A complete collection of mutant library screening technologies for enzyme directed evolution
In the last issue, we introduced the mutant library construction technology for enzyme directed evolution. High-quality mutant libraries are the basis for directed evolution, matched by efficient and sensitive mutant screening techniques. Depending on where the mutant enzyme participates in the reaction, screening technology can be divided into in vivo and in vitro screening. The throughput and accuracy of screening are improving with the continuous upgrading of technology.
In Vivo Screening
The more traditional in vivo screening is derived from strain screening. The activity of the target enzyme is related to the growth of the strain and the degree of substrate metabolism in the environment. Enzymes with defective performance are not enough to support the formation of a single clone in the host, or are unable to significantly change the substrate. This method is suitable for screening enzymes related to key metabolic pathways such as bacterial growth and antibiotic resistance, or proteins that can cause significant color changes in substrates. However, since microorganisms can adjust their metabolic flow to adapt to the environment, and there are multiple compensation pathways, the advantage of strain performance may not be solely the contribution of the target enzyme.
In 2011, David Liu's team developed the PACE system (Phage-assisted continuous evolution) for domestication of T7 RNA polymerase. PACE uses M13 filamentous phage as a vector to correlate the activity of the target enzyme with the number of progeny phages. The design principle is shown in Figure 1b. The T7 RNA polymerase gene replaces the phage capsid protein gene gIII and is controlled by the gIII promoter. The gIII gene is installed on the helper plasmid and is controlled by the T7 promoter. After the recombinant phage infects the host, the gIII promoter opens and the level of T7 RNA polymerase increases, which in turn mediates the massive expression of GIII protein. The T7 RNA polymerase gene is packaged in the progeny phage. The higher the polymerase activity, the more active progeny phage particles are produced, and the corresponding mutant sequences are gradually enriched as the generations increase. Not only that, the PACE system also includes mutagenesis plasmid MP (Mutagenesis plasmid). Multiple mutagenesis genes, induced by L-arabinose, mediate base mutations throughout the genome, including the target gene. In the continuous fed-batch culture system, the E. coli that have accumulated mutations in the genome are continuously diluted and replaced by fresh cells to provide high-quality hosts for progeny phages, while the phages carrying mutations continue to infect and superimpose new mutations, achieving Continuous screening and evolution of target genes (Fig. 1a). Using the PACE system, the project team successfully domesticated a T7 RNA polymerase mutant that can recognize the T3 promoter.
Figure1. PACE evolution system
（From：doi: 10.1038/s41596-020-00410-3.）
The PACE system has been rapidly promoted due to its advantages of speed and automation, and is suitable for the evolution of RNA polymerase, protease, tRNA synthetase and other enzymes, as well as the screening of some proteins that interact with DNA and proteins (Figure 1c). At the same time, with the assistance of the GIII antagonist protein GIII-neg, the PACE system can be more flexibly used to improve the different performances of multiple enzymes (Figure 1d). Nonetheless, proteins that cannot be linked to gIII or other phage gene expression processes remain difficult to screen using the PACE system, and all selective pressures that would lead to abnormal E. coli growth are difficult to apply.
In Vitro Screening
Different from in vivo screening, in vitro screening requires the use of lysozyme, ultrasonic fragmentation or secretion expression strategy to release the target protein and react with the substrate, with the help of detectable changes in the amount of substrate and product or changes in the physical and chemical properties of the system. to select mutants of interest. Traditional in vitro screening uses test tubes and multi-well plates as culture vessels. The mutant enzymes are separated, purified, and concentrated, and then their performance is evaluated. The advantage of well plate screening is that the test is accurate and reproducible. With the support of high-throughput purification technology and fully automatic sampling robots, the test throughput is also increasing. Well plate screening can be used to screen random libraries and saturated mutation libraries.
In addition to conventional test tube or well plate screening, cell diameter water-in-oil droplets are also a tool for separating different mutants. This strategy was first used in methyltransferase screening tests in 1998. Each DNA molecule and the buffer required for protein expression are wrapped in a single droplet, and the active methyltransferase is expressed. DNA can be modified to protect it from cleavage by endonucleases, ensuring that the biotin tag is intact at the 3' end, and can then be purified and enriched. The methyltransferase finally stood out among the 10^7 simulation libraries (Figure 2).
Figure 2. Screening of HaeIII methyltransferase
（From：doi: org/10.1038/nbt0798-652.）
Inspired by the above system, a CSR (Compartmentalized Self-Replication) system tailored for DNA polymerase was developed. E. coli replaces the cell-free expression system and is responsible for expressing the polymerase and providing the DNA template. The buffer, dNTPs and primers required for PCR are added to the droplets. The primers are used to amplify the coding region of the mutant. The high temperature causes the cells to break, and the thermostable DNA polymerase is released to complete the PCR reaction. The higher the activity of the DNA polymerase, the more DNA sequences the corresponding mutant has, and the more clones containing the mutant will be included in the progeny library, ultimately forming a significant quantitative advantage (Figure 3). CSR has been used in the evolution of DNA polymerases such as Taq, pfu, KOD, and Bst, as well as some proteins that can be associated with DNA polymerase expression (such as T7 RNA polymerase, tRNA synthetase, and tRNA). It is difficult to meet the needs in the evolution.
Figure 3. Separate self-replicating systems
（From：doi: 10.1073/pnas.071052198.）
In addition to methods such as PACE and CSR, which enrich dominant mutants by accumulating numbers, directed evolution of enzymes can also adopt mutant sorting strategies.
Taking flow sorting technology as an example, as long as the enzyme performance is related to the size, shape, fluorescence intensity or surface modification status of the cells, when the cells flow through the monitoring port, the instrument can collect relevant signals and use the action of electric charges and magnetic fields to achieve Sorting of target cells. FADS (Fluorescence-Activated Droplet Sorting) and its derivative systems (AADS, BADS, MADS) suitable for micron-scale microorganism sorting are another technology, which is a combination of flow sorting and droplet technology. Microbial cells and substrates are dispersed in the reaction buffer and flow into the PDMS chip as the water phase. The oil phase enters from the vertical direction. By adjusting the flow rates of the two liquids, the water phase is evenly divided into droplets of 20-50 μm in size. . Unlike the heat-resistant CSR system, the cell disruption of the droplet sorting system can only rely on lysozyme or other chemical reagents. To prevent cells from lysing before entering the droplet, it is recommended that the lysing reagent be injected into the droplet by reinjection, or use an alternative method to first mix the lysis component with the cells and then inject the reaction substrate into the droplet. The droplets are then incubated offline under appropriate conditions. The catalytic reaction will cause significant changes in the turbidity, pH value, fluorescence signal intensity, and amount of characteristic compounds within the droplets. Droplets exceeding the set threshold will be exposed to the electric field or air flow. It is deflected under the action and flows into the collection tube (Figure 4). The collected intact cells can be directly spread and cultured, while the broken cells can only amplify and recover DNA for subsequent experiments.
Figure 4. Droplet sorting
（From：doi: 10.1016/j.tibs.2021.11.001.）
The droplet sorting system has the following advantages: direct detection of products can accurately reflect changes in enzymatic properties; pico-level droplet reactors can significantly save reagent costs (~10^6 times); more can achieve 10^8 /day droplet sorting; the chip structure can be designed and the module combination can be adjusted to adapt to the screening scenarios of multiple enzymes.
The emergence of ultra-high-throughput screening technology has greatly improved the efficiency of enzyme directed evolution and met the screening needs of large mutant libraries. In particular, droplet-based in vitro screening technology further shortens the iteration cycle of enzymes, can quickly fill in the defects of enzyme performance in special application scenarios, and promote the development of modern life sciences and industrial applications.