Presentation Information
[P02-209]Directed Evolution of Feedback-Resistant Enzymes in the Lysine Biosynthetic Pathway via Phage-Assisted Selection
○Daisuke Yonemoto1, Teppei Niide1, Takuya Matsumoto1, Mao Oota1, Yoshihiro Toya1, Hiroshi Shimizu1 (1. Osaka Univ. (Japan))
Keywords:
M13 phage,Directed evolution,Metabolic pathway
[Purpose]
In microbial chemical production, the feedback inhibition of metabolic enzymes is a primary factor that decreases productivity; therefore, its alleviation is essential. However, conventional approaches targeting allosteric regulation, such as rational design and random mutagenesis, have been limited by the difficulty of predicting structural changes and low throughput, respectively. This study aims to establish a novel directed evolution method to alleviate the feedback inhibition of metabolic enzymes by applying Phage-Assisted Continuous Evolution (PACE), which enables highly efficient, continuous mutagenesis and selection.
[Method]
We targeted Escherichia coli aspartate kinase III (LysC) and 4-hydroxy-tetrahydrodipicolinate synthase (DapA), which are allosterically inhibited by L-lysine. A novel evolutionary system was constructed to link the amino acid supply, governed by feedback inhibition, with the translation of phage-derived proteins (phage propagation). Specifically, we designed an evolutionary experiment in a glycerol sole-carbon-source medium in which the metabolic fluxes of both enzymes were suppressed using the L-lysine analogue S-(2-aminoethyl)-L-cysteine (AEC). Consequently, the supply of L-lysine, L-threonine, and L-methionine from the host functioned as the selection pressure for phage propagation.
[Results]
After 6 days of laboratory evolution using the constructed method, the phage formation titer per unit time increased by more than 100-fold. Furthermore, evaluation of the acquired mutants revealed that, particularly for LysC, variants were generated that exhibited no decrease in enzyme activity even in the presence of a high lysine concentration of 200 mM.
[Consideration]
The substantial increase in the phage titer indicates that the newly designed, state-dependent selection pressure based on amino acid supply operated properly within the system. Moreover, the rapid acquisition of LysC mutants that evade difficult-to-predict allosteric inhibition suggests that this method is highly effective at applying evolutionary pressure through metabolic network fluctuations, independent of structural information. This approach successfully overcomes the application limits of conventional PACE, which has historically been difficult to adapt for metabolic regulatory enzymes.
[Conclusion]
This system, which links phage propagation to amino acid supply, was demonstrated to be highly effective at alleviating feedback inhibition of metabolic enzymes. This method is expected to resolve metabolic bottlenecks in microbial chemical production in a high-throughput manner, thereby broadly improving product yields.
In microbial chemical production, the feedback inhibition of metabolic enzymes is a primary factor that decreases productivity; therefore, its alleviation is essential. However, conventional approaches targeting allosteric regulation, such as rational design and random mutagenesis, have been limited by the difficulty of predicting structural changes and low throughput, respectively. This study aims to establish a novel directed evolution method to alleviate the feedback inhibition of metabolic enzymes by applying Phage-Assisted Continuous Evolution (PACE), which enables highly efficient, continuous mutagenesis and selection.
[Method]
We targeted Escherichia coli aspartate kinase III (LysC) and 4-hydroxy-tetrahydrodipicolinate synthase (DapA), which are allosterically inhibited by L-lysine. A novel evolutionary system was constructed to link the amino acid supply, governed by feedback inhibition, with the translation of phage-derived proteins (phage propagation). Specifically, we designed an evolutionary experiment in a glycerol sole-carbon-source medium in which the metabolic fluxes of both enzymes were suppressed using the L-lysine analogue S-(2-aminoethyl)-L-cysteine (AEC). Consequently, the supply of L-lysine, L-threonine, and L-methionine from the host functioned as the selection pressure for phage propagation.
[Results]
After 6 days of laboratory evolution using the constructed method, the phage formation titer per unit time increased by more than 100-fold. Furthermore, evaluation of the acquired mutants revealed that, particularly for LysC, variants were generated that exhibited no decrease in enzyme activity even in the presence of a high lysine concentration of 200 mM.
[Consideration]
The substantial increase in the phage titer indicates that the newly designed, state-dependent selection pressure based on amino acid supply operated properly within the system. Moreover, the rapid acquisition of LysC mutants that evade difficult-to-predict allosteric inhibition suggests that this method is highly effective at applying evolutionary pressure through metabolic network fluctuations, independent of structural information. This approach successfully overcomes the application limits of conventional PACE, which has historically been difficult to adapt for metabolic regulatory enzymes.
[Conclusion]
This system, which links phage propagation to amino acid supply, was demonstrated to be highly effective at alleviating feedback inhibition of metabolic enzymes. This method is expected to resolve metabolic bottlenecks in microbial chemical production in a high-throughput manner, thereby broadly improving product yields.
Comment
To browse or post comments, you must log in.Log in