Design the adenosine phosphate hydrolysis (APH) pathway multienzyme cascade system for the biosynthesis of nicotinamide mononucleotide (NMN) in vitro.

Background: Itaconic acid, an unsaturated C5 dicarbonic acid, has significant market demand and prospects. It has numerous biological functions, such as anti-cancer, anti-inflammatory, and anti-oxidative in medicine, and is an essential renewable platform chemical in industry. However, the development of industrial itaconic acid production by Aspergillus terreus, the current standard production strain, is hampered by the unavoidable drawbacks of that species. Developing a highly efficient cell factory is essential for the sustainable and green production of itaconic acid. Results: This study employed combinatorial engineering strategies to construct Escherichia coli cells to produce itaconic acid efficiently. Two essential genes (cis-aconitate decarboxylase (CAD) encoding gene cadA and aconitase (ACO) encoding gene acn) employed various genetic constructs and plasmid combinations to create 12 recombination E. coli strains to be screened. Among them, E. coli BL-CAC exhibited the highest titer with citrate as substrate, and the induction and reaction conditions were further systematically optimized. Subsequently, employing enzyme evolution to optimize rate-limiting enzyme CAD and synthesizing protein scaffolds to co-localize ACO and CAD were used to improve itaconic acid biosynthesis efficiency. Under the optimized reaction conditions combined with the feeding control strategy, itaconic acid titer reached 398.07 mM (51.79 g/L) of engineered E. coli BL-CAR470E-DS/A-CS cells as a catalyst with the highest specific production of 9.42 g/g_(DCW) among heterologous hosts at 48 h. Conclusions: The excellent catalytic performance per unit biomass shows the potential for high-efficiency production of itaconic acid and effective reduction of catalytic cell consumption. This study indicates that it is necessary to continuously explore engineering strategies to develop high-performance cell factories to break through the existing bottleneck and achieve the economical commercial production of itaconic acid.

Phospholipase B (PLB) harbors three distinct activities with broad substrate specificities and application fields. Its hydrolyzing of sn-1 and sn-2 acyl ester bonds enables it to catalyze the production of L-alpha-glycerylphosphorylcholine (L-α-GPC) from phosphatidylcholine (PC) without speed-limiting acyl migration. This work was intended to obtain high-level active PLB and apply it to establish an efficient system for L-α-GPC synthesis. PLB from Pseudomonas fluorescens was co-expressed with five different molecular chaperones, including trigger factor (Tf), GroEL-GroES (GroELS), DnaK-DnaJ-GrpE (DnaKJE), GroELS and DnaKJE, or GroELS and Tf or fused with maltose binding protein (MBP) in Escherichia coli BL21(DE3) to improve PLB expression. PLB with DnaKJE-assisted expression exhibited the highest catalytic activity. Further optimization of the expression conditions identified an optimal induction OD₆₀₀ of 0.8, IPTG concentration of 0.3 mmol/L, induction time of 9 h, and temperature of 25 °C. The PLB activity reached a maximum of 524.64 ± 3.28 U/mg under optimal conditions. Subsequently, to establish an efficient PLB-catalyzed system for L-α-GPC synthesis, a series of organic-aqueous mixed systems and surfactant-supplemented aqueous systems were designed and constructed. Furthermore, the factors of temperature, reaction pH, metal ions, and substrate concentration were further systematically identified. Finally, a high yield of 90.50 ± 2.21% was obtained in a Span 60-supplemented aqueous system at 40 °C and pH 6.0 with 0.1 mmol/L of Mg²⁺. The proposed cost-effective PLB production and an environmentally friendly PLB-catalyzed system offer a candidate strategy for the industrial production of L-α-GPC.