Heteroexpression and biochemical characterization of a glucose-6-phosphate dehydrogenase from oleaginous yeast Yarrowia lipolytica

Mingjie Bian; Shan Li; Huanhuan Wei; Shiping Huang; Feng Zhou; Youming Zhu; Guoping Zhu

doi:10.1016/j.pep.2018.03.007

Heteroexpression and biochemical characterization of a glucose-6-phosphate dehydrogenase from oleaginous yeast Yarrowia lipolytica

Protein Expr Purif. 2018 Aug:148:1-8. doi: 10.1016/j.pep.2018.03.007. Epub 2018 Mar 23.

Authors

Mingjie Bian¹, Shan Li¹, Huanhuan Wei¹, Shiping Huang¹, Feng Zhou¹, Youming Zhu², Guoping Zhu³

Affiliations

¹ Institute of Molecular Biology and Biotechnology and the Research Center of Life Omics and Health, Anhui Normal University, No.1 Beijing East Road, Wuhu 241000, Anhui, China.
² Department of Oral and Maxillofacial Surgery, School of Stomatology, Stomatological Hospital, Anhui Medical University, No.81 Mei Shan Road, Hefei 230032, Anhui, China. Electronic address: zym88888@mail.ustc.edu.cn.
³ Institute of Molecular Biology and Biotechnology and the Research Center of Life Omics and Health, Anhui Normal University, No.1 Beijing East Road, Wuhu 241000, Anhui, China. Electronic address: gpz2012@ahnu.edu.cn.

PMID: 29580928
DOI: 10.1016/j.pep.2018.03.007

Abstract

Yarrowia lipolytica, a nonpathogenic, nonconventional, aerobic and dimorphic yeast, is considered an oleaginous microorganism due to its excellent ability to accumulate large amounts of lipids. Glucose-6-phosphate dehydrogenase (G6PD) is one of two key enzymes involved in the lipid accumulation in this fungi, which catalyzes the oxidative dehydrogenation of glucose-6-phosphate to 6-phosphoglucono-δ-lactone with the reduction of NADP⁺ to NADPH. In this study, the full-length gene of G6PD from Y. lipolytica (YlG6PD) was cloned without intron and heterogeneously expressed in E. coli. Then, YlG6PD was purified and biochemically characterized in details. Kinetic analysis showed that YlG6PD was completely dependent on NADP⁺ and its apparent K_m for NADP⁺ was 33.3 μM. The optimal pH was 8.5 and the maximum activity was around 47.5 °C. Heat-inactivation profiles revealed that it remained 50% of maximal activity after incubation at 48 °C for 20 min YlG6PD activity was competitively inhibited by NADPH with a K_i value of 56.04 μM. Most of the metal ions have no effect on activity, but Zn²⁺ was a strong inhibitor. Furthermore, the determinants in the coenzyme specificity of YlG6PD were investigated. Kinetic analysis showed that the single mutant R52D completely lost the ability to utilize NADP⁺ as its coenzyme, suggesting that Arg-52 plays a decisive role in NADP⁺ binding in YlG6PD. The identification of Y. lipolytica G6PD may provide useful scientific information for metabolic engineering of this yeast as a model for bio-oil production.

Keywords: Biochemical characterization; Coenzyme specificity determinants; Glucose-6-phosphate dehydrogenase; Hetero-expression; Kinetics; Yarrowia lipolytica.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Escherichia coli / genetics
Gene Expression Regulation, Enzymologic
Glucosephosphate Dehydrogenase / chemistry
Glucosephosphate Dehydrogenase / genetics*
Glucosephosphate Dehydrogenase / metabolism
Kinetics
Lipid Metabolism / genetics*
Metabolic Engineering
Yarrowia / enzymology*
Yarrowia / genetics

Substances

Glucosephosphate Dehydrogenase