Efficient Overproduction of Active Nitrile Hydratase by Coupling Expression Induction and Enzyme Maturation via Programming a Controllable Cobalt-Responsive Gene Circuit.

Laichuang Han,Qiao Lin,Zhemin Zhou,Ke Ma,Wenjing Cui,Yang Wang,Zhongyi Cheng,Feiya Suo,Qiaoqing Chen,Wenliang Hao

doi:10.3389/fbioe.2020.00193

Laichuang Han, Qiao Lin + Show 8 more

Open Access

https://doi.org/10.3389/fbioe.2020.00193

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Abstract

A robust and portable expression system is of great importance in enzyme production, metabolic engineering, and synthetic biology, which maximizes the performance of the engineered system. In this study, a tailor-made cobalt-induced expression system (CIES) was developed for low-cost and eco-friendly nitrile hydratase (NHase) production. First, the strong promoter Pveg from Bacillus subtilis, the Ni(II)/Co(II) responsive repressor RcnR, and its operator were reorganized to construct a CIES. In this system, the expression of reporter green fluorescent protein (GFP) was specifically triggered by Co(II) over a broad range of concentration. The performance of the cobalt-induced system was evolved to version 2.0 (CIES 2.0) for adaptation to different concentrations of Co(II) through programming a homeostasis system that rebalances cobalt efflux and influx with RcnA and NiCoT, respectively. Harnessing these synthetic platforms, the induced expression of NHase was coupled with enzyme maturation by Co(II) in a synchronizable manner without requiring additional inducers, which is a unique feature relative to other induced systems for production of NHase. The yield of NHase was 111.2 ± 17.9 U/ml using CIES and 114.9 ± 1.4 U/ml using CIES 2.0, which has a producing capability equivalent to that of commonly used isopropyl thiogalactoside (IPTG)-induced systems. In a scale-up system using a 5-L fermenter, the yielded enzymatic activity reached 542.2 ± 42.8 U/ml, suggesting that the designer platform for NHase is readily applied to the industry. The design of CIES in this study not only provided a low-cost and eco-friendly platform to overproduce NHase but also proposed a promising pipeline for development of synthetic platforms for expression of metalloenzymes.

Highlights

Nitrile hydratase (NHase), a kind of metalloprotein catalyzing the hydration of a broad scope of nitriles to corresponding amides, has been widely used in chemical engineering to produce bulk chemicals and highly valuable medical intermediates, such as acrylamide, nicotinamide, and (S)-mandelamide (Kobayashi and Shimizu, 1998; Gong et al, 2017; Cheng et al, 2018; Cobalt-Induced Expression System for nitrile hydratase (NHase)Supreetha et al, 2019)
We developed a tailor-made cobalt-induced expression system (CIES) in E. coli to overproduce highly active Co-NHase by coupling the Co-dependent induction and enzyme maturation through reprogramming the native rcn operon
To authenticate the designed function mediated by the hybrid promoter, we constructed plasmid pEVOGFP harboring Pvco equipped with a strong ribosome binding site (RBS) and green fluorescent protein (GFP) downstream

Summary

Introduction

Nitrile hydratase (NHase), a kind of metalloprotein catalyzing the hydration of a broad scope of nitriles to corresponding amides, has been widely used in chemical engineering to produce bulk chemicals and highly valuable medical intermediates, such as acrylamide, nicotinamide, and (S)-mandelamide (Kobayashi and Shimizu, 1998; Gong et al, 2017; Cheng et al, 2018; Cobalt-Induced Expression System for NHaseSupreetha et al, 2019). Nitrile hydratase (NHase), a kind of metalloprotein catalyzing the hydration of a broad scope of nitriles to corresponding amides, has been widely used in chemical engineering to produce bulk chemicals and highly valuable medical intermediates, such as acrylamide, nicotinamide, and (S)-mandelamide Engineered NHases that possess higher thermostability, activity, and stereoselectivity were developed in various ways such as subunit fusion and semi-rational design (Xia et al, 2016; Cheng et al, 2017, 2018). Compared with R. rhodochrous, Escherichia coli is a more ideal host to efficiently produce NHase, due to its clear genetic background, fast growth, simple genetic manipulation, and strong ability of protein expression (Baneyx, 1999). Chemical inducers in these induced expression systems are unable to be eliminated in the culture medium, becoming a potential detriment to the environment

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