Recombinant Ralstonia eutropha engineered to utilize xylose and its use for the production of poly(3-hydroxybutyrate) from sunflower stalk hydrolysate solution.
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.
Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, 335 Gwahagno, Yuseong-gu, Daejeon, 34141, Republic of Korea.
Hee Su
HS
Kim
Center for Bio-based Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseong-gu, Daejeon, 34602, Republic of Korea.
Young Hoon
YH
Oh
Center for Bio-based Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseong-gu, Daejeon, 34602, Republic of Korea.
Young-Ah
YA
Jang
Center for Bio-based Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseong-gu, Daejeon, 34602, Republic of Korea.
Kyoung Hee
KH
Kang
Department of Environmental Engineering and Energy, Myongji University, 116 Myongji-ro, Cheoin-gu, Yongin, Gyeonggido, 17058, Republic of Korea.
Yokimiko
Y
David
Center for Bio-based Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseong-gu, Daejeon, 34602, Republic of Korea.
Ju Hyun
JH
Yu
Center for Bio-based Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseong-gu, Daejeon, 34602, Republic of Korea.
Bong Keun
BK
Song
Department of Biotechnology and Bioengineering, Chonnam National University, 77 Yongbong-ro, Buk-gu, Gwangju, 61186, Republic of Korea.
Jong-il
JI
Choi
Department of Chemical and Biomolecular Engineering (BK21 Plus Program), KAIST, 335 Gwahagno, Yuseong-gu, Daejeon, 34141, Republic of Korea.
Yong Keun
YK
Chang
Center for Bio-based Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, P.O. Box 107, 141 Gajeong-ro, Yuseong-gu, Daejeon, 34602, Republic of Korea. jcjoo@krict.re.kr.
Jeong Chan
JC
Joo
Department of Environmental Engineering and Energy, Myongji University, 116 Myongji-ro, Cheoin-gu, Yongin, Gyeonggido, 17058, Republic of Korea. parksj93@mju.ac.kr.
Si Jae
SJ
Park
D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism; D002849:Chromatography, Gas; D002851:Chromatography, High Pressure Liquid; D020118:Cupriavidus necator / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006368:Helianthus / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D060847:Metabolic Engineering; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011091:Polyesters / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D014994:Xylose / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
D019747:Aldose-Ketose Isomerases; D029968:Escherichia coli Proteins; D006885:Hydroxybutyrates; D017853:Phosphotransferases (Alcohol Group Acceptor); D011091:Polyesters; D014994:Xylose; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C032700:xylulokinase; C023298:xylose isomerase
Lignocelluloses; Poly(3-hydroxybutyrate); Ralstonia eutropha; Sunflower stalk; Xylose