Abstract:

Abstract:Yeast surface display (YSD) is an important technological platform integrating protein engineering, synthetic biology, and industrial biocatalysis. Its catalytic efficiency and stability highly depend on the dynamic synergy of the three-tier microenvironments: the cell wall, intracellular environment, and extracellular compartments. This review systematically elaborates on the core components of the YSD microenvironment, including the cell wall, anchor proteins, intracellular secretion pathways, and extracellular physicochemical properties, and clarifies their critical roles in the folding, activity, and anchoring efficiency of displayed proteins. In addition, this review summarizes targeted regulation strategies, including cell wall gene engineering, optimization of anchor proteins and linkers, enhancement of intracellular secretion pathways, adaptation of extracellular culture conditions, and spatial arrangement of multienzyme co-display. It reveals that synergistic optimization of the three-tier microenvironments can significantly improve display efficiency and catalytic performance. YSD technology based on microenvironment regulation has achieved substantial application progress in industrial production, biomedicine, and environmental remediation. However, current microenvironment regulation still faces challenges in terms of precision, compatibility, and stability. Future research should focus on developing corresponding regulation strategies to break through technical bottlenecks and promote the industrial application of YSD. Microenvironment regulation provides important theoretical and technical support for the performance optimization and industrial translation of YSD.

Abstract:Ethylene, a fundamental feedstock in the petrochemical industry, is conventionally produced via energy-intensive steam cracking of fossil fuels, a process associated with high energy consumption and carbon emissions. Constructing photosynthetic cyanobacterial cell factories for the direct, solar-driven conversion of CO₂ to ethylene offers a promising route for the green production of ethylene. This review systematically summarizes recent advances in this field, highlighting the core catalytic mechanism of the ethylene-forming enzyme (EFE), the screening and optimization of key genetic elements, metabolic engineering strategies (including gene copy number amplification, precursor supply enhancement, rewiring of carbon flux, and alleviation of by-product inhibition), and the optimization of cultivation processes. Furthermore, we identify the key challenges currently impeding cyanobacterial production of ethylene, such as low yields and efficiencies, insufficient genetic stability, and bottlenecks in large-scale cultivation. Finally, we outline future development directions—advancing synthetic biology tools, enabling precise regulation of metabolic networks, designing efficient photobioreactors, and integrating industrial technologies, with the aim of providing a theoretical foundation for establishing robust and high-performance cyanobacterial cell factories for ethylene production and facilitating their practical application.

Abstract:Microbial fermentation technology is crucial in modern biomanufacturing processes, such as biopharmaceutical production. However, traditional fermentation process control faces bottlenecks including offline monitoring delay, reliance on empirical experience, and a lack of precise and real-time regulation. Machine learning (ML), with its powerful data extraction and predictive modeling capabilities, has emerged as a key tool for driving the transition from traditional fermentation processes to intelligent paradigms. This paper systematically reviewed the latest research progress on the closed-loop intelligent fermentation control systems, spanning from underlying data perception to high-level decision-making. First, the specific applications of ML in fermentation optimization were elaborated, with a focus on systematic modeling workflows in response to the inherent “black-box” nature and data-lag limitations of purely data-driven models. Second, by comparing the monitoring characteristics of various process analytical technologies, the critical supporting role of Raman spectroscopy in real-time fermentation monitoring was elucidated, and cutting-edge approaches employing semi-supervised learning and data augmentation strategies to address the scarcity of high-quality labeled samples were summarized. Furthermore, the ML-driven “intelligent perception-feedback closed-loop” control mechanism was thoroughly analyzed, and the transformation of fermentation control from traditional “macroscopic feeding regulation” to “microscopic metabolic precise guidance” was expounded. Finally, the future development trajectories for intelligent fermentation was envisioned from two perspectives, namely the construction of cross-genus universal predictive models and the application of digital twin technology. This review aims to provide valuable technical references for the intelligent upgrading of the biomanufacturing sector and the efficient development of fermentation processes.

Abstract:Cordycepin is a natural nucleoside antibiotic isolated from the fruiting bodies of Cordyceps militaris. It possesses diverse health-beneficial and medicinal values. However, the conventional fermentation processes for cordycepin production suffer from inherent drawbacks, including low yields, prolonged production cycles, and batch-to-batch inconsistency. These problems result in the exorbitant market price of cordycepin and severely restrict the potential for market application. The advent of synthetic biology has offered a revolutionary approach to address this challenge. This article systematically reviews the development trajectory of cordycepin production, spanning from conventional fermentation to modern synthetic biology-based manufacturing. It focuses on analyzing the breakthrough advancements in the construction of microbial cell factories such as Saccharomyces cerevisiae and cordycepin synthesis. Finally, it provides perspectives on the future development of more efficient, eco-friendly, and intelligent cordycepin biomanufacturing technologies by leveraging enzyme engineering and synthetic biology. And this study offers theoretical support and technical references for constructing high-cordycepin-producing strains, which is critical to facilitate the industrialization of cordycepin.

Abstract:Algal-bacterial symbiotic and mutualistic systems enhance community stability and functional performance through interspecies cooperation and signal exchange, demonstrating considerable potential in environmental remediation and sustainable biomanufacturing. This review systematically summarizes the fundamental interaction types and underlying mechanisms of algal–bacterial symbiosis and mutualism, with a particular focus on enabling technologies for the rational reconstruction of engineered mutualistic consortia and their emerging applications in environmental remediation and biomanufacturing. Different interaction modes, including mutualism, commensalism, parasitism, and endosymbiosis, are comparatively discussed, together with key regulatory mechanisms such as chemical signaling, nutrient exchange, horizontal gene transfer, and homeostasis maintenance under abiotic stress conditions. At the technical level, this paper introduces the recent advances in single-cell dynamic monitoring approaches, artificial intelligence-driven design platforms, and synthetic community engineering strategies integrating both top-down and bottom-up methodologies. Furthermore, this paper discusses the potential applications of algal-bacterial mutualistic systems in wastewater remediation, aquaculture, bioenergy recovery, and sustainable bioproduction and points out the current research challenges related to mechanism understanding, species specificity, long-term system stability, and ecological risk assessment. Finally, this review proposes that interdisciplinary approaches integrating multi-omics analyses, computational modeling, and controllable environmental validation will facilitate the development of efficient, robust, and sustainable algal-bacterial symbiotic systems, thereby providing theoretical guidance and technical support for advancing synthetic biology-driven resource utilization and green biomanufacturing.

Abstract:The construction of microbial cell factories frequently encounters bottlenecks due to the complexity of metabolic pathways, insufficient enzyme functionality, and poor environmental tolerance. Directed evolution, as a pivotal technology for overcoming these engineering constraints, can significantly enhance the titer, productivity, and overall robustness of target-producing strains. Its development originated from traditional random mutagenesis, a method that generates genetic diversity through the imposition of artificial growth pressures and is currently in widespread use. However, owing to the uncontrollable direction of mutagenesis and the low frequency of beneficial mutations, this approach suffers from low screening efficiency and is both time-consuming and labor-intensive. To accelerate the evolutionary rate, global accelerated evolution strategies based on tools such as DNA repair deficiencies and cytidine deaminases have emerged, achieving orders-of-magnitude increases in genome-wide mutation rates. Targeted continuous evolution strategies centered on CRISPR/Cas and T7 RNA polymerase systems enable the precise introduction of mutations within predefined genomic regions, thereby greatly improving the enrichment efficiency of beneficial mutations while minimizing non-productive mutations. This review will focus on three core strategies—traditional random evolution, global accelerated evolution, and targeted continuous evolution—systematically elucidating their developmental trajectories, applications in the construction of microbial cell factories, and future directions. Collectively, this review aims to provide methodological guidance for overcoming the engineering bottlenecks in the construction of microbial cell factories.

Abstract:With the development of DNA synthesis and genome engineering technology, life science research has gradually evolved from gene editing to higher-level genome writing. By designing and constructing large-scale DNA fragments and even complete genomes from scratch, researchers have been able to reconstruct the genetic structures of life systems at the genome scale, which enables the systematical study of the relationship between genome structure and function. Related studies have achieved remarkable progress in various biological systems such as prokaryotes, Saccharomyces cerevisiae, and mammalian cells and have shown broad application prospects of this technology in functional gene screening, biological manufacturing, medical research, and basic life science research. This paper systematically reviews the main progress in large-scale DNA design and assembly technology, summarizes the research progress of synthetic genomes in different biological systems and their potential in functional gene screening and biotechnology applications, and discusses the current technical challenges and future development directions, aiming to provide a reference for the research and application of synthetic genome engineering.

Abstract:Curdlan is a water-insoluble β-1,3-glucan synthesized by microorganisms under nitrogen-limited conditions. It possesses unique physicochemical properties, such as gelation ability, freeze-thaw stability, water-holding capacity, and thickening capability, and is widely used in the food and pharmaceutical industries. In recent years, with the rapid development of the alternative protein (NeoProtein) sector, curdlan has demonstrated outstanding application potentials by significantly improving the fidelity and texture quality of meat analogues through matrix texture modification, animal fat mimicry, and flavor release modulation, and its efficient bio-manufacturing technology has thus become a research hotspot. This review analyzes and summarizes the structure and function, application areas, biosynthesis and regulatory mechanisms, and advances in biosynthesis technology of curdlan, and proposes future directions for trends for the development of curdlan biosynthesis technology, thereby providing theoretical support for future strain improvement, fermentation process optimization, and application exploration for curdlan biomanufacturing.

Abstract:Environmental functional microorganisms play a key role in environmental engineering processes such as pollutant degradation, wastewater treatment, and resource utilization. Conventional enrichment and directed domestication technologies often have limitations such as long operational cycles and low throughput, being difficult to meet the demands for rapid screening, enhancement, and engineering application of functional microorganisms under complex environmental conditions. In recent years, advances in continuous cultivation, high-throughput cultivation, microfluidic chips, intelligent bioreactors, and automated adaptive laboratory evolution (ALE) have provided new approaches for the precise screening, targeted enhancement, and feedback-based process control of environmental functional microorganisms. This review systematically summarizes the basic principles and key platforms of automated enrichment and directed domestication technologies, as well as their application progress in the development of microorganisms for petroleum hydrocarbon and polycyclic aromatic hydrocarbon degradation, wastewater denitrification, heavy metal tolerance, and emerging contaminant degradation. In addition, by integrating our research group’s work on targeted environmental microorganism isolation devices, programmable logic controller (PLC)-driven ALE systems, and the screening of functional strains for halogenated organic pollutant degradation, this review analyzes the current limitations of these technologies in in situ simulation, maintenance of mixed microbial community interactions, screening of non-growth-coupled phenotypes, and engineering scale-up. Finally, future directions are proposed for the application of automated enrichment and directed acclimation technologies in the exploration, functional enhancement, and engineering application of environmental functional microbial resources. This review is expected to provide theoretical and methodological support for improving the technological framework of environmental functional microorganism development and facilitating its translation into engineering practice.

Abstract:Ergothioneine (ERG), as a natural potent antioxidant, possesses excellent physicochemical properties and diverse physiological activities, thereby exhibiting broad application prospects. However, engineering of existing genetically modified strains is prone to encountering bottlenecks in yield improvement. To identify key regulatory genes involved in ERG biosynthesis and enhance its heterologous production efficiency in Escherichia coli, this study employed E. coli BL21(DE3) as the chassis and constructed a heterologous biosynthetic pathway comprising Trichoderma reesei-derived Tregt1 and Tregt2, yielding a foundational engineered strain P1 with a shake-flask fermentation titer of 650 mg/L. Subsequently, a genome-wide mutant strain library was constructed using a random integration system mediated by Himar1C9 transposase, and combined with a high-throughput screening method based on the mBBr fluorescent probe, four positive mutant strains exhibiting significant phenotypic differences were successfully obtained. Sequencing analysis revealed that the key mutated genes encode phosphoenolpyruvate synthase PpsA, vitamin B₁₂ transporter permease BtuC, DNA helicase RecG, and a bifunctional nicotinamidase/pyrazinamidase PncA. Shake-flask fermentation validation showed that overexpression of recG significantly increased the ERG titer to 797 mg/L, whereas overexpression of the other three genes reduced the yield. This study establishes a systemic strategy combining random transposon integration and high-throughput screening, thereby overcoming the limitations of conventional targeted engineering and laying a foundation for the construction of efficient ERG-producing engineered strains and the advancement of their industrial production.

Abstract:In natural ecosystems, bacteriophages play a crucial role in bacterial lysis. Bacteriophage contamination frequently leads to fermentation failure, resulting in compromised product quality and purity, as well as substantial economic loss. Consequently, effective strategies are required in the fermentation industry to mitigate bacteriophage contamination. The use of a single anti-phage strategy is readily evaded by spontaneous phage mutations, resulting in limited and poorly applicable protection against phage infection. Synergistic defense mechanisms involving multiple systems suppress the spontaneous mutations that enable phage escape, thereby broadening phage resistance. This study is designed to tackle the bottleneck associated with single anti-phage strategies, which are susceptible to escape via spontaneous phage mutations, exhibit limited protective efficacy, and suffer from poor applicability. By integrating receptor engineering with synergistic defense mechanisms, a highly efficient, broad-spectrum, and non-toxic phage defense network system is constructed. This system is subsequently applied to industrial fermentation strains, with the aim of achieving effective resistance against phage contamination and improving the stability of fermentation processes. Initially, a synergistic resistance mechanism between Shedu and Septu was identified through laboratory screening of defense mechanisms. Further investigation revealed that the endonuclease activity of Shedu and the ATPase and HNH nuclease activities of Septu jointly contribute to synergistic resistance. Optimization of promoters was employed to adapt the Shedu and Septu mechanisms, thereby conferring resistance to phages T4, λ, P251, and P255. Subsequently, receptor engineering was applied to modify fhuA, a key adsorption site for phages T1 and T7, to achieve resistance against these phages and complete the construction of the anti-phage network system. Growth curve analysis demonstrated that the anti-phage network system exhibits minimal toxicity and high resistance efficiency, highlighting its strong industrial application potential. Finally, phage resistance was introduced into riboflavin- and ?-leucine-producing strains, enabling them to effectively counter phage contamination during fermentation. In a 5 L bioreactor, riboflavin production reached 11.2 g/L and ?-leucine production reached 64.0 g/L, thereby confirming the industrial potential of the anti-phage system. The resistance strategy reported herein facilitates the development of engineered Escherichia coli strains with robust and broad-spectrum phage resistance for using in the industrial fermentation of natural amino acids and high-value compounds.

Abstract:Chondroitin and its derivatives are essential glycosaminoglycans in articular cartilage and connective tissue, with broad applications in the nutraceutical and pharmaceutical industries. Conventional chemical and enzymatic synthesis approaches are often limited by process complexity, low efficiency, and high substrate costs. In contrast, microbial fermentation offers a promising alternative for the sustainable production of chondroitin due to its shorter production cycle, lower cost, and scalability. To obtain a genetically stable chondroitin-producing strain, in this study, an efficient genome-integrated Escherichia coli strain for chondroitin production was developed based on the strain GZ17 previously constructed in our laboratory. First, a chondroitin synthase from Moraxella canis (McCS) was identified through phylogenetic analysis and stably expressed under a strong promoter via genomic integration. Subsequently, key amino acid residues were identified through structural modeling and molecular dynamics simulations, followed by iterative mutagenesis to obtain a highly active mutant, McCS^{S602A/N632S/K637L}. Finally, two copies of the mutant McCS^{S602A/N632S/K637L} gene were integrated into the genome of GZ17, which significantly enhanced chondroitin biosynthesis. The resulting strain, GZ-2ASLM, achieved a chondroitin titer of 7.41 g/L, a glycerol yield of 0.03 g/g, and a productivity of 0.10 g/(L·h) by fed-batch fermentation in a 5-L fermenter. Notably, this engineered strain utilizes glycerol as the sole carbon source for chondroitin production without depending on plasmids. This study provides a robust strategy for developing efficient microbial cell factories for the production of glycosaminoglycans.

Abstract:Bovine enterokinase light chain (EK_L), a serine protease that specifically recognizes the Asp-Asp-Asp-Asp-Lys (DDDDK) sequence, is widely used for the specific cleavage of affinity tags in recombinant proteins. However, EK_L contains multiple cysteine residues and four disulfide bonds, making it difficult to fold correctly, and it tends to form inactive inclusion bodies when expressed in Escherichia coli. To achieve efficient soluble expression of EK_L in E. coli and screen high-activity mutants, co-expression of the DnaK-DnaJ-GrpE and GroES-GroEL molecular chaperones was adopted to enhance its solubility. Subsequently, the DDDDK sequence was site-specifically introduced into the Loop region of superfolder green fluorescent protein (sfGFP) to generate the fluorescent substrate sfGFP^M for EK_L. Based on the principle of fluorescence loss, a high-throughput screening method for highly active EK_L variants was established. Using error-prone PCR, a random mutagenesis library of the EK_L gene was constructed, from which the EK_L-S42M/I139D mutant was obtained. This mutant showed a 2.56-fold increase in catalytic efficiency and significantly improved thermostability. Molecular dynamics simulations revealed that the S42M/I139D mutations synergistically optimized the stability and activity of EK_L via distal allosteric regulation. Meanwhile, fed-batch fermentation further enhanced the soluble expression level of EK_L-S42M/I139D, with a yield of 203 mg/L and an enzymatic activity of 7.45×10? U/mL. This study not only achieves the soluble expression of EK_L in E. coli and obtains a highly active mutant, but also provides a visual tool for the rapid detection of EK_L activity.

Abstract:β-carotene is a natural lipid-soluble carotenoid widely used in the food and pharmaceutical industries. Although metabolic engineering efforts for the production of β-carotene via microbial fermentation have achieved certain progress, there remains room for further improvement. Compared with Saccharomyces cerevisiae or Escherichia coli, Yarrowia lipolytica exhibits a pronounced lipid-flux orientation in its core metabolic logic, conferring a natural advantage for the production of lipid-soluble compounds. This study aims to engineer Y. lipolytica strains for efficient β-carotene production. First, heterologous synthesis of β-carotene was achieved by replacing native promoters with strong ones, attenuating competing pathways, and introducing a bifunctional enzyme derived from Mucor circinelloides. Subsequently, β-carotene production was enhanced through morphological engineering to inhibit hyphal formation, optimization of key gene copy numbers, and improvement of acetyl-CoA supply. The resulting engineered strain LY20 achieved a β-carotene titer of 346.2 mg/L in shake-flask fermentation. Furthermore, in fed-batch fermentation with glucose as the carbon source in a 7.5-L bioreactor, the β-carotene titer reached 3.56 g/L. This study systematically optimizes the biosynthetic pathway for β-carotene through systems metabolic engineering, highlighting the potential of Y. lipolytica for the engineered synthesis of highly hydrophobic natural products and providing guidance for the efficient production of other lipid-soluble terpenoids.

Abstract:Astaxanthin, a high-value terpenoid secondary metabolite that exhibits excellent antioxidant activity, has broad application potential in the pharmaceutical and nutraceutical industries. However, its sophisticated biosynthetic pathway poses a great challenge for efficient production and industrial-scale application of astaxanthin. In this study, the unconventional oleaginous yeast Yarrowia lipolytica was employed as the chassis organism, and a multidimensional metabolic engineering strategy combined with fermentation process optimization was used to construct a microbial cell factory for astaxanthin production. Firstly, a heterologous β-carotene biosynthetic pathway was reconstituted, the carbon flux supply of the mevalonate (MVA) pathway was systematically enhanced, and endogenous branch metabolism was blocked, leading to the initial construction of an astaxanthin-producing strain. Subsequently, a RIDD-RIAD protein interaction tag was introduced to optimize the expression patterns of the key rate-limiting enzymes of β-carotene ketolase (CrtW) and β-carotene hydroxylase (CrtZ), thereby effectively improving the efficiency of astaxanthin biosynthesis. Following this, six rounds of iterative targeted metabolic modification combined with shake-flask fermentation significantly enhanced the biosynthetic potential of the engineered strains. The results showed that the engineered strain YAX11 achieved an astaxanthin titer of 107.4 mg/L in shake-flask fermentation, and the titer was increased to 1 148.4 mg/L in a 5 L bioreactor. Through the deep integration of metabolic network remodeling and fermentation process optimization, this study successfully constructed a Y. lipolytica cell factory for astaxanthin production, providing a theoretical reference for the green and large-scale manufacturing of astaxanthin, as well as metabolic engineering strategies for the heterologous synthesis of other terpenoid compounds.

Abstract:As an essential amino acid, l-lysine is widely used in the feed, food, and pharmaceutical industries. However, the production of l-lysine via Escherichia coli fermentation is still plagued by inadequate substrate utilization, severe carbon atom waste, and a low sugar-to-acid conversion rate. To address these issues, this study used E. coli M2019435 as the chassis strain to generate strain HM-01 by knocking out the global regulatory factor gene mlc and heterologously expressing malp and glvA derived from Bacillus subtilis as well as the sucrose metabolism gene cluster scrKYAB. On this basis, carbon-13 labeled metabolic flux analysis (¹³C-MFA) was employed to identify the bottlenecks of pyruvate accumulation and carbon atom loss. The pyruvate carboxylase gene pyc and carbonic anhydrase gene cynT were then introduced to construct a CO₂ refixation loop. Meanwhile, feedback inhibition was relieved and the l-lysine degradation gene cadA was knocked out, resulting in the generation of strain HM-05. Further optimization of the residual sugar concentration (0.50 g/L) and carbon-to-nitrogen ratio (1:1 to 1:2) during the fermentation of strain HM-05 was conducted in a 5-L fermenter to achieve the precise supply of carbon and nitrogen sources. After 30 h of fermentation, the l-lysine titer, sugar-to-acid conversion rate, and productivity reached 210.8 g/L, 0.768 g/g, and 7.02 g/(L·h), which were 44.8%, 27.5%, and 42.7% higher than those of the original strain, respectively. The strategy of enhanced substrate utilization-targeted carbon flux optimization-upgraded process optimization developed in this study provides important support for the industrial production of l-lysine.

Abstract:Poly-γ-glutamic acid (γ-PGA) is a biopolymer polymerized from l-glutamic acid (l-Glu) and/or d-glutamic acid monomers, exhibiting broad application prospects in pharmaceuticals, cosmetics, and other fields. The low yields remain a key factor limiting large-scale production of γ-PGA in heterologous expression systems. To enhance the biosynthetic efficiency, this study employed Corynebacterium glutamicum as the chassis cell and utilized the γ-PGA synthase PgsBCA from Bacillus licheniformis to catalyze γ-PGA synthesis. First, a polycistron expression system was constructed, increasing the γ-PGA yield by 20.6% compared with the monocistron system. After site-directed and saturation mutagenesis of PgsA, the mutant K76C was identified, increasing the γ-PGA yield by 75.3%. On this basis, a Bayesian model was employed to optimize seven medium components including glucose and urea. The optimized medium achieved a γ-PGA yield of 14.52 g/L, which represented a 28.6% increase over that in the initial medium. Finally, the recombinant strain was scaled up in a 5 L fermenter. After 48 hours of fermentation, the γ-PGA yield reached 64.13 g/L, with a glucose conversion rate of 0.52 g/g and the l-Glu content of 100%. This study developed a high-yielding γ-PGA-producing engineered strain and established an efficient fermentation process, providing a novel strategy for the efficient production of γ-PGA.

Abstract:d-phenylalanine is an important d-amino acid with a wide range of applications in the pharmaceutical and chemical industries. meso-diaminopimelate dehydrogenase (DAPDH) catalyzes the asymmetric reductive amination of α-keto acids to produce d-amino acids with high optical purity, serving as a key enzymatic module for constructing multi-enzyme cascade systems. However, the strict dependence of wild-type DAPDH on NADPH results in high cofactor costs, limiting its industrial application. To address this issue, this study focused on a previously engineered yet still NADPH-preferring type I DAPDH from Corynebacterium glutamicum ( CgDAPDH). Based on structural analysis of the protein, a three-pronged engineering strategy involving steric hindrance reduction, local electrostatic potential modulation, and conformational flexibility enhancement was proposed. Through site-directed and combinatorial mutagenesis of residues at the cofactor-binding pocket entrance (Ser35, Arg36, Arg37) and conserved regions (Ser68, Thr88, Asp120), a mutant S35E/R36E/R37A/S68T/T88A was obtained. This mutant exhibited a catalytic efficiency ( k _cat/ K _m) of 274.87 mmol/(L·s) toward NADH, representing a 16.9-fold improvement over the starting enzyme, while achieving comparable catalytic competence with both NADH and NADPH. This mutant was coupled with formate dehydrogenase (FDH) to construct an NADH self-regeneration system for whole-cell catalytic reductive amination using phenylpyruvate as the substrate. After 6 h of reaction, the titer of d-phenylalanine reached 36.22 g/L with a conversion rate of 90.12%. When this system was applied to three-enzyme cascade employing l-phenylalanine as the substrate, the d-phenylalanine titer reached 16.25 g/L, which was comparable to that obtained with a glucose dehydrogenase/NADPH regeneration system (17.21 g/L). This study demonstrates that cofactor engineering can alleviate the NADPH dependence of DAPDH and achieve efficient conversion of phenylpyruvate to d-phenylalanine, thereby providing a new enzymatic module and a technical route for cost-effective biomanufacturing of d-amino acids.

Abstract:Galacto-N-biose (GNB), a core structural unit of O-glycan and a key component of gastrointestinal mucins, plays important physiological roles in regulating intestinal microecology and maintaining the mucosal barrier. Traditional enzymatic routes for the synthesis of GNB rely on the supplies of ATP and UDP-sugars. To address this disadvantage, we designed an in vitro ATP-free and UDP-sugar-free enzymatic pathway to synthesize GNB directly from lactose and N-acetylgalactosamine (GalNAc). This pathway consisted of two core enzymes, lactose phosphorylase (LacP) and lacto-N-biose phosphorylase (LnbP), as well as one auxiliary enzyme, polyphosphate glucokinase (PPGK). By incorporating PPGK to remove the byproduct glucose, this pathway not only pushed the reaction equilibrium toward the biosynthesis of GNB, but also achieved the regeneration of inorganic phosphate. Through the systematic optimization of key parameters such as reaction temperature, pH, and enzyme dosage, a molar yield of 76% was achieved with 10 mmol/L GalNAc. This in vitro biotransformation pathway provides a technical basis for the low-cost, large-scale green production of GNB and other high-value functional oligosaccharides.

Abstract:(1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one[(1R,5S)-1a] is the key chiral starting material for the synthesis of mirogabalin. Existing chemical and biocatalytic resolution processes both suffer from cumbersome post-processing procedures and high production costs. To achieve the efficient and green resolution of (1R,5S)-1a, the present study employed racemic ketone 1a as the substrate and identified LfAmDH(M0) as the template for directed evolution through enzyme library screening. The substrate binding site and the key amino acid residues around the substrate access channel were selected, and site-directed mutagenesis and iterative mutagenesis were performed in turn to construct a multi-round mutant library. Following library screening, the specific activities of the mutants were elevated to 1.3- to 5.9-fold that of the starting enzyme, demonstrating significantly optimized catalytic performance. The substrate spectrum characteristics of the starting enzyme M0 and the dominant mutants were investigated, revealing that the mutants exhibited appreciable catalytic activity and stereoselectivity toward aliphatic ketones, cyclic ketones, arylalkyl ketones and aromatic ketones. In a 50 mL reaction system, the optimal mutant L40T/L125R/V291A(M3) converted 150 mmol/L of substrate. After the reaction was completed, the product was subjected to extraction and acid washing, affording the chiral ketone with an ee value of 97.1% and a yield of 45.4%. This study successfully developed a novel amine dehydrogenase-catalyzed resolution approach for (1R,5S)-1a, which simplifies the post-processing workflow of the product and reduces the consumption of organic reagents, thereby providing robust technical support for the efficient industrial-scale biomanufacturing of (1R,5S)-1a.

Abstract:Biocatalysis, as a fundamental enabling technology of biomanufacturing, is indispensable for promoting the green and efficient transformation of industrial processes. The catalytic disorder of enzymes provides key support for the development of new chemical conversions that do not exist in nature. Therefore, we urgently need to expand the diverse new functions of enzymes. In this study, we report a case of an unnatural intermolecular Stetter reaction catalyzed by natural benzaldehyde lyase (pfBAL). Benzaldehyde lyase activates benzaldehyde to undergo an addition reaction with the imine substrate formed in situ, and thus an unnatural amino ketone is obtained. Through systematic screening and optimization of reaction conditions, the yield of the target product was enhanced, and a certain range of substrate applicability was demonstrated. This study demonstrates that the benzaldehyde lyase pfBAL exhibits catalytic activity in mediating the Stetter reaction between unnatural molecules, laying a theoretical foundation for the future expansion of diverse catalytic functions of this enzyme.

Abstract:To achieve efficient enzymatic preparation of specific high-degree-of-polymerization chito-oligosaccharides (degree of polymerization≥5, DP≥5), rational molecular engineering of chitosanase Csn46-Mut4 was performed in this study. Through site-directed mutagenesis coupled with DNS enzyme activity-HPLC product distribution screening, a superior mutant, C41A/T47A/V144A, was obtained. Under conditions of 55 ℃ and pH 5.5, the catalytic efficiency and thermostability of this enzyme were remarkably enhanced, reaching 2.56-fold and 1.67-fold those of Csn46-Mut4, respectively. Peak area normalization analysis revealed that the relative content of chitopentaose (DP5) increased to 22.8%, representing a 12-fold increase compared with Csn46-Mut4, while the total proportion of chitooligosaccharides with DP≥5 reached 27.33%, corresponding to a 6.68-fold increase. Moreover, the total yield of DP≥5 chitooligosaccharides by the mutant reached 64.18% (5.776 g/L), which was 3.14-fold that of Csn46-Mut4 (20.41%, 1.837 g/L). Further molecular dynamics simulations revealed that the three mutation sites acted synergistically via the C41A and T47A substitutions in the catalytic flexible region and the V144A substitution in the substrate-binding channel region. This synergy strengthened the global hydrogen-bonding network and improved the structural rigidity while inducing characteristic dynamic changes in the substrate-binding pocket. In the initial simulation stage, pocket expansion facilitated the entry of long-chain substrates, whereas in the later stage, the enhanced hydrogen-bonding network, combined with the introduction of local critical flexibility, drove the accumulation of medium- and long-chain products, particularly leading to a notable increase in chitopentaose. This study not only yields a novel chitosanase capable of efficiently and specifically producing high-DP chitooligosaccharides (DP≥5) but also offers a new strategy for optimizing enzyme catalytic performance through multi-site synergistic design across different protein regions.

Abstract:The β-glucosidase from Bacillus thermoamylovorans (Bgl52) has been identified as a thermophilic and acidophilic β-glucosidase, which constitutes the most critical component in the cellulolytic enzyme system for cellulose hydrolysis. Although Bgl52 is a thermophilic enzyme, its poor thermal stability at high temperatures restricts its long-term industrial application under elevated-temperature conditions. In this study, rational design strategies, including two free energy calculation programs (Fireprot and FoldX) and homologous sequence alignment, were integrated to perform single-point and multi-point combinatorial mutagenesis on Bgl52. The enzymatic kinetic stability assay and molecular dynamics simulation were conducted to further investigate the catalytic performance and structural variations of positive mutants. After screening, the optimal stacked mutant M5 exhibited increases in T_m, optimal temperature, and T₅₀ by 8.7, 10 and 8 ℃, respectively, compared with Bgl52 (wild type, WT), and its half-life t_1/2 at 80 ℃ was enhanced by 32.5 folds. The catalytic efficiency (K_cat/K_m) of M5 showed a slight decrease compared with that of the wild-type enzyme. Furthermore, M5 hydrolyzed native cellulose into glucose more efficiently at the high temperature of 85 ℃. Structural analysis and molecular dynamics simulation revealed that the improved thermostability was attributed to increased hydrogen bonds, the proline effect, additional salt bridges, and enhanced hydrophobic interactions. The results indicate that the multi-strategy integrated rational design approach demonstrates outstanding efficiency and reliability in enzyme engineering modification, enhancing the industrial application potential of β-glucosidase.

Abstract:Replacing grain-derived carbon sources with non-grain biomass carbon sources for fermentation is a core research direction in the field of biomanufacturing. However, inhibitory byproducts present in the enzymatic hydrolysate of non-grain biomass severely restrict its practical application. We aim to reduce the dependence of xanthan gum fermentation on grain-based carbon sources, clarify the impacts of typical inhibitors from non-grain biomass enzymatic hydrolysate on xanthan gum synthesis, break through the existing application bottlenecks, and develop a green, low-loss and high-efficiency detoxification technology to overcome the inherent defects of traditional detoxification processes. In this study, we used sugarcane bagasse enzymatic hydrolysate as the fermentation carbon source, and the xanthan gum-producing strain Xanthomonas campestris 1.178 as the research object. We investigated the effects of furfural (FFR) and 5-hydroxymethylfurfural (HMF) on xanthan gum biosynthesis. To address the defects of high sugar loss and massive byproduct generation in the conventional CaO-H₃PO₄ detoxification process, we developed an innovative CaO-CO₂ combined detoxification process, and comparatively analyzed the physicochemical properties of xanthan gum produced via the two detoxification processes. We identified that acetic acid and isovaleric acid are the key inhibitory factors in sugarcane bagasse enzymatic hydrolysate that limit xanthan gum synthesis. After CaO-CO₂ treatment, we achieved retention rates of 98.23% for total sugar, 95.63% for reducing sugar, and 95.08% for glucose in the hydrolysate. We obtained a xanthan gum yield as high as 19.76 g/L, which was 19.75% higher than the yield from the CaO-H₃PO₄ treatment. The xanthan gum prepared in this study shared consistent infrared spectrum characteristics with commercially available xanthan gum, and we confirmed its excellent tolerance under low temperature, alkaline and high-salt conditions. The CaO-CO₂ detoxification process developed in this study realizes efficient conversion of sugarcane bagasse enzymatic hydrolysate to xanthan gum, and provides a brand-new green solution for the high-value utilization of non-grain biomass.

Abstract:Filamentous microorganisms—encompassing filamentous fungi and actinomycetes—are versatile cell factories for producing proteins and bioactive natural products, playing a critical role in the food and pharmaceutical industries. These microorganisms exhibit slow growth and complex morphological differentiation. Conventional screening methods for strain engineering are hindered by low throughput and an inability to resolve heterogeneity in strain cultivation and growth. Droplet-based microfluidics offers an attractive platform for high-throughput screening at the single-cell level, yet its utility is limited by poor droplet stability and low post-sorting viability. Given the aforementioned technical bottlenecks, this study aims to compare the compatibility between filamentous fungi and actinomycetes and droplet microfluidic technology, optimize post-sorting strain viability, and thereby establish a droplet microfluidic screening platform applicable to diverse filamentous microorganisms. Here, we systematically evaluated and optimized droplet-based microfluidic screening for filamentous fungi—Acremonium chrysogenum and two actinomycetes, Streptomyces lividans and Saccharopolyspora erythraea. Monodisperse spore-suspension preparation protocols and droplet incubation time were optimized for each organism, which revealed that actinomycete-generated droplets were significantly more stable than those of filamentous fungi, with optimal sorting windows dictated by strain-specific growth and morphological traits. Subsequently, we varied sorting voltage and recovery regimens (no treatment, demulsification, or demulsification followed by shaking incubation). Lower sorting voltage markedly improved viability, while demulsification effectively boosted viability. These targeted optimizations establish a practical workflow that converts droplet-based microfluidics into a robust tool for high-throughput screening of filamentous microorganisms.

Abstract:With the advancement of online monitoring and automation in industrial fermentation, large volumes of production operation data have been accumulated, while their potential value in fermentation regulation and process optimization has not been fully exploited. This study aims to investigate the key causes of titer variation in industrial-scale penicillin fermentation, thereby guiding the optimization of the feeding process. To this end, industrial historical data was analyzed to elucidate the relationships between key process variables and production performance, and to further investigate and validate their physiological regulatory significance. The production titer at harvest, maximum specific production rate (qP,max), and initiation timing of secondary metabolism were selected as characterization indicators. Curve similarity and correlation analyses were performed for staged screening of the data of 325 production batches from a 156 m³ industrial fermenter. The results indicated that cumulative glucose feeding amount and dilution rate were the key factors contributing to variations in fermentation performance. Further analysis revealed that a high dilution rate during the late fermentation stage maintained a high specific growth rate (μ), thereby limiting the accumulation of penicillin, a typical secondary metabolite. In contrast, low glucose feeding and reduced dilution rates promoted a decline in μ and enhanced production capacity, quantitatively demonstrating, at the industrial scale, the regulatory role of μ in secondary metabolism. On the basis of these findings, a viable cell electrode was introduced into a 50-L fermentation system to enable online biomass monitoring, and physiological metabolic parameters were used to reproduce industrial fermentation states. Subsequently, dynamic regulation of μ during the production phase was achieved by increasing the feed glucose concentration and optimizing the feeding rate to reduce late-stage dilution rate. Laboratory-scale experiments showed that the glucose conversion efficiency, titer, and fermentation index increased by 22.06%, 16.10%, and 16.42%, respectively. Preliminary scale-up results based solely on increasing feed glucose concentration demonstrated a 6.56% increase in titer, a 4%-5% improvement in overall production, and a 2.20% increase in fermentation index. This study developed a data–mechanism integrated strategy for fermentation process optimization, providing a new approach for precise regulation and optimization of large-scale biomanufacturing processes.

Abstract:Rhamnolipids, a class of biosurfactants, are widely applied in industries such as petrochemicals, environmental agriculture, daily chemicals and pharmaceuticals. However, their industrial application is severely constrained by the low rhamnolipid yield of natural strains and high overall production costs. To obtain microbial strains with high rhamnolipid-producing capacity and to enhance their fermentation yields, this study developed a screening method for rhamnolipid-producing microorganisms assisted by the oil spreading technique based on machine vision (MV). Using this approach, a Pseudomonas aeruginosa strain PA022 with favorable rhamnolipid-producing capacity was isolated from an environmental soil sample. Based on single-factor preliminary experiments, Bayesian optimization (BO) coupled with Latin hypercube sampling (LHS) was applied to perform global optimization of key fermentation parameters, thereby determining the medium composition and culture conditions for PA022 as follows: rapeseed oil 30 g/L, NaNO₃ 12 g/L, peptone 3 g/L, phosphate at a ratio of 3:1 with a total concentration of 1.2 g/L, initial pH 6.8, temperature 35 ℃, and inoculum size 2.5%. Under the optimal fermentation conditions, strain PA022 achieved a rhamnolipid titer of 20.01 g/L in shake-flask fermentation, and the yield reached 30.52 g/L at the 5 L fermenter scale. This study demonstrates that the small-sample global optimization strategy combining MV and BO can enhance the efficiency of strain screening and fermentation process development, thereby providing a reference for the optimization of rhamnolipid and analogous fermentation processes.

Abstract:Clostridium ljungdahlii can reduce CO₂ to acetic acid via the Wood-Ljungdahl pathway using H₂ as an energy source and CO₂ as a carbon source, and it is a key chassis for realizing resource utilization of CO₂. Acetic acid, as a bulk basic organic chemical raw material, has a large market demand and wide application scenarios. Moreover, the syngas fermentation route is green and low-carbon, with mild reaction conditions, making it an ideal direction for replacing traditional petroleum-based processes. However, during the fermentation process, it encountered problems such as slow growth, low biomass, and low acetic acid production. Therefore, in this study, the strain C. ljungdahlii SL40, which was obtained through laboratory adaptive evolution, was selected as the object of investigation. Through optimization of the culture medium and regulation of key fermentation processes, its acetic acid synthesis capacity was enhanced. Single-factor experiments integrated with response surface methodology were used to optimize the culture conditions. The optimal parameters were determined as follows: H₂: CO₂=60%: 40% (V/V), yeast extract 4.0 g/L, FeSO₄ 16 mg/L, culture temperature 37 ℃. Further experiments were conducted in a 5 L fermentation tank to investigate the effects of aeration rate and pH on the product. The results showed that increasing the aeration rate from 0.8 L/min to 1.5 L/min significantly promoted the production of acetic acid, raising the acetic acid concentration from 20.740 g/L to 25.950 g/L. When pH was controlled at 5.85, the acetic acid concentration was further increased to 36.570 g/L. Through systematic optimization, this study effectively broke through the key bottlenecks such as low biomass and low gas utilization rate in the autotrophic fermentation of the strain and significantly improved the acetic acid yield. These findings provide a feasible path for the directed conversion of CO₂ to produce high value-added chemicals and has good industrial application potential.

Abstract:Improving the production of polyethylene terephthalate (PET) depolymerases is essential for reducing the cost of enzymatic PET recycling and facilitating its industrial application. To enhance the production of PET hydrolases, the fungal-derived hydrolase HiC and the bacterial-derived hydrolase LCC^ICCG were employed as model enzymes. Strategies including recombinant strain construction, gene copy number optimization, molecular chaperone-assisted expression, fermentation scale-up, and crude enzyme stabilization were systematically investigated. The strains with secretory expression were generated by chromosomal integration, and multicopy transformants were obtained through high-stringency zeocin selection. To further improve protein production, we introduced PDI, ERO1, SEC53, SEC1, HAC1, and GCN4 as helper factors. GCN4 showed the strongest enhancement effect on HiC production, whereas ERO1 and HAC1 were more effective for LCC^ICCG. No significant synergistic effect was observed when ERO1 and HAC1 were co-expressed. In 5-L bioreactors, the optimized strains achieved the titers of 2.36 g/L for HiC and 1.72 g/L for LCC^ICCG. Furthermore, in a 50 L fermenter, the maximum extracellular protein concentration of HiC reached 8.5 g/L. A crude enzyme stabilization system composed of 0.1% Kathon, 10% glycerol, and 0.5% phenoxyethanol was subsequently established. After storage at room temperature for 30 days, HiC and LCC^ICCG retained 56.43% and 78.11% of their initial activities, respectively. The results demonstrate that coordinated optimization of expression and formulation can improve both the production and storage stability of PET depolymerases, thereby supporting their practical application in enzymatic PET recycling.

Abstract:Nicotiana benthamiana suspension cells, exhibiting advantages such as short growth cycles, facile genetic manipulation, ease of product purification, and suitability for large-scale cultivation, are emerging as a novel industrial biosynthesis platform for plant-derived natural products. Transient expression and stable transformation are core technologies for validating gene function and constructing plant cell factories. However, current methods have been developed primarily for whole-plant systems, systematic investigations in plant suspension cells are lacking, let alone a technological framework for integrating both approaches. Plant suspension cells, by virtue of cultivating characteristics, are suitable to develop integrated systems for transient expression and stable transformation. This study constructed an integrated system for transient expression and stable transformation (ISTS) by incorporating an efficient expression module, a transient expression enhancement module, and a stable integration screening module. ISTS enabled higher transient transformation efficiency and expression intensity in N. benthamiana suspension cells than classical pCAMBIA1300. Moreover, upon selection and cultivation of transiently expressing cells, stably transformed cell lines with efficient target gene integration and high expression levels can be obtained readily. In this study, tHMGR, tGGPPS, and tTS were cloned into ISTS vector individually and co-transformed into N. benthamiana suspension cells, which enabled the one-step multigene transient expression and stable integration. This approach successfully yielded taxadiene-producing tobacco cell lines. ISTS offers advantages such as simplified vector construction, fewer procedural steps, and shorter operational cycle. This work provides key technical support for establishing efficient, stable plant suspension cell factories and facilitating sustainable biosynthesis of complex natural products.

Abstract:Paclitaxel is an active anticancer ingredient with extremely high medicinal value, whereas its natural plant resources are scarce. Taxadiene, as the first committed precursor in the biosynthesis of paclitaxel, is of great significance for the production of paclitaxel. This study aimed to establish an efficient heterologous synthesis system for taxadiene, so as to provide a theoretical basis and experimental support for its large-scale production. Using the wild-type tobacco BY-2 cell line and the plant expression vector TS-pk7FGW2, we constructed a tobacco BY-2 cell line heterologously expressing taxadiene synthase (TS) for synthesis of taxadiene. The accumulation of taxadiene in the BY-2 cell line heterologously expressing TS was the highest [0.55 μg/g fresh weight (FW)] under dark culture and reached 1.34 μg/g FW under light treatment, which was 1.4-fold higher than that under dark culture. Transcriptomic analysis was performed on 7-day-old wild-type and TS-expressing tobacco BY-2 cell line to identify differentially expressed genes and associated metabolic pathways. A total of 6 121 differentially expressed genes (DEGs) were identified in the TS-expressing line in comparison with the wild type. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that the MVA and MEP pathways in the terpenoid backbone biosynthesis pathway were affected in the tobacco BY-2 cells heterologously expressing TS. This study provides a theoretical basis for optimizing the efficiency of heterologous taxadiene synthesis in BY-2 cells and offers theoretical and data support for elucidating the molecular mechanism by which TS regulates the growth and development of BY-2 cells and the heterologous biosynthesis of taxadiene.