Multisite Cre-lox recombination enables regulatory mechanism elucidation and systematic engineering of echinocandin B biosynthesis in Aspergillus nidulans.
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| Title: | Multisite Cre-lox recombination enables regulatory mechanism elucidation and systematic engineering of echinocandin B biosynthesis in Aspergillus nidulans. |
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| Authors: | Ma, Youchu1,2 (AUTHOR), Zhang, Yue1,2 (AUTHOR), Zhang, Dongfang1 (AUTHOR), Tang, Yue1 (AUTHOR), Zhang, Wanyu1 (AUTHOR), Yue, Liyuan1 (AUTHOR), Lou, Wenqing1 (AUTHOR), Meng, Chao1 (AUTHOR), Li, Yanling1 (AUTHOR) lingl816@163.com, Chen, Xiulai1,3 (AUTHOR) xlchen@jiangnan.edu.cn, Zhao, Fanglong1,2 (AUTHOR) zhaofanglong@sdfmu.edu.cn |
| Source: | Metabolic Engineering. Apr2026, Vol. 95, p15-27. 13p. |
| Subjects: | Aspergillus nidulans, Recombinant DNA, Bioreactors, Genetic regulation, Biochemical engineering, Filamentous fungi, Gene clusters, Biosynthesis |
| Abstract: | Echinocandin B (ECB), a cyclic lipohexapeptide for synthesizing antifungal drugs, is produced by the nonribosomal peptide synthetase gene cluster in Aspergillus nidulans. However, industrial production remains limited by the inefficiency of production capacity, primarily due to the complexity of the biosynthetic pathway and the absence of multi-gene regulatory tools in filamentous fungi. Here, we established an orthogonal Cre- lox -based platform enabling single-site insertion of up to 30 kb and simultaneous dual-site integration of 10 kb DNA fragments in A. nidulans. Through precursor supplementation and targeted gene overexpression, we identified key enzymatic bottlenecks in the precursor biosynthetic pathway, including the oxygenases AniF , AniK , AniG, and the acyl-AMP ligase AniI. Combinatorial overexpression of these genes acted synergistically to increase ECB titers. We further addressed bottlenecks in natural amino acid biosynthesis by overexpressing feedback-resistant mutants of Hom3 (L-Thr pathway) and LeuC (L-Leu pathway). Additionally, we uncovered a temperature-dependent regulation mechanism whereby low temperature (25 °C) concurrently upregulates both the ECB biosynthetic gene cluster and odeA gene, encoding Δ 12-oleic acid desaturase, thereby increasing linoleic acid availability for ECB production. Leveraging our multisite DNA-integration platform to rewire expression of these key genes, we increased ECB production to 3.5 ± 0.2 g/L in a 5-L fed-batch bioreactor, a 2.3-fold improvement that represents the highest titer reported in the literature to date. Our orthogonal dual-site integration strategy and the systematic optimization approach provide a valuable framework for metabolic engineering of complex natural products in filamentous fungi. • Orthogonal Cre- lox platform enabling dual-site integration in A. nidulans. • Systematic identification of seven rate-limiting steps in ECB biosynthesis. • Temperature controls ECB yield by regulating fatty acid desaturase expression. • Multisite genome integration enabled refactoring of the ECB biosynthetic pathway. • Achieved a record-high ECB titer of 3.5 ± 0.2 g/L in a 5-L bioreactor. [ABSTRACT FROM AUTHOR] |
| Copyright of Metabolic Engineering is the property of Academic Press Inc. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) | |
| Database: | Engineering Source |
| FullText | Text: Availability: 0 |
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| Header | DbId: egs DbLabel: Engineering Source An: 192152336 AccessLevel: 6 PubType: Academic Journal PubTypeId: academicJournal PreciseRelevancyScore: 0 |
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| Items | – Name: Title Label: Title Group: Ti Data: Multisite Cre-lox recombination enables regulatory mechanism elucidation and systematic engineering of echinocandin B biosynthesis in Aspergillus nidulans. – Name: Author Label: Authors Group: Au Data: <searchLink fieldCode="AR" term="%22Ma%2C+Youchu%22">Ma, Youchu</searchLink><relatesTo>1,2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Zhang%2C+Yue%22">Zhang, Yue</searchLink><relatesTo>1,2</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Zhang%2C+Dongfang%22">Zhang, Dongfang</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Tang%2C+Yue%22">Tang, Yue</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Zhang%2C+Wanyu%22">Zhang, Wanyu</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Yue%2C+Liyuan%22">Yue, Liyuan</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Lou%2C+Wenqing%22">Lou, Wenqing</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Meng%2C+Chao%22">Meng, Chao</searchLink><relatesTo>1</relatesTo> (AUTHOR)<br /><searchLink fieldCode="AR" term="%22Li%2C+Yanling%22">Li, Yanling</searchLink><relatesTo>1</relatesTo> (AUTHOR)<i> lingl816@163.com</i><br /><searchLink fieldCode="AR" term="%22Chen%2C+Xiulai%22">Chen, Xiulai</searchLink><relatesTo>1,3</relatesTo> (AUTHOR)<i> xlchen@jiangnan.edu.cn</i><br /><searchLink fieldCode="AR" term="%22Zhao%2C+Fanglong%22">Zhao, Fanglong</searchLink><relatesTo>1,2</relatesTo> (AUTHOR)<i> zhaofanglong@sdfmu.edu.cn</i> – Name: TitleSource Label: Source Group: Src Data: <searchLink fieldCode="JN" term="%22Metabolic+Engineering%22">Metabolic Engineering</searchLink>. Apr2026, Vol. 95, p15-27. 13p. – Name: Subject Label: Subjects Group: Su Data: <searchLink fieldCode="DE" term="%22Aspergillus+nidulans%22">Aspergillus nidulans</searchLink><br /><searchLink fieldCode="DE" term="%22Recombinant+DNA%22">Recombinant DNA</searchLink><br /><searchLink fieldCode="DE" term="%22Bioreactors%22">Bioreactors</searchLink><br /><searchLink fieldCode="DE" term="%22Genetic+regulation%22">Genetic regulation</searchLink><br /><searchLink fieldCode="DE" term="%22Biochemical+engineering%22">Biochemical engineering</searchLink><br /><searchLink fieldCode="DE" term="%22Filamentous+fungi%22">Filamentous fungi</searchLink><br /><searchLink fieldCode="DE" term="%22Gene+clusters%22">Gene clusters</searchLink><br /><searchLink fieldCode="DE" term="%22Biosynthesis%22">Biosynthesis</searchLink> – Name: Abstract Label: Abstract Group: Ab Data: Echinocandin B (ECB), a cyclic lipohexapeptide for synthesizing antifungal drugs, is produced by the nonribosomal peptide synthetase gene cluster in Aspergillus nidulans. However, industrial production remains limited by the inefficiency of production capacity, primarily due to the complexity of the biosynthetic pathway and the absence of multi-gene regulatory tools in filamentous fungi. Here, we established an orthogonal Cre- lox -based platform enabling single-site insertion of up to 30 kb and simultaneous dual-site integration of 10 kb DNA fragments in A. nidulans. Through precursor supplementation and targeted gene overexpression, we identified key enzymatic bottlenecks in the precursor biosynthetic pathway, including the oxygenases AniF , AniK , AniG, and the acyl-AMP ligase AniI. Combinatorial overexpression of these genes acted synergistically to increase ECB titers. We further addressed bottlenecks in natural amino acid biosynthesis by overexpressing feedback-resistant mutants of Hom3 (L-Thr pathway) and LeuC (L-Leu pathway). Additionally, we uncovered a temperature-dependent regulation mechanism whereby low temperature (25 °C) concurrently upregulates both the ECB biosynthetic gene cluster and odeA gene, encoding Δ 12-oleic acid desaturase, thereby increasing linoleic acid availability for ECB production. Leveraging our multisite DNA-integration platform to rewire expression of these key genes, we increased ECB production to 3.5 ± 0.2 g/L in a 5-L fed-batch bioreactor, a 2.3-fold improvement that represents the highest titer reported in the literature to date. Our orthogonal dual-site integration strategy and the systematic optimization approach provide a valuable framework for metabolic engineering of complex natural products in filamentous fungi. • Orthogonal Cre- lox platform enabling dual-site integration in A. nidulans. • Systematic identification of seven rate-limiting steps in ECB biosynthesis. • Temperature controls ECB yield by regulating fatty acid desaturase expression. • Multisite genome integration enabled refactoring of the ECB biosynthetic pathway. • Achieved a record-high ECB titer of 3.5 ± 0.2 g/L in a 5-L bioreactor. [ABSTRACT FROM AUTHOR] – Name: AbstractSuppliedCopyright Label: Group: Ab Data: <i>Copyright of Metabolic Engineering is the property of Academic Press Inc. and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.) |
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| RecordInfo | BibRecord: BibEntity: Identifiers: – Type: doi Value: 10.1016/j.ymben.2026.01.009 Languages: – Code: eng Text: English PhysicalDescription: Pagination: PageCount: 13 StartPage: 15 Subjects: – SubjectFull: Aspergillus nidulans Type: general – SubjectFull: Recombinant DNA Type: general – SubjectFull: Bioreactors Type: general – SubjectFull: Genetic regulation Type: general – SubjectFull: Biochemical engineering Type: general – SubjectFull: Filamentous fungi Type: general – SubjectFull: Gene clusters Type: general – SubjectFull: Biosynthesis Type: general Titles: – TitleFull: Multisite Cre-lox recombination enables regulatory mechanism elucidation and systematic engineering of echinocandin B biosynthesis in Aspergillus nidulans. Type: main BibRelationships: HasContributorRelationships: – PersonEntity: Name: NameFull: Ma, Youchu – PersonEntity: Name: NameFull: Zhang, Yue – PersonEntity: Name: NameFull: Zhang, Dongfang – PersonEntity: Name: NameFull: Tang, Yue – PersonEntity: Name: NameFull: Zhang, Wanyu – PersonEntity: Name: NameFull: Yue, Liyuan – PersonEntity: Name: NameFull: Lou, Wenqing – PersonEntity: Name: NameFull: Meng, Chao – PersonEntity: Name: NameFull: Li, Yanling – PersonEntity: Name: NameFull: Chen, Xiulai – PersonEntity: Name: NameFull: Zhao, Fanglong IsPartOfRelationships: – BibEntity: Dates: – D: 01 M: 04 Text: Apr2026 Type: published Y: 2026 Identifiers: – Type: issn-print Value: 10967176 Numbering: – Type: volume Value: 95 Titles: – TitleFull: Metabolic Engineering Type: main |
| ResultId | 1 |
