Total synthesis of Escherichia coli with a recoded genome
Authors: Yonka Christova
Chayasith Uttamapinant
Julius Fredens
Daniel de la Torre
Wolfgang H Schmied
Wesley E Robertson
Kaihang Wang
Thomas S Elliott
Andres Gonzalez Llamazares
Daniel L Dunkelmann
Jason W Chin
Louise F H Funke
Tiongsun Chia
V√°clav Ber√°nek
Journal: Nature (2019)
Discipline: Multidisciplinary
Keywords: codons; synonymous substitutions; genome-wide substitution; Escherichia coli; synthetic genome
Affiliations: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK., Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, USA., School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Rayong, Thailand., Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. .

Abstract:  Nature uses 64 codons to encode the synthesis of proteins from the genome, and chooses 1 sense codon-out of up to 6 synonyms-to encode each amino acid. Synonymous codon choice has diverse and important roles, and many synonymous substitutions are detrimental. Here we demonstrate that the number of codons used to encode the canonical amino acids can be reduced, through the genome-wide substitution of target codons by defined synonyms. We create a variant of Escherichia coli with a four-megabase synthetic genome through a high-fidelity convergent total synthesis. Our synthetic genome implements a defined recoding and refactoring scheme-with simple corrections at just seven positions-to replace every known occurrence of two sense codons and a stop codon in the genome. Thus, we recode 18,214 codons to create an organism with a 61-codon genome; this organism uses 59 codons to encode the 20 amino acids, and enables the deletion of a previously essential transfer RNA. 

Retracted: No

PubPeer comments:
 Sup Data 18 - Syn61 genome.txt is where I pick the genome (and annotation). I have only used ORFs from the reference genome. The problem is that they have not annotated many genes [That way I can delete as many codons as you want - just not annotate the genes. ] As concrete final proof, here is a protein from their proteomic data (Supp 20) which has the supposed deleted stop codon. This protein NP_415930.1 FMN dependent NADH:quinone oxidoreductase [Escherichia coli str. K-12 substr. MG1655] MSKVLVLKSSILAGYSQSNQLSDYFVEQWREKHSADEITVRDLAANPIPVLDGELVGALRPSDAPLTPRQQEALALSDEL IAELKAHDVIVIAAPMYNFNISTQLKNYFDLVARAGVTFRYTENGPEGLVTGKKAIVITSRGGIHKDGPTDLVTPYLSTF LGFIGITDVKFVFAEGIAYGPEMAAKAQSDAKAAIDSIVSA Here is the line from their proteomic data ... P41407 FMN-dependentNADH-azoreductase azoR 1 1.1835 7620500 2 22.7406 It has a 100% match - 201 aa out of 201 aa. And has the  deleted stop codon. Here is the ORF - you can see its not annotated in the GenBank file (Supp 18) Syn61 [1198585 - 1197983] (REVERSE SENSE) Here is the UNIPROT entry - exact same protein. sp|P41407|AZOR_ECOLI FMN-dependent NADH-azoreductase OS=Escherichia coli (strain K12) OX=83333 GN=azoR PE=1 SV=3 MSKVLVLKSSILAGYSQSNQLSDYFVEQWREKHSADEITVRDLAANPIPVLDGELVGALR PSDAPLTPRQQEALALSDELIAELKAHDVIVIAAPMYNFNISTQLKNYFDLVARAGVTFR YTENGPEGLVTGKKAIVITSRGGIHKDGPTDLVTPYLSTFLGFIGITDVKFVFAEGIAYG PEMAAKAQSDAKAAIDSIVSA , 
 #2 It is likely that the still-present codons you have identified are in the ectopic selection marker they introduce: https: twitter.com theosanderson status 1130883809212342272 (so not, to me, a major source of concern). , 
 #4 No, thats not the only case - I have given[ more examples here...] , 
 This paper says it has removed all  TAG stop codons  There are still 6 left. Its a small number, undoubtedly. But not gone completely. These are the protein annotations. So their claim of reducing the codon space from 64 to 61 is not correct. NEWGENOME_12056 id=NP_418083.1 protein=UDP-glucose:(glucosyl)LPS alpha-1,2-glucosyltransferase 60 411 8e-148 NEWGENOME_17220 id=NP_415930.1 protein=FMN dependent NADH:quinone oxidoreductase 100 409 3e-149 NEWGENOME_17103 id=NP_415979.2 protein=flavin reductase-like protein YddH 48 158 6e-52 NEWGENOME_10867 id=YP_001165333.1 protein=uncharacterized protein YjbS 119 132 9e-44 NEWGENOME_18463 id=YP_009029995.1 protein=small protein MntS 100 87.4 1e-26 NEWGENOME_16892 id=YP_009518794.1 protein=protein YnfP 105 54.7 7e-14 Here are their coordinates. The sequence of the Ecoli can be found in the SI Data 2. NEWGENOME_10867 [3700828 - 3700589] (REVERSE SENSE) NEWGENOME_12056 [3233076 - 3232459] (REVERSE SENSE) NEWGENOME_16892 [1335388 - 1335275] (REVERSE SENSE) NEWGENOME_17103 [1241584 - 1241309] (REVERSE SENSE) NEWGENOME_17220 [1196319 - 1195717] (REVERSE SENSE) NEWGENOME_18463 [716318 - 716193] (REVERSE SENSE) , 
 Not sure where Sandeep has sourced his genome annotation from. He may be predicting his own ORFs, which may explain these errors. There is no annotated CDS with these co-ordinates: 3700828 - 3700589 -- however, 3699824..3700903 is a alr gene... The Sup Data 18 - Syn61 genome.txt is an annotated GenBank file. I found 0 annotated CDS sequences with a TAG stop codon, there were 4 TCG s and 6 TCA s, not sure which genes these were housed in. There was one other error relative to the parent: ```[ATGATGAAAAAGTTTATCGCCCCCTTGTTGGCTTTACTGGTTAGCGGATGTCAGATTGATCCT``` ```...``` ```ATGAAAATGCCAGCACAATACGATTAAATTAA]``` ```length is not a factor of 3!``` Which is the DUF2799 domain-containing protein [Escherichia coli] Some minor corrections needed to correct these. ,