was manually curated for primary and secondary metabolism pathways and consists of 1,482 reactions (2,075 genes) and 1,646 metabolites. As part of the model validation, we explored the potential benefits of supplying amino acids and identified five amino acids “compatible” with erythromycin production, whereby if glucose is supplemented with this amino acid on a carbon mole basis, the in silico model predicts that high erythromycin yield is possible without lowering biomass yield. Increased erythromycin titre was confirmed for four of the five amino acids, namely valine, isoleucine, threonine and proline. In bioreactor experiments, supplementation with 2.5?% carbon mole of valine increased the growth rate by 20?% and simultaneously the erythromycin yield on biomass by 50?%. The model presented here can be used as a framework for the future integration of high-throughput biological data sets in S. erythraea and ultimately to realise strain designs capable of increasing erythromycin production closer to the theoretical yield.     

Effect of small scale culture vessel type on hyphal fragment size and erythromycin production in Saccharopolyspora erythraea

Erythromycin production dynamics in stirred, baffled shaken and non-baffled shaken flasks was strongly correlated with the different distributions of hyphal particle diameters observed. Production only took place when hyphal fragments with diameters greater than 88 m were observed. Results are consistent with significant hyphal breakage rates, even in non-baffled shaken flasks.     

Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea

In carbohydrate-based fermentations of Saccharopolyspora erythraea, a polar knockout of the methylmalonyl-CoA mutase (MCM) gene, mutB, improved erythromycin production an average of 126% (within the range of 102–153% for a 0.95 confidence interval). In oil-based fermentations, where erythromycin production by the wild-type strain averages 184% higher (141–236%, 0.95 CI) than in carbohydrate-based fermentations, the same polar knockout in mutB surprisingly reduced erythromycin production by 66% (53–76%, 0.95 CI). A metabolic model is proposed where in carbohydrate-based fermentations MCM acts as a drain on the methylmalonyl-CoA metabolite pool, and in oil-based fermentations, MCM acts in the reverse direction to fill the methylmalonyl-CoA pool. Therefore, the model explains, in part, how the well-known oil-based process improvement for erythromycin production operates at the biochemical level; furthermore, it illustrates how the mutB erythromycin strain improvement mutation operates at the genetic level in carbohydrate-based fermentations.     

Isolation and characterization of Escherichia coli mutants blocked in production of membrane-derived oligosaccharides.

We report a new procedure for the facile selection of mutants of Escherichia coli that are blocked in the production of membrane-derived oligosaccharides. Four phenotypic classes were identified, including two with a novel array of characteristics. The mutations mapped to two genetic loci. Mutations in the mdoA region near 23 min are in two distinct genes, only one of which is needed for the membrane-localized glucosyltransferase that catalyzes the synthesis of the beta-1,2-glucan backbone of membrane-derived oligosaccharides. Another set of mutations mapped near 27 min closely linked to osmZ; these appear to be in the galU gene.