Biosensor-Coupled In Vivo Mutagenesis and Omics Analysis Reveals Reduced Lysine and Arginine Synthesis To Improve Malonyl-Coenzyme A Flux in Saccharomyces cerevisiae
ABSTRACT Malonyl-coenzyme A (malonyl-CoA) is an important precursor for producing various chemicals, but its low availability limits the synthesis of downstream products in Saccharomyces cerevisiae. Owing to the complexity of metabolism, evolutionary engineering is required for developing strains wi...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-04-01
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| Series: | mSystems |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/msystems.01366-21 |
| _version_ | 1828349405980262400 |
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| author | Chenxi Qiu Mingtao Huang Yishan Hou Huilin Tao Jianzhi Zhao Yu Shen Xiaoming Bao Qingsheng Qi Jin Hou |
| author_facet | Chenxi Qiu Mingtao Huang Yishan Hou Huilin Tao Jianzhi Zhao Yu Shen Xiaoming Bao Qingsheng Qi Jin Hou |
| author_sort | Chenxi Qiu |
| collection | DOAJ |
| description | ABSTRACT Malonyl-coenzyme A (malonyl-CoA) is an important precursor for producing various chemicals, but its low availability limits the synthesis of downstream products in Saccharomyces cerevisiae. Owing to the complexity of metabolism, evolutionary engineering is required for developing strains with improved malonyl-CoA synthesis. Here, using the biosensor we constructed previously, a growth-based screening system that links the availability of malonyl-CoA with cell growth is developed. Coupling this system with in vivo continuous mutagenesis enabled rapid generation of genome-scale mutation library and screening strains with improved malonyl-CoA availability. The mutant strains are analyzed by whole-genome sequencing and transcriptome analysis. The omics analysis revealed that the carbon flux rearrangement to storage carbohydrate and amino acids synthesis affected malonyl-CoA metabolism. Through reverse engineering, new processes especially reduced lysine and arginine synthesis were found to improve malonyl-CoA synthesis. Our study provides a valuable complementary tool to other high-throughput screening method for mutant strains with improved metabolite synthesis and improves our understanding of the metabolic regulation of malonyl-CoA synthesis. IMPORTANCE Malonyl-CoA is a key precursor for the production a variety of value-added chemicals. Although rational engineering has been performed to improve the synthesis of malonyl-CoA in S. cerevisiae, due to the complexity of the metabolism there is a need for evolving strains and analyzing new mechanism to improve malonyl-CoA flux. Here, we developed a growth-based screening system that linked the availability of malonyl-CoA with cell growth and manipulated DNA replication for rapid in vivo mutagenesis. The combination of growth-based screening with in vivo mutagenesis enabled quick evolution of strains with improved malonyl-CoA availability. The whole-genome sequencing, transcriptome analysis of the mutated strains, together with reverse engineering, demonstrated weakening carbon flux to lysine and arginine synthesis and storage carbohydrate can contribute to malonyl-CoA synthesis. Our work provides a guideline in simultaneous strain screening and continuous evolution for improved metabolic intermediates and identified new targets for improving malonyl-CoA downstream product synthesis. |
| first_indexed | 2024-04-14T01:09:59Z |
| format | Article |
| id | doaj.art-1c7f01ee523d4b1fa2457f97c46d19a8 |
| institution | Directory Open Access Journal |
| issn | 2379-5077 |
| language | English |
| last_indexed | 2024-04-14T01:09:59Z |
| publishDate | 2022-04-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSystems |
| spelling | doaj.art-1c7f01ee523d4b1fa2457f97c46d19a82022-12-22T02:21:07ZengAmerican Society for MicrobiologymSystems2379-50772022-04-017210.1128/msystems.01366-21Biosensor-Coupled In Vivo Mutagenesis and Omics Analysis Reveals Reduced Lysine and Arginine Synthesis To Improve Malonyl-Coenzyme A Flux in Saccharomyces cerevisiaeChenxi Qiu0Mingtao Huang1Yishan Hou2Huilin Tao3Jianzhi Zhao4Yu Shen5Xiaoming Bao6Qingsheng Qi7Jin Hou8State Key Laboratory of Microbial Technology, Shandong University, Qingdao, People’s Republic of ChinaSchool of Food Science and Engineering, South China University of Technology, Guangzhou, People’s Republic of ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, People’s Republic of ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, People’s Republic of ChinaState Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qi Lu University of Technology, Jinan, People’s Republic of ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, People’s Republic of ChinaState Key Laboratory of Biobased Material and Green Papermaking, School of Bioengineering, Qi Lu University of Technology, Jinan, People’s Republic of ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, People’s Republic of ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao, People’s Republic of ChinaABSTRACT Malonyl-coenzyme A (malonyl-CoA) is an important precursor for producing various chemicals, but its low availability limits the synthesis of downstream products in Saccharomyces cerevisiae. Owing to the complexity of metabolism, evolutionary engineering is required for developing strains with improved malonyl-CoA synthesis. Here, using the biosensor we constructed previously, a growth-based screening system that links the availability of malonyl-CoA with cell growth is developed. Coupling this system with in vivo continuous mutagenesis enabled rapid generation of genome-scale mutation library and screening strains with improved malonyl-CoA availability. The mutant strains are analyzed by whole-genome sequencing and transcriptome analysis. The omics analysis revealed that the carbon flux rearrangement to storage carbohydrate and amino acids synthesis affected malonyl-CoA metabolism. Through reverse engineering, new processes especially reduced lysine and arginine synthesis were found to improve malonyl-CoA synthesis. Our study provides a valuable complementary tool to other high-throughput screening method for mutant strains with improved metabolite synthesis and improves our understanding of the metabolic regulation of malonyl-CoA synthesis. IMPORTANCE Malonyl-CoA is a key precursor for the production a variety of value-added chemicals. Although rational engineering has been performed to improve the synthesis of malonyl-CoA in S. cerevisiae, due to the complexity of the metabolism there is a need for evolving strains and analyzing new mechanism to improve malonyl-CoA flux. Here, we developed a growth-based screening system that linked the availability of malonyl-CoA with cell growth and manipulated DNA replication for rapid in vivo mutagenesis. The combination of growth-based screening with in vivo mutagenesis enabled quick evolution of strains with improved malonyl-CoA availability. The whole-genome sequencing, transcriptome analysis of the mutated strains, together with reverse engineering, demonstrated weakening carbon flux to lysine and arginine synthesis and storage carbohydrate can contribute to malonyl-CoA synthesis. Our work provides a guideline in simultaneous strain screening and continuous evolution for improved metabolic intermediates and identified new targets for improving malonyl-CoA downstream product synthesis.https://journals.asm.org/doi/10.1128/msystems.01366-21malonyl-CoA biosensorgrowth-based screeningin vivo mutagenesisomics analysisSaccharomyces cerevisiae |
| spellingShingle | Chenxi Qiu Mingtao Huang Yishan Hou Huilin Tao Jianzhi Zhao Yu Shen Xiaoming Bao Qingsheng Qi Jin Hou Biosensor-Coupled In Vivo Mutagenesis and Omics Analysis Reveals Reduced Lysine and Arginine Synthesis To Improve Malonyl-Coenzyme A Flux in Saccharomyces cerevisiae mSystems malonyl-CoA biosensor growth-based screening in vivo mutagenesis omics analysis Saccharomyces cerevisiae |
| title | Biosensor-Coupled In Vivo Mutagenesis and Omics Analysis Reveals Reduced Lysine and Arginine Synthesis To Improve Malonyl-Coenzyme A Flux in Saccharomyces cerevisiae |
| title_full | Biosensor-Coupled In Vivo Mutagenesis and Omics Analysis Reveals Reduced Lysine and Arginine Synthesis To Improve Malonyl-Coenzyme A Flux in Saccharomyces cerevisiae |
| title_fullStr | Biosensor-Coupled In Vivo Mutagenesis and Omics Analysis Reveals Reduced Lysine and Arginine Synthesis To Improve Malonyl-Coenzyme A Flux in Saccharomyces cerevisiae |
| title_full_unstemmed | Biosensor-Coupled In Vivo Mutagenesis and Omics Analysis Reveals Reduced Lysine and Arginine Synthesis To Improve Malonyl-Coenzyme A Flux in Saccharomyces cerevisiae |
| title_short | Biosensor-Coupled In Vivo Mutagenesis and Omics Analysis Reveals Reduced Lysine and Arginine Synthesis To Improve Malonyl-Coenzyme A Flux in Saccharomyces cerevisiae |
| title_sort | biosensor coupled in vivo mutagenesis and omics analysis reveals reduced lysine and arginine synthesis to improve malonyl coenzyme a flux in saccharomyces cerevisiae |
| topic | malonyl-CoA biosensor growth-based screening in vivo mutagenesis omics analysis Saccharomyces cerevisiae |
| url | https://journals.asm.org/doi/10.1128/msystems.01366-21 |
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