Metabolic engineering of Pseudomonas taiwanensis for heterologous production of myxochelin A.

Natural compounds are promising molecules with pharmaceutical, environmental and biotechnological applications. The catecholate siderophore myxochelin A displays promising pharmaceutical characteristics, inhibiting human 5-lipoxygenase thereby impacting inflammatory reactions and cancer development. It is naturally produced by myxobacteria, but only in low amounts. We established its heterologous production in the versatile and robust host Pseudomonas taiwanensis VLB120. Different production modules were screened but initial production of myxochelin A from glucose was low (0.6 mg/L). Furthermore, the introduction of the biosynthetic genes for myxochelin A resulted in the unexpected formation of the structurally similar siderophore azotochelin. We showed that myxochelin A is converted to azotochelin and that the myxochelin A biosynthetic pathway is involved in this process. Consequently, cultivation time is important to prevent product losses. First improvements in myxochelin A and azotochelin production were achieved with a co-feed of 40 mM glycerol and 12 mM octanoate, yielding 1.5 mg/L myxochelin A and 8.1 mg/L azotochelin. Overexpression of additional copies of the mxcEFG genes further increased production to 151.2 mg/L from which 55.5 mg/L accounts for myxochelin A. Subsequently, de novo synthesis of 2,3-dihydroxybenzoate was identified as a bottleneck and alleviated by external supplementation of this precursor, resulting in a considerably boosted production. Overall, metabolic engineering and medium optimization, including 2,3-dihydroxybenzoate supplementation, increased myxochelin A production by >300-fold to 226.4 mg/L, while additionally yielding 56.6 mg/L azotochelin.