5.1 Rapid prototyping and screening of novel biotemplates
Despite the exciting future for VLP development, the production and characterization of newly engineered VLPs are time-consuming processes, spanning several days. Thus, new screening tools are necessary to expedite the development of newly engineered VLPs. Cell-free systems that contain the transcriptional and translational machinery needed for VLP production could become integral to rapid VLP prototyping and characterization without laborious purification. Cell-free systems can be used to produce engineered VLP candidates in a highly parallel or high-throughput manner with minimal inputs due to their microliter scale reaction volumes [78]. VLPs derived from non-plant viruses are already being developed via cell-free technologies. For example, a cell-free system was used to incorporate noncanonical amino acids in bacteriophage MS2 and bacteriophage Qβ for click-chemistry functionalization [79].
Rapid screening is also needed to identify engineered mutants with desirable properties. Advanced DNA synthesis methods such as Gibson and Golden-gate assemblies can produce thousands of DNA-encoding VLPs variants in a single reaction [80–82]. However, screening of these variants for improved function requires characterization of individual mutants, which makes the screening process slow and inefficient. Instead of screening every variant, directed evolution can be applied to evolve mutants with desirable traits [83]. Directed evolution is a process where mutants are propagated and those with desirable properties are selected for. The ability of a given mutant to propagate or replicate is linked to the property that is desired, allowing surviving mutants to ‘select’ for enhanced properties. Thus, thousands of variants can be simultaneously evaluated in hours without screening of every single variant. The selected variants can then be mutated to introduce additional genetic diversity and subject to another round of selection. The iteration of these processes can generate protein mutants with optimized or even new properties such as producing VLPs with enhanced structural stability [84]. Although directed evolution has not been widely used for VLP engineering, the recent development of new directed evolution methods, including assisted machine learning [85], which identifies more promising mutations to be constructed and screened, andin vivo continuous directed evolution [86], which streamlines the genetic engineering process, will accelerate the engineering of VLPs.