Metabolic engineering of Halomonas for effective production of tryptophan-derived compounds

• Halomonas TD was engineered to produce 4.79 g L −1 violacein and 1.85 g L −1 indigo in lab-scale fed-batch study. • Gluconate used as a sole carbon source was found to facilitate the synthesis of indigo and violacein. • A QS-based inducer-free system was developed for precise auto-induction control of violacein and indigo synthesis. Halomonas TD able to grow under extreme condition is becoming a rising-star host for industrial biomanufacturing. In addition to the high-yield production of polyhydroxyalkanoates (PHA), the potential for amino acid derivates synthesis has been gradually developed in recent studies. Violacein and indigo derived from tryptophan are valuable natural pigments of various commercial uses, especially in pharmaceutical and cosmetic areas. Therefore, Halomonas TD was engineered to be a tryptophan derivate-producing platform for effective production of violacein and indigo. Firstly, prototyping production of violacein and indigo from tryptophan was achieved and optimized, respectively, by fine-tuning the expression of related key pathways. Secondly, two strategies, including using gluconate as feedstock to boost nicotinamide adenine dinucleotide phosphate (NADPH) synthesis and overexpressing NADPH-regeneration genes encoded by pntAB , were designed to obtain reinforced cofactor supply, leading to 46 % and 18 % titer improvement of violacein and indigo, respectively. Moreover, the de novo synthesis of violacein and indigo was explored by introducing the tryptophan synthesis module together with bypass deletion. Finally, the resultant recombinants were able to produce 1.85 g L −1 indigo and 4.79 g L −1 violacein under fed-batch fermentation condition. Besides, a quorum sensing (QS)-based (LuxR-LuxI) inducer-free expression system was developed to automatically control the synthesis of violacein and indigo with maximum titers reaching 2.64 g L −1 and 1.30 g L −1 , respectively, in fed-batch studies. Therefore, our study not only further expands the biosynthesis capacity of Halomonas TD, but also provides an effective metabolic engineering strategy and gene expression control tool for versatile cell factory design based on Halomonas .