Article(id=1280817611491492444, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1280817479555462000, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20250938, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1765814400000, receivedDateStr=2025-12-16, revisedDate=null, revisedDateStr=null, acceptedDate=1768924800000, acceptedDateStr=2026-01-21, onlineDate=1783300315020, onlineDateStr=2026-07-06, pubDate=1783094400000, pubDateStr=2026-07-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1783300315020, onlineIssueDateStr=2026-07-06, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1783300315020, creator=13701087609, updateTime=1783300315020, updator=13701087609, issue=Issue{id=1280817479555462000, tenantId=1146029695717560320, journalId=1192105938417971205, year='2026', volume='66', issue='7', pageStart='3121', pageEnd='3677', issueExtLink='null', onlineDate='null', pubDate='1783094400000', pubDateStr='2026-07-04', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1783300283564, creator='13701087609', updateTime=1783326087324, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1280925708813832745, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1280817479555462000, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1280925708813832746, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1280817479555462000, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=3508, endPage=3525, ext={EN=ArticleExt(id=1280817611885757021, articleId=1280817611491492444, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Investigating the tiered responses and tolerance mechanisms of Penicillium oxalicum Z2 to licorice flavonoids based on transcriptomics, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

Objective To elucidate the tolerance mechanisms of Penicillium oxalicum Z2 to flavonoids present in licorice residues and to analyze its transcriptional responses under gradient stress concentrations, thereby providing a theoretical basis for breeding resistant strains and improving fermentation efficiency. Methods P. oxalicum Z2 was treated with different concentrations of licorice flavonoids (0, 0.25, 0.50, 1.00, and 2.00 mg/mL). Physiological indicators were systematically measured. Transcriptome sequencing was conducted to identify differentially expressed genes and enriched KEGG/GO pathways, revealing the molecular adaptation mechanisms of P. oxalicum Z2 under flavonoid stress. Results As the flavonoid concentration increased, P. oxalicum Z2 maintained high biomass and cellulase activity. Under the highest flavonoid concentration (2.00 mg/mL), its biomass reached 4.840 g/L, and the activities of endoglucanase, β-glucosidase, filter paper enzyme, and xylanase reached 74.78, 3.24, 6.99, and 562.5 U/mL, respectively, which were more than ten folds of those in the conventional P. oxalicum strain. Transcriptome analysis showed that the number of differentially expressed genes increased in a concentration-dependent manner, from 818 in the Low group to 3 945 in the Super group. Under low stress (≤1.00 mg/mL), the strain reprogrammed carbohydrate metabolism and ABC transporter pathways to adjust carbon utilization and sustain essential cellular activities. Under high stress (2.00 mg/mL), it further activated the ribosome and oxidative phosphorylation pathways, with 65 genes upregulated in the ribosome pathway. Conclusion This study identified key genes (e.g., EF-Tu, SecY,and FtsY) and core pathways (e.g., oxidative phosphorylation and ABC transporter), demonstrating that P. oxalicum Z2 employed tiered responses to licorice flavonoid stress. Specifically, the strain remodels sugar metabolism and transmembrane transport under low flavonoid stress to maintain survival and activates ribosome and energy metabolism compensation mechanisms under high flavonoid stress.

, authors=Zhihao HE1, Bingqian YANG1, Xirui WANG1, Chen YU1, Siyuan CHANG1, 2, Sen ZHANG1, authorsList=Zhihao HE, Bingqian YANG, Xirui WANG, Chen YU, Siyuan CHANG, Sen ZHANG, authorCompany=null, correspAuthors=Siyuan CHANG, Sen ZHANG, authorNote=null, correspAuthorsNote=
E-mail: CHANG Siyuan,
ZHANG Sen,
, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1280817614201012850, articleId=1280817611491492444, tenantId=1146029695717560320, journalId=1192105938417971205, language=CN, title=基于转录组学探究草酸青霉Z2响应甘草黄酮胁迫的分层响应耐受机制, columnId=1192149544164012138, journalTitle=微生物学报, columnName=研究报告, runingTitle=null, highlight=null, articleAbstract=

目的 揭示草酸青霉(Penicillium oxalicum) Z2对甘草药渣中黄酮成分的耐受机制,解析其在不同浓度黄酮胁迫下的转录响应规律,为选育抗性菌株、提升发酵效率提供理论依据。 方法 以草酸青霉Z2为研究对象,设置不同浓度甘草黄酮处理组(0、0.25、0.50、1.00、2.00 mg/mL),系统测定其生理指标,并结合转录组技术,揭示草酸青霉Z2在黄酮胁迫下的分子适应机制。 结果 随着黄酮浓度升高,草酸青霉Z2的生物量与纤维素酶活性维持在较高水平,在Super组条件下其生物量达4.840 g/L,内切酶、β-葡聚糖酶、滤纸酶和木聚糖酶活力分别达74.78、3.24、6.99、562.5 U/mL,是普通草酸青霉的10倍以上。转录组分析表明,差异表达基因数量呈浓度依赖性递增,从Low组的818个增至Super组的3 945个。在低浓度(≤1.00 mg/mL)下,菌株通过重编程碳水化合物代谢与ABC转运体通路,调整碳源利用策略以维持基本生命活动;而在高浓度(2.00 mg/mL)下,进一步激活核糖体、氧化磷酸化通路,其中核糖体通路有65个基因上调。 结论 研究筛选出EF-TuSecYFtsY等关键基因以及氧化磷酸化、ABC转运等核心通路,揭示草酸青霉Z2通过分层响应甘草黄酮胁迫:低浓度下重塑糖代谢与跨膜转运以维持生存,高浓度下激活核糖体与能量代谢补偿机制。

, authors=何志豪1, 杨秉乾1, 王玺瑞1, 鱼晨1, 常思源1, 2, 张森1, authorsList=何志豪, 杨秉乾, 王玺瑞, 鱼晨, 常思源, 张森, authorCompany=null, correspAuthors=常思源, 张森, authorNote=

作者贡献声明

何志豪:完成实验,撰写文章;杨秉乾:数据分析;王玺瑞:协助实验;鱼晨:图片制作;常思源:提供思路;张森:提供基金,修改文章。

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Journal of Fungi, 2022, 8(2): 111., articleTitle=A combined analysis of transcriptome and proteome reveals the inhibitory mechanism of a novel oligosaccharide ester against Penicillium italicum, refAbstract=null)], funds=[Fund(id=1280925078372200495, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, awardId=2025YFC3509100, language=EN, fundingSource=The Key Technology Research and Industrialization Demonstration Project for the Recycling and Utilization of Traditional Chinese Medicine Resources(2025YFC3509100), fundOrder=null, country=null), Fund(id=1280925078439309360, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, awardId=2025YFC3509100, language=CN, fundingSource=中药资源循环利用关键技术研发及产业化示范项目(2025YFC3509100), fundOrder=null, country=null), Fund(id=1280925078514806833, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, awardId=82574719, language=EN, 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articleId=1280817611491492444, language=EN, label=Figure 2, caption=The growth curves changes of Penicillium oxalicum under different concentrations of flavonoids stress. A: Growth curves of Penicillium oxalicum G4; B: Growth curves of Penicillium oxalicum G7; C: Growth curves of Penicillium oxalicum Z2. The concentrations of flavonoids in different groups are as follows: CK (0 mg/mL), Low (0.25 mg/mL), Middle (0.50 mg/mL), High (1.00 mg/mL), Super (2.00 mg/mL)., figureFileSmall=AXlY7Y+1h8xTH0ATnIHJBA==, figureFileBig=v6li4jSQpE9fcnKdRpJPCw==, tableContent=null), ArticleFig(id=1280925077046800414, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=CN, label=图2, caption=不同黄酮浓度胁迫下草酸青霉生长曲线变化, figureFileSmall=AXlY7Y+1h8xTH0ATnIHJBA==, figureFileBig=v6li4jSQpE9fcnKdRpJPCw==, tableContent=null), ArticleFig(id=1280925077130686495, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=EN, label=Figure 3, caption=The changes in extracellular hydrolase activities of Penicillium oxalicum under different flavonoid concentrations stress. A: Changes in endo-enzyme activity of different Penicillium oxalicum under the influence of different flavonoid concentrations; B: Changes in β-glucosidase activity of different Penicillium oxalicum under the influence of different flavonoid concentrations; C: Changes in filter paper enzyme activity of different Penicillium oxalicum under the influence of different flavonoid concentrations; D: Changes in xylanase activity of different Penicillium oxalicum under the influence of different flavonoid concentrations. The data are presented as mean±SD (n=3). Compared with the CK group, **: P<0.01; ***: P<0.001., figureFileSmall=JQvumUJpz1uf2cVl0AUWAg==, figureFileBig=2dLejI2j1PMsHBFtQoNg3g==, tableContent=null), ArticleFig(id=1280925077231349792, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=CN, label=图3, caption=不同黄酮浓度胁迫下草酸青霉胞外水解酶活力变化, figureFileSmall=JQvumUJpz1uf2cVl0AUWAg==, figureFileBig=2dLejI2j1PMsHBFtQoNg3g==, tableContent=null), ArticleFig(id=1280925077294264353, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=EN, label=Figure 4, caption=Changes in extracellular conductivity and protein concentration of Penicillium oxalicum under different flavonoid concentrations stress. A: Changes in extracellular conductivity over time at different flavonoid concentrations; B: Changes in extracellular protein concentration over time under the influence of different flavonoid concentrations., figureFileSmall=+ozLKiVPWsVGrLAmAO3uSg==, figureFileBig=PQYKjkskAOddTbgbW9FKKA==, tableContent=null), ArticleFig(id=1280925077373956130, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=CN, label=图4, caption=不同黄酮浓度胁迫下草酸青霉胞外电导率和蛋白浓度变化, figureFileSmall=+ozLKiVPWsVGrLAmAO3uSg==, figureFileBig=PQYKjkskAOddTbgbW9FKKA==, tableContent=null), ArticleFig(id=1280925077432676387, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=EN, label=Figure 5, caption=Analysis results of differential genes under different concentrations of flavonoids. A: Upregulation and downregulation of differentially expressed genes in different flavonoid treatment groups; B: Volcano plot of differentially expressed genes in different flavonoid treatment groups; C: Venn diagram of differential gene expression in different flavonoid concentration treatment groups., figureFileSmall=um0OxTd4J2blxGNXpHP/xw==, figureFileBig=h+/5OjZol3ZB2OaewzNIQw==, tableContent=null), ArticleFig(id=1280925077508173860, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=CN, label=图5, caption=不同黄酮浓度胁迫下差异基因分析结果, figureFileSmall=um0OxTd4J2blxGNXpHP/xw==, figureFileBig=h+/5OjZol3ZB2OaewzNIQw==, tableContent=null), ArticleFig(id=1280925077566894117, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=EN, label=Figure 6, caption=Cluster analysis results. A: Differential gene clustering analysis. The darker the color, the higher the expression level of the gene in that sample; Conversely, the bluer the color, the lower the expression level of the gene in that sample (Horizontally, the genes are represented. Each column represents a sample); B: Trend analysis of differential genes., figureFileSmall=fR4THTrLxPok58oYaBTSmg==, figureFileBig=t9gcPGD14ghLmpPwWcZ48w==, tableContent=null), ArticleFig(id=1280925077642391590, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=CN, label=图6, caption=聚类分析结果, figureFileSmall=fR4THTrLxPok58oYaBTSmg==, figureFileBig=t9gcPGD14ghLmpPwWcZ48w==, tableContent=null), ArticleFig(id=1280925077709500455, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=EN, label=Figure 7, caption=KEGG enrichment analysis results. A: The KEGG enrichment analysis result of CK_vs._Low; B: The KEGG enrichment analysis result of CK_vs._Middle; C: The KEGG enrichment analysis result of CK_vs._High; D: The KEGG enrichment analysis result of CK_vs._Super., figureFileSmall=YK+N+N5Z7TJBnKwQnnJiUg==, figureFileBig=aGkNXROoznWSJD056xA5Hw==, tableContent=null), ArticleFig(id=1280925077776609320, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=CN, label=图7, caption=KEGG富集分析结果, figureFileSmall=YK+N+N5Z7TJBnKwQnnJiUg==, figureFileBig=aGkNXROoznWSJD056xA5Hw==, tableContent=null), ArticleFig(id=1280925077860495401, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=EN, label=Figure 8, caption=GO enrichment analysis results. A: The GO enrichment analysis result of CK_vs._Low; B: The GO enrichment analysis result of CK_vs._Middle; C: The GO enrichment analysis result of CK_vs._High; D: The GO enrichment analysis result of CK_vs._Super., figureFileSmall=23qBskpOZsaZcpgQBDofQQ==, figureFileBig=Jqa/CS59jux0WugrLF0tRw==, tableContent=null), ArticleFig(id=1280925077940187178, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=CN, label=图8, caption=GO富集分析结果, figureFileSmall=23qBskpOZsaZcpgQBDofQQ==, figureFileBig=Jqa/CS59jux0WugrLF0tRw==, tableContent=null), ArticleFig(id=1280925078007296043, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=EN, label=Figure 9, caption=Single nucleotide variation analysis results. Homo (homozygous-variant) indicates a homozygous variant, meaning that both alleles at this locus have mutated and the mutations are the same; Hete (heterozygous-variant) indicates a heterozygous variant, meaning that at least one of the alleles at this locus has mutated and the mutated alleles are different., figureFileSmall=e2bxQN7iAZmu9yFtjYxT7Q==, figureFileBig=VIxDJqQcG5tZTM6h9Y09lw==, tableContent=null), ArticleFig(id=1280925078091182124, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=CN, label=图9, caption=单核苷酸变异分析结果, figureFileSmall=e2bxQN7iAZmu9yFtjYxT7Q==, figureFileBig=VIxDJqQcG5tZTM6h9Y09lw==, tableContent=null), ArticleFig(id=1280925078166679597, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1280817611491492444, language=EN, label=Figure 10, caption=Ribosome pathway diagram. 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基于转录组学探究草酸青霉Z2响应甘草黄酮胁迫的分层响应耐受机制
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何志豪 1 , 杨秉乾 1 , 王玺瑞 1 , 鱼晨 1 , 常思源 1, 2 , 张森 1
微生物学报 | 研究报告 2026,66(7): 3508-3525
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微生物学报 |研究报告 2026 , 66 (7) : 3508 -3525
基于转录组学探究草酸青霉Z2响应甘草黄酮胁迫的分层响应耐受机制
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何志豪1, 杨秉乾1, 王玺瑞1, 鱼晨1, 常思源1, 2 , 张森1
作者信息
  • 1.南京中医药大学,江苏省中药资源产业化过程协同创新中心,江苏 南京
  • 2.南京科技职业学院,江苏省生物基平台化合物催化工程技术研究开发中心,江苏 南京
作者简介:

作者贡献声明

何志豪:完成实验,撰写文章;杨秉乾:数据分析;王玺瑞:协助实验;鱼晨:图片制作;常思源:提供思路;张森:提供基金,修改文章。

Investigating the tiered responses and tolerance mechanisms of Penicillium oxalicum Z2 to licorice flavonoids based on transcriptomics
Zhihao HE1, Bingqian YANG1, Xirui WANG1, Chen YU1, Siyuan CHANG1, 2 , Sen ZHANG1
Affiliations
  • 1.Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, China
  • 2.Bio-based Platform Chemicals Catalysis Engineering Technology Research and Development Center of Jiangsu Province, Nanjing Polytechnic Institute, Nanjing, Jiangsu, China
出版时间: 2026-07-04 doi: 10.13343/j.cnki.wsxb.20250938
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目的 揭示草酸青霉(Penicillium oxalicum) Z2对甘草药渣中黄酮成分的耐受机制,解析其在不同浓度黄酮胁迫下的转录响应规律,为选育抗性菌株、提升发酵效率提供理论依据。 方法 以草酸青霉Z2为研究对象,设置不同浓度甘草黄酮处理组(0、0.25、0.50、1.00、2.00 mg/mL),系统测定其生理指标,并结合转录组技术,揭示草酸青霉Z2在黄酮胁迫下的分子适应机制。 结果 随着黄酮浓度升高,草酸青霉Z2的生物量与纤维素酶活性维持在较高水平,在Super组条件下其生物量达4.840 g/L,内切酶、β-葡聚糖酶、滤纸酶和木聚糖酶活力分别达74.78、3.24、6.99、562.5 U/mL,是普通草酸青霉的10倍以上。转录组分析表明,差异表达基因数量呈浓度依赖性递增,从Low组的818个增至Super组的3 945个。在低浓度(≤1.00 mg/mL)下,菌株通过重编程碳水化合物代谢与ABC转运体通路,调整碳源利用策略以维持基本生命活动;而在高浓度(2.00 mg/mL)下,进一步激活核糖体、氧化磷酸化通路,其中核糖体通路有65个基因上调。 结论 研究筛选出EF-TuSecYFtsY等关键基因以及氧化磷酸化、ABC转运等核心通路,揭示草酸青霉Z2通过分层响应甘草黄酮胁迫:低浓度下重塑糖代谢与跨膜转运以维持生存,高浓度下激活核糖体与能量代谢补偿机制。

甘草药渣  /  草酸青霉  /  转录组学  /  耐受机制

Objective To elucidate the tolerance mechanisms of Penicillium oxalicum Z2 to flavonoids present in licorice residues and to analyze its transcriptional responses under gradient stress concentrations, thereby providing a theoretical basis for breeding resistant strains and improving fermentation efficiency. Methods P. oxalicum Z2 was treated with different concentrations of licorice flavonoids (0, 0.25, 0.50, 1.00, and 2.00 mg/mL). Physiological indicators were systematically measured. Transcriptome sequencing was conducted to identify differentially expressed genes and enriched KEGG/GO pathways, revealing the molecular adaptation mechanisms of P. oxalicum Z2 under flavonoid stress. Results As the flavonoid concentration increased, P. oxalicum Z2 maintained high biomass and cellulase activity. Under the highest flavonoid concentration (2.00 mg/mL), its biomass reached 4.840 g/L, and the activities of endoglucanase, β-glucosidase, filter paper enzyme, and xylanase reached 74.78, 3.24, 6.99, and 562.5 U/mL, respectively, which were more than ten folds of those in the conventional P. oxalicum strain. Transcriptome analysis showed that the number of differentially expressed genes increased in a concentration-dependent manner, from 818 in the Low group to 3 945 in the Super group. Under low stress (≤1.00 mg/mL), the strain reprogrammed carbohydrate metabolism and ABC transporter pathways to adjust carbon utilization and sustain essential cellular activities. Under high stress (2.00 mg/mL), it further activated the ribosome and oxidative phosphorylation pathways, with 65 genes upregulated in the ribosome pathway. Conclusion This study identified key genes (e.g., EF-Tu, SecY,and FtsY) and core pathways (e.g., oxidative phosphorylation and ABC transporter), demonstrating that P. oxalicum Z2 employed tiered responses to licorice flavonoid stress. Specifically, the strain remodels sugar metabolism and transmembrane transport under low flavonoid stress to maintain survival and activates ribosome and energy metabolism compensation mechanisms under high flavonoid stress.

licorice residue  /  Penicillium oxalicum  /  transcriptomics  /  tolerance mechanisms
何志豪, 杨秉乾, 王玺瑞, 鱼晨, 常思源, 张森. 基于转录组学探究草酸青霉Z2响应甘草黄酮胁迫的分层响应耐受机制. 微生物学报, 2026 , 66 (7) : 3508 -3525 . DOI: 10.13343/j.cnki.wsxb.20250938
Zhihao HE, Bingqian YANG, Xirui WANG, Chen YU, Siyuan CHANG, Sen ZHANG. Investigating the tiered responses and tolerance mechanisms of Penicillium oxalicum Z2 to licorice flavonoids based on transcriptomics[J]. Acta Microbiologica Sinica, 2026 , 66 (7) : 3508 -3525 . DOI: 10.13343/j.cnki.wsxb.20250938
随着中医药的快速发展,中药的需求量逐年上升[1]。甘草作为中药产业的代表性植物,其加工过程每年会产生超过600万t的药渣。这些药渣富含纤维素、半纤维素、黄酮类等活性成分,是极具潜力的生物质资源[2]。目前,甘草药渣的处理方式包括焚烧、堆肥等,资源化利用途径单一,造成了资源的严重浪费。因此,亟需探索高值化的利用途径。
微生物法因其绿色环保、高效低耗的特点,逐渐成为生物质资源循环利用的研究热点[3]。中药废弃物的微生物转化不仅能够实现资源的高值化利用,还能减少环境污染,符合可持续发展的理念[4]。其中,青霉属[5]、木霉属[6]等因其具有强大的纤维素酶与半纤维素酶分泌能力,在木质纤维素类废弃物的生物转化中展现出显著优势。研究表明,利用高产纤维素酶的草酸青霉菌株对甘草药渣进行液体发酵可高效转化其中的多糖成分,生成具有工业应用价值的糖类产物[7]。然而,药渣中富含的黄酮类化合物对微生物发酵过程造成了一定的挑战。
黄酮类化合物是一类广泛存在于植物中的天然活性成分[8]。研究发现,在发酵过程中药渣中的黄酮类化合物对微生物的菌丝生长及代谢活性存在显著抑制作用,导致发酵效率降低、生产成本增加[9]。黄酮类化合物的广谱抑菌活性已在多种微生物(如金黄色葡萄球菌[10]、枯草芽孢杆菌[11]等)中得到验证,提示其在发酵体系中对生产菌株可能产生潜在抑制作用。为缓解这一制约,目前研究多集中在菌种选育[12]、发酵工艺优化[13]等应用策略。然而,这些研究多停留在表型观察与工艺调整层面,对于黄酮类物质在工业发酵环境中对生产菌株的具体作用机制仍不明确[14]。特别是关于黄酮类物质在发酵过程中的动态抑制效应、关键细胞靶点及分子调控网络,目前仍缺乏系统解析。
课题组前期从甘草根际土壤中筛选出了多株草酸青霉菌株,其中草酸青霉Z2对甘草黄酮具有高耐受性,该菌株通过原位酶解可实现11.45 g/L的还原糖产率[15]。本研究以高耐受菌株草酸青霉Z2为研究对象,通过设置甘草黄酮浓度梯度测定其生理指标,重点关注生物量和胞外酶活,分析其耐受性能。结合转录组学分析,揭示草酸青霉Z2应对黄酮胁迫的耐受机制,为后续构建抗逆高产菌株以及优化甘草药渣的微生物转化工艺提供关键的理论依据与基因靶点。
本研究采用的甘草药渣(Glycyrrhiza uralensis residue)来自江苏天晟药业股份有限公司,甘草黄酮提取自甘草药渣。PDB液体培养基(g/L):马铃薯200.0,葡萄糖20.0,充分混合后,121 ℃灭菌20 min。PDA固体培养基:在PDB液体培养基配方基础上,添加20.0 g/L琼脂粉,在121 ℃条件下灭菌20 min。发酵培养基:KH2PO4 3.0 g/L,(NH4)2SO4 2.0 g/L,MgSO4·7H2O 0.5 g/L,尿素5.0 g/L,FeSO4·7H2O 7.5 mg/L,MnSO4·7H2O 2.5 mg/L,ZnSO4·7H2O 3.6 mg/L,CoCl2·6H2O 3.7 mg/L,CaCl2 0.5 g/L,微晶纤维素10.0 g/L,吐温-80 5.0 mL/L。
将甘草药渣粉碎,加入10倍量浓度为70%的乙醇,浸泡30 min后,回流提取2次,每次60 min,过滤,合并滤液,浓缩,用大孔树脂AB-8进行纯化,浓缩冻干即得甘草总黄酮提取物。将甘草黄酮溶于甲醇,精准量取4.0 mL样品溶液置于50 mL容量瓶中,首先边摇边滴入0.4 mL 5% NaNO2溶液,静置6 min,再边摇边加入0.4 mL 10% Al(NO3)3溶液,随后静置6 min,再边摇边加入5.0 mL、4% NaOH溶液,再次静置15 min,最后用甲醇溶液定容,在510 nm波长下测量吸光度,代入标准曲线计算其浓度。
配制含有不同甘草黄酮浓度(0、0.25、0.50、1.00、2.00 mg/mL)的PDA培养基,每个浓度设置3个平行。取5 mL在PDA液体培养基中培养好的菌丝体悬液,在高速离心机中8 000 r/min离心10 min,弃去上清液,收集菌丝体沉淀。用去离子水清洗沉淀3次,每次均以8 000 r/min离心10 min,收集菌丝体并置于50 ℃烘箱中烘至恒重,用于计算每升培养基中的细胞干重(dry cell weight, DCW),计算如公式(1)所示。
DCW (g/L)=(器具及细胞烘干后的质量-器具质量)/培养基体积
以3 g不含抑菌成分的普通农业废物玉米芯为碳源,在发酵培养基中进行培养,向培养基中加入不同质量的甘草黄酮,使其浓度达到0、0.25、0.50、1.00、2.00 mg/mL。采用Chang等[16]的方法测定羧甲基纤维素酶(CMCase)、滤纸酶(FPase)和木聚糖酶(xylanase)活力,酶活性单位(U)表示为每毫升酶溶液每分钟释放的葡萄糖或木糖的微摩尔数。
向培养3 d的草酸青霉液体PDB培养基中加入甘草黄酮,使其浓度为0、0.25、0.50、1.00、2.00 mg/mL,28 ℃、180 r/min培养。分别于2、4、6、8、10、12 h取1 mL菌液,6 500 r/min离心10 min后收集上清液。根据Bradford蛋白浓度测定试剂盒操作说明书制作标准曲线。最后取20 μL待测样品,测定其在595 nm波长处的吸收值,代入标准曲线,计算上清液中的蛋白浓度,试验重复3次。于2、4、6、8、10、12 h吸取PDB培养基上清液,使用电导率仪测量其电导率变化。
使用TRIzol试剂(赛默飞世尔科技公司)提取样品总RNA,采用NanoDrop 2000 (ThermoFisher Scientific公司)检测其浓度及纯度,利用Agilent 2100 Bioanalyzer (安捷伦科技有限公司)检测其完整性。选择总量≥1 μg的总RNA,使用链特异性建库试剂盒NEBNext Ultra Directional RNA Library Prep Kit for Illumina,通过oligo(dT)磁珠富集带有polyA尾的mRNA,随后通过离子打断的方式使用二价阳离子将mRNA随机打断。以片段化的mRNA为模板,随机寡核苷酸为引物,合成cDNA。对双链cDNA进行纯化,之后进行双末端修复及3′端引入“A”碱基并连接测序接头。用AMPure XP beads筛选400-500 bp左右的cDNA,进行PCR扩增并再次使用AMPure XP beads纯化PCR产物,最终获得文库。
使用DESeq2 (v1.38.3)对比较组之间的基因表达进行差异分析。依据STRING数据库(https://string-db.org/)进行蛋白互作分析,以揭示目的基因之间的作用关系。使用clusterProfiler (v4.6.0)进行富集分析,通过超几何分布方法计算P-value (一般显著富集的标准为P<0.05),找出差异基因(all/up/down)显著富集的GO term/KEGG pathway,从而确定差异基因行使的主要生物学功能。采用VarScan (v2.3.9)程序获取SNP和InDel位点。
所有试验均设置3个重复组,结果用平均值±标准差表示,采用OriginPro 2021作图。数据结果以P<0.05为差异显著,P<0.01为差异极显著。
图1所示,以芦丁为标准品制备标准曲线,将甘草黄酮溶于甲醇制成1.00 mg/mL的溶液,稀释至5×10-3 mg/mL后采用比色法测定其黄酮含量。结果表明,OD510为0.5,原浓度甘草黄酮中总黄酮含量为96.24%,所制备的甘草黄酮提取物纯度较高,适用于后续发酵胁迫实验。
图2所示,对3株不同草酸青霉(Penicillium oxalicum)生物量的动态监测结果显示,在对照组中,草酸青霉呈现典型的指数生长模式,菌体干重(DCW)在0 h至72 h内迅速增加至9 g/L以上。草酸青霉菌株G4对甘草黄酮的耐受性较弱,在High组和Super组处理下其生长受到显著抑制,基本处于停滞状态。草酸青霉菌株G7与G4的生长响应较为相似,但在Low组条件下能够恢复至正常生长水平。相比之下,草酸青霉Z2表现出较强的黄酮耐受能力,即使在Middle组处理下,其菌体干重仍可恢复至接近对照组的水平;而在更高黄酮浓度条件下,该菌株仍能维持一定的生长能力,菌体干重达到(4.840±0.241) g/L。上述结果表明,草酸青霉Z2对甘草黄酮具有显著耐受性,能够在较高质量浓度黄酮构成的胁迫环境中维持菌体生长。
图3所示,发酵周期监测结果表明,甘草黄酮的添加显著改变了草酸青霉胞外水解酶系的合成动态。在CK组中,内切葡聚糖酶、β-葡糖苷酶、滤纸酶和木聚糖酶的活力均维持在较高水平,分别达到(129.800±4.870)、(7.040±0.383)、(11.940±0.297)、(960.410±29.200) U/mL;但随着黄酮浓度升高,所有试验组的酶活力均呈现下降趋势,且这种抑制效应与黄酮浓度呈正相关。具体而言,Low组处理下酶活力下降不明显,而在Super组中抑制最为显著。
菌株间比较发现,G4和G7对黄酮较为敏感,其在高浓度处理下的胞外酶活力较CK组下降了90%以上,处于极低水平。与之相反,菌株Z2则表现出卓越的耐受性:在Middle组黄酮条件下,其酶活力可维持在与CK组相当的水平;即使处于更高浓度的黄酮环境中,它依然能保持相对较高的胞外水解酶活力。
当真菌处于正常生长状态时细胞膜结构完整,仅允许少量蛋白质渗出以维持基本物质交换与代谢活动,因此胞外可溶性蛋白含量维持相对稳定。如图4所示,电导率与胞外蛋白浓度测定结果显示,在整个监测周期内,CK组的胞外蛋白浓度与培养液电导率均维持稳定,波动范围较小。经甘草黄酮处理后,草酸青霉的胞外蛋白浓度与培养液电导率均呈现时间与浓度依赖性上升。其中,Low组与CK组差异不显著;随着黄酮浓度升高,Middle组、High组与Super组的胞外蛋白泄漏逐渐加剧,尤以Super组最为显著,其电导率和胞外蛋白浓度分别达到(520.30±18.25) μs/cm和(0.034 3±0.001 8) mg/mL。上述结果表明,甘草黄酮影响了草酸青霉Z2的细胞膜完整性。
如图5A5B所示,对差异基因进行分析,结果表明Low组、Middle组、High组和Super组与CK组相比分别有818个(448个上调和370个下调)、1 241个(470个上调和771个下调)、2 030个(1 141个上调和899个下调)以及3 945个(1 870个上调和2 075个下调)差异基因,随着处理组黄酮浓度升高,差异基因数目逐渐增多,其中Super组拥有最多的差异基因数量。如图5C所示,从维恩图可以发现,8 034个差异基因中只有292个基因呈现相同的表达模式。如图6所示,把差异基因分成9个cluster进行聚类分析和趋势分析,其中Low组与CK组基因的表达模式比较相近,在cluster 3和cluster 4中展现出高表达。High组、Super组与CK组基因表达模式显著不同,Super组在cluster 8和cluster 9中展现出高表达。本研究发现黄酮处理显著影响了基因的表达模式,不同组别的相同基因分别展现不同的表达水平。
图7所示,利用KEGG数据库对信号通路进行分析,结果表明与CK组相比,Low组富集在代谢通路和环境信息处理通路。代谢通路包括:碳水化合物代谢(丙酮酸代谢、乙醛酸和二羧酸代谢、戊糖和葡糖醛酸相互转化、糖酵解/糖异生、淀粉和蔗糖代谢、果糖和甘露糖代谢、抗坏血酸和醛糖代谢、半乳糖代谢、丁酸盐代谢)、脂质代谢(角质、木栓质和蜡质生物合成、脂肪酸降解、醚脂质代谢、亚油酸代谢)、氨基酸代谢(酪氨酸代谢、精氨酸和脯氨酸代谢、色氨酸代谢、苯丙氨酸代谢)等。环境信息处理通路包括:膜转运(ABC转运蛋白)。Middle组和High组与Low组结果相似,大多富集在碳水化合物代谢、脂质代谢、氨基酸代谢以及膜转运等。然而,Super组与其他组别完全不同,与对照组相比,其富集在遗传信息处理、新陈代谢、细胞过程以及环境信息处理通路。遗传信息处理通路包括:翻译(核糖体);代谢通路包括:能量代谢(氧化磷酸化)、氨基酸代谢(精氨酸合成、缬氨酸、亮氨酸和异亮氨酸合成)、脂质代谢(脂肪酸合成);细胞过程通路包括:细胞生长与死亡;环境信息处理通路包括:MAPK信号通路。
图8所示,利用GO数据库进行分析,与CK组相比,Low组有12个MF术语、7个BP术语和1个CC术语富集,其中氧化还原酶活性、碳水化合物代谢过程以及细胞外区域分别是MF、BP和CC中最富集的术语。Middle组有14个MF术语、3个BP术语和3个CC术语富集,其中氧化还原酶活性、跨膜转运以及膜的内在成分分别是MF、BP和CC中最富集的术语。High组有12个MF术语、5个BP术语和3个CC术语富集,其中MF、BP和CC中最富集的术语分别是氧化还原酶活性、跨膜转运以及细胞外区域。与KEGG结果一致,Low组、Middle组和High组富集的术语同样相似。Super组与其他组别最为不同,其有7个MF术语、9个BP术语和4个CC术语,初级主动跨膜转运活性、氮循环代谢过程以及线粒体膜间空间分别是MF、BP和CC中最富集的术语。
KEGG与GO富集分析表明,甘草黄酮对草酸青霉的抑制作用呈现显著的浓度依赖性分层响应调控。在低浓度下,黄酮主要干扰初级代谢通路;而当浓度升高时,则转而破坏核糖体生物合成等遗传信息处理过程。
采用VarScan (v2.3.9)程序获取SNP位点,过滤标准为:(1) SNP位点碱基Q>20;(2) 覆盖该位点的reads数目>8;(3) 支持突变位点的reads数目>2;(4) SNP位点的P<0.01。变异位点分析如图9所示,Super组拥有最多的SNP数量,Super组3个样本纯合子变异超过250个,杂合子变异超过1 000个。这说明高浓度的甘草黄酮会增加核苷酸变异的概率,低浓度甘草黄酮影响较小。根据突变类型统计,transition占据了74.85%,是transversion的3倍以上。
代谢稳态是微生物在胁迫环境下维持生存与生长的核心机制[17]。本研究观察到,在低浓度(≤1.00 mg/mL)甘草黄酮胁迫下,草酸青霉菌株Z2通过启动代谢来应对环境压力,其生物量积累与胞外水解酶的合成均未受到显著影响,细胞结构也无明显损伤,这表明其代谢系统在此条件下能够有效维持稳态。这一耐受现象与腐败希瓦氏菌(Shewanella putrefaciens)[18]及意大利青霉(Penicillium italicum)[19]中报道的黄酮类物质破坏细胞膜结构,影响氨基酸代谢并导致其生长迟缓的结果有所不同。草酸青霉Z2对黄酮类物质具有良好的耐受性,这种差异表明外部的胁迫信号可能触发了草酸青霉Z2内部的代谢网络重塑,使其能够调控关键通路,从而在黄酮胁迫下保持正常的生长与生理功能。
本研究通过KEGG富集分析从基因表达层面初步揭示了草酸青霉Z2在低浓度黄酮胁迫下维持代谢稳态的机制。差异表达基因显著富集于碳水化合物代谢相关通路。其中,丙酮酸代谢作为糖酵解的终末途径和三羧酸循环的关键枢纽,其适应性调整保障了核心代谢中ATP的供应[20]。同时,乙醛酸和二羧酸代谢通路的激活表明细胞能够利用脂肪酸等替代碳源,通过合成葡萄糖以满足能量与生物合成需求[21]。该循环与戊糖磷酸途径协同作用,共同维持了细胞在胁迫下的氧化还原稳态。此外,ABC转运蛋白通路的富集提示,细胞可能通过调控跨膜转运过程以限制黄酮摄入或增强底物利用效率,从而优化代谢过程[22]。这一系列代谢通路的调整表明,草酸青霉Z2能够通过代谢调整应对黄酮引发的碳源利用压力,这为其在胁迫下维持菌体生长与酶活提供了基础。
当抑菌胁迫强度超过微生物的承受极限时,细胞会启动更为紧急的应激程序以维持生存。本研究中,在高浓度(2.00 mg/mL,Super组)甘草黄酮胁迫下,草酸青霉Z2的生长受到了一定程度的抑制,其DCW与CK组相比下降54.6%,内切葡聚糖酶、β-葡糖苷酶、滤纸酶和木聚糖酶活力分别下降42.4%、53.8%、41.5%和41.4%,并伴随细胞膜完整性受损。
本研究的KEGG富集分析显示,高浓度黄酮激活了核糖体与氧化磷酸化通路。如图10所示,在核糖体通路中,65个基因上调,1个基因下调,其中EF-TuSecYFtsY等参与蛋白质翻译、转运与折叠的关键基因普遍上调。EF-Tu能够增强生物对极端环境的敏感性,从而提高抗性[23]SecYFtsY能够调控细胞膜的修复[24]。这表明细胞正在补偿因细胞膜损伤和毒性干扰造成的蛋白质功能损失,维持生命活动所需蛋白组的合成。同时,氧化磷酸化通路中ATP合成酶相关基因的全面上调揭示了细胞正面临严重的氧化应激情况[25]。试图通过最大化产能效率来应对由膜系统损伤导致的能量耗散,为上述高耗能的补偿行为提供动力。
生长曲线与生物量是评估微生物胁迫响应的关键指标[26]。通常,在抑菌物质胁迫下微生物生长会受到抑制,即便在亚抑制浓度下也可能出现生物量下降[27]。丝状真菌作为微生物中的一类代表,广泛分布于自然界,常通过分泌多种水解酶降解植物组织[28]。这类酶系是其利用纤维素类资源的核心工具[29],胞外酶活力的大小也反映了真菌在抑菌物质下抵抗外界胁迫的能力。
以往研究表明,天然提取物可显著抑制真菌胞外酶活与生物量积累[30-31],其机制多与活性成分干扰代谢、破坏细胞结构有关[32]。目前青霉对天然提取物较为敏感,张甜甜等[33]研究表明,1 mg/mL五味子提取物即可显著抑制青霉的生长与孢子萌发。Guo等[34]研究发现,天然化合物在15.63 mg/L的浓度下即可显著抑制青霉生长,通过多组学分析表明其主要干扰了核糖体合成等通路。本研究中,草酸青霉Z2对甘草黄酮表现出区别于普通菌株的响应行为:在1.00 mg/mL黄酮处理下仍能维持接近正常的生物量积累,其胞外酶活力保持高水平,显示出较强的耐受能力;即便在2.00 mg/mL的高浓度胁迫下,虽生长受到显著抑制,但菌体并未丧失全部生理活性。
Feng等[35]通过转录组分析发现,青霉面对抗真菌成分时核糖体生物合成、氨基酸代谢相关通路富集;Lin等[36]研究发现差异基因表达主要涉及遗传信息过程和能量代谢等;Feng等[37]通过转录组和蛋白组联合分析发现,抑菌成分调控并参与了青霉初级代谢、细胞壁代谢等基因或蛋白质。以上研究表明青霉在面对外部抑菌成分胁迫时会启动代谢和遗传信息相关的通路来抵抗外部条件的压力。然而,本研究针对耐受菌株草酸青霉Z2发现,针对不同阶段的抑菌浓度,真菌展现出对外部胁迫的分层响应机制。本研究发现的结果对甘草药渣的直接发酵具有指导性作用,在工艺设计中应当依据胁迫程度采取分段策略。例如,在发酵初期或使用高黄酮含量药渣时可先通过低浓度条件对菌株进行适应性驯化,激活其代谢调整能力;进入主发酵阶段后,关注能量代谢水平,可通过补料或调节通气来缓解高浓度胁迫下的代谢负担。同时,可通过组成型或诱导型强启动子驱动EF-TuSecYFtsY等基因在草酸青霉中过表达,旨在提前或增强其保护效应,从而构建耐受性更强的基础底盘菌株。这种基于菌株内在应答规律的工艺调控,有望实现发酵效率与菌株抗逆性的同步提升,为甘草药渣的规模化生物发酵提供切实可行的操作依据。
本研究基于KEGG与GO富集分析结果发现,草酸青霉Z2对甘草黄酮具有“浓度依赖性的分层响应耐受机制”。在低浓度(≤1.00 mg/mL)胁迫下,Z2主要通过代谢层面的适应性重编程维持生长与酶活,生物量受影响不大;而在高浓度(2.00 mg/mL)胁迫下,菌体启动了核糖体生物合成与能量代谢相关通路进行补偿,以维持细胞在严重胁迫下的生存能力。
本研究系统揭示了草酸青霉对甘草黄酮的浓度依赖性分层响应耐受机制。在低浓度胁迫下,菌株通过重塑碳水化合物代谢与激活ABC转运体,通过代谢调整维持生存与生长;而在高浓度胁迫下,则启动以核糖体生物合成增强和氧化磷酸化补偿为特征的耐受机制,维持细胞基本功能。研究表明,草酸青霉能够通过从“代谢适应”到“应激补偿”的基因表达策略应对黄酮胁迫:低浓度时调整碳代谢过程,高浓度时则保障遗传信息处理与能量供应。
  • 中药资源循环利用关键技术研发及产业化示范项目(2025YFC3509100)
  • 国家自然科学基金(82574719)
  • 国家中医药管理局中医药创新团队及人才支持计划(ZYYCXTD-D-202005)
  • 山东省重点研发计划(2021SFGC1203)
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doi: 10.13343/j.cnki.wsxb.20250938
  • 接收时间:2025-12-16
  • 首发时间:2026-07-06
  • 出版时间:2026-07-04
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  • 收稿日期:2025-12-16
  • 录用日期:2026-01-21
基金
The Key Technology Research and Industrialization Demonstration Project for the Recycling and Utilization of Traditional Chinese Medicine Resources(2025YFC3509100)
中药资源循环利用关键技术研发及产业化示范项目(2025YFC3509100)
The National Natural Science Foundation of China(82574719)
国家自然科学基金(82574719)
The Project of the National Administration of Traditional Chinese Medicine for Supporting Innovation Teams and Talents in Traditional Chinese Medicine(ZYYCXTD-D-202005)
国家中医药管理局中医药创新团队及人才支持计划(ZYYCXTD-D-202005)
The Shandong Provincial Key Research and Development Program(2021SFGC1203)
山东省重点研发计划(2021SFGC1203)
作者信息
    1.南京中医药大学,江苏省中药资源产业化过程协同创新中心,江苏 南京
    2.南京科技职业学院,江苏省生物基平台化合物催化工程技术研究开发中心,江苏 南京

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2种不同金属材料的力学参数

Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)

Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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