Recombinant Ralstonia eutropha engineered to utilize xylose and its use for the production of poly(3-hydroxybutyrate) from sunflower stalk hydrolysate solution

Abstract

Background: Lignocellulosic raw materials have extensively been examined for the production of bio-based fuels, chemicals, and polymers using microbial platforms. Since xylose is one of the major components of the hydrolyzed lignocelluloses, it is being considered a promising substrate in lignocelluloses based fermentation process. Ralstonia eutropha, one of the most powerful and natural producers of polyhydroxyalkanoates (PHAs), has extensively been examined for the production of bio-based chemicals, fuels, and polymers. However, to the best of our knowledge, lignocellulosic feedstock has not been employed for R. eutropha probably due to its narrow spectrum of substrate utilization. Thus, R. eutropha engineered to utilize xylose should be useful in the development of microbial process for bio-based products from lignocellulosic feedstock.

Results: Recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes encoding xylose isomerase and xylulokinase respectively, was constructed and examined for the synthesis of poly(3-hydroxybutyrate) [P(3HB)] using xylose as a sole carbon source. It could produce 2.31 g/L of P(3HB) with a P(3HB) content of 30.95 wt% when it was cultured in a nitrogen limited chemically defined medium containing 20.18 g/L of xylose in a batch fermentation. Also, recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes produced 5.71 g/L of P(3HB) with a P(3HB) content of 78.11 wt% from a mixture of 10.05 g/L of glucose and 10.91 g/L of xylose in the same culture condition. The P(3HB) concentration and content could be increased to 8.79 g/L and 88.69 wt%, respectively, when it was cultured in the medium containing 16.74 g/L of glucose and 6.15 g/L of xylose. Further examination of recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes by fed-batch fermentation resulted in the production of 33.70 g/L of P(3HB) in 108 h with a P(3HB) content of 79.02 wt%. The concentration of xylose could be maintained as high as 6 g/L, which is similar to the initial concentration of xylose during the fed-batch fermentation suggesting that xylose consumption is not inhibited during fermentation. Finally, recombinant R. eutorpha NCIMB11599 expressing the E. coli xylAB gene was examined for the production of P(3HB) from the hydrolysate solution of sunflower stalk. The hydrolysate solution of sunflower stalk was prepared as a model lignocellulosic biomass, which contains 78.8 g/L of glucose, 26.9 g/L of xylose, and small amount of 4.8 g/L of galactose and mannose. When recombinant R. eutropha NCIMB11599 expressing the E. coli xylAB genes was cultured in a nitrogen limited chemically defined medium containing 23.1 g/L of hydrolysate solution of sunflower stalk, which corresponds to 16.8 g/L of glucose and 5.9 g/L of xylose, it completely consumed glucose and xylose in the sunflower stalk based medium resulting in the production of 7.86 g/L of P(3HB) with a P(3HB) content of 72.53 wt%.

Conclusions: Ralstonia eutropha was successfully engineered to utilize xylose as a sole carbon source as well as to co-utilize it in the presence of glucose for the synthesis of P(3HB). In addition, R. eutropha engineered to utilized xylose could synthesize P(3HB) from the sunflower stalk hydrolysate solution containing glucose and xylose as major sugars, which suggests that xylose utilizing R. eutropha developed in this study should be useful for development of lignocellulose based microbial processes.

Keywords: Lignocelluloses; Poly(3-hydroxybutyrate); Ralstonia eutropha; Sunflower stalk; Xylose.

Fig. 1

Time profiles of flask-cultures of a wild-type R. eutropha NCIMB11599, b Recombinant R. eutropha (pKM212-XylAB) in sole carbon of xylose based medium for the synthesis of P(3HB). (Symbols are: filled circle, xylose concentration; open triangle-up, cell growth or cell dry weight; open square, P(3HB) concentration; open diamond, P(3HB) content)

Fig. 2

Metabolic pathways for biosynthesis of P(3HB) in recombinant R. eutropha strain from glucose and xylose as carbon sources used in this study. The overall metabolic pathway is shown together with the introduced metabolic pathways for the production of P(3HB). Xylulose 5-phosphate is generated by E. coli xylose isomerase (xylA) and xylulokinase (xylB). 3-Hydroxybutyryl-CoA is generated by R. eutropha β-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB). In R. eutropha strain, all the genes involved in PHA biosynthesis are in the chromosomal DNA except the xylAB gene encoding E. coli xylose isomerase and xylulokinase, which is additionally expressed by the introduction of pKM212-XylAB

Fig. 3

Time profiles of flask cultures of recombinant R. eutropha NCIMB11599 (pKM212-XylAB) in MR medium containing mixed sugars of glucose and xylose as carbon sources. a 9.25 g/L of glucose and 10.97 g/L of xylose were used as carbon sources b 13.76 g/L of glucose and 5.78 g/L of xylose were used as carbon sources. (Symbols are: filled square, glucose concentration; filled circle, xylose concentration; open triangle-up, cell growth)

Fig. 4

Time profiles of batch fermentations of a recombinant R. eutropha NCIMB11599 (pKM212-XylAB) in MR-B medium containing 20.18 g/L of xylose as a sole carbon source, b recombinant R. eutropha NCIMB11599 (pKM212-XylAB) in MR-B medium containing 10.05 g/L of glucose and 10.91 g/L of xylose as carbon sources and c recombinant R. eutropha NCIMB11599 (pKM212-XylAB) in MR-B medium containing 16.94 g/L of glucose and 6.15 g/L of xylose as carbon sources. (Symbols are: filled square, glucose concentration; filled circle, xylose concentration; open triangle-up, dry cell weight; open square, P(3HB) concentration; open diamond, P(3HB) content)

Fig. 5

Time profiles of fed-batch fermentations of recombinant R. eutropha NCIMB11599 (pKM212-XylAB) in MR-B medium containing 16.01 g/L of glucose and 7.48 g/L of xylose as carbon sources. (Symbols are: filled square, glucose concentration; filled circle, xylose concentration; open triangle-up, dry cell weight; open square, P(3HB) concentration; open diamond, P(3HB) content)

Fig. 6

Time profiles of flask cultures of recombinant R. eutropha NCIMB11599 (pKM212-XylAB) in MR medium containing sunflower stalk hydrolysate solution as a carbon source. The initial XGM is composed of 85.13 % xylose, 0.23 % galactose, and 14.64 % mannose. (Symbols are: filled square, glucose concentration; filled circle, xylose, galactose, and mannose (XGM) concentration; open triangle-up, cell growth)

Fig. 7

Time profiles of batch fermentation of recombinant R. eutropha NCIMB11599 (pKM212-XylAB) in MR-B medium containing sunflower stalk hydrolysate. The initial XGM is composed of 85.13 % xylose, 0.23 % galactose, and 14.64 % mannose. (Symbols are: filled square, glucose concentration; filled circle, xylose, galactose, and mannose (XGM) concentration; open triangle-up, dry cell weight; open square, P(3HB) concentration; open diamond, P(3HB) content)