Engineering a thermostable highly active glucose 6-phosphate dehydrogenase and its application to hydrogen production in vitro

Applied Microbiology and Biotechnology
Rui HuangY-H Percival Zhang

Abstract

Glucose 6-phosphate dehydrogenase (G6PDH) is one of the most important dehydrogenases responsible for generating reduced NADPH for anabolism and is also the rate-limiting enzyme in the Entner-Doudoroff pathway. For in vitro biocatalysis, G6PDH must possess both high activity and good thermostability due to requirements of efficient use and low expense of biocatalyst. Here, we used directed evolution to improve thermostability of the highly active G6PDH from Zymomonas mobilis. Four generations of random mutagenesis and Petri-dish-based double-layer screening evolved the thermolabile wild-type enzyme to the thermostable mutant Mut 4-1, which showed a more than 124-fold increase in half-life time (t1/2) at 60 °C, a 3.4 °C increase in melting temperature (T m ), and a 5 °C increase in optimal temperature (Topt), without compromising the specific activity. In addition, the thermostable mutant was conducted to generate hydrogen from maltodextrin via in vitro synthetic biosystems (ivSB), gaining a more than 8-fold improvement of productivity rate with 76% of theoretical yield at 60 °C. Thus, the engineered G6PDH has been shown to effectively regenerate NADPH at high temperatures and will be applicable for NAD(P)H regeneration in numer...Continue Reading

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Citations

Jun 2, 2020·Frontiers in Bioengineering and Biotechnology·Pingping HanChun You
Jul 16, 2019·Metabolic Engineering·Ting ShiYi-Heng P Job Zhang
Oct 15, 2020·Chemical Reviews·Hui ChenShelley D Minteer
Nov 2, 2021·ACS Synthetic Biology·Biaobiao LuoXuebo Hu

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Datasets Mentioned

BETA
AY218838.1
AKE30817.1
BAD85297.1
JQ040550.1
AAL81018.1
AAL81015.1
AAL81016.1
AAL81017.1

Methods Mentioned

BETA
PCR

Software Mentioned

Autodock
MODEL
Origin
Chemdraw
PyMOL
SWISS
SigmaPlot

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