Multienzyme co-immobilization-based bioelectrode:Design of principles and bioelectrochemical applications

Enzyme cascade reactions play significant roles in bioelectrochemical processes because they permit more complex reactions. Co-immobilization of multienzyme on the electrode could help to facilitate substrate/intermediate transfer among different enzymes and electron transfer from enzyme active sites to the electrode with high stability and retrievability. Different co-immobilization strategies to construct multienzyme bioelectrodes have been widely reported, however, up to now, they have barely been reviewed. In this review, we focus on recent state-of-the-art techniques for constructing co-immobilized multienzyme electrodes including random and positional co-immobilization. Particular attention is given to strategies such as multienzyme complex and surface display. Cofactor co-immobilization on the electrode is also crucial for the enhancement of catalytic reaction and electron transfer, yet, few studies have been reported. The up-to-date advances in bioelectrochemical applications of multienzyme bioelectrodes are also presented. Finally, key challenges and future perspectives are discussed.

MeNA, Controlled by Reversible Methylation of Nicotinate, Is an NAD Precursor that Undergoes Long-Distance Transport in Arabidopsis

Nicotinamide adenine dinucleotide （NAD） biosynthesis, including synthesis from aspartate via the de novo pathway and from nicotinate （NA） via the Preiss-Handler pathway, is conserved in land plants. Diverse spe-cies of NA conjugates, which are mainly involved in NA detoxification, were also found in all tested land plants. Among these conjugates, MeNA （NA methyl ester） has been widely detected in angiosperm plants, although its physiological function and the underlying mechanism for its production in planta remain largely unknown. Here, we show that MeNA is an NAD precursor undergoing more efficient long-distance trans-port between organs than NA and nicotinamide in Arabidopsis. We found that Arabidopsis has one meth- yltransferase （designated AtNaMT1） capable of catalyzing carboxyl methylation of NA to yield MeNA and one methyl esterase （MES2） predominantly hydrolyzing MeNA back to NA. We further uncovered that the transfer of [^14C]MeNA from the root to leaf was significantly increased in both MES2 knockdown and NaMTl-overexpressing lines, suggesting that both NaMT1 and MES2 fine-tune the long-distance transport of MeNA, which is ultimately utilized for NAD production. Abiotic stress （salt, abscisic acid, and mannitol） treatments, which are known to exacerbate NAD degradation, induce the expression of NaMT1 but sup-press MES2 expression, suggesting that MeNA may play a role in stress adaption. Collectively, our study indicates that reversible methylation of NA controls the biosynthesis of MeNA in Arabidopsis, which pre-sumably functions as a detoxification form of free NA for efficient long-distance transport and eventually NAD production especially under abiotic stress, providing new insights into the relationship between NAD biosynthesis and NA conjugation in plants.

Stoichiometric Conversion of Maltose for Biomanufacturing by In Vitro Synthetic Enzymatic Biosystems

Maltose is a natural α-(1,4)-linked disaccharide with wide applications in food industries and microbial fermentation. However,maltose has scarcely been used for in vitro biosynthesis, possibly because its phosphorylation by maltose phosphorylase (MP)yields β-glucose 1-phosphate (β-G1P) that cannot be utilized by α-phosphoglucomutase (α-PGM) commonly found in in vitrosynthetic enzymatic biosystems previously constructed by our group. Herein, we designed an in vitro synthetic enzymaticreaction module comprised of MP, β-phosphoglucomutase (β-PGM), and polyphosphate glucokinase (PPGK) for thestoichiometric conversion of each maltose molecule to two glucose 6-phosphate (G6P) molecules. Based on this syntheticmodule, we further constructed two in vitro synthetic biosystems to produce bioelectricity and fructose 1,6-diphosphate (FDP),respectively. The 14-enzyme biobattery achieved a Faraday efficiency of 96.4% and a maximal power density of 0.6mW/cm^(2),whereas the 5-enzyme in vitro FDP-producing biosystem yielded 187.0mM FDP from 50 g/L (139mM) maltose by adopting afed-batch substrate feeding strategy. Our study not only suggests new application scenarios for maltose but also provides novelstrategies for the high-efficient production of bioelectricity and value-added biochemicals.