Development of a Gene-specific Mutator for Rapid Continuous Evolution of Proteins in vivo

Abstract

Continuous directed evolution has emerged enabling the fast and efficient evolution of proteins in cells. Additionally, diverse in vivo mutagenesis methods have been developed. However, they still have limitations such as a low mutation rate, no targeting ability, or narrow editing window. Here, we report a mutator, eMutaT7, with a high mutation rate and and high gene-specificity in Escherichia coli. eMutaT7, a cytidine deaminase fused to T7 RNA polymerase, can introduce up to ~4 mutations per 1 kb per day, which is comparable to the rate in traditional in vitro mutagenesis. eMutaT7 enables the rapid continuous evolution of proteins for antibiotic resistance and allosteric activation.

지속적인 지향적 진화(Continuous Directed Evolution)는 세포에서 단백질의 빠르고 효율적인 진화를 가능하게 했다. 이를 위해 다양한 생체 내 돌연변이 유발 방법이 개발되었지만, 여전히 낮은 돌연변이율, 표적 불가, 좁은 편집 영역과 같은 한계가 있습니다. 우리는 높은 돌연변이 속도와 대장균에서 높은 유전자 특이성을 가진 eMutaT7이라는 뮤테이터를 개발했다. T7 RNA 중합효소에 cytidine deaminase를 융합한 eMutaT7은 하루에 1kb 당 최대 4 개의 돌연변이를 도입 할 수 있고, 이는 기존의 시험관 내 돌연변이 유발 속도와 비슷한 수준이다. 이후, eMutaT7으로 항생제 내성 단백질과 단백질 분해 효소의 알로스테릭 활성화를 했고, 이는 eMutaT7이 신속한 연속 진화에 활용될 수 있다는 것이다.

DegP, a member of the highly conserved HtrA protease family, performs elaborately regulated degradation of misfolded proteins in the periplasm of Gram-negative bacteria. The allosteric conversion between the inactive and active conformations is a principal mechanism for careful controlling the proteolytic activity of DegP and maintaining optimal cellular fitness, but few molecules except the substrates are known as the allosteric modulators of DegP. Here, we show that a mutant variant of the outer membrane lipoprotein, Lpp+Leu, can act as a novel allosteric effector that alters the dynamic range of DegP activity. The three leucines at the C-termini of the Lpp+Leu trimer are key components for activity regulation. Selection experiments using the Lpp variant library show that Lpp variants with varying sequences at or near the C-terminus, especially Lpp variants with hydrophobic residues at the C-terminus, function similarly to Lpp+Leu. Interestingly, Lpp variants with different residues at the C-terminal, penultimate, or antepenultimate position exhibit dramatically different patterns of activation and inhibition effects, indicating that their interactions with DegP differentially stabilize distinct active or inactive forms. We suggest that the tripodal structure with three hydrophobic ends that mimics the Lpp+Leu can be a new platform for allosteric effectors, which can be applicable to develop new antibiotics against DegP or homologous HtrA protease.