Engineering xylose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Kurosawa, Kazuhiko, Sandra J Wewetzer, and Anthony J Sinskey. 2013 Engineering Xylose Metabolism in Triacylglycerol-producing Rhodococcus Opacus for Lignocellulosic Fuel Production. Biotechnology for Biofuels 6(1): 134. As Published http://dx.doi.org/10.1186/1754-6834-6-134 Publisher BioMed Central Ltd. Version Final published version Citable link http://hdl.handle.net/1721.1/81356 Terms of Use Creative Commons Attribution Detailed Terms http://creativecommons.org/licenses/by/2.0 Kurosawa et al. Biotechnology for Biofuels 2013, 6:134 http://www.biotechnologyforbiofuels.com/content/6/1/134 RESEARCH Open Access Engineering xylose metabolism in triacylglycerol- producing Rhodococcus opacus for lignocellulosic fuel production Kazuhiko Kurosawa1, Sandra J Wewetzer1,4 and Anthony J Sinskey1,2,3* Abstract 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. Keywords: Rhodococcus opacus, Streptomyces padanus, Triacylglycerol, Lipid-based biofuel, Lignocellulosic fuel, Lignocellulosic biomass, Xylose, High-cell-density fermentation, xylA, xylB Background from lignocellulosic biomass, has occurred [5,6]. Ad- The rises in petroleum prices along with depleting vanced liquid (lipid-based) fuels such as gasoline, petroleum reserves have kindled a great interest in kerosene, diesel and jet fuels are of greatest interest the production of bio-based alternative fuels [1-3]. and are expected to play a crucial role in the future Extensive discussion towards the development of global energy infrastructure [7,8]. Triacylglycerols clean and sustainable energy sources [4], especially (TAGs) are esters in which three molecules of fatty acids are linked to glycerol, and are exploited as pre- * Correspondence: [email protected] cursors for the production of the lipid-based biofuels 1Department of Biology, Massachusetts Institute of Technology, [9-11]. Recently, an F-22 Raptor successfully flew on 77 Massachusetts Avenue, Cambridge, MA 02139, USA a 50/50 blend of the renewable (hydroprocessed esters 2Engineering Systems Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA and fatty acids) jet fuel and conventional petroleum- Full list of author information is available at the end of the article based JP-8 [6]. Many sources of TAGs have been © 2013 Kurosawa et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Kurosawa et al. Biotechnology for Biofuels 2013, 6:134 Page 2 of 13 http://www.biotechnologyforbiofuels.com/content/6/1/134 successfully converted to aviation biofuel and the con- to engineer a robust xylose-fermenting Rhodococcus sensus is that generally any TAGs (with the exception strain. of those with very short chain fatty acids) are accept- able as precursors for fuel production [12]. Therefore, Results these energy-rich TAG molecules have attracted much Metabolic engineering of R. opacus PD630 to produce attention for developing sustainable and high-quality TAGs on xylose fuels. Currently the main sources for TAGs are vege- In order to engineer a xylose utilizing strain of R. opacus table oils, animal fats or waste cooking oils [13,14]. PD630, we sought to transfer the genes encoding en- Biofuels produced from crop seeds have come under zymes essential for xylose metabolism. Streptomyces major scrutiny due to the food vs. fuel competition padanus MITKK-103, an actinomycete that is closely re- issue [15]. In addition, although algae have been con- lated to Rhodococcus, efficiently utilizes xylose as a sole sidered as an attractive biocatalyst for lipid-based carbon source [32]. S. padanus genomic DNA was par- fuels owing to their ability to produce substantial tially digested with the restriction enzyme Sau3AI and amounts of TAGs [16,17], it is currently estimated the DNA fragments were ligated into the BamHI site of that large-scale production of biofuels from algae is the pAL358 plasmid bearing a gentamicin resistance cas- unsustainable using existing technologies [18,19]. sette and an origin of replication that allows for propa- Presently, the limited supply of bioresources for gation in R. opacus. R. opacus PD630 was transformed obtaining TAGs is a major obstacle for the produc- by electroporation using the resulting plasmid libraries tion of lipid-based biofuels, in spite of the advanta- from S. padanus. A total of 42 transformants were iso- geous impacts that commercialization of TAG-based lated and grown in a defined medium containing xylose biofuels could provide. as a sole carbon source. Although the cell growth and R. opacus PD630, an oleaginous hydrocarbon-degrading morphology of those strains on the plates were robust bacterium, is able to utilize long-chain-length alkanes, acet- and similar, curiously, the capabilities of utilizing xylose ate, phenylacetic acid, phenyldecane, propionate and gluco- and producing TAGs when grown in the flasks were dif- nate and produces remarkably high amounts of TAGs as a ferent from one another. Eight strains were capable of storage material [20-22]. Although TAG production of R. growing in the flasks, reaching an OD660 greater than opacus PD630 on glucose as a carbon source had not been 10, and only 4 of those, termed Xsp1, 8, 10 and 12, accu- shown until recently, we have demonstrated that the strain mulated over 30% of the cell dry weight (CDW) as TAGs has the rare ability to accumulate large amounts of TAGs (Additional file 1: Table S1). in batch-cultivations containing high concentrations of glu- cose under defined conditions [23,24]. While lignocellulosic Growth of R. opacus Xsp8 with high xylose concentrations carbohydrate fractions are composed primarily of glucose, High-cell-density cultivation is a prerequisite to maximize xylose represents a non-negligible portion of the sugar frac- volumetric productivity of microbial oil fermentation, and tion [25]. R. opacus PD630 is not able to utilize xylose the media used should be typically composed of highly con- naturally. The effective fermentation of xylose is necessary centrated sugars [33-35]. We examined the growth kinetics to develop economically feasible processes for fuel of strain Xsp8, the transformant that revealed the highest productions from lignocellulosic biomass [26,27]. Express- level of fatty acid production, on defined medium with ini- ing xylA, encoding xylose isomerase, and xylB,encoding tial xylose concentrations of 40, 120, 160, 180 and 200 g L-1 xylulokinase, has proven to be a successful strategy to en- in flask cultures (Figure 1). Xsp8 grew on media containing able growth on xylose for improvement of xylose metabol- up to 180 g L-1 of xylose, reaching stationary phase after 4 ism in bacteria [28-30]. Indeed, it has recently been days on 40 and 120 g L-1 and 5 days on 160 g L-1, reported that the two well-characterized genes xylA and respectively. xylB from Streptomyces lividans TK23 were expressed in R. jostii RHA1 and R. opacus PD630 to provide them with a TAG production of R. opacus Xsp8 on xylose and/or xylose utilization capability and the