Biosensor-Based Multigene Pathway Optimization for Enhancing the Production of Glycolate.

Glycolate is widely used in industry, especially in the fields of chemical cleaning, cosmetics, and medical materials, and has broad market prospects for the future. Recent advances in metabolic engineering and synthetic biology have significantly improved the titer and yield of glycolate. However, an expensive inducer was used in previous studies, which is not feasible for use in large-scale industrial fermentations. To constitutively biosynthesize glycolate, the expression level of each gene of the glycolate synthetic pathway needs to be systemically optimized. The main challenge of multigene pathway optimization is being able to select or screen the optimum strain from the randomly assembled library by an efficient high-throughput method within a short time. To overcome these challenges, we firstly established a glycolate-responsive biosensor and developed agar plate- and 48-well deep-well plate-scale high-throughput screening methods for the rapid screening of superior glycolate producers from a large library. A total of 22 gradient-strength promoter-5'-untranslated region (UTR) complexes were randomly cloned upstream of the genes of the glycolate synthetic pathway, generating a large random assembled library. After rounds of screening, the optimum strain was obtained from 6 × 10 transformants in a week, and it achieved a titer of 40.9 ± 3.7 g/liter glycolate in a 5-liter bioreactor. Furthermore, high expression levels of the enzymes YcdW and GltA were found to promote glycolate production, whereas AceA has no obvious impact on glycolate production. Overall, the glycolate biosensor-based pathway optimization strategy presented in this work provides a paradigm for other multigene pathway optimizations. The use of strong promoters, such as pTrc and T7, to control gene expression not only needs the addition of expensive inducers but also results in excessive protein expression that may result in unbalanced metabolic flux and the waste of cellular building blocks and energy. To balance the metabolic flux of glycolate biosynthesis, the expression level of each gene needs to be systemically optimized in a constitutive manner. However, the lack of high-throughput screening methods restricted glycolate synthetic pathway optimization. Our work firstly established a glycolate-response biosensor, and agar plate- and 48-well plate-scale high-throughput screening methods were then developed for the rapid screening of optimum pathways from a large library. Finally, we obtained a glycolate-producing strain with good biosynthetic performance, and the use of the expensive inducer isopropyl-β-d-thiogalactopyranoside (IPTG) was avoided, which broadens our understanding of the mechanism of glycolate synthesis.