• Volume 41,Issue 1,2025 Table of Contents
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    • >Commentary
    • Biomanufacturing driven by engineered organisms

      2025, 41(1):1-78. DOI: 10.13345/j.cjb.241014

      Abstract (118) HTML (145) PDF 1.17 M (194) Comment (0) Favorites

      Abstract:This article reviews the review articles and research papers related to biomanufacturing driven by engineered organisms published in the Chinese Journal of Biotechnology from 2023 to 2024. The content covers 26 aspects, including chassis cells; gene (genome) editing; facilities, tools and methods; biosensors; protein design and engineering; peptides and proteins; screening, expression, characterization and modification of enzymes; biocatalysis; bioactive substances; plant natural products; microbial natural products; development of microbial resources and biopesticides; steroidal compounds; amino acids and their derivatives; vitamins and their derivatives; nucleosides; sugars, sugar alcohols, oligosaccharides, polysaccharides and glycolipids; organic acids and monomers of bio-based materials; biodegradation of polymeric materials and biodegradable materials; intestinal microorganisms, live bacterial drugs and synthetic microbiomes; microbial stress resistance engineering; biodegradation and conversion utilization of lignocellulose; C1 biotechnology; bioelectron transfer and biooxidation-reduction; biotechnological environmental protection; risks and regulation of biomanufacturing driven by engineered organisms, with hundreds of technologies and products commented. It is expected to provide a reference for readers to understand the latest progress in research, development and commercialization related to biomanufacturing driven by engineered organisms.

    • >Review
    • Research progress in the design and application of whole-cell biosensors for antibiotics

      2025, 41(1):79-91. DOI: 10.13345/j.cjb.240400

      Abstract (121) HTML (143) PDF 659.67 K (180) Comment (0) Favorites

      Abstract:Antibiotics are chemicals with bactericidal or bacteriostatic activity produced by microorganisms and artificially synthesized. Since the discovery of penicillin by Alexander Fleming in 1928, antibiotics have been widely used in clinical treatments as well as in the animal husbandry and aquaculture, leading to antibiotic residues in soil, water, food and other environments. At the same time, antibiotic resistance is increasingly serious, which necessitates the discovery of novel antibiotics. In recent years, with the development of synthetic biology, researchers have developed a variety of whole-cell biosensors that can respond to antibiotics. These whole-cell biosensors use microbial cells to convert antibiotic signals into readable signals, which can not only perform dynamic detection of antibiotics simply, quickly, sensitively and accurately but also effectively discover novel antibiotics. This review comprehensively summarizes the reported whole-cell biosensors for antibiotics, classifies them into two types (specific and general), and elaborates on the design principles and applications of the two types of antibiotic biosensors. This review will provide reference for the construction and application of other whole-cell biosensors for antibiotics.

    • Research progress and application of nanopores in single-molecule protein sensing

      2025, 41(1):92-116. DOI: 10.13345/j.cjb.240237

      Abstract (81) HTML (125) PDF 1.78 M (174) Comment (0) Favorites

      Abstract:Proteins are fundamental carriers as the structural elements and biochemically active entities responsible for catalysis, transport, and regulation. These functions are depending on the protein folding into precise three-dimensional structures, interacting with ligands, and conformational changes. This article reviews the recent progress of nanopores in single-molecule protein sensing, involving the identification of polypeptides and proteins, the conformation changes of protein folding, the molecular structure responsible to the pH of solutions, the molecular interactions, and protein sequencing. These studies provide clues to understand life activities and facilitate the early diagnosis of diseases and design of drugs for precise treatment.

    • Visualization of flagella and its applications in research on flagellar functions

      2025, 41(1):117-130. DOI: 10.13345/j.cjb.240227

      Abstract (61) HTML (132) PDF 969.58 K (151) Comment (0) Favorites

      Abstract:Flagella are important protein structures on the cell surface of bacteria and the main appendage for bacterial swimming. Flagella play a crucial role in bacterial motility, chemotaxis, pathogenicity, and environmental sensing. With the development of microscopic tracking technology and flagellum visualization tools, new forms of flagellar motility and increasing roles of flagella in the physiological activities of bacteria have been discovered. This review introduces the visualization methods of flagella and the applications of these methods in studying flagellar functions, giving insights into exploring the functions of flagella and laying a theoretical foundation for its future applications in inhibiting bacterial transmission and treating bacterial infections.

    • Advances in the catalytic promiscuity of nitrilases

      2025, 41(1):131-147. DOI: 10.13345/j.cjb.240265

      Abstract (69) HTML (128) PDF 1.16 M (135) Comment (0) Favorites

      Abstract:As important biocatalysts, nitrilases can efficiently convert nitrile groups into acids and ammonia in a mild and eco-friendly manner, being widely used in the synthesis of important pharmaceutical intermediates. Early studies reported that nitrilases only had the hydrolysis activity of catalyzing the formation of corresponding carboxylic acid products from nitriles, showing catalytic specificity. However, recent studies have shown that some nitrilases exhibit the hydration activity for catalyzing the formation of amides from nitriles, showing catalytic promiscuity. The catalytic promiscuity of nitrilases has dual effects. On the one hand, the presence of amide by-products increases the difficulties and costs of subsequent separation and purification of carboxylic acid products. On the other hand, however, if the catalytic reaction pathways of nitrilases can be precisely regulated to reshape enzyme functions, the reactions catalyzed by nitrilases can be broadened to provide new ideas for the biosynthesis of high-value amides, which is crucial for the development of artificial enzymes and biocatalysis. This review summarized the research progress in the catalytic promiscuity of nitrilases and discussed the key regulatory factors that may affect the catalytic promiscuity of nitrilases from the evolutionary origin, catalytic domains, and catalytic mechanisms, hoping to provide reference and inspiration for the application of nitrilases in biocatalysis.

    • Application of biomanufacturing in polymer flooding

      2025, 41(1):148-172. DOI: 10.13345/j.cjb.240257

      Abstract (78) HTML (147) PDF 1.14 M (140) Comment (0) Favorites

      Abstract:In China, the crude oil supply is highly dependent on overseas countries, and thus strengthening crude oil self-sufficiency has become an important issue of the national energy security. Tertiary oil recovery, especially polymer flooding, has been widely applied in large oil fields in China, which can increase the recovery rate by 15%–20% compared with water flooding. However, the widely used oil flooding polymers show poor thermal stability and salinity tolerance, complicated synthesis ways of monomers, and environmental unfriendliness. Moreover, the polymer flooding induces problems including pore plugging, heterogeneity intensification, high dispersion of remaining oil resources, pressure rise in injection wells, and low efficiency circulation of injection medium, which restrict the subsequent recovery of old oil fields. Here, we systematically review the developing and current situations of polymer flooding, introduce the innovative biomanufacturing of oil flooding polymers and their monomers or precursors as well as low-cost bio-based chemical raw materials for multiple compound flooding. The comprehensive study of the relationships between microbial fermentation metabolites and polymer flooding will reveal the green and low-carbon paths for polymer flooding. Such study will enable the application of enzymes produced by microorganisms in polymer production and polymer plugging removal after polymer flooding as well as the application of microbial metabolites such as biosurfactants, organic acids, alcohols, biogas, and amino acids in enhancing oil recovery. This review suggests that incorporating biomanufacturing into polymer flooding will ensure the high productivity and stability for crude oil production in China.

    • Effects of exogenous additives on growth and high-value bioproducts accumulation of microalgae

      2025, 41(1):173-198. DOI: 10.13345/j.cjb.240335

      Abstract (54) HTML (156) PDF 723.94 K (134) Comment (0) Favorites

      Abstract:With the rapid development of synthetic biology, genetic engineering, and molecular manipulation methods in recent years, microalgae, as representatives of microbial cell factories, have been widely used as hosts in the production of high-value bioproducts, such as oils, pigments, proteins, and biofuels, demonstrating promising prospects of application in biochemical energy, food and drugs, and environmental protection. Despite these advancements, the low production efficiency of microalgae limits their industrial application. In addition to strain improvement and culture condition optimization, the regulation by exogenous chemical additives serves as a promising optimization strategy. This method relies on straightforward phenotypic screening and circumvents the necessity for intricate understanding of molecular targets in the metabolic and catabolic pathways involved in the synthesis of bioproducts. It enables rapid yield increasing of high-value bioproducts from microalgae and obtaining the required phenotypes. Although studies have reported the use of alternatives means such as exogenous additives to improve the growth of microalgae and the yield of high-value bioproducts, the classification and summarization of the types, applications, targeted strains, and molecular mechanisms of these additives are not comprehensive. Here, we review the studies using chemical inducers or enhancers to improve cell growth and high-value bioproduct accumulation in microalgae in recent years. This paper focuses on the types of exogenous additives, the effects of exogenous additives and their combinations on microalgae growth and high-value bioproduct accumulation, and the molecular mechanisms of related effects. We aim to provide information for researchers to use methods of synthetic biology to develop suitable cell chassis and harness microalgae for industrial production.

    • Research progress in tolerance of petroleum hydrocarbon pollutant-degrading strains

      2025, 41(1):199-215. DOI: 10.13345/j.cjb.240350

      Abstract (54) HTML (138) PDF 815.83 K (149) Comment (0) Favorites

      Abstract:Petroleum hydrocarbon pollution has become one of the global environmental problems, posing a serious threat to the environment and human health. Microbial remediation plays an important role in the remediation of petroleum hydrocarbon-contaminated environment. Nevertheless, the stress factors present in the environment polluted by petroleum hydrocarbons limit the effectiveness of microbial remediation. This paper reviews the common stress factors in petroleum hydrocarbon-polluted environment and the response mechanisms of microorganisms to these factors. Furthermore, we introduce the methods to improve microbial tolerance, such as irrational modification, rational modification based on systems biology tools or tolerance mechanisms, and the construction of microbial consortia. The application of these methods is expected to improve the viability and remediation efficiency of microorganisms in petroleum hydrocarbon-contaminated environment and provide new perspectives and technical support for environmental remediation.

    • Regulatory roles of DGAT and PDAT genes in plant oil synthesis

      2025, 41(1):216-229. DOI: 10.13345/j.cjb.240376

      Abstract (57) HTML (126) PDF 590.91 K (158) Comment (0) Favorites

      Abstract:There is a large gap between production and demand of plant oil in China, which leads to the heavy reliance on imports. Diacylglycerol acyltransferase (DGAT) and phospholipid: diacylglycerol acyltransferase (PDAT) are two key enzymes responsible for the synthesis of triacylglycerol, thereby affecting the yield and quality of plant oil. This paper comprehensively reviews the research progress in DGAT and PDAT in terms of their biological functions in plant oil synthesis, the molecular mechanisms of regulating plant lipid metabolism, growth, and development under stress, and their roles in driving oil synthesis under the background of synthetic biology. Furthermore, future research and application of DGAT and PDAT are prospected. This review aims to provide a basis for deeply understanding the molecular mechanism of plant oil synthesis and improving the quality and productivity of oil crops by the utilization of DGAT and PDAT genes.

    • >Industrial Biotechnology
    • Promotion of Stenotrophomonas sp. on the photosynthetic growth of microalgae exposed to high concentrations of formate

      2025, 41(1):230-241. DOI: 10.13345/j.cjb.240242

      Abstract (52) HTML (143) PDF 869.04 K (132) Comment (0) Favorites

      Abstract:Formate is an important solar fuel, with large application potential in bioconversion. Especially, the win-win collaboration is achieved when formate is applied to the cultivation of microalgae, which combines the advantages from both artificial and natural photosynthesis. However, the inhibition of formate on the photosynthetic electron transport hinders the application of formate at high concentrations. The engineering or directed evolution of the regulation pathway is a case-by-case and time-consuming strategy. Here, we developed a new strategy by introducing a Stenotrophomonas sp. strain which was isolated and identified from the long-term self-evolution process of Chlamydomonas reinhardtii for adapting to high concentrations of formate. The co-culture with the strain or the fermentation broth relieved the inhibition of formate (50 mmol/L) on C. reinhardtii and promoted the growth of the microalga. Especially, the protein content increased significantly to nearly 50% of the dried weight. In addition, the co-culture also benefited the growth of both Chlorella pyrenoidesa and Synechocystis sp. PCC 6803 exposed to formate, which indicated broader applicability of this strategy. This strategy provides the opportunity to overcome the bottleneck in the formate-mediated artificial-natural hybrid photosynthesis and to aid the development of technologies for solar energy-driven production of bulk biomass, including proteins, by carbon dioxide reduction.

    • Metabolic engineering of Escherichia coli for efficient biosynthesis of l-citrulline

      2025, 41(1):242-255. DOI: 10.13345/j.cjb.240190

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      Abstract:l-citrulline is a nonprotein amino acid that plays an important role in human health and has great market demand. Although microbial cell factories have been widely used for biosynthesis, there are still challenges such as genetic instability and low efficiency in the biosynthesis of l-citrulline. In this study, an efficient, plasmid-free, non-inducible l-citrulline-producing strain of Escherichia coli BL21(DE3) was engineered by combined strategies. Firstly, a chassis strain capable of synthesizing l-citrulline was constructed by block of l-citrulline degradation and removal of feedback inhibition, with the l-citrulline titer of 0.43 g/L. Secondly, a push-pull-restrain strategy was employed to enhance the l-citrulline biosynthesis, which realized the l-citrulline titer of 6.0 g/L. Thirdly, the NADPH synthesis and l-citrulline transport were strengthened to promote the synthesis efficiency, which achieved the l-citrulline titer of 11.6 g/L. Finally, fed-batch fermentation was performed with the engineered strain in a 3 L fermenter, in which the l-citrulline titer reached 44.9 g/L. This study lays the foundation for the industrial production of l-citrulline and provides insights for the modification of other amino acid metabolic networks.

    • Metabolic engineering of Escherichia coli for the biosynthesis of O-acetyl-l-homoserine

      2025, 41(1):256-270. DOI: 10.13345/j.cjb.240205

      Abstract (57) HTML (121) PDF 965.53 K (178) Comment (0) Favorites

      Abstract:O-acetyl-l-homoserine (OAH) is a promising platform compound for the production of l-methionine and other valuable compounds, while its low yield and low conversion rate limit the industrial application. To solve these problems, we constructed a strain for high OAH production with the previously constructed l-homoserine producer Escherichia coli HS33 as the chassis by systematic metabolic engineering. Firstly, PEP accumulation, pyruvate utilization, and OAH synthesis pathway (overexpressing aspB, aspA, and thrAC1034T) were enhanced to obtain an initial strain accumulating 13.37 g/L OAH. Subsequently, the co-factor synthesis genes were integrated to supply reducing power and energy, which increased the yield to 15.79 g/L. The OAH yield of the engineered strain OAH28 was further increased to 17.49 g/L by strengthening the acetic acid reuse pathway, improving the supply of acetyl-CoA, and regulating the expression of MetX from different sources. Finally, in a 5 L fermenter, OAH28 achieved an OAH titer of 47.12 g/L, with a glucose conversion rate of 32% and productivity of 0.59 g/(L·h). The results lay a foundation for increasing the OAH production by metabolic engineering and give insights into the industrial production of OAH.

    • Engineering of CmpLs enhances l-glutamate production ofCorynebacterium glutamicum

      2025, 41(1):271-287. DOI: 10.13345/j.cjb.240306

      Abstract (57) HTML (137) PDF 1008.28 K (138) Comment (0) Favorites

      Abstract:The efficient production of l-glutamate is dependent on the product’s rapid efflux, hence researchers have recently concentrated on artificially modifying its transport system and cell membrane wall structure. Considering the unique composition and structure of the cell wall of Corynebacterium glutamicum, we investigated the effects of CmpLs on l-glutamate synthesis and transport in SCgGC7, a constitutive l-glutamate efflux strain. First, the knockout strains of CmpLs were constructed, and it was confirmed that the deletion of CmpL1 and CmpL4 significantly improved the performance of l-glutamate producers. Next, temperature-sensitive l-glutamate fermentation with the CmpL1 and CmpL4 knockout strains were carried out in 5 L bioreactors, where the knockout strains showcased temperature-sensitive characteristics and enhanced capacities for l-glutamate production under high temperatures. Notably, the CmpL1 knockout strain outperformed the control strain in terms of l-glutamate production, showing production and yield increases of 69.2% and 55.3%, respectively. Finally, the intracellular and extracellular metabolites collected at the end of the fermentation process were analyzed. The modification of CmpLs greatly improved the l-glutamate excretion and metabolic flux for both l-glutamate production and transport. Additionally, the CmpL1 knockout strain showed decreased accumulation of downstream metabolites of l-glutamate and intermediate metabolites of tricarboxylic acid (TCA) cycle, which were consistent with its high l-glutamate biosynthesis capacity. In addition to offering an ideal target for improving the stability and performance of the industrial strains for l-glutamate production, the functional complementarity and redundancy of CmpLs provide a novel target and method for improving the transport of other metabolites by modification of the cell membrane and cell wall structures in C. glutamicum.

    • Pseudomonas monteilii ZMU-T06 produces 2-substituted quinolines by oxidative dehydroaromatization

      2025, 41(1):288-295. DOI: 10.13345/j.cjb.240359

      Abstract (50) HTML (141) PDF 525.04 K (127) Comment (0) Favorites

      Abstract:2-substituted quinolines are the building blocks for the synthesis of natural products and pharmaceuticals. In comparison with classical methods, dehydroaromatization of 2-substituted-1,2,3,4-tetrahydroquinolines has emerged in recent years as an efficient and straightforward method to synthesize quinolines due to its high atom economy and sustainability. However, existing chemical methods need transition metal catalysts and harsh reaction conditions. Biocatalysis with high efficiency, high selectivity, and mild reaction conditions has become an important method of organic synthesis. We mined a strain Pseudomonas monteilii ZMU-T06 capable of producing monoamine oxidase for the dehydroaromatization of 2-substituted-1,2,3,4-tetrahydroquinolines to synthesize 2-substituted quinolines (8 substrates, yields of 45.7%–48.4%) and then hypothesized the catalytic mechanism, providing a new method for green synthesis of 2-substituted quinolines.

    • Improvement of catalytic activity and thermostability of glucose oxidase from Aspergillus heteromorphus

      2025, 41(1):296-307. DOI: 10.13345/j.cjb.240458

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      Abstract:Glucose oxidase (GOD) is an oxygen-consuming dehydrogenase that can catalyze the production of gluconic acid hydrogen peroxide from glucose, and its specific mechanism of action makes it promising for applications, while the low catalytic activity and poor thermostability have become the main factors limiting the industrial application of this enzyme. In this study, we used the glucose oxidase AtGOD reported with the best thermostability as the source sequence for phylogenetic analysis to obtain the GOD with excellent performance. Six genes were screened and successfully synthesized for functional validation. Among them, the glucose oxidase AhGODB derived from Aspergillus heteromorphus was expressed in Pichia pastoris and showed better thermostability and catalytic activity, with an optimal temperature of 40 ℃, a specific activity of 112.2 U/mg, and a relative activity of 47% after 5 min of treatment at 70 ℃. To improve its activity and thermal stability, we constructed several mutants by directed evolution combined with rational design. Compared with the original enzyme, the mutant T72R/A153P showcased the optimum temperature increasing from 40 to 50 ℃, the specific activity increasing from 112.2 U/mg to 166.1 U/mg, and the relative activity after treatment at 70 ℃ for 30 min increasing from 0% to 33%. In conclusion, the glucose oxidase mutants obtained in this study have improved catalytic activity and thermostability, and have potential for application.

    • Directed evolution improves the catalytic activity of laccase in papermaking

      2025, 41(1):308-320. DOI: 10.13345/j.cjb.240387

      Abstract (48) HTML (150) PDF 1.11 M (129) Comment (0) Favorites

      Abstract:As a biocatalyst, laccase has been widely studied and applied in the papermaking industry. However, the low catalytic efficiency and poor stability of natural laccase limit its application in the pulping process. To develop the laccase with high activity and strong tolerance, we carried out directed evolution for modification of the laccase derived from Bacillus pumilus and screened out the mutants F282L/F306L and Q275P from the random mutant library by high-throughput screening. The specific activities of F282L/F306L and Q275P were 280.87 U/mg and 453.94 U/mg, respectively, which were 1.42 times and 2.30 times that of the wild-type laccase. Q275P demonstrated significantly improved thermal stability, with the relative activity 20% higher than that of the wild-type laccase after incubation at 40 ℃, 50 ℃, and 70 ℃ for 4 h. F282L/F306L and Q275P showed greater tolerance to metal ions and organic solvents than the wild-type laccase. The Km value of the wild-type laccase was 374.97 μmo/L, and those of F282L/F306L and Q275P were reduced to 318.96 μmo/L and 360.71 μmo/L, respectively, which suggested that the substrate affinity of laccase was improved after mutation. The kcat values of F282L/F306L and Q275P for the substrate ABTS were 574.00 s–1 and 898.03 s–1, respectively, which were 1.1 times and 1.7 times that of the wild-type laccase, indicating the improved catalytic efficiency. Q275P demonstrated better performance than the wild-type laccase in pulping, as manifested by the reduction of 0.82 in the Kappa number and the increases of 2.00% ISO, 7.8%, and 7.2% in whiteness, tensile index, and breaking length, respectively. This work lays a foundation for improving the adaptation of laccase to the environment of the papermaking industry.

    • A novel carbonyl reductase for the synthesis of (R)-tolvaptan

      2025, 41(1):321-332. DOI: 10.13345/j.cjb.240238

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      Abstract:Screening carbonyl reductases with the ability to catalyze the reduction of complex carbonyl compounds is of great significance for the biosynthesis of R-tolvaptan(R-TVP). In this study, the target carbonyl reductase in the crude enzyme extract of rabbit liver was separated, purified, and identified by ammonium sulfate precipitation, gel-filtration chromatography, ion exchange chromatography, affinity chromatography, and protein mass spectrometry. With the rabbit liver genome as the template, the gene encoding the carbonyl reductase rlsr5 was amplified by PCR and the recombinant strain was successfully constructed. After RLSR5 was purified by affinity chromatography, its enzymatic properties were characterized. The results indicated that the gene sequence of rlsr5 was 972 bp, encoding a protein with a molecular weight of 40 kDa. RLSR5 was a dimeric protein, and each monomer was composed of a (α/β)8-barrel structure. RLSR5 could asymmetrically reduce 7-chloro-1-[2-methyl-4-[(2- methylbenzoyl)amino]benzoyl]-5-oxo-2,3,4,5-tetrahydro-1H-1-benzazepine (prochiral ketone, PK) to synthesize R-TVP. The specific activity of the enzyme was 36.64 U/mg, and the optical purity of the product was 99%. This enzyme showcased the optimal performance at pH 6.0 and 30 ℃. It was independent of metal ions, with the activity enhanced by Mn2+. This study lays a foundation for the biosynthesis of tolvaptan of optical grade.

    • Mining, characterization, and expression of a fructan sucrase for efficient conversion of soybean oligosaccharides

      2025, 41(1):333-351. DOI: 10.13345/j.cjb.240362

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      Abstract:The high content of sucrose and raffinose reduces the prebiotic value of soybean oligosaccharides. Fructan sucrases can catalyze the conversion of sucrose and raffinose to high-value products such as fructooligosaccharides and melibiose. To obtain a fructan sucrase that can efficiently convert soybean oligosaccharides, we first mined the fructan sucrase gene from microorganisms in the coastal areas of Xisha Islands and Bohai Bay and then characterized the enzymatic and catalytic properties of the enzyme. Finally, recombinant extracellular expression of this gene was carried out in Bacillus subtilis. The results showed that a novel fructan sucrase, BhLS 39, was mined from Bacillus halotolerans. With sucrose and raffinose as substrates, BhLS 39 showed the optimal temperatures of 50 ℃ and 55 ℃, optimal pH 5.5 for both, and Kcat/Km ratio of 3.4 and 6.6 L/(mmol·s), respectively. When 400 g/L raffinose was used as the substrate, the melibiose conversion rate was 84.6% after 30 min treatment with 5 U BhLS 39. Furthermore, BhLS 39 catalyzed the conversion of sucrose to produce levan-type-fructooligosaccharide and levan. Then, the recombinant extracellular expression of BhLS 39 in B. subtilis was achieved. The co-expression of the intracellular chaperone DnaK and the extracellular chaperone PrsA increased the extracellular activity of the recombinant BhLS 39 by 5.2 folds to 17 U/mL compared with that of the control strain. BhLS 39 obtained in this study is conducive to improving the quality and economic benefits of soybean oligosaccharides. At the same time, the strategy used here to enhance the extracellular expression of BhLS 39 will also promote the efficient recombinant expression of other proteins in B. subtilis.

    • Expression and enzymatic characterization of a chitosanase with tolerance to a wide range of pH from Bacillus atrophaeus

      2025, 41(1):352-362. DOI: 10.13345/j.cjb.240178

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      Abstract:To screen and identify a chitosanase with high stability, we cloned the chitosanase gene from Bacillus atrophaeus with a high protease yield from the barren saline-alkali soil and expressed this gene in Escherichia coli. The expressed chitosanase of B. atrophaeus (BA-CSN) was purified by nickel-affinity column chromatography. The properties including optimal temperature, optimal pH, substrate specificity, and kinetic parameters of BA-CSN were characterized. The results showed that BA-CSN had the molecular weight of 31.13 kDa, the optimal temperature of 55 ℃, the optimal pH 5.5, and good stability at temperatures below 45 ℃ and pH 4.0–9.0. BA-CSN also had good stability within 4 h of pH 3.0 and 10.0, be activated by K+, Na+, Mn2+, Ca2+, Mg2+, and Co2+, (especially by Mn2+), and be inhibited by Fe3+, Cu2+, and Ag+. BA-CSN showcased the highest relative activity in the hydrolysis of colloidal chitosan, and it had good hydrolysis ability for colloidal chitin. Under the optimal catalytic conditions, BA-CSN demonstrated the Michaelis constant Km and maximum reaction rate Vmax of 9.94 mg/mL and 26.624 μmoL/(mL·min), respectively, for colloidal chitosan. In short, BA-CSN has strong tolerance to acids and alkali, possessing broad industrial application prospects.

    • >Synthetic Biotechnology
    • Construction and application of an inducible transcriptional regulatory tool fromMedicago truncatulain Saccharomyces cerevisiae

      2025, 41(1):363-375. DOI: 10.13345/j.cjb.240210

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      Abstract:Transcriptional regulation based on transcription factors is an effective regulatory method widely used in microbial cell factories. Currently, few naturally transcriptional regulatory elements have been discovered from Saccharomyces cerevisiae and applied. Moreover, the discovered elements cannot meet the demand for specific metabolic regulation of exogenous compounds due to the high background expression or narrow dynamic ranges. There are abundant transcriptional regulatory elements in plants. However, the sequences and functions of most elements have not been fully characterized and optimized. Particularly, the applications of these elements in microbial cell factories are still in the infancy stage. In this study, natural regulatory elements from Medicago truncatula were selected, including the transcription factors MtTASR2 and MtTASR3, along with their associated promoter ProHMGR1, for functional characterization and engineering modification. We constructed an inducible transcriptional regulation tool and applied it in the regulation of heterologous β-carotene synthesis in S.cerevisiae, which increased the β-carotene production by 7.31 folds compared with the original strain. This study demonstrates that plant-derived transcriptional regulatory elements can be used to regulate the expression of multiple genes in S.cerevisiae, providing new strategies and ideas for the specific regulation and application of these elements in microbial cell factories.

    • Enzymatic MBH reaction catalyzed by an artificial enzyme designed with the introduction of an unnatural tertiary amine cofactor

      2025, 41(1):376-384. DOI: 10.13345/j.cjb.240223

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      Abstract:As the chip of synthetic biology, enzymes play a vital role in the bio-manufacturing industry. The development of diverse functional enzymes can provide a rich toolbox for the development of synthetic biology. This article reports the construction of an artificial enzyme with the introduction of a non-natural cofactor. By introducing the 4-dimethylaminopyridine (DMAP) cofactor into the optimal protein skeleton via covalent bonds based on a click-chemistry strategy, we successfully constructed a novel artificial enzyme with the DMAP cofactor as the catalytic center. The artificial enzyme successfully catalyzed an unnatural asymmetric Morita-Baylis- Hillman (MBH) reaction between cycloketenone and p-nitrobenzaldehyde, with a conversion rate of 90% and enantioselectivity (e.e.) of 38%. This study not only provides an effective strategy for the design of new artificial enzymes but also establishes a theoretical basis for the development of unnatural biocatalytic MBH reactions.

    • An efficient assembly method for a viral genome based on T7 endonuclease I-mediated error correction

      2025, 41(1):385-396. DOI: 10.13345/j.cjb.240302

      Abstract (54) HTML (130) PDF 743.91 K (112) Comment (0) Favorites

      Abstract:Gene synthesis is an enabling technology that supports the development of synthetic biology. The existing approaches for de novo gene synthesis generally have tedious operation, low efficiency, high error rates, and limited product lengths, being difficult to support the huge demand of synthetic biology. The assembly and error correction are the keys in gene synthesis. This study first designed the oligonucleotide sequences by reasonably splitting the virus genome of approximately 10 kb by balancing the parameters of sequence design software ability, PCR amplification ability, and assembly enzyme assembly ability. Then, two-step PCR was performed with high-fidelity polymerase to complete the de novo synthesis of 3.0 kb DNA fragments, and error correction reactions were performed with T7 endonuclease I for the products from different stages of PCR. Finally, the virus genome was assembled by 3.0 kb DNA fragments from de novo synthesis and error correction and then sequenced. The experimental results showed that the proposed method successfully produced the DNA fragment of about 10 kb and reduced the probability of large fragment mutations during the assembly process, with the lowest error rate reaching 0.36 errors/kb. In summary, this study developed an efficient de novo method for synthesizing a viral genome of about 10 kb with T7 endonuclease I-mediated error correction. This method enabled the synthesis of a 10 kb viral genome in one day and the correct plasmid of the viral genome in five days. This study optimized the de novo gene synthesis process, reduced the error rate, simplified the synthesis and assembly steps, and reduced the cost of viral genome assembly.

    • >Methods in Biotechnology
    • Establishment and application of a genetic operating system in Wickerhamomyces ciferrii for the synthesis of tetraacetyl phytosphingosine

      2025, 41(1):397-415. DOI: 10.13345/j.cjb.240385

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      Abstract:Wickerhamomyces ciferrii (W.c), an unconventional heterothallic yeast species, is renowned for its high production of tetraacetyl phytosphingosine (TAPS). Due to its excellent performance in TAPS production, this study aimed to construct a genetic operating system of W.c to enhance the production of TAPS and to screen high-yielding strains by mutagenesis and genetic engineering, thus laying the foundation for further development of industrial production of sphingolipid metabolites. In this study, we selected two autonomous replication elements (CEN, 2μ) and mined 11 endogenous promoter elements to establish a genetic operating system in W. ciferrii. The overexpression of Syr2 and Lcb2 in the sphingolipid metabolism pathway significantly increased the production of TAPS. Meanwhile, we established a method for the identification of haploid mating types of W. ciferrii by combining RT-PCR and flow cytometry. Five strains of W. ciferrii with different mating types constructed from the standard diploid W. ciferrii ATCC 14091 were screened out. A-type haploid W.c 140 showcased the highest production of TAPS with a yield of 4.74 mg/gand a titer of 32.61 mg/L. Mutant strains W.c 140-A9 and W.c 140-A11 were induced by atmospheric pressure room temperature plasma mutagenesis. The recombinant strains W.c 140 OELcb2 and W.c 140 OESyr2 with overexpression were constructed with the genetic operating system established in this study. The TAPS yields of the mutant strains increased by 61.39% and 67.09%, respectively, compared with that of starting strain W.c 140. The recombinant strains cultured in the LCBNB medium achieved yields of 10.60 mg/gand 12.14 mg/g, respectively, representing 2.24 and 2.56 times of that in strain W.c 140. Moreover, the yields of the two recombinant strains were significantly higher than that of the diploid strain ATCC 14091. The genetic operating system and the haploid strain W.c 140 established in this study provide a basis for the subsequent establishment of genetic engineering tools for W. ciferrii.

    • Transaminases: high-throughput screening via a ketone-fluorescent probe and applications

      2025, 41(1):416-426. DOI: 10.13345/j.cjb.240216

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      Abstract:Transaminases are a class of enzymes that catalyze the transfer of amino between amino acids and keto acids, playing an important role in the biosynthesis of organic amines and the corresponding derivatives. However, natural enzymes often have low catalytic efficiency against non-natural substrates, which limits their widespread applications. Enzyme engineering serves as an effective approach to improve the catalytic properties and thereby expand the application scope of transaminases. In this study, a high-throughput screening method for transaminases was established based on the fluorescent color reaction between methoxy-2-aminobenzoxime (PMA) and ketones. According to the changes in fluorescence intensity, the concentration changes of ketones could be easily monitored. The efficiency, sensitivity, and accuracy of the screening method were improved by optimization of the system. With 4-hydroxy-2-butanone as the substrate, the mutant library of the transaminase fromActinobacteria sp. was established and a mutant with increased activity was successfully obtained, which improved the production efficiency of (R)-3-aminobutanol by enzyme-catalyzed synthesis. This study laid an important foundation for efficient screening, modification, and application of transaminase.

    • Mining and characterization of new enzymes based on Phi29 DNA polymerase

      2025, 41(1):427-436. DOI: 10.13345/j.cjb.240222

      Abstract (51) HTML (116) PDF 967.34 K (154) Comment (0) Favorites

      Abstract:In recent years, the bacteriophage Φ29 (Phi29) DNA polymerase has garnered increasing attention due to its high-fidelity amplification capacity at constant temperatures. To advance the industrial application of this type of isothermal polymerases, this study mined and characterized new enzymes from the microbial metagenome based on the known Phi29 DNA polymerase sequence. The results revealed that a new enzyme, Php29 DNA polymerase, was identified in the microbial metagenome with plants as the hosts. This enzyme exhibited higher strand displacement activity, with a 59.5% similarity to bacteriophage Φ29. Experimental validation demonstrated that the enzyme had 3ʹ→5ʹ exonuclease activity, and its amplification products can serve as substrates for further catalytic reactions. The discovery and validation of Php29 DNA polymerase gives insights into the future industrial application of isothermal polymerases.

    • Construction and optimization of 1,4-butanediamine biosensor based on transcriptional regulator PuuR

      2025, 41(1):437-447. DOI: 10.13345/j.cjb.240158

      Abstract (53) HTML (143) PDF 926.27 K (134) Comment (0) Favorites

      Abstract:Biosensors have become powerful tools for real-time monitoring of specific small molecules and precise control of gene expression in biological systems. High-throughput sensors for 1,4-butanediamine biosynthesis can greatly improve the screening efficiency of high-yielding 1,4-butanediamine strains. However, the strategies for adapting the characteristics of biosensors are still rarely studied, which limits the applicability of 1,4-butanediamine biosensors. In this paper, we propose the development of a 1,4-butanediamine biosensor based on the transcriptional regulator PuuR, whose homologous operator puuO is installed in the constitutive promoter PgapA of Escherichia coli to control the expression of the downstream superfolder green fluorescent protein (sfGFP) as the reporter protein. Finally, the biosensor showed a stable linear relationship between the GFP/OD600 value and the concentration of 1,4-butanediamine when the concentration of 1,4-butanediamine was 0–50 mmol/L. The promoters with different strengths in the E.coligenome were used to modify the 1,4-butanediamine biosensor, and the functional properties of the PuuR-based 1,4-butanediamine biosensor were explored and improved, which laid the groundwork for high-throughput screening of engineered strains highly producing 1,4-butanediamine.

    • >Biotechnological Breeding and Process Optimization
    • Preparation of multi-layer compound microcapsules and their application in self-healing of concrete cracks

      2025, 41(1):448-460. DOI: 10.13345/j.cjb.240406

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      Abstract:Concrete is widely used in building construction, civil engineering, roads, bridges, etc., but concrete cracking remains a major issue in the engineering industry. To develop an effective and feasible concrete repair technology, this study combined microbial and microencapsulation technologies to prepare a multi-layer compound microcapsule using the piercing method. The formulation and drying method of microcapsules were optimized by taking their embedding rate and mechanical properties as evaluation criteria. The calcium transcrystallization process of microcapsules and the crystal form of products were characterized and compared with the calcium transcrystallization process in free cells. Finally, the effects of microcapsule incorporation on mechanical properties, impermeability, and self-healing performance of concrete specimens were then tested. The results showed that the air-dried multi-layer compound microcapsules, formulated with 1.0% wet cells of Bacillus cereus, 1.5% calcium chloride, 3.0% sodium alginate, 5.0% nutrients, 6.0% glycerol, 0.6% chitosan, and 2.0% urea, achieved an embedding rate of 95.3%, a rupture force of 60.0 N and a hardness of 150.8 N. These microcapsules can transform from a solid state to a flowing colloidal state when the microorganisms inside undergo a calcium formation reaction. Both the microcapsules and free cells produced stable calcite crystal forms of calcium carbonate through the calcium conversion reaction, with the microcapsules producing more uniform-sized particles, which are more conducive to accumulation in cracks, thereby enhancing the stability of repair. When microcapsules were incorporated into the concrete specimen at a content of 0.45%, the flexural strength of the specimen increased by 17.3%, and the compressive strength increased by 12.3%. In the water impermeability test, specimens with microcapsules demonstrated better impermeability compensation for the cement concrete than those with free cells. The self-healing effect of cracks proved that multi-layer compound microcapsules could completely repair cracks up to 0.7 mm wide, and a repair rate of 95% for 0.8 mm wide cracks. In this study, a multi-layer compound microcapsule was developed to protect microorganisms in concrete and provide nutrients required for their growth, which provided a new idea for microbial induced calcium carbonate precipitation in concrete crack repair.

    • Efficient synthesis of polydatin by a two-enzyme coupled with one-pot method

      2025, 41(1):461-473. DOI: 10.13345/j.cjb.240398

      Abstract (50) HTML (133) PDF 954.81 K (121) Comment (0) Favorites

      Abstract:Traditional Chinese medicine of Polygonum cuspidatum has been utilized in China for thousands of years. Its primary active compound, polydatin, exhibits a variety of pharmacological effects including the regulation of glucose and lipid metabolism, suppression of cough and asthma, as well as antibacterial and anti-inflammatory properties. However, conventional methods for polydatin production are inadequate to satisfy the market demand. This study aims to explore the green and efficient preparation of polydatin. With resveratrol as the substrate, we efficiently synthesized polydatin by using the triple mutant IGW (Y14I/I62G/M315W) of the glycosyltransferase UGTBS based on a strategy of two-enzyme coupled with one-pot and realized the recycling of uridine diphosphate-glucose (UDPG). The conditions of the two-enzyme reaction were optimized. Under the conditions of 35 ℃, pH 8.0, IGW:AtSuSy1 activity ratio of 3:4, dimethyl sulfoxide (DMSO) volume fraction of 5%, uridine diphosphate (UDP) concentration of 0.10 mmol/L, and sucrose concentration of 0.6 mol/L, the conversion of 2 mmol/L resveratrol reached 80.6% within 1 h, and the proportion of polydatin was over 90%. This study achieved the recycling of UDPG via a two-enzyme coupling system and shortened the reaction time. At the same time, the fed-batch strategy was adopted, and the yield of polydatin reached 6.28 g/L after 24 h in the one-pot coupling reaction, which provided a new strategy for green and efficient preparation of polydatin.

    • A flavin-containing monooxygenase from Schizosaccharomyces pombe: characterization and application in the synthesis of S-methyl-l-cysteine sulfoxide

      2025, 41(1):474-485. DOI: 10.13345/j.cjb.240324

      Abstract (49) HTML (129) PDF 923.44 K (131) Comment (0) Favorites

      Abstract:S-methyl-l-cysteine sulfoxide (SMCO) is a non-protein sulfur-containing amino acid with a variety of functions. There are few reports on the enzymes catalyzing the biosynthesis of SMCO from S-methyl-l-cysteine (SMC). In this study, the flavin-containing monooxygenase gene derived from Schizosaccharomyces pombe(spfmo) was heterologously expressed in Escherichia coli BL21(DE3) and the enzymatic properties of the expressed protein were analyzed. The optimum catalytic conditions of the recombinant SpFMO were 30 ℃ and pH 8.0, under which the enzyme activity reached 72.77 U/g. An appropriate amount of Mg2+ improved the enzyme activity. The enzyme kinetic analysis showed that the Km and kcat/Km of SpFMO on the substrate SMC were 23.89 μmol/L and 61.71 L/(min·mmol), respectively. Under the optimal reaction conditions, the yield of SMCO synthesized from SMC catalyzed by SpFMO was 12.31% within 9 h. This study provides reference for the enzymatic synthesis of SMCO.


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