Construction and iterative redesign of synXVI a 903 kb synthetic Saccharomyces cerevisiae chromosome

Abstract

The Sc2.0 global consortium to design and construct a synthetic genome based on the Saccharomyces cerevisiae genome commenced in 2006, comprising 16 synthetic chromosomes and a new-to-nature tRNA neochromosome. In this paper we describe assembly and debugging of the 902,994-bp synthetic Saccharomyces cerevisiae chromosome synXVI of the Sc2.0 project. Application of the CRISPR D-BUGS protocol identified defective loci, which were modified to improve sporulation and recover wild-type like growth when grown on glycerol as a sole carbon source when grown at 37˚C. LoxPsym sites inserted downstream of dubious open reading frames impacted the 5' UTR of genes required for optimal growth and were identified as a systematic cause of defective growth. Based on lessons learned from analysis of Sc2.0 defects and synXVI, an in-silico redesign of the synXVI chromosome was performed, which can be used as a blueprint for future synthetic yeast genome designs. The in-silico redesign of synXVI includes reduced PCR tag frequency, modified chunk and megachunk termini, and adjustments to allocation of loxPsym sites and TAA stop codons to dubious ORFs. This redesign provides a roadmap into applications of Sc2.0 strategies in non-yeast organisms.

Fig. 1. Schematic construction and debugging of synXVI.

Five haploid strains, 5, 6, 8, 10 and 11 were constructed on BY4741 backgrounds (coloured in blue) with contiguous synthetic DNA (coloured in red). Switching mating types facilitated meiotic crossover exploitation to generate chromosome arms. Strain 9, bearing all synXVI synthetic DNA for the left chromosome arm to megachunk O, was generated by crossing Strains 5 and 6 to yield Strain 7, which was then crossed with Strain 8. Strain 12, bearing all synthetic DNA from synXVI right chromosome arm plus megachunks O to Q was generated by crossing Strain 10 and Strain 11. Strain 15 bearing synthetic DNA from chunk B3 to BB4 was generated by crossing Strain 12 and Strain 9. Debugging, discrepancies, and reinsertion of missing synthetic chunks and megachunks were completed in Strain 15. Subsequently the missing sub-telomeric regions on synXVI L-arm were reinserted, resulting in fully synthetic Strain 30. The centromere CEN16 is marked in magenta on chunk Q4.

Fig. 2. Strain harbouring synthetic megachunk exhibits reduced growth.

BY4741 and Strain 15 harbouring synthetic chunks B3, B4 and megachunks from C to BB4 exhibited reduced growth on non-fermentable carbon source (glycerol) and at 37 °C, when compared to a BY4741 control.

Fig. 3. Identification of defective loci using backcrossing and validation using transcriptional reporter assays.

A Synthetic sequence shown is depicted in red and wildtype sequence in blue. From 30 strains, 86% of fit F3 progeny had no synthetic DNA between loci F-I, 100% had no synthetic DNA on chunks X3-X4, while 73% and 63% of F3 unfit progeny had synthetic DNA between megachunks F-I and X3-X4, respectively. B Strain 11/BY4742 cross progeny analysis yielded 19 strains with a crossover between megachunks V and Y. Strains bearing synthetic tags for X4 were unfit. A high proportion of strains with synthetic DNA at AXL1 (YPR122W) and YLH47 (YPR125W) showed poor fitness. C Schematic diagrams depicting the loxPsym sites in five strains encoded on BY4741 chromosome XVI, transcription start sites (TSS) shown as a red line and corresponding spot assays on various media to the right. From top to bottom, BY4741, BY4741 with synthetic CTR1 (YPR124W; Strain 29), BY4741 with a YPR123C loxPsym in the 5’ UTR of CTR1 in duplicate (Strain 30), BY4741 with the synthetic coding sequence for CTR1 (Strain 31), BY4741 with the CTR1 loxPsym (Strain 32). D Six BY4741 strains with GFP constructs in pRS413 with 1 kb upstream from CTR1 or GIP3, and corresponding fluorescence data. Fluorescence data shows the median fluorescence intensity of GFP⁺ gated cells of biological triplicates in mid exponential growth represented by a square, triangle and circle (raw data in Supplementary Fig. 4). From top to bottom, a GFP construct with BY4741 CTR1 5’UTR sequence with GFP, CTR1, and AXL1 loxPsym sites, the YPR123C loxPsym site only, the AXL1 loxPsym only, the GIP3 loxPsym, and the BY4741 sequence of GIP3 followed by GFP.

Fig. 4. Effect of pRS413-tRNA array on growth of synXVI.

BY4741 and strain 20 harbouring synthetic megachunks A2 to BB4 were transformed with a pRS413 empty vector, and a pRS413 vector harbouring 17 tRNAs removed in the design of chromosome XVI (Strains 20 and 21, respectively). Strains demonstrated impaired growth on non-fermentable carbon sources at 37 ˚C. Introduction of the 17 tRNA genes shows improved fitness in the synthetic haploid strain compared to the vector-only control at 37 ˚C, which is especially pronounced on YP-Glycerol medium.

Fig. 5. Potential design feature changes as a result of lessons learned in the design and construction of Sc2.0.

The synthetic chromosome XVI sequence originally designed in BioStudio was modified with Geneious software to account for changes in technology and throughput of DNA sequencing. Chunk and megachunk boundaries were moved to avoid possible insertion into non-chromosome XVI loci during construction. LoxPsym sites assigned to dubious ORFs have been removed. ORFs now defined as putative or dubious ORFs have had the TAG → TAA stop codons recoded, when they overlap with coding sequences of other genes. PCR tags were minimised, due to the developments in long read sequencing technologies now ubiquitously available. This redesigned chromosome is included as a supplementary file.