A supernumerary designer chromosome for modular in vivo pathway assembly in Saccharomyces cerevisiae

Abstract

The construction of microbial cell factories for sustainable production of chemicals and pharmaceuticals requires extensive genome engineering. Using Saccharomyces cerevisiae, this study proposes synthetic neochromosomes as orthogonal expression platforms for rewiring native cellular processes and implementing new functionalities. Capitalizing the powerful homologous recombination capability of S. cerevisiae, modular neochromosomes of 50 and 100 kb were fully assembled de novo from up to 44 transcriptional-unit-sized fragments in a single transformation. These assemblies were remarkably efficient and faithful to their in silico design. Neochromosomes made of non-coding DNA were stably replicated and segregated irrespective of their size without affecting the physiology of their host. These non-coding neochromosomes were successfully used as landing pad and as exclusive expression platform for the essential glycolytic pathway. This work pushes the limit of DNA assembly in S. cerevisiae and paves the way for de novo designer chromosomes as modular genome engineering platforms in S. cerevisiae.

Figure 1.

Modular genome engineering strategy. Schematic representation of the design and construction of neochromosomes (A), as well as screening based on fluorescence, size and sequencing (B). The data in panel B represent the results pertaining to the assembly of the 100 kb neochromosome in strain IMF6. The red bar in the top left of the fluorescence microscopy pictures (40x) represents 10 μm. The CHEF gel used to determine the size of the neochromosomes, contains two transformants (#3 and #4) of the 100 kb neochromosome transformation. The neochromosomes are either undigested (undig.) by I-SceI and therefore in their circular form, or in-plug linearized by I-SceI digestion (dig.) and therefore in linear form, which can be visualised on CHEF gel.

Figure 2.

50 and 100 kb neochromosome designs. The outer circle depicts the in vivo assembly of the 100 kb neochromosome from 44 fragments present in strain IMF6. The inner circle depicts the in vivo assembly of the 50 kb neochromosome from 23 fragments present in strain IMF2.

Figure 3.

Transformation efficiency and fidelity. (A) Transformation efficiencies for the in vivo assembly of 100 and 50 kb synthetic chromosomes. Both neochromosomes have been assembled with 2.5 kb (dark blue), 5 kb (lighter blue) and 10 kb (lightest blue) fragments. The number above each bar indicates the number of assembled fragments. Data were normalized to 10⁸ cells using controls on YPD plates, performed in biological duplicates (replicate 1 and 2) in technical triplicates for replicate 1 and technical duplicates for replicate 2. The asterisks indicates that transformation with 5 kb fragments resulted in a significantly different transformation efficiency compared to both neighbouring efficiencies (two-tailed paired homoscedastic t-test P< 0.05). (B) Assembly fidelity. In vivo assembly of the 100 kb NeoChr12 from 43 fragments. Eleven colonies that grew on selective medium were tested for mRuby2 and mTurquoise2 fluorescence. Neochromosomes were subsequently screened based on their size on CHEF gel and lastly by Illumina sequencing. The percentage of correct colonies for each screening round is indicated.

Figure 4.

Neochromosomes as landing pads. (A) 35 kb of non-coding DNA was simultaneously in vivo assembled and inserted by CRISPR/Cas9 at the mTurquoise2 locus of the 100 kb neochromosome in IMF6 and of the 50 kb neochromosome in IMF2, resulting in 135 kb (IMF11) and 85 kb (IMF12) neochromosomes, respectively. (B) 35 kb of glycolytic genes were simultaneously in vivo assembled and inserted using CRISPR/Cas9 at the mTurquoise2 locus of the 100 kb neochromosome in IMF6 and of the 50 kb neochromosome in IMF2 resulting in 135 kb (IMF13) and 85 kb (IMF14) neochromosomes, respectively. As control, the same glycolytic cassettes were integrated at the native CAN1 locus of chromosome V resulting in strain IMX1959.

Figure 5.

Physiological characterization of strains carrying synthetic chromosomes. (A and B) Specific growth rate of strains carrying empty neochromosomes (Panel A: IMF2, IMF12, IMF6, IMF11 and IMF23) and glycolytic neochromosomes (Panel B: IMF18 and IMF17). Growth rates represent the average and mean deviation of biological duplicates except for the parental strain which cultures were performed in biological quadruplicate. (C and D) Viability measured as number of colonies on YPD (C) or SMD (D) sorted by FACS, divided by the total number of possible colonies (96), for the empty and glycolytic neochromosomes. Data represent biological duplicates and are averaged from 4 days of measurement for all strains except for the 100 kb improved design (two samples at day 1 and day 4). (E) Stability measured as the number of transformants on selective plates (SMD) divided by the number of colonies on non-selective plates (YPD). For each strain, the stability represents the average of 4 days of measurement in biological duplicates, except for 100 kb improved design (IMF23) for which 2 days of measurement were used (day 1 and day 4). (F) Transcript levels of the glycolytic genes from IMF17 (135 kb glycolysis neochromosome) and control strain IMX2109 expressing glycolysis from native chromosome V, grown in aerobic batch cultures. Transcript levels and standard deviations are from biological triplicates. (G) Specific activity of glycolytic enzymes in IMF17 and control strain IMX2109 from aerobic batch cultures. Activities were measured at least in biological duplicates. For panels A, B, C, D and E all significant differences with respect to the first bar are indicated with an asterisk (one-way ANOVA with Post-Hoc Tukey–Kramer, P< 0.05). For panels F and G, the asterisk indicates whether transcript levels or enzyme activities of IMF17 are significantly different with respect to IMX2109 (two-tailed paired homoscedastic t-test P< 0.05).

Figure 6.

Quantification of neochromosome copy number. (A) neochromosome copy number as determined by whole genome sequencing. (B) neochromosome copy number estimation based on mRuby2 fluorescence. CEN.PK113-7D with no copies of mRuby2 was used as negative control and IMX2224 with a single copy of mRuby2 integrated in the genome as positive control. (C) Schematic overview of the potential segregation of circular neochromosome upon cell division.

Figure 7.

Strain construction overview. The parental strain IMX1338 is framed by a filled grey box from which its direct decedents are indicated with black arrows. The schematic representations of linear or circular chromosomes indicate the relevant native or synthetic chromosomes that were modified in this study, and the significance of the symbols is explained in the top left box. Strains characterized in this study are framed by a dashed gray box.