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National accession the in deposited the of sequence The deposition: Data BY-NC-ND) (CC 4.0 NoDerivatives License distributed under is article access open This Submission.y Direct PNAS a is article This etn fsnhtcgnms ..adBC odsae rmGgbssSizradAG. Switzerland Gigabases functional from shares covers hold that B.C. and inventors M.C. as genomes. B.C. synthetic of and testing M.C. with Eidgen (WO2017085249A1) statement: and application M.C., interest paper.y the L.D.M., of wrote J.E.V., Y.B., B.C. Conflict P.S., research; and M.C., A.W., performed J.E.V., L.D.M., and S.D. data; J.E.V., and analyzed research; B.C. R.G., designed M.v.K., C.E.F.-T., B.C. F.T., and D.A., M.C. contributions: Author rc,C-03Z CH-8093 urich, ¨ owo orsodnemyb drse.Eal [email protected]. 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BIOPHYSICS AND SYSTEMS BIOLOGY COMPUTATIONAL BIOLOGY A synthesis methods have been used for the rewriting of gene . crescentus e C gen cassettes in conjunction with genomic replacement strategies tiv om E. coli a e (15, 20). Ongoing de novo synthesis toward a 57-codon n genome was reported (21), with the complete genome synthesis 2.0 2.4 underway. 1.6 Despite this progress, the underlying rewriting design prin-

2.8 ciples have remained ill defined, and debugging has remained challenging (17, 19). It has been speculated that presence of 1.2 embedded transcriptional and translational control signals at 676 rewritten the termini of coding sequences (CDSs) as well as imprecise 3.2 essential genes genome annotations are the underlying cause. We hypothesized

0.8 that massive synonymous rewriting in conjunction with a system-

atic investigation of error causes will shed light onto the general 3.6 sequence design principles of how biological functions are pro- gramed into genomes. However, while some progress has been

0.4 made to study recoding schemes using individual genes and gene

4.0 clusters (21), the field currently lacks a broadly applicable high- 0 throughput error diagnosis approach to probe the rewriting of 0 0.2 0.4 0.6 entire genomes. Here, we report the chemical synthesis of Caulobacter ethensis- 2.0 (C. eth-2.0), a bacterial minimized genome composed of the C e . eth nom most fundamental functions of a bacterial cell. We present a -2.0 ge broadly applicable design–build–test approach to program the most fundamental functions of a cell into a customized genome sequence. By rebuilding the essential genome of Caulobacter crescentus (Caulobacter hereafter) through the process of chemi- B Essential C. eth-1.0 DNA part cal synthesis writing, we studied the genetic information content at the level of its essential genes. Tn hits begin end Results Essential Part List to Build C. eth-1.0. We conceived a bac- ORFs terial genome design encoding the entire set of essential DNA sequences from the freshwater bacterium Caulobacter (Fig. 1A). Caulobacter is recognized as an exquisite cell cycle model organism (22–25) for which multidimensional omics (26) and transcriptome- (27) and ribosome-profiling measure- ments have been integrated into a well-annotated genome model (28, 29). We computationally generated the entire list of essential DNA parts for building a from a previously published high-resolution transposon sequencing DNA dataset (30) that identified with resolution the pre- sequence rewriting cise coordinates of essential genes, including endogenous pro- TSS 500 bp moter sequences. DNA parts were extracted from the native RBS Caulobacter NA1000 genome sequence [National Center for Biotechnology Information (NCBI) accession no. NC 011916.1] according to predefined design rules and concatenated into a rewritten essential C. eth-2.0 gene digital genome design preserving gene organization and orien- Fig. 1. Part design, compilation, and chemical synthesis rewriting of the tation (Fig. 1A and SI Appendix) (31). The resulting 785,701- C. eth-1.0 genome. (A) Schematic representation of the digital design pro- bp genome design termed Caulobacter ethensis-1.0 (C. eth-1.0) cess; 1,745 DNA parts were extracted from the native Caulobacter NA1000 encodes for the most fundamental functions of a bacterial genome (gray) and reorganized into a rewritten genome design (blue) cell. Cumulatively, C. eth-1.0 consists of 1,761 DNA parts, comprising the entire list of essential genes required to run the basic oper- including 676 protein-coding, 54 noncoding, and 1,015 inter- ating system of a bacterial cell. Lines (blue) connect positions of DNA parts genic sequences. To select for faithful assembly and per- between native and rewritten genomes. (B) Workflow of the part identifi- mit stable maintenance in S. cerevisiae, auxotrophic marker cation and chemical synthesis rewriting process. Transposon sequencing was used to identify the entire set of essential DNA parts of Caulobacter at a genes (TRP1, HIS3, MET14, LEU2, ADE2) and a set of 10 resolution of a few base pairs. Absence of transposon insertions [transpo- autonomous replicating sequences (ARSs) were seeded across son (Tn) hits are plotted as gray lines] pinpoints the nondisruptable DNA the genome design (Table 1 and SI Appendix). Furthermore, regions within the native Caulobacter genome. Such essential DNA parts the pMR10Y (31) shuttle vector sequence, permitting stringent may encode for putative alternative ORFs, TSSs, or ribosome binding sites low copy replication in S. cerevisiae, E. coli, and Caulobacter, (RBSs) that are not required for functionality of the essential DNA part itself. was inserted at the native location of the Caulobacter origin of Computational sequence rewriting (Materials and Methods) was used to replication. erase putative sequence features that have not been assigned to a specific biologic function. The resulting rewritten DNA parts are fully defined and Sequence Rewriting of C. eth-1.0 to Enable de Novo Genome Syn- only encode for their desired function. thesis. We were unable to obtain 3- to 4-kb DNA building blocks of C. eth-1.0 from commercial DNA suppliers due to events that occurred primarily in the vicinity of 50 and 30 termini a multitude of synthesis constraints. Synthesis constraints are of protein-coding sequences (18, 19). Recently, to investigate a common problem of natural genome sequences, which have the impact of more complex rewriting schemes, de novo DNA evolved to maintain biological information rather than facilitate

2 of 10 | www.pnas.org/cgi/doi/10.1073/pnas.1818259116 Venetz et al. Downloaded by guest on September 29, 2021 Downloaded by guest on September 29, 2021 N atcategory part DNA erte ee eanfntoa ilietf ee nwhich in genes identify will whether all functional Testing ribo- 2). remain of (Table predicted genes 1,331) 95.3% all of rewritten of S2C), (1,021 76.7% motifs Fig. and stalling 1,730), putative Appendix, some of all (SI (1,648 of TSSs 3,229) 87.4% internal of removed (2,822 we 1B (Fig. Overall, ORFs rates Methods). and tune or rials fine (predicted motifs may sequence that and cryptic) (TSSs), sites elements tran- start internal genetic scriptional gene predicted of hypothetical ORFs, alternative sequences include of elements protein-coding number enabled within rewriting the maintained, present minimize was to genes annotated us sequence 676 acid amino the the of While versions. synonymous In by (33). codons 2) rewritten (Table the design yield to substitutions sequences base protein-coding 123,141 within add design deliberately for to sequence introduced 32) computational (31, substitutions algorithms used base we be the streamlining, can to synthesis genomes addition essential In the funda- identified. within information Furthermore, encoded code. of functions acid amino layers mental primary additional the beyond where exist study genome existing and of accuracy annotations powerful Features. the a probe Genetic offers to approach rewriting of experimental synthesis Number chemical that the reasoned Minimize We to chromosome Rewriting 785-kb Sequence the into blocks building DNA assem- of the standardized facilitate of to bly 1,045 sites additional erased restriction we hinder endonuclease Moreover, to known synthesis. 2), DNA of (Table chemical of composed content regions (GC) 4,342 are guanine-cytosine and high These stretches, homopolymeric 2). removed 93 repeats, and (Table 1,233 substitutions constraints base synthesis 10,172 5,668 introduced generated we termed and latively, design 32) genome reported (31, synthesis-optimized previously algorithms a our used design We DNA func- S1A). computational biological Fig. the Appendix, encoded (SI of the tions maintaining synthesis while to chemical genome easy facilitate 785-kb an would into sequence rewriting synthesize synonymous hypothe- computational We synthesis. that low-cost sized genome for showed bacterial amenable deposited not (31) all are sequences of work three-quarters than bioinformatics more that Recent synthesis. chemical yeast. in selec- replication and kanamycin selection for a AJ606312.1), Caulobacter no. and accession marker, tion (GenBank replicon copy RNA. noncoding and 2.5 ncRNA, assembly yeast. the in design direct † genome to the used of were maintenance that markers selection *Auxotrophic 19,143 16 assembly and replication Genome sequences Intergenic sequences Protein-coding the build to used list Part 1. Table eeze al. et Venetz ocdn sequences Noncoding h M1Ysutevco otisabodhs-ag K-ae low RK2-based host-range broad a contains vector shuttle pMR10Y The pMR10Y ARS markers* Click ncRNA rRNA tRNA Semiessential Essential Nonessential Redundant .eth-2.0. C. oa o fDAprs171785,701 1,761 parts DNA of no. Total † swl sUA akr R,adcnrmr CN elements (CEN) centromere and ARS, marker, URA3 as well as oriT ucinfrcnuainltase from transfer conjugational for function erpae 61 fall of 56.1% replaced we eth-2.0, C. uniySz b)Fato (%) Fraction (bp) Size Quantity ,1 60312.2 96,043 1,015 .eth-1.0 C. 1 1,7 14.5 59.8 83.9 114,270 471,072 113 660,789 462 676 02110.3 2,121 10 0.4 3,455 44 66,7 7. 1.9 60,477 14,970 86 15 49761.2 9,726 54 0601.4 10,670 1 ,5 0.8 6,352 0.2 0.6 5 1,884 4,387 7 3 eoedesign genome Cumu- eth-2.0. C. These eth-2.0. C. and .eth-2.0 C. .coli E. Mate- to lentv eei features genetic Alternative N ytei constraints synthesis DNA Type features genetic of of reduction rewriting massive Sequence 2. Table these corrected We prevented chromosome. which full-length ARS1213), the and of (ARS416 replication elements ARS tive yeast partial of prototrophy Sequencing with successful. reconstitute clones not were and megasegments 16 the genes from assemble to attempts functional Initial 2B). form (Fig. mark- will click (YJV04), ers genes yeast marker megaseg- auxotrophic engineered all adjacent an lacking in strain between assembly HIS3, chromosome split (TRP1, correct On ADE2) genes ments. and yeast auxotrophic LEU2, five MET14, introducing strat- 2A by marker (Fig. click egy a size) applied we in assembly, chromosome kb size) complete (38–65 in megasegments kb (19–22 16 and segments into chromosome further 236 37 initial and into these required blocks assembled block progressively DNA DNA We single a synthesis. man- only custom successfully and were Appendix), blocks (SI 236 ufactured of 235 rewriting, synthesis genome-scale sequence of on ease the Demonstrating 2A). complete assem- (Fig. the (32) 4-kb build to to 3- blocks from bly starting strategy assembly DNA four-tier defined of Synthesis fully Chemical a to lead ultimately will cell. knowl- artificial genes repair precise subsequent nonfunctional the The and of functional elements. remain genes genetic which of of of composed edge number genes artificial minimized defined a fully provide functional will Achieving necessary however, is functioning. code proper acid amino for the beyond information additional Codons Nme fsnnmu oo usiuin nrdcdo sequence on introduced substitutions † codon synonymous rewriting. of *Number rewriting Sequence *ubro N ytei osrit of constraints synthesis DNA of gel **Number field of PacI pulsed linearization by and analysis facilitate PmeI subsequent electrophoresis. to for unique chromosome backbone two assembled pMR10Y final the the the that within Note remained I-SceI). sites I-CeuI, PmeI, PacI, BspQI, k ¶ § ‡ eth-2.0. C. # eth-2.0. C. ein fhg Ccontent GC high of Regions ubro lentv Rsrsdn ihnte66CS of CDSs 676 the within residing ORFs alternative of Number ubro iooebnigsts(Bs nenlt CDSs. to internal (RBSs) sites binding ribosome of CDSs. Number to internal TSSs of Number oa ubro yeISrsrcinststa eermvd(aI BsaI, (AarI, removed were that sites restriction IIS type of number Total ubro eann eei etrswti D of CDS within features genetic remaining of Number RBS TSS ORFs ihG regions GC High etito sites Restriction Homopolymers Hairpins repeats Direct TAG TTA TTG erte codons* Rewritten substitutions Base eann eei features genetic Remaining ytei osrit* ,1 301 7,014 constraints** Synthesis sn es rnfrain oslc o the for select To transformation. yeast using Appendix) SI ‡ § † bp ≥8 bp ≥8 k # .eth-2.0 C. .eth-2.0. C. > . ihna10b window. 100-bp a within 0.8 ¶ sebisietfidtodefec- two identified assemblies .eth-1.0 C. .eh10C eth-2.0 C. eth-1.0 C. .eth-2.0 C. ecmuainlydvsda devised computationally We 1,154 ,3 295.3 82 1,730 ,3 1 76.7 87.4 310 407 1,331 3,229 ,9 799 4,342 6,290 1,047 oe135256.1 17.0 123,562 133,313 None None 3 666.9 46 139 94.2 10 173 0 4 76.9 140 606 8 1 87.2 113 880 46 .eth-1.0 C. NSLts Articles Latest PNAS .eth-2.0 C. into hoooei yeast in chromosome and .eth-2.0 C. .eth-2.0 C. .eth-2.0. C. 100 100 0 0 100 0 99.8 2 .eth-1.0 C. chromosome .eth-1.0 C. rcin(%) Fraction ed to leads | genes, f10 of 3 and and

BIOPHYSICS AND SYSTEMS BIOLOGY COMPUTATIONAL BIOLOGY A B C 16 Mega-segments URA3, ARS209 PmeI, PacI YJV04 YJV04 ARS1323 + C. eth-2.0 C. eth-2.0

TRP1 YJV04 ARS_Max2 Selective medium SD medium ADE2 Ura-, Trp-, His- Ura+, Trp+, His+ - - - + + + -Ura, -Trp, -His, ARS4 Met , Leu , Ade Met , Leu , Ade -Met, -Leu, -Ade

ARS C. eth-2.0 785,701 bp D E 727 16 Mega-segments Segment level in E. coli Marker YJV04 C. eth-2.0 3.0 37 Segments digest digest 1.0 236 Blocks

HIS3 945 kb 3.0 Mega-segment level in E. coli LEU2 ARS416 C. eth-2.0 1.0 ARS_HI 825 kb 771 kb ARS1018 750 kb C. eth- 2.0 3.0 ARS516 680 kb ARS1113 1.0 MET14 565 kb 0100 200 300 400 500 600 700 ARS1213 Sequencing coverage [log10] Genome coordinates [kb]

Fig. 2. Assembly of C. eth-2.0 in S. cerevisiae. (A) Schematic representation of the circular 785,701-bp C. eth-2.0 chromosome with six auxotrophic selection markers (red), 11 ARSs (black), and the restriction sites for PmeI and PacI (blue); 236 DNA blocks (green boxes) were assembled into 37 genome segments (blue boxes) and 16 megasegments (orange boxes) and further assembled into the complete C. eth-2.0 genome (outermost gray track). (B) The complete C. eth-2.0 chromosome was assembled in a single reaction from 16 megasegments by yeast spheroplast transformation and subsequent growth selection for auxotrophic TRP1 and LEU2 markers. (C) Growth selection on medium lacking Ura, Trp, His, Met, Leu, and Ade identified yeast clone 2 (C. eth-2.0) positive for all auxotrophic markers, while the parental strain (YJV04) fails to grow and requires synthetic defined (SD) medium. (D) Size validation of the 785-kb C. eth-2.0 chromosome by pulsed field gel electrophoresis. Digestion with PmeI and PacI releases a 771-kb portion of the C. eth-2.0 chromosome (arrow) from the shuttle vector pMR10Y. Undigested (marker) and PmeI- and PacI-digested yeast chromosomes (YJV04 digest) serve as controls. (E) DNA sequencing coverage at segment level (Top) and megasegment level (Middle) and the complete chromosome assembly (Bottom) are shown.

design errors and added five additional ARS sequences to pro- occurred in the final assembly of the C. eth-2.0 chro- mote efficient replication of the GC-rich C. eth-2.0 chromosome mosome, indicating a high sequence fidelity in the genome build in yeast. process. One-step transformation of the 16 corrected megasegments into yeast spheroplasts yielded two clones, one of which restored Mapping of Toxic Genes. It was previously reported in clone-based prototrophy for all six auxotrophic click markers, indicating genome sequencing studies that natural microbial genomes complete assembly of C. eth-2.0 (Fig. 2C). We subsequently con- contain genes encoding for toxic and dosage-sensitive expression firmed the presence of C. eth-2.0 as a single circular chromosome products (36, 37). We speculated that toxic genes residing on by pulsed field gel electrophoresis (Fig. 2D), diagnostic PCR C. eth-2.0 would prevent chromosomal maintenance in (SI Appendix, Fig. S3), and whole-genome sequencing (Fig. 2E). Caulobacter. Therefore, we tested the design in the form of the C. eth-2.0 has a high GC content exceeding 57%, while previ- 37 individual C. eth-2.0 chromosome segments for the presence ous chemically synthesized chromosomes (5, 8, 34) exhibit low of toxic genes. Quantification of conjugational transfer from E. GC contents closely matching the native yeast genome. So far, coli to Caulobacter in conjunction with sequencing demonstrated attempts to clone high GC sequences in yeast have proven to be that toxic genes were absent in 25 of 37 chromosome segments difficult (35). (SI Appendix, Fig. S5 and Table S4). However, we observed a To assess whether C. eth-2.0 is stably maintained in yeast, we drastic reduction in transfer efficiency for 12 segments, suggest- performed whole-genome sequencing on prolonged cultivation. ing presence of toxic genes that collectively cover 18.9 ± 3.6 kb After propagation for over 60 generations, we found no occur- in sequence (SI Appendix, Table S4). We carried out genetic rences of adaptive mutations or chromosomal rearrangements suppressor analysis and identified evolved strains that tolerated within C. eth-2.0, indicating stable maintenance in YJV04 (SI formerly toxic genome segments (Materials and Methods). Appendix, Fig. S4A). In agreement with this observation, elec- Whole-genome sequencing of suppressor strains led to the tron micrographs showed normal yeast cell morphologies for identification of 14 toxic genetic loci (SI Appendix, Table S5) parental cells and YJV04 bearing the C. eth-2.0 chromosome (SI that bear mutations alleviating toxicity. An additional three Appendix, Fig. S4B). chromosome segments acquired small deletions on selection for We sequence verified C. eth-2.0 at each assembly level to assess fast growth (SI Appendix, Table S6). Among the toxic genes, we the performance of the genome synthesis process (Fig. 2E). found three chromosome replication genes (dnaQ, dnaB, rarA), Across the 785-kb genome design, we detected a total of 21 six genes involved in LPS and fatty acid (fabB, nonsynonymous mutations (SI Appendix, Table S3). Thereof, 17 lptD, lpxD, accC, murU, waaF), two genes encoding interacting emanated from nonsequence perfect DNA blocks that were pro- RNA polymerase components (rpoC, topA), the S10-spc-alpha vided by one of two commercial suppliers. Only four additional ribosomal protein gene cluster (CETH 01304-01323), and the missense mutations within the genes argS (arginyl-tRNA syn- sodium-proton antiporter nhaA. Multiple of the identified toxic thetase) and fabI (acyl-carrier protein) and the ribosomal genes genes encode for interacting that form complexes, S7P and L12P were introduced during segment and megaseg- suggesting an imbalance in subunit dosage as a likely cause for ment assembly in yeast and E. coli, respectively. No additional toxicity. Overall, the observed fraction of 1.9% (14 of 730) toxic

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3) utemr,msaacdexpression misbalanced Furthermore, (37). .eth-2.0 C. TnSeq strain ee hog eei complementation. genetic through genes .eth-2.0 C. ± swl nareetwt h previously the with agreement in well is non-functional .%o oi ee dnie among identified genes toxic of 0.8% absence ofTninsertions accC ee.Fiuei complementation in Failure genes. hoooesget oag n lecrl)aesbetdt rnpsnsqecn TSq.Peec ftransposon of Presence (TnSeq). sequencing transposon to subjected are circle) blue and (orange segments chromosome .eth-2.0 C. a rvosybe reported been previously has ee,sc naayi will analysis an such genes, segments 1-37 chromosome .eth-2.0 C. ee,peiul essen- previously genes, .eth-2.0 C. .eth-2.0 C. C. eth-2.0 C. eth-2.0 .eth-2.0 C. .eth-2.0. C. .eth-2.0 C. eoewas genome chromosome B ee,we genes, 160K .eth-2.0 C. ucinlt a fthe of map Functionality (B) arrow). (orange gene 320K ucinlt seseto the of assessment Functionality (A) chromosome. 480K While 640K 20K 180K 340K 500K ob ipnal eetetrefrel sindantisense assigned formerly three the CCNA (sRNAs) were found transcripts features dispensable genetic be the Among to function. do essential features adopt genetic predicted not and annotated observed previously 6,290 81.5% the of level 6,290 rewritten functionality genome the high from within The CDS current 2). within (Table the features 799 genetic of of to of process refinement number the design to within reduced the lead During functionality will annotation. it features, biologic genetic hence, these and of dispensability of suggests genes rewritten Maintenance wild-type the cryptic, annotation. with (annotated, compared features predicted) genetic of or number the in semiessential reduction and rewritten essential all Functional of eth-2.0 530) of C. native (432 81.5% the found we to insertions and genome transposon assign ously sequence and the of rewriting optimization on introduced function- substitutions the assessed of to and ality conditions chromosome noncomplementing complementing native between and obtained the patterns compared insertion We along functionality. transposon gene test copies to 37 mutagenesis episomal transposon subjected as We bearing ments introduced. strains massive test modification the merodiploid sequence despite genes of native genes level to rewritten equivalent whether functionally asked We are functioning. gene proper for vr infiatnme fnnyoyossubstitutions nonsynonymous of intro- number were significant of substitutions sequences a base protein-coding ever, 133,313 within the duced of majority Sequences. Protein-Coding an large Beyond elicit Flexibility Design not Sequence do 43) sRNAs (42, encoded analysis function. chromosomally transcripts essential transcriptome of antisense by majority assigned identified the formerly that the suggests 4A). of (Fig. respectively Dispensability process, rewriting the during substitutions to 660K rpoC, 40K 200K 360K .eth-2.0 C. 520K and sufB, 680K ee ob ucinl(i.3B (Fig. functional be to genes .eth-2.0 C. 60K 220K 380K 540K .eth-2.0 C. aeil n Methods and (Materials .eth-2.0 C. atpD .eth-2.0 C. 80K hoooesget.Cumulatively, segments. chromosome 240K htaqie 6 7 n 2base 62 and 17, 16, acquired that 400K 00,R11 n 09 internal R0194 and R0151, R0109, 560K ugssta h ag aoiyof majority large the that suggests eoealwdu ounambigu- to us allowed genome .eth-2.0 C. .eth-2.0 C. 100K .eth-2.0 C. ee nops drastic a encompass genes 260K 420K Non-functional genes Functional genes 580K Non-essential genes .eth-2.0 C. NSLts Articles Latest PNAS hoooewt functional with chromosome hoooe Merodiploid chromosome. Caulobacter 120K ehave we eth-2.0, C. 280K hoooeseg- chromosome and .eth-2.0 C. 440K ee(learrow), (blue gene n al 3). Table and 600K aae S1). Dataset Caulobacter 140K 300K genome How- . | 460K f10 of 5 620K The

BIOPHYSICS AND SYSTEMS BIOLOGY COMPUTATIONAL BIOLOGY Table 3. Functionality of C. eth-2.0 genes according to cellular ity. For example, the two tRNA genes tRNATrp and tRNATyr processes remained functional despite base changes that were introduced Category Functional C. eth-2.0 genes* P value† within the anticodon arm to erase DNA synthesis constraints present within the wild-type sequences (Fig. 4B). In the case ‡ Translation 73.6% (81/110) 5.49E-03 of tRNATrp, we removed a Type IIS restriction site, and in ‡ Ribosomal proteins 60.6% (20/33) 2.01E-03 tRNATyr, a homopolymeric sequence pattern hindering DNA tRNA synthetases 81.8% (18/22) 6.14E-01 synthesis was removed. Both rewritten tRNA genes retained tRNAs 67.8% (19/28) 4.73E-02 their function as revealed by our transposon-based complemen- Translation factors 88.9% (24/27) 2.22E-01 tation measurements (Fig. 4B). These findings suggest that, 86.7% (13/15) 4.51E-01 even apart from protein-coding sequences, a high level of DNA replication 83.9% (26/31) 4.69E-01 sequence design flexibility exists to imprint biological functions Cellular processes 87.2% (123/141) 1.12E-02 into DNA. Cell cycle 87.5% (28/32) 2.52E-01 Cell envelope 86.9% (73/84) 8.48E-02 Level of Gene Functionality Among Cellular Processes. We reasoned Protein turnover 88.0% (22/25) 2.81E-01 that the analysis of gene functionality among different cellular Energy production 73.9% (34/46) 1.03E-01 processes would permit identification of gene classes harboring § 90.3% (121/134) 2.60E-04 ‡ high levels of transcriptional and translational control elements Hypothetical proteins 64.2% (34/53) 7.45E-04 within CDS. Assignment of gene functionality among cellular Total 81.5% (432/530) processes revealed that metabolic genes were enriched with over *Fraction of functional C. eth-2.0 genes as assessed by transposon sequenc- 90.3% functionality (P value of 2.60E-4) (Table 3). This supports ing. Numbers of functional genes vs. total gene numbers per class are shown the idea that metabolic genes contain a low level of transcrip- in parentheses. tional and translational control elements embedded within their † P values for functionality enrichment and deenrichment of different gene CDS. This finding correlates with the observation that regulation categories. ‡ of bacterial metabolism mainly occurs at the enzymatic level (44, Categories of genes that display a significant decrease in functionality. 45). However, hypothetical and ribosomal genes were underrep- §Categories of genes that display a significant increase in functionality. resented, with 64.2 and 60.6% functional C. eth-2.0 genes, respec- tively (P values of 5.45E-4 and 2.01E-3, respectively) (Table 3). were inserted within intergenic and noncoding regions, such as Based on these findings, we estimated that one-third of the tRNA genes, to facilitate the de novo DNA synthesis process. hypothetical essential genes encode for genetic features other We found that base substitutions within noncoding sequences than the annotated protein-coding sequence. Likewise, close to were frequently tolerated and did not impair gene functional- 40% of all ribosomal genes likely contain additional regulatory

AB C

Fig. 4. Sequence design flexibility within rewritten C. eth-2.0 genes. (A) Dispensability of antisense RNAs. Schematic depicting dispensable antisense tran- scripts embedded with CDSs of genes rpoC, sufB, and atpD (blue arrows). On synonymous rewriting, antisense transcripts CCNA R0109, CCNA R0151, and CCNA R0194 (doted arrows) internal to rpoC, sufB, and atpD acquired 16, 17, and 62 base substitutions, respectively. Essential chromosomal genes rpoC, sufB, and atpD carry disruptive transposon insertion (blue marks) in the presence of complementing C. eth-2.0 chromosome segments (blue marks) compared with the transposon insertion pattern of the wild-type control strain (green marks), indicating that antisense transcripts are nonessential. (B) Schematic depiction of the secondary structure of the rewritten tRNATrp and tRNATyr. Type IIS restriction sites (red letters; Left) and homopolymeric sequences (red letters; Right) hindering chemical synthesis of tRNA genes were erased by introducing base substitutions (blue) in the anticodon arms while maintaining the anticodons (gray box). Transposon testing reveals functionality of C. eth-2.0 tRNA genes. (C) Functionality testing of C. eth-2.0 . On complementation with C. eth-2.0 operons, chromosomal genes tolerate disruptive transposon insertions (blue marks) throughout the native , leading to simultaneous inactivation of multiple native genes.

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TSSs implies impre- incorrect This and of regions ancestral instances promoter the misannotated 52 of to nonfunctional annotation pinpointed cise 98 Methods) Error features, the and efforts. (Materials genetic of sug- annotation rewriting classification essential genome previous on cause additional evaded function of have their which presence lost the genome. that organism’s gested genes an of of accuracy Discovery annotation the pow- genetic validate sequence a and unknown protein-coding the hitherto offers within encoded map rewriting elements regulatory to synthesis approach chemical experimental of erful CDS. process Within the Features that Genetic Essential of Discovery synthesis chemical using by DNA. chromosomes build rewritten the artificial combined simplify of are ultimately genes will process which synthetic modules, multiple functional when into arise likely effects fitness not additive that do idea the support multiple findings These of genes. inactivation the simultaneous to throughout leading insertions sequence, native transposon operon disruptive chaperone tolerated protein Both the membrane for the obtained and were insertions poson function the the dis- that insertions chromosomal suggesting the transposon mRNA, of of found polycistronic maintenance We the and (48). rupting generation shape the cell for bacterial critical machinery. is biosynthesis complex peptidoglycan This wall cell of coordination exam- One the counterparts. includes native ple their com- of modules function biological the functional plementing encompass formerly fully indeed rewritten 41 may synthesis chemical within the that insertions suggesting transposon transcript. such essential mRNA polycistronic gene observed a multiple of We truncation of to inactivation due simultaneous products to for leading searched operons essential when thus within We arise insertions combined. transposon might chromosomal were effects genes fitness synthetic additive multiple that hypothesized we rewritten Modules. Rewritten modules cell. core bacterial multigene the of other within and embedded genes are How- ribosomal elements genes. regulatory coexpressed of metabolic number high of a sequences ever, protein-coding embedded the is elements a within regulatory that essential suggests additional analysis of our level sum, low In many (47). of genes control operon com- transcriptional ribosomal in concerted ribosome the energy functional on of depends cellular plexes protein biogenesis of as the consumer Furthermore, surprising, major (46). not the is is this synthesis perspective, regulatory From gene sequence. a protein-coding their within embedded elements .eth-2.0 C. lhuhasgicn muto hmclsynthesis chemical of amount significant a Although Caulobacter .eth-2.0 C. .eth-2.0 C. .Smlrpten ftrans- of patterns Similar 4C). (Fig. counterparts mreBCD-rodA prn nops ul ucinlBiological Functional Fully Encompass Operons ee r ucinlo nidvda basis, individual an on functional are genes mreBCD-rodA prngns(i.4C (Fig. genes operon prn hc sivle nthe in involved is which operon, Caulobacter Caulobacter prni opeetdby complemented is operon groEL-ES eoe nol 13 only In genome. eoeincluding genome and .eth-2.0 C. .eth-2.0 C. yidC-yidA ereasoned We aae S3), Dataset prn(49) operon Caulobacter genes genes (50). h idtp euneeeet ptemo ofntoa eldivision cell by nonfunctional measured lacZ as of expression gene upstream restores elements genes sequence wild-type sig- the coupling translational (murC (RBSs; including regions sites cluster, binding present ribosome gene elements internal (murG ), division control nals cell genetic reveals the arrows). (blue rewriting within functional synthesis are genes Chemical rewritten (B) of majority genes large the nonfunctional arrows), four the of tion netosi h idtp oto genmrs n ncomplementation on and marks) (green control with wild-type the in insertions the across diagnosis Fault 5. Fig. eddt nae h xc oeua ucin fteegenetic these of functions molecular are exact studies the additional unravel While to 5B). needed (Fig. conditions control metabolic attenu- may the transcriptional This a element. hairpin resembles ation a which contains structure, sequence wild-type secondary the that revealed nonfunctional (29) analysis CDS the annotated short of a found upstream we Finally, for 5B). 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BIOPHYSICS AND SYSTEMS BIOLOGY COMPUTATIONAL BIOLOGY control elements within the cell division gene cluster, we found ers. These include alternative reading frames as well as hidden that repair of the sequence upstream of the ftsZ gene restored control elements embedded within protein-coding sequences of the ftsZ expression levels (Fig. 5C). Similarly, insertion of the essential genes. wild-type sequence elements into the C. eth-2.0 genes murC, ftsQ, Rewriting of 56% of all codons resulted in complete rewriting and murG also restored gene expression (Fig. 5C). This suggests of the essential Caulobacter transcriptome. Despite incorporat- that, after missing essential genetic elements are identified, error ing such drastic changes at the level of mRNA, our functionality causes can rapidly be deduced to allow for rational repair of analysis revealed that over 432 of the transcribed essential genes genome designs. Furthermore, identification and error diagnosis of C. eth-2.0 corresponding to 81.5% of all rewritten essential of noncomplementing genes will provide a formidable opportu- genes are equal in functionality to natural counterparts to sup- nity to uncover DNA design principles that will further improve port viability. This result suggests that, in most essential genes, our capabilities in programing biological functions into synthetic the primary mRNA sequence, the secondary structure, or the chromosomes. codon context has no significant influence on biological function- ality. This finding is surprising given the fact that previous studies Discussion on individual genes reported that codon translation in vivo is con- C. crescentus has emerged as an important model organism for trolled by many factors, including codon context (56). Further- understanding the regulation of the bacterial cell cycle (25, 51, more, our findings suggest that the vast majority of the probed 52). A notable feature of Caulobacter is that the regulatory events ORFs encode exclusively for proteins and that other layers of that control polar differentiation and cell cycle progression are genetic control do not seem to play a significant role. Among the highly integrated and occur in a temporally restricted order (53). 134 enzyme-encoding genes that make up the metabolic core net- The advent of genomic technologies has enabled global analy- work of C. eth-2.0, the level of functional genes is even over 90%, ses that have revolutionized our understanding of Caulobacter suggesting that rewritten biosynthetic pathways retain their func- genetic core networks that control the lifecycle (26–29). In recent tionality in most cases. A possible explanation for the high pro- years, many components of the regulatory circuit have been iden- portion of functional metabolic genes might be the fact that regu- tified, and simulation of the circuitry has been reported (25, 54). lation of essential metabolic functions occurs rather by allosteric More recent experimental work using transposon sequencing interactions at the level of enzymes than at the level of gene has shown that 12% of the Caulobacter genome is essential for expression. survival under laboratory conditions (30). The identified set of In addition to 432 functional rewritten genes, our study pre- essential sequences included not only protein-coding sequences cisely mapped 98 genes that lost functionality on synonymous but also, regulatory regions and noncoding elements that collec- rewriting as detected by our transposon-based functionality tively store the genetic information necessary to run a living cell. assessment. Since retaining solely the protein-coding sequences Of the individual DNA regions identified as essential, 91 were of these genes is not sufficient for their functionality, it is reason- noncoding regions of unknown function, and 49 were genes pre- able to conclude that these genes are misannotated or contain sumably coding for hypothetical proteins with function that is hitherto unknown essential genetic elements embedded within unknown. their CDS. Alternatively, it is also possible that a subset of Although classical genetic approaches dissect the functioning these genes encode for RNA rather than protein-coding func- of biological systems by analyzing individual native genes, uncov- tions. Taken together, our genome rewriting approach can be ering the function of essential genes has remained very chal- used to experimentally validate the annotation fidelity of entire lenging. Herein, we show that the rewriting of entire genomes genomes. through the process of chemical synthesis provides a power- Altogether, the identified set of 98 nonfunctional genes corre- ful and complementary research concept to understand how sponds to less than 20% of the essential genome of C. eth-2.0 and essential functions are programed into genomes. Contempo- precisely revealed where we currently have gaps in our knowl- rary synthetic genome projects (3, 5, 8) have largely maintained edge that persisted despite previous omics-informed genome natural genome sequences, implementing only modest design reannotation efforts. In the future, it will be interesting to changes to increase the likelihood of functionality. However, unravel why rewriting renders particular genes nonfunctional. conservative genome design misses a key opportunity of chemical These studies will shed light onto hitherto unknown transcrip- DNA synthesis: the rewriting of DNA to advance our under- tional and translational control layers embedded within protein- standing of how fundamental biological functions are encoded coding sequences that are of fundamental importance for proper within genomes. Indeed, synthetic autonomous bacteria, such as gene functioning. Targeted repair of identified nonfunctional M. mycoides strain JCVI-syn3.0 made up of 473 genes within a C. eth-2.0 genes, as exemplified within the subset of the four 531-kb genome (5), resulted in the creation of a replicative cell. faulty cell division genes murG, murC, ftsQ, and ftsZ, will lead However, it also encompasses 149 genes with unknown func- to the discovery of genetic features, such as the essential attenu- tions (84 labeled as “generic,” and 65 labeled as “unknowns”) ator element identified upstream of the ftsZ gene, the function of (55). This corresponds to over one-third of its gene set. While which is currently unknown. We acknowledge that the 98 iden- these studies were highly valuable to experimentally deter- tified nonfunctional genes are still poorly understood, yet our mine the core set of genes for an independently replicating findings on C. eth-2.0 serve as an excellent starting point to close cell, they did not probe the genetic information content of its current knowledge gaps in essential genome functions toward essential genes. rational construction of a synthetic organism with a fully defined By rebuilding the essential genome of Caulobacter through genetic blueprint. the process of chemical synthesis rewriting, we assessed the On the level of de novo DNA synthesis, we herein demonstrate essential genetic information content of a bacterial cell on the how chemical synthesis rewriting facilitates the genome synthe- level of its protein-coding sequences. Within the 785,701-bp sis process. To simplify the entire genome build process, we used genome of C. eth-2.0, we used sequence rewriting to reduce sequence design algorithms (31, 32) and collectively introduce the number of genetic features present within protein-coding 10,172 base substitutions to remove 5,668 DNA synthesis con- sequences from 6,290 to 799. Overall, we introduced 133,313 straints, including 1,233 repeats, 93 homopolymeric stretches, base substitutions, resulting in the synonymous rewriting of and 4,342 regions of high GC content. Successful low-cost syn- 123,562 codons. We speculated that synonymous rewriting of thesis and subsequent higher-order assembly of C. eth-2.0 into protein-coding sequences maintains the encoded the complete chromosome exemplify the utility of our approach sequences but likely erases additional genetic information lay- to rapidly produce designer genomes.

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Transposon by Caulobacter 113:E6859–E6867. 10:e1004463. albce ethensis Caulobacter ee,tasoo eunigwsapplied was sequencing transposon genes, uli cd Res Acids Nucleic 11:e1004831. 328:1295–1297. 304:983–987. osrc a eie sn h Illumina the using verified was construct 105:11340–11345. rcNt cdSiUSA Sci Acad Natl Proc etStrains. Test .coli E. NSLts Articles Latest PNAS 45:6971–6980. 646(oBC) h work The B.C.). (to 166476 EH. eoesequence. genome CETH2.0 1- into S17-1 a Biotechnol Nat Sequence-confirmed .eth-2.0 C. obnhakthe benchmark To 113:E5588–E5597. o ytBiol Syst Mol .eth-2.0 C. C yt Biol Synth ACS Caulobacter .eth-2.0 C. | ee,a genes, 25:584– genes. f10 of 9 7:528– urich ¨

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