SynBac:

: minimal synthetic baculovirus genomes

Abstract

The baculovirus expression vector system (BEVS) is among one of the favourites in protein
expression systems due to the high protein yields attainable and relative ease of use and
availability. The development of the system has been an ongoing task since its discovery as a
successful protein production method. To realise the full potential of the system, key
improvements are still necessary, notably to reduce DNA instability due to passage effects that impede industrial applications, as well as inherent proteolysis of produced recombinant target proteins. Through data mining and phylogenetic analysis, a putative synthetic baculoviral genome, SynBac, has been designed. In an ultimate embodiment, this SynBac genome will be devoid of all elements, which negatively affect or serve no obvious purpose in target protein expression and virus replication and infection in a laboratory environment. The hypothesis is that elimination of some or all of these presumably non-essential elements will improve the characteristics of the baculovirus, as a production tool for important biomolecules in academic and industrial research.

The aim of this thesis is to iteratively create a minimal synthetic baculovirus genome, SynBac,
and validate SynBac for genomic stability and protein expression properties. The first approach involved the generation of hybrid baculoviral genomes by deletion of wild-type DNA and replacement with synthetic fragments devoid of non-essential genes. Rewiring of a first fragment provided a functional initial genome, SynBac1. Addition of further synthetic fragments resulted in virus with impaired virus replication. Thus, necessitating careful identification of individual genes that were necessary to restore virus function. To streamline genome engineering, different
recombinases were tested resulting in an efficient and marker-less protocol to engineer the baculoviral genome. 67 single gene deletions of the SynBac1 genome were generated, providing critical information about the requirement of individual genes. Unlike previously thought, only several genes, ac7, ac20, ac32, ac45, ac56, ac87, ac110 and ac112/113, could be deleted without
negatively affecting the replication and protein expression of the virus. The Tn7 attachment site
was relocated and VloxP and RoxP sites inserted into identified non-essential genes, resulting in
functional viruses, thus further confirming that these genes were dispensable. The stability of the SynBac variants was analysed and suggested the SynBac genomes are more stable compared to EMBacY. The viruses appeared to develop lower titers during amplification, however this data remains to be corroborated by obtaining precise virus titer information. Notwithstanding, the protein expression capacity of the SynBac variants proved to be equal to EMBacY, suggesting the SynBac baculoviruses may exhibit superior protein productivity, on a per virion basis.

Date of Award	28 Sept 2021
Original language	English
Awarding Institution	University of Bristol
Supervisor	Ian R Collinson (Supervisor) & Imre Berger (Supervisor)