Development of a genetic toolkit for Sphingomonas and its application in tailored welan gum fermentation from bagasse hydrolysate.

Sphingomonas represent a significant microbial resource with considerable industrial applicability. However, non-model microorganisms often lack efficient genetic modification tools, which hinders the development of microbial cell factories. In this study, using the welan gum-producing strain Sphingomonas sp. HT-1 as an example, we developed a stable E. coli-Sphingomonas shuttle plasmid pHTK based on endogenous plasmid mining, and the transformation efficiency was enhanced more than 1000-fold by optimizing the transformation conditions. A library of sixty endogenous promoters characterized by distinct strengths and expression dynamics was established. Then, red fluorescent protein expression was successfully achieved in various Sphingomonas species, demonstrating the cross-species applicability of this genetic toolkit. To further improve welan gum synthesis through molecular weight regulation, this toolkit was employed to modulate the expression of a sphingan lyase (SpnR). We demonstrated that the timing of SpnR expression is critical for determining the molecular weight and homogeneity of welan gum. Adaptive domestication enabled S. sp. HT‑1 to efficiently utilize sugarcane bagasse hydrolysate. Expression driven by the strong, late-growth promoter P3 in S. sp. HT-1 resulted in 28.53 g/L of reduced molecular weight welan gum (255.01 kDa) in a 5 L bioreactor fed with bagasse hydrolysate. This study establishes a genetic toolkit for Sphingomonas and presents an efficient one-step fermentation process for producing low-molecular-weight welan gum, expanding its application prospects.