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Engineering of a xylose metabolic pathway in Rhodococcus strains.

ABSTRACT: The two metabolically versatile actinobacteria Rhodococcus opacus PD630 and R. jostii RHA1 can efficiently convert diverse organic substrates into neutral lipids mainly consisting of triacylglycerol (TAG), the precursor of energy-rich hydrocarbon. Neither, however, is able to utilize xylose, the important component present in lignocellulosic biomass, as the carbon source for growth and lipid accumulation. In order to broaden their substrate utilization range, the metabolic pathway of d-xylose utilization was introduced into these two strains. This was accomplished by heterogenous expression of two well-selected genes, xylA, encoding xylose isomerase, and xylB, encoding xylulokinase from Streptomyces lividans TK23, under the control of the tac promoter with an Escherichia coli-Rhodococcus shuttle vector. The recombinant R. jostii RHA1 bearing xylA could grow on xylose as the sole carbon source, and additional expression of xylB further improved the biomass yield. The recombinant could consume both glucose and xylose in the sugar mixture, although xylose metabolism was still affected by the presence of glucose. The xylose metabolic pathway was also introduced into the high-lipid-producing strain R. opacus PD630 by expression of xylA and xylB. Under nitrogen-limited conditions, the fatty acid composition was determined, and lipid produced from xylose by recombinants of R. jostii RHA1 and R. opacus PD630 carrying xylA and xylB represented up to 52.5% and 68.3% of the cell dry weight (CDW), respectively. This work demonstrates that it is feasible to produce lipid from the sugars, including xylose, derived from renewable feedstock by genetic modification of rhodococcus strains.

SUBMITTER: Xiong X

PROVIDER: S-EPMC3406118 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Engineering of a xylose metabolic pathway in Rhodococcus strains.

Xiong Xiaochao X Wang Xi X Chen Shulin S

Applied and environmental microbiology 20120525 16

The two metabolically versatile actinobacteria Rhodococcus opacus PD630 and R. jostii RHA1 can efficiently convert diverse organic substrates into neutral lipids mainly consisting of triacylglycerol (TAG), the precursor of energy-rich hydrocarbon. Neither, however, is able to utilize xylose, the important component present in lignocellulosic biomass, as the carbon source for growth and lipid accumulation. In order to broaden their substrate utilization range, the metabolic pathway of d-xylose ut ...[more]

PMID: 22636009

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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.

Project description:BackgroundLimonene is a cyclic monoterpene that has applications in the food, cosmetic, and pharmaceutical industries. The industrial production of limonene and its derivatives through plant extraction presents important drawbacks such as seasonal and climate issues, feedstock limitations, low efficiency and environmental concerns. Consequently, the implementation of efficient and eco-friendly bioprocesses for the production of limonene and other terpenes constitutes an attractive goal for microbial biotechnology. In this context, novel biocatalysts with the ability to produce limonene from alternative carbon sources will help to meet the industrial demands of limonene.ResultsEngineered strains of the industrial fungus Ashbya gossypii have been developed to produce limonene from xylose. The limonene synthase (LS) from Citrus limon was initially overexpressed together with the native HMG1 gene (coding for HMG-CoA reductase) to establish a limonene-producing platform from a xylose-utilizing A. gossypii strain. In addition, several strategies were designed to increase the production of limonene. Hence, the effect of mutant alleles of ERG20 (erg20F95W and erg20F126W) were evaluated together with a synthetic orthogonal pathway using a heterologous neryl diphosphate synthase. The lethality of the A. gossypii double mutant erg20F95W-F126W highlights the indispensability of farnesyl diphosphate for the synthesis of essential sterols. In addition, the utilization of the orthogonal pathway, bypassing the Erg20 activity through neryl diphosphate, triggered a substantial increase in limonene titer (33.6 mg/L), without critically altering the fitness of the engineered strain. Finally, the overexpression of the native ERG12 gene further enhanced limonene production, which reached 336.4 mg/L after 96 h in flask cultures using xylose as the carbon source.ConclusionsThe microbial production of limonene can be carried out using engineered strains of A. gossypii from xylose-based carbon sources. The utilization of a synthetic orthogonal pathway together with the overexpression of ERG12 is a highly beneficial strategy for the production of limonene in A. gossypii. The strains presented in this work constitute a proof of principle for the production of limonene and other terpenes from agro-industrial wastes such as xylose-rich hydrolysates in A. gossypii.

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Engineering of a xylose metabolic pathway in Rhodococcus strains.

Publications

Engineering of a xylose metabolic pathway in Rhodococcus strains.

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