Abstract:

Abstract:NeoProtein is an emerging protein source developed through advanced biomanufacturing technologies, independent of traditional livestock or fisheries. With advantages such as resource efficiency, low-carbon sustainability, and high production efficiency, it holds potential to partially replace conventional animal protein and is becoming a key driver of economic growth. Its development is crucial for accelerating new quality productive forces, ensuring national food security, and implementing the “big food concept” strategy. This paper systematically analyzes the opportunities, challenges, and current status of NeoProtein, aiming to provide insights for industry research, policy formulation, and industrial practices, thereby promoting high-quality development in China’s NeoProtein industry.

Abstract:Artificial cells, designed to mimic the structure and functions of natural cells, play a crucial role in various research domains. Artificial cells can be constructed through “top-down” and “bottom-up” strategies, and the latter aligns closely with synthetic biology ideology of creating or mimicking the complex pathways in natural cells. Increasingly synthetic biologists are utilizing “bottom-up” approaches to engineer artificial cells for applications in biotechnology, medicine, and disease therapy. This paper explores the applications of artificial cells in synthetic biology and medicine, with a particular focus on their development as drug delivery systems and their utilization in tissue engineering and regenerative medicine. Additionally, this paper examines how novel engineering methods can enhance the activity and stability of artificial cells, thus bridging the gap between artificial and natural cells. This review is expected to accelerate the research on multifunctional artificial cells, broadening their applications across various fields such as biotechnology, medicine, and targeted drug therapy. This expansion holds the potential to offer significant advancements in the sustainability and improvement of the human society.

Abstract:A biosensor is an analytical device composed of a biometric element and a physical transducer to convert the concentration of a target compound into a measurable signal. It has been widely used in the medicine, environment, and food domains. At present, tunable l-tryptophan biosensors are essential in the metabolic engineering and synthetic biology of l-tryptophan and its derivatives. Different l-tryptophan biosensors have their own advantages in practical applications owing to their different working principles. This article reviews the research progress of l-tryptophan biosensors, including their types, working principles, and optimization strategies. Moreover, this article introduces the typical applications of l-tryptophan biosensors in synthetic biology. At last, it proposes possible solutions to the problems faced in the design and construction and prospects the development trend of l-tryptophan biosensors.

Abstract:Actinomycetes are a class of microorganisms with robust secondary metabolism. Rationally engineering actinomycete strains to enhance their production of natural products is of significant importance. The CRISPR/Cas system, known for its high efficiency, ease of operation, and excellent targeting precision, has become a vital tool for genetic modification and functional studies in actinomycetes. This article provides a comprehensive overview and comparison of various CRISPR/Cas tools for gene editing and expression regulation in actinomycetes, including DNA double-strand break-based CRISPR/Cas systems, CRISPR interference systems, CRISPR activation systems, and CRISPR base editing systems. In addition, this article summarizes the applications of these tools in the biosynthesis of natural products in actinomycetes. The development and application of the CRISPR/Cas toolkit in actinomycetes have greatly promoted the research of microbiology and natural products of actinomycetes.

Abstract:Cupriavidus necrotor is a facultative autotrophic bacterium belonging to Betaproteobacteria. It can grow and produce PHA and other products using CO₂. This bacterium has received widespread attention in the context of global CO₂ reduction and is believed to play an important role in the production of key bio-based products such as organic acids, PHA, and secondary metabolites using CO₂. The proposal of metabolic engineering in the 1990s, especially the innovation and development of metabolic engineering enabling technologies, has greatly promoted the construction of C. necator cell factories and the application of the cell factories in industrial fermentation. The article systematically introduces the recent progress in the metabolic engineering of C. necator H16 and the construction of cell factories for producing bio-based chemicals. Finally, the key issues and future development prospects in the metabolic engineering of C. necator H16 are discussed. The purpose of the article is to offer valuable insights and research ideas to the metabolic engineering studies of C. necator H16, and therefore, promote more extensive applications of this strain in carbon dioxide conversion and bio-manufacturing.

Abstract:Yeast surface display technology, which enables the expression and display of target proteins on the surface of yeast cells, has been successfully constructed in various yeast species like Saccharomyces cerevisiae, Pichia pastoris, and Yarrowia lipolytica. This technology provides highly efficient platforms for protein high-throughput screening, enabling rapid screening and evaluation of pharmaceutical and antibody candidates, as well as recombinant strains with improved secretion efficiency. It has been widely applied in biopharmaceutical production, vaccine development, enzyme engineering, and biomaterial production, significantly contributing to advancements in biotechnology. This review delves into optimization strategies for yeast surface display and its applications in high-throughput screening. In addition, it discusses current limitations and explores future directions of this technology, laying the groundwork for enhancing the accuracy and enlarging the scope of applications.

Abstract:Yeast is a valuable host organism for protein, chemical, and plant natural product production in metabolic engineering. The development of neutral sites in yeast holds great significance for the construction and optimization of efficient cell factories. This review summarizes the screening standards, characterization methods, influencing factors, and applications of neutral sites in synthetic biology. Additionally, it discusses the future research directions, providing theoretical support and practical guidance for the construction and optimization of yeast cell factories utilizing neutral sites.

Abstract:Hyaluronic acid (HA) is a highly polymerized linear polysaccharide composed of d-glucuronic acid and N-acetylglucosamine. Hyaluronic acid of different molecular weights exhibits various functions. With the advancements of science and technology, low molecular weight HA has been increasingly employed in the medical field. For example, it can serve as a carrier for targeted anti-tumor drugs, production of wound dressings, and drug delivery systems, demonstrating a broad market prospect and a high application value. This paper comprehensively reviewed the research progress in low molecular weight HA in recent years, encompassing the preparation of low molecular weight HA by physical and chemical degradation methods and the mutation breeding of new strains for the production of low molecular weight HA. In addition, this paper discussed in depth the application of emerging strategies of synthetic biology in the production of low molecular weight HA, such as the modification of hyaluronan synthase and the regulation of substrate balance via manipulating the synthetic metabolic pathway. This paper aims to provide new ideas and methods for the efficient production of low molecular weight HA and point out the challenges to be addressed in the future research.

Abstract:Halogenated compounds are widely used in agricultural chemical, pharmaceutical, and material industries. The chemical synthesis of these compounds ordinarily needs harsh conditions and noxious reagents, generating harmful by-products and products lacking regioselectivity. Halogenase-catalyzed halogenation with high efficiency, mild reaction conditions, outstanding stereoselectivity, and environmental friendliness demonstrates a promising prospect. Different types of halogenases have been characterized, including heme-dependent haloperoxidases, vanadium-dependent haloperoxidases, and flavin-dependent halogenases catalyzing electrophilic halogenation, non-heme iron/α-ketoglutarate-dependent halogenases catalyzing radical halogenation, and S-adenosine-l-methionine-dependent halogenases catalyzing nucleophilic halogenation. This review outlines the catalytic mechanisms of various halogenases and the engineering modifications for application and briefs the industrial application status of these enzymes. Halogenases can efficiently achieve enzymatic as well as chemoenzymatic synthesis of a variety of halogenated compounds, and significantly ameliorate the environmental issues caused by traditional chemical synthesis. Relevant mechanism and engineering research hold important value in the field of synthesis.

Abstract:Nerve agents are among the most toxic compounds ever discovered, and the efficient and environmentally friendly degradation of nerve agents remains a challenge and a hot topic in the research on decontamination technology. Compared with chemical incineration and physical adsorption, enzyme catalysis shows great potential in environmental purification and medical treatment due to its high specificity and mild reaction conditions. Phosphotriesterase (PTE) exhibits a wide range of substrate adaptability and is the only enzyme known to degrade VX, providing an important potential approach for the biodegradation of nerve agents. This article comprehensively reviewed the properties of nerve agents and the three-dimensional structure of PTE, elaborated on the research progress in the theoretical calculation and enzyme engineering in the degradation of nerve agents, looked forward to the application prospect of enzymatic degradation of nerve agents, and proposed the future research directions and challenges. The review aims to provide a theoretical foundation and practical guidance for designing and developing more efficient enzyme systems for nerve agent degradation, ultimately advancing green degradation technologies for nerve agents.

Abstract:Polycyclic aromatic hydrocarbons (PAHs) with two or more benzene rings have caused serious environmental problems due to their carcinogenic, mutagenic, and teratogenic properties. Microbial degradation is one of the methods for the remediation of PAH contamination, offering advantages such as low costs, high efficiency, and environmental friendliness. Compared with single strains in degradation, mixed microbial consortia exhibit improved degradation performance, adaptability, and resistance to adverse conditions. This paper reviews various natural PAH-degrading mixed microbial consortia obtained from natural environments, as well as artificially constructed mixed microbial consortia based on the degradation capabilities or other characteristics of different individual strains. It also discusses the optimization methods for these consortia and the research progress in their application to the remediation of actual contaminated sites. Finally, the paper provides an outlook on the future development of mixed microbial systems for the degradation of polycyclic aromatic hydrocarbons.

Abstract:In response to climate change, the world has begun to promote a green and low-carbon transition, with the goals of carbon neutrality and carbon peaking. Carbonic anhydrases (CAs) can efficiently catalyse the reversible hydration of CO₂ and form carbonate deposits with the participation of metal ions (e.g., Ca²⁺, Mg²⁺). The generated carbonate deposits are stable and do not emit CO₂ again in the nature, and thus be used as cementitious materials in crack repair and soil reinforcement. Therefore, using CAs to convert CO₂ into carbonate is a promising method for CO₂ sequestration. This paper provides an overview of the types and catalytic mechanisms of CAs, mineralisation of CO₂ into CaCO₃ by CAs, immobilisation of CAs, and CA-mediated biomineralisation in civil engineering applications such as crack repair and sandy soil reinforcement. Furthermore, we make an outlook on the ways for developing highly active and stable CAs and the development trend of the application of CA-mediated biomineralisation in the field of civil engineering.

Abstract:The global depletion of fossil fuels and the greenhouse effect pose a threat to human beings. Microorganisms can utilize one-carbon (C1) compounds to produce value-added chemicals, which is of great significance to improve resource utilization and reduce carbon emissions. This paper summarizes four C1 fixation pathways that have been successfully constructed in model microorganisms, including the RuMP pathway, XuMP pathway, CBB cycle, and rGly pathway. Meanwhile, examples of adaptive laboratory evolution as a powerful strategy for the development of C1-utilizing strains are discussed. Finally, this paper prospected the challenges and opportunities for the utilization of C1 compounds by microorganisms, aiming to shed lights for further research on the utilization of C1 compounds.

Abstract:The interactions between DNA and proteins play a pivotal role in cellular processes, underpinning the basis for DNA replication and transcriptional regulation, which are essential for cell growth, development, and adaptation to environmental changes. These interactions primarily occur through the specific recognition and binding of DNA sequences by the DNA-binding domains of proteins. A comprehensive understanding of these interactions is not only critical for elucidating biological mechanisms but also holds significant implications for the research on diseases, drug discoveries, and engineering of industrial strains. This review highlights both traditional and recently developed methods for studying DNA-protein interactions, analyzing their principles, advantages, limitations, and application cases. Additionally, it discusses potential avenues for improvement and explores emerging application scenarios in this rapidly evolving field. Therefore, this review provides researchers with a quick and comprehensive understanding of the advantages, challenges, and future outlooks of various methods of DNA-protein interaction research as well as valuable insights for the further innovation and integration of relevant techniques.

Abstract:Eicosapentaenoic acid (EPA) is an ω-3 long-chain polyunsaturated fatty acid valued for its diverse physiological activities. The production of EPA by extraction from marine fish oils is becoming increasingly unsustainable due to depleting fish stocks and rising environmental pollution. Consequently, alternative sources for EPA production are necessary. In this study, we constructed a Pichia pastoris cell factory for heterologous production of EPA. To enhance the accumulation of total fatty acids (TFAs) in the engineered strain, we knocked out the downstream metabolic pathway of fatty acids and the competitive pathway. Simultaneously, the expression of C16/18 elongase, C18/20 elongase, and desaturase were strengthened. With glucose as the carbon source, the engineered strain accumulated 677.34 mg/L TFAs and 60.81 mg/L EPA. The three key enzymes were further overexpressed to increase the EPA production, which reached a titer of 68.89 mg/L. Furthermore, we replaced the glucose promoter P_GAPof three key enzymes in the EPA synthesis pathway with the promoter P_AOX1 of the alcohol oxidase AOX1 to explore the methanol-induced EPA synthesis, which further increased the EPA titer to 75.21 mg/L. This study lays a foundation for the biosynthesis of long-chain polyunsaturated fatty acids in P. pastoris and the construction of P. pastoris cell factories for producing EPA with methanol, a one-carbon resource.

Abstract:Resveratrol is a valuable plant polyphenol. It is currently obtained primarily through plant extraction, which is difficult to obtain in large quantities over a long period of time due to the scarcity of natural raw materials. Therefore, it is necessary to develop a simple and cost-saving method for producing resveratrol. In this study, the genes encoding key enzymes for resveratrol synthesis (HaTAL1, AtPAL2, AtC4H, At4CL2, and VvSTS) were introduced into the wild-type strain of Scheffersomyces stipitis to construct strain Ss05, which achieved the resveratrol yield of 55.28 mg/L. Subsequently, the supply of p-coumaric acid and malonyl-CoA precursors was enhanced by overexpression of the feedback-insensitive 3-deoxy-d-arabino-heptulonate-7-phosphate synthase mutant SsARO4^K221L and chorismate mutase mutant SsARO7^G139S, knockout of the pyruvate decarboxylase gene (PDC1), and overexpression of the acetyl-CoA carboxylase mutant SsACC1^{S650A, S1152A}. On this basis, the copy number of key genes was increased to create the engineered strain Ss17, which achieved the resveratrol yield of 150.56 mg/L. Finally, 558.40 mg/L resveratrol was produced by fed-batch fermentation of glucose with strain Ss17 in a 5-L fermenter for 128 h. In this study, we employed synthetic biology to construct an engineered strain of S. stipitis for the synthesis of resveratrol from a simple carbon source and then scaled up the microbial fermentation in a bioreactor, providing an important reference for the biosynthesis of aromatic compounds in S. stipitis.

Abstract:Gallic acid is a plant-derived phenolic compound with various medicinal values, including antioxidant properties. The primary method for producing gallic acid at present involves the chemical or enzymatic hydrolysis of tannins extracted from plants, which is associated with high costs and serious environmental pollution. Green synthesis based on microbial cell factories offers an effective alternative route for the production of gallic acid. However, the current yields of green synthesis are insufficient for industrial-scale requirements. Therefore, the development of a de novo synthesis strategy for gallic acid using low-cost substrates holds significant potential for industrial applications. In this study, an Escherichia coli strain capable of efficiently synthesizing 3-dehydroshikimic acid was used as the chassis organism. Initially, the key enzymes for the optimal synthesis pathway of gallic acid were identified as 3-dehydroshikimate dehydratase (AroZ) and 4-hydroxybenzoate hydroxylase (PobA), with an optimal expression ratio of 1:20. The optimal pathway was constructed within the chassis strain through plasmid copy number and promoter engineering. Protein engineering was further employed to obtain a mutant of the rate-limiting enzyme, PobA^M2/A45S/V47P, which enhanced the shake flask production level of gallic acid to 3.6 g/L. Finally, strain stability and fermentation condition optimization were conducted in a 5 L fermentor, resulting in a gallic acid yield of 26.7 g/L and a sugar-to-acid conversion rate of 0.15 g/g. This study achieves a breakthrough in the de novo synthesis of gallic acid from glucose, providing an important reference for enhancing the biological synthesis of gallic acid and related phenolic acids from plants.

Abstract:Glycolic acid, the simplest α-hydroxy acid in structure, is extensively utilized in pharmaceuticals, chemicals, and other fields. However, the strains currently employed for the production of glycolic acid by fermentation typically possess plasmids or necessitate the addition of chemical inducers, which hampers large-scale industrial production. In order to construct a glycolic acid producing strain that does not require plasmids or chemical inducers. In this study, we utilized the wild-type strain Escherichia coli ATCC 8739 as the starting strain. Initially, we screened isocitrate lyase and glyoxylate reductase from different sources and constructed an engineered strain GA01 by genomic integration. Fermentation with this strain in a fermenter produced glycolic acid at a titer of 10.12 g/L. Following optimization of the copy number of pathway enzymes, the titer of glycolic acid was further increased to 15.25 g/L. Additionally, by blocking the branched metabolic pathways in the glycolic acid synthesis route and enhancing precursor supply, we effectively redirected carbon flow towards glycolic acid production, and fermentation with the optimized strain GA07-1 achieved the glycolic acid titer of 41.69 g/L. Finally, fermentation optimization was carried out with strain GA07-1 in a 5 L fermenter, achieving a glycolic acid titer of 50.62 g/L, a yield of 0.49 g/g, and productivity of 1.20 g/(h·L). These results realized the de novo fermentation of glycolic acid from glucose without antibiotics and chemical inducers.

Abstract:d-tagatose, a functional rare sugar, has garnered increasing attention because of its low calories, blood glucose-lowering, anti-caries, and intestinal flora-improving properties. The existing methods of producing d-tagatose still have problems of low production efficiency and high costs. In this study, we achieved whole-cell catalytic synthesis of d-tagatose from lactose by constructing a dual-enzyme efficient expression system in Escherichia coli. l-arabinose isomerase (l-AI) is a key enzyme for the isomerization of galactose to d-tagatose in biosynthesis. In this study, we screened the l-AIs from different sources and found that the l-AI from Lactobacillus fermentum CGMCC 2921 had better catalytic ability. The optimal mutant D390V/V468L was obtained by rational design of LfAI. Its half-life was extended to 72 h, and the enzyme activity was increased by 36.68% under the optimum temperature of 40 ℃. Then, with pET28a as a vector, the optimal mutant gene (LfaraA^D390V/V468L) and the β-galactosidase gene from E. coli (EclacZ) were co-expressed in E. coli BL21(DE3) through promoter optimization. Finally, after optimization of the catalytic system, 115.21 g/L d-tagatose was obtained after fermentation under the optimal conditions (pH 7.0, 50 ℃, and 2.5 mmol/L Mn²⁺) in a 5 L fermenter with 500 g/L lactose as the substrate for 48 h, with a conversion rate of 23.09%. This study has a good industrial application value in the one-step whole-cell catalytic synthesis of d-tagatose from lactose.

Abstract:Succinic acid, an important high-value platform compound, is widely used in chemical, food, and medicine fields. The chemical production of succinic acid has the defects of high pollution and high carbon emissions. Therefore, the fermentation method has become the main direction of succinic acid production at present. Actinobacillus succinogenes, a naturally succinic acid-producing strain, is praised for the high titer, high osmotic pressure tolerance, and short fermentation time, thus serving as an ideal strain for industrial production. Improving the succinic acid titer of A. succinogenes and thus reducing the production cost is an urgent need for industrial production. In this study, we developed a simple high-throughput screening method by combining bromothymol blue with atmospheric room-temperature plasma mutagenesis to direct the evolution of wild type A. succinogenes. A high-yielding mutant strain, A4-K74, was screened out. After 72 hours of anaerobic fermentation in a shake flask, this strain accumulated a maximum of 56.3 g/L of succinic acid, which represented a 40.8% increase compared with that of the wild type strain. Moreover, A4-K74 demonstrated good genetic stability. The transcriptome analysis revealed that the enhancement of sulfur metabolic pathway and amino acid (such as cysteine) synthesis pathway was the potential reason for the increase in succinic acid titer, which provided a reference for the future metabolic engineering of A. succinogenes and was conducive to the further promotion of the industrial production of succinic acid by bioprocessing.

Abstract:l-lactic acid is a vital organic acid with broad applications in food, pharmaceutical, and bioplastic industries. The conventional production of l-lactic acid predominantly relies on lactic acid bacteria, which are limited in the industrial application due to their strict substrate requirements and poor tolerance to acidic environments. Kluyveromyces marxianus has gained attention as a potential host for lactic acid production due to its rapid growth, acid tolerance, and ability to utilize inexpensive substrates. In this study, we aimed to enhance the lactic acid production capacity of K. marxianus through metabolic engineering and process optimization. We first overexpressed the lactate dehydrogenase (LDH) gene and employed CRISPR to knock out the pyruvate decarboxylase gene (PDC1), thus obtaining the engineered strain LA2.1, which achieved the l-lactic acid production of 72.55 g/L. By screening and combining acid tolerance targets, we improved the acid tolerance and lactic acid production of this strain. Through process optimization, the strain LA4.3 achieved the l-lactic acid production of 112.41 g/L and a yield of 84.00% within 72 h of fermentation. Additionally, in the case of reducing the calcium carbonate dosage, the l-lactic acid production reached 101.50 g/L, with pH 3.52 after fermentation. These findings highlight the potential of K. marxianus as a promising host for efficient l-lactic acid production and provide a solid theoretical and technical foundation for the industrial application of K. marxianus in bio-based chemical production.

Abstract:γ-glutamyltranspeptidase (GGT) is widely used in clinical diagnosis, food processing, and pharmaceutical manufacturing, whereas the low expression limits its application expansion. To improve the expression of GGT, we employed MūtCompute to engineer the GGT derived from Bacillus licheniformis and obtained the mutant GGT^A339C with improved catalytic efficiency. By analyzing the substrate-binding pocket, we found that the mutant GGT^A339C had a more open substrate-binding region and a substantially straighter tunnel than the control, with a 255% increase in activity. Subsequently, with B. licheniformis BL11 as the starting strain, we constructed the strain BL11::prsA/pP_ykzA+rbs6-SP_SacC-GGT^A339C with efficient GGT secretion by integrating the expression of chaperone PrsA, and the enzyme activity reached 22.1 U/mL. Finally, after optimization of the fermentation process in a 5 L fermenter, the GGT activity reached 180.14 U/mL, marking the highest level of GGT activity reported to date. In conclusion, the mutant GGT^A339C with high catalytic performance was successfully obtained, and the strain B. licheniformis BL11::prsA/pP_ykzA+rbs6-SP_SacC-GGT^A339C was attained with high production of GGT, which provided an excellent strain for the industrial production and catalytic application of GGT.

Abstract:Cinnabarinic acid (CA) is a high-value nitrogen-containing tricyclic phenoxazinone widely used in dyeing, chemical, and pharmaceutical industries. However, the efficient CA synthetic system for large-scale CA production has not been constructed until now. To achieve efficient CA biosynthesis, we constructed an in vitro enzymatic synthetic system and a whole-cell biocatalytic platform for CA biosynthesis in this study. Firstly, targeting the main limiting factor of the CA synthesis, we identified a superoxide dismutase SodA_Prm capable of efficiently converting 3-hydroxyanthranilate to CA. Subsequently, the in vitro catalytic parameters of SodA_Prm were optimized, after which (176.6±14.3) mg/L CA was efficiently synthesized. Then, based on the selected SodA_Prm, an Escherichia coli whole-cell catalytic system BL-sodA_Prmwas constructed for CA biosynthesis. Finally, a maximal titer of 312.3 mg/L CA was synthesized after optimization of the reaction conditions of whole-cell catalytic system BL-sodA_Prm. In summary, this study lays a profoundly foundation for the biosynthesis and application of CA and its derivatives in the future.

Abstract:Aminopeptidases are commonly used co-enzymes for the deep hydrolysis of proteins in food and medicine fields. However, their industrial application is limited due to the low fermentation activity and poor thermal stability. In this study, we employed rational design to enhance the thermal stability of the aminopeptidase 168AP derived from Bacillus subtilis and achieve its efficient heterologous expression in engineered strains, thereby improving and broadening its application potential. First, online prediction software was used to identify mutation hotspots that could potentially improve the thermal stability of 168AP. The enzymatic properties of two aminopeptidase mutants, which exhibited enhanced thermal stability after preliminary screening, were subsequently evaluated. Molecular docking was employed to simulate the interactions between the aminopeptidase mutant R39I and its substrate, and molecular dynamics simulation was conducted to elucidate the mechanism behind the thermal stability enhancement of R39I. Finally, the efficient heterologous expression of the aminopeptidase mutant with enhanced thermal stability was achieved in Bacillus amyloliquefaciens. We successfully identified two aminopeptidase mutants with improved thermal stability. Notably, the residual enzyme activity of R39I after incubation at 60 ℃ for 30 min was 1.61 times that of the wild type. Molecular docking results revealed that R39I had the lowest binding free energy of −4.93 kcal/mol with the substrate among all mutants. Furthermore, molecular dynamics simulation results indicated that the change in hydrophobic interactions before and after mutagenesis was the primary reason for the improved thermal stability. Finally, the enzyme activity of Δ6/pLY-3-R39I in a 7 L fermentation tank reached 43 131.57 U/mL, 5.11 times that of flask fermentation. This study successfully identified aminopeptidase mutants with enhanced thermal stability and achieved their efficient heterologous expression in engineered strains, thus broadening the industrial application range of aminopeptidases.

Abstract:γ-aminobutyric acid (GABA) is a non-protein amino acid that has been used as a new functional factor in the fields of food, medicine, chemical engineering, and agriculture. Glutamate decarboxylase (GAD) is a key rate-limiting enzyme that catalyzes the formation of GABA from l-glutamic acid (l-Glu). However, GADs from different sources have a common problem of limited thermal stability, which affects their industrial applications. In order to obtain GADs with high activity and thermal stability, and reduce the production cost of GABA, herein, a novel thermostable GAD (EgGAD) derived from Enterococcus gallinarum was obtained through ancestral sequence reconstruction of a candidate gene directly mined with the GAD of Lactobacillus brevis (LbGAD) as the initial template. The recombinant EgGAD exhibited the maximal activity at 60 ℃ and pH 5.0. The Michaelis constant (K_m) and catalytic efficiency (k_cat/K_m) of EgGAD with l-Glu as the substrate were 10.94 mmol/L and 2.07 L/(s∙mmol), respectively. Notably, EgGAD exhibited a larger shift in thermostability, with about 4-fold improvement of half-life (t_1/2) at 55 ℃ and a 4.94 ℃ increase in semi-inactivation temperature (T₅₀¹⁵) compared with that of LbGAD. Furthermore, a high-efficiency synthesis system for GABA was developed with dormant engineered Escherichia coli Nissle 1917 cells as biocatalysts. When E. coli Nissle (T7)/pET28a- EggadB cells were concentrated to reach the optical density (OD)₆₀₀ of 20 in 3 mol/L l-Glu solution, the GABA yield reached 300.94 g/L, with more than 99.5% conversion ratio, after reaction at 40 ℃ and 150 r/min for 4 h. Overall, this study emphasizes the value of an ancestral sequence reconstruction technique for direct gene mining to improve the thermal stability of GAD, provides a functional component for the biosynthesis of GABA, and sheds light on improving the thermal stability of other enzymes.

Abstract:Truncated hemoglobin is a type of hemoglobin found in bacteria. The amino acid sequence of the truncated hemoglobin YjbI derived from Bacillus amyloliquefaciens differs from those of higher organisms and has garnered significant interest due to its distinctive structural configuration and functional characteristics. In this study, Escherichia coli BL21(DE3) was used as the host for heterologous expression of yjbI with optimized codon and connected to the expression vector pET-28a(+). High-purity YjbI was obtained after purification with the His affinity tag. The purified truncated hemoglobin was analyzed by SDS-PAGE and circular dichroism assay, and its heme-binding rate and oxygen-binding capacity were determined. After optimization, the highest yield was achieved at the expression time of 26 h and 2% 5-aminolevulinic acid (ALA) addition, and the expression level of YjbI increased from 122.02 mg/L to 133.19 mg/L. Circular dichroism and AlphaFold3 structure prediction results showed that YjbI formed α-helical structures and folds to generate the heme-binding site, ultimately assembling the complete three-dimensional conformation of the protein. The results from full wavelength scanning and calculation based on the Beer-Lambert law showed that the heme-binding rate of YjbI increased from 13.18% to 22.78% after the addition of ALA. The oxygen-binding capacity was determined by the redox method, which indicated that YjbI had a high oxygen affinity. This study successfully achieved heterologous expression of truncated hemoglobin in E. coli, systematically analyzed its structural and functional characteristics, and provided a theoretical and technical basis for the application of microbial hemoglobin.

Abstract:The biocatalytic synthesis of atropisomeric scaffolds remains challenging. Herein, to expand the biosynthetic approaches to axial chiral compounds, we present the alcohol dehydrogenase-catalyzed stereodivergent reduction of N−N axially chiral aldehydes with pyrroles and indoles, two strains with opposite stereoselectivity were obtained by screening 92 alcohol dehydrogenases, affording both R- and S-configured products with moderate to excellent selectivity (up to 72% conversion and 98% e.e.). The conformational stability was confirmed, and upscaled synthesis and synthetic transformations demonstrated the utility of the current methodology. This study provides the first enzymatic strategy to access N−N axially chiral compounds with potential pharmaceutical applications.

Abstract:Daqu plays a role as an agent of saccharification and fermentation in the traditional production of Baijiu. Investigating the saccharifying enzymes in Daqu and their synergistic applications is vital for the modernization of the brewing industry. However, the enzyme resource mining focusing solely on Daqu faces challenges such as a broad target range, heavy workload, and limited applicability. To address these issues, we mined 17 dominant starch-saccharifying enzymes from the metaproteomic and metagenomic data of the Baijiu fermentation process and designed primers based on their gene sequences. A total of eight starch-saccharifying enzyme genes, originating from bacteria, fungi, and insects, were successfully cloned from Daqu. Additionally, an α-1,4-glucosidase KeGA5 from Kroppenstedtia eburnea and an α-amylase RpAM11 from Rhizomucor pusillus were successfully expressed, with KeGA5 representing the first reported functional enzyme from K.eburnea. KeGA5 and RpAM11 demonstrated the highest activities at 55 ℃ and 75 ℃ and pH 5.0 and 7.0–8.0, respectively. Both enzymes exhibited good stability below 50 ℃ and within the range of pH 5.0–8.0. The temperature and ethanol concentration in the fermentation process of Baijiu significantly affected enzyme activities but had little impact on enzyme stability, while the acidic environment had considerable impacts on the activities and stability of both enzymes. Furthermore, KeGA5 and RpAM11 exhibited synergistic effects in hydrolyzing the substrates containing α-1,4-glucosidic bonds, with the highest hydrolytic activity observed on soluble starch and dextrin. The incubation experiment with sorghum as the substrate demonstrated that the pure enzyme combination had similar effects to the Daqu crude enzyme system in releasing maltose, glucose, and glucuronic acid. This study presents a novel strategy for the rational exploration and functional characterization of saccharifying enzymes in Daqu, providing scientific insights for developing enzyme preparations and microorganism-enzyme mixtures in the future Baijiu industry.

Abstract:The metabolism of tryptophan by gut microbiota can produce a variety of indole derivatives to improve human health. As beneficial bacteria in the gut, some strains of lactobacilli have been developed as probiotics and confirmed to have tryptophan metabolism, while the tryptophan metabolism of most strains remains unclear. The aim of this study is to reveal the potential tryptophan metabolism of lactobacilli using genomic methods, which enables the quick identification of strains with the ability to produce specific tryptophan metabolites. Based on the constructed tryptophan metabolism gene dataset, this study analyzed the genome sequences of 2 235 strains of 16 species of lactobacilli in the List of Strains that Can Be Used in Food in China. The results showed that strains of lactobacilli, except Latilactobacillus, carried rich genes associated with tryptophan metabolism, especially the genes encoding enzymes related to indole-3 lactic acid production. More than 92% of strains carried ArAT, FLDH and LDH with 1–11, 1–7, and 1–11 copies, respectively, showing high indole-3-lactic acid production potential. A phylogenetic tree was conducted to study the sequence similarity of ArAT, FLDH, and LDH encoding enzymes involved in indole-3-lactic acid production. The results showed that ArAT, FLDH, or LDH sequences could be clustered into 3–4 groups, and the gene sequences of each cluster were from different strains, indicating that the indole-3-lactic acid production potential of lactobacilli was influenced by genotypes. In this study, we employed the newly constructed tryptophan metabolism gene dataset to study the distribution and phylogenetic relationship of tryptophan metabolism genes, especially indole-3-lactic acid metabolism genes, in lactobacilli. By characterizing the tryptophan metabolism of lactobacilli, this study provides a theoretical basis and screening method for the development of functional strains by genomics methods.

Abstract:Botulinum neurotoxins (BoNTs) are neurotoxic proteins that can hydrolyze soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) involved in the synaptic vesicle fusion. Synaptobrevin-2 (Sb-2) is a SNARE targeted by BoNTs. This study aims to develop a yeast cell-based assay system for Sb-2-targeting BoNTs. SNAREs are conserved in eukaryotes and Snc1 and Snc2 are homologues of Sb-2 in Saccharomyces cerevisiae. We constructed a functional chimeric SNARE in which a part of Snc2 was replaced with that of Sb-2. The Snc2/Sb2-4 chimera, instead of Snc1 and Snc2, was recognized and cleaved by BoNTs. Since Snc1 and Snc2 are required for sporulation of yeast cells, in snc1∆snc2∆cells harboring the Snc2/Sb2-4 chimera, the sporulation efficiency was significantly decreased by expression of Sb-2-targeting BoNTs including BoNT/B. However, in wild-type yeast cells, the effects of BoNTs expression were negligible. Thus, sporulation efficiency was used as an indicator of the activity of BoNTs in this study. One advantage of this method is that BoNTs activities can be assessed by colorimetric measurement of sporulation efficiency. Our yeast cell-based BoNTs assay provides easy and rapid analysis of BoNTs, which is useful to characterize and engineer the neurotoxins. Furthermore, the assay will be useful for mining of uncharacterized BoNTs and BoNTs inhibitors. BoNTs are widely used for clinical and cosmetic purposes; thus our assay would be useful to find and modify useful BoNTs.

Abstract:Two-component systems (TCS) are signal transduction systems ubiquitous in bacteria, effectively performing signal recognition, transduction, and gene regulation to achieve transmembrane signal transmission and amplification. Gene expression regulation tools based on TCS have been extensively applied in synthetic biology and environmental monitoring. The traditional gene induction systems, T7 and P_BAD systems, have problems such as high cost, ease of inducer utilisation by cells, and poor linearity between inducer concentration and gene expression level. In order to develop low-cost induction systems with high linearity between inducer concentration and gene expression level, in this study, we developed two gene expression systems induced by Cu²⁺ and Ni²⁺ based on the CusS-CusR TCS and the chimeric NrsS/CusS-CusR TCS. By optimizing the expression levels of histidine kinase (CusS or NrsS/CusS) and the response regulator (CusR), we reduced the background fluorescence intensity of the Cu²⁺-inducible system from 2 400 a.u. to 852 a.u. and improved its dynamic range from 1.2 folds to 8.7 folds. The Ni²⁺-inducible system with a similar structure demonstrated a background fluorescence intensity of 2 711 a.u. and a dynamic range of 5.6 folds. Subsequently, we increased the ribosome binding site (RBS) strength and plasmid copy number, increasing dynamic ranges of 50.0 folds and 14.3 folds for the Cu²⁺- and Ni²⁺-inducible systems, respectively. Compared with T7- and P_BAD-inducible systems, the Cu²⁺- and Ni²⁺-inducible systems developed in this study exhibit improved induction gradients and decreased induction costs, providing robust complements to existing expression systems and offering versatile options for diverse experimental applications.

Abstract:Pyruvate is an important organic acid applied in food, chemical, and pharmaceutical fields. The enzymatic synthesis of pyruvate faces the challenge of low enzyme activity, which seriously limits the industrial production of pyruvate. The low-throughput screening methods are the bottleneck for screening high-performance enzymes. Biosensors serve as a high-throughput screening method for molecular modification of enzymes. This study constructs biosensors responsive to pyruvate based on the pyruvate dehydrogenase complex regulator (PdhR) from Escherichia coli. Firstly, the binding site of PdhR was inserted into different sites of three promoters of E. coli to construct hybrid promoters, among which the hybrid promoters P_tacD, P_ticM, and P_trpD exhibited active responses to pyruvate. Secondly, the response performance of biosensors was improved by optimizing the expression level of PdhR. Among the constructed biosensors, B2-1 sensitively responded to changes in the concentration of pyruvate, with a wide ligand detection range (0–12 g/L pyruvate) and a luciferase induction fold change of 14. Finally, this study analyzed the ligand specificity of biosensor B2-1. The results showed that biosensor B2-1 only responded to pyruvate and was not sensitive to other ketone acids, indicating that this biosensor had strong ligand specificity. Therefore, the pyruvate biosensor constructed in this study can be used for high-throughput screening of highly active enzyme mutants, providing an efficient and sensitive screening tool for molecular modification of enzymes and strains, and further laying the foundation for the industrial production of pyruvate.

Abstract:This study aims to develop a low-cost, portable, and quantitative digital nucleic acid detection chip to address the issues of high cost, sample loss, complex operation, and strong equipment dependency associated with existing digital nucleic acid detection methods. To achieve this, an innovative chip design was proposed. The fluidic channels were decoupled and separated from the vacuum channels, and a fractal structure was employed to design the fluidic channels, enabling efficient automatic sample distribution and 100% sample utilization. Meanwhile, the vacuum channels effectively resolved the challenges of pre-degassing and maintaining negative pressure in fractal structure chips, eliminating the need for time-consuming pre-degassing operations, and allowing detection to be performed at any time. Additionally, a digital recombinase polymerase amplification (dRPA) chip based on black polydimethylsiloxane (PDMS) material was developed, which exhibited excellent optical imaging capabilities and strong resistance to background fluorescence interference, providing an ideal platform for optical detection. A novel anti-evaporation strategy was also proposed, where each microchamber was surrounded by an aqueous solution to prevent reagent evaporation, further optimizing detection performance. Ultimately, a portable dRPA nucleic acid detection solution was successfully developed, requiring only simple manual operation and a smartphone to complete the detection. This solution not only retains the advantages of simplicity, low cost, scalability, and absolute quantification but also significantly enhances the convenience and practicality of detection, laying the foundation for the widespread application of portable nucleic acid detection. This innovative detection solution is expected to enable rapid and accurate nucleic acid detection in resource-limited settings and promote the development of on-site rapid diagnostic technologies.

Abstract:Synthetic biology aims to understand and design life by building artificial biological systems. It is a new engine for the next round of scientific and technological revolution and industrial revolution. To adapt to the integration between cutting-edge knowledge of synthetic biology and conventional biological science frameworks, we proposed the combination of theoretical teaching and practical operation of Foldit in the teaching of component development and design in the newly developed Synthetic Biology course. The combined teaching mode significantly enhanced students’ understanding of protein folding and design. Further, we discussed the course design and teaching methods and put forward the idea of using intelligent software to assist in the teaching of difficult points in this course. This innovative approach significantly fosters the students' ability and hands-on experience in the domain of protein design, which play pivotal roles in nurturing next-generation talents to meet the demands of forthcoming technological challenges.

Abstract:With the continuous advancement of the globalization reform wave and the rapid development of science and technology, the internationalization of engineering education has become an important way to promote the reform of higher education. This paper takes the Sino-British cooperative education project (bioengineering major—brewing and distillation) in the School of Biological Engineering of Tianjin University of Science and Technology as an example to discuss the existing problems and solutions of Sino-foreign cooperative education under the internationalization of engineering education. By innovating the philosophy of international talent cultivation, improving the level of international teachers, optimizing the international curriculum systems, implementing diversified teaching models, and establishing continuous supervision and improvement mechanisms, the comprehensive reform forms a new model of “five-element integration” for talent cultivation. This model meets the international engineering education certification standards and the international market demand and can cultivate international outstanding engineering talents with patriotism and a global vision. This study aims to provide practical references for transnational cooperative education programs in other universities, facilitating their cultivation of more high-quality talents during the internationalization process of engineering education.

Abstract:The setting and teaching of interdisciplinary courses is one of the effective ways to cultivate high-quality composite new engineering talents with strong practical ability, strong innovation ability, and international competitiveness. However, at present, it is difficult to achieve effective and satisfactory teaching effect by simply using stacking method of multiple subjects. Based on the development and cross-integration of biological engineering and textile engineering in Jiangnan University, this paper takes the interdisciplinary course Textile Biotechnology as an example to carry out the reform and innovation. We constructed an interactive teaching mode, reorganized multi-dimensional integrated teaching contents, established a cyclic upward teaching quality control system, optimized the assessment system aimed at evaluating students’ interdisciplinary thinking and innovation ability, and adjusted the proportions of teachers with different educational background to form a qualified teaching staff team. Finally, this paper summarizes the practical teaching achievements from the aspects of course development, students’ ability improvement, and teaching staff team building, providing references for improving the teaching effects of interdisciplinary courses.

Abstract:The paradigm of higher engineering education in China has transformed from emphasizing knowledge foundation and ability training to emphasizing quality shaping. How to cultivate engineering talents with solid professional knowledge, innovative thinking, and practical ability under the background of emerging engineering education has become a core issue and urgent problem to be solved in the education of various engineering majors. This paper takes Genetic Engineering, a main course in the curriculum system of bioengineering, as an example to explain the reform ideas of implementing the problem-based learning (PBL) model in online-offline blended teaching. Practice has proved that this reform idea not only significantly improves the teaching quality of the course and the learning effect of students but also provides a reference model for cultivating engineering talents with innovative thinking, practical ability, and correct values.