Sheng Xu

Efficient production of biobased monomers from lignocellulosic hydrolysates is considered an important approach to reducing product costs and achieving sustainable biomanufacturing. However, the low conversion efficiency leads to a low yield and extended fermentation cycles. Here, we enhanced the utilization efficiency of xylose by strengthening the isomerase pathway. Subsequently, we successfully developed an efficient strain capable of synchronous glucose-xylose utilization for cadaverine production, thereby reducing the fermentation time from 30 to 24 h. Through optimization of the fermentation process, cadaverine production reached the highest level of 60.74 g/L in glucose-xylose mixture fermentation, with a yield of 0.34 g/g_sugar and a total sugar utilization rate of 3.65 g/L/h in a 5 L fermenter. When applied to the fermentation of corn stalk hydrolysate, 24.42 g/L cadaverine was obtained, with a yield of 0.32 g/g_sugar. This work has laid the foundation for the large-scale production of biobased monomers from lignocellulosic hydrolysates.

Nicotinamide mononucleotide, known as NMN, is an important nicotinamide adenine dinucleotide (NAD⁺) precursor. It is integral in cellular metabolism, energy generation, and processes associated with aging. Since NMN provides healthy value, it becomes a major focus for the biotechnological industry. This study presents a new biosynthetic pathway for producing NMN without limits on intracellular PRPP (5′-phosphoribosyl pyrophosphate) metabolic flux. The route started by converting cytidine into 1-phosphoribose via pyrimidine-nucleoside phosphorylase (PyNP), after transforming into nicotinamide riboside (NR) through either purine-nucleoside phosphorylase (XapA) or nicotinate riboside kinase (NRK). NR was phosphorylated by NRK in the presence of nicotinamide (NAM) to produce NMN. We established an in vitro enzyme activity verification system for the feasibility check. The optimization of multienzyme cascade reactions was figured out for the NMN biosynthesis. Finally, the enzymes of PyNP and NRK were expressed in the cytidine-producing strain; we established a de novo biosynthesis pathway from glucose to NMN, achieving a production titer of 33.71 mg/L at a shake-flask scale.

The industrial applications of enzymes are limited due to the activity-stability trade-off, which implies that the improvement of thermostability often accompanies decreased activity. This study presents a dual-strategy approach to simultaneously improve the catalytic efficiency and thermostability of α-galactosidase galV from Anoxybacillus vitaminiphilus WMF1. Our integrated method combines computational analysis with enzyme property prediction to selectively target and modify the catalytic region and residues that are distant from the active site. We identified and experimentally validated mutations that improve activity without compromising stability and further increased thermostability through additional distant-site mutations. The resulting mutant enzyme variant N549Q/T550N/Y634F demonstrated a 6.2-fold increase in catalytic efficiency and a 3.2-fold improvement in the half-life at 65 °C. Molecular dynamics (MD) simulations supported the structural basis for the observed enhancements. This approach offers a refined strategy for engineering α-galactosidases with improved industrial applicability, overcoming the traditional trade-offs between enzyme activity and stability. Hydrolytic activity toward raffinose family oligosaccharides (RFOs) was validated using soymilk as a model substrate, demonstrating significant practical potential.

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.

Background: Polyamide (nylon) is an important material, which has aroused plenty of attention from all aspects. PA 5.4 is one kind of nylon with excellent property, which consists of cadaverine and succinic acid. Due to the environmental pollution, bio-production of cadaverine and succinic acid has been more attractive due to the less pollution and environmental friendliness. Microbes, like Escherichia coli, has been employed as cell factory to produce cadaverine and succinic acid. However, the accumulation of cadaverine will cause severe damage on cells resulting in inhibition on cell growth and cadaverine production. Herein, a novel two stage co-production of succinic acid and cadaverine was designed based on an efficient thermos-regulated switch to avoid the inhibitory brought by cadaverine. Results: The fermentation process was divided into two phase, one for cell growth and lysine production and the other for cadaverine and succinic acid synthesis. The genes of ldhA and ackA were deleted to construct succinic acid pathway in cadaverine producer strain. Then, a thermal switch system based on pR/pL promoter and CI857 was established and optimized. The fermentation conditions were investigated that the optimal temperature for the first stage was determined as 33 ℃ and the optimal temperature for the second stage was 39 ℃. Additionally, the time to shifting temperature was identified as the fermentation anaphase. For further enhance cadaverine and succinic acid production, a scale-up fermentation in 5 L bioreactor was operated. As a result, the titer, yield and productivity of cadaverine was 55.58 g/L, 0.38 g/g glucose and 1.74 g/(L·h), respectively. 28.39 g/L of succinic acid was also obtained with yield of 0.19 g/g glucose. Conclusion: The succinic acid metabolic pathway was constructed into cadaverine producer strain to realize the co-production of succinic acid and cadaverine. This study provided a novel craft for industrial co-production of cadaverine and succinic 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.

Microbial cells utilizing electricity to produce high-value fuels and chemicals are the foundation of the biocathodic bioelectrochemical system. However, molecular mechanisms of electron transfer and utilization have not been elucidated. In this work, Escherichia coli engineered by introducing the Mtr pathway from Shewanella oneidensis exhibited stronger electrochemical activity than control and could utilize exogenous electrons to stimulate metabolite profiles and boost succinate production in the bioelectrochemical system. Proteomic analysis and real-time PCR were performed to investigate the effect of exogenous electrons on electroactive E. coli. Bioinformatics analysis suggested that the proteins of molecular function associated with oxidoreductase activity, 4 iron, 4 sulfur([4Fe-4S]) cluster binding, iron-sulfur cluster binding, and metal cluster binding were positively affected by exogenous electrons. Moreover, mapping to the Kyoto Encyclopedia of Genes and Genomes pathway database showed that the up-regulated proteins were mainly involved in metabolic pathways of tricarboxylic acid cycle, pyruvate metabolism, and nitrogen metabolism pathway, providing support for the metabolic balance of microbial cells shifting toward reduced end-products due to electron utilization. Using a biochemical method, the ompF-overexpressed strain was employed to investigate the function of the channel protein. These findings provided a theoretical basis for further improving electron transfer and utilization efficiency, and contributed to the potential applications of the bioelectrochemical system.