Creating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide

Xueying Wang,Zongbao K Zhao,Qing Li,Junting Wang,Qian Wang,Lei Wang,Xiaojia Guo,Siyang Ning,Yanbin Feng

doi:10.1038/s41467-021-22357-z

Abstract

Nicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. Diverse NAD mimics have been developed for applications in chemical and biological sciences. Nicotinamide cytosine dinucleotide (NCD) has emerged as a non-natural cofactor to mediate redox transformations, while cells are fed with chemically synthesized NCD. Here, we create NCD synthetase (NcdS) by reprograming the substrate binding pockets of nicotinic acid mononucleotide (NaMN) adenylyltransferase to favor cytidine triphosphate and nicotinamide mononucleotide over their regular substrates ATP and NaMN, respectively. Overexpression of NcdS alone in the model host Escherichia coli facilitated intracellular production of NCD, and higher NCD levels up to 5.0 mM were achieved upon further pathway regulation. Finally, the non-natural cofactor self-sufficiency was confirmed by mediating an NCD-linked metabolic circuit to convert L-malate into D-lactate. NcdS together with NCD-linked enzymes offer unique tools and opportunities for intriguing studies in chemical biology and synthetic biology.

Highlights

Nicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life
We engineered a number of NAD-dependent enzymes, including phosphite dehydrogenase, formate dehydrogenase, and D-lactate dehydrogenase to favor NCD11–14, and found wild-type nucleotide transporter NDT2 form Arabidopsis thaliana and NTT4 originated from Protochlamydia amoebophila UWE25 were efficient to shuttle Nicotinamide cytosine dinucleotide (NCD) into E. coli cells[15]
We further demonstrate that NCD self-sufficient cells were able to support NCD-linked metabolic circuit for orthogonal redox chemistry

Summary

Introduction

Nicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. We engineered a number of NAD-dependent enzymes, including phosphite dehydrogenase, formate dehydrogenase, and D-lactate dehydrogenase to favor NCD11–14, and found wild-type nucleotide transporter NDT2 form Arabidopsis thaliana and NTT4 originated from Protochlamydia amoebophila UWE25 were efficient to shuttle NCD into E. coli cells[15] By using these genetic parts, we have successfully devised metabolic circuits for pathway-selective chemical energy transfer, suggesting NCD potentially as a non-natural cofactor to establish more orthogonal redox chemistry. If the binding pockets of ATP and NaMN of NaMNAT are redesigned to favor cytidine triphosphate (CTP) and NMN, respectively (Fig. 1b), the engineered enzyme is expected to make NCD following a similar condensation mechanism, and can be designated as NCD synthetase (NcdS) As both CTP and NMN are endogenous metabolites, no auxiliary nutrients are required to synthesize NCD by those NcdS-empowered cells. This study provides NcdS to enable NCD biosynthesis and should promote further applications of NCD as a non-natural cofactor in chemical biology and synthetic biology

Methods

Results

Conclusion