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J Biotechnol. 2019 Aug 20;302:1-9. doi: 10.1016/j.jbiotec.2019.06.005. Epub 2019 Jun 11.

Characterization of the biosynthetic pathway of nucleotide sugar precursor UDP-glucose during sphingan WL gum production in Sphingomonas sp. WG.

Author information

1
State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, People's Republic of China.
2
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, People's Republic of China.
3
College of Chemistry and Materials Science, Fujian Normal University, 32 Shangsan Road, Fuzhou 350007, People's Republic of China.
4
State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, People's Republic of China. Electronic address: jqwang@upc.edu.cn.
5
State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, People's Republic of China; College of Chemistry and Materials Science, Fujian Normal University, 32 Shangsan Road, Fuzhou 350007, People's Republic of China. Electronic address: zhuhu@fjnu.edu.cn.

Abstract

To elucidate the possible biosynthetic pathway of a precursor UDP-glucose of the sphingan WL gum produced by Sphingomonas sp. WG, two enzymes phosphoglucomutase (PGM) and UDP-glucose pyrophosphorylase (UGPase) were bioinformatically analysed, expressed in Escherichia coli BL21 (DE3) and characterized. PGM was in the phosphoglucomutase/phosphomannomutase subclass and UGPase was predicted to be a UDP-glucose pyrophosphatase in a tetrameric structure. Both enzymes were expressed in soluble form, purified to near homogeneity with high activity at 1159 and 796 U/mg, exhibited folding with reasonable secondary structures, and existed as monomer and tetramer, respectively. The optimal pH and temperature of PGM were 9.0 and 50 °C, respectively, and this protein was stable at pH 8.0 and at temperatures ranging from 40 to 50 °C. The optimal pH and temperature of UGPase were 9.0 and 45 °C, respectively, and the protein was stable at pH 8.0 and at temperatures ranging from 30 to 55 °C. A small-scale one-pot biosynthesis of UDP-glucose by combining PGM and UGPase using glucose-6-phosphate and UTP as substrates was also performed, and formation of UDP-glucose was observed by HPLC detection, which confirmed the biosynthetic pathway of UDP-glucose in vitro. PGM and UGPase will be ideal targets for the metabolic engineering to improve WL gum yields in industrial production.

KEYWORDS:

Phosphoglucomutase; Sphingan WL gum; Sphingomonas sp. WG; UDP-glucose; UDP-glucose pyrophosphorylase

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