Correlation between genotype and phenotype in enzyme directed evolution
Directed evolution mainly includes two parts: library construction and mutant screening. The key point is to establish the physical correspondence between genotype and phenotype (ie, mutant performance). In the previous two issues, we have introduced the mutant library construction technology and mutant screening technology for enzyme directed evolution. Today I will introduce the correlation between genotype and phenotype.
The premise of an efficient screening method is to establish a reliable correlation between genotype and phenotype, which involves two levels: first, the genotypes of mutants that conform to the expected phenotype can be traced back, that is, the connection between the amino acid sequence and genetic information is established; Secondly, the phenotypic changes of the mutant compared to the parent can be captured by equipment or the naked eye, and it is better to quantify the degree of performance improvement of the mutant.
01 Direct correlations that rely on physical space or molecular interactions
The key to the physical connection between the mutant's genetic material and the protein is to lock the two in a fixed space. The changes in physical and chemical properties caused by the enzyme reaction serve as the basis for screening, and the genetic material in the same space serves as the molecular tag of the mutant enzyme. The microbial cells used in in vivo screening are naturally occurring and ideal physical spaces. The desired phenotype can be directly traced back to the corresponding genotype, and individuals with sustainable reproduction can be directly obtained. The microdroplets in CSR and FADS technologies construct a physical barrier outside the cells to ensure that their DNA and proteins still coexist after the cells are broken. For droplet screening technology based on a cell-free reaction system (Figure 1), the "internal packaging" of microorganisms is missing, and nucleic acids (mRNA or DNA) and proteins in homogeneous solutions can only rely on intermolecular interactions. Direct coupling (such as relying on puromycin and puromycin linker to establish contact) forms a structure similar to an mRNA-ribosome-protein ternary complex to ensure one-to-one correspondence between the enzyme and the corresponding nucleic acid.
02 Indirect correlation relying on clone encoding and backup
Another method is to back up a single clone of the mutant in advance. After the performance evaluation of the mutant enzyme is completed, it can be traced back to the corresponding seed solution through the test tube number for re-cultivation and subsequent testing. In the field of droplet technology, as the accuracy, speed, and intelligence of equipment improve, droplet coding marking and single droplet separation technology will also be able to realize the backup of droplets in the FADS system.
Figure 1.Enzyme reaction process monitoring
（From：doi: 10.1016/j.tibtech.2020.01.001）
The types of enzymes vary widely, and different enzymes have diverse performance improvement needs. Therefore, the criteria for directed evolution screening need to be accurate, objective, and easy to quantify. More importantly, performance changes can be identified directly or indirectly.
03 Indirect detection
In the in vivo screening system, changes in the performance of enzyme proteins involved in the transcription and translation processes can be converted into differences in the levels of reporter genes, such as methylases, RNA polymerases, transcription factors (Figure 1c1), regulatory factors involved in transcription, involving Translating ribozymes, tRNA and tRNA synthetase. In addition, some transcription factors need to cooperate with specific metabolic intermediates (such as LacZ and galactose, AraC and arabinose). The metabolic pathway-related enzymes involved in these small molecules also have the potential to establish an intracellular screening system based on reporter genes. . A riboswitch is a special RNA sequence that can change its secondary structure under the induction of small molecules. This type of small molecules can also be associated with specific metabolic pathways, and the expression of reporter genes downstream of the riboswitch indicates the enzymes in the pathway. Performance changes (Figure 1 c3, Figure 1 c4).
04 Direct detection
The product is a better enzyme reaction marker. In the in vitro screening system, the product can be directly quantified using mass spectrometry equipment (Figure 1e), or the changes in turbidity, pH, potential, and fluorescence intensity of the reaction solution caused by product accumulation (Figure 1e) can be monitored. 1a&d). However, during high-throughput screening, the signal in the system is often interfered by cell debris and contents, and the extremely low total amount of product in the microdroplets also poses a great challenge to the sensitivity of the equipment. Therefore, some specific recognition, labeling techniques and cascade amplification strategies are used to assist in the screening of mutants. An aptamer is a nucleic acid sequence (DNA or RNA) that folds into a special secondary structure to specifically recognize and bind to the target molecule, and even excites fluorescence by forming a stable complex (Figure 1c5), which is characteristically used by devices. Recognition thereby improves the specificity and sensitivity of detection. Aptamers can also serve as reactants for RNA polymerase, directly indicating the activity of mutants (Figure 2). Labeling groups used in DNA product detection can be used in various combinations to test the activity of polymerases, ligases, restriction endonucleases, fluorescent groups and quenching groups caused by changes in substrate structure or integrity. The change in spatial distance gives the fluorophore the potential to emit fluorescence and be captured by the device (Figure 3).
Figure 2. RNA aptamer
（From：doi: 10.1016/j.saa.2022.121760.）
Figure 3. FRET quantitative detection of DNA
（From：doi: 10.1021/acssynbio.9b00103.）
The sequence composition of DNA determines the primary structure and catalytic performance of the enzyme protein. The physical connection between the dominant mutants and the corresponding genotype is the key to people's ultimate deciphering of the amino acid sequence. At least until the advent of efficient and convenient protein sequencing technology, one-to-one correlation of nucleic acids and proteins is indispensable for the directed evolution of enzymes.
At this point, the mutant library construction technology of enzyme directed evolution, mutant screening technology, and the correlation between genotype and phenotype have been introduced. In summary, ultra-high-throughput screening is crucial to fully realize the potential of directed evolution, but there is still huge room for improvement in the development of signal amplification strategies and equipment that match efficient screening (i.e., fast, accurate, and sensitive). It is believed that with the continuous advancement of research on enzyme reaction mechanisms and detection technologies, directed evolution technology will be able to iterate faster in the future.