Abstract
BACKGROUND: The establishment of synthetic microbial communities comprising complementary auxotrophic strains requires efficient transport processes for common goods. With external supplementation of the required metabolite, most auxotrophic strains reach wild-type level growth. One exception was the L-trypton auxotrophic strain phaCorynebacterium glutamicum ΔTRP ΔtrpP, which grew 35% slower than the wild type in supplemented defined media. C. glutamicum ΔTRP ΔtrpP lacks the whole L-tryptophan biosynthesis cluster (TRP, cg3359-cg3364) as well as the putative L-tryptophan transporter TrpP (Cg3357). We wanted to explore the role of TrpP in L-tryptophan transport, metabolism or regulation and to elucidate the cause of growth limitation despite supplementation. RESULTS: Mutants lacking either TRP or trpP revealed that the growth defect was caused solely by trpP deletion, whereas L-tryptophan auxotrophy was caused only by TRP deletion. Notably, not only the deletion but also the overexpression of trpP in an L-tryptophan producer increased the final L-tryptophan titer, arguing against a transport function of TrpP. A transcriptome comparison of C. glutamicum ΔtrpP with the wild type showed alterations in the regulon of WhcA, that contains an [Fe-S] cluster. Through evolution-guided metabolic engineering, we discovered that inactivation of SufR (Cg1765) partially complemented the growth defect caused by ΔtrpP. SufR is the transcriptional repressor of the suf operon (cg1764-cg1759), which encodes the only system of C. glutamicum for iron‒sulfur cluster formation and repair. Finally, we discovered that the combined deletion of trpP and sufR increased L-tryptophan production by almost 3-fold in comparison with the parental strain without the deletions. CONCLUSIONS: On the basis of our results, we exclude the possibility that TrpP is an L-tryptophan transporter. TrpP presence influences [Fe-S] cluster formation or repair, presumably through a regulatory function via direct interaction with another protein. [Fe-S] cluster availability influences not only certain enzymes but also targets of the WhiB-family regulator WhcA, which is involved in oxidative stress response. The reduced growth of WT ΔtrpP is likely caused by the reduced activity of [Fe-S]-cluster-containing enzymes involved in central metabolism, such as aconitase or succinate: menaquinone oxidoreductase. In summary, we identified a very interesting link between L-tryptophan biosynthesis and iron sulfur cluster formation that is relevant for L-tryptophan production. CLINICAL TRIAL NUMBER: Not applicable.