Synthetic genome

Abstract

The current invention provides a synthetic prokaryotic genome comprising 5 or fewer occurrences of one or more sense codons; and/or a synthetic prokaryotic genome derived from a parent genome, wherein the synthetic prokaryotic genome comprises less than 10%, 5%, 2%, 1%, 0.5%, 0.1% of the occurrences of one or more sense codons, relative to the parent genome; and/or a synthetic prokaryotic genome comprising 100 or more, 200 or more, or 1000 or more genes with no occurrences of one or more sense codons.

1 . A synthetic prokaryotic genome comprising 5 or fewer occurrences of one or more sense codons. 
     
     
         2 . The synthetic prokaryotic genome according to  claim 1 , wherein the synthetic prokaryotic genome comprises 100 or more genes. 
     
     
         3 . The synthetic prokaryotic genome according to  claim 1 , wherein:
 (i) the synthetic prokaryotic genome is a synthetic bacterial genome;   (ii) the one or more sense codons consist of one sense codon or two sense codons;   (iii) the synthetic prokaryotic genome comprises no occurrences of two or more sense codons;   (iv) the one or more sense codons are selected from TCG, TCA, TCT, TCC, AGT, AGC, GCG, GCA, GCT, GCC, CTG, CTA, CTT, CTC, TTG, and TTA; and/or   (v) the synthetic prokaryotic genome comprises 10 or fewer occurrences, or no occurrences, of the amber stop codon (TAG).   
     
     
         4 . The synthetic prokaryotic genome according to  claim 1 , wherein the synthetic prokaryotic genome is viable. 
     
     
         5 . A synthetic prokaryotic genome derived from a parent prokaryotic genome, wherein the synthetic prokaryotic genome comprises less than 10% of the occurrences of one or more sense codons, relative to the parent prokaryotic genome, or wherein the synthetic prokaryotic genome comprises no occurrences of one or more sense codons. 
     
     
         6 . The synthetic prokaryotic genome according to  claim 5 , wherein:
 (i) the synthetic prokaryotic genome is a synthetic bacterial genome;   (ii) the one or more sense codons are selected from TCG, TCA, TCT, TCC, AGT, AGC, GCG, GCA, GCT, GCC, CTG, CTA, CTT, CTC, TTG, and TTA;   (iii) 90% or more of the occurrences of the one or more sense codons in the parent prokaryotic genome are replaced with synonymous sense codons;   (iv) the synthetic prokaryotic genome comprises 10 or fewer occurrences, or no occurrences, of the amber stop codon (TAG);   (v) 99.9% or more of the occurrences of two or more sense codons in the parent prokaryotic genome are replaced with synonymous sense codons, and wherein all of the occurrences of TAG in the parent prokaryotic genome are replaced with TAA;   (vi) one or more pairs of genes which share an overlapping region comprising the one or more sense codons in the parent prokaryotic genome are refactored; and/or   (vii) the synthetic prokaryotic genome is 100 kb to 10 Mb in size.   
     
     
         7 . The synthetic prokaryotic genome according to  claim 6 , wherein for pairs of genes in opposite orientations, a synthetic insert is inserted between the genes, wherein the synthetic insert comprises the overlapping region; and/or
 wherein for pairs of genes in the same orientation, a synthetic insert is inserted between the genes, wherein the synthetic insert comprises: (i) a stop codon; (ii) about 20-200 bp from upstream of the overlapping region; and (iii) the overlapping region.   
     
     
         8 . A polynucleotide comprising twenty or more essential genes with no occurrences of one or more sense codons. 
     
     
         9 . The polynucleotide according to  claim 8 , wherein:
 (i) the one or more sense codons consist of one sense codon or two sense codons;   (ii) the one or more sense codons are selected from TCG, TCA, TCT, TCC, AGT, AGC, GCG, GCA, GCT, GCC, CTG, CTA, CTT, CTC, TTG, and TTA;   (iii) the occurrences of the one or more sense codons in the genes are replaced with synonymous sense codons; and/or   (iv) the essential genes comprise essential genes selected from one or more of the list consisting of: ribF, lspA, ispH, dapB, folA, imp, yabO, ftsL, ftsI, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, lpxC, secM, secA, can, folK, hemL, yadR, dapD, map, rpsB, tsf, pyrH, frr, dxr, ispU, cdsA, yaeL, yaeT, lpxD, fabZ, lpxA, lpxB, dnaE, accA, tilS, proS, yafF, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, lpxH, cysS, folD, entD, mrdB, mrdA, nadD, holA, rlpB, leuS, lnt, ginS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, lpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, me, fabD, fabG, acpP, tmk, holB, IC, lolD, lolE, purB, minE, minD, pth, prsA, ispE, lolB, hemA, prfA, prmC, kdsA, topA, ribA, fabI, tyrS, ribC, ydiL, pheT, pheS, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, ligA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, rnc, bepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, csrA, ispF, ispD, ftsB, eno, pyrG, chpR, lgt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, parE, ribB, cca, ygjD, tdcF, yraL, yhbV, infB, nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsI, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, rplW, rplD, rpbC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, yhhQ, bcsB, glyQ, gpsA, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spoT, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, yihA, ftsN, murI, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, bexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, and dnaC.   
     
     
         10 . A prokaryotic host cell comprising the synthetic prokaryotic genome according to  claim 1 . 
     
     
         11 . The prokaryotic host cell according to  claim 10 , wherein:
 (i) the prokaryotic host cell is viable; and/or   (ii) the prokaryotic host cell is a bacterial cell.   
     
     
         12 . The prokaryotic host cell according to  claim 11 , wherein the cell is an  Escherichia coli, Salmonella enterica , or  Shigella dysenteriae  cell. 
     
     
         13 . A prokaryotic host cell comprising the polynucleotide according to  claim 8 . 
     
     
         14 . A method for production of polypeptides comprising one or more non-proteinogenic amino acids, the method comprising culturing the prokaryotic host cell according to  claim 10  under conditions and for a time sufficient for production of polypeptides comprising one or more non-proteinogenic amino acids. 
     
     
         15 . A method for producing a synthetic genome comprising:
 (a) providing a parent genome;   (b) carrying out one or more rounds of recombination-mediated genetic engineering on the parent genome, to produce two or more different partially synthetic genomes; and   (c) carrying out one or more rounds of directed conjugation with the two or more different partially synthetic genomes to produce a synthetic genome;
 wherein the partially synthetic genomes each comprise a synthetic region that has 50 or fewer occurrences, or 0 occurrences, of each of one or more sense codons; or wherein the partially synthetic genomes each comprise a synthetic region that has less than 10% of the occurrences of each of one or more sense codons, relative to the corresponding region in the parent genome. 
   
     
     
         16 . The method according to  claim 15 , wherein:
 (i) the synthetic regions collectively cover 90% or greater of the parent genome;   (ii) the synthetic regions are 10-1000 kb in size;   (iii) the viability of the partially synthetic genomes is tested after each round of recombination-mediated genetic engineering and/or after each round of directed conjugation;   (iv) the two or more different partially synthetic genomes comprise at least one partially synthetic donor genome and at least one partially synthetic recipient genome; and/or   (v) the one or more rounds of recombination-mediated genetic engineering comprise one or more rounds of replicon excision for enhanced genome engineering through programmed recombination (REXER).   
     
     
         17 . The method according to  claim 16 , wherein the at least one partially synthetic donor genome comprises a synthetic region and a first selectable marker flanked by two homology regions immediately downstream of an origin of transfer; and wherein the at least one partially synthetic recipient genomes comprise a second selectable marker flanked by two corresponding homology regions. 
     
     
         18 . The method according to  claim 17 , wherein the synthetic region present in the at least one partially synthetic recipient genomes is outside the region flanked by the homology regions. 
     
     
         19 . The method according to  claim 17 , wherein the method further comprises one or more rounds of selection for the selectable markers. 
     
     
         20 . A synthetic prokaryotic genome produced by the method according to  claim 15 .