Engineering Pseudomonas taiwanensis VLB120 for regio- and stereospecific hydroxylation of l-lysine fueled by the Weimberg pathway

Abstract Background Hydroxy- l -lysines are versatile chiral building blocks and can be obtained by hydroxylation of the amino acid l -lysine. The conversion is catalyzed by α-ketoglutarate-dependent lysine dioxygenases (KDOs), which belong to the superfamily of Fe 2+ /α-ketoglutarate-dependent oxygenases. These enzymes are highly regio- and stereoselective; however, they require α-ketoglutarate (α-KG) as a cosubstrate. Apart from the costly direct addition of α-KG, it can be generated via cellular metabolism from inexpensive and renewable carbon sources, such as d -xylose. Therefore, we engineered a Pseudomonas taiwanensis VLB120 chassis to efficiently convert l -lysine to hydroxy- l -lysine using KDOs with the supply of α-KG from d -xylose as the sole carbon source via the Weimberg pathway. Results For the generation of a suitable whole-cell biocatalyst, we investigated the l -lysine catabolism of P. taiwanensis VLB120 and created a mutant strain that is deficient in l -lysine catabolism to minimize l -lysine degradation and to facilitate complete conversion via the biotransformation reaction. Next, a library of KDO genes was heterologously expressed in the engineered chassis strain P. taiwanensis VLB120∆C∆3. The hydroxylation of l -lysine was assessed in biotransformations with growing cells and d -xylose to supply α-KG via the Weimberg pathway. Hydroxy- l -lysine was successfully produced by strains harboring KDOs that hydroxylate the C-4 position of l -lysine. We further explored the three most promising whole-cell biocatalysts and investigated the influence of increased concentrations of the substrate l -lysine and the metal cofactor Fe 2+ . Finally, the engineered strain expressing a KDO from Flavobacterium species was grown in stirred-tank bioreactors and was able to produce 8.7 ± 0.3 g L −1 hydroxy- l -lysine with a space-time yield of 98.6 ± 3.4 mg L h −1 and a specific product yield on biocatalyst (Y Hyl/X ) of 1.68 ± 0.07 g g CDW −1 . The supply of α-KG via the Weimberg pathway proved very efficient, as approximately every second molecule of d -xylose which was converted and entered the central carbon metabolism was used for the biotransformation reaction (Y Hyl/Xyl,net = 0.48 ± 0.02 mol mol −1 ). Conclusions We successfully established a whole-cell biocatalyst for the synthesis of hydroxy- l -lysine from l -lysine and d -xylose and demonstrated multigram-scale production with our engineered strain. Our work lays the foundation for whole-cell bioprocesses utilizing Fe 2+ /α-ketoglutarate-dependent oxygenases fueled by the Weimberg pathway.