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Article(id=1320166882880106854, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1320166565862027513, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2026-0478, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1777046400000, receivedDateStr=2026-04-25, revisedDate=1781539200000, revisedDateStr=2026-06-16, acceptedDate=null, acceptedDateStr=null, onlineDate=1792681912392, onlineDateStr=2026-10-22, pubDate=1789142400000, pubDateStr=2026-09-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1792681912392, onlineIssueDateStr=2026-10-22, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1792681912392, creator=13701087609, updateTime=1792681912392, updator=13701087609, issue=Issue{id=1320166565862027513, tenantId=1146029695717560320, journalId=1189982191388893191, year='2026', volume='61', issue='9', pageStart='1', pageEnd='3490', issueExtLink='null', onlineDate='null', pubDate='1789142400000', pubDateStr='2026-09-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1792681836810, creator='13701087609', updateTime=1792681836810, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext=null, issueFiles=null, downloadFileDto=null}, startPage=3475, endPage=3483, ext={EN=ArticleExt(id=1320166883098210663, articleId=1320166882880106854, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Construction and metabolic engineering optimization of a Saccharomyces cerevisiae chassis strain for high-level production of α-humulene, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

α-Humulene is a natural sesquiterpenoid with multiple pharmacological activities including anti-inflammatory, antibacterial and antitumor effects, and also serves as the key precursor for zerumbone biosynthesis. In this study, Saccharomyces cerevisiae was used as the chassis for heterologous production of α-humulene. One novel α-humulene synthase gene, AsTPS8, was mined from the transcriptome of Aquilaria sinensis. Compared with the reported ZSS1 from Zingiber zerumbet, AsTPS8 exhibited higher catalytic efficiency. Furthermore, fusion expression of ERG20 with AsTPS8 via a (GGGS)2 linker combined with multi-copy integration at the TY1 locus increased the α-humulene titer to 1.41 g·L-1 in shake flasks. On this basis, the bypass phosphatase gene LPP1 was knocked out using CRISPR/Cas9, and the transcriptional regulators IME4 and UPC2-1 were simultaneously introduced, further elevating the titer to 1.80 g·L-1. Finally, fed-batch high-density fermentation in a 5 L bioreactor achieved an α-humulene titer of 22.02 g·L-1 at 144 h, representing the highest reported level to date. This study provides an efficient chassis strain and technical support for the industrial production of α-humulene and downstream zerumbone.

, authors=Gui-li ZHANG1, Si-qi LAI2, Ting-ting CHEN2, Chang-yu GOU2, Ruo-lan SHI1, Xin-ye MA2, Li-xin DUAN2, *, Feng WANG1, *, authorsList=Gui-li ZHANG, Si-qi LAI, Ting-ting CHEN, Chang-yu GOU, Ruo-lan SHI, Xin-ye MA, Li-xin DUAN, Feng WANG, authorCompany=null, correspAuthors=Li-xin DUAN, Feng WANG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2026 Acta Pharmaceutica Sinica. All rights reserved., 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=1320166885849674102, articleId=1320166882880106854, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=高产α-石竹烯酿酒酵母菌株的构建与代谢工程优化, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

α-石竹烯(α-humulene) 是具有抗炎、抗菌、抗肿瘤等多种药理活性的天然倍半萜类化合物, 亦是花姜酮生物合成的关键前体。本研究以酿酒酵母为底盘, 从白木香(Aquilaria sinensis) 转录组中挖掘到一条新型α-石竹烯合酶基因AsTPS8, 与已报道的红球姜来源的ZSS1相比, AsTPS8表现出更高的催化效率。进一步通过ERG20与AsTPS8的(GGGS)2融合表达, 结合TY1位点多拷贝整合, 使α-石竹烯摇瓶产量达到1.41 g·L-1。在此基础上, 利用CRISPR/Cas9系统敲除旁路磷酸酶基因LPP1并同步引入转录调控因子IME4和UPC2-1, 使摇瓶产量提升至1.80 g·L-1。最终在5 L发酵罐中进行分批补料高密度发酵, 144 h时α-石竹烯产量达到22.02 g·L-1, 为目前报道的最高水平。本研究为α-石竹烯及花姜酮的工业化生产提供了高效底盘菌株和技术支撑。

, authors=张桂丽1, 赖思琪2, 陈婷婷2, 苟昌雨2, 时若兰1, 马新业2, 段礼新2, *, 王峰1, *, authorsList=张桂丽, 赖思琪, 陈婷婷, 苟昌雨, 时若兰, 马新业, 段礼新, 王峰, authorCompany=null, correspAuthors=段礼新, 王峰, authorNote=

# 共同第一作者.

作者贡献:

张桂丽负责实验设计、实验操作、数据分析和论文撰写; 赖思琪负责数据分析; 陈婷婷负责生信分析; 苟昌雨和时若兰参与实验操作; 马新业提供白木香转录组测序数据; 王峰及段礼新负责实验指导和文章修改。

, correspAuthorsNote=
段礼新，E-mail:
王峰，E-mail:
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A: Total ion chromatograms of ZE01 and α-humulene standard; B: Mass spectra of the 16.3 min peak from ZE02 (upper) and the standard (lower), figureFileSmall=My4avT627Y3mPy/4ED5q3A==, figureFileBig=lMa3QxgaIUAO9T+JSr9X6A==, tableContent=null), ArticleFig(id=1320166892082409910, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1320166882880106854, language=EN, label=null, caption=null, figureFileSmall=kBRpvGpyRGCp7gfxYEZ8bQ==, figureFileBig=lvElUGHvUkUJxDHSdtouWA==, tableContent=null), ArticleFig(id=1320166892162101687, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1320166882880106854, language=CN, label=Fig.4, caption=Detection results of 50 mL shake-flask fermentation of strains ZE02 and ZE03. A: GC-MS total ion chromatograms showing the characteristic α-humulene peak in fermentation products of strains ZE02 (AsTPS8) and ZE03 (ZSS1); B: α-Humulene titers (mg·L-1) produced by different synthases in shake-flask cultures. n = 3, $ \overline{x} $ ± s. **P < 0.01, figureFileSmall=kBRpvGpyRGCp7gfxYEZ8bQ==, figureFileBig=lvElUGHvUkUJxDHSdtouWA==, tableContent=null), ArticleFig(id=1320166892220821944, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1320166882880106854, language=EN, label=null, caption=null, figureFileSmall=YDQ9efRWOv49HHamxQX2sA==, figureFileBig=lpH6EOZcKLdVg2H/uN/IVA==, tableContent=null), ArticleFig(id=1320166892317290937, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1320166882880106854, language=CN, label=Fig.5, caption=Effect of enzyme fusion expression on α-humulene biosynthesis. A: Schematic diagram of strain construction; B: HPLC chromatograms of α-humulene standard and fermentation products of strains ZE04 and ZE05; C: Comparison of α-humulene titers (g·L-1) produced by ZE04 and ZE05 in shake-flask cultures. n = 3, $ \overline{x} $ ± s. ****P < 0.000 1, figureFileSmall=YDQ9efRWOv49HHamxQX2sA==, figureFileBig=lpH6EOZcKLdVg2H/uN/IVA==, tableContent=null), ArticleFig(id=1320166892392788410, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1320166882880106854, language=EN, label=null, caption=null, figureFileSmall=MQnmsI+PN+IaJk65Zrtxew==, figureFileBig=CCOnv3R+ruVTPUV6H72sjA==, tableContent=null), ArticleFig(id=1320166892485063099, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1320166882880106854, language=CN, label=Fig.6, caption=Effects of metabolic pathway optimization on α-humulene biosynthesis. A: Schematic diagram of metabolic engineering strategies; B: HPLC chromatograms of fermentation products from strains ZE05 and ZE06, with the retention time of the α-humulene standard indicated by the dashed line; C: Comparison of α-humulene titers (g·L-1) produced by ZE05 and ZE06 in shake-flask cultures. n = 3, $ \overline{x} $ ± s. ****P < 0.000 1, figureFileSmall=MQnmsI+PN+IaJk65Zrtxew==, figureFileBig=CCOnv3R+ruVTPUV6H72sjA==, tableContent=null), ArticleFig(id=1320166892560560572, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1320166882880106854, language=EN, label=null, caption=null, figureFileSmall=vXc+ODmXq2H+k2gnJHT2RA==, figureFileBig=60ykWe0OgN4nBF2izA4hSw==, tableContent=null), ArticleFig(id=1320166892627669437, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1320166882880106854, language=CN, label=Fig.7, caption=Growth and α-humulene production of strain ZE06 during fed-batch fermentation in a 5 L bioreactor, figureFileSmall=vXc+ODmXq2H+k2gnJHT2RA==, figureFileBig=60ykWe0OgN4nBF2izA4hSw==, tableContent=null), ArticleFig(id=1320166892728332734, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1320166882880106854, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Plasmids and strainsDescriptionSource
pESC-URAYeast expression vector with URA selection makerStored in the lab
pESC-URA-AsTPS8pESC-URA-GAL1p-AsTPS8-CYC1tThis study
pESC-URA-ZSS1pESC-URA-GAL1p-ZSS1-CYC1tThis study
pCfb-2988-TRPYeast expression vector with TY1 integration sites and TRP selection makerStored in the lab
pCfb-2988-TRP-ERG20pCfb-2988-TRP; GAL1p-ERG20-CYC1tStored in the lab
pESC-URA-ERG20-(GGGS)2-AsTPS8pESC-URA-GAL1p-ERG20-(GGGS)2-AsTPS8-CYC1tThis study
pESC-URA-IME4-UPC2-1pESC-URA-PGK1p-IME4-ADH1t-GAL1p-UPC2-1-CYC1tStored in the lab
pCRCT-GTR-URA3-ΔLPP1Cas9 plasmid (ΔLPP1)Stored in the lab
yYF601CEN.PK2-1C; erg9:: KanMX-CTR3p-ERG9; leu2-3, 112:: His3MX6-GAL1p-ERG19/GAL10p-ERG8; ura3-52:: GAL1p-EfMvaSA110G/GAL10p-EfMvaE; his3Δ1:: hphMX4-GAL1p-ERG12/GAL10p-IDI1; 308a: : GAL1p-ERG20F96W-N127WPresented by Xiaozhou Luo team
ZE01yYF601; GAL80ΔStored in the lab
ZE02ZE00; pESC-URA-AsTPS8This study
ZE03ZE00; pESC-URA-ZSS1This study
ZE04ZE00; TY1:: TRP-GAL1p-AsTPS8-CYC1tThis study
ZE05ZE00; TY1:: TRP-GAL1p-ERG20-(GGGS)2-AsTPS8-CYC1tThis study
ZE06ZE05; LPP1Δ: : URA3-PGK1p-IME4-ADH1t-GAL1p-UPC2-1-CYC1tThis study
), ArticleFig(id=1320166894007595455, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1320166882880106854, language=CN, label=Tab.1, caption=

Plasmids and strains used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
Plasmids and strainsDescriptionSource
pESC-URAYeast expression vector with URA selection makerStored in the lab
pESC-URA-AsTPS8pESC-URA-GAL1p-AsTPS8-CYC1tThis study
pESC-URA-ZSS1pESC-URA-GAL1p-ZSS1-CYC1tThis study
pCfb-2988-TRPYeast expression vector with TY1 integration sites and TRP selection makerStored in the lab
pCfb-2988-TRP-ERG20pCfb-2988-TRP; GAL1p-ERG20-CYC1tStored in the lab
pESC-URA-ERG20-(GGGS)2-AsTPS8pESC-URA-GAL1p-ERG20-(GGGS)2-AsTPS8-CYC1tThis study
pESC-URA-IME4-UPC2-1pESC-URA-PGK1p-IME4-ADH1t-GAL1p-UPC2-1-CYC1tStored in the lab
pCRCT-GTR-URA3-ΔLPP1Cas9 plasmid (ΔLPP1)Stored in the lab
yYF601CEN.PK2-1C; erg9:: KanMX-CTR3p-ERG9; leu2-3, 112:: His3MX6-GAL1p-ERG19/GAL10p-ERG8; ura3-52:: GAL1p-EfMvaSA110G/GAL10p-EfMvaE; his3Δ1:: hphMX4-GAL1p-ERG12/GAL10p-IDI1; 308a: : GAL1p-ERG20F96W-N127WPresented by Xiaozhou Luo team
ZE01yYF601; GAL80ΔStored in the lab
ZE02ZE00; pESC-URA-AsTPS8This study
ZE03ZE00; pESC-URA-ZSS1This study
ZE04ZE00; TY1:: TRP-GAL1p-AsTPS8-CYC1tThis study
ZE05ZE00; TY1:: TRP-GAL1p-ERG20-(GGGS)2-AsTPS8-CYC1tThis study
ZE06ZE05; LPP1Δ: : URA3-PGK1p-IME4-ADH1t-GAL1p-UPC2-1-CYC1tThis study
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高产α-石竹烯酿酒酵母菌株的构建与代谢工程优化
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张桂丽 1 , 赖思琪 2 , 陈婷婷 2 , 苟昌雨 2 , 时若兰 1 , 马新业 2 , 段礼新 2, * , 王峰 1, *
药学学报 | 研究论文 2026,61(9): 3475-3483
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药学学报 |研究论文 2026 , 61 (9) : 3475 -3483
高产α-石竹烯酿酒酵母菌株的构建与代谢工程优化
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张桂丽1, 赖思琪2, 陈婷婷2, 苟昌雨2, 时若兰1, 马新业2, 段礼新2, * , 王峰1, *
作者信息
  • 1广东药科大学中药学院, 广东 广州 510006
  • 2广州中医药大学中药学院, 广东 广州 510006
通讯作者:
段礼新，E-mail:
王峰，E-mail:
作者简介:

# 共同第一作者.

作者贡献:

张桂丽负责实验设计、实验操作、数据分析和论文撰写; 赖思琪负责数据分析; 陈婷婷负责生信分析; 苟昌雨和时若兰参与实验操作; 马新业提供白木香转录组测序数据; 王峰及段礼新负责实验指导和文章修改。

Construction and metabolic engineering optimization of a Saccharomyces cerevisiae chassis strain for high-level production of α-humulene
Gui-li ZHANG1, Si-qi LAI2, Ting-ting CHEN2, Chang-yu GOU2, Ruo-lan SHI1, Xin-ye MA2, Li-xin DUAN2, * , Feng WANG1, *
Affiliations
  • 1School of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China
  • 2School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou 510006, China
出版时间: 2026-09-12 doi: 10.16438/j.0513-4870.2026-0478
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α-石竹烯(α-humulene) 是具有抗炎、抗菌、抗肿瘤等多种药理活性的天然倍半萜类化合物, 亦是花姜酮生物合成的关键前体。本研究以酿酒酵母为底盘, 从白木香(Aquilaria sinensis) 转录组中挖掘到一条新型α-石竹烯合酶基因AsTPS8, 与已报道的红球姜来源的ZSS1相比, AsTPS8表现出更高的催化效率。进一步通过ERG20与AsTPS8的(GGGS)2融合表达, 结合TY1位点多拷贝整合, 使α-石竹烯摇瓶产量达到1.41 g·L-1。在此基础上, 利用CRISPR/Cas9系统敲除旁路磷酸酶基因LPP1并同步引入转录调控因子IME4和UPC2-1, 使摇瓶产量提升至1.80 g·L-1。最终在5 L发酵罐中进行分批补料高密度发酵, 144 h时α-石竹烯产量达到22.02 g·L-1, 为目前报道的最高水平。本研究为α-石竹烯及花姜酮的工业化生产提供了高效底盘菌株和技术支撑。

α-石竹烯  /  酿酒酵母  /  代谢工程  /  萜烯合酶  /  高密度发酵

α-Humulene is a natural sesquiterpenoid with multiple pharmacological activities including anti-inflammatory, antibacterial and antitumor effects, and also serves as the key precursor for zerumbone biosynthesis. In this study, Saccharomyces cerevisiae was used as the chassis for heterologous production of α-humulene. One novel α-humulene synthase gene, AsTPS8, was mined from the transcriptome of Aquilaria sinensis. Compared with the reported ZSS1 from Zingiber zerumbet, AsTPS8 exhibited higher catalytic efficiency. Furthermore, fusion expression of ERG20 with AsTPS8 via a (GGGS)2 linker combined with multi-copy integration at the TY1 locus increased the α-humulene titer to 1.41 g·L-1 in shake flasks. On this basis, the bypass phosphatase gene LPP1 was knocked out using CRISPR/Cas9, and the transcriptional regulators IME4 and UPC2-1 were simultaneously introduced, further elevating the titer to 1.80 g·L-1. Finally, fed-batch high-density fermentation in a 5 L bioreactor achieved an α-humulene titer of 22.02 g·L-1 at 144 h, representing the highest reported level to date. This study provides an efficient chassis strain and technical support for the industrial production of α-humulene and downstream zerumbone.

α-humulene  /  Saccharomyces cerevisiae  /  metabolic engineering  /  terpene synthase  /  high-density fermentation
张桂丽, 赖思琪, 陈婷婷, 苟昌雨, 时若兰, 马新业, 段礼新, 王峰. 高产α-石竹烯酿酒酵母菌株的构建与代谢工程优化. 药学学报, 2026 , 61 (9) : 3475 -3483 . DOI: 10.16438/j.0513-4870.2026-0478
Gui-li ZHANG, Si-qi LAI, Ting-ting CHEN, Chang-yu GOU, Ruo-lan SHI, Xin-ye MA, Li-xin DUAN, Feng WANG. Construction and metabolic engineering optimization of a Saccharomyces cerevisiae chassis strain for high-level production of α-humulene[J]. Acta Pharmaceutica Sinica, 2026 , 61 (9) : 3475 -3483 . DOI: 10.16438/j.0513-4870.2026-0478
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萜类化合物是植物特化代谢产物中结构最丰富、生物活性最广泛的天然产物家族, 在医药、食品、日化等领域具有重要应用价值。倍半萜类化合物因其独特的生理功能而备受关注。α-石竹烯(α-humulene) 作为一种典型的天然倍半萜, 广泛存在于生姜、啤酒花、沉香等多种植物中, 具有抗炎、抗菌、抗肿瘤、镇痛等多重药理活性[1, 2]。更为重要的是, α-石竹烯是花姜酮(zerumbone) 生物合成的关键前体, 依赖α-石竹烯的氧化修饰转化为花姜酮[3]。花姜酮对多种肿瘤细胞有显著的抑制作用, 但植物中含量极低, 严重制约其临床开发[4, 5]。因此, 实现α-石竹烯的高效合成, 对花姜酮的规模化生产及相关医药产业具有重要支撑意义。
目前, α-石竹烯的获取主要依赖植物提取法, 但其在植物中含量极低且受生长周期、气候等环境因素影响显著, 提取过程复杂、成本高昂, 难以满足工业化生产需求[6, 7]。随着合成生物学技术的快速发展, 构建微生物细胞工厂成为实现天然产物高效、可持续合成的理想途径。酿酒酵母(Saccharomyces cerevisiae) 因具有遗传背景清晰、遗传操作简便、发酵过程易于控制、自身具备完整的甲羟戊酸(mevalonate, MVA) 代谢途径可合成萜类前体法尼基焦磷酸(farnesyl pyrophosphate, FPP) 等优势, 已成为异源合成植物源萜类化合物的优选底盘菌株[8, 9]。
构建高产α-石竹烯的酿酒酵母底盘菌株, 核心在于解决三个关键问题: 一是筛选高效的α-石竹烯合酶, 实现前体FPP向α-石竹烯的高效转化; 二是优化代谢途径, 强化前体FPP的供应, 减少旁路代谢的消耗; 三是调控代谢流分配, 进一步提升目标产物的合成效率。α-石竹烯的合成依赖萜烯合酶(terpene synthases, TPSs) 的催化作用, TPSs是萜类化合物合成的关键限速酶, 不同来源的TPSs催化效率存在显著差异, 挖掘新型高效的α-石竹烯合酶基因是提升产量的基础[10]。
在代谢流调控过程中, 多个基因参与酿酒酵母萜类合成途径的调控。UPC2-1基因是酿酒酵母中重要的转录调控因子, 可特异性结合MVA途径关键基因的启动子区域, 显著上调ERG10、ERG13、HMGR等基因的表达, 强化MVA途径通量, 增加FPP积累[11]。IME4基因编码的N6-腺苷甲硫氨酸依赖的甲基转移酶, 参与酵母的RNA m6A甲基化修饰, 其过表达可显著上调糖酵解、乙酰辅酶A合成及MVA途径中甲基化基因的转录水平, 增加异戊二烯类化合物的产量[12]。此外, 酿酒酵母存在旁路代谢途径, LPP1和DPP1基因编码的磷脂酸磷酸酶可将FPP转化为法尼醇, 导致FPP的无效消耗, 敲除这些基因已被证明可增强倍半萜类产物的合成[13]。
本研究以酿酒酵母为底盘, 围绕α-石竹烯的高效合成开展系统性代谢工程改造。首先, 从白木香转录组数据中挖掘新型α-石竹烯合酶基因, 通过系统发育分析与保守结构域鉴定筛选候选基因; 其次, 构建合酶与前体合成关键酶ERG20的融合表达体系, 并结合TY1多拷贝位点整合增强基因表达量; 而后利用CRISPR/Cas9基因编辑技术敲除旁路磷酸酶基因LPP1, 同时引入转录调控因子IME4和UPC2-1以强化MVA途径通量; 最后, 通过5 L发酵罐分批补料高密度发酵验证最优菌株的生产性能, 为α-石竹烯的工业化生产及花姜酮的后续合成提供技术支撑和理论依据。
材料与方法
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实验材料  植物材料: 白木香(Aquilaria sinensis) 采自广州中医药大学药王山, 红球姜(Zingiber zerumbet) 购自广西贵港。菌株与质粒: 大肠杆菌(Escherichia coli) DH5α购自北京全式金生物技术股份有限公司。酿酒酵母起始菌株yYF601由罗小舟团队提供[14], 课题组在此基础上构建了敲除GAL80基因的菌株ZE01。本研究所用的质粒及系列酵母工程菌株汇总见表 1。
主要试剂与仪器  高保真DNA聚合酶Phanta Max Super-Fidelity、同源重组酶ClonExpress Ⅱ购自南京诺唯赞生物科技股份有限公司。限制性内切酶FastDigest系列购自美国Thermo Fisher Scientific公司。植物总RNA提取试剂盒RNAprep Pure Plant Kit、植物基因组DNA提取试剂盒DP321购自北京天根生化科技有限公司。质粒小提试剂盒、PCR产物纯化试剂盒、胶回收试剂盒购自上海生工生物工程技术服务有限公司。Evo M-MLV Plus cDNA合成试剂盒购自艾科瑞生物科技有限公司。α-石竹烯(CAS: 6753-98-6) 标准品购自Macklin公司。正十二烷购自安耐吉化学。气相色谱-质谱联用仪(GC-MS) 为日本Shimadzu公司QP2010SE型。高效液相色谱仪使用Waters HPLC仪器结合Agilent EC-C18。平行生物反应器购自上海迪必尔生物工程有限公司。
植物总RNA提取与cDNA合成  取白木香根、茎、叶组织及红球姜根茎, 液氮研磨后使用RNA prep pure多糖多酚植物总RNA提取试剂盒提取总RNA。以提取的总RNA为模板, 使用Evo M-MLV Plus cDNA合成试剂盒, 按照说明书步骤合成cDNA。
α-石竹烯合酶基因的挖掘与克隆  白木香转录组测序数据由马新业提供, 以已知功能的厚叶沉香(Aquilaria crassna) α-石竹烯合酶蛋白序列(GenBank: AMQ67165.1) 等为诱饵进行本地BLAST同源比对。利用MEGA11软件的ClustalW程序进行多序列比对, 采用邻接法(neighbor-joining) 构建系统发育树(bootstrap = 1 000)。通过NCBI保守结构域数据库(CDD) 和MEME在线工具进行保守基序分析。根据筛选出的候选基因ORF序列设计特异性引物, 以白木香cDNA为模板进行PCR扩增。ZSS1基因以红球姜cDNA为模板进行扩增。PCR产物经纯化后, 与BamHI/HindⅢ双酶切的pESC-URA载体通过同源重组连接, 转化DH5α感受态细胞, 阳性转化子由上海生工生物工程有限公司进行测序验证。
融合表达质粒构建  以pESC-URA-AsTPS8和pCfb-2988-TRP-ERG20为模板, 设计引物扩增AsTPS8和ERG20基因片段, 在ERG20的C端和AsTPS8的N端之间引入编码(GGGS)2柔性连接肽的序列。采用重叠延伸PCR法将两片段拼接为ERG20-(GGGS)2-AsTPS8融合基因, 与BamHI/HindⅢ双酶切的pESC-URA载体同源重组, 获得融合表达质粒pESC-URA-ERG20-(GGGS)2-AsTPS8。
酵母转化与菌株构建  酿酒酵母感受态细胞的制备与转化采用醋酸锂化学转化法[15]。质粒转化在SD-URA营养缺陷型平板上筛选。基因组整合采用多片段共转化策略: 以pCfb-2988-TRP、pESC-URA-AsTPS8和pESC-URA-ERG20-(GGGS)2-AsTPS8为模板, 分别扩增TY1位点整合所需的上游同源臂(2988-TRP-UP)、表达盒(AsTPS8或ERG20-(GGGS)2-AsTPS8) 和下游同源臂(2988-TRP-DOWN) 三个片段, 共同转入菌株ZE00中, 在SD-TRP平板上筛选转化子。对于LPP1位点编辑, 以pESC-URA-IME4-UPC2-1和CEN.PK2-1C基因组DNA为模板, 分别扩增LPP1位点上游同源臂(LPP1-UP)、PGK1p-IME4-CYC1t-GAL1p-UPC2-1-ADH1t表达盒及下游同源臂(LPP1-DOWN), 与pCRCT-GTR-URA3-ΔLPP1质粒共转至菌株ZE05中, 在SD-URA平板上筛选。所有阳性转化子经菌落PCR验证。
摇瓶发酵  挑取阳性转化子单菌落接种于2 mL SD筛选液体培养基(含20 g·L-1葡萄糖), 30 ℃、200 r·min-1过夜培养作为种子液。以1%接种量转接至装有50 mL SD培养基的摇瓶中, 在发酵10 h后加入5 mL正十二烷, 30 ℃、200 r·min-1诱导培养5 d。每组设置3个生物学重复。
5L发酵罐高密度发酵   将菌株ZE06经三级种子活化(2 mL→50 mL→200 mL YPD培养基, 30 ℃、200 r·min-1, 各培养12~16 h) 后, 全部接种至装有3 L YPD发酵培养基的5 L发酵罐中。发酵培养基初始葡萄糖浓度为25 g·L-1, 添加10 mL·L-1微量元素混合液和12 mL·L-1维生素混合液。培养温度30 ℃, 通气量3 L·min-1, 初始搅拌转速400 r·min-1, 通过级联控制使溶氧(DO) 维持在30%以上。通过自动流加5 mol·L-1氨水控制pH为5.2。发酵12 h后加入300 mL正十二烷(10%) 进行萃取。当DO值升至50%以上时, 自动补料600 g·L-1葡萄糖溶液。发酵144 h, 每24 h取样检测OD600和α-石竹烯产量。
GC-MS分析  发酵液12 000 r·min-1离心3 min分层, 取上层正十二烷相。样品经正十二烷适当稀释后, 进行GC-MS分析。色谱条件: Rxi-5HT毛细管柱(30 m × 0.25 mm × 0.25 μm); 载气为高纯氦气, 恒流模式流速1.0 mL·min-1; 不分流进样, 进样量1 μL, 进样口温度250 ℃; 升温程序: 40 ℃保持3 min, 10 ℃·min-1升至130 ℃, 3 ℃·min-1升至160 ℃, 40 ℃·min-1升至250 ℃保持2 min。质谱条件: EI电离源70 eV, 离子源温度250 ℃, 接口温度170 ℃, 全扫描模式m/z 50~600。谱图经NIST17标准谱库检索匹配(匹配度 > 85%)。标准曲线的制备: 以正十二烷为溶剂, 配制浓度为10 mg·mL-1的α-石竹烯标准品母液, 之后按不同的稀释比例配制成浓度分别为0.02、0.04、0.06、0.08、0.1 mg·mL-1的α-石竹烯标准品溶液。通过对α-石竹烯浓度与峰面积进行处理得到α-石竹烯的标准曲线, 用于后续α-石竹烯样品定量分析。
HPLC检测方法   采用Waters HPLC仪器结合Agilent EC-C18 (150 mm × 4.6 mm, 2.7 μm) 90%乙腈等度洗脱, 流速1 mL·min-1, 柱温30 ℃。标准曲线的制备: 以无水乙醇为溶剂, 配制浓度为10 mg·mL-1的α-石竹烯标准品母液, 之后按不同的稀释比例配制成浓度分别为0.02、0.04、0.06、0.08、0.1 mg·mL-1的α-石竹烯标准品溶液, 将它们放入进样小瓶中进行HPLC检测。通过对α-石竹烯浓度与峰面积进行处理得到α-石竹烯的标准曲线, 用于后续α-石竹烯样品定量分析。
统计学分析  统计学分析使用GraphPad Prism 9软件完成。所有实验均设置3个生物学重复, 数据以均值±标准差($ \overline{x} $ ± s) 表示。组间比较采用独立样本t检验, P < 0.05认为差异具有统计学意义。
结果
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1　 α-石竹烯合酶基因的挖掘与功能鉴定
以已知功能的萜烯合酶蛋白序列为诱饵, 对本课题组白木香转录组数据进行本地BLAST同源比对, 结合系统发育树分析, 如图 1所示, 筛选获得了一条候选TPS基因, 命名为AsTPS8。系统发育分析表明, 此基因聚类于TPS-a亚家族, 说明其可能是编码倍半萜合酶的基因, 且与来自厚叶沉香(Aquilaria crassna) 的α-石竹烯合酶具有高度相似性, 氨基酸序列一致性为96.7%, 猜测AsTPS8能产生α-石竹烯。
图 2保守基序分析显示, AsTPS8含有Ⅰ类萜烯合酶特征性保守结构域, 包括C端的DDxxD和DTE金属结合基序以及N端的RRX8W基序, 符合功能性萜烯合酶的典型特征[16-18]。
将AsTPS8克隆至pESC-URA表达载体, 转化至酿酒酵母底盘菌株ZE01中, 获得菌株ZE02。50 mL摇瓶诱导表达5 d后, GC-MS分析发酵产物。总离子流色谱图(TIC) 显示, 在保留时间16.3 min处有一明显色谱峰, 结果如图 3所示, 经NIST17谱库检索与α-石竹烯匹配度达98%。与α-石竹烯标准品比对确认发酵产物为α-石竹烯, 表明AsTPS8为α-石竹烯合酶。
2　AsTPS8与已知α-石竹烯合酶ZSS1的催化活性比较
为评估新挖掘的α-石竹烯合酶AsTPS8的催化效率, 选择红球姜(Zingiber zerumbet) 来源的α-石竹烯合酶ZSS1 (GenBank: BAG12020.1) 作为对照。ZSS1是首个被功能鉴定的α-石竹烯合酶, 其在植物体内和异源宿主中均具有较高的α-石竹烯合成活性[19], 在大肠杆菌中异源表达时可催化生成95%以上的α-石竹烯[20], 在酿酒酵母中亦实现了α-石竹烯及下游花姜酮的从头合成[21]。ZSS1是目前应用广泛且催化性能较为稳定的α-石竹烯合酶之一, 因此, 本研究选择ZSS1作为对照以评价AsTPS8的催化能力。
从红球姜根茎中克隆ZSS1基因(ORF 1 644 bp), 构建pESC-URA-ZSS1表达载体并转化ZE01, 获得菌株ZE03。摇瓶发酵5天后取样用GC-MS进行检测, 结果如图 4所示, ZE02 (AsTPS8) 和ZE03 (ZSS1) 的α-石竹烯产量分别为0.83、0.42 g·L-1。白木香来源的AsTPS8在酿酒酵母中表现出显著优于红球姜来源ZSS1的催化性能, 后续研究以AsTPS8为核心合酶进行代谢工程优化。
3　ERG20与AsTPS8融合表达提高α-石竹烯产量
FPP合成酶ERG20是MVA途径中将香叶基焦磷酸(GPP) 转化为FPP的关键酶, 也是萜类合成途径的重要限速步骤。为提高AsTPS8对前体FPP的催化效率, 将ERG20与AsTPS8通过柔性连接肽(GGGS)2进行融合表达。构建融合表达质粒pESC-URA-ERG20-(GGGS)2-AsTPS8, 并通过多片段同源重组将AsTPS8单基因表达盒和ERG20-(GGGS)2-AsTPS8融合表达盒分别整合至ZE00基因组的TY1多拷贝位点(图 5A), 获得菌株ZE04和ZE05。
摇瓶发酵结果显示, 经TY1位点多拷贝整合的ZE04菌株(单基因) α-石竹烯产量为1.25 g·L-1, 较游离质粒表达的ZE01 (0.83 g·L-1) 提高约51%; 融合表达菌株ZE05产量达到1.41 g·L-1 (图 5C), 较ZE04提高约13%。结果表明, 融合表达策略可有效增强底物通道效应, 提高FPP向α-石竹烯的转化效率。
4　 LPP1的敲除与IME4/UPC2-1的过表达协同强化代谢通量
FPP在酿酒酵母细胞内除作为萜烯合酶的底物外, 还可被LPP1p和DPP1p磷酸酶水解为法尼醇, 构成前体无效消耗的主要旁路途径。同时, MVA途径关键基因的表达受到多重转录调控。为进一步提升α-石竹烯产量, 利用CRISPR/Cas9系统敲除LPP1基因, 并在该位点同步整合IME4和UPC2-1过表达盒(图 6A), 获得菌株ZE06。
摇瓶发酵结果显示, ZE06的α-石竹烯产量达到1.80 g·L-1 (图 6C）, 较菌株ZE05 (1.41 g·L-1) 提高约28%。敲除旁路磷酸酶基因减少了FPP向法尼醇的分流, 而IME4的过表达上调了MVA途径中甲基化基因的转录水平, UPC2-1进一步激活ERG10、ERG13等基因的表达。三者协同作用有效强化了FPP供给通量, 显著提升了α-石竹烯的合成效率。
5　5 L发酵罐分批补料高密度发酵
为评估工程菌株ZE06在规模化生产条件下的性能, 进行5 L发酵罐分批补料高密度发酵验证。初始葡萄糖浓度为25 g·L-1, 发酵12 h后加入300 mL正十二烷(10%) 作为萃取剂。设置溶氧关联补料, 当溶氧超过50%时自动补入600 g·L-1葡萄糖溶液。发酵周期内持续监测生物量(OD600) 和α-石竹烯产量。
如图 7所示, 0~24 h为菌体适应期, α-石竹烯开始合成但积累量较低。24~72 h为快速生长期和产物快速积累期, 菌体密度持续增加至OD600约85, α-石竹烯产量呈近似线性增长。72 h后, 菌体生长速率减慢, 但OD600和α-石竹烯产量仍持续上升直至144 h。发酵至144 h时, α-石竹烯产量达到22.02 g·L-1, 平均产率为0.15 g·L-1·h-1。由于发酵罐运行周期限制, 未继续延长培养时间; 但由图 7可见, 144 h时产物积累曲线尚未进入平台期, 进一步延长发酵时间可能获得更高产量。表明工程菌株ZE06在高密度发酵条件下仍能保持高效的代谢通量, 具备工业化规模生产的潜力。
讨论
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本研究从基因挖掘、途径构建、旁路消除及全局调控四个层面, 系统构建了高产α-石竹烯的酿酒酵母底盘菌株。从白木香转录组中挖掘到的新型合酶AsTPS8, 催化效率显著优于已报道的ZSS1, 为后续改造提供了高效催化元件。通过ERG20-(GGGS)2-AsTPS8融合表达结合TY1位点多拷贝整合, 摇瓶产量提升至1.41 g·L-1, 证实底物通道效应与拷贝数增加存在协同增效。在此基础上, 敲除LPP1以阻断FPP向法尼醇的无谓分流, 并同步过表达UPC2-1和IME4, 从转录调控和RNA表观修饰两个层面强化MVA途径通量, 产量进一步提高至1.80 g·L-1。最后通过5 L发酵罐分批补料发酵, 实现了α-石竹烯的高效合成, 产量达到22.02 g·L-1。
与文献中倍半萜类产物的微生物合成相比, 本研究在产量和产率方面均具优势。该高产底盘菌株的获得, 为α-石竹烯的下游氧化产物花姜酮的规模化制备奠定了基础。本研究为植物源萜类化合物的微生物合成提供了可借鉴的代谢工程策略。
本研究主要基于活细胞产量比较不同合酶的催化效率, 后续将开展体外酶动力学实验, 测定AsTPS8和ZSS1的催化参数。此外, 发酵终点(144 h) 尚未达到产物峰值, 优化补料策略或延长发酵周期有望进一步提高产量。

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2026年第61卷第9期
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doi: 10.16438/j.0513-4870.2026-0478
  • 接收时间：2026-04-25
  • 首发时间：2026-10-22
  • 出版时间：2026-09-12
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  • 收稿日期：2026-04-25
  • 修回日期：2026-06-16
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    1广东药科大学中药学院, 广东 广州 510006
    2广州中医药大学中药学院, 广东 广州 510006

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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
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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