Synthetic genome

Abstract

The present invention provides a synthetic genome in which one or more sense codons have been removed. [Solution] A synthetic prokaryotic genome is provided that includes the appearance of five or four or fewer sense codons, one or more sense codons; and / or a synthetic prokaryotic genome derived from a parent genome, in which the synthetic prokaryotic genome includes the appearance of one or more sense codons in amounts of less than 10%, 5%, 2%, 1%, 0.5%, or 0.1% compared to the parent genome; and / or a synthetic prokaryotic genome containing 100 or 101 or more, 200 or 201 or more, or 1000 or 1001 or more genes, without the appearance of one or more sense codons.

Description

[Technical Field]

[0001] This invention relates to a synthetic genome and a method for producing it. [Background technology]

[0002] Genome design and synthesis offer a powerful approach to understanding and modifying biology. Genome synthesis has the potential to accelerate metabolic engineering. In particular, genome synthesis may elucidate the function of synonymous codons and facilitate the synthesis of genetically encoded non-natural polymers (Wang, K., et al., 2016. Nature, 539(7627), 59-64).

[0003] The standard genetic code uses 61 sense codons to code for 20 canonical amino acids, 18 of which are coded by more than one synonymous codon. Nature selects one sense codon from up to six synonyms to code for each amino acid at each position within a gene. The selection of synonymous codons may influence mRNA folding, transcription and translation regulatory sequences, translation rate, co-translational folding, and protein levels, and has novel, yet-to-be-understood roles (Wang, K., et al., 2016. Nature, 539(7627), 59-64; and Cambray, G., et al., 2018. Nature biotechnology, 36(10), 1005-1015).

[0004] Genome-wide substitution of target codons with synonymous codons (synonymous codon compression) can provide a basis for reassigning sense codons to non-canonical amino acids (or other monomers) to facilitate the in vivo biosynthesis of genetically encoded non-canonical biopolymers (Chin, JW, 2017. Nature, 550(7674), 53-60).

[0005] Site-directed mutagenesis approaches have been used to replace up to 321 amber stop codons in the *E. coli* genome (Mukai, T., et al., 2015. Scientific reports, 5, p.9699). However, sense codons are generally several orders of magnitude more numerous than stop codons, and genome synthesis, rather than mutagenesis, may often be a preferred method for addressing sense codon removal.

[0006] Genome synthesis has made it possible to create mycoplasmas with synthetic genomes (Gibson, DG, et al., 2010. Science, 329(5987), 52-56), with one or two of the 16 chromosomes being synthesized. The development of nine strains of S. cerevisiae in which the original DNA has been replaced with synthetic DNA. This made production possible (Zhang, W., et al., 2017. Science, 355(6329), eaaf3981; and Richardson, SM, et al., 2017. Science, 355(6329), 1040-1044). These experiments were conducted individually. In the strains, up to 1 Mb of DNA has been replaced (0.99 Mb in yeast; 1.08 Mb in mycoplasma). Replicon excision for enhanced genome engineering through programmed recombination (REXER) has been reported to replace more than 100 kb of E. coli genome with synthetic DNA in a single step. Furthermore, it has been shown that REXER can be replicated by genome stepwise interchange synthesis (GENESIS) to replace a 220 kb E. coli genome with 230 kb of synthetic DNA. (Wang, K., et al., 2016. Nature, 539(7627), 59-64; International Publication No. 2018) (Brochure No. 020248).

[0007] Genome synthesis involves synonymous codons in individual genes (Napolitano, MG, et al., 2016. PNAS, 113(38), E5588-E5597), genomic regions, and essential operons (Wang, K., et al., 2016. Nature, 539(7627), 59-64; and Lau, YH, et al. 2017. Nucleic acids research, 4) It has been used to modify 5(11), 6971-6980). For example, Wang et al. used a defined “rewrite scheme” to replace a 20kb region of the E. coli genome that is abundant in both essential genes and target codons.

[0008] However, these studies mutated only a small fraction (up to 4.7%) of the targeted sense codons in the genome of a single strain. As a result, it is unclear whether applying these methods to genome-wide synonymous codon compression can produce viable genomes. For example, it is unclear whether the defined rewriting scheme tested by Wang et al. can be applied genome-wide to create an organism in which a small number of sense codons are used to encode 20 canonical amino acids. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] International Publication No. 2018 / 020248 Brochure [Non-patent literature]

[0010] [Non-Patent Document 1] Wang, K., et al., 2016. Nature, 539(7627), 59-64 [Non-Patent Document 2] Cambray, G., et al., 2018. Nature biotechnology, 36(10), 1005-1015 [Non-Patent Document 3] Chin, JW, 2017. Nature, 550(7674), 53-60 [Non-Patent Document 4] Mukai, T., et al., 2015. Scientific reports, 5, p.9699 [Non-Patent Document 5] Gibson, DG, et al., 2010. Science, 329(5987), 52-56 [Non-Patent Document 6] Zhang, W., et al., 2017. Science, 355(6329), eaaf3981 [Non-Patent Document 7] Richardson, SM, et al., 2017. Science, 355(6329), 1040-1044 [Non-Patent Document 8] Napolitano, MG, et al., 2016. PNAS, 113(38), E5588-E5597 [Non-Patent Document 9] Lau, YH, et al. 2017. Nucleic acids research, 45(11), 6971-6980 [Overview of the project] [Problems that the invention aims to solve]

[0011] Therefore, synthetic genomes from which one or more sense codons have been removed are needed. Improved methods for producing synthetic genomes are also desired. [Means for solving the problem]

[0012] The inventors have surprisingly found that viable synthetic prokaryotic genomes can be produced in which one or more sense codons are removed. In particular, the inventors produced a viable synthetic genome in which the number of codons used to encode cellular proteins was reduced from 64 to 61 by genome-wide rewriting of two sense codons and one stop codon. The inventors also produced E. coli host cells containing the synthetic genome.

[0013] The inventors also found, surprisingly, that the defined rewriting and refactoring schemes can enable genome-wide synonymous codon compression for more than 99.9% of target codons. The inventors also found that alternative rewriting and refactoring at unacceptable locations enables genome-wide synonymous codon compression.

[0014] The inventors have also surprisingly found that gene modification via recombination (e.g., REXER and / or GENESIS) can be combined with directed conjugation to effectively produce synthetic genomes. For example, they found that at least about 4 Mb of DNA can be effectively replaced by the method, and that failures (unacceptable positions) in the design of synthetic DNA can be identified with codon-level resolution.

[0015] Accordingly, in one embodiment, the present invention provides a synthetic prokaryotic genome comprising five or four or fewer occurrences of one or more sense codons. In some embodiments, the synthetic prokaryotic genome comprises or does not comprise four, three or fewer, three or two or fewer, two or one or fewer, one or zero occurrences of one or more sense codons. In some embodiments, the one or more sense codons consist of one sense codon or two sense codons, preferably two sense codons. In some embodiments, the synthetic prokaryotic genome does not comprise the occurrence of two or three or more sense codons, preferably two sense codons, and does not comprise the occurrence of one stop codon, preferably an amber stop codon (TAG).

[0016] The synthetic prokaryotic genome may be a synthetic bacterial genome, preferably a synthetic Escherichia coli genome, a Salmonella enterica genome, or a Shigella dysenteriae genome. In some embodiments, the synthetic prokaryotic genome The genome is 100kb to 10Mb, or 1Mb to 10Mb, or 2Mb to 6Mb in size. The synthetic prokaryotic genome may be viable. In some embodiments, the synthetic prokaryotic genome contains 100 or 101 or more, 200 or 201 or more, or 1000 or 1001 or more genes, and the genes may not have the appearance of one or more sense codons, and preferably the genes are essential genes.

[0017] In some embodiments, 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, preferably one or more sense codons are selected from TCG, TCA, AGT, AGC, GCG, GCA, CTG, CTA, TTG, and TTA, more preferably one or more sense codons are selected from TCG, TCA, AGT, AGC, TTG, TTA, GCG, and GCA, and most preferably one or more sense codons are TCG and / or TCA.

[0018] In some embodiments, the synthetic prokaryotic genome includes or does not include 10 or 9 or fewer amber stop codons (TAGs), 5 or 4 or fewer amber stop codons (TAGs).

[0019] In further embodiments, the present invention provides a synthetic prokaryotic genome comprising 100 or more, 200 or more, or 1000 or more genes, wherein the genes comprise a total of five or four occurrences of one or more sense codons, and preferably the genes are essential genes. In some embodiments, the genes comprise a total of four or three or fewer, three or two or fewer, two or one or fewer, one or zero occurrences of one or more sense codons, or no occurrences at all. In some embodiments, the one or more sense codons consist of one sense codon or two sense codons, preferably two sense codons.

[0020] The synthetic prokaryotic genome may be a synthetic bacterial genome, preferably a synthetic Escherichia coli genome, a Salmonella enterica genome, or a Shigella genome. In some embodiments, the synthetic prokaryotic genome is 100kb to 10Mb, or 1Mb to 10Mb, or 2Mb to 6Mb in size. The synthetic prokaryotic genome may be viable.

[0021] In some embodiments, one or more sense codons are TCG, TCA, TCT, TCC, AGT, AGC, GCG, GCA, GCT, GCC, CTG, CTA, CTT, The sense codon is selected from CTC, TTG, and TTA, preferably one or more sense codons are selected from TCG, TCA, AGT, AGC, GCG, GCA, CTG, CTA, TTG, and TTA, more preferably one or more sense codons are selected from TCG, TCA, AGT, AGC, TTG, TTA, GCG, and GCA, and most preferably one or more sense codons are TCG and / or TCA.

[0022] In some embodiments, the synthetic prokaryotic genome includes or does not include 10 or 9 or fewer amber stop codons (TAGs), 5 or 4 or fewer amber stop codons (TAGs).

[0023] In further embodiments, the present invention provides a synthetic prokaryotic genome derived from a parent prokaryotic genome, wherein the synthetic prokaryotic genome contains the appearance of one or more sense codons in amounts of less than 10%, 5%, 2%, 1%, 0.5%, or 0.1% compared to the parent prokaryotic genome, or the synthetic prokaryotic genome does not contain the appearance of one or more sense codons. In some embodiments, the one or more sense codons consist of one sense codon or two sense codons, preferably two sense codons.

[0024] The synthetic prokaryotic genome may be a bacterial genome, preferably an Escherichia coli genome, a Salmonella enterica genome, or a Shigella genome. In some embodiments, the synthetic prokaryotic genome is 100kb to 10Mb, or 1Mb to 10Mb, or 2Mb to 6Mb in size. The synthetic prokaryotic genome may be viable.

[0025] In some embodiments, 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, preferably one or more sense codons are selected from TCG, TCA, AGT, AGC, GCG, GCA, CTG, CTA, TTG, and TTA, more preferably one or more sense codons are selected from TCG, TCA, AGT, AGC, TTG, TTA, GCG, and GCA, most preferably one or more sense codons are TCG and / or TCA, where TCG and / or TCA may be substituted with synonymous sense codons.

[0026] Preferably, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100% of the occurrences of one or more sense codons in the parent prokaryotic genome are replaced with synonymous sense codons. In some embodiments, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100% of the occurrences of TCG and / or TCA in the parent prokaryotic genome are replaced with AGC and / or AGT, most preferably 90% or more, 95% or more, 98% or more, 99% or more, or 99% or more of the occurrences of TCG in the parent prokaryotic genome. 9.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100% are replaced with AGC, and / or 90%, 95%, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100% of TCA occurrences in the parent prokaryotic genome are replaced with AGT.

[0027] In some embodiments, the synthetic prokaryotic genome has 10 amber stop codons (TAGs) Alternatively, it may include 9 or fewer occurrences, 5 or 4 or fewer occurrences, or no occurrences at all, preferably with 90% or more, 95% or more, 98% or more, 99% or more, or all of the TAG occurrences in the parent prokaryotic genome being replaced by TAAs.

[0028] In some embodiments, 99.9% or more, or even 100%, of the occurrences of two or more sense codons, preferably two sense codons, in the parent prokaryotic genome are replaced with synonymous sense codons, and all occurrences of TAGs in the parent prokaryotic genome are replaced with TAAs.

[0029] One or more gene pairs sharing an overlapping region containing one or more sense codons in the parent prokaryotic genome may be refactored, preferably one or more gene pairs in which substitution of one or more synonymous sense codons alters the encoded protein sequence of both or one of the gene pairs.

[0030] In some embodiments, with respect to a pair of reversed genes, the synthetic insert is inserted between the genes and includes an overlapping region, and / or with respect to a pair of same-oriented genes, the synthetic insert is inserted between the genes and includes (i) a stop codon, (ii) about 20 to 200 bp upstream of the overlapping region, and (iii) the overlapping region.

[0031] In further embodiments, the present invention provides polynucleotides containing 20 or 21 or more, 30 or 31 or more, 40 or 41 or more, 50 or 51 or more, 100 or 101 or more essential genes, without the appearance of one or more sense codons. In some embodiments, the one or more sense codons consist of one sense codon or two sense codons, preferably two sense codons.

[0032] In some embodiments, 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, preferably one or more sense codons are selected from TCG, TCA, AGT, AGC, GCG, GCA, CTG, CTA, TTG, and TTA, more preferably one or more sense codons are selected from TCG, TCA, AGT, AGC, TTG, TTA, GCG, and GCA, and most preferably one or more sense codons are TCG and / or TCA.

[0033] The appearance of one or more sense codons in a gene may be substituted with synonymous sense codons, preferably the TCG codon being substituted with AGC and / or the TCA codon being substituted with AGT.

[0034] Essential genes are ribF, lspA, ispH, dapB, folA, imp, yabQ, ftsL, ftsI, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, lpxC, sec M, secA, can, folK, hemL, yadR, dapD, map, rpsB, tsf, pyrH, frr, dxr, ispU, cdsA, yaeL, yaeT, lpxD, fabZ, lpxA, lpxB, dnaE, accA, til S, proS, yafF, hemB, secD, secF, ribD, ribE, thiL, dxs, ispA, dnaX, adk, hemH, lpxH, cysS, folD, entD, mrdB, mrdA, nadD, holA, rlpB, leuS, lnt, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, lpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, rne, fabD, fa bG、acpP、tmk、holB、lolC、lolD、lolE、purB、minE、minD、pth、prsA、ispE、l olB、hemA、prfA、prmC、kdsA、topA、ribA、fabI、tyrS、ribC、ydiL、pheT、phe S、rplT、infC、thrS、nadE、gapA、yeaZ、aspS、argS、pgsA、yefM、metG、folE、 yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, ligA, zipA, dapE, dapA r、hisS、ispG、suhB、tadA、acpS、era、rnc、lepB、rpoE、pssA、yfiO、rplS、tr mD、rpsP、ffh、grpE、csrA、ispF、ispD、ftsB、eno、pyrG、chpR、lgt、fbaA、pg k、yqgD、metK、yqgF、plsC、ygiT、parE、ribB、cca、ygjD、tdcF、yraL、yhbV、i nfB、nusA、ftsH、obgE、rpmA、rplU、ispB、murA、yrbB、yrbK、yhbN、rpsI、rpl M、degS、mreD、mreC、mreB、accB、accC、yrdC、def、fmt、rplQ、rpoA、rpsD、rp sK、rpsM、secY、rplO、rpmD、rpsE、rplR、rplF、rpsH、rpsN、rplE、rplX、rplN rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, rplW, rplD, rplC, rpsJ, fusA, r psG、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、f tsN、murI、murB、birA、secE、nusG、rplJ、rplL、rpoB、rpoC、ubiA、plsB、lex A、dnaB、ssb、alsK、groS、psd、orn、yjeE、rpsR、chpS、ppa、valS、yjgP、yjgQ、It may also include essential genes selected from one or more of the list consisting of and dnaC.

[0035] In a further embodiment, the present invention provides a synthetic prokaryotic genome or a prokaryotic host cell containing a polynucleotide according to the present invention.

[0036] The prokaryotic host cell may be viable. The prokaryotic host cell may be a bacterial cell, preferably an Escherichia coli cell, a Salmonella enterica cell, or a Shigella cell. Preferably, the host cell is suitable for use in the production of a polypeptide containing one or more non-proteinogenic amino acids, preferably two or more non-proteinogenic amino acids, most preferably three or more non-proteinogenic amino acids.

[0037] In a further embodiment, the present invention provides the use of a prokaryotic host cell according to the present invention for producing a polypeptide comprising one or more non-proteinogenic amino acids, preferably two or three or more non-proteinogenic amino acids, most preferably three or four or more non-proteinogenic amino acids.

[0038] In a further embodiment, the present invention relates to a method for producing a synthetic genome, (a) Steps to prepare the parent genome, (b) A step of performing one or more rounds of genetic modification via recombination on the parent genome to produce two or more different partial synthetic genomes, (c) A step of producing a synthetic genome by performing one or more rounds of induction conjugation with two or more different partial synthetic genomes and Each of the partial synthetic genomes includes a synthetic region having 50 or 49 or fewer occurrences of each of one or more sense codons, 20 or 19 or fewer occurrences, 10 or 9 or fewer occurrences, 5 or 4 or fewer occurrences, or 0 occurrences of each of the partial synthetic genomes, The present invention provides a synthetic region comprising one or more sense codons having appearances of less than 10%, 5%, 2%, 1%, 0.5%, and 0.1% respectively, compared to the corresponding region in the parent genome.

[0039] The synthetic region may occupy 90% or more, 95% or more, 99% or more, or 100% of the parent genome in total. In some embodiments, the synthetic region is 10–1000kb, 50–1000kb, 100–1000kb, or 100–500kb in size.

[0040] The method may further include a step of testing the viability of the partially synthetic genome after each round of genetic modification via recombination and / or after each round of inducible conjugation.

[0041] Two or more different partial synthetic genomes may comprise at least one partial synthetic donor genome and at least one partial synthetic recipient genome. In some embodiments, at least one partial synthetic donor genome comprises a first selectable marker consisting of a synthetic region and two homologous regions adjacent to the origin of transmission, and at least one partial synthetic recipient genome comprises a second selectable marker consisting of two corresponding homologous regions adjacent, the first selectable marker may comprise a positive selectable marker, and / or the second selectable marker may comprise a negative selectable marker. In some embodiments, the synthetic region present in at least one partial synthetic recipient genome is outside the region where the homologous regions are adjacent. In some embodiments, the method further comprises one or more rounds of selection for the selectable markers.

[0042] One or more rounds of recombination-mediated gene modification may include one or more rounds of replicon excision (REXER) for enhanced genome modification by programmed recombination.

[0043] The synthetic genome may be a synthetic prokaryotic genome according to the present invention.

[0044] In a further embodiment, the present invention provides a synthetic prokaryotic genome produced by the method of the present invention. [Brief explanation of the drawing]

[0045] [Figure 1-1] ~ [Figure 1-3]This figure shows the design of a synthetic genome that implements a defined rewriting scheme for synonymous codon compression. a. Defined rewriting scheme for synonymous codon compression. It shows synonymous serine codons and three stop codons used in the genome of WT E. coli. By systematically implementing the defined rewriting scheme for synonymous codon compression, target codons are rewritten to defined synonyms and the amber stop codon TAG is replaced with the ochre stop codon TAA. This creates an organism with a rewritten genome that uses a reduced number of serine and stop codons. b. Refactoring of 3', 3' duplications enables their independent rewriting. Duplications between two open reading frames (ORF-1 and ORF-2) are replicated to create synthetic insertions. This enables independent rewriting of ORFs. c. Refactoring of 5', 3' duplications. 20 bp upstream of the duplication is added and replicated to generate synthetic insertions. If the duplication is longer than 1 bp at the end of the upstream ORF, an in-frame TAA is introduced at the beginning of the synthetic insertion, and this in-frame stop codon ensures the termination of translation from the original RBS. Therefore, translation of the entire length of the downstream ORF begins from the reconstructed RBS in the synthetic insertion. d. Map of the synthetic genome design with all TCG, TCA, and TAG codons removed. Outer ring: All 18,218 positions of TCG→AGC, TCA→AGT, and TAG→TAA rewrites. Gray ring: 12 positions of designed silent mutations in the duplication, 21 refactorings of 3', 3' duplications (b) and 58 refactorings of 5', 5' duplications (c). Two inner rings illustrate genomic compartments. Outer ring: 8 genomic compartments of the synthetic genome design (A-H). Inner ring: 37 fragments, each approximately 100 kb. Fragments 37 are shown as 37a and 37b to reflect the final assembly. oriC: Origin of the copy. [Figure 2-1] ~ [Figure 2-2]This figure shows the retrosynthesis of a synthetic genome. a. The genome is cut into eight compartments. The synthetic genome is cut into compartments A-H, with each compartment corresponding to approximately 0.5 Mb (Step 1). The location of the replication origin oriC is shown (orange square). The compartments were assembled into a genome completely rewritten by induction conjugation (in the forward sense, in the opposite direction of the retrosynthesis arrow) (Figures 10 and 11). b. The genome compartments are cut into 100 kb fragments. Each compartment is further cut into 4-5 fragments of approximately 100 kb each. Compartment A is shown, and the other compartments were processed similarly. Almost all compartments were completely reconstructed by GENESIS (Figure 4) via a series of REXER steps (Figure 3). Each step replaced approximately 100 kb of wild-type genome sequence with a 100 kb synthetic fragment (Steps 2 and 3). A double-selection marker consisting of negative selection marker-1 (rpsL), positive selection marker+1 (KanR), negative selection marker-2 (SacB), and positive selection marker+2 (CmR) was used in alternating rounds of REXER to achieve GENESIS. c) Each 100kb synthetic fragment was cleaved into 10kb synthetic stretches. Each 100kb synthetic fragment was further cleaved into 9 to 14 short synthetic stretches approximately 10kb in length (step 4). BACs with 100kb synthetic fragments were assembled by homologous recombination in yeast. Each BAC contains a Cas9 cleavage site (black triangle) that enables in vivo excision of synthetic DNA, homologous regions (HR1 and HR2) for targeting recombination, a suitable dual-selection cassette (indicated as +2 and -2) for selection between REXER and GENESIS, a negative selection marker (indicated as -1) to enable scaffold loss after REXER, a BAC YAC origin, and a URA3 marker for maintenance in Escherichia coli and S. cerevisiae. [Figure 3]This figure illustrates the use of a 100kb fragment of synthetic DNA to replace a corresponding region in the genome by REXER. REXER (Replicon Excision for Enhanced Genome Modification by Programmed Recombination) utilizes CRISPR / Cas9 and lambda-red-mediated recombination to replace genomic DNA with synthetic DNA provided from the episome (BAC). This allows for the replacement of large regions of the genome (over 100kb) with synthetic DNA (Wang, K., et al., 2016. Nature, 539(7627), 59-64; International Publication No. 2018 / 020248). The black triangles indicate the locations of CRISPR protospacers where homologous regions (HR) are cleaved by Cas9 to release synthetic DNA (pink) cassettes from adjacent BACs. Homologous regions 1 and 2 (HR1, HR2) program the sites of recombination into the E. coli genome. The selection cassette -1 / +1 ensures the integration of synthetic DNA, while the selection cassette -2 / +2 on the genome ensures the removal of the corresponding wt DNA. In the example shown in the figure, +1 is KanR, -1 is rpsL, +2 is CmR, and -2 is sacB. [Figure 4]This figure shows that GENESIS enables stepwise replacement of genomic DNA with synthetic DNA to generate rewritten compartments. Genome stepwise exchange synthesis (GENESIS) is made possible by repeating cycles of REXER (see Figure 3), which alternately selects positive and negative selection cassettes (Wang, K., et al., 2016. Nature, 539(7627), 59-64). This allows for the assembly of large compartments of the synthetic genome by repeating additions of fragments that replace corresponding genomic sequences in a clockwise direction. The first REXER of a 100kb synthetic fragment of DNA leaves a -1 / +1 selection cassette on the genome, which acts as a landing site for downstream integration of a second fragment of synthetic DNA containing a -2 / +2 selection cassette. In the example shown, +1 is KanR, -1 is rpsL, +2 is CmR, and -2 is sacB, but the same logic can be used with different permutations of markers on the genome and BAC. [Figure 5]This figure shows the rewriting of ftsI-murE and map in fragment 1. a. Rewriting of the landscape of fragment 1. The inventors sequenced six clones after REXER. Each point represents the frequency of rewriting (y axis) in the sequenced clones for the target codon at the indicated position in the genome (x axis). Black points indicate positions where the inventors did not observe rewriting. Four codons and refactorings in ftsI-murE and one codon in map were rejected. b. Refactoring of a 14 bp ftsI-murE duplication. Codons and duplications are gray, scaled by their post-REXER substitution frequency in the sequenced clones. Using the inventors' initial refactoring scheme (1), the duplication was replicated with 20 bp of the upstream sequence added, and the inventors did not observe substitution of the duplication with synthetic DNA (in the six clones sequenced after REXER). Refactoring scheme 2, which replicates a duplicate with 182 bp of the upstream sequence added, resulted in complete rewriting of this region in 12 of the 16 REXER-post-sequenced clones. c. Testing of alternative codons at Ser4 in map. The double-selection marker pheS*-HygR on the constitutive EM7 promoter was introduced upstream of map, followed by RBS. The inventors replaced the cassette with linear double-stranded DNA introducing an alternative codon at position 4 (as shown) by negative selection for lambda red recombination and loss of pheS*. DNA with AGC and AGT was not incorporated (0 / 16 clones); the inventors recovered one clone for AGC, but sequencing revealed that it contained a mutant AAC(Asn) codon. TCT(6 / 8), TCC(6 / 16), ACA(6 / 8), and TTA(4 / 8) were acceptable. d. Refactoring scheme 2 for ftsI-murE duplication and rewriting the landscape across the genomic region shown in (a) after REXER in BAC containing a TCT at position 4 of the map. Two out of seven REXER clones were completely refactored and rewritten, with each target codon replaced in at least five out of seven clones. Data from (a) are shown for comparison. [Figure 6-1] ~ [Figure 6-2]This figure shows the rewriting of rne and yceQ in fragment 9. a. Rewriting landscape of fragment 9. The synthetic sequence of fragment 9 designed by the inventors was incorporated into the genome by REXER, and 19 clones were fully sequenced by NGS. The rewriting landscape graph shows the frequency of rewriting each target codon across the 19 clones. Most codon substitutions were accepted, but rewriting of the 26kb region was consistently rejected; codon positions with a rewriting frequency of zero in all sequenced clones are shown as black dots. To accurately show the problematic sequence, a 10kb stretch of the genome (G2-7) was deleted in the presence of an episodic copy of synthetic fragment 9. The synthetic sequence was sufficient to support the deletion of all stretches except G4 (dark gray box), suggesting that the underlying problem lies within this stretch. 0 / 19 clones were fully rewritten. b. Rewriting landscape of stretch G4. After sequencing REXER and 10 clones across a 10kb stretch "G4," the rewriting landscape shown was generated. This revealed minimal, clear rewriting in yceQ, the "gene" encoding the predicted protein, but there is no evidence of transcription, protein synthesis, or homology for this gene (Pundir, S., et al., 2017. Methods Mol Biol, 1558, 41-55). All target codons in yceQ were rewritten at least once in each individual clone, but never simultaneously; therefore, the minimal rewriting landscape was not zero, with 0 out of 10 clones being completely rewritten. This is consistent with epistasis between targeted locations. The map below the rewriting landscape shows sequences and target codons annotated as essential. Sequence positions (x-axis) are relative to panel a. c, design changes in the region around rne in fragment 9. The upper part shows the original design of the yceQ rewrite and rne (coding RNAse E) regulatory sequences. Target codons are indicated. Prne1, 2, and 3 are promoters for the essential gene rne; these are found within and around the hypothetical gene yceQ.The -10 sequence of the primary promoter P1rne is mutated in our initial design. The sequence contains hairpins 1 (hp1) and 2 (hp2) that bind to RNAse E to mediate transcript degradation; this sequence also contains the remaining target codons and is mutated in our initial design. At the bottom, the second codon in yceQ is replaced with a stop codon, while the remaining target codons retain their original sequences. Sequence positions (x-axis) are relative to panel a. This modified fragment 9 from d and c was incorporated into the genome, and the sequenced 4 / 5 clones were completely rewritten. The graph axes are the same as those in panel a. The rewritten landscape for the modified fragment 9, derived from the 5 sequenced clones, is shown in purple. Data from panel a is duplicated for comparison. [Figure 7-1] ~ [Figure 7-3]This figure shows the rewriting of yaaY in fragment 37a. a. Rewriting landscape of fragment 37a. The synthetic sequence of fragment 37a designed by the inventors was incorporated into the genome by REXER, and six clones were fully sequenced by NGS. Most codon substitutions were accepted, but rewriting of the 6.5kb region was consistently rejected. Target codon positions that were never rewritten in the six sequenced clones are shown as black dots. b. Identification of the problematic target codon. Within the identified 6.5kb problematic region, the inventors first focused on codons in essential genes (dark gray arrows) rather than non-essential genes (light gray arrows). Sanger sequencing of 24 clones (black bars) showed that two clones were rewritten at all six target codons within the essential gene subcompartment. Sanger sequencing of the remaining target codons in the essential gene of these two clones revealed that one clone was rewritten at all 17 target codons. This clone was fully sequenced by NGS and used to generate a rewrite landscape, where each target codon was either rewritten or not. This allowed the inventors to identify the problematic region 1.8kb upstream of ribF in combination with the rewrite landscape in (a). Here, the inventors focused on four target codons in genes rpsT and yaaY as the codons closest to the essential ribF gene. Sanger sequencing of 33 clones across this sequence revealed only one codon that was never rewritten: the codon for Ser70 in the hypothetical gene yaaY (the sequencing results are shown as scaled gray on the gene maps of rspT and yaaY). Therefore, the inventors investigated alternative codon substitutions in yaaY. c, Alternative codon substitution in the hypothetical gene yaaY. Substitution of TCA at AGT at Ser70 in this gene was unsuccessful.To investigate alternative codon substitution schemes, the double-selection marker pheS*-HygR, followed by RBS, on the constitutive EM7 promoter was introduced into yaaY, 12 bp upstream of the codon at Ser70. A negative selection marker was then used to select clones in which the cassette was substituted using linear double-stranded DNA introducing an alternative codon at position 70 by lambda red recombination. While linear double-stranded DNA with AGT did not incorporate (0 / 16 clones), the incorporation of dsDNA with TCC (2 / 16), TCG (2 / 16), TCT (6 / 16), and AGC (9 / 16) proved viable. d. REXER rewrite landscape in BAC containing fragment 37a of the exact type, carrying AGC at position 70 in the hypothetical gene yaaY. When incorporated by REXER, the inventors identified 1 / 7 completely rewritten clones. The AGC ranked 70th in Ser in yaaY was introduced into clones 4 / 7. [Figure 8] This figure shows the substitution of a hypothetical gene yceQ duplication in the regulatory element of rne, which encodes the essential protein RNAse E. a. In our original design, a programmed substitution from TCA to AGT in the hypothetical gene yceQ results in a mutation in the -10 promoter element of P1rne (circled). The transcription start site (tss) of this promoter for rne transcription is indicated by an arrow; this is the major promoter for rne transcription. b. The target codon substitution may overlap with and disrupt key regulatory hairpins hp2 and hp3 in the long 5'UTR of the rne transcript. hp2 and hp3 mediate a regulatory feedback loop in which RNAse E is recruited to mRNA to facilitate the degradation of its own transcript. A schematic diagram of the wild-type secondary structure of the rne 5'UTR is shown (Diwa, A., et al., 2000 Genes Dev 14, 1249-1260). The target codon for synonymous substitution is highlighted. [Figure 9-1] ~ [Figure 9-2]This figure shows the completion of sections A-B and H. a. GENESIS started with fragment 4 and proceeded smoothly to fragment 9, in which the inventors were unable to rewrite yceQ. The identification and correction of problems with the inventors' initial design of fragment 9 was carried out as shown in Figure 6 by introducing a stop codon at the start of the predicted yceQ ORF. After replacing the sacB-CmR(sC) double-selection cassette at the end of fragment 9 with the pheS*-HygR(pH) double-selection cassette, this strain was prepared to act as a recipient for a conjugation to assemble a strain in which fragments 4-13 (sections A+B) are completely rewritten. In parallel, the inventors continued to rewrite the strain containing the rewritten fragment 4 with GENESIS to the incomplete fragment 9; this resulted in a second strain for an assembly in which fragments 4-8 and 10-13 are completely rewritten and fragment 9 is partially rewritten. Next, the inventors incorporated oriT 3kb upstream of the start of fragment 10 in a second strain to generate a donor for a conjugation to assemble a strain in which fragments 4-13 (section A+B) are completely rewritten. The conjugation of the donor and recipient strains yielded a strain in which sections A and B are completely rewritten. Individual REXERs of fragments 37a and 1 resulted in incomplete rewriting. The inventors performed both troubleshooting independently (Figures 5 and 7). The repairs are shown. Each strain then served as a starting point for two independent sets of GENESIS, one generating 37a-37b (left side) and ending with the rpsL-KanR(rK) cassette, and the other generating 1-3 (right side) and ending with the sacB-CmR cassette. The inventors incorporated oriT 3kb upstream of the start of fragment 1, and this strain served as a donor for the induction conjugation of 1-3 to 37a-37b. The exact product was selected by the acquisition of CmR and the loss of rpsL. This resulted in the completion of compartment H in a single strain. [Figure 10]This figure shows the assembly of an organism with a fully synthetic genome obtained by conjugation of rewritten genomic compartments. Synthetic genomic compartments from multiple individual partially rewritten genomes were assembled into a single fully rewritten genome by conjugation (Ma, NJ, et al., 2014. Nat Protoc 9, 2285-2300). The donor (d) and recipient (r) strains each possess unique rewritten genomic compartments; rewritten overlapping homologous regions (3kb-400kb) were used to seamlessly recombine the strains. Small homologous regions in the range of 3-5kb are indicated by an asterisk (*). Conjugations in which the inventors used homology (HR) greater than 5kb are indicated by letters. For assembly, the rewritten genomic contents from the donor were conjugated clockwise to replace the corresponding wt genomic compartments in the recipient. The origins of strains AB and H are described in detail in Figure 9, but all other individual synthetic genomes were generated by GENESIS (Figure 4). Conjugation was followed by recombination until the final, fully rewritten A-H strains were assembled, and the sequences were validated by NGS sequencing. [Figure 11]This figure shows the assembly of a rewritten genomic compartment into a fully rewritten organism. a) Schematic assembly of a partially synthetic donor and recipient genome into a fully synthetic genome by conjugation. In recipient cells, the rewritten genomic compartment is extended with rewritten DNA, typically 3-4 kb, by lambda red-mediated recombination and positive and negative selection; this step utilizes genomic markers at the ends of the rewritten sequence introduced by GENESIS to provide homologous regions with the ends of the rewritten fragment in the donor strain. The donor strain is prepared by incorporating an origin of transfer (oriT) at the ends of the rewritten DNA. The shown positive and negative selection ensures the survival of the recipient strain and selects recipients that successfully incorporate the synthetic DNA from the donor. An F' plasmid containing mutations in the oriT sequence that make transfer impossible was used to facilitate the conjugation of the donor genome into the recipient. +2, CmR;-2, SacB;+3, HygR;-3, pheS*;+4 Gentamicin R;+5, Tetracycline R. b. Synthetic genomic compartments from multiple individual partially rewritten genomes were assembled into a single fully rewritten genome by the sequences indicated by the conjugation. The donor (d) and recipient (r) strains possess unique rewritten genomic compartments. The rewritten genomic contents from the donor were conjugated clockwise to replace the corresponding WT genomic compartments in the recipient. Conjugation was carried out until the final fully rewritten strains A-H were assembled. Figure 10 shows the process including all homologous regions in more detail. [Figure 12]This figure shows the functional results of synonymous codon compression in Syn61. Synonymous codon compression and deletion of a, prfA, serU, and serT. The gray boxes show serine codons and stop codons along with tRNAs and termination factors that decode them in WT E. coli (WT genome). The tRNA anticodons and termination factors are linked to the codons they read, indicated by the black lines. The tRNA and termination factor genes are shown within the black boxes. serT is the only tRNA that decodes the TCA codon in WT E. coli and is essential. Synonymous codon compression (Syn.Codon.Comp.) results in a rewritten genome where i) tRNAs with the CGA anticodon should not have a cognitive codon, and ii) serT should be non-essential. All factors that read target codons should be non-essential in Syn61. b. Co-translational incorporation of the non-canonical amino acid (ncAA), Nε-(((2-methylcycloprop-2-en-1-yl)methoxy)carbonyl)-L-lysine (CYPK), using orthogonal MmPylRS / tRNAPyl CGA pairs was toxic in MDS42 but not in Syn61. When CYPK is provided, this pair incorporates ncAA in a dose-dependent manner in response to the TCG codon. In MDS42, this incorporation results in erroneous synthesis of the proteome and toxicity. However, in Syn61, which does not contain the TCG codon, this is non-toxic. The line follows the mean of three biological copies (each shown as a point) at each [CYPK] (0 mM, 0.5 mM, 1 mM, 2.5 mM, and 5 mM). "Maximum growth %" was determined by the final OD600, calculated by dividing the CYPK concentration shown in the absence of CYPK by the final OD600. The final OD600 was determined after 600 minutes. c) Synonymous codon compression allows for the deletion of serT at Syn61. PCR adjacent to the serT locus before (-) and after (clones 1 and 2) substitution with the PheS*-HygR cassette.See also Figure 14. Complete gel in Figure 16. [Figure 13]This figure shows the characterization of organisms with a fully synthetic genome. a) Doubling times for Syn61 and MDS42. Our fully synthetically rewritten E. coli Syn61 has a doubling time 1.6 times longer than that of the parent strain MDS42 (Posfai, G. et al., 2006. Science 312, 1044-1046) when grown under standard medium conditions (LB + 2% glucose, 90.1 min vs. 57.6 min). The ratio of growth rates between Syn61 and MDS42 is 1.7 in LB (with reduced inhibition of carbon catabolism) at 37°C, 1.7 in M9 minimal medium, 1.4 in richer medium (2XTY), 2.5 in LB at 25°C, and 1.3 in LB at 42°C. The doubling times for MDS42 and Syn61 under different media conditions are listed: LB at 37°C, 58.3 min and 100.6 min; LB + 2% glucose, 57.6 min and 90.1 min; M9 minimal medium, 130.5 min and 221.1 min; 2XTY, 68.2 min and 92.6 min; LB at 25°C, 86.3 min and 218.4 min; LB at 42°C, 77.4 min and 99.7 min. Syn61 carrying plasmids with or without serV showed a growth rate ratio of 0.99 (138.3 min vs. 136.2 min). Doubling times represent the mean ± standard deviation from the mean of 10 independently grown biological replicas for each strain (see Methods). b. Representative microscopic images of E. coli strains MDS42 and Syn61. Samples were imaged using an upright Zeiss Axiophot phase-contrast microscope with a 63X 1.25NA Plan Neofluar relative object lens (see Methods). c. Histograms of quantified cell lengths from microscopic images of strains MDS42 and Syn61. The mean cell length for MDS42 was 1.97±0.57 μm, and for Syn61 it was 2.3±0.74 μm. Images of n=500 cells were taken during the exponential growth phase for both strains. Cell length measurements were performed using Nikon NIS Elements software (see Methods). d. Label-free quantification of the MDS42 and Syn61 proteomes. Each strain was grown in three biological replications. Each biological replication was analyzed by tandem mass spectrometry of the technical replicas.Technical replicas of biologically replicated proteins were fused. A total of 1,084 proteins were quantified across the entire sample. P-values ​​for differences in abundance were calculated using a two-sample t-test for proteins quantified in at least two biological replicas. The data showed that the abundance of three proteins differed significantly (P=0.01) between strains: aminopeptidase N (P04825) and peptidase T (P29745) were present in large proportions in Syn61, while 30S ribosomal protein S20 (P0A7U7) was present in small proportions. Based on LFQ values, the protein abundance did not differ by more than 1.14 times between strains. [Figure 14-1] ~ [Figure 14-4]This figure shows the results of synonymous codon compression in Syn61. a. Synonymous codon compression and deletion of prfA, serU, and serT in E. coli. The gray boxes show the serine codons and stop codons of E. coli, along with the tRNAs and termination factors that decode them in WT E. coli (WT genome). The tRNA anticodons and termination factors are linked to the codons they read with black lines. The tRNA and termination factor genes are shown in the black boxes. Synonymous codon compression (Syn.Codon.Comp.) yields Syn61 cells with a rewritten genome from which the TCG and TCA codons have been removed. The abundance of each codon is indicated in its box. b. Similar to Figure 12b, except for UGA, which is the MmPylRS / tRNAPyl anticodon shown. Since there are fewer cognitive codons for this tRNA in Syn61 than in MDS42, CYPK addition can be predicted to be less toxic in Syn61, as observed. c. Similar to Figure 12b, except for the MmPylRS / tRNAPyl anticodon GCU shown. Since there are more cognitive codons for this tRNA at Syn61 than at MDS42, CYPK addition can be predicted to be more toxic at Syn61, as observed. d. serT (dark gray) is deleted by insertion of the PheS*-HygR cassette (black) via lambda-red recombination. Recombination results in new junctions 1 and 2, as shown. For each recombination, both junctions were sequence-validated by Sanger sequencing. Arrows above the Sanger chromatogram indicate the exact location of the junction, the sequence corresponding to the selection cassette, and bars correspond to genomic sequences adjacent to the selection cassette. Primers used to generate selection cassettes with appropriate homology for serU, serT, and prfA for recombination are provided in Figure 23. e, prfA (dark gray) is removed by insertion of rpsL-KanR (black) via homologous recombination through lambda-red. The agarose gel is annotated as shown in Figure 12c, and the rest of the data is annotated as shown in panel d.The complete gel is available in Figure 16. f, serU (dark gray) has been removed by insertion of a PheS*-HygR cassette (black) via recombination through lambda-red. The agarose gel is annotated as shown in Figure 12c, and the rest of the data is annotated as shown in panel d. The complete gel is available in Figure 16. [Figure 15]This figure shows the scale of genome synthesis, the scale of rewriting, and the fidelity. a) Genome and chromosome synthesis. The size (Mb) of the synthetic genomes produced for M. genitalium and M. mycoides (Gibson, DG et al., 2008. Science 319, 1215-1220; and Gibson, DG et al., 2010. Science 329, 52-56) and several S. cerevisiae chromosomes (Shen, Y. et al., 2017. Science 355, aaf4791; Annaluru, N. et al., 2014. Science 344, 55-58; Xie, ZX et al., 2017. Science 355, aaf4704; Mitchell, LA et al., 2017. Science 355, aaf4831; Dymond, JS et al., 2011. Nature) are also included. (477, 471-476; Wu, Y. et al., 2017. Science 355, aaf4706; Zhang, W. et al., 2017. Science 355, aaf3981; and Richardson, SM et al., 2017. Science 355, 1040-1044) are shown in light gray. The sizes of the synthetic E. coli genomes presented here are shown in dark gray. b. Attempts at genome rewriting.Attempts to rewrite the target codons TTA and TTG in S. typhimurium (Lau, YH et al., 2017. Nucleic Acids Res 45, 6971-6980); AGC, AGT, TTG, TTA, AGA, AGG, and TAG in E. coli (Ostrov, N. et al., 2016. Science 353, 819-822); AGA and AGG in E. coli (Napolitano, MG et al., 2016. Proc Natl Acad Sci USA 113, E5588-5597), as well as the rewriting of all TAGs in E. coli (Lajoie, MJ et al., 2013. Science 342, 357-360), are shown in light gray. A comparison with the complete removal of TCA, TCG, and TAG in E. coli is presented here (dark gray). The total number of rewritten codons in a single strain is shown graphically, and the maximum percentage of rewritten target codons in a single strain for each trial is shown. The number of reported unprogrammed mutations and indels as a function of the number of rewritten target codons for the experiments shown in c and b. [Figure 16] This figure shows a complete gel for Figure 12. The complete gel is shown in the corresponding panel of the figure. Molecular size standards are annotated, and the regions shown in the relevant figures are indicated by white outlines. [Figure 17-1] ~ [Figure 17-2]This figure shows codon-anticodon interactions in the *E. coli* genome. 28 sense codons are highlighted in gray along with the amber stop codon. Genome-wide removal of these sense codons, rather than other sense codons, allows for the deletion of all their cognitive tRNAs without removing the ability to decode one or more remaining sense codons in the genome. This is necessary, but insufficient, for reassigning sense codons to non-natural monomers. The codon boxes for serine, leucine, and alanine are highlighted because the endogenous aminoacyl-tRNA synthetases for these amino acids do not recognize the anticodons of their cognitive tRNAs. This facilitates the reassignment of codons within these boxes to new amino acids by introducing tRNAs carrying cognitive anticodons that do not lead to incorrect aminoacylation by the endogenous synthetases. This study reports the total number of all 64 triplet codons in the MDS42 genome (Genbank accession number AP012306), all known codon-anticodon interactions via both Watson-Crick base pairing and fluctuations, tRNA anticodon base modifications, tRNA genes, and tRNA relative abundances measured in vivo. This analysis identifies 10 codons from the serine, leucine, and alanine groups (serine codons TCG, TCA, AGT, AGC; leucine codons CTG, CTA, TTG, TTA; and alanine codons GCG, GCA), satisfying both codon-anticodon interactions and aminoacyl-tRNA synthetase recognition criteria for codon reassignment. [Figure 18] This figure shows the designed synthetic E. coli genome (SEQ ID NO: 1). It is a type of E. coli MDS42 genome in which the serine codons TCG and TCA and the stop codon TAG within the open reading frames (ORF) are systematically replaced by their synonyms AGC, AGT, and TAA, respectively. Using defined rules for synonymous codon compression and refactoring, the genome is designed in which all 18,218 target codons are rewritten with their target synonyms. [Figure 19]This figure shows the final synthesized E. coli genome (Syn61) (Sequence ID 2). The sequence of E. coli Syn61 has all 1.8 × 10⁴ target codons in the genome rewritten. Our synthesis of the rewritten genome introduced only 8 unprogrammed mutations (Table 6), of which 4 occurred during the preparation of 100kb BAC and 4 occurred during the rewriting process. [Figure 20-1] ~ [Figure 20-13] This figure shows BACs for assembling synthetic genomes. A. BAC-sacB-CmR-rpsL. Nucleotide sequence for an annotated BAC vector containing a sacB-CmR selective cassette with a 5' homologous region (HR) and a CRISPR / Cas9 protospacer sequence (spacer 1) adjacent upstream. The sacB-CmR cassette has a 3' homologous region, a CRISPR / Cas9 protospacer sequence (spacer 2), and an rpsL selective marker adjacent downstream. B.-BAC-rpsL-KanR-sacB. Nucleotide sequence for an annotated BAC vector containing an rpsL-KanR selective cassette with a 5' homologous region (HR) and a CRISPR / Cas9 protospacer sequence (spacer 1) adjacent upstream. The rpsL-KanR cassette has a 3' homologous region, a CRISPR / Cas9 protospacer sequence (spacer 2), and a sacB selection marker adjacent downstream. C, BAC-rpsL-KanR-pheS*-HygR. Nucleotide sequences for an annotated BAC vector containing an rpsL-KanR selection cassette with a 5' homologous region (HR) and a CRISPR / Cas9 protospacer sequence (spacer 1) adjacent upstream. The rpsL-KanR cassette has a 3' homologous region, a CRISPR / Cas9 protospacer sequence (spacer 2), and a pheS*-HygR selection marker adjacent downstream. D, Table of BAC constructions. Oligonucleotides and selection markers used to construct BAC using homologous regions between synthetic DNA and synthetic DNA fragments for REXER. The second tab lists the plasmid backbone and protospacer sequences used for REXER. [Figure 21-1] ~ [Figure 21-2] This figure shows exemplary spacer plasmid maps. A. Spacer plasmid map. An exemplary map of pKW1_MB1amp_spacer_REXER2 containing a CRISPR insert having a spacer sequence used as a linear or circular spacer for REXER. B. Second-generation spacer plasmid map. An exemplary map of pKW3_MB1amp_spacer_REXER2 containing a CRISPR insert having a spacer sequence used as a circular second-generation spacer for REXER. [Figure 22-1] ~ [Figure 22-4] This figure shows the construct for conjugation. A. Gentamicin-resistant OriT cassette. B. Primers for the conjugation construct. Oligonucleotide primers used for conjugation. C. pJF146. Non-autotransmitting F' plasmid. [Figure 23] This figure shows primers for deletion experiments. Oligonucleotide primers used for the deletion of tRNA serT and serU, as well as the termination factor prfA, in Syn61. [Modes for carrying out the invention]

[0046] Detailed explanation As used herein, "comprising", "comprises", and " The term "comprised of" is equivalent to "including" or "Includes," or "contains," is synonymous with "includes," and is comprehensive or open-ended, not excluding additional unlisted components, elements, or steps. (Also known as "comprising" or "comprises") And the term "composed of" is equivalent to "consisting of". This also includes the term "."

[0047] Synthetic genome genome As used herein, “genome” refers to the genetic material of an organism, encompassing both genes and non-coding DNA. As used herein, “synthetic genome” refers to a genome constructed synthetically. Typically, a synthetic genome is produced by genetic modification of an existing (i.e., “parent”) genome. Therefore, a synthetic genome may be derived from a parent genome, i.e., it may be identical to a parent genome except for containing one or more genetic modifications. Those skilled in the art will be able to readily identify the parent genome on which a synthetic genome is based and the genetic modifications performed. As used herein, “parent genome” may be any naturally occurring, commercially available, deposited, cataloged, or otherwise well-known genome, or derivatives thereof.

[0048] The synthetic genome of the present invention is a synthetic prokaryotic genome. Prokaryotes are single-celled organisms that lack a membrane-bound nucleus, mitochondria, or any other membrane-bound organelles. Prokaryotes are divided into two regions: archaea and bacteria. The genome of a prokaryotic organism is generally a circular double-stranded DNA fragment, and multiple copies thereof can exist at any given time.

[0049] Preferably, the synthetic genome of the present invention is a synthetic bacterial genome. Preferably, the synthetic bacterial genome is suitable for heterologous protein production, particularly the production of polypeptides containing one or more non-proteinogenic amino acids (e.g., those described in Ferrer-Miralles, N. and Villaverde, A., 2013. Microbial Cell Factories, 12:113). Suitable bacterial genomes include Escherichia (e.g., Escherichia coli), Caulobacteria (e.g., Caulobacter crescentus), photosynthetic bacteria (e.g., Rodobacter sphaeroides), and cold-adapted bacteria. Type bacteria (e.g., Pseudoalteromonas haloplanktis, Shewanella sp. strain Ac10), Pseudomonads (e.g., Pseudomonas fluorescens) Pseudomonas putida, Pseudomonas aeruginosa, halophilic bacteria (e.g., Halomonas elongate, Chromohalobacter salexigens) Streptomycetes (for example, Streptomyces lividans, Streptomyces griseus) ), Nocardia (for example, Nocardia lactamjurance) lactamdurans), mycobacteria (for example, Mycobacterium) • Mycobacterium smegmatis), coryneform bacteria (e.g., Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum), Bacilli (e.g., Bacillus subtilis, Bacillus brevis, Bacillus megaterium, Bacillus lichen) Bacillus licheniformis, Bacillus amyloliquefaciens, and lactic acid bacteria (e.g., Lactococcus lactis, Lactobacillus plantarum, Lactobacillus) Examples include the genomes of Lactobacillus casei, Lactobacillus reuteri, and Lactobacillus gasseri. In some embodiments, the synthetic genome is a synthetic Gram-negative bacterial genome.

[0050] Bacterial genomes can range in size from approximately 130kb to over 14Mb. Therefore, in some embodiments, the synthetic prokaryotic genomes of the present invention are 100kb to 20Mb, or 130kb to 15Mb, or 200kb to 15Mb, or 300kb to 15Mb, or 500kb to 15Mb, or 1Mb to 15Mb, or 1Mb to 10Mb, or 1Mb to 8Mb, or 1Mb to 6Mb, or 2Mb to 6Mb, or 2Mb to 5Mb, or 3Mb to 5Mb, or approximately 4Mb. A synthetic prokaryotic genome may contain 100 or more genes, 200 or more genes, 300 or more genes, 400 or more genes, 500 or more genes, 600 or more genes, 700 or more genes, 800 or more genes, 900 or more genes, 1000 or more genes, 1001 or more genes, 1500 or more genes, or 2000 or more genes, preferably 1000 or more genes. A synthetic prokaryotic genome may contain 100 or more genes, 200 or more genes, 300 or more genes, 400 or more genes, 500 or more genes, 600 or more genes, 700 or more genes, 800 or more genes, 900 or more genes, 1000 or more genes, 1500 or more genes, or 2000 or more genes, with evidence of translation and / or predicted protein products for those genes, preferably 1000 or more genes. Preferably, the synthetic prokaryotic genome contains 100 or 101 or more, 200 or 201 or more, 300 or 301 or more, 400 or 401 or more, 500 or 501 or more essential genes, preferably 300 or 301 or more essential genes.

[0051] Preferably, the synthetic genome of the present invention is a synthetic Escherichia coli genome, a Salmonella enterica genome, or a Shigella genome. These are described in Lukjancenko, O., et al., 2010. Microbial ecology, 60(4), pp.708-720; and Karberg, KA, et al., 2011. PNAS, 108(50). They are phylogenetically related species, as disclosed on pp. 20154-20159.

[0052] More preferably, the synthetic genome of the present invention is a synthetic E. coli genome. The parent genome may be any suitable E. coli genome encompassing MDS42, K-12, MG1655, BL21, BL21(DE3), AD494, Origami, HMS174, BLR(DE3), HMS174(DE3), Tuner(DE3), Origami2(DE3), Rosetta2(DE3), Lemo21(DE3), NiCo21(DE3), T7 Express, SHuffle Express, C41(DE3), C43(DE3), and m15 pREP4 or derivatives thereof (Rosano, GL and Ceccarelli, EA, 2014. Frontiers in microbiology, 5, p.172). Most preferably, the parent genome is MDS42, MG1655, or BL21 or a derivative thereof. MG1655 is considered a wild-type strain of E. coli. The GenBank ID for the genome sequence of this strain is U00096. BL21 is widely available commercially. For example, it can be purchased from New England BioLabs under catalog number C2530H (https: / / www.neb.com / products / c2530-bl21-competent-e-coli).

[0053] In some embodiments, the synthetic genome is a small synthetic genome or a minimal synthetic genome. A "small genome" is one in which the size of the parent genome is reduced by removing non-essential genes and / or non-coding regions. A "minimal genome" is a genome that has been reduced to its minimum size while maintaining viability, for example, by deleting all non-essential regions of the genome.

[0054] The synthetic genome of the present invention may be a viable genome. As used herein, “viable genome” means a genome containing nucleic acid sequences sufficient to cause and / or maintain the viability of a cell, for example, a genome encoding molecules required for replication, transcription, translation, energy production, transport, production of membrane and cytoplasmic components, and cell division.

[0055] Preferably, one or more tRNAs or termination factors may be deleted from the synthetic genome, and the synthetic genome may remain viable. For example, a tRNA that decodes only the one or more substituted (or deleted) sense codons may be non-essential. Similarly, if the remaining sense codons decoded by the tRNA can also be decoded by alternative tRNAs, a tRNA that decodes the one or more substituted (or deleted) sense codons may be non-essential. For example, tRNA Ser UGA The serT gene, which encodes the TCA codon, is usually essential because it is the only tRNA that decodes the TCA codon in E. coli. However, if the synthetic genome does not contain the TCA codon, serT may be non-essential.

[0056] Sense codon The present invention provides a synthetic prokaryotic genome containing five or four or fewer occurrences of one or more sense codons; and / or a synthetic prokaryotic genome derived from a parent genome, in which the synthetic prokaryotic genome contains the occurrence of one or more sense codons in amounts of less than 10%, 5%, 2%, 1%, 0.5%, or 0.1% compared to the parent genome; and / or a synthetic prokaryotic genome containing 100 or 101 or more, 200 or 201 or more, or 1000 or 1001 or more genes, without the occurrence of one or more sense codons.

[0057] One or more sense codons may consist of one, two, three, four, five, six, seven, or eight sense codons. Preferably, one or more sense codons consist of one sense codon or two sense codons, most preferably two sense codons.

[0058] A synthetic prokaryotic genome may include, or may not include, five or four occurrences (e.g., five, four, three, two, one) of one or more (e.g., one, two, three, four, five, six, seven, or eight) sensecodons. In some embodiments, the synthetic prokaryotic genome includes five or four occurrences (e.g., five, four, three, two, one, zero) of each of one or more (e.g., one, two, three, four, five, six, seven, or eight) sensecodons. In other embodiments, the synthetic prokaryotic genome comprises five or four sense codons (e.g., five, four, three, two, one, or zero) in total (i.e., one, two, three, four, five, six, seven, or eight). In preferred embodiments, the synthetic prokaryotic genome does not contain the appearance of one sense codon. In other preferred embodiments, the synthetic prokaryotic genome does not contain the appearance of two sense codons.

[0059] The synthetic prokaryotic genome may be derived from the parent genome and may include or not include five or four occurrences (e.g., five, four, three, two, one) of one or more (e.g., one, two, three, four, five, six, seven, or eight) of natural sense codons. In some embodiments, the synthetic prokaryotic genome includes five or four occurrences (e.g., five, four, three, two, one, or zero) of each of one or more (e.g., one, two, three, four, five, six, seven, or eight) natural sense codons. In other embodiments, the synthetic prokaryotic genome comprises five or four (e.g., five, four, three, two, one, or zero) natural sense codons in total (i.e., five, four, three, two, one, or eight). Preferred Embodiments In this embodiment, the synthetic prokaryotic genome is derived from the parent genome and does not include the appearance of one natural sense codon. In another preferred embodiment, the synthetic prokaryotic genome is derived from the parent genome and does not include the appearance of two natural sense codons.

[0060] In some embodiments, the synthetic prokaryotic genome includes 100 or 101 or more, 200 or 201 or more, 300 or 301 or more, 400 or 401 or more, 500 or 501 or more, 600 or 601 or more, 700 or 701 or more, 800 or 801 or more, 900 or 901 or more, 1000 or 1001 or more, 1500 or 1501 or more, or 2000 or 2001 or more genes, preferably 1000 or 1001 or more genes. In some embodiments, the genes are those for which evidence of translation and / or predicted protein products exists. For example, a synthetic prokaryotic genome may contain 100 or more genes, 200 or more genes, 300 or more genes, 400 or more genes, 500 or more genes, 600 or more genes, 700 or more genes, 800 or more genes, 900 or more genes, 1000 or more genes, 1001 or more genes, 1500 or more genes, or 2000 or more genes, preferably 1000 or more genes, and evidence of translation and / or predicted protein products exists for those genes. Preferably, the synthetic prokaryotic genome contains 100 or 101 or more, 200 or 201 or more, 300 or 301 or more, 400 or 401 or more, 500 or 501 or more essential genes, preferably 300 or 301 or more essential genes. Preferably, the (essential) genes do not have the appearance of one or more sense codons.

[0061] The synthetic prokaryotic genome may include the appearance of one or more sense codons (e.g., one, two, three, four, five, six, seven, or eight) in amounts less than 10%, 5%, 2%, 1%, 0.5%, or 0.1% compared to the parent genome. In some embodiments, the synthetic prokaryotic genome includes the appearance of one or more sense codons (e.g., one, two, three, four, five, six, seven, or eight) in amounts less than 10%, 5%, 2%, 1%, 0.5%, or 0.1% compared to the parent genome. In other embodiments, the synthetic prokaryotic genome includes the appearance of one or more sense codons (e.g., one, two, three, four, five, six, seven, or eight) in amounts less than 10%, 5%, 2%, 1%, 0.5%, or 0.1% combined compared to the parent genome. In preferred embodiments, the synthetic prokaryotic genome contains one sense codon in amounts less than 10%, 5%, 2%, 1%, 0.5%, or 0.1% compared to the parent genome. In other preferred embodiments, the synthetic prokaryotic genome contains two sense codons in amounts less than 10%, 5%, 2%, 1%, 0.5%, or 0.1% compared to the parent genome.

[0062] A synthetic prokaryotic genome may contain 100 or 101 or more, 200 or 201 or more, or 1000 or 1001 or more genes, each lacking one or more (e.g., one, two, three, four, five, six, seven, or eight) sense codon occurrences. Preferably, all or substantially all genes in the synthetic prokaryotic genome lack one or more (e.g., one, two, three, four, five, six, seven, or eight) sense codon occurrences. In a preferred embodiment, all or substantially all genes in the synthetic prokaryotic genome lack one sense codon occurrence. In another preferred embodiment, all or substantially all genes in the synthetic prokaryotic genome lack two sense codon occurrences. "Substantially all" means that all genes, with the exception of 10 or 9 or fewer (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0), contain one or more sense codons.

[0063] Synthetic prokaryotic genomes lack the appearance of one or more (e.g., one, two, three, four, five, six, seven, or eight) natural sense codons, and have 100 or 101 genomes. The genome may contain 10 or more genes, 200 or 201 or more genes, or 1000 or 1001 or more genes. Preferably, all or substantially all genes in the synthetic prokaryotic genome do not have the appearance of one or more (e.g., one, two, three, four, five, six, seven, or eight) native sense codons. In a preferred embodiment, all or substantially all genes in the synthetic prokaryotic genome do not have the appearance of one native sense codon. In another preferred embodiment, all or substantially all genes in the synthetic prokaryotic genome do not have the appearance of two native sense codons. Substantially all means that all genes except for 10 or 9 or fewer (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0) contain the appearance of one or more native sense codons.

[0064] Preferably, the gene codes for a protein (for example, the gene is one for which evidence of translation and / or predicted protein product exists), and / or the gene is an essential gene. Therefore, in a more preferred embodiment, the synthetic prokaryotic genome includes genes that code for 100 or more proteins, 200 or more proteins, or 1000 or more proteins, and / or essential genes that do not contain one or two sense codon occurrences. In another more preferred embodiment, all or substantially all protein-coding genes and / or essential genes in the synthetic prokaryotic genome do not contain one or two sense codon occurrences.

[0065] In preferred embodiments, the protein is not translated from any of the remaining occurrences of one or more sense codons, and / or the gene containing the remaining occurrences of one or more sense codons is a putative or non-coding gene. In some embodiments, translation of the gene containing the remaining occurrences of one or more sense codons is reduced and / or blocked (for example, the gene may contain a stop codon in its 5' sequence).

[0066] The presence of any remaining sense codons may be necessary to ensure the viability of the synthetic prokaryotic genome. For example, one or more, preferably all, of the remaining sense codons in one or more sense codons in a synthetic prokaryotic genome may be present in the regulatory elements of essential genes, and / or one or more, preferably all, of the remaining sense codons may be present in genes for which there is no evidence of translation or predicted protein products (i.e., putative or non-coding genes).

[0067] As used herein, "sense codon" is a nucleotide triplet that codes for an amino acid. Therefore, sense codons can be identified within a genome by gene prediction, that is, by identifying protein-coding regions of the genome (i.e., genes) and their corresponding open reading frames (ORFs). Typically, the genome naturally contains 61 sense codons: GCT, GCC, GCA, GCG, CGT, CGC, CGA, CGG, AGA, AGG, AAT, AAC, GAT, GAC, TGT, TGC, CAA, CAG, GAA, GAG, GGT, GGC, GGA, GGG, CAT, CAC, ATT, ATC, ATA, TTA, TTG, CTT, CTC, CTA, CTG, AAA, AAG, ATG, TTT, TTC, CCT, CCC, CCA, CCG, TCT, TCC, TCA, TCG, AGT, AGC, ACT, ACC, ACA, ACG, TGG, TAT, TAC, GTT, GTC, GTA, and GTG (read from 5' to 3' on the coding strand of DNA). The standard genetic code uses 61 triplet codons to encode 20 canonical amino acids. Eighteen of the 20 amino acids are encoded by more than one synonymous codon (see Figure 17). One or more sense codons are one or more native sense codons, i.e., sense codons present in the parent genome. obtain.

[0068] The 61 sense codons in DNA are transcribed into corresponding mRNA, which is then sequenced by one or more tRNAs. The tRNAs deliver amino acids to the ribosomes as directed by the sense codons in the mRNA. The tRNAs can recognize one or more sense codons through complementary anticodons. Subsequently, the sense codon sequences are translated into polypeptides (i.e., amino acid sequences). Codon-anticodon interactions in the E. coli genome are shown in Figure 17.

[0069] Preferably, genome-wide removal of one or more sense codons, rather than other sense codons, can remove all cognitive tRNAs corresponding to the one or more sense codons without removing their ability to decode the one or more sense codons remaining in the genome. Therefore, the one or more sense codons may be selected from TCG, TCA, AGT, AGC, GCG, GCA, GTG, GTA, CTG, CTA, TTG, TTA, ACG, ACA, CCG, CCA, CGG, CGA, CGT, CGC, AGG, AGA, GGG, GGA, GGT, GGC, ATT, and ATC.

[0070] The aminoacyl-tRNA synthetases for serine, leucine, and alanine do not recognize the anticodons of their cognitive tRNAs. This makes it possible to facilitate the assignment of codons in these boxes to new amino acids by introducing tRNAs carrying cognitive anticodons that do not lead to incorrect aminoacylation by endogenous synthetases. Therefore, one or more sense codons can be selected from TCG, TCA, TCT, TCC, AGT, AGC, GCG, GCA, GCT, GCC, CTG, CTA, CTT, CTC, TTG, and TTA.

[0071] Preferably, one or more sense codons satisfy both of these criteria, so one or more sense codons may be selected from TCG, TCA, AGT, AGC, GCG, GCA, CTG, CTA, TTG, and TTA. More preferably, one or more sense codons may be selected from TCG, TCA, AGT, AGC, TTG, TTA, GCG, and GCA. Most preferably, one or more sense codons are TCG and / or TCA.

[0072] Preferably, one or more sense codons are removed so that the genome is compatible with codon reassignment to non-proteinogenic amino acids. Therefore, one or more sense codons may include one or more of TCA, CTA, or TTA. Alternatively, two or more sense codons are removed, and those two or more sense codons include one or more sense codon pairs selected from the group consisting of GCG and GCA; GCT and GCC; TCG and TCA; AGT and AGC; TCT and TCC; CTG and CTA; TTG and TTA; and CTT and CTC. Preferably, two or more sense codons are removed, and those two or more sense codons include one or more sense codon pairs selected from the group consisting of GCG and GCA; TCG and TCA; AGT and AGC; CTG and CTA; and TTG and TTA. More preferably, two or more sense codons include TCG and TCA.

[0073] To achieve the removal of sense codons, they may be replaced with synonymous sense codons. This is preferable to ensure that the encoded protein sequence remains unchanged. For example, the present invention may be used to remove 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100% of the occurrence of one or more sense codons in the parent genome. We provide a synthetic prokaryotic genome that is substituted with synonymous sense codons. Those skilled in the art can deduce appropriate synonymous sense codon substitutions. For example, in Escherichia coli, typically TCG, TCA, TCT, TCC, AGT and AGC all encode serine; typically GCG, GCA, GCT and GCC all encode alanine; and typically CTG, CTA, CTT, CTC, TTG and TTA all encode leucine.

[0074] In some embodiments, the substitution is a defined substitution, i.e., one sense codon is replaced by a single synonymous sense codon. Preferably, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100% of the occurrences of one or more sense codons in the parent genome are replaced by a defined (i.e., single) synonymous sense codon.

[0075] For example, a defined substitution may be GCG substituted with either GCT or GCC; GCA substituted with either GCT or GCC; TCG substituted with any one of TCT, TCC, AGT, or AGC; TCA substituted with any one of TCT, TCC, AGT, or AGC; AGT substituted with any one of TCG, TCA, TCT, or TCC; AGC substituted with any one of TCG, TCA, TCT, or TCC; CTG substituted with any one of CTT, CTC, TTG, or TTA; CTA substituted with any one of CTT, CTC, TTG, or TTA; TTG substituted with any one of CTG, CTA, CTT, or CTC; or TTA substituted with any one of CTG, CTA, CTT, or CTC. Preferably, one or more defined sense codon substitutions are selected from one or more of the following: GCG to GCT or GCC; GCA to GCT or GCC; TCG to AGT or AGC; TCA to AGT or AGC; AGT to TCA or TCT; AGC to TCG, TCC or TCA; TTG to CTT; and TTA to CTC. More preferably, TCG and / or TCA are substituted with AGC and / or AGT. Most preferably, TCG is substituted with AGC and / or TCA is substituted with AGT.

[0076] Preferably, the defined substitutions are such that the genome is compatible with codon reassignment to nonproteinogenic amino acids. For example, (i) GCG may be substituted with either GCT or GCC, and GCA may be substituted with either GCT or GCC; (ii) TCG may be substituted with either TCT, TCC, AGT, or AGC, and TCA may be substituted with either TCT, TCC, AGT, or AGC; (iii) AGT may be substituted with either TCG, TCA, TCT, or TCC (iv) CTG may be substituted with any of CTT, CTC, TTG, or TTA, and CTA may be substituted with any of CTT, CTC, TTG, or TTA; or (v) TTG may be substituted with any of CTG, CTA, CTT, or CTC, and TTA may be substituted with any of CTG, CTA, CTT, or CTC.

[0077] Preferably, the defined substitution scheme is one or more of those listed in the table below:

[0078] [Table 1] JPEG2026094253000002.jpg18965JPEG2026094253000003.jpg7162

[0079] Preferably, none of these codon substitutions affect the ribosome-binding site (AGGAGG), a highly conserved regulatory sequence in E. coli. Selected codon substitutions may be tested in small test regions (e.g., 20kb regions of the genome rich in both essential target genes and target codons) to assess viability. If codon substitutions are not viable in the small test regions, they may be ignored.

[0080] If the substitution of one or more sense codons in the parent genome with a defined substituted synonymous sense codon does not result in a viable genome, alternative substituted synonymous sense codons may be used. For example, 99.9% of the occurrences of one or more sense codons in the parent genome may be substituted with a defined (i.e., single) synonymous sense codon, and the remaining 0.1% may be substituted with alternative synonymous sense codons. For example, 99.9% of TCG occurrences may be substituted with AGC, and 0.1% may be substituted with TCT, TCC, AGT, or AGC; and / or 99.9% of TCA occurrences may be substituted with AGT, and 0.1% may be substituted with TCT, TCC, AGT, or AGC.

[0081] As used herein, a “stop codon” is a nucleotide triplet that codes for the termination of translation into a protein. Typically, a genome has three stop codons: TAA (“ochre”), TGA (“opal” or “umber”), and TAG (“unclear”). It contains naturally occurring "bars".

[0082] In some embodiments, the synthetic prokaryotic genome further includes, or does not include, 10 or 9 or fewer occurrences of one or two stop codons, 5 or 4 or fewer occurrences, preferably including, or not including 10 or 9 or fewer occurrences of amber stop codons (TAGs), 5 or 4 or fewer occurrences. Preferably, 90% or more, 95% or more, 98% or more, 99% or more, or all of the occurrences of TAGs in the parent prokaryotic genome are replaced with TAAs (ochre stop codons). In preferred embodiments, the synthetic prokaryotic genome may not include any occurrences of amber stop codons (TAGs), and all of the occurrences of TAGs in the parent prokaryotic genome may be replaced with TAAs (ochre stop codons).

[0083] Therefore, in a preferred embodiment, the synthetic prokaryotic genome of the present invention does not include the appearance of one or more sense codons, or two or more sense codons, and does not include the appearance of one stop codon, preferably an amber stop codon (TAG). In a more preferred embodiment, the synthetic prokaryotic genome of the present invention includes two sense codons, preferably TCG and TCA. Excluding occurrences and occurrences of amber stop codons (TAGs), TCG, TCA, and TAG in the parent prokaryotic genome may be replaced with synonymous codons. For example, 99.9% or more of TCG occurrences in the parent prokaryotic genome are replaced with AGC, 99.9% or more of TCA occurrences in the parent prokaryotic genome are replaced with AGT, and all TAG occurrences in the parent prokaryotic genome are replaced with TAA.

[0084] In some embodiments, the synthetic prokaryotic genome includes a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, or 99.9% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

[0085] The present invention provides a synthetic prokaryotic genome that is at least 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 2.

[0086] Sequence comparison may be performed by visual estimation, or more commonly, by utilizing readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences.

[0087] Sequence identity can be calculated over consecutive sequences; that is, one sequence is aligned with another, and each amino acid in one sequence is directly compared to its corresponding amino acid in the other sequence, one residue at a time. This is called a "gapless" alignment. Typically, such gapless alignments are performed only over a relatively small number of residues (e.g., fewer than 50 consecutive amino acids).

[0088] While this is a very simple and consistent method, it does not take into account, for example, that a single insertion or deletion in an otherwise identical sequence pair may exclude subsequent amino acid residues from the alignment, and therefore can result in a significant decrease in homology percentage when performing a global alignment. Thus, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without excessively penalizing the overall homology score. This is achieved by attempting to maximize local homology by inserting "gaps" into the sequence alignment.

[0089] However, these more complex methods assign a "gap penalty" to each gap that occurs during alignment, so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible (reflecting a higher relevance between the two comparison sequences) scores higher than one with many gaps. Typically, an "affine gap cost" is used, which imposes a relatively high cost on the presence of gaps and a smaller penalty on each subsequent residue within the gap. This is the most commonly used gap scoring system. A high gap penalty naturally produces an optimized alignment with fewer gaps. Most alignment programs allow you to change the gap penalty. However, when using such software for sequence comparison, it is preferable to use the default value. For example, when using the GCG Wisconsin Bestfit package (see below), the default gap penalty for amino acid sequences is... Narti's value is -12 for a single gap and -4 for each extension.

[0090] Therefore, calculating the maximum percentage of sequence identity first requires generating an optimal alignment that takes gap penalties into account. A suitable computer program for performing such alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12:387). Other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410), and the GENEWORKS comparison tools suite. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, it is preferable to use the GCG Bestfit program.

[0091] Appropriately, sequence identity can be determined over the entire sequence. Appropriately, sequence identity can be determined over the entire range of candidate sequences compared to the sequences listed herein.

[0092] While final sequence identity can be measured in terms of identity, the alignment process itself is not typically based on all-or-nothing pairwise comparisons. Instead, a scaled similarity score matrix is ​​commonly used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. A commonly used example of such a matrix is ​​the BLOSUM62 matrix (the default matrix for the BLAST program suite). The GCG Wisconsin program generally uses either public default values ​​or, if provided, custom symbol comparison tables (see the user manual for further details). Preferably, GCG The package uses public default values, or in the case of other software, default matrices, such as BLOSUM62.

[0093] Once the software generates the optimal alignment, the sequence identity percentage can be calculated. Typically, this software performs this as part of a sequence comparison, yielding a numerical result.

[0094] Refactoring The genome contains numerous overlapping open reading frames (ORFs), which can be classified as 3', 3' (between reversed ORFs) or 5', 3' (between same-oriented ORFs). One or more sense codons (i.e., those to be substituted) may be found within both classes of overlap in the parent genome.

[0095] If the substitution of one or more sense codons in each ORF within the duplication can be achieved without altering the encoded protein sequence of any of the ORFs (i.e., by introducing synonymous codons), then editing (e.g., refactoring) the parent genome may not be necessary. However, if the encoded protein sequence is altered by the substitution of one or more sense codons (i.e., one or more synonymous sense codons are not introduced into one or both ORFs), then editing (e.g., refactoring) the parent genome may be necessary.

[0096] Therefore, in some embodiments, one or more gene pairs that share an overlapping region containing one or more sense codons in the parent genome are refactored. "Refactored" means that the genes are rearranged to prevent changes to the encoded protein sequence. Preferably, the gene pairs are refactored such that a sense codon substitution (e.g., a defined synonymous codon substitution) alters the encoded protein sequence of both or either of the gene pairs. Most preferably, all gene pairs that share an overlapping region containing one or more sense codons in the parent genome are refactored such that a sense codon substitution (e.g., a defined synonymous codon substitution) alters the encoded protein sequence of both or either of the gene pairs.

[0097] With respect to 3',3' duplication (i.e., reversed gene pairs), synthetic inserts may be inserted between the genes. With respect to 3',3' duplication, synthetic inserts may include the overlapping region.

[0098] With respect to 5', 3' duplications (i.e., gene pairs of the same orientation, including an upstream gene and a downstream gene), a synthetic insertion may be inserted between the genes. With respect to 5', 3' duplications, the synthetic insertion may include (i) a stop codon; (ii) about 20–200 bp, or 20–100 bp, or 20–50 bp upstream of the duplication region; and (iii) the duplication region. Preferably, the synthetic insertion includes (i) a stop codon; (ii) about 20 bp upstream of the overlapping region; and (iii) the overlapping region. This allows for the distribution of RBS for the downstream ORF. The distance between the sequence and this RBS and its start codon is stored.

[0099] In a preferred embodiment, the stop codon is in-frame with the original start site for downstream genes. Preferably, the stop codon is TAA.

[0100] Apart from the specific mutations described above, namely mutations aimed at reducing the amount of one or more sense codons (e.g., substitution and / or refactoring of one or more sense codons) and mutations aimed at reducing the amount of amber stop codons, the synthetic prokaryotic genome may contain 1000 or fewer, 999 or fewer, 100 or fewer, 99 or fewer, 50 or fewer, 49 or fewer, 20 or fewer, 19 or fewer, or 10 or fewer, unprogrammed mutations compared to the parent genome. Preferably, the synthetic prokaryotic genome contains 2 × 10⁶ mutations per target codon (i.e., per occurrence of one or more sense codons in the parent genome). -4 Includes one or fewer additional or unprogrammed mutations.

[0101] Polynucleotides The present invention provides a polynucleotide comprising one or more genes that lack the appearance of one or more sense codons. The polynucleotide may contain two or three or more, three or four or more, four or five or more, five or six or more, ten or eleven or more, twenty or 21 or more, thirty or 31 or more, forty or 41 or more, fifty or 51 or more, 100 or 101 or more, 200 or 201 or more, 500 or 501 or more, 600 or 601 or more, 700 or 701 or more, 800 or 801 or more, 900 or 901 or more, 1000 or 1001 or more, 1500 or 1501 or more, or 2000 or 2001 or more genes that lack the appearance of one or more sense codons. Preferably, the polynucleotide contains 100 or 101 or more genes that do not contain one or more sense codons. More preferably, the polynucleotide contains 1000 or 1001 or more genes that do not contain one or more sense codons.

[0102] One or more sense codons may consist of one, two, three, four, five, six, seven, or eight sense codons. Preferably, one or more sense codons consist of one or two sense codons, most preferably two sense codons. Therefore, in a preferred embodiment, the polynucleotide contains 100 or 101 or more genes that do not contain one or two sense codons. In another preferred embodiment, the polynucleotide contains 1000 or 1001 or more genes that do not contain one or two sense codons.

[0103] One or more sense codons may be selected from TCG, TCA, AGT, AGC, GCG, GCA, GTG, GTA, CTG, CTA, TTG, TTA, ACG, ACA, CCG, CCA, CGG, CGA, CGT, CGC, AGG, AGA, GGG, GGA, GGT, GGC, ATT, and ATC. Alternatively, one or more sense codons may be TCG, TCA, TCT, TCC, AGT, AGC, GCG, GCA, GCT, The sense codons may be selected from GCC, CTG, CTA, CTT, CTC, TTG, and TTA. Preferably, one or more sense codons are selected from TCG, TCA, AGT, AGC, GCG, GCA, CTG, CTA, TTG, and TTA. More preferably, one or more sense codons are selected from TCG, TCA, TTG, TTA, GCG, and GCA. Most preferably, one or more sense codons are TCG and / or TCA.

[0104] One or more sense codons in a gene may be replaced with synonymous sense codons. Preferably, the substitution is a defined substitution, i.e., one sense codon is replaced with a single synonymous sense codon.

[0105] For example, GCG may be replaced with GCT or GCC; GCA may be replaced with GCT or GCC; TCG may be replaced with TCT, TCC, AGT, or AGC; TCA may be replaced with TCT, TCC, AGT, or AGC; AGT may be replaced with TCG, TCA, TCT, or TCC; AGC may be replaced with TCG, TCA, TCT, or TCC; CTG may be replaced with CTT, CTC, TTG, or TTA; CTA may be replaced with CTT, CTC, TTG, or TTA; TTG may be replaced with CTG, CTA, CTT, or CTC; or TTA may be replaced with CTG, CTA, CTT, or CTC. Preferably, one or more defined sense codon substitutions are selected from GCG to GCT or GCC; GCA to GCT or GCC; TCG to AGT or AGC; TCA to AGT or AGC; AGT to TCA or TCT; AGC to TCG, TCC or TCA; TTG to CTT; and TTA to CTC. More preferably, TCG and / or TCA are substituted with AGC and / or AGT. Most preferably, TCG is substituted with AGC and / or TCA is substituted with AGT.

[0106] In some embodiments, the gene is one for which evidence of translation and / or predicted protein products exists.

[0107] In a preferred embodiment, the gene is an essential gene. The essential genes are ribF, lspA, ispH, dapB, folA, imp, yabQ, 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, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, lpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, rne, fabD, fabG, acpP, tmk, holB, lolC, 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, lepB, 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, rplC, 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, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, and may be selected from one or more of the list consisting of dnaC.

[0108] RibF, lspA, ispH, dapB, folA, imp, yabQ, lpxC, secM, secA can、folK、hemL、yadR、dapD、map、rpsB、tsf、pyrH、frr、dxr、ispU、cdsA、yae L、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、glnS、fldA、cydA、infA、cydC、fts K、lolA、serS、rpsA、msbA、lpxK、kdsB、mukF、mukE、mukB、asnS、fabA、mviN、r ne、fabD、fabG、acpP、tmk、holB、lolC、lolD、lolE、purB、minE、minD、pth、p rsA、ispE、lolB、hemA、prfA、prmC、kdsA、topA、ribA、fabI、tyrS、ribC、ydiL 、pheT、pheS、rplT、infC、thrS、nadE、gapA、yeaZ、aspS、argS、pgsA、yefM、m etG、folE、yejM、gyrA、nrdA、nrdB、folC、accD、fabB、gltX、ligA、zipA、dapE 、dapA、der、hisS、ispG、suhB、tadA、acpS、era、rnc、lepB、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、rp sI、rplM、degS、mreD、mreC、mreB、accB、accC、yrdC、def、fmt、rplQ、rpoA、rp sD、rpsK、rpsM、secY、rplO、rpmD、rpsE、rplR、rplF、rpsH、rpsN、rplE、rplX、rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, rplW, rplD, rplC, 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, ub, One or more of the following may be selected from the list consisting of iD, hemG, yihA, ftsN, murI, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, and dnaC.

[0109] It is also a TCG certificate / TCA certificate 1. 1. 2. 1. 1. 1. 1. 1. 1. 1. 2. 1. 1. 1. One of the 1 and 2 of these are ribF, lspA, ispH, dapB, folA, imp, yabQ, lpxC, and secM. secA, can, folK, hemL, yadR, dapD, map, rpsB, tsf, pyrH, frr, dxr, ispU, cd sA, 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, glnS, fldA, cydA, infA, cyd C, ftsK, lolA, serS, rpsA, msbA, lpxK, kdsB, mukF, mukE, mukB, asnS, fabA. mviN, rne, fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, minE, minD. pth、prsA、ispE、lolB、hemA、prfA、prmC、kdsA、topA、ribA、fabI、tyrS、rib C, ydiL, pheT, pheS, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA. yefM、metG、folE、yejM、gyrA、nrdA、nrdB、folC、accD、fabB、gltX、ligA、zi pA、dapE、dapA、der、hisS、ispG、suhB、tadA、acpS、era、rnc、lepB、rpoE、pss A, yfiO, rplS, trmD, rpsP, ffh, grpE, csrA, ispF, ispD, ftsB, eno, pyrG, ch pR, lgt, fbaA, pgk, yqgD, metK, yqgF, plsC, ygiT, parE, ribB, cca, ygjD, tdc F、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, rp lD, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, yhhQ, bcsB, glyQ, gpsA, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spo Selected from the list consisting of T, 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, lexA, dnaB, ssb, alsK, groS, psd, orn, yjeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, and dnaC. Preferably, the polynucleotide contains two or three or more, three or four or more, four or five or more, five or six or more, ten or eleven or more, twenty or 21 or more, thirty or 31 or more, forty or 41 or more, fifty or 51 or more, one hundred or 101 or more, or two hundred or 201 or more essential genes that lack TCG codons and / or TCA codons.

[0110] In some embodiments, the polynucleotide is relative to SEQ ID NO: 1 or SEQ ID NO: 2 It comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, or 99.9%, or 100% identical to a fragment of either SEQ ID NO: 1 or SEQ ID NO: 2, and preferably the fragment is at least 10kb, 20kb, 50kb, 100kb, or 500kb in length.

[0111] Preferably, the polynucleotides are viable. That is, polynucleotides can be incorporated into a genome such that the genome is a viable genome. Preferably, the polynucleotides can replace corresponding regions of the parent genome and maintain the viability of the genome. As used herein, “viable genome” means a genome containing sufficient nucleic acid sequences to cause and / or maintain the viability of a cell, for example, a genome encoding molecules required for replication, transcription, translation, energy production, transport, production of membrane and cytoplasmic components, and cell division. Therefore, the present invention also provides a viable synthetic prokaryotic genome (e.g., a viable synthetic E. coli genome) comprising the polynucleotides of the present invention.

[0112] The present invention provides a polynucleotide that is at least 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 2, or to a fragment of either SEQ ID NO: 1 or SEQ ID NO: 2, preferably the fragment having a length of at least 10kb, 20kb, 50kb, 100kb, or 500kb.

[0113] Host cells and their use host cell The present invention also provides a host cell comprising the synthetic prokaryotic genome or polynucleotide of the present invention. The host cell may be an isolated host cell.

[0114] The host cell of the present invention is a prokaryotic cell. More preferably, the host cell is a bacterial cell. Preferably, a bacterial host cell is suitable for heterologous protein production, in particular for the production of polypeptides containing one or more non-proteinogenic amino acids (e.g., those described in Ferrer-Miralles, N. and Villaverde, A., 2013. Microbial Cell Factories, 12:113). Suitable bacterial host cells include Escherichia (e.g., E. coli), Caulobacteria (e.g., Caulobacter crescentus), photosynthetic bacteria (e.g., Rhodobacter spheroides), cold-adapted bacteria (e.g., Pseudoalteromonas haloplanchtis, Shewanella strain Ac10), Pseudomonas (e.g., Pseudomonas fluorescein, Pseudomonas putida, Pseudomonas erginosa), halophilic bacteria (e.g., Halomonas elongata, Chromohalobacter salexigens), Streptomisetes (e.g., Streptomyces lividans, Streptomyces grizeus), and Nocardia. Examples include bacteria (e.g., Nocardia lactamjurans), mycobacteria (e.g., Mycobacterium smegmatis), Corynebacteria (e.g., Corynebacterium glutamicum, Corynebacterium ammoniagenes, Brevibacterium lactofermentum), Bacillus (e.g., Bacillus satiris, Bacillus brevis, Bacillus megatherium, Bacillus licheniformis, Bacillus amyloricephaciens), and lactic acid bacteria (e.g., Lactococcus lactis, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus gasseri). In some embodiments, the bacterial host cell is a Gram-negative bacterium.

[0115] Preferably, the host cell is Escherichia coli, Salmonella enterica, or Shigella. More preferably, the host cell is Escherichia coli. Suitable Escherichia coli host cells include MDS42, K-12, MG1655, BL21, BL21(DE3), AD494, Origami, HMS174, BLR(DE3), HMS174(DE3), Tuner(DE3), Origami2(DE3), Rosetta2(DE3), and Lemo21(DE3). Examples include NiCo21(DE3), T7 Express, SHuffle Express, C41(DE3), C43(DE3), and m15 pREP4 or their derivatives (Rosano, GL and Ceccarelli, EA, 2014. Frontiers in microbiology, 5). (p.172). Most preferably, the host cell is MDS42, MG1655, or BL21 or a derivative thereof. MG1655 is considered the wild-type strain of E. coli. The Genbank ID for the genome sequence of this strain is U00096. BL21 is widely commercially available. For example, it can be purchased from New England BioLabs under catalog number C2530H. It is possible.

[0116] The host cell may preferably be the same as the one from which the synthetic prokaryotic genome or polynucleotide was present (or from which it originated). For example, if the synthetic prokaryotic genome is a synthetic E. coli genome, the host cell is preferably E. coli. If the parent genome of the cell has been modified to produce the synthetic prokaryotic genome of the present invention, the host cell is preferably the same cell; that is, the host cell containing the synthetic prokaryotic genome is preferably the same as the host cell of the parent genome (parent host cell).

[0117] The host cells may be viable, that is, they may be able to proliferate and replicate.

[0118] When the cell genome is modified to produce the synthetic prokaryotic genome of the present invention, the synthetic prokaryotic genome, when present in the parental host cell, preferably does not substantially reduce the growth rate. Therefore, preferably, host cells containing the synthetic prokaryotic genome do not substantially reduce the growth rate compared to host cells containing the parental genome. In some embodiments, host cells containing the synthetic prokaryotic genome have a doubling time that is 4 times, 3 times, 2 times, or less than about 1.6 times slower than host cells containing the parental genome. The doubling time can be determined by any method known to those skilled in the art. In some embodiments, the doubling time is determined in LB medium at 37°C, 25°C, or 42°C.

[0119] When the cell genome is modified to produce the synthetic prokaryotic genome of the present invention, the synthetic prokaryotic genome, preferably, does not cause any substantial phenotypic changes when present in the parental host cell. Therefore, preferably, a host cell containing the synthetic prokaryotic genome has no substantial phenotypic changes compared to a host cell containing the parental genome. In some embodiments, a host cell containing the synthetic prokaryotic genome has an average cell length that is 100%, 50%, or less than about 20% longer than a host cell containing the parental genome. For example, the cell length may be about 1.5 to 3 microns. The cell length can be determined by any method known to those skilled in the art. In some embodiments, a host cell containing the synthetic prokaryotic genome has a proteome that is substantially the same as the proteome of a host cell containing the parental genome. The proteome can be determined by any method known to those skilled in the art.

[0120] Reallocation to alternative canonical amino acids In some embodiments, one or more sense codons (i.e., those removed from the parent genome) are reassigned to encode alternative canonical amino acids. For example, if TCG and TCA are removed, one or both may be reassigned to encode a canonical amino acid other than serine (e.g., alanine).

[0121] For example, the synthetic prokaryotic genome of the present invention substantially or completely lacks one or more sense codons. Therefore, one or more tRNAs or termination factors may be deleted from the synthetic genome. For example, the tRNA that decodes one or more sense codons that have been substituted (or deleted) may be deleted from the synthetic prokaryotic genome. The tRNA that decodes one or more sense codons that have been substituted (or deleted) may be deleted, and if the tRNA decodes only one or more of the substituted (or deleted) sense codons or, alternatively, if the tRNA decodes one or more of the substituted (or deleted) sense codons and one or more of the non-substituted (or deleted) sense codons, and the tRNA is non-essential for one or more of the non-substituted (or deleted) sense codons (i.e., one or the remaining sense codons decoded by the tRNA are decoded by one or more alternative tRNAs), the synthetic prokaryotic genome remains viable. For example, if the synthetic prokaryotic genome lacks the TCA sense codon, the serT that encodes the tRNA Ser UGA may be deleted, and / or if the synthetic prokaryotic genome lacks the TCG sense codon, the serU that encodes the tRNA Ser CGA may be deleted. The deletion of one or more tRNAs can be used, for example, in combination with re-assigned endogenous tRNAs or orthogonal aminoacyl-tRNA synthetase / tRNA pairs to reassign one or more sense codons to alternative amino acids.

[0122] For example, if TCG and TCA have been removed from the synthetic prokaryotic genome, the serT that encodes the tRNA Ser UGA and the serU that encodes the tRNA Ser CGA may be deleted from the synthetic prokaryotic genome, and any of the tRNAs CGA can be (e.g., tRNA Ala CGAIt can be reassigned to orthogonal aminoacyl-tRNA synthetase / tRNA CGA The pair can be introduced into a host cell (for example, by heterologous nucleic acid or by incorporation into a synthetic prokaryotic genome) to reassign the TCG to an alternative canonical amino acid. Therefore, in some embodiments of the present invention, the host cell of the present invention receives one or more reassigned tRNAs and / or one or more heterologous nucleotides encoding one orthogonal aminoacyl-tRNA synthetase (aaRS, aminoacyl-tRNA synthetase)-tRNA pair. The present invention further includes a gene (e.g., a plasmid). In some embodiments, the host cell of the present invention further includes a plasmid encoding an orthogonal aminoacyl-tRNA synthetase (aaRS)-tRNA pair. Alternatively, the orthogonal aminoacyl-tRNA synthetase (aaRS)-tRNA pair can be introduced into the host cell by incorporation into a synthetic prokaryotic genome. Therefore, in some embodiments, the synthetic prokaryotic genome encodes an orthogonal aminoacyl-tRNA synthetase (aaRS)-tRNA pair, and preferably the gene encoding the native tRNA has been removed from the parent prokaryotic genome. In preferred embodiments, the host cell of the present invention further includes one or more reassigned tRNAs. Methods for reassigning tRNAs are well known to those skilled in the art.

[0123] Reallocation to encode alternative canonical amino acids can increase biosafety. Therefore, in some embodiments, the host cells of the present invention exhibit increased biosafety. Thus, the present invention provides host cells with improved biosafety.

[0124] For example, reassignment to encode alternative canonical amino acids can make host cells containing synthetic prokaryotic genomes resistant to bacteriophage infection. Since one or more bacteriophage genes typically contain one or more sense codons, if one or more bacteriophage genes are translated, alternative canonical amino acids can be incorporated into the corresponding bacteriophage proteins. The incorporation of alternative canonical amino acids can destabilize, disrupt, or reduce the activity of the proteins, thereby reducing the infectivity of bacteriophages and making host cells resistant to bacteriophage infection.

[0125] Therefore, in some embodiments, the host cells of the present invention are resistant to phage infection. For example, if the cell genome is modified to produce the synthetic prokaryotic genome of the present invention, the synthetic prokaryotic genome, when present in the parental host cell, may increase resistance to phage infection. Therefore, in some embodiments, the host containing the synthetic prokaryotic genome The cells exhibit increased phage resistance compared to host cells containing the parent genome.

[0126] Therefore, the present invention provides phage-resistant host cells and host cells in which phage resistance is increased.

[0127] Furthermore, reallocation to encode alternative canonical amino acids makes it possible to design genetic material, such as antibiotic resistance genes, so that they function in the reprogrammed strain rather than the wild-type strain. For example, genetic material can be incorporated into the host cells of the present invention (e.g., by heterologous nucleic acids or by incorporation into a synthetic prokaryotic genome) so that the host cells proliferate under certain conditions (e.g., in the presence of antibiotics) but other host cells (e.g., parental host cells) do not. Therefore, in some embodiments, the host cells of the present invention can make compositions containing the host cells more resistant to contamination by other host cells (e.g., other prokaryotes).

[0128] Reassignment to non-protein amino acids In some embodiments, one or more sense codons (i.e., those removed from the parent genome) are reassigned to encode non-canonical amino acids (non-proteinogenic amino acids).

[0129] Therefore, the present invention provides the use of host cells according to the present invention for producing polypeptides comprising one or more non-proteinogenic amino acids, preferably two or more non-proteinogenic amino acids, most preferably three or more non-proteinogenic amino acids.

[0130] The present invention also provides polypeptides obtained or obtainable by using host cells according to the present invention. In some embodiments, the polypeptide comprises one or more non-proteinogenic amino acids, preferably two or more non-proteinogenic amino acids, most preferably three or more non-proteinogenic amino acids. Therefore, the present invention also provides polypeptides comprising two or more non-proteinogenic amino acids and polypeptides comprising three or more non-proteinogenic amino acids.

[0131] As used herein, “non-proteinogenic amino acids” (also known as “non-coding amino acids” or “non-canonical amino acids”) are amino acids that are not naturally coded or found in the genetic code. Despite the use of only 22 amino acids by the translational mechanism for building proteins (proteinogenic amino acids, i.e., the 20 in the standard genetic code plus two additional that can be incorporated by special translational mechanisms), more than 140 amino acids are known to exist naturally in proteins, and thousands more may exist naturally or be synthesized in the laboratory. Therefore, non-proteinogenic amino acids may include L-alanine, L-cysteine, L-aspartic acid, L-glutamic acid, L-phenylalanine, glycine, L-histidine, L-isoleucine, L-lysine, L-leucine, L-methionine, L-asparagine, L-proline, L-glutamine, L-arginine, L-serine, L-threonine, L-valine, L-tryptophan, and L-tyrosine, as well as any amino acids optionally excluding L-pyrrolidine and L-selenocysteine.

[0132] In some embodiments, the non-proteinogenic amino acids are unnatural amino acids (UAAs).

[0133] Non-protein amino acids or UAAs are not particularly limited. Suitable non-protein amino acids and UAAs are well known to those skilled in the art, for example, as disclosed in Neumann, H., 2012. FEBS letters, 586(15), pp.2057-2064; and Liu, CC and Schultz, PG, 2010. Annual review of biochemistry, 79, pp.413-444. In some embodiments, non-protein... Particle amino acids and / or UAAs include p-acetylphenylalanine, m-acetylphenylalanine, O-allyl tyrosine, phenylselenocysteine, p-propargyloxyphenylalanine, p-azidophenylalanine, p-boronophenylalanine, O-methyltyrosine, p-aminophenylalanine, p-cyanophenylalanine, m-cyanophenylalanine, p-fluorophenylalanine, p-iodophenylalanine, p-bromophenylalanine, p-nitrophenylalanine, L-DOPA, 3-aminotyrosine, 3-iodotyrosine, p-isopropylphenylalanine, 3-(2-naphthyl)alanine, biphenylalanine, homoglutamic acid One or more of the following are selected: amine, D-tyrosine, p-hydroxyphenyllactic acid, 2-aminocaprylic acid, bipyridylalanine, HQ-alanine, p-benzoylphenylalanine, o-nitrobenzylcysteine, o-nitrobenzylserine, 4,5-dimethoxy-2-nitrobenzylserine, o-nitrobenzyllysine, o-nitrobenzyltyrosine, 2-nitrophenylalanine, dansylalanine, p-carboxymethylphenylalanine, 3-nitrotyrosine, sulfotyrosine, acetyllysine, methylhistidine, 2-aminononanoic acid, 2-aminodecanoic acid, pyrrolidine, Cbz-lysine, Boc-lysine, and allyloxycarbonyllysine.

[0134] Prokaryotes, such as E. coli, typically cannot incorporate most eukaryotic post-translational modifications, including ubiquitination, glycosylation, and phosphorylation. Furthermore, they typically cannot undergo other eukaryotic maturation processes, including proteolytic protein maturation. Additionally, precise disulfide bond formation and lipopolysaccharide contamination can be problematic (see Ovaa, H., 2014. Frontiers in Chemistry, 2, p.15). However, anti Therapeutic proteins such as proteins, enzymes, and cytokines typically retain post-translational modifications and disulfide bonds, and often require proteolytic maturation to achieve their precisely folded state. Therefore, the vast majority of therapeutic proteins are produced in eukaryotic and mammalian cell lines. However, expression in prokaryotic host cells, such as E. coli, is generally inexpensive, readily modifiable, offers broad applications in mutation library development, and is suitable for industrial-scale fermentation (Ovaa, H., 2014. Frontiers in Chemistry, 2, p.15). ).

[0135] Therefore, in some embodiments, the polypeptide is a therapeutic polypeptide, preferably in which a mammalian protein modification is introduced by one or more non-proteinogenic amino acids. For example, Amber codon repression has been previously used to incorporate one or more non-proteinogenic amino acids (i.e., mammalian protein modifications) into a therapeutic polypeptide. The present invention enables the incorporation of two or more non-proteinogenic amino acids. Therefore, the present invention provides a therapeutic polypeptide comprising two or more non-proteinogenic amino acids.

[0136] Since the synthetic prokaryotic genome of the present invention substantially or completely lacks one or more sense codons, one or more tRNAs or termination factors may be deleted from the synthetic genome. For example, tRNAs that decode only the one or more substituted (or deleted) sense codons may be deleted from the synthetic prokaryotic genome. For example, if the synthetic prokaryotic genome lacks the TCA sense codon, tRNAs may be deleted. Ser UGA The setT encoding may be deleted, and / or if the synthetic prokaryotic genome lacks the TCG sense codon, tRNA Ser CGA The serU encoding may be deleted. The synthetic prokaryotic genome may then be used (in conjunction with orthogonal aminoacyl-tRNA synthetase-tRNA pairs) to guide the incorporation of non-proteinogenic amino acids into proteins.

[0137] Genetic coding extensions respond to unassigned codons (e.g., amber stop codons, UAGs) introduced at desired sites in the desired gene, resulting in non-proteinoid ami. Orthogonal aminoacyl-tRNA synthetase (aaRS)-tRNA pairs are used to guide the uptake of amino acids into proteins. Orthogonal synthetases do not recognize endogenous tRNAs and specifically aminoacylate orthogonal-cognitive tRNAs (which are not effective substrates for endogenous synthetases) with non-proteinogenic amino acids provided to (or synthesized by) cells (Chin, JW, 2017. Nature, 550(7674), 53-60). Those skilled in the art can identify and / or generate suitable orthogonal aminoacyl-tRNA synthetase (aaRS)-tRNA pairs (e.g., Elliott, TS et al., 2014. Nat Biotechnol 32, 465-472; Elliott, TS, et al., 2016. Cell Chem Biol 23, 805-815; and Krogager, TP et al., 2018. Nat Biotechnol 36, 156-159). Therefore, in some embodiments, the host cell of the present invention further comprises one or more heterologous nucleotides (e.g., plasmids) encoding a single orthogonal aminoacyl-tRNA synthetase (aaRS)-tRNA pair. In preferred embodiments, the host cell of the present invention further comprises a plasmid encoding an orthogonal aminoacyl-tRNA synthetase (aaRS)-tRNA pair. Alternatively, the orthogonal aminoacyl-tRNA synthetase (aaRS)-tRNA pair can be introduced into the host cell by incorporating it into a synthetic prokaryotic genome. Therefore, in some embodiments, the synthetic prokaryotic genome encodes an orthogonal aminoacyl-tRNA synthetase (aaRS)-tRNA pair, and preferably, the gene encoding the native tRNA has been removed from the parent prokaryotic genome.

[0138] Therefore, in some embodiments, the host cell of the present invention further comprises one or more heterologous nucleotides (e.g., plasmids) comprising one or more genes comprising the sense codon. In preferred embodiments, the host cell further comprises a plasmid comprising the gene comprising the sense codon. One or more sense codons may be located at a desired site on the gene, preferably at the desired site, which allows for the incorporation of one or more non-proteinogenic amino acids (i.e., mammalian protein modifications) into a polypeptide, preferably a therapeutic polypeptide.

[0139] In other embodiments, the sense codon may be present in one or more genes in a synthetic prokaryotic genome (for example, heterologous nucleotides may be incorporated into a synthetic prokaryotic genome). The sense codon may be present in a desired location in the gene, preferably the desired location, which allows for the incorporation of one or more non-proteinogenic amino acids (i.e., mammalian protein modifications) into a polypeptide, preferably a therapeutic polypeptide.

[0140] For example, if TCG and TCA are removed from the synthetic prokaryotic genome, tRNA Ser UGA serT and tRNA that encode serT and tRNA Ser CGA The serU encoding orthogonal aminoacyl-tRNA synthetase / tRNA may be deleted from the synthetic prokaryotic genome. CGA The pair may be used in combination with a (heterogeneous) gene containing a TCG codon so that it encodes a polypeptide containing one or more nonproteinogenic amino acids. Therefore, the host cell of the present invention may, for example, (i) orthogonal aminoacyl-tRNA synthetase / tRNA CGA (ii) a plasmid encoding a pair; and (ii) a plasmid further comprising a gene containing one or more TCG codons. Similarly, if AGT and AGC are removed, tRNA Ser GCUThe serV encoding may be deleted from the synthetic prokaryotic genome, orthogonal aminoacyl-tRNA synthetase / tRNA ACU Paired and / or orthogonal aminoacyl-tRNA synthetase / tRNA GCU Pairs may be used. Similarly, if CTG and CTA are removed, tRNA Leu CAG leuP, Q, T, V, and tRNA, which encode these molecules. Leu UAG The leuW encoding may be deleted from the synthetic prokaryotic genome, orthogonal aminoacyl-tRNA synthetase / tRNA CAG Pairs may be used. Similarly, if TTG and TTA are removed, tRNA Leu CAA leuX, which codes for tRNA, and tRNA Leu UAA The leuZ encoding may be deleted from the synthetic prokaryotic genome, orthogonal aminoacyl-tRNA synthetase / tRNA CAA Pairs and / or orthogonal pairs Minoacyl-tRNA synthetase / tRNA UAA Pairs may be used. Similarly, if GCG and GCA are removed, tRNA Ala UGC The alaT, U, and V encoding orthogonal aminoacyl-tRNA synthetase / tRNA may be deleted from the synthetic prokaryotic genome. CGC They may be used in pairs.

[0141] In some embodiments, the synthetic prokaryotic genome lacks a gene encoding a termination factor (e.g., RF1), and / or the host cell lacks a termination factor (e.g., RF1) to increase the efficiency of non-proteinogenic amino acid incorporation.

[0142] Methods for producing synthetic genomes In one aspect, the present invention is a method for producing a synthetic genome, (a) Steps to prepare the parent genome, (b) A step of performing one or more rounds of genetic modification via recombination on the parent genome to produce two or more different partial synthetic genomes, (c) A step of producing a synthetic genome by performing one or more rounds of induction conjugation with two or more different partial synthetic genomes and This provides a method that includes this.

[0143] Genetic modification via recombination Preferably, one or more rounds of recombination-mediated gene modification are used to edit 10–1000kb, 50–1000kb, 100–1000kb, or 100–500kb of the parental genome to provide two or three or more different partial synthetic genomes. Therefore, in a preferred embodiment, each round of recombination-mediated gene modification inserts or replaces 10kb or more, 50kb or more, 100kb or more, or about 100kb of the DNA of the parental genome.

[0144] As used herein, the term “recombination-mediated genetic modification” (also known as “recombination”) refers to methods for genetic modification (i.e., genome editing) based on homologous recombination systems. Typically, recombination is based on homologous recombination in Escherichia coli mediated by bacteriophage proteins, specifically RecE / RecT from Rac prophage or Red αβδ from bacteriophage lambda. Any suitable method of recombination-mediated genetic modification may be used. Methods for recombination-mediated genetic modification are well known to those skilled in the art.

[0145] In "classical recombination" (exemplified by lambda red-mediated recombination in E. coli), short regions of synthetic DNA can be inserted into the genome, or a two-step process can be used: i) transformation of the cell with linear double-stranded DNA (dsDNA) that maintains the stretch of synthetic DNA, is linked to a positive selection marker, and has homologous regions (HRs) adjacent to each end of the target region of the genome; and (ii) replacement of genomic DNA in homologous region-mediated recombination, followed by selection for genomic integration by a positive selection marker. This approach can be used to insert or replace 2-3 kb of genomic DNA. Therefore, when classical recombination is used, many rounds of recombination-mediated gene modification are required to edit 100-500 kb of the parent genome.

[0146] Therefore, in a preferred embodiment, one or more rounds of gene modification via recombination include one or more rounds of replicon excision (REXER) for enhanced genome modification by programmed recombination.

[0147] REXER is described in International Publication No. 2018 / 020248 (incorporated herein by reference). Each round of REXER is based on the DNA of the parent genome. It can be used to insert or replace approximately 50kb to 250kb, or approximately 100kb.

[0148] Therefore, one or more rounds of genetic modification via recombination are i) A step of preparing a host cell (e.g., E. coli), wherein the host cell comprises an episome replicon (e.g., a plasmid or bacterial artificial chromosome) and a target nucleic acid (e.g., a genome), the episome replicon comprising a donor nucleic acid sequence (i.e., a synthetic region), the donor nucleic acid sequence comprising, in order, 5'-homologous recombination sequence 1-desired sequence-homologous recombination sequences 2-3', the desired sequence comprising a positive selectable marker, and the target nucleic acid comprising, in order, 5'-homologous recombination sequence 1-negative selectable marker-homologous recombination sequences 2-3', ii) A step of preparing a helper protein (e.g., lambda red protein) that can support nucleic acid recombination in the host cell, iii) Helper proteins that can support nucleic acid excision in the host cells and / or a step to prepare RNA (e.g., CRISPR / Cas9 protein / RNA), iv) A step of inducing the excision of the donor nucleic acid sequence, v) Incubating to enable recombination between the excised donor nucleic acid and the target nucleic acid, and vi) Step of selecting a recombinant organism in which the donor nucleic acid has been incorporated into the target nucleic acid. It may include.

[0149] Preferably, the step of selecting a recombinant in which the donor nucleic acid has been incorporated into the target nucleic acid includes the selection of a positive selectable marker for the donor nucleic acid and the selection of a negative selectable marker for the target nucleic acid. Preferably, the selection of a positive selectable marker for the donor nucleic acid and the selection of a negative selectable marker for the target nucleic acid are performed simultaneously. Preferably, the desired sequence includes both a positive selectable marker and a negative selectable marker. Preferably, the negative selectable marker is sacB (sucrose sensitive), rpsL (S12 ribosomal protein-streptomycin sensitive), or phe ST251A_A294G The group is selected from those (sensitive to 4-chlorophenylalanine). Appropriately, the positive selectable marker is Cm R (Chloramphenicol resistance), Kan R (Kanamycin resistance), Hyg R (Hygromycin resistance), Gentamicin R (Resistant to gentamicin), or tetracycline R Selected from the group consisting of (tetracycline resistance). Preferably, the step of selecting a recombinant includes sequential selection of the positive and negative markers, or sequential selection of the negative and positive markers. Preferably, the step of selecting a recombinant includes simultaneous selection of the positive and negative markers.

[0150] Preferably, the method described above further includes the step of inducing at least one double-strand break in a target nucleic acid sequence, wherein the double-strand break is between homologous recombination sequence 1 and homologous recombination sequence 2. Preferably, at least two double-strand breaks are induced in the target nucleic acid sequence, each of which double-strand breaks is between homologous recombination sequence 1 and homologous recombination sequence 2.

[0151] Ideally, the excised donor nucleic acid begins with homologous recombination sequence 1 and ends with homologous recombination sequence 2.

[0152] Preferably, the episome replicon includes a negative selectable marker independent of the donor nucleic acid sequence. Preferably, the method includes a further step of selecting the loss of the episome replicon by selecting the loss of the negative selectable marker independent of the donor nucleic acid sequence. Preferably, the episome replicon includes, in order, excision site 1 - donor nucleic acid sequence - excision site 2. Preferably, the target nucleic acid functions within the host cell. It possesses its own unique origin of replication. Appropriately, the episome replicon is a plasmid nucleic acid. Appropriately, the episome replicon is a bacterial artificial chromosome (BAC). Appropriately, the target nucleic acid is the host cell genome. ru.

[0153] Episome replicons (e.g., BACs) are homologous in S. cerevisiae, as described, for example, in Kouprina, N., et al., 2004. Methods Mol Biol 255, 69-89. The assembly can be assembled by substitution. The assembly can combine 7 to 14 stretches of synthetic DNA, each 6 to 13 kb in length; a selection construct (containing a negative selection marker and / or a positive selection marker); and a BAC shuttle vector backbone. The stretches of synthetic DNA may correspond holistically to the donor nucleic acid sequence (i.e., the synthetic region) in the episome replicon, with each stretch containing 80 to 200 bp of mutually overlapping DNA sequences, where the overlapping region contains no rewritten targets. The stretches can be fed into a pSC101 or pST vector adjacent to an appropriate restriction site (e.g., BsaI, AvrII, SpeI, or XbaI). Therefore, during assembly, the synthetic DNA stretches can be excised by digestion with the corresponding restriction enzymes. The assembly of the episome replicon can be validated by sequencing.

[0154] Appropriately, the two homologous regions may be 30–100 bp, 40–50 bp, or approximately 50 bp in length.

[0155] A CRISPR / Cas9 mechanism may be used for excision. In some embodiments, the CRISPR / Cas9 mechanism comprises Cas9, tracrRNA, and two spacer RNAs, the spacer RNAs targeting two homologous regions for excision. In preferred embodiments, the spacer RNAs are linear double-stranded spacers. In other embodiments, the CRISPR / Cas9 mechanism comprises Cas9 and two sgRNAs, the sgRNAs targeting two homologous regions for excision.

[0156] A lambda-red recombination mechanism may be used for recombination. The lambda-red recombination mechanism may include lambda-alpha / beta / gamma.

[0157] The method may include the steps of performing one or more rounds of REXER, i.e., the above steps using a first donor nucleic acid sequence, selecting a further donor sequence consecutive to the first donor nucleic acid sequence, and repeating the above steps using the further donor nucleic acid sequences until a partially synthetic genome is assembled. This is known as genome stepwise exchange synthesis (GENESIS), described by Wang, K. et al., 2016. Nature 539, 59-64, and is schematically shown in Figure 4.

[0158] In a preferred embodiment, the donor sequence corresponds to a region of the synthetic genome according to the present invention and / or a polynucleotide according to the present invention.

[0159] Therefore, the donor sequence (i.e., the synthetic region) may contain 20 or 19 occurrences of one or more sense codons, and / or the donor sequence may contain 10 or 11 or more, 20 or 21 or more, or 100 or 101 or more genes that do not contain one or more sense codons.

[0160] The donor sequences (i.e., synthetic regions) must have 50 or fewer, 49 or fewer, 20 or fewer, 19 or fewer, 10 or fewer, 9 or fewer, 5 or fewer, or 0 occurrences of each of one or more sense codons, and / or have less than 10%, 5%, 2%, 1%, 0.5%, or 0.1% of the corresponding region in the parent genome. Alternatively, the sequence may be identical to that of the parent genome (i.e., the non-synthetic region), except that it includes the appearance of each of two or more sense codons, and / or includes 10 or 11 or more, 20 or 21 or more, or 100 or 101 or more genes that do not include the appearance of one or more sense codons.

[0161] The donor sequence (i.e., the synthetic region) may also be refactored relative to the parent genome sequence (i.e., the non-synthetic region). With respect to 3',3' duplication (i.e., reversed gene pairs), the synthetic insertion may be inserted between the genes. With respect to 3',3' duplication, the synthetic insertion may include the overlapping region. With respect to 5',3' duplication (i.e., same-oriented gene pairs), the synthetic insertion may be inserted between the genes. With respect to 5',3' duplication, the synthetic insertion may include (i) a stop codon; (ii) approximately 20–200 bp, or 20–100 bp, or 20–50 bp upstream of the overlapping region; and (iii) the overlapping region. Preferably, the synthetic insertion includes (i) a stop codon; (ii) about 20 bp upstream of the overlapping region; and (iii) the overlapping region. In a preferred embodiment, the stop codon is downstream of the gene. The child is in frame with the original starting point. Preferably, the stop codon is TAA.

[0162] Preferably, the donor sequences (i.e., the synthetic regions) are 50–10000kb, 100–5000kb, 100–2000kb, 100–1000kb, or 100–500kb in total size. Preferably, each donor sequence is 50–300kb, 100–200kb, or about 100kb in size.

[0163] Therefore, donor sequences may be approximately 100kb in size each, may be identical to the corresponding sequences in the parent genome, except that they do not contain the appearance of one or more sense codons, and all gene pairs that share overlapping regions containing one or more sense codons in the parent genome are refactored, and the sense codon substitution alters the encoded protein sequence of both or one of the gene pairs.

[0164] In preferred embodiments, genome viability is tested after each round of genetic modification via recombination. In some embodiments, genome sequencing is verified after each round of genetic modification via recombination.

[0165] Partially synthetic genome The present invention provides two or more different partial synthetic genomes.

[0166] As used herein, “partially synthetic genome” is a genome in which one or more contiguous regions of the parent genome have been edited (i.e., the partially synthetic genome includes one or more synthetic regions), and the one or more contiguous (synthetic) regions do not constitute the entire parent genome. Preferably, the partially synthetic genome of the present invention has one contiguous (synthetic) region. In contrast, “synthetic genome” may include genome editing that constitutes substantially the entire parent genome.

[0167] The partially synthetic genome of the present invention may be a prokaryotic genome. Preferably, the partially synthetic genome of the present invention is a bacterial genome. More preferably, the partially synthetic genome of the present invention is an Escherichia coli, Salmonella enterica, or Shigella genome. Most preferably, the partially synthetic genome of the present invention is an Escherichia coli genome. In some embodiments, the partially synthetic genome is small or minimal. In preferred embodiments, the partially synthetic genome is a viable genome.

[0168] In some embodiments, the partially synthetic genome of the present invention is 100kb to 20Mb, or 130kb to 15Mb, or 200kb to 15Mb, or 300kb to 15Mb, or 500kb to 15Mb, or 1Mb to 15Mb, or 1Mb to 10Mb, or 1Mb to 8Mb, or 1Mb to 6Mb, or 2Mb to 6Mb, or 2Mb to 5Mb, or 3Mb to 5Mb, or about 4Mb in size.

[0169] A partially synthetic genome may include synthetic regions having 50 or 49, 20 or 19, 10 or 9, 5 or 4, or 0 occurrences of each of one or more sense codons, or a partially synthetic genome may include synthetic regions having 10%, 5%, 2%, 1%, 0.5%, or 0.1% occurrences of each of one or more sense codons compared to the corresponding region in the parent genome.

[0170] Preferably, the synthesis region is 50-10000kb, 100-5000kb, or 100-500kb in size.

[0171] Therefore, a partial synthetic genome may include one or more contiguous regions of 100-5000kb having occurrences of 10 or 9 or fewer, 5 or 4 or fewer, or 0 of each of one or more sense codons, and / or a partial synthetic genome may include one or more contiguous regions of 100-5000kb having occurrences of one or more sense codons in amounts of less than 10%, 5%, 2%, 1%, 0.5%, or 0.1% compared to the corresponding region in the parent genome, and / or a partial synthetic genome may include one or more contiguous regions of 100-5000kb having 10 or 11 or more, 20 or 21 or more, or 100 or 101 or more genes that do not have occurrences of one or more sense codons.

[0172] The remainder of the partially synthetic genome (i.e., the non-synthetic region) may contain unaltered sense codons. Therefore, the partially synthetic genome may include one or more non-synthetic regions having 100% or 99% of the occurrence of each sense codon compared to the corresponding region in the parent genome, and / or the partially synthetic genome may include one or more non-synthetic regions having 100 or 101 or more genes, each having the occurrence of each sense codon. The non-synthetic region may be 500kb to 20Mb, or 500kb to 10Mb, or 500kb to 5Mb, or about 3.5Mb in size.

[0173] For example, a partially synthetic genome may include one contiguous region of 100-5000kb (i.e., a synthetic region) having 10 or 11 or more, 20 or 21 or more, or 100 or 101 or more genes that do not contain one or more sense codons, and one contiguous region of 500kb-10000kb having 100 or 101 or more genes that contain each sense codon (i.e., a non-synthetic region).

[0174] Two or more different partial synthetic genomes may originate from the same parent genome, that is, they may contain substantially the same sequences, for example, two or more different partial synthetic genomes may share 90%, 95%, 99%, or 99.5% sequence identity.

[0175] Two or more different partial synthetic genomes may contain one or more synthetic regions such that the synthetic regions collectively occupy 90% or more, 95% or more, 99% or more, or 100% of the parent genome. Preferably, each of the two or more different partial synthetic genomes contains one or more synthetic regions, and these synthetic regions do not substantially overlap (for example, the overlap between synthetic regions is 10kb or less, preferably about 3-4kb). Therefore, each of the two or more different partial synthetic genomes contains one It may include a specific or substantially specific synthetic region.

[0176] Therefore, in a preferred embodiment, each of two or more different partial synthetic genomes includes one contiguous synthetic region of 100-5000kb having 10 or 11 or more, 20 or 21 or more, or 100 or 101 or more genes without the appearance of one or more sense codons, and one contiguous non-synthetic region of 500kb-10000kb having 100 or 101 or more genes with the appearance of each sense codon, wherein the synthetic regions collectively constitute substantially the entire parent genome, and the synthetic regions do not substantially overlap.

[0177] Two or more different partial synthetic genomes may be suitable for inducible conjugation. Therefore, in a preferred embodiment, the two or more different partial synthetic genomes comprise at least one partial synthetic donor genome and at least one partial synthetic recipient genome. The method of the present invention may further include one or more rounds of genetic modification via recombination, preferably via lambda red (prior to inducible conjugation), to provide at least one partial synthetic donor genome and at least one partial synthetic recipient genome. The method may further include one or more rounds of selection for at least one partial synthetic donor genome and at least one partial synthetic recipient genome.

[0178] At least one partially synthetic donor genome may include a first selectable marker comprising a synthetic region and two homologous regions immediately downstream of the origin of transmission adjacent to each other, and at least one partially synthetic recipient genome may include a second selectable marker comprising two corresponding homologous regions adjacent to each other, the first selectable marker may include a positive selectable marker, and / or the second selectable marker may include a negative selectable marker.

[0179] Appropriately, negative selectable markers include sacB (sucrose sensitive), rpsL (S12 ribosomal protein-streptomycin sensitive), or phe ST251A_A294G The group is selected from those (sensitive to 4-chlorophenylalanine). Appropriately, the positive selectable marker is Cm R (Chloramphenicol resistance), Kan R (Kanamycin resistance), Hyg R (Hygromycin resistance), Gentamicin R (Resistant to gentamicin), or tetracycline R The markers are selected from the group consisting of (tetracycline resistance). The selectable markers may differ from those in one or more steps of the recombination-mediated gene modification.

[0180] Preferably, the synthetic regions present in at least one partial synthetic recipient genome are outside the regions where homologous regions are adjacent, i.e., the synthetic regions do not substantially overlap. Preferably, the homologous regions are 3 kb to 500 kb in length, most preferably about 3 to 5 kb.

[0181] Inductive conjugation One or more rounds of inductive conjugation may be performed on two or more different partial synthetic genomes of the present invention to produce a synthetic genome.

[0182] Each round of inductive conjugation can be used to provide a partial synthetic genome with larger contiguous synthetic regions. For example, after one or more rounds of genetic modification via recombination, there may be eight partial synthetic genomes each having a contiguous synthetic region of about 500 kb. After the first round of inductive conjugation, two of the partial synthetic genomes may be combined to provide six partial synthetic genomes each having a contiguous synthetic region of about 500 kb and one partial synthetic genome having a contiguous synthetic region of about 1 Mb. The second round can provide five partial synthetic genomes each having a contiguous synthetic region of about 500 kb and one partial synthetic genome having a contiguous synthetic region of about 1.5 Mb; or four partial synthetic genomes each having a contiguous synthetic region of about 500 kb and two partial synthetic genomes each having a contiguous synthetic region of about 1 Mb. After several rounds of inductive conjugation, a complete synthetic genome (i.e., one having a contiguous synthetic region of about 4 Mb) can be provided. Examples are schematically shown in FIGS. 10 and 11b. Any suitable method of inductive conjugation may be used. Methods of inductive conjugation are well known to those skilled in the art, for example, Ma, N.J., Moonan, D.W. and Isaacs, F.J.,

[0183] Preferably, the synthetic regions present in at least one partial synthetic recipient genome are outside the regions where homologous regions are adjacent, i.e., the synthetic regions do not substantially overlap. Preferably, the homologous regions are 3 kb to 500 kb in length, most preferably about 3 to 5 kb. It is described in 2014. Nature Protocols, 9(10), p.2285. The route to the synthetic genome is not limited.

[0184] Therefore, one or more rounds of induced conjugation may involve i) preparing a first host cell containing a partially synthetic recipient genome and a second host cell containing a partially synthetic donor genome and a conjugative plasmid, ii) conjugating the partially synthetic recipient genome and the partially synthetic donor genome, and iii) selecting recombinants in which the synthetic region of the donor genome has been incorporated into the partially synthetic recipient genome step. may include.

[0185] The partially synthetic donor genome may include a first selectable marker in which two homologous regions immediately downstream of the synthetic region and the transfer origin are adjacent, and the partially synthetic recipient genome may include a second selectable marker in which two corresponding homologous regions are adjacent. The first selectable marker may include a positive selectable marker and / or the second selectable marker may include a negative selectable marker. Therefore, step (iii) may include selection of the said selectable able markers, i.e., selection of acquisition of the first selectable marker and loss of the second selectable marker.

[0186] Suitably, the negative selectable marker is selected from the group consisting of sacB (sucrose sensitivity), rpsL (S12 ribosomal protein - streptomycin sensitivity), or phe ST251A_A294G (4-chlorophenylalanine sensitivity). Suitably, the positive selectable marker is Cm R (chloramphenicol resistance), Kan R (kanamycin resistance), Hyg R (hygromycin resistance), gentamicin R (gentamicin resistance), or tetracycline RThe markers are selected from the group consisting of (tetracycline resistance). The selectable markers may differ from those in one or more steps of the recombination-mediated gene modification.

[0187] Preferably, the homologous region is 3kb to 500kb in length, most preferably about 3 to 5kb. Preferably, if the induction conjugation step is the last step of the induction conjugation, the homologous region is 50kb to 500kb.

[0188] Step (ii) may include a step of incubating the first host cells and the second host cells. For example, the first host cells and the second host cells may be mixed, transferred to a suitable medium (e.g., an agar plate), and incubated at approximately 37°C for approximately 1 to 3 hours.

[0189] The conjugate plasmid may be an F plasmid, and preferably the conjugate plasmid does not contain an origin of transfer (e.g., Figure 22c).

[0190] In a preferred embodiment, genome viability is tested after each round of induction conjugation. Advantageously, this allows verification that genome editing (e.g., sense codon substitution) results in a viable genome and enables correction of unapproved edits. In some embodiments, genome sequencing is validated after each round of induction conjugation.

[0191] Those skilled in the art will understand that all features of the present invention disclosed herein can be combined without departing from the scope of the invention, as disclosed herein.

[0192] Preferred features and embodiments of the present invention are described herein as non-limiting examples.

[0193] The implementation of this invention, unless otherwise indicated, utilizes the prior arts of chemistry, biochemistry, molecular biology, microbiology, and immunology, which are within the scope of those skilled in the art. Such arts are described in the literature. For example, Sambrook, J., Fritsch, EF and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory. Press; Ausubel, FM et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, JM and McGee, J.O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, MJ (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, DM and Dahlberg, JE (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. [Examples] [Examples]

[0194] Genome design using synonymous codon compression The inventors first designed a type of *E. coli* MDS42 genome (Uniprot accession number AP012306.1) in which the serine codons TCG and TCA and the stop codon TAG in the open reading frame (ORF) are systematically replaced with their synonyms AGC, AGT, and TAA, respectively (Figure 1a, Figure 18, Sequence ID No. 1). The inventors previously showed that this defined rewriting scheme for synonymous codon compression is possible in a 20kb region of the *E. coli* genome rich in essential genes (Wang, K. et al., 2016. Nature 539, 59-64). However, this region accounts for only 0.46% of the target codons in the genome.

[0195] E. coli contains numerous overlapping open reading frames (ORFs), which the inventors classify into 3',3' (between reversed ORFs) or 5',3' (between same-oriented ORFs). Targeted codons are found within both classes of overlap. If rewriting each ORF within a 3',3' overlap can be achieved without altering the encoded protein sequence of any of the ORFs, i.e., by introducing a synonymous codon, the overlapping structure is maintained and the sequence is directly rewritten. However, if this is not possible, the inventors duplicated the overlapping region and rewrote each ORF individually (Figure 1b, Table 1).

[0196] Regarding 5' and 3' overlaps, the inventors separated the ORFs by duplicating both the overlapping region between ORFs and the 20bp sequence upstream of the overlap. This refactoring allows the inventors to rewrite each ORF independently (Figure 1c, Table 1). The inventors' strategy preserves the RBS sequence for downstream ORFs and the distance between this RBS and its start codon.

[0197] Using defined rules and refactoring techniques for synonymous codon compression, we designed a genome in which all 18,218 target codons are rewritten with their target synonyms (Figure 1d).

[0198]

Table 2

Example

[0199] Synthesis of Rewritten Sections The inventors performed a retrosynthesis similar to that commonly used to design synthetic pathways to small molecules on the designed genome (Figure 2). The inventors cleaved the genome into approximately 0.5 Mb A to H, which are eight sections (Figure 1d, Figure 2a, Figure 18, SEQ ID NO: 1), and then cleaved each section into 4 to 5 fragments (Figure 2b). Thereby, 37 fragments of 91 kb to 136 kb were obtained (Figure 1d, Table 2). The inventors placed boundaries between fragments and between sections in the intergenic regions between non-essential genes. The fragments were further cleaved into 9 to 14 stretches of approximately 10 kb (Figure 2c, Table 2).

[0200] The inventors assembled BACs for REXER containing each fragment by homologous recombination in S. cerevisiae (Figure 2c, Figure 20) (Wang, K. et al., 2016. Nature 539, 59-64; and Kouprina, N., et al., 2004. Methods Mol Biol 255, 69-89). For 36 of the fragments, BAC assembly proceeded smoothly (Table 3). Since fragment 37 was difficult to assemble, the inventors split it into two 50 kb fragments (37a and 37b) and straightened them for assembly (Table 3).

[0201] The inventors initiated genome substitution in seven different strains using REXER. The starting point for REXER in each strain corresponded to the beginning of compartments A, C, D, E, F, G, or H (Figures 1d, 2b, 3), with compartment B later being established in compartment A, as described below. The inventors marked the starting points of genome substitution in each strain by introducing cassettes carrying positive and negative selection markers. The inventors used Cas9 (Jiang, W., et al., 2013. Nat Biotechnol 31, 233-239) and the lambda red recombination mechanism (Datsenko, KA). (& Wanner, BL, 2000. Proc Natl Acad Sci USA 97, 6640-6645), and for each compartment, a BAC containing the fragment initially rewritten to the relevant strain was introduced, encoding the relevant Cas9 spacer (Jiang, W., et al., 2013. Nat Biotechnol 31, 233-239). Genomic DNA replacement was initiated by adding NA to cells. Cas9-mediated excision of the rewritten DNA from the BAC and lambda-red-mediated recombination of this DNA into the genome resulted in the replacement of genomic DNA compartments with the rewritten DNA, the removal of positive and negative selection markers from the genome, and the introduction of new orthogonal positive and negative selection markers. Clones recombined across the target region were selected based on the loss of negative selection markers from the genome and the acquisition of positive selection markers from the BAC.

[0202] In each strain, the positive and negative selection markers introduced in the initial REXER provide a template for the next round of REXER, enabling genome-level exchange synthesis (GENESIS) (Figures 2b and 4). The inventors used plasmids encoding spacers for the initial round of REXER (Table 4, Figures 20d and 21). However, the inventors subsequently found that REXER can be initiated by electroporation of linear double-stranded spacers generated by PCR (Table 4, Figure 21a). Since these spacers do not propagate through cell division, this makes it possible to use cells from one step of REXER more quickly for the next step of REXER. This advance accelerated GENESIS. For compartments A, C, D, E, F, and G, the inventors proceeded with GENESIS clockwise through 4–5 steps of REXER until approximately 0.5 Mb of genomic DNA was replaced with synthetic DNA. Since section A was started first and completed before the other sections, the inventors proceeded with GENESIS through section B once the end of section A was reached.

[0203] After each REXER, the inventors sequenced the resulting genomes to identify cells that had been completely rewritten across targeted regions of the genome (Table 4). In parallel, the inventors performed numerous single-step REXERs (Table 4) to rapidly identify 100kb regions of the genome that may be difficult to rewrite, and then, via GENESIS, the inventors... And so they reached them. For 35 of the 38 steps, including all of compartments A, C, D, E, F, and G, the inventors were able to completely rewrite the targeted genome sequence by GENESIS. For fragment 9 in compartment B, and for fragments 37a and 1 in compartment H, the inventors observed only incomplete substitution of the corresponding genome region by synthetic DNA (Table 4).

[0204] [Table 3] JPEG2026094253000008.jpg255165JPEG2026094253000009.jpg25593

[0205] [Table 4]

[0206] [Table 5]

[0207] [Table 6] [Examples]

[0208] Identification and repair of design defects By sequencing several clones after REXER, the inventors were able to score the frequency of rewriting each target codon, thereby aggregating the rewriting landscape for genomic regions. From the rewriting landscape in fragment 1, the inventors directly identified the fourth codon (Ser4, TCA) in map, an essential gene encoding methionine aminopeptidase, which is difficult to rewrite using the scheme defined by the inventors (Figure 5a). The inventors also identified a 14 bp duplication of the essential genes ftsI and murE, as well as a second region in ftsI and murE containing several serine codons, which had not been replaced by the inventors' rewritten and refactored sequences. Since the inventors had previously rewritten this region with the same rewriting scheme, they used 182 bp for the duplication instead of the 20 bp used here. When the added material was replicated (Wang, K. et al., 2016. Nature 539, 59-64) (Figure 1c), the inventors concluded that the defect in the synthetic DNA for this region was in its refactoring, not its rewriting. A new REXER using the BAC of fragment 1, containing both the extended refactoring (Figure 5b) and the TCA-to-TCT mutation at Ser4 in the map (Figure 5c, Table 5), enabled complete rewriting of the targeted 100kb region of the genome (Figure 5d).

[0209] From the rewritten landscape after REXER of fragment 9, the inventors identified a 26kb genomic region that had not been rewritten (Figure 6). By attempting to delete 10kb genomic regions within and around this region in the presence of the BAC containing the rewritten fragment 9, the inventors narrowed down the region that was difficult to rewrite to 10kb of the genome. REXER across the 10kb genomic region revealed the minimum value in the rewritten landscape obtained at yceQ. This identified that the problem lay in rewriting five target codons within yceQ. Similarly, from the rewritten landscape after REXER of fragment 37a, and subsequent further sequencing, the inventors were able to identify a single codon at the 3' end of yaaY that had not been rewritten (Figure 7).

[0210] Both yceQ and yaaY encode "predictive proteins," and multiple insertions in yceQ are viable, with no evidence of mRNA production and / or protein synthesis from these predictive genes (Pundir, S., et al., 2017. Methods Mol Biol 1558, 41-55). In particular, all unrewriting codons in yceQ and yaaY are located within the 5' untranslated region (UTR) of essential genes. The inventors of yceQ and yaaY encode "predictive proteins," and multiple insertions in yceQ are viable, with no evidence of mRNA production and / or protein synthesis from these predictive genes (Pundir, S., et al., 2017. Methods Mol Biol 1558, 41-55). This suggests that the sequence changes introduced by rewriting aY adversely affect the regulation of adjacent essential genes. In fact, target codons in yceQ were mapped to the RNA secondary structure and the promoter element within the 5'UTR of rne (encoding the essential ribonuclease RNase E) (Figure 8), and these sequences are essential for regulating RNase E homeostasis (Schuck, A., et al. 2009. Mol Microbiol 72, 470-478).

[0211] The inventors modified fragment 9 by introducing a stop codon to the 5' sequence of yceQ, thereby minimizing any potential translation while preserving the native sequence for regulating rne transcription (Figure 6, Table 5). This novel BAC-based REXER completely rewrote the corresponding 100kb genomic region (Figure 6, Table 5). A novel BAC-based REXER containing fragment 37a, in which the problematic codon in yaaY was replaced from TCA to AGC, completely rewrote the corresponding region of the genome (Figure 7, Table 5).

[0212] By identifying and correcting all the initial problematic sequences, the inventors completed assemblies of strains in which compartments A and B are completely rewritten (Figure 9), and assemblies of strains in which compartment H is completely rewritten (Table 5, Figure 9). This completed the assembly of all compartments in seven different strains.

[0213] [Table 7] JPEG2026094253000014.jpg25589 [Examples]

[0214] Rewritten genome assembly The inventors have developed a conjugation-based strategy to assemble rewritten compartments into a single genome (Isaacs, FJ et al., 2011. Science 333, 348-353; Ma, NJ, et al., 2014. Nat Protoc 9, 2285-2300; and Lederberg, J. & Tatum, EL, 1946. Nature 158, 558). The inventors' strategy assembles a clockwise rewritten genome by conjugating rewritten "donor" compartments containing the origin of transmission (oriT) to their adjacent rewritten "recipient" compartments that are extended to provide homology with the donor (Figures 10, 11a, 22a, b). This generates a new genome containing rewritten compartments from both the donor and recipient. The cells containing this new genome can then be used as recipients for the next donor to be rewritten, and through iterative processes, the rewritten genome can be assembled by gradually adding the rewritten compartments to the rewritten recipient (Figures 10, 11a, and 11b). The donor cells contained a type of F' plasmid that facilitates the transfer of the donor genome to the recipient cells, but unlike standard F' plasmids, they did not have the ability to transfer themselves to the recipient cells (Figure 22c), and as a result, this F' plasmid did not need to be lost from the recipient cells after all conjugation. This accelerated our workflow.

[0215] The inventors initiated conjugation by mixing donor and recipient cells and varied the conjugation time and conditions to control the degree of genome transfer from donor to recipient. After conjugation between donor and recipient cells, the inventors selected recipient cells and then selected those recipients that had acquired a positive marker at the end of the rewritten sequence from the donor and lost a negative marker at the end of the recipient's elongation (Figure 11a).

[0216] The inventors performed convergent synthesis of rewritten genomes through compartments A to E (Figures 10 and 11b). Next, the inventors used strains A to E as recipients for F to generate rewritten strains A to F. Then, A to F were used as recipients for F to G to generate A to G. This conjugation utilized a fairly long shared rewritten sequence (0.4 Mb) between the donor and recipient strains to increase conjugation efficiency.

[0217] To create a completely rewritten genome, the inventors first created a recipient strain by introducing 37a and 37b into A-G to create A-G-37ab (providing a 115kb homologous region to the final donor). The inventors then created the final donor strain by conjugation between strain H and strain AB, thereby obtaining strain HA-09, in which fragments 9 from H, A, and compartment B are rewritten (Figures 10 and 11b). By adding additional sequences from A and B to H, the inventors ensured that the rewriting of A in the final conjugation was not erased. The final conjugation between the HA-09 donor strain and the A-G-37ab recipient strain resulted in the synthesis of E. coli, which the inventors named E. coli Syn61, and in E. coli Syn61, 1.8 × 10¹⁶ kb of genome 4 All target codons have been rewritten (Figure 19, Sequence ID No. 2). Our synthesis of the rewritten genome introduced only eight unprogrammed mutations (Table 6), of which four occurred during the preparation of the 100kb BAC and four occurred during the rewriting process.

[0218] [Table 8] JPEG2026094253000016.jpg25550 [Examples]

[0219] Results of synonym codon compression in Syn61 Syn61 doubled only 1.6 times slower than MDS42 when glucose was added to LB at 37°C, with this rate increasing at 25°C and decreasing at 42°C (Figure 13a). Syn61 contains 65% more AGT and AGC codons than MDS42, but provides additional copies of serV, the tRNA that decodes these codons (Figure 12a), without increasing proliferation (Figure 13a), suggesting that serV is not restricted. Imaging of Syn61 cells suggests they are slightly longer than MDS42 cells (Figures 13b, c). The proteome of Syn61 was comparable to that of MDS42 (Figure 13d). TCG codon-targeted orthogonal aminoacyl-tRNA synthetase / tRNA CGA Co-translational incorporation of non-canonical amino acids using pairs was highly toxic in MDS42 but completely non-toxic in Syn61, providing phenotypic validation for TCG codon removal in Syn61 (Figure 12b). This approach also provided further insights (Figures 14a, b, c). tRNA Ser UGA The serT encoding the TCA codon is essential because it is the only tRNA that decodes the TCA codon in E. coli. Since Syn61 does not contain the TCA codon, serT should be non-essential in our strain. In fact, we demonstrated that serT (Figures 12c, 14d, 23), as well as serU and prfA (Figures 14e, f, 23), can be easily removed in Syn61. These data provide functional confirmation of our removal of the target codon from the genome, demonstrating that tRNA and termination factors that decode the target codon can be removed in Syn61, and illustrating the unique properties of Syn61 resulting from this rewriting. [Examples]

[0220] Consideration The inventors created Escherichia coli in which the entire 4 Mb genome was replaced with synthetic DNA. In the inventors' experiments, the scale of genome replacement was approximately four times larger than that previously reported for genome replacement in Mycoplasma or chromosome replacement in a single strain of S. cerevisiae (Figure 15a).

[0221] The inventors have found that all known 1.8 × 10⁶ values ​​in a single strain of E. coli are... 4 We demonstrated genome-wide removal of 100 target codons (two sense codons, TCG and TCA, and the amber codon, TAG). Our study removes 60 times more codons than experiments using site-directed mutagenesis to remove the amber stop codon (Figure 15b). Furthermore, this demonstrates complete, genome-wide rewriting of all targeted sense codons (Figure 15b). Therefore, we created a synthetic organism that uses 61 codons instead of the usual 64. The new organism uses fewer sense codons to encode 20 canonical amino acids.

[0222] The inventors' synthetic genome has 2 × 10 per target codon. -4 It contains only 1 unprogrammed mutation (Figure 15c). This is advantageously comparable to 1.05 unprogrammed mutations per target codon reported for ambir codon substitution by site-directed mutagenesis (Lajoie, MJ et al., 2013. Science 342, 357-360) (Figure 15c).

[0223] The inventors' final synthetic genome was rewritten using a defined refactoring and rewriting scheme, employing rewriting rules previously determined by the inventors for only 83 target codons (0.43%) of the genome (Wang, K. et al. 2016. Nature 539, 59-64). The rewriting rules are 1.8 × 10⁻¹⁰ of the genome. 4 Despite performing the test on 99.9% of individual target codons, The refactoring rules were then applied to 99% of the code.

[0224] The inventors' initial modification of the rewriting scheme resulted in a 1.8 × 10⁶ modification of the entire genome. 4 Only seven of the target codons were required. One of these codons was in the essential gene, but the other six were within the 5'UTR of the essential gene. Therefore, with the exception of one of the alterations to the rewriting scheme as defined by the inventors, all correct unintended alterations to the 5'UTR of the essential gene, rather than having a direct effect of the altered synonym on translation.

[0225] The strategies developed by the inventors to cleave designed genomes into compartments, fragments, and stretches, and to achieve convergent, seamless, and robust integration of REXER, GENESIS, and inducible conjugations, provide a blueprint for future genome synthesis. In future research, the inventors will further characterize the results of synonymous codon compression in E. coli Syn61 and test further rewriting schemes in E. coli and other organisms. Furthermore, the inventors will test sense codon reassignment for non-canonical biopolymer synthesis. [Examples]

[0226] method Rewritten genome design The inventors based their synthetic genome design on the sequence of the E. coli MDS42 genome (accession number AP012306.1, published October 7, 2016), which has 3547 annotated CDSs. The inventors manually curated the annotations of the start genome to remove three CDSs and add twelve others. The three predicted CDSs removed were htgA, ybbV, and yzfA, for which there is no evidence that these sequences encode proteins (Pundir, S., et al., 2017. Methods Mol Biol 1558, 41-55). These sequences completely or largely overlap with well-characterized genes, making it difficult to rewrite them without disrupting those overlapping genes or creating large repeat regions. Conversely, the pseudogenes ydeU, ygaY, pbl, yghX, yghY, agaW, yhiK, yhjQ, rph, ysdC, glvG, and cybC were recommended for CDS. To enable negative selection in rpsL, the inventors used a genome copy of rpsL. K43R The gene was mutated. Finally, deep sequencing of the inventors' in-house MDS42 revealed a 51 bp insertion between mrcB and hemL that had not been reported in AP012306.1. The inventors manually introduced and annotated this insertion in their start genome sequence.

[0227] The inventors created a custom Python script to i) identify and rewrite all target codons, and ii) identify and degrade duplicate gene sequences containing target codons. Using the inventors' curated MDS42 start sequences, the inventors used the script to generate a new synthetic genome in which all TCG, TCA, and TAG codons were replaced with AGC, AGT, and TAA, respectively. This script reported 91 CDSs with duplicates containing target codons. In 33 cases, the genes were tail-to-tail (3', 3') duplicated (Table 1), of which 12 could be rewritten by introducing silent mutations into the duplicated genes, while the remaining 21 were replicated to separate the genes (Figure 1b). 58 examples of head-to-tail (5', 3') duplicated genes were degraded by replicating the duplicate with a 20 bp upstream sequence added to allow endogenous expression of the downstream gene (Figure 1c). For duplications longer than 1 bp, in-frame TAA was introduced to terminate the expression of downstream genes from the original RBS. Because prfB (termination factor RF-2) was not annotated as a CDS in our initiation MDS42 genome due to its regulatory internal stop codon, we introduced a gene within... All target codons were manually rewritten, thereby preserving the internal stop codons. The resulting genome design contained 3,556 CDSs with 1,156,625 codons, of which 18,218 were rewritten (Figure 18, Sequence ID No. 1).

[0228] Rewritten stretch retrosynthesis The inventors divided the designed genome into 37 fragments ranging from 91 to 136 kb. The inventors determined that i) the boundary sequence consists of a 5'-NGG-3'PAM so that REXER4 can be used for incorporation if necessary, and ii) the PAM is located within 50 bp of the target codon. iii) PAM is located between non-essential genes, and iv) PAM is located in a promoter or similar To avoid interfering with any of the annotated features, boundary sequences were selected to demarcate these fragments. The inventors refer to the approximately 50–100 bp regions upstream and downstream of these boundaries as "landing regions" and annotate them as Lxx, where xx is the number of the upstream fragment; for example, L01 is the landing region between fragments 1 and 2. In the inventors' design, the landing region sequences are contained at the 3' end and the following 5' end of the fragments, and as a result, all 37 fragments contain 54–155 bp of overlapping homology with their adjacent fragments.

[0229] Each fragment was further broken down into 7 to 14 stretches of 4 to 15 kb each. The inventors designed the stretches to contain 80 to 200 bp of overlap with each other, defining the overlapping regions in intergeneric regions that did not contain any rewrite targets. A total of 409 stretches were synthesized (GENEWIZ, USA) and fed into pSC101 or pST vectors adjacent to BsaI, AvrII, SpeI, or XbaI restriction sites. The synthetic stretches contain fewer of these restriction sites in nature. None of them contained any of the specified ingredients.

[0230] Construction of selection cassettes and plasmids for REXER / GENESIS The cloning procedure described in this section was performed in *E. coli* DH10b resistant to streptomycin due to the rpsLK43R mutation. The plasmid pKW20_CDFtet_pAraRedCas9_tracrRNA used throughout this study was, as previously described, Under the control of the arabinose-inducible promoter, it encodes Cas9 and the alpha / beta / gamma recombinant components of lambda-red, and under its native promoter, it encodes tracrRNA (Wang, K. et al., 2016. Nature 539, 59-64).

[0231] The protospacer for REXER is plasmid pKW1_MB1 AmpThe spacer is encoded in (Figure 21a), which, as previously described, contains the pMB1 origin of replication, an ampicillin resistance marker, and a protospacer array under the control of its endogenous promoter (Wang, K. et al., 2016. Nature 539, 59-64). From this plasmid, we have derived the derivative pKW3_MB1 Amp _Tracr K A spacer was constructed (Table 5), which further contains tracrRNA upstream of the protospacer array. For this purpose, the inventors assembled the PCR product containing tracrRNA together with its modified endogenous promoter into pKW1_MB1 by Gibson assembly using NEBuilder HiFi Master Mix. Amp _Space It was introduced into the BamHI region of the plasmid. From this plasmid, and also by Gibson assembly We constructed derivatives that further encode Cas9, pKW5_MB1 Amp _Tracr K I named it _Cas9_spacer.

[0232] For each REXER step, a derivative of one of these three plasmids was constructed to possess a protospacer / direct repeat array containing two (REXER2) or four (REXER4) protospacers corresponding to the target sequences for cleaving the BAC and genome. Different protospacer arrays were constructed from duplicate oligos by multiple rounds of PCR, and the products were used to create pKW1_MB1 Amp Spacer, pKW3_MB1 Amp _Tracr K _Spacer or pKW5_MB1 Amp _Tracr K Restriction area AccI in the _Cas9_ spacer skeleton Inserted between and EcoRI by Gibson Assembly. Pro obtained from each assembly The spacer array was verified to be free of mutations by Sanger sequencing.

[0233] The positive-negative selection cassettes used in REXER and GENESIS were -1 / +1 (rpsL-Kan R ), -2 / +2 (sacB-Cm R ), and -3 / +3 (pheS T251A_A294G -Hyg R ). -1 / +1 and -2 / +2 are as previously described (Wang, K. et al., 2016. Nature 539, 59-64). For -3 / +3, pheS T251A_A294G is dominant lethal in the presence of 4-chlorophenylalanine, and Hyg R confers resistance to hygromycin. Both proteins are expressed polycistronically under the control of the EM7 promoter. The -3 / +3 cassette was synthesized de novo. The -3 / +3 cassette is also referred to as pheS * / Hyg R .

[0234] Construction of Escherichia coli strains containing a double selection cassette at the genomic landing site. According to the inventors' design, for each region of the genome targeted for substitution by a synthetic fragment, an upstream landing site and a downstream landing site are adjacent, and these genomic landing site sequences are the same as the above-mentioned landing site sequences. To initiate REXER / GENESIS, insertion of a double selection cassette into the upstream genomic landing site is required. The inventors inserted a double selection cassette into the landing site by recombination via lambda-red. Briefly, sacB-Cm R or rpsL-Kan ROne of the cassettes was PCR-amplified using primers containing homologous regions to the desired genome landing site. For the recombinant experiment, the inventors prepared electrocompetent cells as previously described (Wang, K. et al., 2016. Nature 539, 59-64) and 3 μg of purified PCR product was transferred to 10 cells containing the pKW20_CDFtet_pAraRedCas9_tracrRNA plasmid expressing the lambda-red alpha / beta / gamma genes. 0 μL of MDS42 rpsLK43R Cells were electroporated. Under the control of the arabinose promoter (pAra), OD 600 Recombination was induced by adding 0.5% L-arabinose at a starting concentration of 0.2 for 1 hour. The pre-induced cells were electroporated and then harvested in 4 mL of super optimal broth (SOB) medium at 37°C for 1 hour. The cells were then treated with 10 μg / mL tetra The cells were diluted in 100 mL of LB medium containing cyclin and grown at 37°C and 200 rpm for 4 hours. Afterward, the cells were centrifuged, resuspended in 4 mL of H2O, serially diluted, seeded, and then treated with 10 μg / mL tetracycline and 18 μg / mL chloramphenicol (sacB-Cm). R (For use) or 50 μg / mL kanamycin (rpsL-Kan R The sample was incubated overnight at 37°C on an LB agar plate containing the specified ingredients.

[0235] BAC assembly and delivery The inventors constructed a bacterial artificial chromosome (BAC) shuttle vector containing 97-136 kb of synthetic DNA. On the 5' side, the synthetic DNA was adjacent to a homology region (HR1) to the genome and a Cas9 cleavage site. On the 3' side, the synthetic DNA was adjacent to a double selection cassette, a homology region (HR2) to the genome, and a second Cas9 cleavage site. The BAC also contained a negative selection marker, a BAC origin, a URA marker, and a YAC origin (CEN6 centromere (CEN / ARS) fused to a self-replicating sequence) (Figure 2c, Figures 20a-c).

[0236] The BAC was assembled by homologous recombination in S. cerevisiae. Each assembly combined i) 7-14 stretches of synthetic DNA, each 6-13 kb in length, ii) a selection construct (see below), and iii) the BAC shuttle vector backbone (Figures 20a-c, Wang, K. et al., 2016. Nature 539, 59-64).

[0237] The synthetic DNA stretches were excised from their source vectors provided by GENEWIZ by digestion with BsaI, AvrII, SpeI, or XbaI restriction sites. In the case of AvrII, SpeI, and XbaI, following restriction digestion, Mung Bean nuclease treatment was performed to remove sticky ends and make them blunt-ended.

[0238] The selection construct consisted of a homology region to the most 3'-side stretch of the fragment, a double selection cassette (sacB-Cm R or rpsL-Kan R ), a homology region (HR2) to the targeted genomic locus, a negative selection marker (rpsL, sacB, or pheS * -Hyg R) and YAC were included. See Figure 20d for specific double-selection cassettes, negative selection markers, and homologous region sequences. The inventors assembled an episomal form of the selection construct in the pSC101 backbone from three PCR fragments using NEBuilder HiFi DNA Assembly Master Mix. This episomal form was designed so that restriction digestion with BsaI would yield DNA fragments for BAC assembly.

[0239] The BAC skeleton, containing the BAC origin and URA3 marker, was amplified by PCR using a previously described BAC template (Wang, K. et al., 2016. Nature 539, 59-64), and the PCR products were used for BAC assembly. The primers used for these PCR assemblies are listed in Figure 20d.

[0240] To assemble the stretch, selective construct, and BAC skeleton, 30–50 fmol of each DNA fragment was transformed into S. cerevisiae ferroplasts, which were prepared as previously described (Kouprina, N., et al., 2004. Methods Mol Biol 255, 69-89). After assembly, the inventors identified yeast clones potentially harboring accurately assembled BACs at the junctions of overlapping fragments and vector insertion junctions by colony PCR. Clones that appeared accurate by colony PCR had sequences validated by NGS after transformation into E. coli, as described below.

[0241] The assembled BAC was extracted from yeast using the Gentra Puregene Yeast / Bact. Kit (Qiagen) according to the manufacturer's instructions. MDS was then extracted by electroporation. 42 rpsLK43RThe assembled BACs were transformed into cells. Due to the large size of the BACs, the inventors sometimes observed inefficient electroporation to target cells. As a result, the inventors introduced oriT-apramycin cassettes, provided as PCR products with a 50 bp homologous region by lambda-red recombination (as described above), into some BACs after assembly (Figures 20a-c). This facilitated the successful transfer of BACs from transformed E. coli to other strains via conjugation.

[0242] Combination of rewritten partitions by REXER and GENESIS The inventors used various genome and plasmid selection markers for serial REXER experiments (GENESIS) (Table 4). The inventors used rpsL-Kan at the genome landing site for selection. R (-1 / +1) or sacB-Cm R A (-2 / +2) cassette was used. The inventors used rpsL-Kan as an episome selection marker. R -sacB(-1 / +1, -2), rpsL-Kan R -pheS * -Hyg R (-1 / +1, -3 / +3) or sacB-Cm R -rpsL(-2 / +2, -1) cassette was used.

[0243] For each REXER, the relevant upstream genomic landing site contains pKW20_CDFtet_pAraRedCas9_tracrRNA and MDS42 containing a double-selection cassette. rpsLK43R The cells were transformed with the relevant BAC. The inventors then transformed the BAC with 2% glucose, 5 μg / ml tetracycline, and an antibiotic of their choice (i.e., 18 μg / ml tetracycline). Cells were seeded on LB agar supplemented with loramphenicol or 50 μg / ml kanamycin. The inventors inoculated individual colonies into LB medium containing 5 μg / ml tetracycline and a BAC-specific antibiotic and grew the cells overnight at 37°C and 200 rpm. The overnight culture was diluted to OD600 = 0.05 with LB medium containing 5 μg / ml tetracycline and a BAC-specific antibiotic and grown at 37°C with shaking for approximately 2 hours until OD600 ≈ 0.2. To induce lambda-red expression, the inventors added arabinose powder to the culture to a final concentration of 0.5% and incubated the culture for a further 1 hour at 37°C with shaking. The inventors collected cells at an OD600 of approximately 0.6 and made the cells electrocompetent as previously described (Wang, K. et al., 2016. Nature 539, 59-64).

[0244] For each REXER experiment, a linear dsDNA protospacer array was PCR-amplified from a pKW1_MB1Amp_spacer using universal primers (Figure 21a). Approximately 5–10 μg of digested DpnI and purified PCR products were transformed into 100 μL of electrocompetent and induced cells. The cells were harvested in 4 ml of SOB medium at 37°C for 1 hour, then diluted in 100 mL of LB supplemented with 5 μg / mL of tetracycline and an antibiotic of choice for BAC, and incubated for a further 4 hours at 37°C with shaking. Alternatively, electrocompetent and induced cells were transformed with a 5 μg circular protospacer array (pKW1_MB1Amp_spacer or pKW3_MB1Amp_spacer plasmid), harvested in SOB medium at 37°C for 1 hour, and then transferred to 100 mL of LB supplemented with 100 μg / mL ampicillin at 37°C for a further 4 hours with shaking (Figure 21a, b). If REXER2 was insufficient, the inventors performed REXER4 using the pKW5_MB1Amp_spacer plasmid as previously described (Wang, K. et al., 2016. Nature 539, 59-64).

[0245] The inventors centrifuged the culture to precipitate it, resuspended it in 4 ml of Milli-Q filtered water, and 5 Selective plates of LB agar were streaked in serial dilutions with μg / ml tetracycline, a drug selected for the negative selective marker, and an antibiotic selected for the positive marker derived from BAC. These plates were incubated overnight at 37°C. Several colonies were selected, resuspended in Milli-Q filtered water, and treated with 50 μg / ml kanamycin and 18 μg / ml chlorambyl. Several LB agar plates were placed in supplement with phenicol, 200 μg / ml streptomycin, 7.5% sucrose, or 2.5 mM 4-chlorophenylalanine. Colony PCR was also performed on colonies resuspended using both primer pairs adjacent to the genomic locus at the landing site and the position of a newly incorporated selection cassette from BAC. Recombination via REXER resulted in a band of approximately 500 bp at the upstream genomic locus, compared to the control MDS42. rk / MDS42 sC A 2.5kb (rk-landing site) or 3.5kb (sC-landing site) band in the strain indicates successful removal of the landing site from the genome. A primer pair adjacent to the 3' end of the substituted DNA shows approximately 2.5kb (rK-selective cassette on pBAC) or 3.5kb (sC-selective cassette on pBAC) bands and successful incorporation of the selection marker in control MDS42. rk / MDS42 sC Generate a 500bp band for the stock.

[0246] When plasmid-based circular protospacer arrays were used in previous REXER experiments, plasmid loss was required before subsequent experiments. Therefore, successful clones from the initial REXER experiment were grown in LB supplemented with 2% glucose, 5 μg / mL tetracycline, and an antibiotic selected for a positive marker within the genome, at 37°C with shaking until a high-density culture was achieved. Two μL of the culture was then streaked onto LB agar plates containing the same supplements and incubated overnight at 37°C. Several colonies were placed on LB agar plates containing replicas and LB agar plates supplemented with 100 μg / mL ampicillin to screen for plasmid loss.

[0247] BAC Editing When loss-of-function mutations were encountered in the selection cassette on BAC in E. coli, the defective cassette was replaced with a suitable double-selection cassette provided as a PCR product, which has a 50 bp homologous region adjacent to it and is incorporated by lambda-red-mediated recombination (Figure 20d).

[0248] To correct spontaneous mutations or alter rewritten codons, alterations to the rewritten sequence of BAC synthesis were introduced via a two-step substitution approach. For BACs containing selection cassettes -2 / +2 and -1 at the ends of the rewritten sequence, the -3 / +3 cassette was provided as a PCR product incorporated by lambda-red-mediated recombination, adjacent to a 50 bp homologous region targeting a desired locus, and subsequently selected for +3. Due to homology between the rewritten DNA and the genome, some of the resulting clones contained -3 / +3 on the BAC, and some contained it on the genome. To identify clones by cassette on the BAC, clones were selected for (1) +3, (2) -3, and (3) +2 and -3, and seeded onto replicas on agar plates. Only clones that survived on plates (1) and (2) but not (3) possessed the -3 / +3 cassette incorporated into the BAC. Cassette locations were determined using QIAprep Spin. Validation was performed by purifying BAC using the Miniprep Kit and then genotyping. In the second step, the -3 / +3 cassette was replaced by a PCR product containing a 50 bp homologous region adjacent to the desired sequence incorporated by lambda-red recombination, followed by selection for +2 and -3. The BAC was genotyped as described above and the sequence was validated by NGS.

[0249] Preparation of nontransferable F' plasmids and conjugate transfer of episomes The inventors have created a type of F' plasmid for use in genomic DNA conjugation and inter-strain BAC transfer, enabling the transfer of oriT-supported sequences without transferring the F' plasmid itself (Figure 22c). The inventors achieved this by deleting the nick site of the origin of transfer (oriT) within the F' plasmid itself, a related approach that has been previously reported (Strand, TA, et al., 2014. PLoS One 9, e90372). The F' plasmid derivative pRK24 (addgene#51950) has a 50 bp homology. The region is modified by incorporating a desired marker into the adjacent PCR product, and the incorporation is performed by Tet R Kan R Using a variant of pKW20 that has lambda- This was carried out by recombinant DNA. First, ampicillin resistance was conferred to pRK24. The β-lactamase gene was replaced with an artificial T5-luxABCDE operon (Bryksin, AV & Matsumura, I., 2010. PLoS One 5, e13244) that generates bioluminescence enabling visual identification of infected bacterial cells. Next, Tet R To select it with 50 μg / mL apramycin For this purpose, it was replaced with T3-aac3, which produces aminoglycoside 3-N-acetyltransferase IV. Finally, the 24 bp deletion of the oriT nick site was performed by incorporating EM7-bsd expressing blastosidine-S deaminase, which can be selected with 50 μg / mL blastosidine in low-salt TYE / LB. The resulting F' plasmid, called pJF146 (Figure 22c), was extracted using the QIAprep Spin Miniprep Kit (QIAgen) and transformed into a donor strain by electroporation for subsequent conjugation.

[0250] Episomal DNA containing oriT was transmitted by conjugation (Isaacs, FJ et al., 2011. Science 333, 348-353; and Ma, NJ, et al. 2014. Nat). (Protoc 9, 2285-2300). Donor strains were double-transformed with assembled BACs containing pJF146 and oriT (see above). Recipient strains were transformed with pKW20. 5 ml of donor and recipient cultures were grown overnight in selected LB medium until saturated, and then... The cells were washed three times with antibiotic-free LB medium. The resuspended donor and recipient strains were combined in a 4:1 ratio, spotted onto TYE agar plates, and incubated at 37°C for 1 hour. The cells were washed from the plates and streaked in serial dilutions onto LB agar plates containing 2% glucose, 5 μg / ml tetracycline selected for the recipient strain, and an antibiotic selected for BAC. Successful BAC transfer was confirmed by colony PCR of the BAC-vector insertion junction.

[0251] Assembling a synthetic genome from rewritten regions The partial synthetic E. coli genome was assembled into a synthetic genome by transferring genomic DNA, followed by recombination via recBCD. In the preparation of donor and recipient strains, rpsL-HygR-oriT or Gm R-oriT cassettes were supplied as PCR products and incorporated into the donor strain genome by lambda-red-mediated recombination (Figure 22a, b). Separately, pheS * -Hyg R The cassette was incorporated approximately 3kb downstream of the donor strain's synthetic DNA. This resulted in 3'pheS * -Hyg R A template genomic DNA was provided for PCR amplification of a 3kb synthetic DNA segment with a selection cassette. This PCR product was supplied to a recipient strain, and the WT DNA was replaced by lambda-red-mediated recombination. This replaced the selection marker at the 3' end of the synthetic segment, generating a 3kb homologous region to the donor synthetic DNA. This strategy ensures that each of these donors has 3kb homology, always with pheS-Hyg at the 3' end. R This helped systematically generate recipient strains possessing [specific trait]. Furthermore, donor strains were transformed with pJF146 to confirm susceptibility to tetracycline. In contrast, pKW20 was maintained in the donor strain to [specific trait]. It induced resistance to lasilein.

[0252] For conjugation, donor and recipient strains were grown overnight in LB medium containing 2% glucose, 5 μg / ml tetracycline, and 50 μg / ml kanamycin or 20 μg / ml chloramphenicol (donor), 50 μg / ml apramycin, and 200 μg / ml hygromycin B (recipient) until saturated. The overnight cultures were diluted 1:10 in the same selective LB medium and OD was used. 600The cells were grown to a concentration of 0.5. 50 ml each of the donor and recipient cultures were washed three times with LB medium containing 2% glucose, and then resuspended in 400 μl of LB medium containing 2% glucose. 320 μl of donor was mixed with 80 μl of recipient, spotted onto TYE agar plates, and incubated at 37°C. Incubation time was varied from 1 to 3 hours, depending on the length of the transmitted synthetic DNA and the doubling time of the recipient strain. The cells were washed from the plates and transferred to 100 ml of LB medium containing 2% glucose and 5 μg / ml tetracycline, and incubated at 37°C for 2 hours with shaking. Subsequently, 50 μg / ml kanamycin or 20 μg / ml chloramphenicol (choice for the positive selection marker transmitted by the donor) was added, and then incubated for a further 2 hours at 37°C. The cultures were centrifuged and resuspended in 4 ml of Milli-Q filtered water, and 2 Selective plates of LB agar containing % glucose, 5 μg / ml tetracycline, 2.5 mM 4-chlorophenylalanine, and 50 μg / ml kanamycin or 20 μg / ml chloramphenicol were streaked in serial dilutions. Success of DNA transfer and recombination was indicated by pheS * -Hyg R Loss of cassettes, inclusion of donor selection cassettes, and absence of Gm-oriT cassettes were determined by colony PCR.

[0253] BAC libraries for whole genome preparation and next-generation sequencing Use the DNEasy Blood and Tissue Kit (QIAgen) according to the manufacturer's instructions. E. coli genomic DNA was purified. BAC was extracted from the cells using the QIAprep Spin Miniprep Kit (QIAgen) according to the manufacturer's instructions. The inventors found that this kit was 13 We found that this method is suitable for purifying BACs greater than 0 kb. The inventors avoided vigorous shaking of the sample throughout the purification process to reduce DNA shear.

[0254] Paired-end Illumina sequencing libraries were prepared using the Illumina Nextera XT Kit according to the manufacturer's instructions. Sequencing data was obtained using Illumina MiSeq by running 2 × 300 or 2 × 75 cycles with the MiSeq Reagent kit v3.

[0255] Sequencing Data Analysis The standard workflow for sequence analysis in this study is consolidated in the iSeq package. In short, sequencing reads are aligned to a reference rewritten genome or wild-type genome using bowtie2 with soft clipping activated. Aligned reads were classified and indexed using samtools (Langmead, B. & Salzberg, SL, 2012. Nat Methods 9, 357-359). A customized Python script was used with samtools. This also resulted in a variant that calls the summary in conjunction with the functionality of igvtools. This script was used to combine mutations with visual analysis in Integrative Genomics Viewer. Delamination and structural changes were evaluated (Thorvaldsdottir, H., et al., 2013. Brief Bioinform 14, 178-192).

[0256] The inventors created a custom Python script to generate a rewritten landscape across a target genomic region. Briefly, the script receives a BAM alignment file, a fasta reference, and a Genbank annotation file as input. This process identifies target codons for rewriting and aggregates reads to align with these target codons in the alignment file. It then outputs the rewrite frequency for each target codon and plots these frequencies over the length of the desired genomic region.

[0257] Measurement and analysis of proliferation rate Bacterial colonies were grown overnight at 37°C in LB containing 2% glucose and 100 μg / mL streptomycin. The overnight cultures were diluted 1:50, and growth was monitored while varying the temperature (25°C, 37°C, or 42°C) and medium conditions (LB, LB containing 2% glucose, M9 minimal medium, 2XTY). OD 600 Measurements were taken every 5 minutes for 18 hours on a Biomek automated workstation platform while performing high-speed linear shaking.

[0258] To determine the doubling time, the growth curve was log2 transformed. The first derivative was determined in the linear phase of the curve during exponential growth (d(log2(x)) / dt), and the doubling time for each replication was calculated using 10 consecutive time points with the largest log2 derivative. A total of 10 independently grown biological replicas were used for the rewritten Syn61 strain and wt MDS42. rpsLK43R The following measurements were taken. The mean doubling time and the standard deviation from the mean were calculated for all n=10 copies.

[0259] Microscopic examination and measurement of cell size The cells were shaken in LB containing 100 μg / mL streptomycin for approximately OD. 600 The bacteria were grown to a concentration of 0.2. A thin layer of bacteria was placed between an agarose pad and a coverslip. Standard microscope slides were prepared using a 1% agarose pad (Sigma-Aldrich A4018-5G). A 2 μl to 4 μl sample of bacterial culture was placed on the top of the pad. The sample was dropped onto the pad. This was covered with a #1 coverslip supported on both sides by glass spacers fitted to a height of approximately 1 mm on the pad. The sample was imaged using an upright Zeiss Axiophot phase-contrast microscope with a 63X 1.25NA Plan Neofluar phase-contrast lens (Zeiss UK, Cambridge, UK). Images were taken using an IDS ueye monochrome camera under the control of ueye cockpit software (IDS Imaging Development Systems GmbH, Obersulm, Germany). The images were captured. Ten fields of view were taken for each sample. For further quantification, the images were loaded into Nikon NIS Elements software (Nikon Instruments, Surrey, UK). General analysis An intensity threshold was applied to segment the bacteria using a tool. A size limit of 1 micron was imposed to remove background particles and dust. Subsequently, the length of the segmented bacteria was measured using a common analytical quantification tool.

[0260] mass spectrometry Three biological replications were performed for each strain. Proteins from each E. coli lysate were solubilized in a buffer containing 6 M urea in 50 mM ammonium bicarbonate, reduced with 10 mM DTT, and alkylated with 55 mM iodoacetamide. After alkylation, the protein was diluted in 1 M urea with 50 mM ammonium bicarbonate and digested with Lys-C (Promega, UK) at a protein-to-enzyme ratio of 1:50 for 2 hours at 37°C, followed by digestion with trypsin (Promega, UK) at a protein-to-enzyme ratio of 1:100 for 12 hours at 37°C. The peptide mixture was acidified by adding formic acid to a final concentration of 2% v / v. Nanoscale capillary LC-MS / Massaging was performed using an Ultimate U3000 HPLC (ThermoScientific Dionex, San Jose, USA) to deliver a flow rate of approximately 300 nL / min. Digestion was analyzed using a double dose (1 ug of initiating protein / injection) with S. C18 Acclaim PepMap100 3 μm, 75 μm × 250 mm nanoViper (ThermoScientific Dionex, San Prior to separation at San Jose, USA, peptides were captured using a 5μm C18 Acclaim PepMap100, 100μm×20mm nanoViper (ThermoScientific Dionex, San Jose, USA). The peptides were eluted with a 100-minute gradient of acetonitrile (2%~60%). The analytical column outlet was directly connected to a hybrid dual-pressure linear ion trap mass spectrometer (Orbitrap Velos, ThermoScientific, San Jose, USA) via a nanoflow electrospray ionization source. The system was connected. Data-dependent analysis was performed using a resolution of 30,000 for the complete MS spectrum, followed by a resolution of 10 for the MS / MS spectrum with a linear ion trap. MS spectra were collected over the 300–2000 m / z range. MS / MS scans were collected using a threshold energy of 35 for collision-induced dissociation. All raw files were processed in MaxQuant 1.5.5.1 using standard settings and searched for E. coli strain K-12 using the Andromeda search engine built into the MaxQuant software suite. Enzyme search The specificity was trypsin / P for both endoproteinases. A maximum of two false cleavages were tolerated for each peptide. Carbamide methylation of cysteine ​​was set as a fixed modification by oxidized methionine, and protein N-acetylation was considered a variable modification. This search was performed with an initial mass tolerance of 6 ppm for the precursor ion and 0.5 Da for the CID MS / MS spectrum. The false detection rate was fixed at 1% at both the peptide and protein levels. Statistical analysis was performed using the Perseus (1.5.5.3) module of MaxQuant. Prior to statistical analysis, proteins identified only by peptides mapped to known contaminants, reverse hits, and site were removed. Of the proteins identified by at least two peptides, one was unique, and two quantitative events were considered for data analysis. For proteins quantified at least once in each strain, the average abundance of each protein over Syn61 replication was divided by the abundance of MDS42 replication, and then log2 transformed. P-values ​​for differences in abundance between strains were calculated using a two-sample t-test (Perseus). .

[0261] Orthogonal aminoacyl-tRNA synthetase tRNA xxx CYPK uptake poison using s (Elliott, TS et al., 2014. Nat Biotechnol 32, 465-472; Elliott, TS, et al., 2016. Cell Chem Biol 23, 805-815; and Krogager, TP et al., 2018. Nat Biotechnol 36, 156-159) PylRS and tRNA in electrocompetent MDS42 and Syn61 cells Pyl xxx To express plasmid pKW1_MmPylS_PylT XXX Transform it, and here, XXX This is the anticodon shown. tRNA Pyl The anticodon is CGA(pKW1_MmPylS_Py lT CGA ), UGA(pKW1_MmPylS_PylT UGA ) or GCU(pKW1_MmPylS_PylT GCU ) mutates Three variants of this plasmid were used. Cells were grown overnight in LB medium containing 75 μg / ml spectinomycin. The overnight cultures were diluted 1:100 in LB medium supplemented with Nε-(((2-methylcyclopropane-2-en-1-yl)methoxy)carbonyl)-L-lysine (CYPK) at 0 mM, 0.5 mM, 1 mM, 2.5 mM, and 5 mM, and growth was measured as described above. "Maximum growth %" was expressed as the final OD in the absence of CYPK. 600 Final OD in the presence of the concentration of CYPK divided by . 600 This was decided. Final OD 600 The decision was made 600 minutes later.

[0262] Deletion of prfA, serU, and serT by homologous recombination To ensure that the expression of the selected protein does not depend on decoding by serU or serT, the rewriting scheme shown in Figure 1a is used to process pheS * -Hyg R and rpsL-Kan R The rewritten cassette type was de novo synthesized. To remove prfA, the rewritten rpsL-Kan R This was amplified using an oligonucleotide containing approximately 50 bp of homology to the prfA adjacent genome sequence. The same was done with a rewritten selection cassette pheS. * -Hyg R This was performed on serU and serT using [a specific method]. Oligonucleotide sequences are provided in Figure 23. Syn61 cells containing plasmid pKW20_CDFtet_pAraRedCas9_tracrRNA were subjected to LB [a specific method]. Competence was achieved using 2xTY instead of the above. Approximately 8 μg of PCR product was electroporated into the cells, collected in 4 mL of SOB for 1 hour, and then transferred to 100 mL of 2xTY supplemented with 5 μg / ml of tetracycline. After 4 hours, the cells were centrifuged and resuspended in 500 μL of H2O, and 5 μg / ml of tetracycline and 200 μg / ml of hygromycin B (pheS) were added. * -Hyg R (For use) or 50 μg / ml kanamycin (rpsL-Kan RSeeds were seeded in serial dilutions on 2xTY agar plates supplemented with (for use). In each case, deletion was verified by colony PCR using primers adjacent to the desired locus.

[0263] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the disclosed methods, cells, compositions and uses of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been disclosed in relation to certain preferred embodiments, it should be understood that the claimed invention should not be excessively limited to such specific embodiments. In fact, various modifications of the disclosed methods for carrying out the invention, which are obvious to those skilled in the art, are intended to be within the scope of the appended claims.

Claims

[Claim 1] A synthetic prokaryotic genome containing five or four occurrences of one or more sense codons.

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