Discovery of novel amino acid production traits by evolution of synthetic co-cultures

Background: Amino acid production features of Corynebacterium glutamicum were extensively studied in the last two decades. Many metabolic pathways, regulatory and transport principles are known, but purely rational approaches often provide only limited progress in production optimization. We recently generated stable synthetic co-cultures, termed Communities of Niche-optimized Strains (CoNoS), that rely on cross-feeding of amino acids for growth. This setup has the potential to evolve strains with improved production by selection of faster growing communities.

Results: Here we performed adaptive laboratory evolution (ALE) with a CoNoS to identify mutations that are relevant for amino acid production both in mono- and co-cultures. During ALE with the CoNoS composed of strains auxotrophic for either L-leucine or L-arginine, we obtained a 23% growth rate increase. Via whole-genome sequencing and reverse engineering, we identified several mutations involved in amino acid transport that are beneficial for CoNoS growth. The L-leucine auxotrophic strain carried an expression-promoting mutation in the promoter region of brnQ (cg2537), encoding a branched-chain amino acid transporter in combination with mutations in the genes for the Na⁺/H⁺-antiporter Mrp1 (cg0326-cg0321). This suggested an unexpected link of Mrp1 to L-leucine transport. The L-arginine auxotrophic partner evolved expression-promoting mutations near the transcriptional start site of the yet uncharacterized operon argTUV (cg1504-02). By mutation studies and ITC, we characterized ArgTUV as the only L-arginine uptake system of C. glutamicum with an affinity of K_D = 30 nM. Finally, deletion of argTUV in an L-arginine producer strain resulted in a faster and 24% higher L-arginine production in comparison to the parental strain.

Conclusion: Our work demonstrates the power of the CoNoS-approach for evolution-guided identification of non-obvious production traits, which can also advance amino acid production in monocultures. Further rounds of evolution with import-optimized strains can potentially reveal beneficial mutations also in metabolic pathway enzymes. The approach can easily be extended to all kinds of metabolite cross-feeding pairings of different organisms or different strains of the same organism, thereby enabling the identification of relevant transport systems and other favorable mutations.