Cover Image,Volume 116, Number 6, June 2019

Metabolic engineering consistently demands to produce the maximum carbon and energy flux to target chemicals. To balance metabolic flux, gene expression levels of artificially synthesized pathways usually fine-tuned using multimodular optimization strategy. However, forward construction is an engineering conundrum because a vast number of possible pathway combinations need to be constructed and analyzed. Here, an iterative high-throughput balancing (IHTB) strategy was established to thoroughly fine-tune the (2S)-naringenin biosynthetic pathway. A series of gradient constitutive promoters from Escherichia coli were randomly cloned upstream of pathway genes, and the resulting library was screened using an ultraviolet spectrophotometry–fluorescence spectrophotometry high-throughput method, which was established based on the interactions between AlCl₃ and (2S)-naringenin. The metabolic flux of the screened high-titer strains was analyzed and iterative rounds of screening were performed based on the analysis results. After several rounds, the metabolic flux of the (2S)-naringenin synthetic pathway was balanced, reaching a final titer of 191.9 mg/L with 29.2 mg/L p-coumaric acid accumulation. Chalcone synthase was speculated to be the rate-limiting enzyme because its expression level was closely related to the production of both (2S)-naringenin and p-coumaric acid. The established IHTB strategy can be used to efficiently balance multigene pathways, which will accelerate the development of efficient recombinant strains.