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.