Construction and screening of L-valine high-yielding Escherichia coli using an artificial screening marker

Bowen Du¹Sheng Gao¹Daixue Kou¹Yinuo Li¹Dan Li¹Yongsheng Cao¹Cuiping Yang²chuanzhuang Guo²Jianbin Wang²Junqing Wang^1*Nan LI^1*

• ¹State Key Laboratory of Biobased Material and Green Papermaking, Qilu University of Technology, Jinan, China
• ²Dongxiao Biotechnology Co, JInan, China

Introduction: L-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.Methods: Using 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 Lvaline 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%.Fermentation 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.Concussion: This approach offers a more comprehensive and effective method for identifying highyielding L-valine bacterial strains.