Refactoring biosynthetic pathways for enhanced secondary metabolite production is a central challenge for synthetic biology. Here we applied advanced DNA assembly methods and a uniform overexpression logic using constitutive promoters to achieve efficient heterologous production of the complex insecticidal macrolide spinosad. We constructed a 79-kb artificial gene cluster in which 23 biosynthetic genes were grouped into 7 operons, each with a strong constitutive promoter. Compared with the original gene cluster, the artificial gene cluster resulted in a 328-fold enhanced spinosad production in Streptomyces albus J1074. To achieve this goal, we applied the ExoCET DNA assembly method to build a plasmid from 13 GC-rich fragments with high efficiency in one step. Together with our previous direct cloning and recombineering tools, we present new synthetic biology options for refactoring large gene clusters for diverse applications.

Background: Biological nitrogen fixation converting atmospheric dinitrogen to ammonia is an important way to provide nitrogen for plants. Pseudomonas stutzeri DSM4166 is a diazotrophic Gram-negative bacterium isolated from the rhizosphere of cereal Sorghum nutans. Endogenous constitutive promoters are important for engineering of the nitrogen fixation pathway, however, they have not been systematically characterized in DSM4166. Results: Twenty-four candidate promoters were identified from DSM4166 by RNA-seq analysis. These 24 promoters were cloned and characterized using the firefly luciferase gene. The strengths of fourteen promoters varied from 100 to 959% of the strength of the gentamicin resistance gene promoter. The strongest P12445 promoter was used to overexpress the biological nitrogen fixation pathway-specific positive regulator gene nifA. The transcription level of nitrogen fixation genes in DSM4166 were significantly increased and the nitrogenase activity was enhanced by 23 folds. When the nifHDK nitrogenase genes and nifA were both overexpressed by endogenous strong constitutive promoters, the nitrogen fixation efficiency of DSM4166 was increased by 51 folds. Conclusions: The endogenous strong constitutive promoters identified in this study will facilitate development of DSM4166 as a microbial cell factory for nitrogen fixation and production of other useful compounds.

Background Biological nitrogen fixation converting atmospheric dinitrogen to ammonia is an important way to provide nitrogen for plants. Pseudomonas stutzeri DSM4166 is a diazotrophic Gram-negative bacterium isolated from the rhizosphere of cereal Sorghum nutans. Endogenous constitutive promoters are important for engineering of the nitrogen fixation pathway, however, they have not been systematically characterized in DSM4166. Results Twenty-six candidate promoters were identified from DSM4166 by RNA-seq analysis. These 26 promoters were cloned and characterized using the firefly luciferase gene. The strengths of nineteen promoters varied from 100 to 959% of the strength of the gentamicin resistance gene promoter. The strongest P12445 promoter was used to overexpress the biological nitrogen fixation pathway-specific positive regulator gene nifA. The transcription level of nitrogen fixation genes in DSM4166 were significantly increased and the nitrogenase activity was enhanced by 4.1 folds determined by the acetylene reduction method. The nifA overexpressed strain produced 359.1 µM of extracellular ammonium which was 25.6 times higher than that produced by the wild-type strain. Conclusions The endogenous strong constitutive promoters identified in this study will facilitate development of DSM4166 as a microbial cell factory for nitrogen fixation and production of other useful compounds.