Hao He

Cadaverine, an essential precursor for bio-based nylon PA5X production, has emerged as a highly valuable compound in the engineered plastics and synthetic fibers industry. Current industrial biomanufacturing of cadaverine depends critically on the enzymatic activity of L-lysine decarboxylase (LDC). In this investigation, we systematically evaluated ten LDC variants originating from six distinct bacterial species, all cloned and expressed in Escherichia coli BL21(DE3). Comprehensive characterization identified SpLDC from Serratia proteamaculans as the most promising candidate, demonstrating exceptional catalytic activity under alkaline conditions. Enzymatic analysis showed that SpLDC established optimal activity at pH 6.5 and 52 °C, with remarkable stability maintained between pH 5.5-8.0 and temperatures of 37–52 °C during prolonged 12-hour incubations. Kinetic studies revealed favorable catalytic parameters (K_m = 11.5 mM, V_max = 1000 U/mg), indicating efficient substrate conversion. To enhance industrial applicability, we developed an immobilized enzyme system by genetically fusing SpLDC with a chitin-binding domain (ChBD) and subsequently immobilizing it on chitin supports. This engineered biocatalyst demonstrated superior performance compared to whole-cell systems, achieving a 3.4-fold enhancement in cadaverine production efficiency. The immobilized preparation exhibited excellent operational stability, retaining over 50% of its initial activity through ten repeated reaction cycles. This work establishes an efficient, stable, and reusable enzymatic platform for cadaverine biosynthesis, offering significant potential for sustainable nylon PA5X production at industrial scales.

Pyridoxal 5′-phosphate (PLP), the active form of vitamin B6, is an essential cofactor, yet its industrial supply still relies largely on multi-step chemical synthesis. Here, using the industrial chassis Corynebacterium glutamicum ATCC 13032, we proposed and validated a strategy based on a minimal heterologous entry coupled to endogenous pathway continuation, resulting in a distinct PLP-producing route. Three engineered strains were constructed and compared: S1 expressing ecepd from Escherichia coli; S2 co-expressing ecepd plus ecpdxB from Escherichia coli (a minimal two-gene module); and S3 carrying an additional ecpdxA from Escherichia coli and smpdxJ from Sinorhizobium meliloti to form a four-gene module as a benchmark for heterologous reconstruction. The wild-type (WT) strain produced a basal PLP level of 10.6 mg/L. Overexpressing ecepd alone increased the titer to 40.4 mg/L (3.8-fold vs WT), whereas the minimal two-gene module in S2 yielded the highest PLP titer of 95.5 mg/L (9.0-fold vs WT; 136.0% higher than S1). Notably, the four-gene module (S3) reached 70.0 mg/L, which was 36.3% lower than S2 under matched conditions. These results indicated that introducing only a minimal two-gene entry could cooperate with the endogenous metabolic network of Corynebacterium glutamicum to establish a new and highly effective PLP biosynthetic route, with production performance exceeding that of a multi-gene heterologous reconstruction in the tested window. This work provides a low-burden and scalable framework for sustainable PLP biomanufacturing and motivates further optimization targeting the endogenous continuation steps and regulatory constraints.

Industrial microbes have become the core of biological manufacturing, which utilized as the cell factory for production of plenty of chemicals, fuels and medicine. However, the challenge that the extreme stress conditions exist in production is unavoidable for cell factory. Consequently, to enhance robustness of the chassis cell lays the foundation for development of bio-manufacturing. Currently, the researches on cell tolerance covered various aspects, involving reshaping regulatory network, cell membrane modification and other stress response. In fact, the strategies employed to improve cell robustness could be summarized into two directions, irrational engineering and rational engineering. In this review, the metabolic engineering technologies on enhancement of microbe tolerance to industrial conditions are summarized. Meanwhile, the novel thoughts emerged with the development of biological instruments and synthetic biology are discussed.

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.