Erythromycin is a macrolide antibiotic produced by Saccharopolyspora erythraea. Its yield is greatly affected by the fermentation conditions and the bioreactor configurations. In this study, a novel scale-up method for erythromycin fermentation was developed based on computational fluid dynamics (CFD) and time constant analysis. Firstly, the dissolved oxygen (DO) was determined as a key parameter according to the physiological properties of S. erythraea cultivated in a 50 L bioreactor. It was found that the time constant of oxygen supply (t_mt) in a 500 m³ bioreactor should be less than 6.25 s in order to satisfy the organism's oxygen uptake rate (OUR). Subsequently, a 500 m³ bioreactor was designed using the time constant method combined with empirical correlations. The impeller combination with one BDT8 impeller at bottom and two MSX4 impellers at upper part was determined, and then validated by numerical simulation. The results indicated that the t_mt of the bioreactor (< 6.25 s) and the fluid properties, including gas hold-up, shear stress and fluid vector, met the requirements of erythromycin fermentation. Finally, the industrial production of erythromycin in the 500 m³ showed the design method was applicable in large scale fermentation.
Erythromycin ; Saccharopolyspora/genetics* ; Bioreactors ; Fermentation ; Anti-Bacterial Agents

In order to study the relationship between lengths of homologous fragments and chromsomic recombination rate in Saccharopolyspora erythraea, three homologous sequences, with mutant loci and different flanking sequences, (26bp + 27bp), (500bp + 576bp) and (1908bp + 1749bp), were synthesized by chemical reaction or PCR amplification, and cloned into pWHM3 to construct homologous recombination plasmids, pWHM1113, pWHM1116 and pWHM1119. When the plasmids were transformed into protoplast of Saccharopolyspora erythraea A226 under PEG mediated, on an average 30, 69 and 170 transformants grew on each plate for the three plasmids respectively, but chromosomic integration frequency were 0, 2% and 19% among corresponding transformants. Both pWHM1116 and pWHM1119 could take double crossover recombination, and exchange the mutant loci in the chromosome. It was concluded that when the flanking sequences were equal or more than (500bp + 576bp), they could take effective single and double recombination with Saccharopolyspora erythraea chromosome.
Chromosomes, Bacterial ; genetics ; Polymerase Chain Reaction ; Recombination, Genetic ; genetics ; Saccharopolyspora ; genetics

Through introducing mutations into ribosomes by obtaining spontaneous drug resistance of microorganisms, ribosome engineering technology is an effective approach to develop mutant strains that overproduce secondary metabolites. In this study, ribosome engineering was used to improve the yield of butenyl-spinosyns produced by Saccharopolyspora pogona by screening streptomycin resistant mutants. The yields of butenyl-spinosyns were then analyzed and compared with the parent strain. Among the mutants, S13 displayed the greatest increase in the yield of butenyl-spinosyns, which was 1.79 fold higher than that in the parent strain. Further analysis of the metabolite profile of S13 by mass spectrometry lead to the discovery of Spinosyn α1, which was absent from the parent strain. DNA sequencing showed that there existed two point mutations in the conserved regions of rpsL gene which encodes ribosomal protein S12 in S13. The mutations occurred a C to A and a C to T transversion mutations occurred at nucleotide pair 314 and 320 respectively, which resulted in the mutations of Proline (105) to Gultamine and Alanine (107) to Valine. It also demonstrated that S13 exhibited genetic stability even after five passages.
Genetic Engineering ; Macrolides ; metabolism ; Point Mutation ; Ribosomal Proteins ; genetics ; Ribosomes ; metabolism ; Saccharopolyspora ; metabolism

Spinosad represents a new class of insecticides produced by Saccharopolyspora spinosa. To understand the transcription of the spinosad biosynthetic gene cluster, two promoter detection plasmids based on different reporter genes were constructed and used to detect 9 promoters in the spinosad biosynthetic gene cluster. In addition, the temporal transcriptional profiles of the corresponding genes controlled by the 9 promoters, together with 4 genes outside of the spinosad cluster but are required for the synthesis of sugars in spinosad, were examined by real-time PCR. The results indicate that the 9 spinosad biosynthetic genes were highly expressed at the stationary phase, which coincides with the accumulation of spinosad in the fermentation broth. Of particular note is that the transcription of the 4 sugar synthetic genes showed higher level at the exponential phase, suggesting the expression of sugar synthetic genes is not correlated with the spinosad synthetic genes. The data suggest that spinosad biosynthesis could be improved by engineering the expression pattern of the sugar synthetic genes that lie outside the spinosad gene cluster.
Drug Combinations ; Insecticides ; metabolism ; Macrolides ; metabolism ; Multigene Family ; Plasmids ; Promoter Regions, Genetic ; Real-Time Polymerase Chain Reaction ; Saccharopolyspora ; genetics ; metabolism

Genetic engineering on macrolide antibiotics was a new field in recent years and more than 100 novel polyketides had been produced until then. Using genomic DNA of S. erythraea A226 as a template, about 3.2 kb DNA fragment without KR6 domain was amplified by overlapping PCR technique and cloned into pWHM3 carrier, which resulted in the construction of homologous recombinant plasmid pWHM2201. Plasmid pWHM2201 was introduced into protoplasts of S. erythraea A226 by PEG-mediated transformation and integrated into the gene locus for erythromycin biosynthesis. After integrants grew for two generations on R3M media without Tsr, they were protoplasted and grown on R3M plates. By PCR identification, 8 mutants without KR6 domain were selected out and named S. erythraea M(1-8). With the identification of mass spectrometry, it was proved that S. erythraea M1 synthesized a novel ketolide compound 3-deoxy-3-oxo-erythronolide B.
Anti-Bacterial Agents ; biosynthesis ; chemistry ; Chromosomes, Bacterial ; Erythromycin ; analogs & derivatives ; biosynthesis ; chemistry ; Genetic Engineering ; Ketolides ; Molecular Structure ; Multienzyme Complexes ; genetics ; Saccharopolyspora ; enzymology ; genetics ; metabolism

To improve spinosyn-producing strain and enhance spinosyns yield, we studied the effects of glycin concentration and the operational time, temperature and lysozyme concentration on protoplast preparation of Saccharopolyspora spinosa SP06081. We also studied different regeneration media and osmotic stabilizing agents. In addition, we compared the change of morphology and spinosyns yield of the regenerated strains. The results showed that the Saccharopolyspora spinosa SP06081 protoplast yield was the highest under these conditions: the collected mycelium from SP06081 grown in Tryptic Soy Broth (TSB) medium with 0.2% glycin for 48 h was treated by 0.1 mg/mL lysozyme at 28 degrees C for 20 min, then plated on the R2YE medium with sucrose as osmotic stabilizer, the number of regeneration protoplast was up to 10(8)/mL. The protoplast-regenerated strains exhibited changes in morphology and antibiotic production, 29.3% protoplast-regenerated strains was characterized by loose mycelium and abundant broken branches as did their parent. Among them, 58.2% strains presented the trend to positive variation in spinosad yield, with the highest spinosad yield of up to 582.0 mg/L, 85.6% higher than that of their parent. There is significant correlation between the morphological differentiation and antibiotic yield of the protoplast-regenerated strains from spinosyn-producing strain.
Culture Media ; pharmacology ; Drug Combinations ; Glycine ; pharmacology ; Insecticides ; metabolism ; Macrolides ; metabolism ; Muramidase ; pharmacology ; Protoplasts ; cytology ; drug effects ; Regeneration ; Saccharopolyspora ; genetics ; metabolism ; physiology