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In the current study, the effect of different available nitrogen sources on erythromycin fermentation by Saccharopolyspora erythraea No. 8 is evaluated. Three different combinations of corn steep liquor and yeast powder were developed to investigate their impacts on erythromycin production. The results indicate that the optimal combination of available nitrogen sources was 10.0 g/L corn steep liquor and 4.0 g/L yeast power, generating a maximum yield of erythromycin of 13672 U/mL. To explore the effects of nitrogen perturbations on cell metabolism, metabolic flux analyses were performed and compared under different conditions. A high flux pentose phosphate pathway provided more NADPH for erythromycin synthesis via nitrogen optimization. Moreover, high n-propanol specific consumption rate enhanced erythromycin synthesis and n-propanol flowed into the central carbon metabolism by methylmalonyl-CoA node. These results indicate that the selection of an appropriate organic nitrogen source is essential for cell metabolism and erythromycin synthesis, and this is the first report of the successful application of available nitrogen source combinations in industrial erythromycin production.

A high erythromycin producing mutant strain Saccharopolyspora erythraea HL3168 E3-ΔmutB was constructed by deleting mutB (SACE_5639) gene encoding the beta subunit of methylmalonyl-CoA mutase of an industrial strain of S. erythraea HL3168 E3. Industrial media and process control strategies were adopted in a 5 L bioreactor for characterizing the physiological parameters. The total erythromycin titer and erythromycin A concentration in mutant were 46.9 (12740.5 μg/mL) and 64.9 % (8094.4 μg/mL) higher than those in original strain, respectively, which were comparable to industrial erythromycin production. The specific glucose and n-propanol consumption rates were increased by 52.4 and 39.8 %, respectively. During the rapid erythromycin synthesis phase, the yield of erythromycin on n-propanol also increased from 24.3 % in control group to 66.9 % in mutant group. Meanwhile, the specific formation rates of methylmalonyl-CoA and propionyl-CoA, two crucial precursors for erythromycin synthesis, were 1.89- and 2.02-folds higher in the mutant strain, respectively.

Saccharopolyspora erythraea E3 is an important industrial strain for erythromycin production and knowledge on its metabolism is limited. In the present work, (13)C labeling experiments were conducted to characterize the metabolism of S. erythraea E3. We found that S. erythraea E3 was difficult to grow on minimal medium with glucose as sole carbon source and the addition of proline remarkably improved the cell growth. The activity of EMP pathway was very low and ED pathway was alternatively the main glucose utilization pathway. The addition of proline resulted in remarkable changes in the fluxes of central metabolism. The fluxes in PP pathway, in TCA cycle and in ED pathway were 90% higher, 64% and 31% lower on Glc/Pro than on Glc, respectively. The maintenance energy on Glc/Pro was 58.4% lower than that on Glc. The energy charge was lower on Glc than on Glc/Pro, indicating that the cells on Glc suffered from energy burden. This study elucidates the impacts of proline on the central metabolism of S. erythraea and deepens the understanding of its metabolism., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of the present study was to optimize the feeding proportion of glucose and propanol for erythromycin biosynthesis by real-time monitoring and exploring its limited ratio by the on-line multi-frequency permittivity measurement. It was found that the capacitance values were sensitive to the variation of biomass concentration and microbial morphology as well as the true state of cell growth. It was most favorable to both cell growth and secondary metabolism to keep the ratio of glucose to propanol at 4.3 (g/g). The specific growth rate calculated by the capacitance measurement correctly and accurately reflected the cell physiological state. An appropriate feed rate of propanol was crucial for cell growth and secondary metabolism, as well as to improve the quality of erythromycin-A. In addition, the erythromycin production titer (10,950 U/mL) was further enhanced by 4 % when the propanol feed was regulated by step-down strategy based on both OUR (oxygen uptake rate) and the on-line monitoring capacitance.

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(-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(3) fermenter, erythromycin titer and erythromycin A concentration at 155 h were reached 9.12 and 7.12 g l(-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(3) fermenter.

In this paper, several different fermentation experiments were designed to address whether modulating glucose and propanol feeds could benefit the production level of erythromycin during pilot plant (30 L) fermentation. Results showed that glucose feed rate (determined by a set high or low culture pH) had no effect on erythromycin production, indicating that glucose was not the limiting factor for erythromycin biosynthesis under these conditions. It was found that decreasing glucose feed could stimulate the consumption of propanol, and the high erythromycin production (12.49 ± 0.50 mg ml⁻¹) was achieved by controlling the feed rates of glucose and propanol. The quantitative metabolic flux analysis disclosed that high propanol consumption increased the pool size of propionyl-CoA (~2.147 mmol g⁻¹ day⁻¹) and methylmalonyl-CoA (~1.708 mmol g⁻¹ day⁻¹). It was also found that 45-77 % of the propanol went into the TCA cycle which strengthened the conclusion that blocking the propionate pathway to TCA cycle could lead to a significant increase in erythromycin production in carbohydrate-based media (Reeves et al. Ind Microbiol Biotechnol 7:600-609, 2006). In addition, the results also suggested that a relative low intracellular ATP level resulting from low glucose feed did not limit the erythromycin biosynthesis, and a relatively high NADPH should be beneficial for erythromycin biosynthesis.

In this study, the effects of nitrogen sources on broth viscosity and glucose consumption in erythromycin fermentation were investigated. By controlling ammonium sulfate concentration, broth viscosity and glucose consumption were decreased by 18.2% and 61.6%, respectively, whereas erythromycin biosynthesis was little affected. Furthermore, erythromycin A production was increased by 8.7% still with characteristics of low broth viscosity and glucose consumption through the rational regulations of phosphate salt, soybean meal and ammonium sulfate. It was found that ammonium sulfate could effectively control proteinase activity, which was correlated with the utilization of soybean meal as well as cell growth. The pollets formation contributed much to the decrease of broth viscosity. The accumulation of extracellular propionate and succinate under the new regulation strategy indicated that higher propanol consumption might increase the concentration of methylmalonyl-CoA and propionyl-CoA and thus could increase the flux leading to erythromycin A., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Erythromycin A (Er-A) is the most potent and clinically important member in the Er family produced by Saccharopolyspora erythraea. Er-B and Er-C, which are biologically much less active and cause greater side effects than Er-A, serve as the intermediates for Er-A biosynthesis and impurities in fermentation processes of many industrial strains. In this study, systematical modulation of the amounts of tailoring enzymes EryK (a P450 hydroxylase) and EryG (an S-adenosylmethionine-dependent O-methyltransferase) was carried out by genetic engineering in S. erythraea, including alterations of gene copy number ratio and organization and integrating the locus on the chromosome by homologous recombination. Introduction of additional eryK and eryG genes into S. erythraea showed significant impacts on their transcription levels and enhanced the biotransformation process from Er-D to Er-A with gene dose effects. At the eryK/eryG copy number ratio of 3:2 as well as their resultant transcript ratio of around 2.5:1 to 3.0:1, Er-B and Er-C were nearly completely eliminated and accordingly converted to Er-A, and the Er titer was improved by around 25% in the recombinant strain ZL1004 (genotype PermK*-K-K-G + PermE*-K + PermA*-G) and ZL1007 (genotype PermK*-K-G-K + PermE*-K + PermA*-G). This study may contribute to the continuous efforts toward further evaluation of the Er-producing system, with the aims of improving Er-A purity and production at the fermentation stage and lowering the production costs and environmental concerns in industry.

An S-adenosylmethionine synthetase (SAM-s) gene from Streptomyces spectabilis was integrated along with vector DNA into the chromosome of a Saccharopolyspora erythraea E2. Elevated production of SAM was observed in the recombinant strain Saccharopolyspora erythraea E1. The results from the bioassay showed that the titer of erythromycin was increased from 920 IU ml(-1) by E2 to approximately 2,000 IU ml(-1) by E1. High performance liquid chromatography (HPLC) analysis revealed that there was a 132% increase in erythromycin A compared with the original strain, while the erythromycin B, the main impurity component in erythromycin, was decreased by 30%. The sporulation process was inhibited, while the SAM-s gene was expressed. The addition of the exogenous SAM also inhibited sporulation and promoted an increase in erythromycin titers.

Abstract: A simple and rapid microplate-based bioassay was developed for the quantitative assay of erythromycin in Saccharopolyspora erythraea fermentation samples. High performance liquid chromatography (HPLC), colorimetric method and agar diffusion assay (ADA) were used as reference methods to evaluate the accuracy of this strategy. S. erythraea T-13 and its derivatives were used to assess the optimum assay conditions for erythromycin measurement and it was verified that this downscaled method was comparable to traditional assay methods for rapid quantification. Large number of isolates could be evaluated simultaneously within 4–5h by using the high-throughput method. The cost effective method reported here demonstrates the successful application of a microplate assay for high throughput analysis, and could be potentially applicable to general antibiotics analysis. [Copyright &y& Elsevier]

A correction to the article "Improved production of erythromycin A by expression of a heterologous gene encoding S-adenosylmethionine synthetase," by Yong Wang and colleagues that was previously published is presented.

• A data enhancement method based on generative adversarial network is proposed. • The method combines the advantages of data augmentation and feature selection. • Case studies on a simulated case and two real industrial processes are provided. • Comparisons to state-of-the-arts show effectiveness of the proposed method. Soft sensor plays an important role in improving product quality; however, practical applications may often face with the problem of small sample size, which is challenging for developing data-driven models in terms of feature selection and good generalization. This paper proposes a data enhancement approach for small sample size data-driven problems based on generative adversarial networks integrated with maximum relevance minimum redundancy (MRMR). First, sample expansion is performed on the initial data by using a generative adversarial network. Second, irrelevant variables are eliminated by the MRMR and optimal features are obtained. Finally, neural networks-based soft sensor modeling is performed using the augmented dataset and the selected features. The proposed method is tested on a simulated penicillin case, an actual penicillin production case and an actual erythromycin production case. Experimental results show that the proposed method outperforms state-of-the-art existing methods, which verify the effectiveness and superiority of the proposed method. [ABSTRACT FROM AUTHOR]