Periodic Reporting for period 4 - SGCR (Systematic Genetic Code Reprogramming)
Berichtszeitraum: 2020-07-01 bis 2022-06-30
We are interested in converting the cell into a factory for discovering and making polymers- potentially including new materials and medicines. This is important for society because the ability to discover make materials and medicines using synthetic processes is often ecologically and economically challenging. The overall objective is to understand how to convert cellular biosynthetic machinery to discover and make new polymers.
We have now demonstrated that cells can be engineered to biosynthesize new polymers including important classes of drug molecules and molecules containing the chemical linkages found in several classes of biodegradeable materials.
Many of the materials in our everyday lives – from plastics and materials to therapeutic molecules – are polymers, in which simpler chemical building blocks are linked together in a chain. Chemistry traditionally makes polymers from materials derived from crude oil using resource and energy intensive processes, and the chemical building blocks in the resulting polymers are often linked by chemical bonds that cannot easily be broken down.
Chemically synthesized polymers are made from a wide range of building blocks, but each polymer is typically only made of one or two types of building blocks. Crucially we cannot control the order of building blocks in the chain of a chemically synthesized polymer. Because we know that the order of building blocks in a chain can control the properties of a polymer, this may limit the properties we can access by current polymer synthesis methods.
Nature can control the sequence of amino acid building blocks in amino acid polymers (proteins, made by protein translation), and the translational machinery of cells provides a unique paradigm in the biological or physical world for making polymers of defined sequence and composition. Proteins composed of the 20 canonical amino acids fold up into defined three dimensional shapes and carry out diverse functions, further exemplifying how diverse functions can arise from distinct sequences.
The goal of our project was to turn living cells into programmable factories for the encoded synthesis of non-canonical polymers. Cells naturally make proteins by protein translation. The sequence of triplet codons within genes in the DNA of an organisms are converted to a messenger RNA tape. There are 64 triplet codons and 61 triplet codons encode the 20 canonical amino acids, while the remaining three triplets encode the termination of protein synthesis. Most amino acids are encoded by more than one triplet codon, codons that encode the same amino acid are described as synonymous codons. The mRNA tape is read by the ribosome, and tRNAs – loaded with amino acids by cognate aminoacyl-tRNA synthetases. Base pairing between the tRNA anticodon and mRNA codon specifies which amino acid is incorporated at each codon and the ribosome polymerises the amino acids to make the polypeptide chain.
In order to create a version of protein translation for encoded polymer synthesis we required 1) codons that can be used for non-canonical monomers in the cells, 2) aminoacyl-tRNA synthetase and tRNA systems that can deliver now monomers to the ribosome for polymerisation.
Since all 64 triplet codons are used for encoding natural protein synthesis in the cell we need to create new coding capacity for encoding new building blocks (monomers). We investigated the creation of cells that use fewer codons for encoding the 20 canonical amino acids, in this approach the synonymous codons that are no longer used for protein synthesis can be reassigned to encoding the building blocks of non canonical polymers. Two key advances were our discovery of ‘recoding rules’ which define which synonymous codons can be used to replace the codons we wish to remove from the DNA genome of the cell and methods for replacing the entire genome of the microbe E. coli. By combining these advances we created a version of E. coli with a completely synthetic genome. The resulting synthetic genome contained more than 18,000 changes with respect to the natural genome and used fewer codons to encode protein synthesis with the canonical amino acids.
Next, we deleted the genes for the, otherwise essential, tRNAs that normally read the deleted codons. This created a cell that no longer contains certain codons in its genome or the machinery to read those codons. We showed that these cells were resistant to a wide range of viruses; since viruses contain the DNA or RNA to make the proteins in their capsid and copies of themselves, but rely on the machinery of host cell to read the full genetic code in their DNA or RNA, they cannot make copies of themselves in our new cells that do not have the tRNAs to correctly read all the codons in their viral genes.
We developed new strategies for discovering aminoacyl-tRNA synthetase/tRNA pairs for incorporating non-canonical monomers. In one approach we discovered several useful pyrrolysyl-tRNA synthetase/tRNA pairs. In another approach we used genome mining and information about how synthetases recognize tRNAs to discover new useful synthetase and tRNA systems. We also developed approaches for changing the specificity of aminoacyl-tRNA synthetases and tRNAs to recognize distinct monomers and decode distinct codons.
Finally, we combined our advances in developing new synthetases and tRNAs, and our advances in providing new codons by genome engineering to enable the encoded cellular synthesis of non-canonical polymers.
We have now demonstrated that cells can be engineered to biosynthesize new polymers including important classes of drug molecules and molecules containing the chemical linkages found in several classes of biodegradeable materials.
Many of the materials in our everyday lives – from plastics and materials to therapeutic molecules – are polymers, in which simpler chemical building blocks are linked together in a chain. Chemistry traditionally makes polymers from materials derived from crude oil using resource and energy intensive processes, and the chemical building blocks in the resulting polymers are often linked by chemical bonds that cannot easily be broken down.
Chemically synthesized polymers are made from a wide range of building blocks, but each polymer is typically only made of one or two types of building blocks. Crucially we cannot control the order of building blocks in the chain of a chemically synthesized polymer. Because we know that the order of building blocks in a chain can control the properties of a polymer, this may limit the properties we can access by current polymer synthesis methods.
Nature can control the sequence of amino acid building blocks in amino acid polymers (proteins, made by protein translation), and the translational machinery of cells provides a unique paradigm in the biological or physical world for making polymers of defined sequence and composition. Proteins composed of the 20 canonical amino acids fold up into defined three dimensional shapes and carry out diverse functions, further exemplifying how diverse functions can arise from distinct sequences.
The goal of our project was to turn living cells into programmable factories for the encoded synthesis of non-canonical polymers. Cells naturally make proteins by protein translation. The sequence of triplet codons within genes in the DNA of an organisms are converted to a messenger RNA tape. There are 64 triplet codons and 61 triplet codons encode the 20 canonical amino acids, while the remaining three triplets encode the termination of protein synthesis. Most amino acids are encoded by more than one triplet codon, codons that encode the same amino acid are described as synonymous codons. The mRNA tape is read by the ribosome, and tRNAs – loaded with amino acids by cognate aminoacyl-tRNA synthetases. Base pairing between the tRNA anticodon and mRNA codon specifies which amino acid is incorporated at each codon and the ribosome polymerises the amino acids to make the polypeptide chain.
In order to create a version of protein translation for encoded polymer synthesis we required 1) codons that can be used for non-canonical monomers in the cells, 2) aminoacyl-tRNA synthetase and tRNA systems that can deliver now monomers to the ribosome for polymerisation.
Since all 64 triplet codons are used for encoding natural protein synthesis in the cell we need to create new coding capacity for encoding new building blocks (monomers). We investigated the creation of cells that use fewer codons for encoding the 20 canonical amino acids, in this approach the synonymous codons that are no longer used for protein synthesis can be reassigned to encoding the building blocks of non canonical polymers. Two key advances were our discovery of ‘recoding rules’ which define which synonymous codons can be used to replace the codons we wish to remove from the DNA genome of the cell and methods for replacing the entire genome of the microbe E. coli. By combining these advances we created a version of E. coli with a completely synthetic genome. The resulting synthetic genome contained more than 18,000 changes with respect to the natural genome and used fewer codons to encode protein synthesis with the canonical amino acids.
Next, we deleted the genes for the, otherwise essential, tRNAs that normally read the deleted codons. This created a cell that no longer contains certain codons in its genome or the machinery to read those codons. We showed that these cells were resistant to a wide range of viruses; since viruses contain the DNA or RNA to make the proteins in their capsid and copies of themselves, but rely on the machinery of host cell to read the full genetic code in their DNA or RNA, they cannot make copies of themselves in our new cells that do not have the tRNAs to correctly read all the codons in their viral genes.
We developed new strategies for discovering aminoacyl-tRNA synthetase/tRNA pairs for incorporating non-canonical monomers. In one approach we discovered several useful pyrrolysyl-tRNA synthetase/tRNA pairs. In another approach we used genome mining and information about how synthetases recognize tRNAs to discover new useful synthetase and tRNA systems. We also developed approaches for changing the specificity of aminoacyl-tRNA synthetases and tRNAs to recognize distinct monomers and decode distinct codons.
Finally, we combined our advances in developing new synthetases and tRNAs, and our advances in providing new codons by genome engineering to enable the encoded cellular synthesis of non-canonical polymers.
We have converted cells into factories for the programmable synthesis of new molecules and materials. In order to do this we have created 1) organisms with synthetic genomes that use fewer codons than extant biology and 2) engineered translational machinery to read the codons we free up. By combining our advances in 1 and 2 we have reprogrammed the genetic code for encoded polymer synthesis.
This work has been disseminated in a number of publications:
A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design. D. L. Dunkelmann, S. B. Oehm, A. T. Beattie, J. W. Chin. Nature Chem. 2021. 13: 1110-1117.
Sense codon reassignment enables viral resistance and encoded polymer synthesis. W. E. Robertson, L. F. H. Funke, D. de la Torre, J. Fredens, T. S. Elliott, M. Spinck, Y. Christova, D. Cervettini, F. L. Boge, K. C. Liu, S. Buse, S. Maslen, G. P. Salmond, J. W. Chin. Science 2021. 372: 1057-1062.
Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS. W. E. Robertson, L. F. H. Funke, D. de la Torre, J. Fredens, K. Wang, J. W. Chin. Nature Protocols 2021. 16: 2345-2380.
Reprogramming the genetic code. D. de la Torre, J. W. Chin. Nature Reviews Genetics. 2021. 22: 169-184.
This work has been disseminated in a number of publications:
A 68-codon genetic code to incorporate four distinct non-canonical amino acids enabled by automated orthogonal mRNA design. D. L. Dunkelmann, S. B. Oehm, A. T. Beattie, J. W. Chin. Nature Chem. 2021. 13: 1110-1117.
Sense codon reassignment enables viral resistance and encoded polymer synthesis. W. E. Robertson, L. F. H. Funke, D. de la Torre, J. Fredens, T. S. Elliott, M. Spinck, Y. Christova, D. Cervettini, F. L. Boge, K. C. Liu, S. Buse, S. Maslen, G. P. Salmond, J. W. Chin. Science 2021. 372: 1057-1062.
Creating custom synthetic genomes in Escherichia coli with REXER and GENESIS. W. E. Robertson, L. F. H. Funke, D. de la Torre, J. Fredens, K. Wang, J. W. Chin. Nature Protocols 2021. 16: 2345-2380.
Reprogramming the genetic code. D. de la Torre, J. W. Chin. Nature Reviews Genetics. 2021. 22: 169-184.
By the end of the project we expect to have new strategies to recode genomes and new strategies to biosynthesize proteins and polymers containing new building blocks.
We have created new methods for genome synthesis, discovered rules for recoding genomes, created the largest and most radically altered synthetic genome. We have created new engineered translational components and methods for their engineering. We have combined these advances for encoded polymer synthesis. All these advances go well beyond the state of the art.
We have created new methods for genome synthesis, discovered rules for recoding genomes, created the largest and most radically altered synthetic genome. We have created new engineered translational components and methods for their engineering. We have combined these advances for encoded polymer synthesis. All these advances go well beyond the state of the art.