Electrocatalytic NAD(P)H regeneration for biosynthesis

The highly efficient chemoselectivity,stereoselectivity,and regioselectivity render enzyme catalysis an ideal pathway for the synthesis of various chemicals in broad applications.While the cofactor of an enzyme is necessary but expensive,the conversed state of the cofactor is not beneficial for the positive direction of the reaction.Cofactor regeneration using electrochemical methods has the advantages of simple operation,low cost,easy process monitoring,and easy product separation,and the electrical energy is green and sustainable.Therefore,bioelectrocatalysis has great potential in synthesis by combining electrochemical cofactor regeneration with enzymatic catalysis.In this review,we detail the mechanism of cofactor regeneration and categorize the common electron mediators and enzymes used in cofactor regeneration.The reaction type and the recent progress are summarized in electrochemically coupled enzymatic catalysis.The main challenges of such electroenzymatic catalysis are pointed out and future developments in this field are foreseen.

Recent advance of chemoenzymatic catalysis for the synthesis of chemicals: Scope and challenge

Chemoenzymatic catalysis can give full play to the advantages of versatile reactivity of chemocatalysis and excellent chemo-,regio-,and stereoselectivities of biocatalysis.These chemoenzymatic methods can not only save resource,cost,and operating time but also reduce the number of reaction steps,and avoid separating unstable intermediates,leading to the generation of more products under greener circumstances and thereby playing an indispensable role in the fields of medicine,materials and fine chemicals.Although incompatible challenges between chemocatalyst and biocatalyst remain,strategies such as biphasic system,artificial metalloenzymes,immobilization or supramolecular host,and protein engineering have been designed to overcome these issues.In this review,chemoenzymatic catalysis according to different chemocatalysis types was classifiably described,and in particular,the classic dynamic kinetic resolutions(DKR)and cofactor regeneration were summarized.Finally,the bottlenecks and development of chemoenzymatic catalysis were summarized,and future development was prospected.

Synergetic engineering of Escherichia coli for efficient production of L-tyrosine

L-Tyrosine,an aromatic non-essential amino acid,is the raw material for many important chemical products,including levodopa,resveratrol,and hydroxytyrosol.It is widely used in the food,drug,and chemical industries.There are many studies on the synthesis of L-tyrosine by microorganisms,however,the low titer of L-tyrosine limited the industrial large-scale production.In order to enhance L-tyrosine production in Escherichia coli,the expression of key enzymes in the shikimate pathway was up-or down-regulated.The L-tyrosine transport system and the acetic acid biosynthesis pathway were modified to further enhance L-tyrosine production.In addition,the phosphoketolase pathway was introduced in combination with cofactor engineering to redirect carbon flux to the shikimate pathway.Finally,after adaptive laboratory evolution to low pH an optimal strain was obtained.The strain can produce 92.5 g/L of L-tyrosine in a 5-L fermenter in 62 h,with a yield of 0.266 g/g glucose.

Iron-doping Accelerating NADH Oxidation over Carbon Nitride

As a state-of-the-art conjugated polymer photocatalyst,graphitic carbon nitride(abbreviated as g-C3Na)has shown sreat potential in photocatalvtic cofactor(reduced form of nicotinamide adenine dinucleotide.NADH)regene-ration.Herein,Fe-doped g-CaNa was engineered for photocatalvtic NADH oxidation.The n-r interaction between the NADH molecule and the conjugated heptazine buildine block facilitates the adsorption of NADH onto the framework as revealed by density functional theory(DFT)calculations,Furthermore,iron doping promoted the oxidation kinetics of NADH under blue LED illumination.The conversion ratio of NADH to its oxidized form could be up to 85.7%in 20 min,comparing with 59.4%for metal-free counterpart.Enzyme assay employing formate dehydrogenase(FDH)further verified the selectivity of the products,with 67.5%+2.6%of enzymatically active 1,4-NADH being regenerated following the oxidation process.Scavenger experiments suggest the dominant role of photo-induced electrons in theoxidation of NADH.This work could shed light on developing a novel cofactor regeneration route through the syner-gistic effect between the metal doping and noncovalent interaction based on the coniugated polymer.

Asymmetric reduction of conjugated C=C bonds by immobilized fusion of old yellow enzyme and glucose dehydrogenase

Asymmetric reduction of the conjugated C=C bonds by the old yellow enzymes(OYEs)presents a promising field in the synthesis of chiral chemicals.Nevertheless,few natural OYEs have been applied in large-scale applications due to the requirement of costly NADPH and low operational stability.Herein,a stable and efficient fusion of YqjM from Bacillus subtilis and glucose dehydrogenase(GDH)from Bacillus megaterium was constructed to stereoselectively reduce the conjugated C=C bonds in a self-sufficient continuous process.The effects of the enzyme order and different linkers on the fusions were investigated by structural analysis and all-atom molecular dynamics simulation.The best fusion YqjM_G_GDH gave 98% conversion of 100 mmol/L 2-methylcyclopentenone with an excellent ee value(>99%)in 3 h,while the mixture of individual enzymes only obtained 68% conversion after more than 8 h.The improved substrate conversion of YqjM_G_GDH fusion was probably attributed to the increased flexibility of each fused enzyme and the shortening of the diffusion distance of NADPH regenerated.A one-pot process was designed to purify and immobilize the fusion on the Ni2t-nitrilotriacetic acid functionalized magnetic mesoporous silica nanoflowers.The resulting immobilized biocatalyst not only catalyzed the asymmetric reduction of various α,β-unsaturated ketones(20 mmol/L)continuously with only 50μmol/L NADPt to initiate the whole process,but also retained more than 82%of the initial activity after seven cycles,serving as a good candidate for the industrial applications.