• Volume 39,Issue 11,2023 Table of Contents
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    • >Briefing and introduction
    • Briefing and introduction

      2023, 39(11):4331-4334. DOI: 10.13345/j.cjb.230651 CSTR: 32114.14.j.cjb.230651

      Abstract (191) HTML (310) PDF 378.94 K (714) Comment (0) Favorites

      Abstract:

    • >Invited Review
    • Biomanufacturing of bioactive compounds: current situation, challenges, and future perspectives

      2023, 39(11):4335-4357. DOI: 10.13345/j.cjb.230334 CSTR: 32114.14.j.cjb.230334

      Abstract (532) HTML (3772) PDF 786.70 K (2408) Comment (0) Favorites

      Abstract:Biomanufacturing uses biological systems, including cells, microorganisms, and enzymes, to produce natural or synthetic molecules with biological activities for use in various industries, such as pharmaceuticals, cosmetics, and agriculture. These bioactive compounds are expected to play important roles in improving the quality of life and prolonging its length. Fortunately, recent advances in synthetic biology and automation technologies have accelerated the development of biomanufacturing, enabling us to create new products and replace conventional methods in a more sustainable manner. As of now, the role of biomanufacturing in the growth and innovation of bioeconomy is steadily increasing, and this techbology becomes a prevalent technology in global markets. To gain a comprehensive understanding of this field, this article presents a retrospective review of Bloomage Biotechnology’s Research and Development and briefly reviews the developments of biomanufacturing and offers insights into the futre prospects. In conclusion, biomanufacturing will continue to be an important, environmentally friendly, and sustainable production mode in the ongoing development of bioeconomy.

    • >Review
    • Advances in the application of yeast surface display technology

      2023, 39(11):4358-4375. DOI: 10.13345/j.cjb.230085 CSTR: 32114.14.j.cjb.230085

      Abstract (785) HTML (1220) PDF 776.95 K (1988) Comment (0) Favorites

      Abstract:Yeast surface display (YSD) is a technology that fuses the exogenous target protein gene sequence with a specific vector gene sequence, followed by introduction into yeast cells. Subsequently, the target protein is expressed and localized on the yeast cell surface by using the intracellular protein transport mechanism of yeast cells, whereas the most widely used YSD system is the α-agglutinin expression system. Yeast cells possess the eukaryotic post-translational modification mechanism, which helps the target protein fold correctly. This mechanism could be used to display various eukaryotic proteins, including antibodies, receptors, enzymes, and antigenic peptides. YSD has become a powerful protein engineering tool in biotechnology and biomedicine, and has been used to improve a broad range of protein properties including affinity, specificity, enzymatic function, and stability. This review summarized recent advances in the application of YSD technology from the aspects of library construction and screening, antibody engineering, protein engineering, enzyme engineering and vaccine development.

    • Engineering and application of Komagataella phaffii as a cell factory

      2023, 39(11):4376-4396. DOI: 10.13345/j.cjb.230095 CSTR: 32114.14.j.cjb.230095

      Abstract (386) HTML (951) PDF 910.17 K (1209) Comment (0) Favorites

      Abstract:Nowadays, engineered Komagataella phaffii plays an important role in the biosynthesis of small molecule metabolites and protein products, showing great potential and value in industrial productions. With the development and application of new editing tools such as CRISPR/Cas9, it has become possible to engineer K. phaffii into a cell factory with high polygenic efficiency. Here, the genetic manipulation techniques and objectives for engineering K. phaffii are first summarized. Secondly, the applications of engineered K. phaffii as a cell factory are introduced. Meanwhile, the advantages as well as disadvantages of using engineered K. phaffii as a cell factory are discussed and future engineering directions are prospected. This review aims to provide a reference for further engineering K. phaffii cell factory, which is supposed to facilitate its application in bioindustry.

    • Advances in the development of covalent small molecule inhibitors of monoacylglycerol lipase

      2023, 39(11):4397-4412. DOI: 10.13345/j.cjb.230143 CSTR: 32114.14.j.cjb.230143

      Abstract (277) HTML (709) PDF 725.25 K (3055) Comment (0) Favorites

      Abstract:Monoacylglycerol lipase (MGL) is a serine hydrolase that plays a major role in the degradation of endogenous cannabinoid 2-arachidonoylglycerol. The role of MGL in some cancer cells has been confirmed, where inhibition of the MGL activity shows inhibition on cell proliferation. This makes MGL a promising drug target for the treatment of cancer. Recently, the development of covalent inhibitors of MGL has developed rapidly. These drugs have strong covalent binding ability, high affinity, long duration, low dose and low risk of drug resistance, so they have received increasing attention. This article introduces the structure and function of MGL, the characteristics, mechanisms and progress of covalent MGL inhibitors, providing reference for the development of novel covalent small molecule inhibitors of MGL.

    • Recent advances in poly phosphate kinase (PPK) and the construction of PPK-mediated ATP regeneration system

      2023, 39(11):4413-4427. DOI: 10.13345/j.cjb.230290 CSTR: 32114.14.j.cjb.230290

      Abstract (435) HTML (640) PDF 1.19 M (1759) Comment (0) Favorites

      Abstract:Adenosine triphosphate (ATP) regeneration systems are essential for efficient biocatalytic phosphoryl transfer reactions. Polyphosphate kinase (PPK) is a versatile enzyme that can transfer phosphate groups among adenosine monophosphate (AMP), adenosine diphosphate (ADP), ATP, and polyphosphate (Poly P). Utilization of PPK is an attractive solution to address the problem of ATP regeneration due to its ability to use a variety of inexpensive and stable Poly P salts as phosphate group donors. This review comprehensively summarizes the structural characteristics and catalytic mechanisms of different types of PPKs, as well as the variations in enzyme activity, catalytic efficiency, stability, and coenzyme preference observed in PPKs from different sources. Moreover, recent advances in PPK-mediated ATP regeneration systems and protein engineering of wild-type PPK are summarized.

    • Synthesis and application of the methyl analogues of S-adenosyl-L-methionine

      2023, 39(11):4428-4444. DOI: 10.13345/j.cjb.230271 CSTR: 32114.14.j.cjb.230271

      Abstract (263) HTML (967) PDF 1.12 M (1055) Comment (0) Favorites

      Abstract:Methylation plays a vital role in biological systems. SAM (S-adenosyl-L-methionine), an abundant cofactor in life, acts as a methyl donor in most biological methylation reactions. SAM-dependent methyltransferases (MTase) transfer a methyl group from SAM to substrates, thereby altering their physicochemical properties or biological activities. In recent years, many SAM analogues with alternative methyl substituents have been synthesized and applied to methyltransferases that specifically transfer different groups to the substrates. These include functional groups for labeling experiments and novel alkyl modifications. This review summarizes the recent progress in the synthesis and application of SAM methyl analogues and prospects for future research directions in this field.

    • Biodegradation of polyethylene terephthalate: a review

      2023, 39(11):4445-4462. DOI: 10.13345/j.cjb.230215 CSTR: 32114.14.j.cjb.230215

      Abstract (913) HTML (894) PDF 823.66 K (977) Comment (0) Favorites

      Abstract:Plastics are widely used in human daily life, which bring great convenience. Nevertheless, the disposal of a large amount of plastic wastes also brings great pressure to the environment. Polyethylene terephthalate (PET) is a polymer thermoplastic material produced from petroleum. It has become one of the most commonly used plastics in the world due to its durability, high transparency, light weight and other characteristics. PET can exist in nature for a long time due to its complex structure and the difficulty in degradation, which causes serious pollution to the global ecological environment, and threatens human health. The degradation of PET wastes has since become one of the global challenges. Compared with physical and chemical methods, biodegradation is the greenest way for treating PET wastes. This review summarizes the recent advances on PET biodegradation including microbial and enzymatic degradation of PET, biodegradation pathway, biodegradation mechanisms, and molecular modification of PET-degrading enzymes. In addition, the prospect for achieveing efficient degradation of PET, searching and improving microorganisms or enzymes that can degrade PET of high crystallinity are presented, with the aimto facilitate the development, application and molecular modification of PET biodegradation microorganisms or enzymes.

    • Construction of gut microbial culture banks: advances, methods and perspectives

      2023, 39(11):4463-4481. DOI: 10.13345/j.cjb.230026 CSTR: 32114.14.j.cjb.230026

      Abstract (406) HTML (573) PDF 679.78 K (1538) Comment (0) Favorites

      Abstract:Recently, the gut microbiota-based live biotherapeutics (LBPs) development, the interaction between gut microbial species and the host, and the mining of new antimicrobial peptides, enzymes and metabolic pathway have received increasing attention. Culturing gut microbial species is therefore of great importance. This review systemically compared the construction advances of gut microbial culture banks and also analyzed the differences of methods used by research groups to give insight into the construction and enrichment of gut microbial resources. Presently, the gut microbial culture banks have included more than 1 000 bacterial species, belonging to 12 phyla, 22 classes, 39 orders, 96 families, and 358 genera. Among these, Firmicutes, Proteobacteria, Bacteroidota, and Actinomycetota exhibited the greatest diversities at the species level. The sequencing data showed that there are more than 2 000 species inhibited in the human gut. Therefore, the cultured gut microbial species are far from saturation. In terms of the construction method, the stool samples were pre-treated with ethanol or directly spread and cultured in the non-selective nutritional rich medium (represented by Gifu anaerobic medium) to obtain single colony. Then single colony was further purified. Generally, a simplified isolation and culture method is sufficient to obtain the most common and important intestinal bacterial species, such as Bifidobacteria-Lactobacillus, Akkermansia muciniphila, Faecalibacterium prausnitzii, Prevotella and S24-7 family strains. Finally, microbial resources with great diversities at the strain level are required for further functional research and product development. Samples covering hosts with distinct physiological status, diets or regions are necessary.

    • Structure and function of human-derived lysozyme: a review

      2023, 39(11):4482-4496. DOI: 10.13345/j.cjb.230241 CSTR: 32114.14.j.cjb.230241

      Abstract (246) HTML (766) PDF 816.49 K (1795) Comment (0) Favorites

      Abstract:Human-derived lysozyme is a general term for a group of naturally occurring alkaline proteins in the human body that are capable of lysing bacterial cell walls. Its action is characterized by its ability to cleave the β-(1,4)-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in peptidoglycan. Human-derived lysozyme has a variety of properties such as antibacterial, anti-inflammatory, antiviral and immune enhancing, and is therefore widely used in the domestic and international pharmaceutical markets. This review summarizes the structural features, expression sites, biological functions of human-derived lysozymes and its market applications.

    • Advances in the biosynthesis of cyclodipeptide type natural products derived from actinomycetes

      2023, 39(11):4497-4516. DOI: 10.13345/j.cjb.230068 CSTR: 32114.14.j.cjb.230068

      Abstract (216) HTML (594) PDF 1.60 M (922) Comment (0) Favorites

      Abstract:Cyclodipeptide (CDP) composed of two amino acids is the simplest cyclic peptide. These two amino acids form a typical diketopiperazine (DKP) ring by linking each other with peptide bonds. This characteristic stable ring skeleton is the foundation of CDP to display extensive and excellent bioactivities, which is beneficial for CDPs’ pharmaceutical research and development. The natural CDP products are well isolated from actinomycetes. These bacteria can synthesize DKP backbones with nonribosomal peptide synthetase (NRPS) or cyclodipeptide synthase (CDPS). Moreover, actinomycetes could produce a variety of CDPs through different enzymatic modification. The presence of these abundant and diversified catalysis indicates that actinomycetes are promising microbial resource for exploring CDPs. This review summarized the pathways for DKP backbones biosynthesis and their post-modification mechanism in actinomycetes. The aim of this review was to accelerate the genome mining of CDPs and their isolation, purification and structure identification, and to facilitate revealing the biosynthesis mechanism of novel CDPs as well as their synthetic biology design.

    • Recent advances in the structure and function of microbial community in anaerobic granular sludge

      2023, 39(11):4517-4533. DOI: 10.13345/j.cjb.230219 CSTR: 32114.14.j.cjb.230219

      Abstract (297) HTML (684) PDF 917.68 K (1111) Comment (0) Favorites

      Abstract:Anaerobic granular sludge (AnGS), a self-immobilized aggregate containing various functional microorganisms, is considered as a promising green process for wastewater treatment. AnGS has the advantages of high volume loading rate, simple process and low excess sludge generation, thus shows great technological and economical potentials. This review systematically summarizes the recent advances of the microbial community structure and function of anaerobic granular sludge, and discusses the factors affecting the formation and stability of anaerobic granular sludge from the perspective of microbiology. Moreover, future research directions of AnGS are prospected. This review is expected to facilitate the research and engineering application of AnGS.

    • Visualization method of type IV pili and its application in the study of pili function

      2023, 39(11):4534-4549. DOI: 10.13345/j.cjb.230082 CSTR: 32114.14.j.cjb.230082

      Abstract (328) HTML (734) PDF 874.47 K (1096) Comment (0) Favorites

      Abstract:As an important protein structure on the surface of bacteria, type IV pili (TFP) is the sensing and moving organ of bacteria. It plays a variety of roles in bacterial physiology, cell adhesion, host cell invasion, DNA uptake, protein secretion, biofilm formation, cell movement and electron transmission. With the rapid development of research methods, technical equipment and pili visualization tools, increasing number of studies have revealed various functions of pili in cellular activities, which greatly facilitated the microbial single cell research. This review focuses on the pili visualization method and its application in the functional research of TFP, providing ideas for the research and application of TFP in biology, medicine and ecology.

    • >Industrial Biotechnology
    • Identification of a new C-23 metabolite in sterol degradation of Mycobacterium neoaurum HGMS2 and analysis of its metabolic pathways

      2023, 39(11):4550-4562. DOI: 10.13345/j.cjb.230177 CSTR: 32114.14.j.cjb.230177

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      Abstract:Mycobacterium neoaurum has the ability to produce steroidal intermediates known as 22-hydroxy-23,24-bisnorchol-4-en-3-one (BA) upon the knockout of the genes for either the hydroxyacyl-CoA dehydrogenase (Hsd4A) or acyl-CoA thiolase (FadA5). In a previous study, we discovered a novel metabolite in the fermentation products when the fadA5 gene was deleted. This research aims to elucidate the metabolic pathway of this metabolite through structural identification, homologous sequence analysis of the fadA5 gene, phylogenetic tree analysis of M. neoaurum HGMS2, and gene knockout. Our findings revealed that the metabolite is a C23 metabolic intermediate, named 24-norchol-4-ene-3,22-dione (designated as 3-OPD). It is formed when a thioesterase (TE) catalyzes the formation of a β-ketonic acid by removing CoA from the side chain of 3,22-dioxo-25,26-bisnorchol-4-ene-24-oyl CoA (22-O-BNC-CoA), followed by spontaneously undergoing decarboxylation. These results have the potential to contribute to the development of novel steroid intermediates.

    • Characterization the response of Chlamydomonas reinhardtii serine/threonine protein kinase mutant to blue light

      2023, 39(11):4563-4579. DOI: 10.13345/j.cjb.230185 CSTR: 32114.14.j.cjb.230185

      Abstract (150) HTML (519) PDF 1.32 M (818) Comment (0) Favorites

      Abstract:In order to investigate the molecular mechanism of silk/threonine protein kinase (STK)-mediated blue light response in the algal Chlamydomonas reinhardtii, phenotype identification and transcriptome analysis were conducted for C. reinhardtii STK mutant strain crstk11 (with an AphvIII box reverse insertion in stk11 gene coding region) under blue light stress. Phenotypic examination showed that under normal light (white light), there was a slight difference in growth and pigment contents between the wild-type strain CC5325 and the mutant strain crstk11. Blue light inhibited the growth and chlorophyll synthesis in crstk11 cells, but significantly promoted the accumulation of carotenoids in crstk11. Transcriptome analysis showed that 860 differential expression genes (DEG) (559 up-regulated and 301 down-regulated) were detected in mutant (STK4) vs. wild type (WT4) upon treatment under high intensity blue light for 4 days. After being treated under high intensity blue light for 8 days, a total of 1 088 DEGs (468 upregulated and 620 downregulated) were obtained in STK8 vs. WT8. KEGG enrichment analysis revealed that compared to CC5325, the crstk11 blue light responsive genes were mainly involved in catalytic activity of intracellular photosynthesis, carbon metabolism, and pigment synthesis. Among them, upregulated genes included psaA, psaB, and psaC, psbA, psbB, psbC, psbD, psbH, and L, petA, petB, and petD, as well as genes encoding ATP synthase α, β and c subunits. Downregulated genes included petF and petJ. The present study uncovered that the protein kinase CrSTK11 of C. reinhardtii may participate in the blue light response of algal cells by mediating photosynthesis as well as pigment and carbon metabolism, providing new knowledge for in-depth analysis of the mechanism of light stress resistance in the algae.

    • Promoting fucoxanthin accumulation in Phaeodactylum tricornutum by multiple nitrogen supplementation and blue light enhancement

      2023, 39(11):4580-4592. DOI: 10.13345/j.cjb.230192 CSTR: 32114.14.j.cjb.230192

      Abstract (191) HTML (550) PDF 1.01 M (780) Comment (0) Favorites

      Abstract:The aim of this study was to promote fucoxanthin accumulation in Phaeodactylum tricornutum by photo-fermentation through optimizing the mode of multiple nitrogen supplementation and blue light enhancement. The results showed that the mixed nitrogen source (tryptone:urea=1:1, N mol/N mol; total nitrogen concentration at 0.02 mol/L) added to the culture system by six times was the best mode in shake flasks. Two-phase culture with light adjustment was then carried out in 5 L photo-fermenter with an enhanced blue light (R:G:B=67.1:16.7:16.3) in the second phase, leading to improved cell density (1.12×108 cells/mL), biomass productivity (330 mg/(d·L)), fucoxanthin content (19.62 mg/g), titer (69.71 mg/L) and productivity (6.97 mg/(d·L)). Compared with one-phase culture under red/blue (R:G:B=70.9:18.3:10.9) light and six-times nitrogen supplementation, the fucoxanthin content was significantly increased by 7.68% (P<0.05) but the productivity did not change significantly (P>0.05). Compared with one-phase culture under red/blue (R:G:B=70.9:18.3:10.9) light and one-time nitrogen supplementation, the content and productivity of fucoxanthin were significantly increased by 45.98% and 48.30% (P<0.05), respectively. This study developed a two-phase culture mode with multiple nitrogen supplementation and blue light enhancement, which effectively promoted the accumulation of fucoxanthin and improved the efficiency of nitrogen source utilization, thus providing a new approach for fucoxanthin accumulation in P. tricornutum by photo-fermentation.

    • Expression of β-xylosidase An-xyl from Aspergillus niger and characterization of its xylose tolerance

      2023, 39(11):4593-4607. DOI: 10.13345/j.cjb.230206 CSTR: 32114.14.j.cjb.230206

      Abstract (208) HTML (474) PDF 1.11 M (684) Comment (0) Favorites

      Abstract:The hydrolysis of xylo-oligosaccharides catalyzed by β-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited by its catalytic product xylose, which severely limits its application. Based on molecular docking, this paper studied the xylose affinity of Aspergillus niger β-xylosidase An-xyl, which was significantly differentially expressed in the fermentation medium of tea stalks, through cloning, expression and characterization. The synergistic degradation effect of this enzyme and cellulase on lignocellulose in tea stems was investigated. Molecular docking showed that the affinity of An-xyl to xylose was lower than that of Aspergillus oryzae β-xylosidase with poor xylose tolerance. The Ki value of xylose inhibition constant of recombinant-expressed An-xyl was 433.2 mmol/L, higher than that of most β-xylosidases of the GH3 family. The Km and Vmax towards pNPX were 3.6 mmol/L and 10 000 μmol/(min·mL), respectively. The optimum temperature of An-xyl was 65 °C, the optimum pH was 4.0, 61% of the An-xyl activity could be retained upon treatment at 65°C for 300 min, and 80% of the An-xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by An-xyl and cellulase produced 19.3% and 38.6% higher reducing sugar content at 2 h and 4 h, respectively, than that of using cellulase alone. This study showed that the An-xyl mined from differential expression exhibited high xylose tolerance and higher catalytic activity and stability, and could hydrolyze tea stem lignocellulose synergistically, which enriched the resource of β-xylosidase with high xylose tolerance, thus may facilitate the advanced experimental research and its application.

    • Adaptation of the electron transport chain improves the biocatalytic efficiency of progesterone 17α hydroxylation

      2023, 39(11):4608-4620. DOI: 10.13345/j.cjb.230283 CSTR: 32114.14.j.cjb.230283

      Abstract (161) HTML (550) PDF 805.42 K (863) Comment (0) Favorites

      Abstract:17α hydroxylase is a key enzyme for the conversion of progesterone to prepare various progestational drug intermediates. To improve the specific hydroxylation capability of this enzyme in steroid biocatalysis, the CYP260A1 derived from cellulose-mucilaginous bacteria Sorangium cellulosum Soce56 and the Fpr and bovine adrenal-derived Adx4-108 derived from Escherichia coli str. K-12 were used to construct a new electron transfer system for the conversion of progesterone. Selective mutation of CYP260A1 resulted in a mutant S276I with significantly enhanced 17α hydroxylase activity, and the yield of 17α-OH progesterone reached 58% after optimization of the catalytic system in vitro. In addition, the effect of phosphorylation of the ferredoxin Adx4-108 on 17α hydroxyl activity was evaluated using a targeted mutation technique, and the results showed that the mutation Adx4-108T69E transferred electrons to S276I more efficiently, which further enhanced the catalytic specificity in the C17 position of progesterone, and the yield of 17α-OH progesterone was eventually increased to 74%. This study provides a new option for the production of 17α-OH progesterone by specific transformation of bacterial-derived 17α hydroxylase, and lays a theoretical foundation for the industrial production of progesterone analogs using biotransformation method.

    • Multicellular coupling fermentation for 3ʹ-sialyllactose conversion using N-acetyl-glucosamine and lactose

      2023, 39(11):4621-4634. DOI: 10.13345/j.cjb.230152 CSTR: 32114.14.j.cjb.230152

      Abstract (257) HTML (528) PDF 1.09 M (944) Comment (0) Favorites

      Abstract:Sialyllactose is one of the most abundant sialylated oligosaccharides in human milk oligosaccharides (HMOs), which plays an important role in the healthy development of infants and young children. However, its efficient and cheap production technology is still lacking presently. This study developed a two-step process employing multiple-strains for the production of sialyllactose. In the first step, two engineered strains, E. coli JM109(DE3)/ pET28a-BT0453 and JM109(DE3)/pET28a-nanA, were constructed to synthesize the intermediate N-acetylneuraminic acid. When the ratio of the biomass of the two engineered strains was 1:1 and the reaction time was 32 hours, the maximum yield of N-acetylneuraminic acid was 20.4 g/L. In the second step, E. coli JM109(DE3)/ pET28a-neuA, JM109(DE3)/ pET28a-nst and Baker’s yeast were added to the above fermentation broth to synthesize 3ʹ-sialyllactose (3ʹ-SL). Using optimal conditions including 200 mmol/L N-acetyl-glucosamine and lactose, 150 g/L Baker’s yeast, 20 mmol/L Mg2+, the maximum yield of 3ʹ-SL in the fermentation broth reached 55.04 g/L after 24 hours of fermentation and the conversion rate of the substrate N-acetyl-glucosamine was 43.47%. This research provides an alternative technical route for economical production of 3ʹ-SL.

    • >Synthetic Biotechnology
    • Theoretical analysis and practical applications of the catalytic mechanism of flavonoid 6-hydroxylase

      2023, 39(11):4635-4646. DOI: 10.13345/j.cjb.230224 CSTR: 32114.14.j.cjb.230224

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      Abstract:Insufficient catalytic efficiency of flavonoid 6-hydroxylases in the fermentative production of scutellarin leads to the formation of at least about 18% of by-products. Here, the catalytic mechanisms of two flavonoid 6-hydroxylases, CYP82D4 and CYP706X, were investigated by molecular dynamics simulations and quantum chemical calculations. Our results show that CYP82D4 and CYP706X have almost identical energy barriers at the rate-determining step and thus similar reaction rates, while the relatively low substrate binding energy of CYP82D4 may facilitate product release, which is directly responsible for its higher catalytic efficiency. Based on the study of substrate entry and release processes, the catalytic efficiency of the L540A mutation of CYP82D4 increased by 1.37-fold, demonstrating the feasibility of theoretical calculations-guided engineering of flavonoid 6-hydroxylase. Overall, this study reveals the catalytic mechanism of flavonoid 6-hydroxylases, which may facilitate the modification and optimization of flavonoid 6-hydroxylases for efficient fermentative production of scutellarin.

    • Production of limonene and its derivative in Saccharomyces cerevisiae via metabolic engineering

      2023, 39(11):4647-4662. DOI: 10.13345/j.cjb.230181 CSTR: 32114.14.j.cjb.230181

      Abstract (343) HTML (405) PDF 942.77 K (882) Comment (0) Favorites

      Abstract:Limonene and its derivative perillic acid are widely used in food, cosmetics, health products, medicine and other industries as important bioactive natural products. However, inefficient plant extraction and high energy-consuming chemical synthesis hamper the industrial production of limonene and perillic acid. In this study, limonene synthase from Mentha spicatawas expressed in Saccharomyces cerevisiae by peroxisome compartmentalization, and the yield of limonene was 0.038 mg/L. The genes involved in limonene synthesis, ERG10, ERG13, tHMGR, ERG12, ERG8, IDI1, MVD1, ERG20ww and tLS, were step-wise expressed via modular engineering to study their effects on limonene yield. The yield of limonene increased to 1.14 mg/L by increasing the precursor module. Using the plasmid with high copy number to express the above key genes, the yield of limonene significantly increased up to 86.74 mg/L, which was 4 337 times higher than that of the original strain. Using the limonene-producing strain as the starting strain, the production of perillic acid was successfully achieved by expressing cytochrome P450 enzyme gene from Salvia miltiorrhiza, and the yield reached 4.42 mg/L. The results may facilitate the construction of cell factory with high yield of monoterpene products by S. cerevisiae.

    • >Environmental Biotechnology
    • An examination of the carbon metabolic pathways in Acinetobacter sp. TAC-1 in the context of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) utilization

      2023, 39(11):4663-4681. DOI: 10.13345/j.cjb.230120 CSTR: 32114.14.j.cjb.230120

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      Abstract:The present study aimed to unravel the carbon metabolism pathway of Acinetobacter sp. TAC-1, a heterotrophic nitrification-aerobic denitrification (HN-AD) strain that utilizes poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as a carbon source. Sodium acetate was employed as a control to assess the gene expression of carbon metabolic pathways in the TAC-1 strain. The results of genome sequencing demonstrated that the TAC-1 strain possessed various genes encoding carbon metabolic enzymes, such as gltA, icd, sucAB, acs, and pckA. KEGG pathway database analysis further verified the presence of carbon metabolism pathways, including the glycolytic pathway (EMP), pentose phosphate pathway (PPP), glyoxylate cycle (GAC), and tricarboxylic acid (TCA) cycle in the TAC-1 strain. The differential expression of metabolites derived from distinct carbon sources provided further evidence that the carbon metabolism pathway of TAC-1 utilizing PHBV follows the sequential process of PHBV (via the PPP pathway)→gluconate (via the EMP pathway)→acetyl-CoA (entering the TCA cycle)→CO2+H2O (generating electron donors and releasing energy). This study is expected to furnish a theoretical foundation for the advancement and implementation of novel denitrification processes based on HN-AD and solid carbon sources.

    • >Food Biotechnology
    • Characterization of a D-mannitol oxidase from Paenibacillus sp. and its application in the preparation of D-mannose

      2023, 39(11):4682-4693. DOI: 10.13345/j.cjb.230078 CSTR: 32114.14.j.cjb.230078

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      Abstract:D-mannose has many functional activities and is widely used in food, medicine, agriculture and other industries. D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose. A D-mannitol oxidase (PsOX) was found from Paenibacillus sp. HGF5. The similarity between PsOX and the D-mannitol oxidase (AldO) from Streptomyces coelicolor was 50.94%. The molecular weight of PsOX was about 47.4 kDa. A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3). The Km and kcat/Km values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s∙mmol). Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35℃, respectively, while its enzyme activity could be stably remained below 60 ℃. The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%. The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively. The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced. PsOX showed a higher catalytic efficiency compared to that of AldO. PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.

    • Expression and characterization of mesophilic GH1 β-glucosidase CdBglA from acidophilic Cuniculiplasma divulgatum

      2023, 39(11):4694-4707. DOI: 10.13345/j.cjb.230302 CSTR: 32114.14.j.cjb.230302

      Abstract (226) HTML (653) PDF 1.60 M (659) Comment (0) Favorites

      Abstract:β-glucosidase has important applications in food, pharmaceutics, biomass conversion and other fields, exploring β-glucosidase with strong adaptability and excellent properties thus has received extensive interest. In this study, a novel glucosidase from the GH1 family derived from Cuniculiplasma divulgatum was cloned, expressed, and characterized, aiming to find a better β-glucosidase. The amino acid sequences of GH1 family glucosidase derived from C. divulgatum were obtained from the NCBI database, and a recombinant plasmid pET-30a(+)-CdBglA was constructed. The recombinant protein was induced to express in Escherichia coli BL21(DE3). The enzymatic properties of the purified CdBglA were studied. The molecular weight of the recombinant CdBglA was 56.0 kDa. The optimum pH and temperature were 5.5 and 55 ℃, respectively. The enzyme showed good pH stability, 92.33% of the initial activity could be retained when treated under pH 5.5-11.0 for 1 h. When pNPG was used as a substrate, the kinetic parameters Km, Vmax and Kcat/Km were 0.81 mmol, 291.99μmol/(mg·min), and 387.50 s-1 mmol-1, respectively. 90.33% of the initial enzyme activity could be retained when CdBglA was placed with various heavy metal ions at a final concentration of 5 mmol/L. The enzyme activity was increased by 28.67% under 15% ethanol solution, remained unchanged under 20% ethanol, and 43.68% of the enzyme activity could still be retained under 30% ethanol. The enzyme has an obvious activation effect at 0-1.5 mol/L NaCl and can tolerate 0.8 mol/L glucose. In conclusion, CdBglA is an acidic and mesophilic enzyme with broad pH stability and strong tolerance to most metal ions, organic solvents, NaCl and glucose. These characteristics may facilitate future theoretical research and industrial production.

    • >Methods in Biotechnology
    • Plasmid DNA extraction by double magnetic beads method and its application

      2023, 39(11):4708-4717. DOI: 10.13345/j.cjb.230277 CSTR: 32114.14.j.cjb.230277

      Abstract (345) HTML (454) PDF 810.83 K (2305) Comment (0) Favorites

      Abstract:Plasmids are the most commonly used gene carriers in the field of gene synthesis and sequencing. However, the main problems faced by traditional plasmid DNA extraction technology are low extraction throughput and high production cost, so they cannot meet the growing demand. In this study, a double-magnetic-bead method (DMBM) for plasmid extraction was developed based on the principle of plasmid extraction. The effects of the input of magnetic beads, the size of plasmid DNA fragments, and the volume of bacterial on plasmid DNA extraction were explored. In addition, the quality, throughput, and cost of plasmid DNA extraction were also compared between this technique and the commercial plasmid DNA extraction kits. The results showed that the DMBM can meet the needs of extracting plasmid DNA with different cell densities and fragment lengths. Moreover, the sensitivity and quality of plasmid extraction by the DMBM method were both superior to those of the centrifugal adsorption column method. In addition, this technique could be applied on a 96-channel automated nucleic acid extractor, resulting in higher purity of the extracted plasmid DNA, 80% reduction in extraction time, and 57.1% reduction in cost. It also reduces manual operations, achieving high-throughput and low-cost plasmid DNA extraction, thus may facilitate gene synthesis and sequencing.

    • >Education
    • Interdisciplinary teaching-assisted education reform in “Principal Biology”

      2023, 39(11):4718-4729. DOI: 10.13345/j.cjb.230205 CSTR: 32114.14.j.cjb.230205

      Abstract (167) HTML (426) PDF 864.80 K (750) Comment (0) Favorites

      Abstract:General education in biological courses such as “Principal Biology” is an essential avenue for gaining an understanding of life science and developing an interest in the field. The reform of biological education teaching mode based on interdisciplinary approaches aims to foster cross-disciplinary talents, which is crucial for the rapid development of China’s bioeconomy. Teaching method that simply superimposes different subjects is difficult to discover the value of interdisciplinary education. To address this, a novel teaching system and an innovative teaching mode were proposed for “Principal Biology” course by integrating science and engineering subjects, based on the cross-disciplinary feature in Beijing Institute of Technology. The system involves the design of cross-disciplinary course content and the integration of multiple disciplines and knowledge points based on students’ majors, taking into account the characteristics of students’ physical and mental development. To improve students’ scientific literacy and interdisciplinary thinking ability, differentiated and major-driven teaching modes were applied by incorporating the “1+N” mixed and immersive cross-thinking training. The effectiveness of tailored cross-disciplinary teaching was evaluated using “in-teaching” and “post-teaching” data feedback models, which promote the optimization of teaching process and enhance the quality of education in cross-disciplinary biological science.

    • Fostering talents in biotechnology major through the university-enterprise collaborative training mode

      2023, 39(11):4730-4741. DOI: 10.13345/j.cjb.230112 CSTR: 32114.14.j.cjb.230112

      Abstract (185) HTML (681) PDF 617.96 K (618) Comment (0) Favorites

      Abstract:With the high-quality development of biotechnology-related industries in China, the demand for talents and training quality in this field have received extensive attention. Several universities in Nanjing have conducted in-depth analysis on the shortcomings of talent training that does not closely match the needs of industries and enterprises. These universities have since effectively leveraged its professional characteristics, deepened university-enterprise cooperation, and encouraged the alignment of professional development with industrial growth. Biotechnology major has always focused on nurturing individuals with “right conduct, good learning, and strong ability”, and capitalized on its comparative disciplinary advantages. These universities vigorously promoted and continuously optimized the model of university-enterprise collaborative training, highlighted the integration of science, industry and education, focused on innovative education teaching methods, as well as practical engineering practice to enhance its quality. The preliminary training results show that this model has promoted students’ engineering practical abilities and comprehensive qualities, garnering recognition from employers and students alike.

    • Exploration and practice of innovative teaching of molecular biology experiment course under the context of new agricultural science

      2023, 39(11):4742-4749. DOI: 10.13345/j.cjb.230242 CSTR: 32114.14.j.cjb.230242

      Abstract (173) HTML (393) PDF 534.50 K (694) Comment (0) Favorites

      Abstract:The development of new agricultural science is a new idea and a new measure that aims to deepen the reform of higher education in agriculture and forestry for the development of new agriculture, new countryside, new farmers and new ecosystem. It is therefore essential for the current undergraduate experimental teaching reform to timely introduce new technologies and new methods used in the development of agriculture and forestry industry into the experimental course teaching, and promote the integration of professional education with innovation and entrepreneurship education, according to the requirements of the new agricultural science. In view of this, the exploration and practice of molecular biology experiment course was carried out from the perspective of experimental projects, teaching modes and evaluation methods, according to the teaching requirements and characteristics of molecular biology experiment and teachers’ scientific research achievements. The results showed that this reform greatly improved the students’ comprehensive quality and innovation ability and may facilitate the innovation experiment teaching of other courses.

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