Team:UChicago/Project

Introduction

Directed evolution is a fundamental technique in bioengineering organisms to express proteins of novel function and to mass produce industrially relevant biomolecules. Generally, directed evolution simulates an algorithmic process in which the entire sequence space is searched for an optimal genotype by increasing the natural mutation rate to artificially speed the process of selective evolution. Current methods of in vivo directed evolution typically rely on physical or chemical mutagens to accomplish stochastic genomic or plasmid mutagenesis. However, the extremely high and uncontrolled mutation rate often accumulates deleterious mutations nonspecific to the process of interest. This can result in a toxic effect to the organism, leading to suboptimal evolved levels of production.

A novel directed evolution system, termed feedback-regulated evolution of phenotype (FREP) (citation http://www.ncbi.nlm.nih.gov/pubmed/24131951), incorporates a dynamic mutation rate to overcome the existing problems of directed evolution by mimicking the plasticity of the mutation rate in natural evolution. This is achieved through dynamic control of a mutator element that is negatively regulated by the desired end product. In this feedback scheme, as more of the desired biomolecule is produced, the rate of mutation decreases and eventually approaches zero, allowing evolution and maintenance of a high level of production while minimizing the accumulation of toxic, nonspecific mutations.

Additionally, many directed evolution systems that incorporate mutator genes often rely on the strongest known mutator, MutD. However, the use of MutD alone in these systems is problematic because like all other individual mutator genes, it may only catalyze certain types of base pair substitutions reflective of its “mutational bias”, limiting the search of sequence space. Diversifying the mutator genes to eliminate mutational bias should increase the efficiency by which the sequence space can be searched. Ultimately, the addition of multiple mutators should lead to higher overall levels of evolved production. Despite a very few number of past studies to incorporate multiple mutators, these efforts have failed to show an increased efficiency of evolution. They fail to encompass a systematic screen of all mutators identified in literature, which would be required for determination of the ideal combination of mutators. In general, there is a surprising lack of studies on the use of different combinations of mutators in directed evolution.

The 2014 UChicago iGEM team has two main goals. The first goal is to implement and optimize FREP in E. coli by evolving mutants with elevated production of a given biomolecule, specifically evolving tyrosine production as a relatively simple pathway to demonstrate proof of concept. The mutator element will be controlled by the tyrosine-sensitive repressible promoter ParoF, suppressing the mutation rate as the tyrosine production of the cell increases. The mutator gene is linked to a fluorescent reporter gene which allows for convenient screening of desired phenotype, as bacteria with low levels of fluorescence should also have the highest levels of production due to the negative feedback loop. The second goal is to enable a deeper exploration in the search space by combining multiple mutators with different biases to increase the initial mutation rate and speed the accumulation of beneficial mutations. We have identified previously characterized mutator genes that function in a diversity of pathways, including polymerases, proofreading enzymes, methylases and topoisomerases. We will characterize the mutation rates of these mutator genes in their active dominant negative forms individually as well as in various combinations, aiming to determine the ideal combination for directed evolution. We hope to introduce this ideal combination of mutators into the FREP system and demonstrate increased levels of evolved tyrosine production compared to the single mutator. If successful, our project should significantly improve the process of directed evolution of any biomolecule of industrial importance.

Mutator Constructs

In order to work towards our goal of finding the ideal multiple mutator gene combination for FREP, we first researched a variety of genes that when overexpressed, had been previously documented in literature to increase the mutation rate. Because of the manner in which FREP is specifically set up, dynamic control can only be achieved in a gene that increases mutation rate when overexpressed (as opposed to mutators that cause an increase in mutation rate from knockdown or full deletion for instance). Since most of the mutator genes we used had little effect from simple overexpression, we instead relied on mutation dominant negative versions of these genes that are documented to cause high increases in mutation rate when expressed in cells. These mutator genes are involved in a variety of pathways, and include polymerase subunits, DNA repair enzymes, and transposable elements. We also kept in mind the various mutational biases of the various mutators we were interested in. The table below lists all various types of mutators that we researched, as well as the point mutations needed to create the active dominant negative genes when necessary.; bolded mutators with an asterisk (*) are the actual mutators we ended up attempting to PCR from the wild-type E. Coli genome. (citation http://www.sciencedirect.com/science/article/pii/S0966842X98014243)