Research

Overexpression and simple purification of the Thermotoga maritima 6-phosphogluconate dehydrogenase in Escherichia coli and its application for NADPH regeneration

Yiran Wang¹ and Y-H Percival Zhang^1,2,3

¹  Biological Systems Engineering Department, 210-A Seitz Hall, Virginia Polytechnic Institute and State University, Blacksburg, Virgina 24061, USA

²  Institute for Critical Technology and Applied Sciences (ICTAS) Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA

³  DOE BioEnergy Science Center (BESC), Oak Ridge, Tennessee 37831, USA

author email corresponding author email

Microbial Cell Factories 2009, 8:30doi:10.1186/1475-2859-8-30

Published:	4 June 2009

Abstract

Background

Thermostable enzymes from thermophilic microorganisms are playing more and more important roles in molecular biology R&D and industrial applications. However, over-production of recombinant soluble proteins from thermophilic microorganisms in mesophilic hosts (e.g. E. coli) remains challenging sometimes.

Results

An open reading frame TM0438 from a hyperthermophilic bacterium Thermotoga maritima putatively encoding 6-phosphogluconate dehydrogenase (6PGDH) was cloned and expressed in E. coli. The purified protein was confirmed to have 6PGDH activity with a molecular mass of 53 kDa. The k_cat of this enzyme was 325 s^-1 and the K_m values for 6-phosphogluconate, NADP⁺, and NAD⁺ were 11, 10 and 380 μM, respectively, at 80°C. This enzyme had half-life times of 48 and 140 h at 90 and 80°C, respectively. Through numerous approaches including expression vectors, hosts, cultivation conditions, inducers, and codon-optimization of the 6pgdh gene, the soluble 6PGDH expression levels were enhanced to ~250 mg per liter of culture by more than 500-fold. The recombinant 6PGDH accounted for >30% of total E. coli cellular proteins when lactose was used as a low-cost inducer. In addition, this enzyme coupled with glucose-6-phosphate dehydrogenase for the first time was demonstrated to generate two moles of NADPH per mole of glucose-6-phosphate.

Conclusion

We have achieved a more than 500-fold improvement in the expression of soluble T. maritima 6PGDH in E. coli, characterized its basic biochemical properties, and demonstrated its applicability for NADPH regeneration by a new enzyme cocktail. The methodology for over-expression and simple purification of this thermostable protein would be useful for the production of other thermostable proteins in E. coli.