Protein engineering of oxidoreductases utilizing nicotinamide-based coenzymes, with applications in synthetic biology

Abstract

Two natural nicotinamide-based coenzymes (NAD and NADP) are indispensably required by the vast majority of oxidoreductases for catabolism and anabolism, respectively. Most NAD(P)-dependent oxidoreductases prefer one coenzyme as an electron acceptor or donor to the other depending on their different metabolic roles. This coenzyme preference associated with coenzyme imbalance presents some challenges for the construction of high-efficiency in vivo and in vitro synthetic biology pathways. Changing the coenzyme preference of NAD(P)-dependent oxidoreductases is an important area of protein engineering, which is closely related to product-oriented synthetic biology projects. This review focuses on the methodology of nicotinamide-based coenzyme engineering, with its application in improving product yields and decreasing production costs. Biomimetic nicotinamide-containing coenzymes have been proposed to replace natural coenzymes because they are more stable and less costly than natural coenzymes. Recent advances in the switching of coenzyme preference from natural to biomimetic coenzymes are also covered in this review. Engineering coenzyme preferences from natural to biomimetic coenzymes has become an important direction for coenzyme engineering, especially for in vitro synthetic pathways and in vivo bioorthogonal redox pathways.

Keywords: Biomimetic coenzymes; Coenzyme engineering; NAD; NADP; Nicotinamide-based coenzymes; Protein engineering; Synthetic biology.

Fig. 1

Structures of nicotinamide-based coenzymes and biomimetic nicotinamide coenzymes. a) Two natural coenzymes, NAD⁺ and NADP⁺, in which the chemical groups in the open red rounded rectangles represent where the redox reaction occurs; these chemical groups are the same in all coenzymes. b) Biomimetic coenzymes derived from natural coenzymes, nicotinamide flucytosine dinucleotide (NFCD⁺), nicotinamide mononucleotide (NMN⁺), and nicotinamide mononucleoside (NR⁺); the chemical group in the shaded area indicates the structural difference between NFCD⁺ and NAD⁺. c) Synthetic biomimetic coenzyme 1-benzyl nicotinamide (BNA⁺).

Fig. 2

Scheme of coenzyme engineering methods, including rational design, semi-rational design and random mutagenesis.

Fig. 3

a) Amino acid sequence alignment of the coenzyme-binding motif of various 6PGDH enzymes. The residues comprising the loop region and responsible for coenzyme recognition are boxed. Red stars represent M. thermoacetica wild-type NADP⁺-preferred 6PGDH and the NAD⁺-preferred 6PGDH mutant. The blue star indicates T. maritima 6PGDH studied in this research. (b) Sub-alignments of key amino acid residues playing an important role in 2′-phosphate interaction. The colors in the sequence logo refer to hydrophobic (black), positive charge (blue), negative charge (red) and polar (green) residues (This figure is a courtesy from Ref. [2]).

Fig. 4

Scheme of double layer-based screening. a) Mechanism of the colorimetric assay. 6PGDH catalyzes the oxidation of 6-phosphogluconate to ribulose 5-phosphate and CO₂, and the reduction of NAD⁺ to NADH. In the presence of PMS, NADH transfers its hydride and reduces the colorless redox dye TNBT to black color TNBT formazan. b) The process of the double layer-based screening method. The mutant library was treated by heat and overlaid by a second agarose layer with reagents. The colonies with a darker color and halo were identified as positive mutants.

Fig. 5

Engineering the coenzyme preference of oxidoreductases in a metabolic pathway by protein engineering in vitro followed by the replacement of the wild-type enzyme with the mutant enzyme to solve the problem of coenzyme un-matching.