Abstract:

Abstract:Pyridoxal phosphate (PLP), the active form of vitamin B6, is an important coenzyme in various enzyme-catalyzed reactions. PLP-dependent enzymes can catalyze a variety of chemical reactions, such as racemization, decarboxylation, β-addition, β-elimination, retro-aldol cleavage, transamination, and α-elimination. They are biologically synthesized a powerful tool for a variety of natural amino acids, non-natural amino acids and their related compounds. This article details the structural features and catalytic mechanisms of typical PLP-dependent enzymes such as ω-transaminase, lysine decarboxylase, threonine aldolase, and L-tyrosine phenol-lyase, and reviews the research progress in molecular modification and industrial applications of these enzymes. Finally, this article provides an outlook on the future development of PLP-dependent enzymes, including in vivo regeneration system and industrial applications of PLP cofactors, and discusses the tremendous potential of these enzymes in biocatalytic applications.

Abstract:2-ketoglutarate (2-KG)/Fe²⁺-dependent dioxygenases can catalyze the highly specific regio- and stereoselective functionalization of C(sp³)-H bond of complex compounds under mild reaction conditions. Hyoscyamine 6β-hydroxylase (H6H), a member of these dioxygenases, catalyzes two consecutive oxidation reactions in the synthesis of scopolamine. The first reaction is the hydroxylation of hyoscyamine to 6β-hydroxyhyoscyamine and the second is epoxidation of 6β-hydroxyhyoscyamine. This paper introduces the catalytic mechanism, substrate scope, and application of H6H and evaluates the possibility of this enzyme as a biocatalyst for the functionalization of C(sp³)-H bond in complex compounds with different structural characteristics via hydroxylation or epoxidation, providing a theoretical basis for modification and application of this enzyme.

Abstract:Bacterial cellulose (BC) is the glucose polymer produced by bacterial metabolism. The bacterial cellulose synthase (BCS) is the key enzyme for catalyzing the formation of BC. The cooperation between different submits of BCS is necessary for the intracellular formation and extracellular secretion of BC. This review summarized the BC-producing strains and the differences of BCS among different strains. Furthermore, we detailed the BC synthesis mechanism, the interactions between BCS subunits, and the relationship between the structural characteristics of strains and the formation of highly ordered fiber structures. A comprehensive insight into the mechanism of BC synthesis and secretion will supply more strategies for optimizing the BC synthesis via methods of synthetic biology.

Abstract:Polyethylene terephthalate (PET) is one of the widely used plastics, but its waste pollution has become a global environmental issue. The discovery of polyethylene terephthalate hydrolase (PETase) has provided a green and environmentally friendly approach for PET degradation. However, PETase produces intermediate products that inhibit the enzyme’s further activity, leading to a decrease in enzyme efficiency. Mono(2-hydroxyethyl) terephthalate hydrolase (MHETase) works synergistically with PETase to further degrade the intermediate product MHET into ethylene glycol (EG) and terephthalic acid (TPA). MHETase exhibits extremely high specificity for MHET and is crucial for the complete degradation of PET. This article comprehensively reviews MHETase from various perspectives, including its three-dimensional structure, substrate binding, and catalytic mechanism. It demonstrates the structural features and key residues associated with the enzyme’s degrading activity and discusses the progress in enzyme engineering modifications. Additionally, the study envisions the development of a two-enzyme PET degradation system by combining MHETase with PETase, aiming to provide valuable references for designing and developing more efficient PET hydrolytic enzyme systems.

Abstract:Gamma-aminobutyric acid (GABA) derivatives are a class of effective inhibitory neurotransmitters for treating neurodegenerative diseases, with an immense market demand. Chemical methods are currently the main synthetic strategies for GABA derivatives, facing challenges such as complex processes, low yields, low atom economy, and environmental burden. In recent years, chemoenzymatic synthesis of GABA derivatives has garnered increasing attention because of the high atom economy, high yields, and environmental friendliness. This article reviews the latest advances in the chemical synthesis and chemoenzymatic synthesis of GABA derivatives. Furthermore, it introduces the progress in the industrial synthesis of representative GABA derivatives such as gabapentin, pregabalin, and brivaracetam and prospects the future development of GABA derivatives.

Abstract:Human milk oligosaccharides (HMOs) are a structurally complex group of unbound polysaccharides, representing the third-largest solid component in breast milk. They play a crucial role in the intestinal health and immune system development of infants. Sialylated HMOs, including 3′-sialactose (3′-SL) and 6′-sialactose (6′-SL), are major components of HMOs, playing significant roles in immune regulation, anti-inflammatory processes, and promotion of probiotic growth. Currently, the cost-effective production of high-value sialactose by microbial fermentation with readily available raw materials has become a research hotspot due to the high nutritional value and potential applications of sialylated HMOs in infant food. This paper summarizes the functions and biosynthesis of 3′-SL and 6′-SL. Furthermore, it reviews the research progress in the synthesis of sialactose by Escherichia coli, offering valuable insights for future industrial production.

Abstract:The utilization of C1 gases (CH₄, CO₂, and CO) for the production of oleochemicals applied in the energy and platform chemicals through microbial engineering has emerged as a promising approach to reduce greenhouse gas emissions and decrease dependence on fossil fuel. C1 gas-utilizing microorganisms, such as methanotrophs, microalgae, and acetogens, are capable of converting C1 gases as the sole substrates for cell growth and oleochemical synthesis with different carbon-chain lengths, garnering considerable attention from both scientific community and industry field for sustainable biomanufacturing. This paper comprehensively reviews recent advancements in the development of engineered cell factories utilizing C1 gases for the production of oleochemicals, elucidating the key metabolic pathways of biosynthesis. Furthermore, this paper highlights the research progress and prospects in optimizing gene expression, metabolic pathway reconstruction, and fermentation conditions for efficient oleochemical production from C1 gases. This review provides valuable insights and guidance for the efficient utilization of C1 gases and the development of carbon cycling-based bioeconomy.

Abstract:Carbon capture, utilization and storage is the vital technology for China to achieve the goals of carbon peaking and carbon neutrality. Microbial activities in situ are an indispensable part in the process of geological CO₂ sequestration. Some microorganisms can convert CO₂ into methane and organics as the resource for utilization or into carbonate to achieve long-term sequestration. These activities contribute to the stable storage of CO₂ and even negative carbon emission. This paper focuses on the processes of bio-methanation, bio-liquefaction, and bio-precipitation that may be involved in CO₂ sequestration in deep stratum and discusses the research progress in the bio-transformation pathways. Bio-methanation and bio-liquefaction can convert CO₂ into methane or high-value organic compounds to realize resource reuse. The two technologies can be used alone or coupled to expand the application range of CO₂ biotransformation. Bio-mineralization can convert CO₂ into calcite by microorganism-induced carbonate precipitation, being a technology of great potential in fixing CO₂ and limiting CO₂ escape. At present, this field is still in the infancy stage, and there is an urgent need to establish and improve the theoretical and technical systems of CO₂ in-situ biotransformation from transformation principle, influencing factors, conversion efficiency, economy, environmental protection, and technological conditions. Moreover, it can be combined with CCUS to establish a technical system integrating capture, transport, displace, storage, transfer, and exploit, so as to promote the value-added application of CCUS and the achievement of carbon peaking and carbon neutrality.

Abstract:The inherent stability and recalcitrance of benzene ring structures render aromatic compounds a major ecological concern and a substantial risk to human health. Hence, developing a facile and efficacious detection technique for aromatic compounds is essential. As our comprehension of aromatic compound characteristics deepens, microbial cell-based biosensors have emerged as increasingly popular tools in the detection of aromatic compounds. This article introduces the operational principles of microbial whole-cell biosensors and elucidates the construction techniques and applications of electroactive biofilm-based microbial whole-cell sensors, transcription factor-based microbial whole-cell sensors, and degradation gene promoter-dependent microbial whole-cell sensors in the detection of aromatic compounds. In addition, we review the methodologies for improving the performance of microbial whole-cell sensors based on surface display, logic gate construction, genetic circuit modification, and quorum sensing signal amplification.

Abstract:The human gut is a complex ecosystem harboring rich microbes that play a key role in the nutrient absorption, drug metabolism, and immune responses. With the continuous development of microfluidics and organ-on-a-chip, gut-on-a-chip has become a powerful tool for modeling host-microbe interactions. The chip is able to mimic the complex physiological environment of the human gut in vitro, providing a unique platform for studying host-microbe interactions. Firstly, we introduce the physiological characteristics of the human gut. Secondly, we comprehensively summarize the advantages of the microfluidic chip in vitro recapitulating the intestinal system by integrating microenvironmental factors, such as complex cell components, dynamic fluids, oxygen gradients, and mechanical mechanics. Thirdly, we expound the key performance indicators for evaluating the construction performance of gut-on-a-chip. In addition, we review the progress of gut-on-a-chip models in the research on gut microecology, disease modeling, and drug evaluation. Finally, we highlight the challenges and prospects in the applications of the emerging technology. The above is summarized with a view to informing the application of gut-on-a-chip for indepth studies of gut microbe-host interactions.

Abstract:Cardiovascular diseases are major diseases, and there is lack of artificial blood vessels with small diameters which can be applied in coronary artery bypass surgery. The conventional vascular scaffold preparation techniques in tissue engineering have shortcomings in regulating the diameter, geometric shape, and interconnectivity of the scaffold. 3D bioprinting can simulate the natural structure of the vascular tissue, accurately print live cells and biomaterials, and regulate the microstructure and porosity of scaffolds on the nanoscale, providing new ideas for vascular tissue engineering. This article systematically evaluates the classification of 3D bioprinting technologies and reviews the latest research progress of 3D bioprinting in vascular tissue engineering. It summarizes the advantages of 3D bioprinting and points out the problems that need to be solved, such as the immune rejection of blood vessel materials, providing reference for the further research.

Abstract:Microalgae, with the ability to harness solar energy to fix CO₂ and convert it into organic compounds, have emerged as promising green cell factories. With the rapid development of cutting-edge biotechnologies, the research and application of photosynthetic microalgae have been expanding, leading to comprehensive and in-depth engineering of microalgae. The synthetic biology and genome editing technologies have enabled the applications of microalgae in medicine, agriculture, food, energy, and the environment. However, the survival and spreading of engineered microalgae in the natural environment pose potential safety risks to ecosystems and human health. To curb the risks caused by the spreading of engineered microalgae in the environment, biosafety policies should be formulated for engineered microalgae and the prevention and control technologies should be developed. Toward this goal, researchers have developed biocontainment systems, including positive strategies such as the design of toxic protein-based kill switches and passive strategies such as knocking out essential genes to construct the strains with nutritional deficiencies, thereby spatially containing engineered microalgae. This article summarizes the application of cutting-edge biotechnologies in the engineering of microalgae, the biosafety risks and management regulations associated with the escape of engineered microalgae, and the progress in novel biocontainment technologies established for engineered microalgae. Finally, this article gives insights into the future development direction of microalgae biocontainment.

Abstract:The major science and technology infrastructure in the field of life science is an indispensable and important content in the large-science facility landscape. It encompasses cutting-edge, strategic, and fundamental aspects. This field differs from traditional facilities such as particle physics, astronomy and nuclear energy. Moreover, it represents a relatively underdeveloped area in China’s facility landscape. Unique characteristics are observed in terms of capital investment, physical form, facility lifespan, digitization degree, organizational structure, project risk, and development effect when compared to traditional facilities. Despite its importance, challenges persist in project establishment, investment, management, and construction. Therefore, it is necessary to strengthen the condensing mechanism for addressing major scientific issues in the life science field, improve the strategic investment layout, facilitate the localization of technical equipment based on original scientific ideas, and strengthen the differentiated management capacity of life science facilities.

Abstract:Bovine chymosin is an essential food enzyme widely used in cheese production in the dairy industry. This study used a codon-optimized prochymosin gene to construct an expression cassette for extracellular expression of bovine chymosin in Kluyveromyces lactis. After integration of the prochymosin gene into the host cell genome, the single-copy integration strain KLUcym showed the clotting activity of 40 U/mL in a shake flask. The CRISPR/Cas9 system was employed to delete amdS and construct the double-copy integration strain and triple-copy integration strain, which achieved the clotting activities of 70 U/mL and 78 U/mL in shake flasks, separately. Subsequently, multiple rounds of UV mutagenesis were performed on the double-copy strain KLUcym^D, and a recombinant K. lactis strain with a high yield of bovine chymosin was obtained. This strain achieved the clotting activity of 270 U/mL in a shake flask and 600 U/mL in a 5 L bioreactor after 76 h. In summary, we construct a strain KLUcym^D-M2 for high production of bovine chymosin, which lays a foundation of industrial fermentation.

Abstract:The organelles in the multi-nucleated filamentous fungus Aspergillus oryzae present polymorphism. To observe the organelle morphology in A. oryzae and provide references for the localization prediction of unknown proteins and the disclosure of biological reaction pathways in A. oryzae, we fused different subcellular localization signals with green fluorescent protein (GFP) to obtain different subcellular localization vectors, which were then transferred into A. oryzae by Agrobacterium tumefaciens-mediated transformation. The A. oryzae reporter strains with fluorescence-labeled nuclei, mitochondria, endoplasmic reticulum, vacuole, lipid droplets, peroxisome, and Golgi apparatus were successfully constructed. Furthermore, staining with small-molecule specific dyes was carried out to validate the co-localization of fluorescence-labeled mitochondria, nuclei, and lipid droplets in the reporter strains, which further confirmed that the reporter strains were successfully constructed. The distribution and morphology of fluorescence-labeled organelles were observed at different growth stages and under different culture conditions. The constructed reporter strains provide basic tools for studying the organelle morphology, localization of unknown target proteins, and subcellular localization in A. oryzae.

Abstract:Neohesperidin is a flavonoid glycoside widely used in the food and pharmaceutical industries. The current production of neohesperidin mainly relies on extraction from plants. Microbial fermentation demonstrates a promising prospect as an environmentally friendly, efficient, and economical method. In this study, we designed and constructed the biosynthetic pathway of neohesperidin in an Escherichia coli strain by introducing the glycosyltransferase UGT73B2 from Arabidopsis thaliana, rhamnose synthase VvRHM-NRS from Vitis vinifera, and rhamnose transferase Cm1, 2RhaT from Citrus maxima. After optimization of the module and the uridine diphosphate (UDP)-glucose synthetic pathway, the engineered strain produced4.64 g/L neohesperidin in a 5 L bioreactor, and the molar conversion rate of hesperetin was 45.8%. This has been the highest titer reported to date for the biosynthesis of neohesperidin in microorganisms. This study lays a foundation for the construction and application of strains with high yields of neohesperidin and provides a potential choice for the microbial production of other flavonoid glycosides.

Abstract:Guanidinoacetic acid, as an energetic substance, has a wide range of applications in the food, pharmaceutical, and feed industries. However, the biosynthesis of guanidinoacetic acid has not been applied in industrial production. In this study, we designed the synthetic route of guanidinoacetic acid in a food-grade strain of Bacillus subtilis. By regulating the expression of key enzymes, lifting feedback inhibition, and increasing membrane permeability, we achieved the efficient synthesis of guanidinoacetic acid by whole-cell catalysis. Firstly, the optimal L-arginine:glycine amidinotransferase was screened based on the phylogenetic tree, and the expression of the key enzyme was enhanced by a strategy combining strong promoter and genome integration. Secondly, the ornithine cycle for L-arginine synthesis in Corynebacterium glutamicum was introduced to alleviate the feedback inhibition of the enzyme by the byproduct L-ornithine, and the L-arginine degradation pathway was knocked down to enhance substrate regeneration. Thirdly, the expression of N-acetylmuramoyl-L-alanine amidase (LytC) was up-regulated to increase the cell membrane permeability. Finally, after optimization of whole-cell production conditions, strain Bs-13 achieved guanidinoacetic acid production at a titer of 13.1 g/L after 24 h, with a proudction rate of 0.54 g/(L·h) and a glycine conversion rate of 92.7%. The above strategy improved the production of guanidinoacetic acid and provided a reference for the biosynthesis of guanidinoacetic acid.

Abstract:Gadusol, an efficient natural ultraviolet (UV) absorbing substance with antioxidant capacity, is ubiquitous in aquatic organisms such as microorganisms, algae, and fish eggs. In order to address issues such as its low natural extraction yield and environmental unfriendliness, we introduced the gadusol synthesis pathway from zebrafish into Komagataella phaffii and successfully constructed the recombinant strain capable of synthesizing gadusol. The xylose assimilation genes derived from Scheffersomyces stipitis were further introduced into the recombinant strain to increase the content of the key substrate sedoheptulose 7-phosphate (S7P). The results showed that the utilization of xylose was an effective strategy to increase the yield of gadusol. In the medium with only xylose as the substrate, the yield of gadusol reached 141.8 mg/L (32.3 mg/g dry cell weight, DCW), which was about 46 times of that in the medium with only glucose as the substrate. The product showed obvious absorption in the range of 275–305 nm, with the maximum absorption at 290 nm. Moreover, the product demonstrated antioxidant capacity. After reaction for 5 h, the ferric ion reducing antioxidant power (FRAP), 2,2′-azino- bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging rates changed by 90.83%, 25.50%, and 131.80%, respectively, which suggested that the product may be used as a long-acting antioxidant. This study demonstrated the great potential of K. phaffii as a chassis for the biosynthesis of natural UV absorbers. The biosynthesized gadusol has good UV absorption and antioxidant properties, which provides a theoretical basis for the industrial production and application of gadusol.

Abstract:Catechol (CA) is an important chemical and pharmaceutical intermediate with wide applications. At present, CA is produced by phenol hydroxylation with non-renewable petrochemical resources, which causes serious environmental pollution. Hence, the biosynthesis of CA attracts much attention recently. However, due to the low activities of protocatechuic acid (PCA) decarboxylases, the production efficiency of biosynthetic catechol is too low to meet the requirements of large-scale industrial production. To improve the yield of CA, we screened 21 PCA decarboxylases from different species. RbAroY originated from Rikenellaceae had the best catalytic performance. The whole-cell biocatalyst ER11 with RbAroY was able to produce CA at a titer of 13.54 g/L. Then, the online tool HotSpot Wizard was employed to measure the enzyme stability, which revealed 10 potential mutation sites causing significant decreases in Gibbs free energy. The whole-cell biocatalyst ERT01 with the mutated RbAroYG99A could produce CA at a titer of 15.16 g/L, which increased by 12% compared with that of the wild-type whole-cell biocatalyst. After optimization of the biocatalytic conditions, the whole-cell biocatalyst ERT01 was able to produce CA at a titer of 25.70 g/L with PCA as the substrate. Finally, with the fermentation broth of 3-dehydroshikimate as the substrate, the whole-cell biocatalyst DER03 expressing both 3-dehydroshikimate dehydratase and PCA decarboxylase realized the production CA at a titer of 29.55 g/L, which is currently the highest biosynthetic titer reported. This study provides a reference for the industrial production of CA by biosynthesis.

Abstract:Dextranase is an enzyme that specifically hydrolyzes the α-1,6 glucoside bond. In order to improve the activity of dextranase from Arthrobacter oxidans KQ11, site-directed mutagenesis was used to modify the amino acids involved in the “tunnel-like binding site”. A saturating mutation at position 507 was carried out on this basis. The mutant enzymes A356G, S357W, W507Y, and W507F with improved enzyme activities and catalytic efficiency were successfully obtained. Compared with wild type (WT), W507Y showed the specific activity increasing by 3.00 times, the k_cat value increasing by 3.62 times, the K_m value decreasing by 54%, and the catalytic efficiency (k_cat/K_m) increasing by 8.98 times. The three-dimensional structure analysis showed that the increase of the number of hydrogen bonds and the distance between “tunnel-like binding sites” were important factors affecting enzyme activity. Compared with WT, W507Y had a shortened distance from the residues on the other side of the “tunnel-like binding site”, which made it easier to generate hydrogen binding forces. Accordingly, the substrate hydrolysis and product efflux were accelerated, which dramatically increased the enzyme activity and catalytic efficiency.

Abstract:Tyrosinase is a copper-containing polyphenol oxidase widely applied in the food, cosmetics, pharmaceutical, and other industries. Currently, the production of commercial tyrosinase primarily relies on extraction from fungi, which has high costs, low purity, low specific activity, and poor stability. The objective of this study is to obtain highly expressed bacterial tyrosinase with potential for industrial applications. The bacterial tyrosinases from five different sources were heterologously expressed in Escherichia coli BL21(DE3), and the tyrosinases TyrBm and TyrVs derived from Bacillus megaterium and Verrucomicrobium spinosum were obtained with the enzyme activities of (16.1±0.2) U/mL and (48.6±0.9) U/mL, respectively. After protein purification, we compared the enzymatic properties of TyrBm and TyrVs, which revealed that TyrVs exhibited better thermal stability and higher substrate specificity than TyrBm. On the basis of characterizing TyrVs with high catalytic performance, we established a biological hair dyeing system based on TyrVs catalysis to achieve in-situ catalytic hair dyeing. The color washing fastness test measured the △E value less than 7.38±0.64 after simulated 14-day cleaning. To facilitate the rapid separation of catalytic products and enzymes, we successfully constructed an immobilized enzyme TyrVs-CipA dependent on self-assembly label CipA and applied this enzyme in the DOPA modification of hydrolyzed silk fibroin (HSF). The immobilized enzyme continuously catalyzed HSF for more than seven cycles, resulting in a single DOPA modification degree exceeding 70.00%. Further investigations demonstrated that DOPA modification enhances the scavenging activity of HSF towards DPPH and O2- radicals by 507.80% and 78.23%, respectively. This study provides a technical foundation for the development of environmentally friendly biological hair dye based on tyrosinase and biomaterials for tissue engineering.

Abstract:The widespread use of non-naturally degradable plastics is causing increasingly serious harm to the environment. Reducing plastic pollutants has become the core of ecological and environment management. Biological methods such as enzymes demonstrate advantages in depolymerizing plastics with mild reaction conditions and recycling of depolymerization products. However, there are few reports on the biological depolymerization of polyamide plastics. In this study, by using 4-nitropropionanilide as the model substrate, we screened against our plastic depolymerase library and obtained a Meiothermus ruber-derived enzyme (MrABH) that can hydrolyze the polyamide bond. We expressed this enzyme in Escherichia coli and purified the protein by affinity chromatography. Furthermore, we investigated the catalytic properties, enzymatic properties, and catalytic products of this enzyme with polyamide as the substrate. MrABH had good stability at pH 8.0–10.0, with the optimal performance at pH 9.0 and 30 ℃. The catalytic performance of this enzyme for ester bonds and amide bonds was similar. MrABH can catalyze the depolymerization of PA6 and PA66 to produce monomers and oligomers, demonstrating the potential to be used in the depolymerization and recycling of polyamide.

Abstract:Corynebacterium glutamicum is a major workhorse in the industrial production of branched-chain amino acids (BCAAs). The acetohydroxyacid synthase (AHAS) encoded by ilvBN is a key enzyme in the biosynthesis of BCAAs. Enhancing AHAS expression is essential for engineering BCAA producers. However, at present, the available studies only used limited promoters to regulate AHAS expression, which is insufficient for achieving efficient regulation. Herein, we first employed a previously developed reporter system to screen out a strong constitutive promoter P_gpmA* from six candidate promoters for expressing ilvBN. P_gpmA* showcased the expression strength 23.3-fold that of the native promoter P_ilvBN. Moreover, three synthetic RBS libraries based on the promoter P_gpmA* were constructed and evaluated by plate fluorescence imaging. The results revealed that “R₍₉₎N₍₆₎” was the best mutant library. A total of 36 RBS mutants with enhanced strength were further screened by evaluation in 96-deep-well plates, and the highest strength reached up to 62.3-fold that of P_ilvBN. Finally, the promoter P_gpmA* was combined with three RBS mutants (WT, RBS18, and RBS36) to fine-tune the expression of ilvBN^S155F for L-valine biosynthesis, respectively. Increased expression strength led to enhanced L-valine production, with titers of 1.17, 1.38, and 2.29 g/L, respectively. The combination of RBS18 strain with the further overexpression of ilvC produced 7.57 g/L L-valine. The regulatory elements obtained in this study can be utilized to modulate AHAS expression for BCAA production in C. glutamicum. Additionally, this strategy can guide the efficient expression regulation of other key enzymes.

Abstract:Salidroside is a functional ingredient with wide applications in food and pharmaceutical fields. It is conventionally produced by extraction from plants, the application of which is limited by the scarcity of raw materials and cumbersome process. This study achieved the efficient production of salidroside by biosynthesis with tyrosol as the substrate. While utilizing glycosyltransferases for tyrosol glycosylation, we introduced sucrose synthase to construct the uridine diphosphate glucose (UDPG) recycling system. The glycosyltransferase UGT33 and sucrose synthase AtSUS were screened out by comparison, and the recombinant strain Escherichia coli BL21/pETDuet-AtSUS-UGT33 was constructed. The copy number of the gene was optimized and the optimal copy number ratio of glycosyltransferase to sucrose synthase was determined to be 3:1. The whole-cell transformation conditions (temperature, pH, inoculum amount, substrate concentration, and concentrations of metal ions) of the recombinant strain were optimized, and the highest yield of salidroside reached 8.17 g/L after fermentation under the optimal conditions in a 5 L fermenter for 24 h. This study provides a reference for the efficient production of salidroside by microorganisms.

Abstract:1,4-butanediol is an important intermediate widely used in chemical, agricultural, and pharmaceutical industries. This study constructed a new short path for the production of 1,4-butanediol with glucose as the substrate by combining enzyme engineering and metabolic engineering. Firstly, a novel path catalyzed by α-ketoglutarate decarboxylase (SucA), carboxylate reductase (Car), and alcohol dehydrogenase (YqhD) was designed by database mining, and the de novo synthesis of 1,4-butanediol was achieved after introduction of the path into Escherichia coli W3110 (K-12) chassis cells. To further improve the synthesis efficiency of this path, we deleted the genes encoding lactate dehydrogenase A (LdhA) and pyruvate formate lyase B (PflB) to block the metabolic bypass. Furthermore, the expression of citrate synthase (GltA^R163L) was up-regulated to increase the α-ketoglutarate metabolic flux. In addition, we improved the synthesis of the key cofactor NADPH and up-regulated the expression of sucA, car, and yqhD by substituting with strong promoters to increase the efficiency of supplying precursors to 1,4-butanediol synthesis. Eventually, the recombinant strain produced up to 770 mg/L of 1,4-butanediol within 48 h in a shake flask, and 4.22 g/L of 1,4-butanediol within 60 h in a 5 L fermenter with a yield of 12.46 mg/g glucose. Compared with the previously reported method, the novel path designed in this study for the de novo synthesis of 1,4-butanediol does not need acetyl coenzyme A and avoids the byproduct acetate or the addition of ammonia. Therefore, the outcome is expected to provide a new idea for the metabolic engineering of microbial chassis for the production of 1,4-butanediol and its high-value derivatives.

Abstract:D-mannose is a natural hexose with great economic and application values in the food, medicine, and cosmetic fields. However, most biosynthesis methods of D-mannose rely on Escherichia coli as the host, which poses safety issues during the production process and imposes limitations on subsequent applications. This study compared the enzyme properties of mannose isomerases from multiple sources to select the most suitable source. B. subtilis 168/pMA5-EcMIaseA was constructed with “generally recognized as safe” (GRAS) Bacillus subtilis as the host and used as a whole-cell catalyst to synthesize D-mannose from D-fructose. Optimizing the conversion conditions such as culture temperature, pH, and substrate concentration increased the yield of D-mannose. The results showed that the conversion rates reached 27.75% and 27.22% and the yields of D-mannose were 138.74 g/L and 163.30 g/L after 6 h whole-cell transformation with D-fructose at the concentrations of 500 g/L and 600 g/L, respectively, in a 5 L fermentor. This study achieves the highest yield of D-mannose produced under the catalysis by recombinant B. subtilis that has ever been reported and provides a basis for the industrial production and application of D-mannose.

Abstract:Hexokinase is a crucial diagnostic reagent in blood glucose testing, which has high requirements for the enzyme activity and thermal stability. The hexokinases in China mainly rely on imports and are primarily sourced from yeast, with high costs and poor thermal stability, which limit the development of blood glucose diagnostic reagents. Therefore, there is an urgent need for the efficient expression of highly active and thermally stable hexokinases. In this study, an ATP-dependent hexokinase (glucokinase, Glk) from a thermophilic bacterium Glk was heterologously expressed in Escherichia coli BL21(DE3). Glk exhibited high specificity for glucose, dependence on Mg²⁺, and the highest activity at pH 8.5 and 80 ℃. It retained over 90% activity after storage at 30–37 ℃ for 7 days, demonstrating thermal stability as an alkaline glucose kinase. Subsequently, the factors influencing Glk expression, including culture medium, OD₆₀₀, final concentration of the inducer, induction temperature, and induction duration, were systematically optimized. The optimization increased the Glk expression by 4.71 folds Glk compared with non-optimized conditions. After purification, Glk exhibited a specific activity of (43.05±2.00) U/mg and the purity ≥98%. In conclusion, the developed expression and purification method for the highly thermostable hexokinase provides more possibilities for overcoming the shortcomings in the preparation of blood glucose diagnostic reagents in China.

Abstract:Ganoderma lucidum is a precious fungus with both edible and medicinal values and has a long history of medical use. Triterpenes as the main active components endow G. lucidum with anti-tumor, antioxidant, and other pharmacological activities. The present study endeavors to establish a proficient liquid-state fermentation technology for the enhanced production of triterpenes. In view of the limitations inherent in conventional submerged fermentation and oscillation-static two-stage cultivation, this study established an oscillation-static cycle cultivation process and optimized the cultivation conditions by building an artificial neural network model based on genetic algorithms. The cultivation conditions for the high-yield production of triterpenes were optimized as follows: 2.8 days of oscillation, 7.3 days of static cultivation, 0.2 day of oscillation, and 0.3 day of static cultivation. Under these conditions, the content of triterpenes reached 20.82 mg/g. The yield of triterpenes reached 129.09 mg/L, showing a remarkable increase of 324.78% compared with that of the Z10J0 method. Moreover, the established method shortened the cultivation cycle by 10.6 days. The mycelia cultivated under this regimen exhibited commendable anti-tumor and antioxidant activities. This study not only presents an economical liquid-state fermentation approach but also streamlines the fermentation flow, reduces fermentation duration, and effectively ameliorates drawbacks associated with conventional cultivation methods. In addition, this study gives valuable insights into the scaled application of liquid-state fermentation in the high-yield production of triterpenes, which showcases broad prospects.

Abstract:L-tryptophan is an indispensable essential amino acid with a wide range of applications, which leads to a high demand. Accordingly, the production of L-tryptophan becomes a much-anticipated direction in research and industrial development. While irrational mutagenesis is an effective means to breed industrial strains, how to screen the strains with desirable phenotypes is still a major challenge. In order to improve the efficiency and accuracy of screening L-tryptophan high-yield strains, we used atmospheric and room temperature plasma mutagenesis to construct a random mutant library and then combined it with high-throughput screening in deep-well plates. Using a pseudo-fluorescent protein sensor capable of responding specifically to L-tryptophan, we successfully screened out a strain producing L-tryptophan at a high yield from a random mutagenesis library. The fermentation with the strain in shake flasks produced L-tryptophan at a yield of 1.99 g/L, which was 41.77% higher than that of the starting strain. Finally, the mechanism of high yield of the strain was deciphered by comparative genomics and transcriptomics. The above strategies provide a solid research foundation for further selection and development of high quality L-tryptophan producing strains.

Abstract:We analyzed the biological and genome characteristics of a phage infecting enteroinvasive Escherichia coli (EIEC), aiming to provide resources and a reference for the prevention and treatment of EIEC. With the EIEC preserved in our laboratory as the host bacterium, one strain of phage was isolated from the effluent sample from a chicken farm in Huzhou, Zhejiang and named ΦEP1. The titer, optimal multiplicity of infection, one-step growth curve, temperature, pH value, chloroform and bile salt sensitivity of ΦEP1 were determined by the double-layer agar plate method. The morphology of the phage was observed by transmission electron microscopy. The biocontrol effects of ΦEP1 in different food matrixes and the protective effect of this phage on Caco-2 cells were tested. The phage ΦEP1 showed the optimal multiplicity of infection of 0.1, the titer of 1.3×10¹⁰ PFU/mL, strong tolerance to temperature, pH, chloroform, and bile salt, and a broad host spectrum. Furthermore, it expressed lysis activity against multiple strains of multiple antibiotic-resistant pathogenic E. coli and Shigella with different serotypes. Phage ΦEP1 had an incubation period of 10 min, an outbreak period of 80 min, and an outbreak volume of 48 PFU/cell. According to the morphology observed by transmission electron microscopy, phage ΦEP1 belonged to the order of Caudovirales, and it had a good protective effect on Caco-2 cells. Phage ΦEP1 had a genome of 87 182 bp with the GC content of 39.80%, 128 putative open reading frames, and no antibiotic resistance genes or virulence genes. ΦEP1 inhibited the growth of EIEC in artificially contaminated milk and beef and eliminated EIEC in cell protection experiments. It significantly increased the survival rate of Caco-2 cells and down-regulated the expression of interleukin (IL)-6 and IL-1β to reduce inflammation. We obtained an EIEC-targeting phage ΦEP1 with a high titer and strong tolerance to the environment, which provided a basis for the application of phages in food preservation and other fields.

Abstract:Value shaping and innovation capability improvement are two important goals of postgraduate course teaching. Biocatalysis and Enzyme Engineering is a core course for postgraduates majoring in bioengineering. Our teaching team established a “dual-drive and dual-guide” teaching model, that is, “double drive” (value-driven + innovation-driven) as the traction, this model integrated professional quality with industry needs, engineering ethics, and scientist spirit, combined theoretical teaching with research frontiers, industrial cases, and engineering practice, and incorporated thematic lectures, group peer evaluation, and review papers into course assessment. The teaching under this model achieved the teaching objectives of “double guide” (guiding ideology and ability), and improved the postgraduates’ value identification, innovation awareness, engineering thinking, and ability to solve practical engineering problems.

Abstract:According to the Bio-economy Development Plan during the 14th Five-Year Plan period, biotechnology has become an effective force to promote future development. More than 220 universities and research institutes in China have got the right to confer master’s degrees in bioengineering. Great attention has been paid to the cultivation of top innovative talents that can serve the innovation-driven development of the national bio-economy. In the last 15 years, the Research Center of Microbial-Manufacturing Engineering in Jiangnan University has built a diversified education platform, recruited high-level faculty members, and innovated the scientific research management. The new concept and method for cultivating the innovation capabilities of postgraduates in a multi-dimension and whole-process manner have been formed, which involved building a curriculum, focusing on major projects, and establishing a supervisor team. This cultivation mode has comprehensively improved the engineering and academic innovation capabilities of postgraduates majoring in bioengineering.

Abstract:Under the background of developing new engineering disciplines, teaching reform is a key strategy applied by higher education institutions to develop new engineering professionals and accomplish the mission of cultivating morality and nurturing talents. As a foundational course for majors of life sciences and food sciences, “Principles of Fermentation Engineering” has a strong scientific, practical, and historical focus. It serves as an excellent resource for changing the way that college students are taught professional courses. To examine the reform and practical route of specialized course teaching combined with innovation and entrepreneurship fostering under the integration production, education, and research, this article takes the teaching of “Principles of Fermentation Engineering” for undergraduates majoring in food science and engineering at Hebei Agricultural University as an example. A new teaching paradigm integrating production, education, and research is developed considering a variety of factors, including instructional content, teaching methods, and evaluation approaches. This paradigm is result-oriented, replaces examination with competition, and promotes learning by research. It achieves the integration of specialized course teaching and innovation and entrepreneurship fostering and lays a foundation for the teaching reform and the development of professional talents in the context of developing new engineering disciplines.

Abstract:In the context of new engineering, it is of great importance to enhance the innovative capabilities of biological science undergraduates receiving higher education. We conducted a questionnaire survey at the School of Life and Environmental Science, Hangzhou Normal University to examine the current situation of undergraduates’ innovative capabilities. We explored the teaching reform in biological majors based on outcome-based education (OBE) from teaching innovation, tutorial system establishment, and dual integration of research-education and production-education. Furthermore, an empirical study adopting the Williams’s Creativity Assessment Packet highlighted that the OBE-oriented teaching reform effectively fostered students’ creativity and laid a foundation for enhancing their innovative capabilities. This study provides valuable insights for cultivating innovative talents majoring in biological sciences in universities.

Abstract:In recent years, artificial intelligence has been employed to empower synthetic biology, demonstrating great potential in the simulation and prediction of protein structures as well as the design and optimization of regulatory elements and metabolic networks. Integrating artificial intelligence into the teaching of Synthetic Biology is in line with the development trends of synthetic biology and can promote the cultivation of interdisciplinary high-level talents and collaborative innovation. This paper expounds the idea of integrating artificial intelligence into the teaching of Synthetic Biology from establishing interdisciplinary course contents and teaching methods, simultaneously considering the fundamentals and application of artificial intelligence in synthetic biology, cultivating independent learning and innovative practice abilities, and enhancing the ethics education related to artificial intelligence. Furthermore, a system integrating artificial intelligence with the teaching contents of Synthetic Biology is designed, which focuses on supplementing fundamentals of artificial intelligence and incorporating artificial intelligence into the classroom and experimental teaching contents of Synthetic Biology. Moreover, with the course of Synthetic Biology in Jiangnan University as an example, this paper presents the pathway of integrating artificial intelligence into the teaching of this course under the background of discipline crossing. Finally, the teaching effects are expected.

Abstract:Synthetic Biology, as an emerging discipline, has gained widespread attention and is developing rapidly, profoundly impacting the fields of life sciences and biotechnology. Concurrently, as emerging engineering education programs take shape, accelerated cultivation of multifaceted innovative talents represents a new mission and imperative for higher education in China. In the context of the flourishing development of Synthetic Biology, East China University of Science and Technology has established a curriculum cluster in Synthetic Biology, focusing on microbiological drug discovery and biomanufacturing. The teaching team initially reviewed the curriculum system related to Synthetic Biology and its upstream and downstream courses. Subsequently, they expanded the core courses in Synthetic Biology, creating a curriculum cluster that encompasses not only the theoretical foundations and cutting-edge technologies but also integrates with related disciplines. Moreover, the curriculum cluster leverages lectures from renowned domestic and international professors in the State Key Laboratory of Bioreactor Engineering, and harnesses the rich resources of the Program of Introducing Talents of Discipline to Universities (the “111 plan”), aiming to enhance students’ innovation capabilities. With the support of this curriculum cluster and teaching team, undergraduate students actively participate in international Synthetic Biology competitions like international genetic engineering machine competition (iGEM), consistently achieving gold awards. Furthermore, many students have applied for patents and made contributions to research paper publications. This work stands as a valuable exemplar for cultivating multifaceted talents with exceptional innovative capabilities.