A semi-synthetic organism with an expanded genetic alphabet

Abstract

Organisms are defined by the information encoded in their genomes, and since the evolution of life, this information has been encoded using a two base pair genetic alphabet (A-T and G-C). In vitro, the alphabet has been expanded to include several unnatural base pairs (UBPs)1–3. We have developed a class of UBPs formed between nucleotides bearing hydrophobic nucleobases, exemplified by the pair formed between d5SICS and dNaM (d5SICS-dNaM, Fig. 1a), which is efficiently PCR amplified1 and transcribed4,5 in vitro, and whose unique mechanism of replication has been characterized6,7. However, expansion of a organism’s genetic alphabet presents new and unprecedented challenges: the unnatural nucleoside triphosphates must enter the cell; endogenous polymerases must be able to faithfully incorporate the unnatural triphosphates into DNA within the complex cellular milieu; and finally, the UBP must be stable in the presence of pathways that maintain the integrity of DNA. Here we show that an exogenously expressed algal nucleotide triphosphate transporter efficiently imports the triphosphates of both d5SICS and dNaM (d5SICSTP and dNaMTP) into E. coli, and that the endogenous replication machinery uses them to accurately replicate a plasmid containing d5SICS-dNaM. Neither the presence of the unnatural triphosphates nor the replication of the UBP introduces a significant growth burden. Lastly, we find that the UBP is not efficiently excised by DNA repair pathways. Thus, the resulting bacterium is the first organism to stably propagate an expanded genetic alphabet.

A semisynthetic organism engineered for the stable expansion of the genetic alphabet

“…To determine whether the optimized transporter system of YZ3 facilitates high UBP retention, we constructed three plasmids that position the UBP within the 75-nt TK1 sequence (14) [with a local sequence context of d(A-NaM-T)]. These include two high-copy pUC19-derived plasmids, pUCX1 [referred to in previous work as pINF (14)] and pUCX2, as well as one low-copy pBR322-derived plasmid, pBRX2 (Fig.…”

“…In our previously reported SSO (hereafter referred to as DM1), the transporter was expressed from a T7 promoter on a multicopy plasmid (pCDF-1b) in E. coli C41(DE3), and its toxicity mandated carefully controlled induction (14). In its native algal cell, PtNTT2's N-terminal signal sequences direct its subcellular localization and are ultimately removed by proteolysis.…”

“…True expansion of the genetic alphabet requires the unrestricted retention of multiple UBPs at any loci and in any sequence context. Moreover, several limitations were already apparent with the nascent SSO (14). First, although expression of the nucleoside triphosphate transporter enabled E. coli to import dNaMTP and d5SICSTP, its expression caused the SSO to grow poorly, with doubling times twice that of the parental strain.…”

“…Despite lacking complementary hydrogen bonding, we demonstrated that the dNaM-d5SICS UBP is well replicated by a variety of DNA polymerases in vitro (7-10), and that this efficient replication is mediated by a unique mechanism that draws upon interbase hydrophobic and packing interactions (11,12). These efforts then culminated in the first progress toward the creation of an SSO in 2014, when we reported that Escherichia coli grown in the presence of the corresponding unnatural nucleoside triphosphates (dNaMTP and d5SICSTP), and provided with a plasmid-encoded nucleoside triphosphate transporter (NTT2) from Phaeodactylum tricornutum (which we denote as PtNTT2) (13), is able to import the unnatural triphosphates and replicate a single dNaM-d5SICS UBP on a second plasmid (14).Although this first SSO represented an important proof-ofconcept, the generality of the expanded genetic alphabet remained unclear, as retention of the UBP was explored at only a single locus and in only a single sequence context. True expansion of the genetic alphabet requires the unrestricted retention of multiple UBPs at any loci and in any sequence context.…”

Exploring the potential impact of an expanded genetic code on protein function

“…However, it remains to be determined whether genetically encoded ncAAs can provide a unique evolutionary advantage that cannot be achieved by either single or multiple canonical amino acid mutations at other sites in a protein or the incorporation of nonproteinogenic cofactors. We expect that further advances in site-specific ncAA incorporation technology in combination with other new mutagenesis techniques, such as TAG codon scanning, multiplex automated genomic engineering, and multiplex iterative plasmid engineering, may provide the opportunity to address this question at a more global whole-organism level (43)(44)(45)(46).…”