Rewriting the genetic code of synthetic bacteria imparts viral resistance & advances biotherapeutics

synthetic bacteria are resistant to viral infections

Date: 8th June 2021

Making bacteria resistant to viruses offers to advance the manufacturing of drugs, making them more reliable and cheaper.  Furthermore, genetic code re-engineering to create designer proteins could open the avenues to synthesise a range of materials such as new and novel medicines, chemicals and even plastics.  One early approach has been to utilise protein making cellular components, enabling them to incorporate unnatural or non-canonical amino acids however, work is still embryonic and often remains theoretical.  Now, researchers have reengineered a synthetic E.coli compressed genome strain, syn61, introducing three distinct non-canonical amino acids.  This represents the first genetically encoded cellular synthesis of completely synthetic polymers, and renders the bacteria completely resistant to viral infection.

Until recently, virtually all organisms use 20 canonical amino acids in the assembly of proteins, encoded by 64 triplet codons.  However, minimising the genome is possible as shown by Craig Venter in 2016, who created artificial life derived from Mycoplasma mycoides.  The latest version, called JCVI-syn 3.0, focused on the minimal genetic requirement for a free living organism, 473 genes in this instance, which was around half of the natural number of genes for this organism.

Then, in 2019 Jason Chin and colleagues at the Medical Research Council’s Laboratory of Molecular Biology in Cambridge, UK,  created the world’s first living organism that had a fully synthetic and radically altered DNA code. Known as Syn61, the team synthesised and recoded the entire genome of Escherichia coli, which is ~ 4 times larger than that of M.mycoides. Removing its superfluous codons, syn61 contained just 61 codons resulting from an impressive number of 18,214 codon replacements, in which two sense codons (serine codons TCG and TCA) and a stop codon (TAG) were replaced with synonymous codons.

Now, Chin and researchers have evolved Syn61 yet further, deleting two transfer RNAs (tRNAs) and a release factor that decode the absent codons (TCG,TCA and TAG) – making their cognate codons unreadable.  The resulting strain, Syn61Δ3, was completely resistant to a cocktail of viruses.  In addition, they reassigned these codons to enable the efficient synthesis of proteins containing three distinct noncanonical amino acids, creating a programmable ‘living’ factory which synthesised non-canonical heteropolymers and macrocycles.

It had been hypothesised that removing cellular transfer RNAs (tRNAs)—which make reading their codons impossible – might create a genetic firewall to viral infection and enable sense codon reassignment.  However, until now scientists have not been able to test the theory however, Syn61 provided the perfect tool, as three codons had already been removed.

To start, the team therefore deleted the associated anticodon tRNAs and the release factor 1, RF1 (which would inhibit the ability to efficiently terminate translation on the TAG stop codon), that were predicted to decode the missing codons that had previously been deleted in Syn61.  They found the resulting Syn61Δ3 was viable and after some optimisation grew at a similar rate to the original synthetic strain.

The researchers then infected Syn61Δ3 with a single phage or even a cocktail of phage and found it ablated virus production, and led to a steady decrease in total phage titre, without affecting bacteria growth.  Furthermore, they found that it was deletion of tRNAs in Syn61Δ3, rather than RF1, that provided resistance to the broad range of phage.

Having freed up the three codons, the team wanted to repurpose them for example coding for synthetic building blocks.  Here, they reassigning the two sense codons and a stop codon to distinct noncanonical amino acid (ncAA) in Syn61∆3, and engineered the bacteria to produce tRNAs coupled with artificial monomers, which recognised the newly available codons. In total, to demonstrate the generality of the approach, they programmed the synthesis of seven distinct version of the ubiquitin protein, each of which incorporated three distinct ncAAs.

To further build on this, the team wanted create synthetic polymers, composed out of the individual distinct monomers.  They genetically encoded 22 synthetic polymer sequences ranging from tetra-, hexa-, and octa-meric in size.  All encoded polymerisations were successful and ncAA-dependent furthermore, they were able to join the ends of these polymers together to make non-natural macrocyles – which are a type of molecule that form the basis of drugs such as antibiotics and cancer drugs.

Conclusions and future applications

The team here have synthetically uncoupled a synthetic bacteria strain from the ability to read the canonical code, rendering it resistant to viral infection.  This advance will have profound effects on bioproduction, eliminating the potential catastrophic risks associated with viral contamination and lysis.  The synthetic codon compression and codon reassignment strategy has produced the first genetically encoded cellular synthesis of completely synthetic polymers.

The team will now expand the principles and system to enable programmable and encoded synthesis of an expanded set of nano-canonical heteropolymers.  They are hoping that by creating long synthetic polymers that fold into structures, they may form new classes of biotherapeutics, opening the avenue to creating vast libraries of potential new drugs.  Furthermore, they will also be exploiting the system to develop new biomaterials with innovative properties.  With many more potential redundant codons still available, this expands the landscapes even further for repurposing.

This is a significant milestone for genetic code re-engineering, and research like this, marrying synthetic and engineering biology, will have a huge impact on future drugs and biomaterial development in both a clinical and industrial setting.

For more information please see the press release from the MRC Laboratory of Molecular Biology

Robertson, W.E., Funke, L.F.H., de la Torre, D., Fredens, J., Elliott, T.S., Spinck, M., Christova, Y., Cervettini, D., Böge, F.L., Liu, K.C., et al. (2021). Sense codon reassignment enables viral resistance and encoded polymer synthesis. Science 372, 1057-1062.