Screening of a high-yield strain of avermectin B1a by colony analysis in situ / Selección in situ de una cepa de alto rendimiento de avermectina B1a mediante análisis de colonias

Avermectin, an agricultural antibiotic, is widely used as an agricultural insecticide and an important lead compound of antibiotics. It is manufactured by Streptomyces avermitilis through fermentation. Manufacturers pay special attention to screening for strains with high fermentation capacity based on morphological properties of the colony and by the result of shake flask fermentation. These traditional screening methods are time-consuming and labor-intensive and require specialized equipment. Moreover, evaluation of colony appearance is highly subjective. To improve and accelerate the screening process, we developed a rapid in situ screening method. Forty-four strains isolated naturally from the spores of industrial high-yielding strains were studied. The data show that the colony fermentation titer is highly correlated with the yield from the shake flask fermentation of avermectin, and the Pearson’s R is 0.990. The total titer of avermectins by shake flask fermentation is also highly correlated with the B1a titer (Pearson’s R is 0.994). This result also shows that strains can be quickly screened by analyzing the colony titer. Pigment rings of the colonies that appeared after growing and maturing on the new medium plate were analyzed. The chosen colonies were directly marked and punched and then extracted with methanol. The fermentation ability can be evaluated by measuring the absorbance at 245 nm. This methodology can be applied in both natural breeding and mutation breeding conditions. By continuously breeding from 2008 to 2020, the flask titer of avermectin B1a increased from 4582 ± 483 to 9197 ± 1134 μg/mL.(AU)

Screening of a high-yield strain of avermectin B1a by colony analysis in situ.

Avermectin, an agricultural antibiotic, is widely used as an agricultural insecticide and an important lead compound of antibiotics. It is manufactured by Streptomyces avermitilis through fermentation. Manufacturers pay special attention to screening for strains with high fermentation capacity based on morphological properties of the colony and by the result of shake flask fermentation. These traditional screening methods are time-consuming and labor-intensive and require specialized equipment. Moreover, evaluation of colony appearance is highly subjective. To improve and accelerate the screening process, we developed a rapid in situ screening method. Forty-four strains isolated naturally from the spores of industrial high-yielding strains were studied. The data show that the colony fermentation titer is highly correlated with the yield from the shake flask fermentation of avermectin, and the Pearson's R is 0.990. The total titer of avermectins by shake flask fermentation is also highly correlated with the B1a titer (Pearson's R is 0.994). This result also shows that strains can be quickly screened by analyzing the colony titer. Pigment rings of the colonies that appeared after growing and maturing on the new medium plate were analyzed. The chosen colonies were directly marked and punched and then extracted with methanol. The fermentation ability can be evaluated by measuring the absorbance at 245 nm. This methodology can be applied in both natural breeding and mutation breeding conditions. By continuously breeding from 2008 to 2020, the flask titer of avermectin B1a increased from 4582 ± 483 to 9197 ± 1134 µg/mL.

Reverse biological engineering of hrdB to enhance the production of avermectins in an industrial strain of Streptomyces avermitilis.

Avermectin and its analogues are produced by the actinomycete Streptomyces avermitilis and are widely used in the field of animal health, agriculture, and human health. Here we have adopted a practical approach to successfully improve avermectin production in an industrial overproducer. Transcriptional levels of the wild-type strain and industrial overproducer in production cultures were monitored using microarray analysis. The avermectin biosynthetic genes, especially the pathway-specific regulatory gene, aveR, were up-regulated in the high-producing strain. The upstream promoter region of aveR was predicted and proved to be directly recognized by sigma(hrdB) in vitro. A mutant library of hrdB gene was constructed by error-prone PCR and selected by high-throughput screening. As a result of evolved hrdB expressed in the modified avermectin high-producing strain, 6.38 g/L of avermectin B1a was produced with over 50% yield improvement, in which the transcription level of aveR was significantly increased. The relevant residues were identified to center in the conserved regions. Engineering of the hrdB gene can not only elicit the overexpression of aveR but also allows for simultaneous transcription of many other genes. The results indicate that manipulating the key genes revealed by reverse engineering can effectively improve the yield of the target metabolites, providing a route to optimize production in these complex regulatory systems.

Assessing the potential of an induced-mutation strategy for avermectin overproducers.

Mutant libraries of avermectin-producing Streptomyces avermitilis strains were constructed by different mutagenesis strategies. A metric was applied to assess the mutation spectrum by calculating the distribution of average phenotypic distance of each population. The results showed for the first time that a microgravity environment could introduce larger phenotype distribution and diversity than UV and N-methyl-N-nitro-N-nitrosoguanidine (NTG) could.

Rational design for over-production of desirable microbial metabolites by precision engineering.

Microbes represent a valuable source of commercially significant natural products that have improved our quality of life. Precision engineering can be used to precisely identify and specifically modify genes responsible for production of natural products and improve this production or modify the genes creating products that would not otherwise be produced. There have been several success stories concerning the manipulation of regulatory genes, pathways, and genomes to increase the productivity of industrial microbes. This review will focus on the strategies and integrated approaches for precisely deciphering regulatory genes by various modern techniques. The applications of precision engineering in rational strain improvement also shed light on the biology of natural microbial systems.

Identification of avermectin-high-producing strains by high-throughput screening methods.

Avermectins produced by Streptomyces avermitilis are potent against a broad spectrum of nematode and arthropod parasites with low-level side effects on the host organisms. This study was designed to investigate a high-throughput screening strategy for the efficient identification of avermectin high-yield strains. The production protocol was miniaturized in 96 deep-well microplates. UV absorbance at 245 nm was used to monitor avermectin production. A good correlation between fermentation results in both 96 deep-well microplates and conventional Erlenmeyer flasks was observed. With this protocol, the production of avermectins was determined in less than 10 min for a full plate without compromising accuracy. The high-yield strain selected through this protocol was also tested in 360 m(3) batch fermentation with 1.6-fold improved outcome. Thus, the development of this protocol is expected to accelerate the selection of superior avermectin-producing strains.

Paraffin oil as a "methane vector" for rapid and high cell density cultivation of Methylosinus trichosporium OB3b.

Slow growth and relatively low cell densities of methanotrophs have limited their uses in industrial applications. In this study, a novel method for rapid cultivation of Methylosinus trichosporium OB3b was studied by adding a water-immiscible organic solvent in the medium. Paraffin oil was the most effective at enhancing cell growth and final cell density. This is at least partially due to the increase of methane gas transfer between gas and medium phases since methane solubility is higher in paraffin than in water/nitrate minimal salt medium. During cultivation with paraffin oil at 5% (v/v) in the medium, M. trichosporium OB3b cells also showed higher concentrations of the intermediary metabolites, such as formic acid and pyruvic acid, and consumed more methane compared with the control. Paraffin as methane vector to improve methanotroph growth was further studied in a 5-L fermentor at three concentrations (i.e., 2.5%, 5%, and 10%). Cell density reached about 14 g dry weight per liter with 5% paraffin, around seven times higher than that of the control (without paraffin). Cells cultivated with paraffin tended to accumulate around the interface between oil droplets and the water phase and could exist in oil phase in the case of 10% (v/v) paraffin. These results indicated that paraffin could enhance methanotroph growth, which is potentially useful in cultivation of methanotrophs in large scale in industry.

Functional expression of the particulate methane mono-oxygenase gene in recombinant Rhodococcus erythropolis.

In order to construct an expression system for the particulate methane mono-oxygenase (pMMO) gene (pmo), the structural gene cluster pmoCAB amplified from Methylosinus trichosporium OB3b was inserted into a shuttle vector pBS305 under the control of a dsz promoter and transformed into Rhodococcus erythropolis LSSE8-1. A stable transformant was successfully obtained using ethane as the sole carbon source. Fluorescence in situ hybridization results showed that the dsz promoter allowed the pmo genes to be transcribed in the recombinant strain. The effects of Cu2+ and Zn2+ concentrations on cell growth and pMMO activity in ethane-containing medium were examined. It was discovered that 7.5 microM Cu2+ and 1.8 microM Zn2+ were suitable to achieve high cell concentration and pMMO activity, but the amount of methanol accumulated during methane oxidation by the recombinant strain was still low.

A simplified method for assay of hydrogenase activities of H2 evolution and uptake in Enterobacter aerogenes.

Assay of hydrogenase activity pertaining to H2 production needs anaerobic conditions. To establish a simplified method for assay of hydrogenase activities by using intact cells of Enterobater aerogenes, different chemicals capable of enhancing the cell-wall permeability to electron mediators were examined. As a result, Triton X-100 and CTAB were found to be appropriate for H2 uptake and evolution activities of the intact cells, respectively. This method enabled H2 uptake and evolution activities of the intact cells to be easily detected. This is also the first report of the presence of H2 uptake hydrogenase activity in E. aerogenes.