“…We recently demonstrated that synthetic CoNoS composed of two complementary amino acid-auxotrophic strains are able to grow based on mutual dependency [ 9 ]. To improve amino acid exchange and thus growth of the CoNoS, the strains were metabolically engineered addressing known targets for increased amino acid production [ 9 ]. In this work, we exploit ALE to improve the fitness of microbial communities (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…ΔLEU ARG+ is derived from the genome-reduced strain C1* with an in frame deletion of argR (cg1585) and produces a sufficient amount of l- arginine for co-culture growth. A CoNoS composed of these two strains has a growth rate resembling almost half of the WT level, leaving potential for improvement by selecting for faster growing cultures [ 9 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…After confirming the beneficial effect of the single identified mutations on CoNoS growth, we wanted to know whether these mutations further improve a CoNoS so far only optimized by rational engineering. In our previous paper, we described the CoNoS C1*∆LEU ARG ++ ↔ WT∆ARG LEU ++ as the best CoNoS available to date [ 9 ]. In the meantime, we also tested a CoNoS with strains WT∆LEU ARG−+ (ΔLEU ARG −+ ) ↔ WT∆ARG LEU ++ where ∆LEU ARG −+ only carries the mutations ArgB A26V M31V but not the deletion of argR (Please note the superscript “− + ” to distinguish this strain from the one that only carries the Δ argR mutation).…”
Section: Resultsmentioning
confidence: 99%
“…Many metabolic pathways as well as regulatory and transport principles are known, but further improvements of amino acid production strains by rational approaches became less and less in the last years. Thus, we decided to use C. glutamicum as model organism for our CoNoS approach [ 9 ]. We established a fast and stable growing community of an l -arginine and an l -leucine auxotrophic strain, both rationally engineered for higher production of the amino acid required by their partner strain.…”
Section: Introductionmentioning
confidence: 99%
“…We established a fast and stable growing community of an l -arginine and an l -leucine auxotrophic strain, both rationally engineered for higher production of the amino acid required by their partner strain. This CoNoS reached a growth rate equivalent to 83% of the wild type, suggesting some remaining bottlenecks in their cross-feeding relationship [ 9 ]. These bottlenecks are most likely related either to amino acid production, which is well studied in C. glutamicum , or to transport processes.…”
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 KD = 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.
“…We recently demonstrated that synthetic CoNoS composed of two complementary amino acid-auxotrophic strains are able to grow based on mutual dependency [ 9 ]. To improve amino acid exchange and thus growth of the CoNoS, the strains were metabolically engineered addressing known targets for increased amino acid production [ 9 ]. In this work, we exploit ALE to improve the fitness of microbial communities (Fig.…”
Section: Resultsmentioning
confidence: 99%
“…ΔLEU ARG+ is derived from the genome-reduced strain C1* with an in frame deletion of argR (cg1585) and produces a sufficient amount of l- arginine for co-culture growth. A CoNoS composed of these two strains has a growth rate resembling almost half of the WT level, leaving potential for improvement by selecting for faster growing cultures [ 9 ]. Fig.…”
Section: Resultsmentioning
confidence: 99%
“…After confirming the beneficial effect of the single identified mutations on CoNoS growth, we wanted to know whether these mutations further improve a CoNoS so far only optimized by rational engineering. In our previous paper, we described the CoNoS C1*∆LEU ARG ++ ↔ WT∆ARG LEU ++ as the best CoNoS available to date [ 9 ]. In the meantime, we also tested a CoNoS with strains WT∆LEU ARG−+ (ΔLEU ARG −+ ) ↔ WT∆ARG LEU ++ where ∆LEU ARG −+ only carries the mutations ArgB A26V M31V but not the deletion of argR (Please note the superscript “− + ” to distinguish this strain from the one that only carries the Δ argR mutation).…”
Section: Resultsmentioning
confidence: 99%
“…Many metabolic pathways as well as regulatory and transport principles are known, but further improvements of amino acid production strains by rational approaches became less and less in the last years. Thus, we decided to use C. glutamicum as model organism for our CoNoS approach [ 9 ]. We established a fast and stable growing community of an l -arginine and an l -leucine auxotrophic strain, both rationally engineered for higher production of the amino acid required by their partner strain.…”
Section: Introductionmentioning
confidence: 99%
“…We established a fast and stable growing community of an l -arginine and an l -leucine auxotrophic strain, both rationally engineered for higher production of the amino acid required by their partner strain. This CoNoS reached a growth rate equivalent to 83% of the wild type, suggesting some remaining bottlenecks in their cross-feeding relationship [ 9 ]. These bottlenecks are most likely related either to amino acid production, which is well studied in C. glutamicum , or to transport processes.…”
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 KD = 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.
The concept of modular synthetic co-cultures holds considerable potential for biomanufacturing, primarily to reduce the metabolic burden of individual strains by sharing tasks among consortium members. However, current consortia often show unilateral relationships solely, without stabilizing feedback control mechanisms, and are grown in a shared cultivation setting. Such ‘one pot’ approaches hardly install optimum growth and production conditions for the individual partners. Hence, novel mutualistic, self-coordinating consortia are needed that are cultured under optimal growth and production conditions for each member. The heterologous production of the antibiotic violacein (VIO) in the mutually interacting E. coli–E. coli consortium serves as an example of this new principle. Interdependencies for growth control were implemented via auxotrophies for L-tryptophan and anthranilate (ANT) that were satisfied by the respective partner. Furthermore, VIO production was installed in the ANT auxotrophic strain. VIO production, however, requires low temperatures of 20–30 °C which conflicts with the optimum growth temperature of E. coli at 37 °C. Consequently, a two-compartment, two-temperature level setup was used, retaining the mutual interaction of the cells via the filter membrane-based exchange of medium. This configuration also provided the flexibility to perform individualized batch and fed-batch strategies for each co-culture member. We achieved maximum biomass-specific productivities of around 6 mg (g h)−1 at 25 °C which holds great promise for future applications.
Synthetic bacterial communities are currently under intensive investigation. Using natural communities as models, we established the CoNoS (Communities of Niche-optimized Strains) approach to create synthetic communities composed of different strains of the same species. By combining CoNoS with adaptive laboratory evolution, we identified new amino acid production traits, thereby demonstrating the high potential for their use in basic research, and applied biotechnology.
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