Discovery and engineering of an endophytic Pseudomonas strain from Taxus chinensis for efficient production of zeaxanthin diglucosided

Background: Endophytic microorganisms are a rich source of bioactive natural products. They are considered as promising biofertilizers and biocontrol agents due to their growth-promoting interactions with the host plants and their bioactive secondary metabolites that can help manage plant pathogens. Identification of new endophytes may lead to the discovery of novel molecules or provide new strains for production of valuable compounds. Results: In this study, we isolated an endophytic bacterium from the leaves of Taxus chinensis, which was identified as Pseudomonas sp. 102515 based on the 16S rRNA gene sequence and physiological characteristics. Analysis of its secondary metabolites revealed that this endophytic strain produces a major product zeaxanthin diglucoside, a promising antioxidant natural product that belongs to the family of carotenoids. A carotenoid (Pscrt) biosynthetic gene cluster was amplified from this strain, and the functions of PsCrtl and PsCrtY in the biosynthesis of zeaxanthin diglucoside were characterized in Escherichia coli BL21(DE3). The entire Pscrt biosynthetic gene cluster was successfully reconstituted in E coil BL21(DE3) and Pseudomonas putida KT2440. The production of zeaxanthin diglucoside in Pseudomonas sp. 102515 was improved through the optimization of fermentation conditions such as medium, cultivation temperature and culture time. The highest yield under the optimized conditions reached 206 mg/L. The engineered strain of P. putida KT2440 produced zeaxanthin diglucoside at 121 mg/L in SOC medium supplemented with 0.5% glycerol at 18 degrees C, while the yield of zeaxanthin diglucoside in E coli BL21(DE3) was only 2 mg/L. To further enhance the production, we introduced an expression plasmid harboring the Pscrt biosynthetic gene cluster into Pseudomonas sp. 102515. The yield in this engineered strain reached 380 mg/L, 85% higher than the wild type. Through PCR, we also discovered the presence of a turnerbactin biosynthetic gene cluster in Pseudomonas sp. 102515. Because turnerbactin is involved in nitrogen fixation, this endophytic strain might have a role in promoting growth of the host plant. Conclusions: We isolated and identified an endophytic strain of Pseudomonas from T chinensis. A zeaxanthin diglucoside biosynthetic gene cluster was discovered and characterized in this bacterium. Through fermentation and genetic engineering, the engineered strain produced zeaxanthin diglucoside at 380 +/- 12 mg/L, representing a promising strain for the production of this antioxidant natural product. Additionally, Pseudomonas sp. 102515 might also be utilized as a plant-promoting strain for agricultural applications.