To our knowledge this is the first report of the cloning of a gram-negative bacterial genome in S. cerevisiae
and the second report of cloning a complete circular genome that uses the standard genetic code (18
). DNA sequence analysis showed that the complete MED4 genome was present in the yeast clones. We observed a few mutational differences (primarily single base substitutions) between two sequences of the native Prochlorococcus
MED4 genome and a yeast clone of that genome. However, because MED4 is maintained by serial passage without clonal isolation, there is no reason to believe that any of these differences arose by mutation during propagation in yeast.
There are two major reasons that the Prochlorococcus
genome may be sustainable in yeast. First, it has a high incidence of ACS sites due to its high AT content, and these may be active as yeast origins of replication. Second, it is likely that most of the Prochlorococcus
genes are not accurately expressed in yeast due to differences between the mechanisms of prokaryotic and eukaryotic transcription and translation. Prokaryotic promoters generally contain defined −35 and −10 regions, typically within 50 bp of the start of a gene. However, transcription in cyanobacteria may be more complicated due to additional RNA polymerase subunits γ and δ. Analysis of transcription start sites in Prochlorococcus
MED4 has shown similar upstream elements to those of yeast (e.g. TATAAT or TATTAT) (28–30
). Thus, transcription initiation of the prokaryotic genome in yeast might be possible. However, other studies involving vaccine development in yeast have shown that transcripts of foreign AT-rich genes are prematurely polyadenylated at unexpected sites (31
). The native 1400 bp fragment C gene, a subunit of the tetanus toxin derived from the bacterium Clostridium tetani
(GC content 28.6%), could not be expressed in its entirety from a yeast GAL
promoter until a synthetic version was engineered with a GC content of 47%. Judging from previous studies expressing constructs up to 3.5 kb, sequences with a GC content below 40% resulted in truncated mRNA transcripts (32
Translation of Prochlorococcus
transcripts would require a high degree of similarity between the ribosome binding sites (Shine–Dalgarno sequences) and the yeast translation initiator consensus sites. This does not seem to be the case for cyanobacteria and yeast. Shine–Dalgarno (S–D) sequences typically have a consensus sequence GGAGG or a degenerate version of this which is within 5–10 bp upstream of the translation start codon. Cyanobacterial and chloroplast genes show very few ribosome binding sites in their 5′-UTR regions that can be classified as S–D consensus sequences even though the 16S rRNA in these organisms still retains the complementary (CCUCC) to the S–D consensus sequence (33
). While the translation initiation site in polycistronic prokaryotic mRNAs is usually selected by base pairing of the S–D sequence with ribosomal RNA, initiation in eukaryotes is facilitated by a scanning mechanism whereby the small 40 S ribosomal subunit and additional co-factors (eIF2, eIF3, eIF4C, Met-tRNAi and Guanosine-5′-triphospate (GTP)) bind the 5′-cap structure of the mRNA and then migrate down the untranslated leader sequence scanning for the first AUG codon (35
). Translation initiation of prokaryotic mRNA in yeast could be misplaced toward downstream AUG codons.
Additionally, there may be translational differences due to differences in codon usage between Prochlorococcus
and yeast. Although the initiation step of mRNA translation is considered rate-limiting, codon usage is known to affect elongation rate and can be a limiting factor in product yield. Codon usage tables showed a bias in codon usage between Prochlorococcus
MED4 and S. cerevisiae
. For example, MED4 uses the glycine GGA codon and the leucine codon TTA twice as much compared to yeast (Supplementary Figure S7
We did not observe a toxic effect as far as growth rates of these yeast clones are concerned. Deep sequencing analysis confirmed the presence of all Prochlorococcus genes inside the yeast host; however, several bacterial genes did not complement the yeast host genotype. We conclude that there is limited expression of Prochlorococcus genes in yeast and that the Prochlorococcus genome is stably maintained under selective conditions as observed for other bacterial genomes cloned in yeast thus far. We see these results as a proof of concept that bacteria using the standard genetic code which remain difficult to isolate or transform can be cloned and maintained in yeast. With the growing number of whole-genome sequences currently available, expanding the range of bacteria that can be co-transformed into yeast is a valuable stepping stone to the study of genetically intractable species.