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

Abstract

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

Renewable Hydrogen Carrier — Carbohydrate: Constructing the Carbon-Neutral Carbohydrate Economy

“…We estimated that the enzyme costs would be very small when every enzyme in the SyPaB cocktails has total turn-over number (TTN) values of more than 10 7 -10 8 [31,33,35]. Based on our experience and data in the literature, it is relatively easy to obtain thermoenzymes meeting with such basic TTN requirement, for example, Clostridium themocellum phosphoglucomutase (PGM) [63], C. themocellum phosphoglucose isomerase (PGI) [64], Thermotoga martima 6-phosphogluconate dehydrogenase (6PGDH) [65], T. martima fructose bisphosphatase (FBP) [66], and immobilized glucose isomerase [62]. Costly enzymes purchased from Sigma-Aldrich and other biotechnology companies (e.g., New England Biolabs) give most researchers an impression that enzymes are very costly.…”

“…Clearly, it is highly operative to obtain numerous high-TTN non-membrane enzymes suitable for the SyPaB projects. With developments in (i) engineered oxidoreductases that can use biomimetic NAD factors [91][92][93] and (ii) stable enzymes as building blocks of SyPaB [63,65,66,73], we estimate that ultimate hydrogen production costs may decrease to ~$1.30 per kg of hydrogen (Figure 9a), where carbohydrate accounts for ~95% of its production costs, in part because hydrogen has very low product separation and purification costs and the other chemicals in the reaction can be recycled.…”

“…For industrial fructose production, ultra-stable immobilized thermophilic glucose isomerase with TTN of ~10 9 leads enzyme cost to a near negligible level. In our laboratory, we have obtained a few thermostable enzymes with TTN values of more than 10 7 , for example, C. thermocellum phosphoglucomutase [63], T. maritima fructose-1,6-bisphosphatase [66], and T. maritima 6-phosphogluconate dehydrogenase [65]. We have found out that free C. thermocellum phosphoglucose isomerase has low TTN values but it becomes ultra-stable (more than 10 9 ) after simple immobilization through adsorption on cellulose surface by using cellulose-binding module [64], as shown in Figure 7.…”

“…For example, expression levels of a recombinant hyperthermophilic T. maritima 6-phosphogluconate dehydrogenase in E. coli have been increased by ~500-fold by optimization of codon usage, expression plasmid and host, inducer type, concentration, and adding time, and so on [65]. Typical values of recombinant enzyme yield based on sugar (Y P/S ) may be up to 0.2 g of protein/g of sugar consumed [35], based on several well-known facts of (i) cell mass yield based on sugar in aerobic fermentation (Y X/S ) = ~0.5-0.6 g cell mass/g sugar [33,36,71], (ii) high intracellular cellular protein content = ~0.5-0.6 g protein/g cell mass [72], and (iii) high recombinant enzyme percentage in a total cellular protein = 50-70% [63,65,66,73]. Based on the rule of thumb of industrial fermentations, it is estimated that the production cost of recombinant protein may be $4.50-7.5/kg of protein = $0.30/kg sugar/(0.2 g protein/g sugar) × 3-5 (a cost adjustment coefficient due to other nutrient costs, labor, etc.…”

“…In reality, several low-cost scalable protein purification approaches are available, for example, simple centrifugation for secretory enzymes, adsorption/desorption on low-cost cellulosic materials [74,75] (Figure 5), heat precipitation for thermostable enzymes [65,76] (Figure 6), and ammonia precipitation [77] (Figure 6). Therefore, the purification costs for bulk enzymes would become relatively minor.…”