Dataset Information

Identification and product optimization of amylolytic Rhodococcus opacus GAA 31.1 isolated from gut of Gryllotalpa africana.

ABSTRACT: An attempt has been made to isolate potent amylase producing gut bacteria from Gryllotalpa africana. Out of 82 isolates, GAA 31.1 was selected as potent producer, having enzyme activity 9.6 ± 0.861 U/ml. The isolate GAA 31.1 was identified as Rhodococcus opacus following morphological, biochemical, physiological characterization and phylogenetic analysis through 16S rRNA gene sequencing. Fatty acid methyl ester profile of the isolate was also studied. The optimized physical cultural conditions for amylase production were found as incubation period 48 h, inoculum volume 2%, initial pH of the fermentation medium 7.0, temperature 38 °C and aeration at 150 rpm. Optimum nutrient conditions were determined as: supplementation of maltose 1.4% and sodium nitrate 1.4%. Surfactants SDS, EDTA, Tween 80 and Triton X-100 showed positive effect on enzyme production. Riboflavin (50 ?g/ml) among the tested vitamins stimulated the production maximally. The isolate was also able to produce amylase using agro-industrial waste. This actinobacterium may be a potent candidate for amylase as it is capable of enhanced production (326.72 ± 6.081 U/ml) by utilizing agro-residues.

SUBMITTER: Banerjee S

PROVIDER: S-EPMC6299904 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Identification and product optimization of amylolytic Rhodococcus opacus GAA 31.1 isolated from gut of Gryllotalpa africana.

Banerjee Sandipan S Maiti Tushar Kanti TK Roy Raj Narayan RN

Journal, genetic engineering & biotechnology 20160609 1

An attempt has been made to isolate potent amylase producing gut bacteria from Gryllotalpa africana. Out of 82 isolates, GAA 31.1 was selected as potent producer, having enzyme activity 9.6 ± 0.861 U/ml. The isolate GAA 31.1 was identified as Rhodococcus opacus following morphological, biochemical, physiological characterization and phylogenetic analysis through 16S rRNA gene sequencing. Fatty acid methyl ester profile of the isolate was also studied. The optimized physical cultura ...[more]

PMID: 30647607

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Project description:Background:Lignin is a recalcitrant aromatic polymer that is a potential feedstock for renewable fuel and chemical production. Rhodococcus opacus PD630 is a promising strain for the biological upgrading of lignin due to its ability to tolerate and utilize lignin-derived aromatic compounds. To enhance its aromatic tolerance, we recently applied adaptive evolution using phenol as a sole carbon source and characterized a phenol-adapted R. opacus strain (evol40) and the wild-type (WT) strain by whole genome and RNA sequencing. While this effort increased our understanding of the aromatic tolerance, the tolerance mechanisms were not completely elucidated. Results:We hypothesize that the composition of lipids plays an important role in phenol tolerance. To test this hypothesis, we applied high-resolution mass spectrometry analysis to lipid samples obtained from the WT and evol40 strains grown in 1 g/L glucose (glucose), 0.75 g/L phenol (low phenol), or 1.5 g/L phenol (high phenol, evol40 only) as a sole carbon source. This analysis identified?>?100 lipid species of mycolic acids, phosphatidylethanolamines (PEs), phosphatidylinositols (PIs), and triacylglycerols. In both strains, mycolic acids had fewer double bond numbers in phenol conditions than the glucose condition, and evol40 had significantly shorter mycolic acid chain lengths than the WT strain in phenol conditions. These results indicate that phenol adaptation affected mycolic acid membrane composition. In addition, the percentage of unsaturated phospholipids decreased for both strains in phenol conditions compared to the glucose condition. Moreover, the PI content increased for both strains in the low phenol condition compared to the glucose condition, and the PI content increased further for evol40 in the high phenol condition relative to the low phenol condition. Conclusions:This work represents the first comprehensive lipidomic study on the membrane of R. opacus grown using phenol as a sole carbon source. Our results suggest that the alteration of the mycolic acid and phospholipid membrane composition may be a strategy of R. opacus for phenol tolerance.

Project description:BACKGROUND:There has been a great deal of interest in fuel productions from lignocellulosic biomass to minimize the conflict between food and fuel use. The bioconversion of xylose, which is the second most abundant sugar present after glucose in lignocellulosic biomass, is important for the development of cost effective bioprocesses to fuels. Rhodococcus opacus PD630, an oleaginous bacterium, accumulates large amounts of triacylglycerols (TAGs), which can be processed into advanced liquid fuels. However, R. opacus PD630 does not metabolize xylose. RESULTS:We generated DNA libraries from a Streptomyces bacterium capable of utilizing xylose and introduced them into R. opacus PD630. Xsp8, one of the engineered strains, was capable of growing on up to 180 g L-1 of xylose. Xsp8 grown in batch-cultures derived from unbleached kraft hardwood pulp hydrolysate containing 70 g L-1 total sugars was able to completely and simultaneously utilize xylose and glucose present in the lignocellulosic feedstock, and yielded 11.0 g L-1 of TAGs as fatty acids, corresponding to 45.8% of the cell dry weight. The yield of total fatty acids per gram of sugars consumed was 0.178 g, which consisted primarily of palmitic acid and oleic acid. The engineered strain Xsp8 was introduced with two heterologous genes from Streptomyces: xylA, encoding xylose isomerase, and xylB, encoding xylulokinase. We further demonstrated that in addition to the introduction and the concomitant expression of heterologous xylA and xylB genes, there is another molecular target in the R. opacus genome which fully enables the functionality of xylA and xylB genes to generate the robust xylose-fermenting strain capable of efficiently producing TAGs at high xylose concentrations. CONCLUSION:We successfully engineered a R. opacus strain that is capable of completely utilizing high concentrations of xylose or mixed xylose/glucose simultaneously, and substantiated its suitability for TAG production. This study demonstrates that the engineered strain possesses a key trait of converters for lipid-based fuels production from lignocellulosic biomass.

Dataset Information

Identification and product optimization of amylolytic Rhodococcus opacus GAA 31.1 isolated from gut of Gryllotalpa africana.

Publications

Identification and product optimization of amylolytic <i>Rhodococcus opacus</i> GAA 31.1 isolated from gut of <i>Gryllotalpa africana</i>.

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