Construction and screening of L-valine high-yielding Escherichia coli using an artificial screening marker
IntroductionL-valine is commonly utilized in cosmetics, pharmaceuticals, food additives, and animal feeds. The selection and breeding of high-yielding, low-cost, and genetically stable production strains have become a key objective in the L-valine production industry.MethodsUsing Escherichia coli DB...
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Frontiers Media S.A.
2025-08-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1627242/full |
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| author | Bowen Du Bowen Du Sheng Gao Sheng Gao Daixue Kou Daixue Kou Yinuo Li Yinuo Li Dan Li Dan Li Yongsheng Cao Yongsheng Cao Cuiping Yang Chuanzhuang Guo Jianbin Wang Junqing Wang Junqing Wang Nan Li Nan Li |
| author_facet | Bowen Du Bowen Du Sheng Gao Sheng Gao Daixue Kou Daixue Kou Yinuo Li Yinuo Li Dan Li Dan Li Yongsheng Cao Yongsheng Cao Cuiping Yang Chuanzhuang Guo Jianbin Wang Junqing Wang Junqing Wang Nan Li Nan Li |
| author_sort | Bowen Du |
| collection | DOAJ |
| description | IntroductionL-valine is commonly utilized in cosmetics, pharmaceuticals, food additives, and animal feeds. The selection and breeding of high-yielding, low-cost, and genetically stable production strains have become a key objective in the L-valine production industry.MethodsUsing Escherichia coli DB-1-1, we developed a screening marker LESG associated with intracellular L-valine levels by choosing GTC, a less common codon for L-valine, in place of all L-valine codons. The artificial LESG was then ligated into pUC-57 and transformed into competent E. coli DB-1-1 cells with the rare L-valine codon. After conducting atmospheric and room-temperature plasma mutagenesis cultures, mutants that displayed elevated fluorescence were sorted using flow cytometry. After sorting the 240 strains. We sorted out 143 highly fluorescent strains, and the sorting efficiency reached 59.5%.ResultsFermentation results showed that the mutant strains with increased fluorescence intensity had an improved L-valine fermentation titer (23.1%) and a higher screening positivity rate (62.5%) than that of the wild-type strain. The maximum titer of valine at 24 h was 84.1 g/L.ConclusionThis approach offers a more comprehensive and effective method for identifying high-yielding L-valine bacterial strains. |
| format | Article |
| id | doaj-art-48a881ccbb4146e38e995efd1b0168c3 |
| institution | Kabale University |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj-art-48a881ccbb4146e38e995efd1b0168c32025-08-20T04:01:08ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-08-011610.3389/fmicb.2025.16272421627242Construction and screening of L-valine high-yielding Escherichia coli using an artificial screening markerBowen Du0Bowen Du1Sheng Gao2Sheng Gao3Daixue Kou4Daixue Kou5Yinuo Li6Yinuo Li7Dan Li8Dan Li9Yongsheng Cao10Yongsheng Cao11Cuiping Yang12Chuanzhuang Guo13Jianbin Wang14Junqing Wang15Junqing Wang16Nan Li17Nan Li18State Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology, Jinan, Shandong, ChinaSchool of Bioengineering, Qilu University of Technology, Jinan, Shandong, ChinaState Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology, Jinan, Shandong, ChinaSchool of Bioengineering, Qilu University of Technology, Jinan, Shandong, ChinaState Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology, Jinan, Shandong, ChinaSchool of Bioengineering, Qilu University of Technology, Jinan, Shandong, ChinaState Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology, Jinan, Shandong, ChinaSchool of Bioengineering, Qilu University of Technology, Jinan, Shandong, ChinaState Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology, Jinan, Shandong, ChinaSchool of Bioengineering, Qilu University of Technology, Jinan, Shandong, ChinaState Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology, Jinan, Shandong, ChinaSchool of Bioengineering, Qilu University of Technology, Jinan, Shandong, ChinaDongxiao Bioengineering (Shandong) Co., Ltd., Zhucheng, ChinaDongxiao Bioengineering (Shandong) Co., Ltd., Zhucheng, ChinaDongxiao Bioengineering (Shandong) Co., Ltd., Zhucheng, ChinaState Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology, Jinan, Shandong, ChinaSchool of Bioengineering, Qilu University of Technology, Jinan, Shandong, ChinaState Key Laboratory of Biobased Material and Green Papermaking (LBMP), Qilu University of Technology, Jinan, Shandong, ChinaSchool of Bioengineering, Qilu University of Technology, Jinan, Shandong, ChinaIntroductionL-valine is commonly utilized in cosmetics, pharmaceuticals, food additives, and animal feeds. The selection and breeding of high-yielding, low-cost, and genetically stable production strains have become a key objective in the L-valine production industry.MethodsUsing Escherichia coli DB-1-1, we developed a screening marker LESG associated with intracellular L-valine levels by choosing GTC, a less common codon for L-valine, in place of all L-valine codons. The artificial LESG was then ligated into pUC-57 and transformed into competent E. coli DB-1-1 cells with the rare L-valine codon. After conducting atmospheric and room-temperature plasma mutagenesis cultures, mutants that displayed elevated fluorescence were sorted using flow cytometry. After sorting the 240 strains. We sorted out 143 highly fluorescent strains, and the sorting efficiency reached 59.5%.ResultsFermentation results showed that the mutant strains with increased fluorescence intensity had an improved L-valine fermentation titer (23.1%) and a higher screening positivity rate (62.5%) than that of the wild-type strain. The maximum titer of valine at 24 h was 84.1 g/L.ConclusionThis approach offers a more comprehensive and effective method for identifying high-yielding L-valine bacterial strains.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1627242/fullEscherichia colihigh-throughput screeningrare codonL-valinefluorescent protein |
| spellingShingle | Bowen Du Bowen Du Sheng Gao Sheng Gao Daixue Kou Daixue Kou Yinuo Li Yinuo Li Dan Li Dan Li Yongsheng Cao Yongsheng Cao Cuiping Yang Chuanzhuang Guo Jianbin Wang Junqing Wang Junqing Wang Nan Li Nan Li Construction and screening of L-valine high-yielding Escherichia coli using an artificial screening marker Frontiers in Microbiology Escherichia coli high-throughput screening rare codon L-valine fluorescent protein |
| title | Construction and screening of L-valine high-yielding Escherichia coli using an artificial screening marker |
| title_full | Construction and screening of L-valine high-yielding Escherichia coli using an artificial screening marker |
| title_fullStr | Construction and screening of L-valine high-yielding Escherichia coli using an artificial screening marker |
| title_full_unstemmed | Construction and screening of L-valine high-yielding Escherichia coli using an artificial screening marker |
| title_short | Construction and screening of L-valine high-yielding Escherichia coli using an artificial screening marker |
| title_sort | construction and screening of l valine high yielding escherichia coli using an artificial screening marker |
| topic | Escherichia coli high-throughput screening rare codon L-valine fluorescent protein |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1627242/full |
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