Chemical synthesis rewriting of a bacterial genome to achieve design flexibility and biological functionality

Abstract

Understanding how to program biological functions into artificial DNA sequences remains a key challenge in synthetic genomics. Here, we report the chemical synthesis and testing of Caulobacter ethensis-2.0 (C. eth-2.0), a rewritten bacterial genome composed of the most fundamental functions of a bacterial cell. We rebuilt the essential genome of Caulobacter crescentus through the process of chemical synthesis rewriting and studied the genetic information content at the level of its essential genes. Within the 785,701-bp genome, we used sequence rewriting to reduce the number of encoded genetic features from 6,290 to 799. Overall, we introduced 133,313 base substitutions, resulting in the rewriting of 123,562 codons. We tested the biological functionality of the genome design in C. crescentus by transposon mutagenesis. Our analysis revealed that 432 essential genes of C. eth-2.0, corresponding to 81.5% of the design, are equal in functionality to natural genes. These findings suggest that neither changing mRNA structure nor changing the codon context have significant influence on biological functionality of synthetic genomes. Discovery of 98 genes that lost their function identified essential genes with incorrect annotation, including a limited set of 27 genes where we uncovered noncoding control features embedded within protein-coding sequences. In sum, our results highlight the promise of chemical synthesis rewriting to decode fundamental genome functions and its utility toward the design of improved organisms for industrial purposes and health benefits.

Keywords: Caulobacter crescentus; chemical genome synthesis; de novo DNA synthesis; genome rewriting; synonymous recoding.

Fig. 1.

Part design, compilation, and chemical synthesis rewriting of the C. eth-1.0 genome. (A) Schematic representation of the digital design process; 1,745 DNA parts were extracted from the native Caulobacter NA1000 genome (gray) and reorganized into a rewritten genome design (blue) comprising the entire list of essential genes required to run the basic operating system of a bacterial cell. Lines (blue) connect positions of DNA parts between native and rewritten genomes. (B) Workflow of the part identification and chemical synthesis rewriting process. Transposon sequencing was used to identify the entire set of essential DNA parts of Caulobacter at a resolution of a few base pairs. Absence of transposon insertions [transposon (Tn) hits are plotted as gray lines] pinpoints the nondisruptable DNA regions within the native Caulobacter genome. Such essential DNA parts may encode for putative alternative ORFs, TSSs, or ribosome binding sites (RBSs) that are not required for functionality of the essential DNA part itself. Computational sequence rewriting (Materials and Methods) was used to erase putative sequence features that have not been assigned to a specific biologic function. The resulting rewritten DNA parts are fully defined and only encode for their desired function.

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.

Fig. 3.

Fault diagnosis and error isolation across the C. eth-2.0 chromosome. (A) Functionality assessment of the C. eth-2.0 chromosome. Merodiploid strains bearing episomal C. eth-2.0 chromosome segments (orange and blue circle) are subjected to transposon sequencing (TnSeq). Presence of transposon insertions (blue marks) in a previously essential chromosomal gene (gray arrows) indicates functionality of the homologous C. eth-2.0 gene (blue arrow), while absence of insertions indicates a nonfunctional C. eth-2.0 gene (orange arrow). (B) Functionality map of the C. eth-2.0 chromosome with functional genes (blue arrows), nonfunctional genes (orange arrows), and nonessential control genes (gray arrows).

Fig. 4.

Sequence design flexibility within rewritten C. eth-2.0 genes. (A) Dispensability of antisense RNAs. Schematic depicting dispensable antisense transcripts 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 tRNA^Trp and tRNA^Tyr. 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 operons. On complementation with C. eth-2.0 operons, chromosomal genes tolerate disruptive transposon insertions (blue marks) throughout the native operon, leading to simultaneous inactivation of multiple native genes.

Fig. 5.

Fault diagnosis and repair across the C. eth-2.0 chromosome. (A) Fault diagnosis across the C. eth-2.0 cell division gene cluster. Transposon insertions in the wild-type control (green marks) and on complementation with C. eth-2.0 cell division genes (blue marks) are shown. With the exception of the four nonfunctional genes murG, murC, ftsQ, and ftsZ (orange arrows), the large majority of rewritten genes are functional (blue arrows). (B) Chemical synthesis rewriting reveals genetic control elements present within the cell division gene cluster, including translational coupling signals (murG), internal ribosome binding sites (RBSs; ftsQ), extended promoter regions (murC), and attenuator sequences upstream of ftsZ. (C) Insertion of the wild-type sequence elements upstream of nonfunctional cell division genes restores gene expression as measured by $\beta$-galactosidase assays using lacZ reporter gen fusions.