biostudies-literatureZhao XNatural Science Foundation of ChinaNational Key Research and Development Program of China689https://www.ebi.ac.uk/biostudies/studies/S-EPMC9146380biostudies-literatureUnknown12(5)Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized <i>Streptomyces</i> hosts. Then, we present an efficient, markerless and multiplex large gene cluster editing method based on in vitro CRISPR/Cas9 digestion and yeast homologous recombination. With this method, we successfully engineered the thaxtomin A cluster by simultaneously replacing the native promoters of the <i>txtED</i> operon, <i>txtABH</i> operon and <i>txtC</i> gene with strong constitutive promoters, and the yield of thaxtomin A improved to 289.5 µg/mL in heterologous <i>Streptomyces coelicolor</i> M1154. To further optimiLife (Basel, Switzerland)Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous <i>Streptomyces</i> Hosts.PMC914638032071412, 12090053, 32088101, 315000692018YFA0900700, 2020YFA0907101, 2021YFF1200500, 2015CB910300Zong YLou CWei WZhao XfalseMultiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous <i>Streptomyces</i> Hosts.Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized <i>Streptomyces</i> hosts. Then, we present an efficient, markerless and multiplex large gene cluster editing method based on in vitro CRISPR/Cas9 digestion and yeast homologous recombination. With this method, we successfully engineered the thaxtomin A cluster by simultaneously replacing the native promoters of the <i>txtED</i> operon, <i>txtABH</i> operon and <i>txtC</i> gene with strong constitutive promoters, and the yield of thaxtomin A improved to 289.5 µg/mL in heterologous <i>Streptomyces coelicolor</i> M1154. To further optimi2022-01-01T00:00:00Z2022 May2025-04-22T18:22:06.163Z2025-04-06T02:23:02.01ZS-EPMC91463803562935810.3390/life12050689