Biotechnology Bulletin

Catalytic Promiscuity-driven Redesign of Enzyme Functions

QU Ge, SUN Zhou-tong

2023, 39(4): 1-9. doi:10.13560/j.cnki.biotech.bull.1985.2022-1305

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As biocatalysts, the reactions directed by enzymes can be conducted in a green and sustainable fashion under mild conditions. However, compared to the conversional chemical catalysts, functional limitations of native enzymes restrict their broad applications in biomanufacturing. Previous studies reveal that enzymes also have catalytic promiscuities in addition to catalytic specificity, which are able to catalyze non-natural reactions. This property sheds light on the enzyme redesign, which can be used to guide the design of artificial enzymes, expand the catalytic boundaries of natural enzymes, achieve novel enzymatic reaction types, and broaden the application scenarios of enzyme catalysis. Based on the evolutionary mechanism of catalytic promiscuity, this review summarized the common strategies used for inducing promiscuities, including directed evolution, conformational dynamics, manipulating reaction conditions, ancestor enzyme reconstruction. This review also explored the molecular mechanism behind the catalytic promiscuity in the view of catalytic mechanism, structure-function relationship and adaptive evolution combined with recent relevant study cases. It may provide a reference for breaking through the limitations of natural enzymatic reactions and creating efficient artificial enzyme components that catalyze unnatural reactions.

Redox Partner Engineering: A Solution to the Low Catalytic Efficiency of P450s

ZHANG Yan-feng, YE Li-dan, YU Hong-wei

2023, 39(4): 10-23. doi:10.13560/j.cnki.biotech.bull.1985.2022-0985

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Cytochrome P450 enzymes（CYPs or P450s）can insert one atom of O₂ into organic substrates and reduce another atom to water. As P450s are widely involved in various anabolic and catabolic processes, they have always been a focus of biotechnology. Oxidation of substrates in the catalytic cycle relies on the transfer of electrons from redox partners to the heme iron, suggesting electron transfer as a rate-limiting step in the catalytic process of P450s. Optimization of protein-protein interactions to improve the electron transfer efficiency of the P450 system, known as “redox partner engineering”, is one of the important means to engineer P450s, and has achieved fruitful progress. This paper will focus on the progress in the replacement and assembly of redox partners, fusion of P450 enzymes with redox partners, and interface modification between P450 enzymes and redox partners, aiming to provide some guidance for future work in this area.

Application of Cytochrome P450 in the Biosynthesis of Flavors and Fragrances

YU Hui-li, LI Ai-tao

2023, 39(4): 24-37. doi:10.13560/j.cnki.biotech.bull.1985.2022-1248

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Cytochromes P450 belong to a large superfamily of hemeproteins, which are widespread in almost all forms of life. P450s are known as “golden catalysts”, and they are mainly involved in the synthesis of endogenous substances, metabolism of xenobiotics, and biosynthesis of natural products in living organisms. P450- catalyzed reactions demonstrate various advantages including wide substrate spectrum, diverse types of catalytic reactions, and high catalytic regio-and stereospecificity, which make them increasingly attractive in drug/toxin metabolism, and engineered product biosynthesis. Among the P450 catalyzed reactions, the introduction of a hydroxyl group at a specific inert C-H bonds under mild conditions makes them highly useful in high value-added fine chemicals biosynthesis. Hereby the mechanism of selective hydroxylation of P450s and related several common types of electron transfer chain system are introduced. The biosynthesis of several natural flavors and fragrances such as limonene oxygenated derivatives, nootkatone, santalol, 4-hydroxyisophorone, and macrocyclic musk, which are catalyzed by single enzyme-catalyzed biotransformation or whole-cell de novo biosynthesis involving protein and metabolic engineering, are reviewed. Finally, the challenges and prospects of their practical applications are discussed.

Advances in the Application of Machine Learning Methods for Directed Evolution of Enzymes

WANG Mu-qiang, CHEN Qi, MA Wei, LI Chun-xiu, OUYANG Peng-fei, XU Jian-he

2023, 39(4): 38-48. doi:10.13560/j.cnki.biotech.bull.1985.2022-0724

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Directed evolution can increase the rate of enzyme evolution by mimicking the natural evolutionary process and has become a key technology for enzyme engineering. Directed evolution has played an important role in biocatalysis and drug design, however the experimental screening is in great challenge due to the large number of mutant libraries caused by the randomness of mutations. In recent years, emerging technologies such as artificial intelligence and big data processing have also become crucial in biocatalysis researches. Machine learning methods are statistical learning approaches to obtain sequence/structure mappings to enzyme function in a data-driven manner, which will improve the efficiency of enzyme engineering. This paper reviews the state-of-the-art technologies involved in machine learning models, especially focusing on the research and application progresses of machine learning methods in enzyme engineering. With the advancement of machine learning algorithms and technologies, it is expected that more accurate and effective models will be proposed in the future to promote screening of new enzymes and accurate design of biocatalysts.

CRISPR-associated Transposases and Their Applications in Bacterial Genome Editing

ZHOU Xiao-jie, YANG Si-qi, ZHANG Yi-wen, XU Jia-qi, YANG Sheng

2023, 39(4): 49-58. doi:10.13560/j.cnki.biotech.bull.1985.2022-1163

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CRISPR-Cas can target a specific sequence of DNA or RNA guided by a short RNA guide. The target sites can be changed with the RNA sequences. A variety of powerful genetic tools have been developed based on this reprogramming property. Recently, it has been found that some CRISPR elements are captured by Tn7 transposons during evolution, the derived CRISPR-associated transposases（CASTs）system has the ability of RNA-guided DNA integration. Some of them has been deployed as programmable genome integration tools, which have broad application prospects in large fragments and multiple gene integration. This review traces the discovery of CASTs, summarizes the characteristics of different types of CASTs loci, the mechanism models of gene integration, and their deployments and applications in a variety of Gram-negative bacteria.

The Role of NAD-dependent Deacetylase SRT in Plant Epigenetic Inheritance Regulation

WEI Ming WANG Xin-yu WU Guo-qiang ZHAO Meng

2023, 39(4): 59-70. doi:10.13560/j.cnki.biotech.bull.1985.2022-0971

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Sirtuin（SRT）is one of nicotinamide adenine dinucleotide（NAD）-dependent protein deacetylases being highly homologous to yeast silencing information regulator 2（Sir2）. In eukaryotes, SRT regulates chromosome stability, gene transcription and the activity status of related proteins by catalyzing the deacetylation of specific or non-histone lysine（Lys）residues. Studies have shown that SRT-mediated deacetylation plays an important role in plant metabolism, growth and development, and stress response. In this review, the identification, structure and classification, biological evolution, epigenetic regulation mechanism and biological processes of SRT in plants are summarized, and its future research directions are also prospected, aiming to provide a basis for future research on SRT-mediated epigenetic biological processes in plant.

Structure and Function Analysis of Novel GH5 Multi-domain Cellulase

YANG Jun-zhao, ZHANG Xin-rui, ZHAO Guo-zhu, ZHENG Fei

2023, 39(4): 71-80. doi:10.13560/j.cnki.biotech.bull.1985.2022-0641

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Cellulase can convert cellulose into fermentable sugars. In order to enrich the sequences and structure resources of cellulase and reveal the relationship between cellulase structure and function, two novel multi-domain endoglucanases belonging to glycosyl hydrolases（GH）family 5 TlCel5 and ReCel5 were cloned and expressed. Their enzymatic properties were determined and the mutation of structure domain was studied. Sequence and structure analysis showed that Tlcel5 and Recel5 encoded 655-residue and 632-residue polypeptides with theoretical molecular weights of 68.3 kD and 65.9 kD respectively. They wee both multi-domain cellulases, comprising a carbohydrate-binding module grouped into family 1（CBM1）and a catalytic module of family 5 glycoside hydrolase（CD）at N-terminal, a family X2 carbohydrate binding module（CBMX2）and an unknown domain（UM）at the C-terminal, which made them significantly different from most single-domain or dual-domain cellulases reported previously. With the purpose of understanding the effect of additional domains on enzyme function, the truncated mutant without the N-terminal CBM1 domain（TM1）and the truncated mutant without the C-terminal unknown domain（TM2）were constructed based on wild-type ReCel5. The assays of enzymatic properties demonstrated that the optimal pH and temperature of TlCel5 and ReCel5 were pH 3.0, 50℃ and pH 4.0, 70℃, respectively. They remained fine stability at 50℃ and 70℃, and showed certain hydrolytic ability to a variety of cellulose and hemicellulose substrates. Their hydrolysis specific viabilities to sodium carboxymethyl cellulose, barley β-glucan, and lichenan reduced by 23% to 68% although the enzymatic properties of TM1 and TM2 did not change significantly compared with the wild-type proteins. These results suggested that there was a close relationship between the additional domains and the hydrolysis ability of multi-domain enzymes.

Molecular Modification of the Short-chain Dehydrogenase Lvchun and Its Application in the Synthesis of Chloromycetin

HAN Hui, ZHANG Jian, REN Yu-hong

2023, 39(4): 81-92. doi:10.13560/j.cnki.biotech.bull.1985.2022-0771

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2-amino-1-（4-nitrophenyl）-1,3-propanediol, commonly known as chloromycetin（ANP）, has a wide range of applications due to its two chiral centers and the good coordination ability of the O and N atoms in its structure. For responding to the many drawbacks of chemical synthesis such as high production cost, low atomic economy and high environmental pressure, here we aim to construct a new pathway for the synthesis of（1R）-ANP using p-nitro-α-acetamido-β-hydroxypropiophenone（p-NAH）as a substrate by combining chemical hydrolysis with biocatalysis. Firstly, 1-（4-nitrophenyl）-2-amino-3-hydroxypropiophenone（AHNA）was prepared by chemical hydrolysis of p-NAH, and a carbonyl reductase with catalytic activity for the hydrolysis product was screened. The catalytic activity of the enzyme was improved by molecular modification, and the enzymatic properties of a mutant mut-V112Y were investigated. Then, a dual enzyme co-expression or fusion expression recombinant strain containing mut-V112Y and formic acid dehydrogenase was constructed, and the catalytic efficiencies of the recombinant strains were verified. Finally, the catalytic reaction conditions were optimized and the preparative reactions were performed. The results showed that the chemical method can be used to hydrolyze p-NAH to AHNA, the screened short-chain dehydrogenase Lvchun calatyzed AHNA to（1R）-ANP, and a mutant mut-V112Y with 3.47-fold increase in catalytic efficiency was obtained by targeted mutation of carbonyl reductase, which presented good temperature and pH stability with an optimal temperature of 30℃ and an optimal pH of 7.5. A dual enzyme co-expression and fusion expression recombinant strain was successfully constructed, and the highest catalytic efficiency was found in strain mut-V112Y-CbFDH. The catalytic reaction conditions were optimized, 14.56 mmol/L（1R）-ANP was produced from 50 mmol/L AHNA in 30 min under optimal conditions, with a yield of 29.12%. The combination of chemical hydrolysis and biocatalysis can effectively catalyze the synthesis of（1R）-ANP from p-NAH, which provides a new way to synthesize optically pure ANP.

Enzymatic Characterization and Degradation Sites of AFB₁ Degradation by the Extracellular Enzyme of Bacillus subtilis Strain WTX1

YANG Dong, TANG Ying

2023, 39(4): 93-102. doi:10.13560/j.cnki.biotech.bull.1985.2022-1384

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This work aims to enrich the resources of biodegradable aflatoxin B₁（AFB₁）and to study the extracellular enzymatic properties of degrading AFB₁ by the screened Bacillus subtilis WTX1 as well as its degradation sites. Liquid chromatography tandem triple quadrupole mass spectrometry（UPLC-MS/MS）was used to detect the degradation solution by degrading three compounds with similar structures to the AFB₁ toxicity site. The two main extracellular enzymes degrading AFB₁ were Ea and Eb after chromatographic separation. The optimal degradation conditions: degradation enzyme Ea was in 50℃ and pH 5, and the degradation rate was up to 88.4%; degradation enzyme Eb was in 37℃ and pH 5, the degradation rate was 73.4%. The action sites of Ea enzyme were the double bond of furan ring, vanillin endo-ring and pentenone ring structure of AFB₁. The action sites of Eb enzyme were the vanillin endo-ring and pentenone ring structure of AFB₁. The degradation pathway of the enzyme group was via hydrolysis, hydrogenation and continuous removal of -CO group. In conclusion, the extracellular enzyme of strain WTX1 destroys the AFB₁ virulence sites by hydrolysis and thus plays a degradation role. The enzyme has good thermal stability, wide temperature and pH adaptability, which meet the harsh conditions of industrial processing.

Investigation on the Complex Mutagenesis Selection of High-yield Nitrate Reductase Strain Staphylococcus simulans ZSJ6 and Its Enzymatic Properties

ZHAO Sai-sai, ZHANG Xiao-dan, JIA Xiao-yan, TAO Da-wei, LIU Ke-yu, NING Xi-bin

2023, 39(4): 103-113. doi:10.13560/j.cnki.biotech.bull.1985.2022-1270

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Nitrate reductase can reduce the nitrate in natural meat products to nitrite, avoiding a sudden increase in its content due to direct addition of nitrite and reducing the chance of producing nitrosamines, which is very important for the quality and safety of natural meat products. Staphylococcus simulans D14 screened from sausage as the starting strain, and the different effects of single mutagenesis condition including UV, microwave, and lithium chloride mutagenesis, and two compound mutagenesis methods of UV and microwave combined with lithium chloride on the strains were investigated to obtain high-yield strains of nitrate reductase, and their enzymatic properties were initially explored by shake flask fermentation in order to provide strain selection for fermenters of natural meat products. The optimal conditions for UV, microwave and lithium chloride mutagenesis were determined: UV irradiation for 60 s, microwave irradiation for 80 s and lithium chloride concentration of 1.5%. The mutagenic strain S. simulans ZSJ6 was screened, and the enzyme activity of the strain reached 603.29 U/mg protein, which was 3.59 times higher that of the starting strain, and there was no significant change in the enzyme activity of the mutagenic strain after 8 consecutive passages（P>0.05）, indicating that its genetic stability was good. From the results of enzymatic properties, its optimal action temperature was 30℃, the optimal action pH was 7.5, and its stability was excellent, and the enzyme activity retained more than 90% after incubation at pH 7.5 for 2 h. Mg²⁺, Ca²⁺, and K⁺ all promoted enzyme activity, of which Ca²⁺ had the largest role in promoting the enzyme activity, which was 1.63 times higher than that blank group. Cu²⁺, Fe²⁺, Hg²⁺ and Mn²⁺ suppressed enzyme activity, among which Cu²⁺and Hg²⁺ had the maximum inhibitory action on the enzyme activity, and the enzyme activity was all inhibited to less than 30%. The above results provide a solution for the transformation of nitrate in natural meat products and the problem of high nitrite concentration brought by the use of nitrite at the initial stage of production, which has research value and potential for application.

Cloning and Expression of 11α Hydroxylase from Aspergillus ochraceus and Analysis of Key Amino Acid Sites

AI Lu, CHEN Wen-hui, SHI Jing-hui, REN Zhi-yuan, SHEN Wen-qi, YANG Jia-ning, LUO Jian-mei, WANG Min

2023, 39(4): 114-123. doi:10.13560/j.cnki.biotech.bull.1985.2022-0572

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11α, 17α-dihydroxy progesterone is an important intermediate of steroid drugs, and is mainly produced by the 11α hydroxylation reaction of 17α-hydroxyprogesterone with molds in industry. In this paper, 11α hydroxylase of Aspergillus ochraceus and its key amino acid sites were investigated, which can provide basic data for further analysis of the catalytic mechanism of 11α hydroxylase. Using the substrate transformation, the conversion of 17α-hydroxyprogesterone by 10 molds with hydroxylation reaction was studied, then the activity of 11α hydroxylase CYP68J5 from A. ochraceus in different expressing systems was evaluated. Finally, the key amino acid sites of CYP68J5 were analyzed by means of structure prediction, molecular docking and site-directed mutation. The results showed that A. ochraceus had the strongest conversion ability, and the maximum molar production rate of 11α, 17α-dihydroxy progesterone reached 78.55% at 60 h. CYP68J5 had the highest activity in Saccharomyces cerevisiae. D118, F216 and M488 near the substrate binding pocket were identified as key amino acid sites of CYP68J5, and they played important roles in maintaining the structural stability of the enzyme and would be the potential targets for further engineering.

Identification, Heterologous Expression and Functional Analysis of a GH61 Family Glycoside Hydrolase from Setosphaeria turcica with the Assisting Function in Degrading Cellulose

MA Yu-qian, SUN Dong-hui, YUE Hao-feng, XIN Jia-yu, LIU Ning, CAO Zhi-yan

2023, 39(4): 124-135. doi:10.13560/j.cnki.biotech.bull.1985.2022-0996

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Glycoside hydrolase 61 family（GH61）belongs to the category of both oxidation and hydrolysis of cellulose degradation enzymes, it has a weak cellulose endonuclease, also can promote cellulase by oxidative damage to cellulose crystal structure on the degradation of lignocellulose, in the use of biomass resources it has potential application value. The GH61 family gene of Setosphaeria turcica was identified and bioinformatics to it was analyzed. Through transcriptome data analysis and fluorescence quantitative PCR verification, the GH61 family gene StGH61-11, induced by the carbon source corn stalk, was screened out. StGH61-11 was heterologous expressed, and its enzyme activity was determined by oxidation reaction with 2,6-dimethoxy-phenol, and the induction conditions were optimized. Its enzymatic properties were explored and the effect of StGH61-11 on promoting cellulase hydrolysis of cellulose was studied. The results showed that there were 21 GH61 family genes in S. turcica genome, and the filter paper enzyme activity of S. turcica significantly increased under the induction of corn stover. Transcriptome analysis of S. turcica showed that the expression levels of 11 genes in GH61 family increased when corn stover was used as carbon source. StGH61-11 was heterologous expressed in Escherichia coli. The optimal induction condition was 1 mmol/L IPTG at 25℃ for 9 h, and the specific activity reached（54.08±1.67）U/g. The optimal catalytic conditions were 50℃ and pH 5 for the recombinant protein StGH61-11 and the recombinant protein significantly improved the activity of cellulase hydrolysis of corn stover, with the highest degree of synergy up to 2.5 and the highest glycosylation rate up to 46.5%.

Genome-wide Identification of Catalase Family Genes and Expression Analysis in Kiwifruit

LAI Rui-lian, FENG Xin, GAO Min-xia, LU Yu-dan, LIU Xiao-chi, WU Ru-jian, CHEN Yi-ting

2023, 39(4): 136-147. doi:10.13560/j.cnki.biotech.bull.1985.2022-0533

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Catalase（CAT）is one of the main antioxidant enzymes in plants. In order to reveal the sequence characteristics and expression patterns of kiwifruit CAT family genes（AcCATs）, genome-wide identification and expression analysis were performed. The genome-wide identification and bioinformatics analysis of AcCATfamily genes were carried out, and its expression changes in different organs and during fruit storage were analyzed. Nine genes of AcCAT family were identified from kiwifruit genome using bioinformatics method and named as AcCAT1-AcCAT9 respectively according to their chromosome location information. There were high similarities in protein physicochemical properties, gene structure, conserved motifs and cis-acting elements in different members. AcCAT genes were unevenly distributed on four chromosomes, five AcCAT genes formed two tandem duplication gene clusters, and six AcCAT genes had segmental duplication. By psRNAtarget analysis, AcCAT genes were predicted to be mainly regulated by miR166 family members. Phylogenetic analysis showed that 23 CAT proteins from kiwifruit, Camellia sinensis, Gossypium hirsutum and Oryza sativa could be classified into three groups, and the classification was not completely conducted by species. Transcriptome analysis of different kiwifruit organs revealed that AcCAT3 expressed highly in the root, AcCAT1 and AcCAT4 expressed highly in the flower, AcCAT2, AcCAT5, AcCAT6, AcCAT8 and AcCAT9 were mainly expressed in the leaf, while AcCAT7 was up-expressed both in the root and flower. During the storage of kiwifruit, the expression levels of AcCAT5and AcCAT6 decreased gradually, but AcCAT1 and AcCAT2 were up-expressed in early stage, while the expression of AcCAT3, AcCAT4 and AcCAT8 increased in late stage. Additionally, the expressions of miR166 family members increased during this process. AcCAT family genes are conservative in evolution, and they play important regulatory roles in the growth and during the storage of kiwifruit.

Cloning and Analysis of Chalcone Synthase Gene and Its Promoter from Euphorbia maculata

GUO San-bao, SONG Mei-ling, LI Ling-xin, YAO Zi-zhao, GUI Ming-ming, HUANG Sheng-he

2023, 39(4): 148-156. doi:10.13560/j.cnki.biotech.bull.1985.2022-1130

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Quercetin is one of the main medicinal ingredients of Euphorbia maculata, and chalcone synthase（CHS）is one of key enzymes in the biosynthesis of quercetin. To explore the mechanism of quercetin biosynthesis in E. maculata, a CHS gene named EmCHS（GenBank access number: ON652865）was cloned from E. maculatabased on transcriptome sequencing results, combined with technologies of 3' RACE and Tail-PCR. The amino acid sequence was aligned, and physicochemical properties, transmembrane region, subcellular localization and phylogenetic relationships of EmCHS protein were analyzed. And the expressions of EmCHS in different tissues during different growth stages were detected with RT-qPCR. The results showed that the ORF of EmCHS gene was 1 194 bp, encoding 397 amino acids. The EmCHS protein was located in the cytoplasm. The theoretical isoelectric point of the protein was 5.96 and the theoretical molecular weight was 43.48 kD, and it did not contain a transmembrane region and was a hydrophilic protein with stable structure. The results of amino acid sequence alignment and phylogenetic relationships analysis revealed that EmCHS had the highest similarity（91.92%）with the amino acid sequence of Manihot esculenta in Euphorbiaceae family, which was consistent with the characteristics of plant taxonomy. The analysis of RT-qPCR confirmed that EmCHS gene was expressed in different tissues during different growth stages, and there were significant differences. The expression of EmCHS gene was the lowest in the leaf at the reproduction stage, while the highest in the root at the reproduction stage. Besides, the promoter（GenBank access number: OP626754）length of EmCHS gene cloned was 971 bp, including TATA-box, CAAT-box, MYB and MYC transcription factor binding sites, multiple light response and hormone response cis-acting elements, and so on. These results will provide a foundation for further research on gene function and expression regulation of EmCHS gene.

Advances in Upstream Open Reading Frame in Plant Genes

XUE Jiao ZHU Qing-feng FENG Yan-zhao CHEN Pei LIU Wen-hua ZHANG Ai-xia LIU Qin-jian ZHANG Qi YU Yang

2023, 39(4): 157-165. doi:10.13560/j.cnki.biotech.bull.1985.2022-0832

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uORF（upstream open reading frame）is a kind of mRNA element that can accurately control protein translation. It is located in the 5' leader region of mRNA. It has been reported to regulate the translation rate of downstream mORF（main open reading frame）by inhibiting the initiation of translation. Currently, the prediction and identification of plant uORF mainly focus on bioinformatics prediction and translatomics identification technology. uORF has regulatory roles on plants in many aspects, such as growth and development, nutrition and metabolism, disease resistance and immunity. In this review, we summarize the classification, functional mechanism, prediction and identification methods of plant uORF, strategies for plants to evade uORF, and the application progress of plant uORF in engineering. The purpose of this paper is to gain a more systematic and in-depth understanding of the function and mechanism of plant uORFs, and to provide a reference for the application of uORF in crop molecular breeding.

Research Progress in the Diversity of Endophytic Bacteria in Seeds and Their Interaction with Plants

LI Shan-jia, LEI Yu-xin, SUN Meng-ge, LIU Hai-feng, WANG Xing-min

2023, 39(4): 166-175. doi:10.13560/j.cnki.biotech.bull.1985.2022-0982

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As an important reproductive organ of plants, seeds are the carriers of a variety of beneficial microorganisms, and there are endophytic communities with rich and diverse species as well as certain effects on plants. Seed endophytic bacteria can directly or indirectly promote seed germination and plant growth, improving plant resistance to various pathogens and environmental stresses, promoting the production and the accumulation of active ingredients, and produce various biological effects. Their distribution and population structure are affected by the genetic characteristics and health status of host plants and the ecology of the surrounding environment. In this paper, the progress in the research field of seed endophytic bacteria was discussed and prospected from the diversity of seed endophytic bacteria, seed endophytic bacteria promoting plant growth, improving plant resistance, promoting the production and the accumulation of active ingredients, and the application prospect in agricultural production and biological control, so as to provide reference for the follow-up research of seed endophytic bacteria.

RNA Modification and Its Research Progress in Caenorhabditis elegans

AN Lei, ZHAO Jin-ling, REN Xiao-liang

2023, 39(4): 176-186. doi:10.13560/j.cnki.biotech.bull.1985.2022-1001

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RNA modification is an important research area in the field of epigenetics. It is involved in a wide variety of complex processes and plays a key role in regulating growth, development and metabolism. Recently, mapping RNA modification has been developed from the traditional quantitative detecting methods to more advanced detecting technologies combined with the next and third-generation sequencing platforms, which leads to better understanding of RNA modification types, related regulatory proteins, as well as their regulatory mechanisms and functions. The current research based on the RNA modification of Caenorhabditis elegansreveals that the degree of RNA modification in C. elegans is closely linked to its lifespan, resistance and fecundity. This review introduced the common types of RNA modification and the advancement of detection techniques, then summarized the current state of RNA modification research in C. elegans, and forecasted future research trends.

Towards the Understanding on the Physiological Functions of Bile Acids and Interactions with Gut Microbiota

XIONG Shu-qi

2023, 39(4): 187-200. doi:10.13560/j.cnki.biotech.bull.1985.2022-0931

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Bile acids（BAs）are produced from the metabolism of cholesterol. In the liver, cholesterol is converted into primary bile acids，and they are metabolized into secondary bile acids by gut microbiota when they reache the intestinal tract. Their homeostasis are maintained in the body through the enterohepatic circulation. The molecules of bile acids have both hydrophilic and lipophilic ends, which can play the role of detergent and help to dissolve and absorb dietary lipids and lipid-soluble vitamins. This also endows bile acids with certain antibacterial properties. The interaction between bile acids and gut microbiota is not only reflected in their own physiological functions of bile acids, but also in the regulations of the gut microbial composition and abundances in bile acid metabolism. Bile acids regulate glucose, lipid and energy metabolisms, and immune and inflammatory reactions by activating different receptor signal pathways. This article reviews the metabolic process of bile acids, and discusses how it regulates host physiological functions through FXR, TGR5 and other receptor signal pathways. It also provides references for maintaining animal health and promoting livestock and poultry production performance through scientifically and reasonably regulating gut microbiota and its metabolites.

Exploring Cultivation Approaches for New Endophytic Bacterial Resource in Oryza sativa

LI Yi-jun, WU Chen-chen, LI Rui, WANG Zhe, HE Shan-wen, WEI Shan-jun, ZHANG Xiao-xia

2023, 39(4): 201-211. doi:10.13560/j.cnki.biotech.bull.1985.2022-0961

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In order to explore approaches of isolating new endophytic bacteria in rice and obtain novel bacterial resources in rice, 3 aspects of material surface sterilization, preparing bacterial suspension, and the formulation of cultural medium were explored while using seeds and seedlings of three rice varieties as materials. The representative pure isolates were identified based on the sequence of the 16S rRNA gene. The results indicated that promising surface sterilization could be achieved by treating seeds with 75% ethanol for 90 s plus NaClO solution（containing 2% available chlorine）for 12 min, or by treating plant tissues with 75% ethanol for 90 s plus NaClO solution for 5 min. In rice seeds, the amount of endophytic bacteria was up to 10⁵-10⁶ CFU per gram，of which 90%-95% were harbored in glumes, and the bacterial community varied according to varieties and localities of growth. The grinding degree should be considered during the preparation of bacterial suspension so as to obtain colonies as diverse as possible, and stripping off glumes was beneficial to isolating species distributed mainly in the brown rice. For the separation of endophytic bacteria in rice root, stem and leave, it was necessary to control the concentration of bacterial suspension according to the material characteristics so as to obtain the trace species harbored inside. The isolation and culture of some endophytic bacteria in rice root, stem and leave could be facilitated by adding tissue extract to the culture medium and by prolonging the cultivation time to over two weeks. Using the modified protocol, we separated and cultivated the endophytic bacteria in seeds as well as roots, stems and leaves of three rice varieties, and identified 71, 14, 10 and 2 pure isolates respectively, by 16S rRNA gene sequencing. A total of 8 isolates, accounting for 8.25% of the total identified isolates, showed a similarity of 97.22%-98.74% to the model strains recorded in the Ezbiocloud database and were putative new species. The results of our study may provide a referenced method to isolate and cultivate new resources of endophytic bacteria in rice.

A New Method for Onion Seed Disinfection and Aseptic Seedling Culture

SUN Ya-ling, LI Rui-ping, WANG Zhen-bao, ZHANG Shu, LIU Bing-jiang, HUO Yu-meng

2023, 39(4): 212-220. doi:10.13560/j.cnki.biotech.bull.1985.2022-0983

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This work aims to obtain a simple, efficient and low-contamination method for onion seed disinfection and aseptic seedling culture. The sterilization of onion seeds and the culture of sterile seedlings were studied by controlling the pH value, disinfection soaking time and culture concentration of plant preservative mixture（PPM）. The results showed that the disinfection effect of soaking seeds in PPM（20 mL/L, pH=2）for 24-48 h reached the level of 10% NaClO disinfection for 10 min, which did not affect seed germination, and the germination potential exceeded 90% in 8 d. The analysis of contaminated microorganisms found that four species of fungi（Alternaria alternata, Fusarium proliferatum, Fusarium oxysporum, and Stemphylium vesicarium）and two species of bacteria（Phytobacter diazotrophicus and Atlantibacter hermannii）were transmitted through onion seeds, and the bacteriostasis test confirmed that PPM was able to effectively inhibit the growth of contaminated microorganisms. The medium added with PPM effectively reduced the contamination rate using PPM for seed soaking disinfection; high concentration PPM（1-9 mL/L）inhibited the growth of onion seedlings, while low concentration PPM（0-0.5 mL/L）had no significant inhibitory effect on the growth of onion seedlings. In sum, a set of simple and efficent methods for onion seed disinfection and aseptic seedling culture are explored and developed.

Inoculation Method for Colletotrichum in Pepper（Capsicum annuum）Seedlings

LI Yue, YU Wan-xian, LI Ning, YAO Ming-hua, LI Feng, DENG Ying-tian

2023, 39(4): 221-226. doi:10.13560/j.cnki.biotech.bull.1985.2022-0994

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Anthracnose is one of the most serious diseases in pepper production, there is an urgent need for reliable methods to screen for Colletotrichum disease resistance in the early pepper development. In this study, by using Colletotrichum strain CD, we have introduced two methods to inoculate pepper ‘ST-8’ seedling: infiltration and needle point, and tried to find the optimal way by comparing the plant infection rate, leave curl rate and the size of necrotic disease spots. The results showed that the results obtained by using the needle point method under the resuspend spore concentration of 1 × 10⁶cells/mL were the best, which is expected to be applied to large-scale screening of pepper being resistant to anthracnose.

Research Progress in Low-input Chromatin Immunoprecipitation Assay

ZHANG Xin-bo, CUI Hao-liang, SHI Pei-hua, GAO Jin-chun, ZHAO Shun-ran, TAO Chen-yu

2023, 39(4): 227-235. doi:10.13560/j.cnki.biotech.bull.1985.2022-0940

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The chromosome immunoprecipitation sequencing technology（ChIP-seq）, which combines the chromatin immunoprecipitation technology（ChIP）with the second generation sequencing technology，is an important method for analyzing the epigenetic changes of the whole genome，and can quickly and effectively detect DNA and protein binding sites，transcription factor binding sites（TFBS），histone post-translation modifications（hPTMs），nucleosome localization and DNA methylation in the whole genome. However，for a long time，ChIP-seq needs a large number of cells to generate high-quality data sets，which limits the application of ChIP in some specific tissue and low-cell samples such as oocytes，early embryonic cells and other research fields. In recent years, based on ChIP，researchers have proposed a series of low-input ChIP-seq methods to reduce the initial sample amount and experimental cost and increase the sequencing quality，which has promoted the development of epigenomics. In this paper, we reviewed the principles of ChIP and the methodological development of ChIP-seq reducing low-input amount，compared several of the more important methods，and summarized the application of ChIP-seq with low-input amount in epigenetic research. Finally，we prospected the application and development of low initial ChIP-seq technology，aiming to provide reference for the selection of different low-input ChIP-seq methods for low cell number samples.

Functional Analysis of SlMYB96 Gene in Tomato Under Cold Stress

HU Ming-yue, YANG Yu, GUO Yang-dong, ZHANG Xi-chun

2023, 39(4): 236-245. doi:10.13560/j.cnki.biotech.bull.1985.2022-1051

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This study is to discuss the function of SlMYB96 in tomato under cold resistance, aiming to provide theoretical basis for the molecular mechanism of cold resistance and breeding of tomato under cold resistance. The SlMYB96 gene was cloned using tomato cDNA as the template. Then the physical and chemical properties of the gene were analyzed by bioinformatic software, and the expression features of SlMYB96 and its role in cold resistance in tomato was studies by real-time quantitative fluorescence（RT-qPCR）technology and virus-induced gene silencing（VIGS）technology. The results showed that SlMYB96 was expressed in the roots, stems, leaves, flowers, and fruit of tomato, with the highest expression in the flowers. And the expression of SlMYB96 increased with increasing time of 4℃ cold treatment, where expression reached the maximum at three hour of low temperature treatment. With the help of VIGS the SlMYB96 gene was silenced. Three different types of tomato plants in wild-type group（WT）, empty load group（CK）and gene transient silencing group（pTRV-MYB96）were treated with low temperature. And the appearance traits showed that plants in the transient gene silencing group（pTRV-MYB96）after cold treatment at 4℃ for five days, presented more obvious cold damage symptoms compared with the wild-type group（WT）and the empty-load group（CK）. The identification results of the physiological level showed that when tomato seedlings were treated with 4℃ at low temperature for five days, the content of chlorophyll, malondialdehyde, soluble protein and superoxide dismutase activity of the gene transient silencing group（pTRV-MYB96）plants were significantly lower, while the activity of soluble sugar, relative conductivity, free proline content, catalase and peroxidase increased. When tomato seedlings treated under cold 4℃ for five days, chlorophyll, malondialdehyde, soluble protein content and superoxide dismutase activity were significantly lower in gene transient silent group（pTRV-MYB96）, while soluble sugar, relative conductivity, free proline content and catalase and peroxidase activity increased, indicating that gene transient silent group（pTRV-MYB96）plants had lower cold resistance compared with wild-type group（WT）and empty load group（CK）. It is confirmed that SlMYB96 gene can respond to cold stress and reduce cold resistance after silencing.

Identification and Interaction Analysis of SMAS Gene Family in Tea Plant（Camellia sinensis）

WANG Yi-qing, WANG Tao, WEI Chao-ling, DAI Hao-min, CAO Shi-xian, SUN Wei-jiang, ZENG Wen

2023, 39(4): 246-258. doi:10.13560/j.cnki.biotech.bull.1985.2022-1045

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S-adenosylmethionine synthase（SAMS）is the only enzyme that catalyzes the synthesis of S-adenosylmethionine（SAM）from methionine and ATP. Research shows that SAMS participates in lignin biosynthesis. In this study, we analyzed the expression patterns and protein interaction networks of the SAMS gene family identified from ‘Huangdan’ tea plant（Camellia sinensis）to explore candidate genes of CsSAMS that may be involved in lignin synthesis. Then we identified the members of CsSAMS gene family by bioinformatics based on the genome of ‘Huangdan’, and further analyzed their protein physicochemical properties, phylogenetic tree, chromosome location, gene structure, protein structure and expression pattern. We also studied the protein interaction network by yeast two-hybrid technology（Y2H）. Finally, we determined the lignin contents of the first bud and the two leaves of ‘Haungdan’, ‘Tieguanyin’, ‘Jinguanyin’ and ‘Fuding Dahao’ by UV spectrophotometer. The results of bioinformatics analysis showed that 4 CsSAMS gene family members were identified in ‘Huangdan’. The number of encoded amino acids was 345-519, and the isoelectric point ranged from 6.12 to 6.47. The prediction results of subcellular localization revealed that CsSAMS1 was located in the chloroplast, CsSAMS2 and CsSAMS3 were located in the cytoplasm, and CsSAMS4 was located in the cytoskeleton. Expression of CsSAMS and lignin content detection of different tea varieties indicated that CsSAMS2, CsSAMS3 and CsSAMS4 might potentially regulate lignin content. In addition, Y2H results showed that CsSAMS4 formed homodimers with itself. In this study, the physicochemical properties of four CsSAMS members are identified and analyzed, and their functions are predicted. The expression patterns of CsSAMS genes in different tissue sites, under nitrogen and fluoride treatments, as well as the potential involvement of CsSAMS in lignin synthesis process, are clarified.

Genetic Transformation and Chilling Resistance Analysis of Maize ZmDHN15 Gene in Tobacco

CHEN Nan-nan, WANG Chun-lai, JIANG Zhen-zhong, JIAO Peng, GUAN Shu-yan, MA Yi-yong

2023, 39(4): 259-267. doi:10.13560/j.cnki.biotech.bull.1985.2022-1083

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Maize（Zea mays L.）originated in the subtropical zone, is a thermophilic crop, and is susceptible to low temperature stress. Dehydrin, group 2 members of the late embryogenesis abundant protein（LEA）, are a class of proteins that play an important role in plant abiotic stress. In this study, the ZmDHN15 gene was cloned and obtained, and the basic characteristics and tissue expression characteristics of the gene were analyzed using bioinformatics methods, real-time fluorescent quantitative PCR and other techniques, and the construction of plant overexpression vector and the genetic transformation of tobacco were carried out. T₂generation plants were used to verify their cold resistance. The results showed that the ZmDHN15 gene was 1 442 bp in length, encoded a total of 290 amino acids, with a molecular weight of 31.44 kD and a theoretical isoelectric point of 6.05. It was a hydrophilic non-transmembrane protein with a conserved domain unique to the dehydratin family. RT-qPCR analysis showed that the ZmDHN15 gene was highly expressed in maize leaves and increased under cold stress conditions.Nine T₂ generation transgenic tobacco plants were obtained. Compared with the wild type, the germination rate of transgenic tobacco increased by 1.40 times, the root length increased by 1.58 times, the wilting degree of its leaves was lower, the content of proline, malondialdehyde and peroxidase reduced by 41.17%, 28.47% and 23.33%, respectively. Soluble sugar content, oxide activity and superoxide dismutase activity increased by 58.97%, 47.85% and 47.53%, respectively, and the accumulation of H₂O₂ and O₂^- decreased by 34.78% and 47.00%, respectively. In conclusion, overexpression of ZmDHN15 gene can effectively improve the tolerance of tobacco plants to cold stress, which lays a foundation for further research on the function of ZmDHN15 gene in maize.

Construction of the Soybean Membrane System cDNA Library and Interaction Proteins Screening for Effector PsAvr3a

HOU Xiao-yuan, CHE Zheng-zheng, LI Heng-jing, DU Chong-yu, XU Qian, WANG Qun-qing

2023, 39(4): 268-276. doi:10.13560/j.cnki.biotech.bull.1985.2022-0972

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The plant plasma membrane is the first barrier in response to pathogen infection, and the receptor proteins on it are the radar for sensing pathogen invasion, which is an important part of the plant immune system. Phytophthora sojae disrupts the host immune activities of plants by secreting effectors, promoting Phytophthora colonization and spread. The construction of soybean split-ubiquitin based membrane yeast two-hybridization cDNA library is significant for exploring the interaction mechanisms between effectors and host cell membrane protein targets. The avirulent effector PsAvr3a was used as a bait protein to screen the library, and 198 candidate host targets were preliminarily obtained. The yeast two-hybrid technique between the interested target proteins and PsAvr3a revealed that five of them interacted with the bait vector. The enzyme that regulates tyrosine breakdown, the vesicle transport protein, the ethylene biosynthesis regulatory enzyme, the cell metabolic binding protein, the stress-resistant osmotic protein, and others were among the prospective host targets. The interactions amony GmACO, GmSec61 and PsAvr3a were verified by Bimolecular Fluorescence Complementation（BIFC）and Luciferase Complementation Assay（LCA）. Therefore, two soybean membrane proteins, ethylene precursor ACC oxidase GmACO and trimeric transposon GmSec61, were identified as the host target of Phytophthora sojae avirulent effector PsAvr3a.

Isolation, Identification and Biocontrol Mechanism of an Antagonistic Bacterium Against Anthracnose on Mango Caused by Colletotrichum gloeosporioides

ZHANG Le-le, WANG Guan, LIU Feng, HU Han-qiao, REN Lei

2023, 39(4): 277-287. doi:10.13560/j.cnki.biotech.bull.1985.2022-0908

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The antagonistic bacteria of mango anthracnose were isolated from the environment, the antagonistic mechanism of the strain RL-LL04 against the pathogen was clarified and the potential of the strain for biological control applications was investigated. Antagonistic bacteria of Colletotrichum gloeosporioides were isolated and screened via dilution and plate confrontation methods, and then were systematically identified. The plate confrontation method was used to study the inhibitory effect of the strains on a variety of pathogens on common tropical fruit. The extracellular enzyme detection and solid-phase micro extraction gas-mass spectrometry were combined to detect the volatile organic components and to study biocontrol mechanism. Moreover, the influence of the strain on the growth of pathogenic mycelium was observed under the light microscope. In addition, in vitro inoculation trials of mangoes were conducted for biological control applications of mango anthracnose. A total of 73 antagonistic bacterial strains against C. gloeosporioides were isolated and the inhibition rates of five strains were above 70.0%. Among these isolates, strain RL-LL04 showed the highest inhibition rate（82.2%）. Strain RL-LL04 was identified as Bacillus velezensis by morphologial, physiological & biochemical characteristics and 16S rRNA, gyrB and rpoB gene analysis. Strain RL-LL04 demonstrated antagonistic ability toward a variety of pathogenic fungi and strain RL-LL04 inhibited the growths of pathogenic fungi by producing volatile organic compounds containing benzaldehyde, 3-methylbutyric acid and phenol as well as extracellular enzymes such as cellulase, protease and xylanase. The pathogenic mycelium was deformed, twisted and broken, while observed by light microscopy. The inhibition rate of in vitro anthracnose on mango by strain RL-LL04 reached 52.7%. This study may provide a microbial resource for the biological control of mango anthracnose as well as a fundamental insight for elucidating the antagonistic mechanism of strain RL-LL04 against mango anthracnose.

Comparative Proteomics Analysis of Aeromonas hydrophila Under Enrofloxacin Stress

YAO Jin-dong, TANG Hua-mei, YANG Wen-xiao, ZHANG Li-shan, LIN Xiang-min

2023, 39(4): 288-296. doi:10.13560/j.cnki.biotech.bull.1985.2022-0917

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In order to understand the mechanism of bacterial drug resistance under the stress of quinolone antibiotics, this study took Aeromonas hydrophila as the research object, quantitative proteomics was used to compare the protein expression differences in A. hydrophila with and without enrofloxacin treatment. The results showed that total 446 differentially expressed proteins were identified by mass spectrometry, including 233 up-regulated proteins and 213 down-regulated proteins. Bioinformatics analysis showed that A. hydrophila survival may be promoted by up-regulating the expressions of most DNA repair and sulfur metabolism related proteins. Through the growth curve determination, cysteine combined with enrofloxacin can better inhibit the growth of bacteria. Meanwhile, qPCR was used to verify the expression levels of sulfur metabolism-related genes at the mRNA level, and it was uncovered that the differential expression levels of most genes at the transcription level were consistent with the protein level. These results indicate that sulfur metabolism plays an important role in the ENR stress of A. hydrophila.

Study on Factors Affecting the Storage Survival Rates of Probiotic Bacteria Powder

YAN Tao, CHEN Ke-ke, YANG Heng-fei, ZHU Jian-guo, XIA Jiu-xue, FANG Shu-guang

2023, 39(4): 296-303. doi:10.13560/j.cnki.biotech.bull.1985.2022-0954

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This work is aimed to study the factors affecting the storage activities of three types of probiotic powder, Lactobacillus, Bifidobacterium and Coccus, so as to provide guidance for the quality index control and application of probiotics powder. Lactobacillus acidophilus LA85, Lactobacillus plantarum Lp90, Lactobacillus rhamnosusLRa05, Lactobacillus casei LC89, Bifidobacterium lactis BLa80, Bifidobacterium longum BL21, Bifidobacterium breve BBr60, Bifidobacterium infantis BI45, Streptococcus thermophilus ST81, Pediococcus lactis PA53, Pediococcus pentosaceus PP06, Lactococcus lactis LLa61 were used as the research objects, and the survival rates of bacteria and protease activities were used as indicators to investigate the quality indicators of probiotic bacteria powder（moisture and water activity）, storage temperature and encapsulation method on the cell survival rate and protease activity during storage. The moisture and water activity of the bacteria powder presented no significant effect on the storage survival rate and protease activity within a certain range（P<0.05）. The storage survival rate and protease activity decreased significantly（P<0.05）with the increasing of moisture and water activity while beyond a certain range. Different storage temperatures（-18℃, 4℃, and 25℃）had significant effects on the storage survival rate and protease activity of bacterial powder（P<0.05）. The survival rate of bacteria in vacuum encapsulation was better than that in ordinary encapsulation（P<0.05）. The optimal moisture content of Lactobacillus, Bifidobacterium and Coccus powders, the quality control index of water activity, storage temperature and encapsulation method are determined to ensure the survival rate of probiotic powder during storage, and to provide guidance for the formulation and application of quality control standards for probiotic powder manufacturers.

Gene Cloning, Expression Pattern, and Promoter Activity Analysis of Chicken BMP15

YANG Lan, ZHANG Chen-xi, FAN Xue-wei, WANG Yang-guang, WANG Chun-xiu, LI Wen-ting

2023, 39(4): 304-312. doi:10.13560/j.cnki.biotech.bull.1985.2022-1224

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The purpose of this study is to investigate the transcriptional regulation mechanism of the bone morphogenetic protein 15（BMP15）gene in chickens, as well as its function, structure, and promoter active region. RACE（rapid amplification of cDNA ends）was used to clone the full-length cDNA of the chicken BMP15gene, and bioinformatics was to analyze the properties and structures of its encoding protein. Quantitative real-time PCR was plied to detect the expressions of chicken BMP15 gene in different tissues. Dual luciferase was used to identify the primary promoter region of the gene. According to the results, the chicken BMP15 gene has a transcript of 1 865 bp in length, encoding 350 amino acids, and its hereditary correlation is the closest to that of turkey. The protein structure primarily consists of disordered coils, there is a signal peptide region and no transmembrane domain. Additionally, BMP15 gene was significantly expressed in both ovaries and granulosa cells of the chicken（P<0.01）according to the constructed tissue-expression profile. Dual-luciferase assay revealed that the core promoter region of chicken BMP15 gene was positioned at -1 bp to -153 bp. This sets the foundations for further research into the BMP15 gene's function and its transcriptional regulatory mechanism.

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2023, 39(4): 313-313.

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