Vitamin B5 [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (R)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel \"PEACES\" system by (1) expressing the NAD+ kinase gene ppnk from Clostridium glutamicum and the NADP+-dependent gapCcae from Clostridium acetobutyricum and (2) knocking-out the endogenous sthA gene, which interacts with ilvC and panE in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products.

A key approach in developing green chemistry involves converting solar energy into chemical energy of biomolecules through photocatalysis. Photocatalysis can facilitate the regeneration of nicotinamide cofactors during redox processes. Nicotinamide cofactor biomimetics (NCBs) are economical substitutes for natural cofactors. Here, photocatalytic regeneration of NADH and reduced NCBs (NCBsred) using graphitic carbon nitride (g-C3N4) was developed. The process involves g-C3N4 as the photocatalyst, Cp*Rh(bpy)H2O2+ as the electron mediator, and Triethanolamine as the electron donor, facilitating the reduction of NAD+ and various oxidative NCBs (NCBsox) under light irradiation. Notably, the highest reduction yield of 48.32 % was achieved with BANA+, outperforming the natural cofactor NAD+. Electrochemical analysis reveals that the reduction efficiency and capacity of cofactors relies on their redox potentials. Additionally, a coupled photo-enzymatic catalysis system was explored for the reduction of 4-Ketoisophorone by Old Yellow Enzyme XenA. Among all the NCBsox and NAD+, the highest conversion ratio of over 99 % was obtained with BANA+. After recycled for 8 times, g-C3N4 maintained over 93.6 % catalytic efficiency. The photocatalytic cofactor regeneration showcases its outstanding performance with NAD+ as well as NCBsox. This work significantly advances the development of photocatalytic cofactor regeneration for artificial cofactors and its potential application.

BACKGROUND: Lactones are important compounds in the field of medicine, material and chemical industry. One of the promising accesses to these flexible scaffolds is NAD(P)+-dependent alcohol dehydrogenases-catalyzed oxidative lactonization of diols, which relies on the construction of an efficient NAD(P)+ regeneration system.
RESULTS: In this study, a novel system combining horse liver alcohol dehydrogenase (HLADH) with the synthetic bridged flavin cofactor was established for biosynthesis of lactones. The reaction conditions of this system were optimized and a variety of lactones including chiral lactones were efficiently obtained from various diols. Compared to the previously reported NAD(P)+-regeneration systems, this system showed better regeneration efficiency and product yield. A two-phase system was further applied to solve the problem of product inhibition, and 80% yield was obtained at the condition of 300 mM substrate.
CONCLUSIONS: This study provides an efficient method to synthesis of lactones from diols under mild conditions. We believe this system will be a promising alternative to promote the synthesis of other valuable compounds.

Electron-rich phenolic substrates can be derived from the depolymerisation of lignin feedstocks. Direct biotransformations of the hydroxycinnamic acid monomers obtained can be exploited to produce high-value chemicals, such as α-amino acids, however the reaction is often hampered by the chemical autooxidation in alkaline or harsh reaction media. Regioselective O-methyltransferases (OMTs) are ubiquitous enzymes in natural secondary metabolic pathways utilising an expensive co-substrate S-adenosyl-l-methionine (SAM) as the methylating reagent altering the physicochemical properties of the hydroxycinnamic acids. In this study, we engineered an OMT to accept a variety of electron-rich phenolic substrates, modified a commercial E. coli strain BL21 (DE3) to regenerate SAM in vivo, and combined it with an engineered ammonia lyase to partake in a one-pot, two whole cell enzyme cascade to produce the l-DOPA precursor l-veratrylglycine from lignin-derived ferulic acid.
Protein and strain engineering combined. The combination of two engineered enzymes (a methyltransferase and an ammonia lyase) and an engineered E. coli strain (for regeneration of the SAM cofactor) has been developed to enable a fully biocatalytic one‐pot methylation–hydroamination cascade. As an example, the synthesis of l ‐veratrylglycine from renewable lignin‐derived ferulic acid has been demonstrated, in high yield and excellent ee.

Effective photolytic regeneration of the NAD(P)H cofactor in enzymatic reductions is an important and elusive goal in biocatalysis. It can, in principle, be achieved using a near-infrared light (NIR) driven artificial photosynthesis system employing H2O as the sacrificial reductant. To this end we utilized TiO2/reduced graphene quantum dots (r-GQDs), combined with a novel rhodium electron mediator, to continuously supply NADPH in situ for aldo-keto reductase (AKR) mediated asymmetric reductions under NIR irradiation. This upconversion system, in which the Ti-O-C bonds formed between r-GQDs and TiO2 enabled efficient interfacial charge transfer, was able to regenerate NADPH efficiently in 64 % yield in 105 min. Based on this, the pharmaceutical intermediate (R)-1-(3,5-bis(trifluoromethyl)phenyl)ethan-1-ol was obtained, in 84 % yield and 99.98 % ee, by reduction of the corresponding ketone. The photo-enzymatic system is recyclable with a polymeric electron mediator, which maintained 66 % of its original catalytic efficiency and excellent enantioselectivity (99.9 % ee) after 6 cycles.

(2S)-Eriodictyol, a polyphenolic flavonoid, has found widespread applications in health supplements and food additives. However, the limited availability of plant-derived (2S)-eriodictyol cannot meet the market demand. Microbial production of (2S)-eriodictyol faces challenges, including the low catalytic efficiency of flavone 3\'-hydroxylase/cytochrome P450 reductase (F3\'H/CPR), insufficient precursor supplementation, and inadequate NADPH regeneration. This study systematically engineered Yarrowia lipolytica for high-level (2S)-eriodictyol production. In doing this, the expression of F3\'H/CPR was balanced, and the supply of precursors was enhanced by relieving feedback inhibition of the shikimate pathway, promoting fatty acid β-oxidation, and increasing the copy number of synthetic pathway genes. These strategies, combined with NADPH regeneration, achieved an (2S)-eriodictyol titer of 423.6 mg/L. Finally, in fed-batch fermentation, a remarkable 6.8 g/L (2S)-eriodictyol was obtained, representing the highest de novo microbial titer reported to date and paving the way for industrial production.

Acetaldehyde is an important carbonyl compound commonly detected in wines. A high concentration of acetaldehyde can affect the flavor of wines and result in adverse effects on human health. Alcohol dehydrogenase I (ADH1) in Saccharomyces cerevisiae catalyzes the reduction reaction of acetaldehyde into ethanol in the presence of cofactors, showing the potential to reduce the content of acetaldehyde in wines. In this study, ADH1 was successfully expressed in Pichia pastoris GS115 based on codon optimization. Then, the expression level of ADH1 was enhanced by replacing its promoter with optimized promoters and increasing the copy number of the expression cassette, with ADH1 being purified using nickel column affinity chromatography. The enzymatic activity of purified ADH1 reached 605.44 ± 44.30 U/mg. The results of the effect of ADH1 on the content of acetaldehyde in wine revealed that the acetaldehyde content of wine samples was reduced from 168.05 ± 0.55 to 113.17 ± 6.08 mg/L with the addition of 5 mM NADH and the catalysis of ADH1, and from 135.53 ± 4.08 to 52.89 ± 2.20 mg/L through cofactor regeneration. Our study provides a novel approach to reducing the content of acetaldehyde in wines through enzymatic catalysis.

NAD+ -dependent formate dehydrogenase (FDH) catalyzes the conversion of formate and NAD+ to produce carbon dioxide and NADH. The reaction is biotechnologically important because FDH is widely used for NADH regeneration in various enzymatic syntheses. However, major drawbacks of this versatile enzyme in industrial applications are its low activity, requiring its utilization in large amounts to achieve optimal process conditions. Here, FDH from Bacillus simplex (BsFDH) was characterized for its biochemical and catalytic properties in comparison to FDH from Pseudomonas sp. 101 (PsFDH), a commonly used FDH in various biocatalytic reactions. The data revealed that BsFDH possesses high formate oxidizing activity with a kcat value of 15.3 ± 1.9 s-1 at 25°C compared to 7.7 ± 1.0 s-1 for PsFDH. At the optimum temperature (60°C), BsFDH exhibited 6-fold greater activity than PsFDH. The BsFDH displayed higher pH stability and a superior tolerance toward sodium azide and H2 O2 inactivation, showing a 200-fold higher Ki value for azide inhibition and remaining stable in the presence of 0.5% H2 O2 compared to PsFDH. The application of BsFDH as a cofactor regeneration system for the detoxification of 4-nitrophenol by the reaction of HadA, which produced a H2 O2 byproduct was demonstrated. The biocatalytic cascades using BsFDH demonstrated a distinct superior conversion activity because the system tolerated H2 O2 well. Altogether, the data showed that BsFDH is a robust enzyme suitable for future application in industrial biotechnology.

Aliphatic ω-amino fatty acids (ω-AFAs) and α,ω-diamines (α,ω-DMs) are essential monomers for the production of nylons. Development of a sustainable biosynthesis route for ω-AFAs and α,ω-DMs is crucial in addressing the challenges posed by climate change. Herein, we constructed an unprecedented thermodynamically favorable multi-enzyme cascade (TherFavMEC) for the efficient sustainable biosynthesis of ω-AFAs and α,ω-DMs from cheap α,ω-dicarboxylic acids (α,ω-DAs). This TherFavMEC was developed by incorporating bioretrosynthesis analysis tools, reaction Gibbs free energy calculations, thermodynamic equilibrium shift strategies and cofactor (NADPH&ATP) regeneration systems. The molar yield of 6-aminohexanoic acid (6-ACA) from adipic acid (AA) was 92.3 %, while the molar yield from 6-ACA to 1,6-hexanediamine (1,6-HMD) was 96.1 %, which were significantly higher than those of previously reported routes. Furthermore, the biosynthesis of ω-AFAs and α,ω-DMs from 20.0 mM α,ω-DAs (C6-C9) was also performed, giving 11.2 mM 1,6-HMD (56.0 % yield), 14.8 mM 1,7-heptanediamine (74.0 % yield), 17.4 mM 1,8-octanediamine (87.0 % yield), and 19.7 mM 1,9-nonanediamine (98.5 % yield), respectively. The titers of 1,9-nonanediamine, 1,8-octanediamine, 1,7-heptanediamine and 1,6-HMD were improved by 328-fold, 1740-fold, 87-fold and 3.8-fold compared to previous work. Therefore, this work holds great potential for the bioproduction of ω-AFAs and α,ω-DMs.

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.