Abstract
The genome of an organism is inherited from its ancestor and continues
to evolve over time, however, the extent to which the current version
could be altered remains unknown. To probe the genome plasticity of
Saccharomyces cerevisiae, here we replace the native left arm of
chromosome XII (chrXIIL) with a linear artificial chromosome harboring
small sets of reconstructed genes. We find that as few as 12 genes are
sufficient for cell viability, whereas 25 genes are required to recover
the partial fitness defects observed in the 12-gene strain. Next, we
demonstrate that these genes can be reconstructed individually using
synthetic regulatory sequences and recoded open-reading frames with a
“one-amino-acid-one-codon” strategy to remain functional. Finally, a
synthetic neochromsome with the reconstructed genes is assembled which
could substitute chrXIIL for viability. Together, our work not only
highlights the high plasticity of yeast genome, but also illustrates
the possibility of making functional eukaryotic chromosomes from
entirely artificial sequences.
Subject terms: Genome, Synthetic biology, Chromosomes
__________________________________________________________________
In Saccharomyces cerevisiae, the left arm of chromosome XII only
requires 12 genes to maintain cell viability, whereas 25 genes are
needed for robust fitness. Here the authors demonstrate that the entire
arm can be replaced by a neochromosome with completely artificial
sequences.
Introduction
Mycoplasma genitalium was previously regarded as the bacterium with the
smallest genome that can grow in axenic culture^[70]1,[71]2, however,
in 2016, the record was broken after the creation of JCVI- syn3.0 with
a genome only 531 kbp in size, reduced from over 1.0 Mbp in the
original Mycoplasma mycoides^[72]3. In this genome, only 473 genes,
including the set of essential and quasi-essential genes, were
retained^[73]3,[74]4. For the model bacterium Escherichia coli, it has
a genome at 4.6 Mbp, encoding 4434 genes. Through rounds of deletion,
this was reduced by up to 15%, constituting the loss of 743 genes,
including mobile DNAs^[75]5. The reduced genome strain was reported to
not only preserve good growth profiles and protein production, but to
also have beneficial properties including high electroporation
efficiency and stability of recombinant plasmids.
Compared to prokaryotes, studies on genome reduction in eukaryotes are
very limited. The genome of Saccharomyces cerevisiae (S. cerevisiae), a
model eukaryotic organism, is 12 Mbp in length and encodes about 6000
genes^[76]6. Using chromosome-splitting and loss techniques, it has
been reported that 5% reduction of the S. cerevisiae genome could
improve the productivity of ethanol and glycerol^[77]7. Since 2006, a
group of researchers have committed to the construction of a designer
version of the yeast genome (known as Sc2.0), in which the yeast genome
size will be reduced about 8% through deletion of the retrotransposon
related sequences, introns and subtelomeric repeat sequences^[78]8. In
the Sc2.0 genome, numerous loxPsym sites were introduced to facilitate
Synthetic Chromosome Rearrangement and Modification by LoxPsym-mediated
Evolution (SCRaMbLE)^[79]9. Upon the activation of SCRaMbLE, large
scale genome rearrangements such as inversion, deletion and duplication
were observed and strains with particular phenotypes including
resistance to higher temperature, for example, were identified^[80]10.
To facilitate genome reduction, we recently developed an iterative
SCRaMbLE-based genome compaction strategy, which allowed us to remove
about 40% of synXIIL while the cells remained viable at 30 °C in rich
medium^[81]11. However, how much more DNA could be further removed from
this chromosome arm without impairing cell viability remains elusive.
Besides the content, regulation of gene expression by non-coding
sequences adds another layer of complexity, particularly in eukaryotic
organisms. Many functional elements such as enhancers, transcription
factor binding sites, the TATA box, the transcription start site and
poly(A) sites reside within these sequences even though these cis
regulatory sequences are not systematically defined for every gene.
Recently, synthetic promoters and terminators have been designed based
on regulatory sequences with varied activity^[82]12–[83]15. One
challenge in synthetic genomics is to understand regulatory networks to
such an extent that it is possible to design artificial sequences that
effectively restore their function. Additionally, due to codon
degeneracy and usage bias, different nucleotide sequences for the same
amino acids can have substantially different functional impacts^[84]16.
Up to now, genome-wide codon compression has only been successfully
applied in E. coli^[85]17–[86]19. Permissive and restrictive synonymous
recoding schemes are largely unexplored in eukaryotes. Recently, using
several essential genes as tested, we employed a codon compression
scheme, in which only one codon is used for each amino acid, and
swapped the regulatory sequences with those of CYC1^[87]20. Notably, we
found that 7 out of the 10 reconstructed genes could complement their
deletion from the haploid genome. However, whether cells can tolerate a
chromosome or even an entire genome with such dramatical sequence
modifications remains unknown.
The left arm of chrXII in S. cerevisiae (chrXIIL) is 150,827 bp long,
ranking as the seventh shortest yeast arm. It contains 74 genes (62
protein coding genes, 9 dubious genes, 2 pseudogenes, 1 tRNA coding
gene), 3 autonomously replicating sequences and 1 Ty1 LTR
(Saccharomyces Genome Database, [88]https://www.yeastgenome.org/).
Among these genes, 10 are defined as essential based on the phenotype
of individual knockout mutations and their ability to support spore
viability in a heterozygous diploid. The length of proteins encoded by
genes on chrXIIL, showed a similar distribution pattern to that of
genome-wide genes (Supplementary Fig. [89]1a). The average number of
directly evidenced gene ontology (GO) terms per gene on chrXIIL was
slightly higher than most of the other chromosome arms, suggesting
higher functional complexity to be considered during reconstruction
(Supplementary Fig. [90]1b). To expand the understanding of genome
plasticity in eukaryotes, we used this chromosome arm to explore the
maximum extent of changes that can be made without affecting basic
functions. A neochromosome was initially designed to facilitate the
relocation of essential genes that are dispersed throughout chrXIIL.
Using a partially synthetic chromosome XII, we systematically probed
sequence essentiality in chrXIIL by targeted DNA deletion, chromosome
truncation and gradual gene replenishment. Eventually, a series of
neochromosomes, capable of substituting for chrXIIL for cell viability
were constructed.
Design of a neochromosome as a flexible carrier of exogenous DNA
Linear artificial chromosomes containing structural elements for
segregation, replication and stability (i.e., centromere, autonomous
replication sequences and telomeres) have been reported to behave like
native chromosomes^[91]21,[92]22. However, de novo construction of a
linear chromosome with capability for future applications remains
limited. To produce a flexible carrier of exogenous DNA, we designed a
neochromosome with unique features allowing it to be assembled easily
in vivo. The overall construct design is shown in Fig. [93]1a.
Fig. 1. A designer neochromosome to harbor essential genes from the left arm
of chrXII.
[94]Fig. 1
[95]Open in a new tab
a, The design of neochromosome. Marker genes, white; TeSS, brown;
Insulators, green; URRs, blue. b Arrangements of the ten essential
genes (pink) in ptWT10 and ptWT10U. Arrows point to the direction of
transcription. The genes with changed transcription direction on
ptWT10U are labeled in red. c PFGE and southern blotting analysis of
the assembled neochromosomes. M: λ DNA-Mono Cut Mix. Source data are
provided as a Source Data file. Images are representative of at least
three independent experiments. d Stability test of neochromosomes.
BY4742 cells containing ptWT10 or ptWT10U were cultured in SC-His
medium for about 125 generations before PCR analysis and whole genome
sequencing. e Fitness analysis of strains on different growth
conditions. 10-fold gradient dilutions were conducted for cells in this
study. WT: BY4741. H[2]O[2]: hydrogen peroxide; Noc: nocodazole; MMS:
methyl methanesulfonate; Rap: rapamycin; Ben: Benomyl. f–i Full-length
transcriptome analysis of BY4742 with ptWT10 or ptWT10U neochromosome.
The transcripts on neochromosome (orange) and native transcripts
(chrXIIL, blue) were mirrored. On the x-axis, negative number
represents distance upstream of ATG and positive values indicate
distance downstream of the stop codon. The Y-axis represents the counts
of detected transcripts. j, k Transcription analysis of the ten
essential genes in ptWT10-10KO or ptWT10U-10KO using DESeq2 to
calculate Benjamini-Hochberg adjusted p values and fold change values.
WT: BY4742. Differential expression in this study was defined as
|log[2]FC| > 1 and -log[10] (Adjusted p-value) >4. FC, fold change.
Dashed lines, the threshold defined above. Red, differentially
expressed essential genes on neochromosomes. l The copy number of
neochromosomes in ptWT10-10KO or ptWT10U-10KO. a,b,c are three
independent biological repeats. The mean read-depth of native
chromosomes (n = 11,845 1 kb-sized bins) was normalized to 1. For the
neochromosomes, n = 36 1 kb-sized bins. The bounds of the box were the
upper and lower quartile with the median value in the center. The
whiskers indicated 5th and 95th percentile. Source data are provided as
a [96]Source Data file. m Normalized expression of genes on ptWT10
(n = 3) and ptWT10U (n = 3). Expression of each gene was normalized to
HIS3 on neochromosomes. The data are presented as the mean and SD. A
two-tail unpaired Welch’s t-test were employed to calculate the p
values, which were adjusted by Benjamini-Hochberg method. *P < 0.05,
**P < 0.01, ***P < 0.001. Source data are provided as a [97]Source Data
file. The exact p value was listed in [98]Source data.
The telomere seed sequences (TeSS), that is, the telomeric TG repeat
plus the Core X element, which have been constructed and functional
tested previously^[99]8, served as the telomeres. To ensure replication
and segregation of the neochromosome, the centromere sequence from
Chromosome II and an origin of replication (ARS208) were placed in the
middle. It is well known that genes placed near the telomeres will be
silenced—a phenotype called the telomere position effect (TPE)^[100]23.
Therefore, to reduce TPE, a short insulator sequence (10 tandem repeats
of TTAGGG) was put adjacent to TeSS to block the spreading of
heterochromatin^[101]24. In addition, to allow robust integration of
exogenous DNA with high specificity, two 500 bp random sequences,
designated as the universal recombination regions (URRs)^[102]25, were
also included in each chromosomal arm. Finally, two auxotrophic
markers, HIS3 and LYS2 were inserted into the left and right arm
respectively, to facilitate the selection of the assembled
neochromosome. The structural elements of the neochromosome were
divided into three parts, segments L, M, and R, which could be
assembled together in yeast by homologous recombination.
Construction of neochromosomes carrying essential genes from chrXIIL
We used the designed neochromosome to carry the 10 known essential
genes of chrXIIL. We positioned these genes in two different formats
(Fig. [103]1b): 1) in the same relative chromosomal position and
transcriptional orientation as on the native chromosome (designated as
ptWT10), and 2) unifying the transcriptional direction to point towards
the telomere (ptWT10U). In ptWT10, the sequences and directions of
essential genes were the same as those in a circular eArray reported to
effectively compensate for the loss of a single native gene^[104]11. In
ptWT10U, four genes YLL003W, YLL004W, YLL008W, YLL011W were flipped
together, leading to a change of their relative position
(Fig. [105]1b). In addition, since YLL035W and YLL036C share a
bi-directional promoter and the intergenic region is unable to sustain
the function of YLL036C^[106]20, we chose the reported CYC1 promoter to
achieve functional expression of both genes and maintain consistent
regulation in ptWT10U. The sequences of ptWT10 and ptWT10U are listed
in Supplementary Data [107]1.
To construct the neochromosomes, the three structural fragments, i.e.,
fragment L, M, and R, together with PCR-amplified essential-gene
fragments were co-transformed into BY4742 (Supplementary Fig. [108]2a).
Subsequently, cells containing assembled chromosomes were selected in a
medium lacking histidine and lysine, randomly isolated and confirmed by
PCR (Supplementary Fig. [109]2b–e). Next, the strains were analyzed by
Pulsed-field gel electrophoresis (PFGE) followed by Southern Blotting.
As shown in Fig. [110]1c, a band of around 40 kb was detected in both
PFGE and Southern Blotting, indicating the successful construction of
the neochromosome. Finally, nanopore sequencing was performed which
revealed that both ptWT10 and ptWT10U are present in the cells with
sequences as designed (Supplementary Fig. [111]2f, g). However, we
observed several single nucleotide substitutions and insertions in the
neochromosome (Supplementary Data [112]1) which do not affect the
function of each essential gene (see below), and therefore, no further
correction was performed. Interestingly, we found that the telomeric TG
repeats, in both neochromosomes, significantly expanded (Supplementary
Fig. [113]2h, i), indicating the TeSS end grew a new telomere
successfully^[114]8,[115]26.
Since the neochromosomes are relatively short (about 40 kb) and contain
loxPsym sites in segments L and R, we firstly tested if such sequences
remain stable in cells as reported^[116]27. As shown in Fig. [117]1d,
we inoculated the strains in selective liquid medium and cultured the
cells by 1000-fold dilution every day for ten consecutive days,
accounting for about 125 mitotic generations. The cells were analyzed
by both PCR analysis and next generation sequencing. For three
independent clones tested, we found that none of them had changes in
PCRtags or nucleotide sequences. These results demonstrate that the
linear neochromosomes are stable in the cells.
Essential genes relocated to the neochromosome could substitute their native
counterparts for viability
To test whether genes relocated to linear neochromosomes function
equivalently to those in native loci, we constructed strains with
ptWT10 or ptWT10U in which the original genomic copies of these
essential genes were deleted sequentially (designated ptWT10-10KO and
ptWT10U-10KO). ptWT10-10KO showed indistinguishable growth from that of
wild type under various conditions (Fig. [118]1e). This indicates that
ptWT10 functions well to replace the native genes. ptWT10U-10KO cells
exhibited a slight growth defect in rich medium (Fig. [119]1e). These
results suggest that the general function of these essential genes is
largely unaffected after direct relocation to the neochromosome, whilst
changes in regulatory sequences or the orientation of transcription
seem to result in fitness defects.
Since we rearranged the position of these essential genes by placing
them side by side, especially in ptWT10U, it might lead to interference
between adjacent transcription units (TUs)^[120]28. Therefore, we
examined whether there were any abnormalities in transcription
initiation and termination sites of each gene in the two neochromosomes
using the isoform-sequencing (Iso-seq) method, a high-throughput method
to identify all full-length transcripts within a cell. From the Iso-seq
results of BY4742 containing either ptWT10 or ptWT10U, the full-length
transcripts of nine essential genes on the neochromosomes, were
identified using specific PCRTags (except for YLL050C, which is too
short to contain PCRTags)^[121]29. For genes with the same
transcription direction, such as YLL018C, the transcription start sites
(TSS) and termination sites (TES) on the neochromosomes are similar to
those in the native transcriptome (Fig. [122]1f, g, Supplementary
Fig. [123]3a, b). The genes that were flipped on the neochromosomes,
such as YLL003W, they were also transcribed similarly to native
transcriptome (Fig. [124]1h, i, Supplementary Fig. [125]3c–e). For
YLL035W and YLL036C, the TES and TSS showed a similar pattern between
ptWT10 and the native genome locus, while the promoter and terminator
of CYC1 used in ptWT10U changed the transcription pattern as expected
(Supplementary Fig. [126]3f–h). In addition, we did not observe any
abnormal transcripts that extended through two adjacent genes
(Supplementary Fig. [127]3i). These results indicated that the
sequences we used for each gene were sufficient to regulate the
transcription initiation and termination processes.
To examine whether the transcription level of genes was altered when
relocated to the neochromosome, the expression of the ten genes in
ptWT10-10KO and ptWT10U-10KO was analyzed by RNA-seq (Fig. [128]1j, k).
Consistent with the above Iso-seq results, the transcripts of most
genes identified on both ptWT10 and ptWT10U showed clear boundaries
between different TUs (Supplementary Fig. [129]4a). We found that in
ptWT10-10KO, all 10 essential genes except for YLL031C were transcribed
at similar levels to their genomic counterparts in BY4742.
Unexpectedly, 9 out of the 10 essential genes, including the two with
altered transcription regulatory sequences, were overexpressed in
ptWT10U-10KO (Fig. [130]1k and Supplementary Data [131]2). Besides,
more other differentially expressed genes were identified in
ptWT10U-10KO than ptWT10-10KO (190 vs 118, Supplementary Fig. [132]4b).
ptWT10U-10KO also showed a more obvious transcriptome-wide perturbation
than ptWT10-10KO when compared to BY4742 (Supplementary Fig. [133]4c).
The neochromosomes exist in cells with variable copy number
The elevated expression of essential genes in ptWT10U-10KO lead us to
ask whether the copy number of neochromosome had changed. Similar to
the method for DNA copy number estimation using read-depth of the
next-generation sequencing data^[134]30, we evaluated long-read
sequencing data and calculated the ratio of the mean depth of the
neochromosome to that of all native chromosomes as the average copy
number of the neochromosome per haploid genome. As shown in
Fig. [135]1l, we found that the neochromosomes exist in cells in one to
two copies.
To eliminate the effects of DNA copy number, we normalized the
expression level of each gene on the neochromosome to that of HIS3,
which is located on the left arms of the neochromosomes. As shown in
Fig. [136]1m, three genes, including the two with changed promoters and
YLL031C, showed obviously different transcription levels between ptWT10
and ptWT10U, while none of the four “flipped” native genes, namely
YLL011W, YLL008W, YLL004W, and YLL003W, showed significantly different
transcription levels. These results suggest that the regulatory
sequences, rather than the orientation of transcription, plays
important roles in local gene regulation.
Only 12 genes are sufficient to replace chrXIIL for viability
Previously, we found over half of the nonessential genes in synXIIL
could be deleted by SCRaMbLE^[137]11. However, further compaction
attempts using the same method failed, which presumably may be
partially due to the extremely slow growth of the final strain
ZLY349^[138]11. To probe the minimal gene set to support cell
viability, two additional strategies were carried out here.
At first, we systematically examined the essentiality of sequences in
the left arm of chrXII using CRISPR/Cas9 technology. The six regions
flanking essential genes, which were either partially deleted or
retained in ZLY349^[139]11, were knocked out individually
(Supplementary Fig. [140]5a). Notably, all deletions generated viable
strains, despite the growth defects exhibited by two of the generated
strains (Supplementary Fig. [141]5b). These results suggested that all
these non-essential regions could be removed.
Next, a chromosome truncation method, which is similar to previous
technique for the replacement of telomere^[142]31, was adopted to
truncate the chromosome arm piece by piece (Fig. [143]2a). In this
method, a fragment containing a universal telomere, a marker gene and a
homologous region is transformed into yeast to create a new telomere at
the left end of chrXII by homologous recombination. Depending on the
location of the homologous region (HR I–IV, Supplementary
Fig. [144]5a), the left-most arm up to this region will be deleted. We
systematically removed the left arm of chrXII in quarterly increments
to a maximum of the entire arm in a heterozygous diploid strain (Region
I-IV, Fig. [145]2b). Due to deletion of essential genes, the diploid
strains with an empty neochromosome backbone (Neo0) produced only two
viable spores upon sporulation (Fig. [146]2b). In contrast, the
presence of ptWT10 rescued the lethality of the two spores in three
strains, except for the one in which the entire chrXIIL was deleted
(chrXIILΔ, Fig. [147]2b), illustrating that the 10 essential genes are
insufficient to substitute for chrXIIL for viability. It is consistent
with the theory that the essential gene set is not sufficient to
construct a viable organism with a minimal genome due to the phenotypic
consequences of complex genetic interactions on fitness^[148]32.
Fig. 2. Only 12 genes are sufficient to replace chrXIIL for viability.
[149]Fig. 2
[150]Open in a new tab
a Schematic diagram of the method for chromosomal truncation. b Tetrad
analysis of heterozygous diploid strains with different chrXIIL
truncations. c Fitness tests of strains carrying yll002w and yll006w
deletion. WT: BY4742. d Tetrad analysis of heterozygous diploid strains
with one chrXIIL removed. YLL002W (green) and YLL006W (purple) were
integrated into the neochromosomes respectively or together. Red
circle, the spores containing chrXIILΔ and the neochromosome.
Then, we systematically examined the viability of strains containing
one more non-essential gene plus the ten essential genes. Among the 64
non-essential genes on chrXIIL, we excluded 9 dubious genes, 2
pseudogenes, 3 genes at the telomere and 1 tRNA gene, leaving 49
protein-coding genes to be tested. However, none of the 11-gene
combinations were able to produce four viable spores from one tetrad
(Supplementary Fig. [151]5c), suggesting that more genes are required
for viability.
Thus, we tried to add two genes. Genetic interactions are quantified by
measuring phenotypes of single and double mutants and calculating an
interaction factor that reflects any deviation from the expected
combined effect of the two single mutants^[152]32. To date, the
investigations of genetic interaction have been extensively conducted
in budding yeast, using genome-wide yeast mutant collections and
automated colony size-scoring methodology^[153]32. It has been reported
that essential genes participate in more genetic interactions than
non-essential genes^[154]32, raising the hypothesis that the higher
importance of the gene, the more genetic interactions may exist.
Therefore, we used the number of genes that showed genetic interaction
with a target gene (GGI) as a quantitative indicator of genetic
interactions. Essential genes did show significantly higher GGI than
non-essential genes (Supplementary Fig. [155]6a). There are 214
non-essential genes (~3.6% in genome) with GGI higher than 489.4
(2-fold to the average GGI of essential genes). Nearly all these genes
(98.5%) showed synthetic lethality with other genes, and more than 80%
showed decreased vegetative growth or absent respiratory growth when
deleted (Supplementary Fig. [156]6b, c), providing additional evidence
for the importance of these genes. Among the nonessential genes on
chrXIIL, YLL002W and YLL006W met this threshold (Supplementary
Table [157]1). YLL002W was deleted in the strain Δ1 which showed severe
growth defects, and it also showed synthetic lethality with 29 other
genes (Supplementary Fig. [158]5, Supplementary Table [159]1). YLL006W
was synthetic lethal with 9 other genes, including another
non-essential gene YLL040C on chrXIIL (Supplementary Table [160]1). So,
we chose YLL002W and YLL006W for further testing.
As expected, single mutants of the two genes exhibited impaired cell
fitness and the double mutant led to a severe growth defect
(Fig. [161]2c). Therefore, we constructed three versions of
neochromosomes containing the 10 essential genes plus either YLL002W,
YLL006W, or both. Excitingly, we found the strain containing ptWT10
plus both YLL002W and YLL006W generated four viable spores
(Fig. [162]2d), suggesting that a neochromosome containing just the 12
genes is sufficient to substitute chrXIIL for yeast viability. We named
the neochromosome with 12 genes as ptWT12 (Supplementary Data [163]1)
and the yeast strain carrying ptWT12and chrXIILΔ as yWT12.
Additional genes are needed to restore robust cell fitness
Although yWT12 is viable, it grew poorly even in rich medium
(Fig. [164]2d). To understand how the fitness of yWT12 was compromised,
we tried to add back additional non-essential genes from chrXIIL,
prioritizing those with high GGI value. Since 91.2% of non-essential
genes with high GGI (244.7 < GGI < 489.4) also showed synthetic
lethality with other genes (Supplementary Fig. [165]6b), the five
genes, YLL049W, YLL039C, YLL043W, YLL040C, and YLL045, were chosen. In
addition, among the genes in region IV, deletion of YLL009C also led to
obvious growth defects (absent respiratory growth, Supplementary
Table [166]1), and therefore, it was also included. The six genes,
including their regulatory sequences, were amplified from the wild type
genome, and incorporated into the neochromosome to construct ptWT18
(Fig. [167]3a) and the corresponding strain yWT18 (ptWT18, chrXIILΔ).
The successful construction of the neochromosome and strains were
verified by sequencing (Supplementary Data [168]1).
Fig. 3. Restoration of cell fitness using a simplified gene set.
[169]Fig. 3
[170]Open in a new tab
a Schematic diagrams of additional genes in the three neochromosomes
besides the essential genes. b Fitness analysis of strains carrying
assembled neochromosomes under various conditions. WT, BY4741. c
Doubling times of corresponding strains in SC medium (n = 3). The
average doubling time of BY4742 was set to 1.0. The data are presented
as the mean and SD. Source data are provided as a Source Data file. d
The logarithmic phenotype index (LPI) of BY4742 (n = 3) and yWT25
(n = 3) under MMS condition. The data are presented as the mean and SD.
Unpaired t-test (two-tail) was used compare the two groups, p = 0.0155.
The LPI[MMS] significantly greater than zero indicates the resistance
phenotype of corresponding strain. Source data are provided as a
[171]Source Data file. e Cell morphology of indicated strains. WT,
BY4742. Scale bars, 5 μm. Images are representative of at least three
independent samples. f Lysine auxotrophy due to yll027w deletion.
YLL027W is expressed in a centromeric plasmid under its native promoter
and terminator. g Transcriptome-wide perturbation of yWT12 and yWT25.
BY4742 was used as the control for normalization. X axis represents
|log[2]FC|. Y axis represents the percentage of genes with |log[2]FC|>
the value of X axis. h, i KEGG pathway enrichment analysis of the
differentially expressed genes in strains, using clusterProfiler
package to calculate Benjamini-Hochberg adjusted p values. All eleven
terms with adjusted p value < 0.05 in yWT12 were shown in (h). All
twelve terms with adjusted p value < 0.05 in yWT25 were shown in (i).
Bubble size indicates the gene count and the color reflects the
adjusted p value.
As shown in Fig.[172]3b, introducing the six genes could obviously
improve the growth of cells, not only on rich medium, but also under
the chemical stresses (DNA damaging agent MMS and mTOR inhibitor
rapamycin). All of the strains showed no obvious sensitivity to the
oxidative stress (H[2]O[2]) or the anti-microtubule drugs (benomyl and
nocodazole). Next, we measured the growth rates of these strains in
synthetic complete (SC) medium. The doubling time of yWT12 was
lengthened to over twice that of the wild type. It was much reduced for
yWT18 but remained longer than BY4742 (Fig. [173]3c).
Besides the genes in ptWT18, there are eight growth-related genes on
chrXIIL (Supplementary Table [174]1). Seven out of the eight genes were
also retained in ZLY348, the strain with a compacted chrXIIL (~40%
removal of synXIIL sequences) but retaining wild type-like growth on
YPD at 30 °C^[175]11. Therefore, we added them to construct ptWT25 and
the strain yWT25 (ptWT25, chrXIILΔ, Fig. [176]3a, b, Supplementary
Data [177]1), and the doubling time of yWT25 was much shorter than
yWT18 (Fig. [178]3c). yWT25 cells also showed higher resistance to
rapamycin than yWT18, and even better resistance to MMS than BY4742
(Fig. [179]3b, d and Supplementary Fig. [180]6d, e).
In addition, we analyzed whether there were morphological changes among
these strains. As shown in Fig. [181]3e, the cells of yWT12, yWT18 and
yWT25 all looked similar, rounder than BY4742. Interestingly, neither
yWT12 nor yWT18 could grow on the synthetic medium without lysine while
yWT25 did (Fig. [182]3f). This phenotype may result from the removal
and subsequent re-incorporation of YLL027W, the null mutant of which
leads to lysine auxotrophy^[183]33,[184]34. Consistently, expression of
YLL027W in yWT12 or yWT18 did recover growth on the selective medium
(Fig. [185]3f). In accordance with above phenotypes, the transcriptomic
perturbations observed in yWT12 were evidently restored in yWT25
(Fig. [186]3g). And the majority of genes within the significantly
enriched pathways of yWT12 did not exhibit differential expression in
yWT25 (Fig. [187]3h, Supplementary Data [188]2).
We also tried to further remedy the fitness of yWT25 by adding more
genes. From the genes remained in ZLY348, we selected the top five
genes exhibiting GGI > 129.6 to construct ptWT31 and the strain yWT31
(ptWT31, chrXIILΔ, Supplementary Fig. [189]6f, Supplementary
Data [190]1). YLL013C (PUF3), a gene that encodes an mRNA binding
protein involved in mRNA decay processes and known to have over 2000
putative mRNA targets^[191]35, was also included (Supplementary
Fig. [192]6f). However, no significant improvements of cell growth in
YPD were observed in yWT31(Supplementary Fig. [193]6g). Subsequently,
we looked into the transcriptomic data and found that the
differentially expressed genes in yWT25 are highly enriched in
oxidative phosphorylation (Fig. [194]3i), including the removed YLL041C
(SDH2). And 25.6% (21/82) of the overlapping differentially expressed
genes in yWT12 and yWT25 were implicated in the interaction network
with YLL041C (SDH2) and YLL013C (PUF3) (Supplementary Fig. [195]6h, i).
Therefore, we incorporated the two genes to build ptWT27 and the strain
yWT27 (ptWT27, chrXIILΔ, Supplementary Fig. [196]6j, Supplementary
Data [197]1). Strikingly, yWT27 exhibited a significant recovery of
growth in YPD (Supplementary Fig. [198]6j). This result highlights the
potential of transcriptomic data for debugging fitness defects.
Together, these results suggest that although only 12 genes are
required for cell survival, additional genes are needed to maintain
relatively robust growth. The principles used in this study are useful
to identify critical genes to improve strain fitness.
Altered metabolic profiles contribute to the growth differences between yWT12
and yWT25
The enrichments of differentially expressed genes in yWT12 and yWT25
across various metabolic pathways signify the profound metabolic
alterations. We employed untargeted metabolomics to investigate the
metabolic profiles of the BY4742, yWT12, and yWT25 strains with
different growth time in SC medium (8 h, 24 h and 48 h, Supplementary
Fig. [199]7a). Principal component analysis (PCA) has been widely used
as a multivariate method in metabolomics analysis, which is a key tool
to identify patterns and outliers in the metabolomics datasets^[200]36.
PCA of the metabolites composition among the three strains showed that
the first principal component (PC1) versus PC2 accounted for over 49%
of the total variation (Fig. [201]4a), which revealed a shift in
metabolite profiles over time. Reliable separations among the three
strains were observed at 24 h and 48 h (Fig. [202]4a), with an
increased number of differential metabolites (DMs) found in the yWT12
and yWT25 along with culture time as compared with BY4742
(Fig. [203]4b). The number of elevated DMs in yWT12 increased
progressively over time, with 160, 575, and 868 increased metabolites
at 8 h, 24 h, and 48 h, respectively, while the number of decreased DMs
declined gradually (Fig. [204]4b). Similar findings were observed in
yWT25, except that more DMs were decreased at 48 h (Fig. [205]4b).
Fig. 4. Untargeted metabolomics reveals altered metabolic profiles among
yWT12, yWT25 and BY4742.
[206]Fig. 4
[207]Open in a new tab
a Principal component analysis of metabolite composition using
untargeted metabolomics in BY4742, yWT12 and yWT25 collected at 8 h,
24 h, and 48 h. b Differentially increased or decreased metabolites at
different growth phases shown in Venn diagrams (|log[2]FC| > 1, and
one-way ANOVA t-test P < 0.05). Three biological replicates were
conducted. c Heatmap of differential metabolites (left) and the
proportion of different types of differential metabolites (right) in
BY4742, yWT12, and yWT25 at 24 h. The color code in heatmap denotes
Z-scaled values of metabolites after correction of confounders. d
Differential metabolites (dots, 8 h) and differentially expressed
metabolic genes (lines) of yWT12 and yWT25 mapped to the yeast
metabolic network using iPath3.0. Red and blue dots/lines represent
significantly upregulated or downregulated metabolites/genes.
Radius/thickness of dots/lines represents |log[2]FC| of
metabolites/genes. e KEGG pathways that were significantly altered in
different strains are indicated. Significantly enriched pathways are
identified with a hypergeometric test’s p-value for given metabolites.
f The abundance of DMs belonging to the ABC transporters-dependent
pathway was assessed. The abundance of each metabolite was compared to
that in BY4742 at 8 h. The color scale indicates the log[2]FC, with
blue indicating increased abundance and red indicating decreased
abundance. g Representative DMs enriched in the ABC
transporters-dependent pathway that foster or limit the growth of
different strains (n = 3). The data are presented as the mean and SD.
Source data are provided as a [208]Source Data file.
Unsupervised hierarchical clustering of the metabolome revealed
distinct clusters of metabolites at each time point, with BY4742
exhibiting more accumulated metabolites at 8 h, and yWT25 displaying
more accumulated metabolites at 24 h and 48 h (Fig. [209]4c and
Supplementary Fig. [210]7b, c). At 24 h, DMs in cluster II was
over-accumulated in yWT25, while DMs in cluster I and III exhibited
high accumulation in yWT12 and BY4742 (Fig. [211]4c). The DMs were
classified into ten superclasses, with amino acid and its metabolites
being the most prevalent DMs and accounting for over 30% of the total
DMs at 24 h (Fig. [212]4c). However, the metabolites within each
cluster were involved in all ten superclasses, revealing a broad range
of metabolic fluctuations during the growth of these strains.
Visualization of the differentially expressed enzymes and DMs (8 h) by
mapping them to the metabolic network using iPath3^[213]37 revealed
that the metabolic alterations observed in yWT12 were markedly restored
in yWT25 (Fig. [214]4d). Notably, while differentially expressed
enzymes in yWT25 highlighted changes in energy metabolism and amino
acid metabolism, DMs in yWT25 were distributed throughout the metabolic
network, similar to those in yWT12 (Fig. [215]4d).
Further analysis of the DMs using KEGG pathway enrichment revealed that
various metabolic pathways were affected during the growth of the
different strains (Fig. [216]4e). ABC transporters, which are involved
in the transport of diverse substrates across membranes, were found to
be significantly affected (Fig. [217]4e). 32 DMs were enriched in ABC
transporters-dependent pathway (Fig. [218]4f). Metabolites in cluster I
of this pathway, such as xanthoside and taurine (Ia and Ib), and
2’-deoxyinosine (Ic), were particularly abundant in yWT12 and yWT25,
respectively (Fig. [219]4f, g, and Supplementary Fig. [220]7d),
indicating a potential defects in their utilization or transport
outside of the cell, which could impede their growth. In contrast,
metabolites in clusters IIa and IIb were scarce in yWT12 or yWT25
(Fig. [221]4f, g, and Supplementary Fig. [222]7d). For instance, serine
was barely detectable in pWT12, while maltotriose was found to be only
minimally accumulated in both pWT12 and pWT25 (Fig. [223]4g), which
indicated a potential disability in biosynthesis or transport inside of
the cell in these strains. Metabolites in clusters IIc-e showed
different abundance levels in different strains (Fig. [224]4f, g, and
Supplementary Fig. [225]7d), suggesting differences in uptake or
metabolic capacity, which could influence growth rates. These findings
highlight the importance of ABC transporters for the growth of the
modified strains. Consistently, differentially expressed genes in yWT25
were also enriched in the ABC transporters pathway (Fig. [226]3i).
Together, introducing genes involved in ABC transport could be a
potential way to further improve cell growth.
Reconstruct transcription units using exogenous artificial sequences
Given that the native genes could be relocated to the neochromosome
without disruption of core function, we next asked if both the coding
and regulatory sequences are able to be reconstructed with completely
synthetic ones. For the 25 genes in ptWT25, we systematically
reconstructed them using the following principles: For coding sequences
(CDS), the optimized DNA sequences were generated using GeneDesign
software as before^[227]20,[228]38, which employs a radical codon
compression scheme, in which only one optimal codon is used for each
amino acid (Supplementary Fig. [229]8a). And the optimal codon for each
amino acid was defined by the highest relative synonymous codon usage
value in highly expressed genes in yeast genome^[230]20,[231]38. Based
on previous studies^[232]12–[233]15, 44 short synthetic promoters and
28 short synthetic terminators with glaring variances in sequence and a
variety of expression activities were selected for the promoter and
terminator (Supplementary Data [234]3). Each part was synthesized and
cloned into the YeastFab vectors we developed previously^[235]25.
To obtain functional combinations of promoter, CDS and terminator for
each gene, we mixed the promoter-, terminator-containing plasmids
together with a particular CDS-containing plasmid to assemble a pool of
TUs, which were subsequently transformed into the corresponding haploid
deletion mutant (Fig. [236]5a). For essential genes, plasmid shuffling
was performed to identify the viable clones. For non-essential genes,
fitness change under a particular growth condition was identified for
each knockout strain and clones that were able to restore growth to
that of wild type were collected. The plasmids were extracted,
transformed into E. coli and subsequently sequenced to obtain the
identity of promoter and terminator. Combinations of promoter and
terminator for each gene were re-tested to confirm their functionality,
either by tetrad-based analysis of reconstructed essential genes for
the ability to support cell survival (Fig. [237]5b) or by
serial-dilution analysis (Fig. [238]5c, Supplementary Fig. [239]8 and
Supplementary Data [240]3). Using promoters and terminators as
different as possible for each gene, we successfully reconstituted 21
TUs except YLL031C, YLL028W, YLL003W and YLL039C. For both YLL031C and
YLL028W, we found the recoded CDS failed to complement the function of
native CDS, either losing cell viability or failing to grow under
stress conditions. In addition, we discovered that YLL003W, a gene
required for G2/M transition, lost function when constitutively
expressed. To obtain a functional TU, the promoter of SWI6, a
transcription factor activating transcription during G1/S transition,
was employed which, luckily, could restore the function of
YLL003W^[241]20. As for YLL039C (UBI4), we failed to assemble the
recoded gene, potentially due to its repetitive nature since it encodes
five head-to-tail ubiquitin repeats within CDS^[242]39.
Fig. 5. Reconstruction of transcription units.
[243]Fig. 5
[244]Open in a new tab
a Strategy to identify functional synthetic promoters and terminators
to support both essential and nonessential genes. eCDS, CDS of
essential gene. neCDS, CDS of nonessential gene. URA3-WT, the CEN
plasmid containing wild-type essential gene and URA3. b Functional
analysis of reconstructed genes by tetrad analysis. The corresponding
heterozygous diploid containing a reconstructed TU (rTU) was sporulated
and dissected. One representative tetrad was shown for each strain. For
YLL035W, the ATG codon was mutated (marked with *) to avoid impact on
the expression of YLL036W. Red circle, the spores containing disrupted
native gene and reconstructed TU. c Functional complementation test of
reconstructed YLL002W and YLL006W. WT, BY4741. + and − indicate strains
with or without the reconstructed TU. d Comparison of sequence identity
in CDS between the 24 rTUs and corresponding genes in Sc2.0 project.
The bounds of the box were the upper and lower quartile with the median
value in the center. The whiskers indicated the minimum and maximum. e
Length comparison of Pro and Ter between the 24 rTUs and the native
ones used in ptWT25. The data are presented as the mean and SD.
Unpaired t test (two-tail) was used for comparison in (d)
(p = 3.57E-11) and (e) (p = 2.15E-06 for Pro, p = 2.77E-05 for Ter).
Source data are provided as a [245]Source Data file.
We compared sequence changes in the 24 TUs after reconstruction to
those in wild type or Sc2.0 strain. At first, significant reduction of
sequence identities in CDS was present. The Sc2.0 sequences share over
95% identity to wild type, whereas the mean identity of the
reconstructed genes dropped below 80% (Fig. [246]5d). As for the
regulatory sequences, not only totally different DNA sequences were
used (Supplementary Data [247]3), the lengths of the synthetic
promoters and terminators were also much shorter than the counterparts
in ptWT25 (Fig. [248]5e).
Replacement of chrXIIL by a completely revamped neochromosome
Given that each refactored gene is able to function similarly to the
native one, we tested whether combinations of these genes could replace
chrXIIL entirely. The ptSYN10 neochromosome with ten refactored
essential genes was constructed (Supplementary Fig. [249]9a) and those
essential genes were deleted from the native chromosomal locations to
get the strain ptSYN10-10KO (Supplementary Fig. [250]9b). As shown in
Supplementary Fig. [251]9b, ptSYN10 was able to support the viability
of the ptSYN10-10KO strain, but the cells showed growth defects on
either YPD or upon treatment of different drugs. RNA-seq analysis
indicated that, in contrast to the neochromosomes made up of essential
genes with native regulatory sequences (Supplementary Fig. [252]4a),
nearly all the intergenic sequences of the synthetic genes on ptSYN10
were highly transcribed (Supplementary Fig. [253]9c). To further look
into the transcriptional fidelity of each gene, ptSYN10 in BY4742 was
also analyzed by Iso-seq (Supplementary Fig. [254]9d). Intrusive
readthrough was identified into adjacent transcription units
(Supplementary Fig. [255]9d). Moreover, many anti-sense transcripts
appeared, especially for syn035w and syn034c (Supplementary
Fig. [256]9d). These results suggested that the short synthetic
terminators might not be effective at terminating transcription, and
insertion of artificial DNA might bring in sequences with unexpected
promoter activities.
We next built a neochromosome (ptSYN12) carrying the same 12 genes as
ptWT12 but using recoded sequences (Supplementary Fig. [257]9e).
Unfortunately, unlike its wild type counterpart, the ptSYN12
neochromosome was unable to substitute chrXIIL for survival, although
it did support several cell divisions after spore germination
(Supplementary Fig. [258]9f). Next, based on what we learned above
using native genes, we constructed the ptSYN24 neochromosome
(Fig. [259]6a), which contains 24 recoded TUs from ptWT25 without UBI4.
Excitingly, we found ptSYN24 could support viability in spores with
chrXIILΔ (Supplementary Fig. [260]9g). These strains were designated as
ySYN24 (Supplementary Data [261]1).
Fig. 6. Functional replacement of chrXIIL with a completely revamped
neochromosome.
[262]Fig. 6
[263]Open in a new tab
a Schematic representation of gene arrangement on ptSYN24
neochromosome. b Fitness of ySYN24 in different growth conditions. WT,
BY4741. c The doubling time of ySYN24 (n = 3) in YPD. WT, BY4742
(n = 3). Source data are provided as a [264]Source Data file. The data
are presented as the mean and SD. d The cell morphology of ySYN24. WT,
BY4742. Scale bars, 5 μm. Images are representative of at least three
independent samples. e FACS analysis after propidine iodide staining on
asynchronous cells. WT, BY4742. f Differentially expressed genes on
ptSYN24 in ySYN24. The nine essential TUs, whose counterpart in
ptWT10U-10KO were also differentially expressed, were labeled in brown.
g Functional enrichment analysis of the differentially expressed genes
in ySYN24. The differential genes were mapped to a global similarity
network annotated using SAFE. The colors represent different function
domains. Triangles with white rims indicate genes located in chrXIIL.
Subsequently, several characterizations on ySYN24 were carried out. At
first, it showed growth defects on YPD plates and severe sensitivity to
rapamycin, while less dramatic fitness defects were detected under
other conditions (Fig. [265]6b). Consistently, its doubling time was
more than twofold longer than that of wild type (Fig. [266]6c). In
addition to the round shape like yWT25, the cell size was slightly also
enlarged in ySYN24 (Fig. [267]6d and Supplementary Fig. [268]9h). The
fraction of cells with 2 N DNA in log-phase population was
significantly higher than that of BY4742 (Fig. [269]6e), suggesting a
cell-cycle defect. In addition, ySYN24 could not be arrested as
efficiently as wild type under nutrient depletion conditions
(Supplementary Fig. [270]9i).
Furthermore, we determined the transcriptional profile of ySYN24
(Fig. [271]6f, g) and found that over three-quarters of genes on the
neochromosome (21/24), including nine essential genes, were
overexpressed (Fig. [272]6f, Supplementary Data [273]2). To visualize
the functional enrichments of the differentially expressed genes in
ySYN24, we mapped them to a yeast global genetic interaction similarity
network annotated by spatial analysis of functional enrichment
(SAFE)^[274]40 and labeled the genes on chrXIIL (Fig. [275]6g). These
genes were enriched in several bioprocesses, such as glycosylation,
cell wall biosynthesis, rRNA processing, DNA replication and repair,
mitosis, morphogenesis, and respiration. Four essential genes (YLL031C,
YLL008W, YLL011W, and YLL004W) and six non-essential genes (YLL040C,
YLL027W, YLL009C, YLL002W, YLL049W, and YLL021W) were clustered in
corresponding bioprocesses. Misexpression or deletion of these genes
may collectively contribute to the defects of ySYN24, such as round
cell shape caused by the removal of YLL021W^[276]41. Fine-tuning the
expression of the TUs for the other nine genes could be a promising
approach to optimize the fitness of ySYN24.
Discussion
The synthetic yeast genome has provided us a foundation to probe the
function of the eukaryotic genome with various design
principles^[277]8. In this study, using chrXIIL as an example, we
explored the possibility of simplifying the yeast genome by targeted
knockout and bottom-up gene replenishment, extended from previous
results of random deletions using SCRaMbLE^[278]11. We successfully
removed about four-fifths of the sequences in the chromosome arm and
generated a strain that could survive with only 12 genes (10 essential
genes plus YLL002W and YLL006W). In addition, we demonstrated that, for
most genes tested in this study (21/24), they could be reconstructed
using synonymously recoded ORFs and synthetic regulatory DNA to retain
their basic function. In particular, we found that aggressive
reprogramming of the coding sequences, that is, to encode each amino
acid with the same codon, can be tolerated, at least on this small
scale by the yeast. However, the fact that two genes with recoded CDSs
and one gene with synthetic promoters resulted in lethality or some
phenotypic changes in the mutants, suggests that caution must be taken
when redesigning synthetic genes with artificial sequences.
The successful synthesis of viral genomes, bacterial genomes and yeast
chromosomes imply that chemically synthesized genomes can support life
as well as wild-type genomes^[279]42–[280]44. Here, we demonstrate that
the functions of wild-type chromosomes can be assigned to
neochromosomes with simplified gene contents, arrangements and
sequences. By utilizing the GGI and growth phenotype of nonessential
genes, we constructed strains resistant to various stresses, such as
yWT25, with only about one-third genes on chrXIIL. Through
transcriptome data comparison, we improved the fitness of yWT25 using
only two additional genes. Combining these principles with multi-omics
data can facilitate the design of more simplified neochromosomes with
preferred features, which can be used to study the central network of
yeast genome and even potentially bypass the need of certain essential
genes. In addition, the results in this study and several previous
studies including relocating the rDNA locus^[281]29,[282]45 and
minimizing chromosome number in yeast^[283]46–[284]48 all suggest that
the specific organizational form of the yeast genome, including the
chromosome number, gene organization and genome 3D structure, might
only play minor roles in the basic functions of the yeast genome. This
might be the consequence of a small genome and mainly short-range gene
regulation in yeast^[285]49.
In nature, the 20 amino acids are encoded by 61 genetic codons, that
is, most amino acids are encoded by more than one codon. Codon usage
differs among organisms, and two of the 61 sense codons were removed
from a synthesized E. coli genome^[286]19, suggesting the number of
codons can be reduced. Theoretically, only 20 codons are needed to
encode 20 amino acids. It may, however, be difficult to achieve this
minimal codon set because of potentially important roles of codon
degeneracy in gene expression regulation^[287]16. It is surprising
that, in this study, most of the synthetic genes using the audacious
rule that one amino acid is encoded by only one codon remain
functional, suggesting the natural genome very plastic.
To prevent unexpected deleterious effects on gene function and to
reserve the wild type-like fitness in the synthetic strains, no
sequence engineering was applied to the promoters and terminators in
Sc2.0^[288]8. Synthetic promoters and terminators designed based on the
architecture of native ones yield diverse but reproducible expression
levels of fluorescent proteins and exogenous enzymes, which helps a lot
in deciphering the eukaryotic gene-regulatory logic and metabolic
engineering^[289]12–[290]14,[291]50,[292]51. In this study, we showed
that, although no sequence homology exists between these synthetic
promoters and terminators and the native ones, they actually can also
be used to build functional TUs, including the essential and
nonessential ones, demonstrating the high plasticity of regulatory
sequences.
In summary, as a pilot of the Sc3.0 project^[293]52, this study
presents the most radical changes we have ever made to a chromosome
arm. It indicates substantial plasticity of the yeast genome and
suggests the feasibility of using computationally designed sequences to
build functional eukaryotic genomes when we acquire enough artificial
regulatory elements and knowledge of the complex co-regulation network
in future. At the same time, the fitness defects of the strain with
totally artificial sequences and the failure to reprogram several
elements suggest that current strategies used in this study will be
risky to scale up to genome-wide redesign at the moment. Natural
sequences from related organisms may provide one of better solutions
for the reprogramming of the whole yeast genome.
Methods
Strains and growth media
The yeast strains used in this paper were derivatives of BY4741/2 or
synXIIL^[294]11. Standard methods for yeast culture and transformation
were applied. Targeted knockout methods were applied for deletion
through homologous recombination. The strains generated in this study
are listed in Supplementary Data [295]4. For phenotypic analysis of
strains, cells were cultured in YPD medium or YPD medium containing
0.9 mM H[2]O[2], 2 μg/mL nocodazole, 10 ng/mL rapamycin, 0.01% methyl
methanesulfonate, or 10 μg/mL benomyl separately. For the function
analysis of reconstructed TUs, cells were cultured on synthetic medium
with or without additional drugs. Particularly, the carbon source in
SCG-Ura was 3% glycerol, other than 2% glucose in other medium.
Construction of neochromosomes
Construction of neochromosomes include four steps: segment preparation,
yeast transformation, PCR identification and nanopore sequencing.
Segment preparation
All segments used for assembly contained at least 40 bp sequences
overlapped with their adjacent segments at each side. The segments were
released from plasmids using NotI digestion or PCR amplified using
synXIIL genome as template. Consistent with previous study^[296]25, the
native promoters were defined as 500 bp or the intergenic regions,
whichever is shorter. Similarly, the native terminators defined as
200 bp downstream or the intergenic regions, whichever is shorter.
(A) A total of 11 segments were used for assembling the ptWT10
neochromosome. Segment L and R were released from the constructed
plasmids using NotI digestion, segment M was PCR amplified using a
yeast strain containing a yeast artificial chromosome (Fig. [297]1A).
Segment 1–7 were amplified using synXIIL genome as template. The
coordinates for these segments in the synXII reference sequence^[298]29
are: segment 1, encoding YLL050C, 27,316-28,330; segment 2, encoding
YLL034C, YLL035W and YLL036C, 54,606–61,379; segment 3, encoding
YLL031C, 64,787–68,394; segment 4, encoding YLL018C, 96,751–100,011;
segment 5, encoding YLL011W, 115,173–117,239; segment 6, encoding
YLL008W, 119,397–122,283; segment 7, encoding YLL003W and YLL004W,
128,529–134,272. In addition, a fused linker containing sequences of
both URR3 and URR4 was also used to ligate segment M and R.
(B) In total, eight segments were used for assembling the ptWT10U
neochromosome. Segment L, R, and M were prepared using the same method
as ptWT10. Since the promoters of YLL035W and YLL036C share common
sequence and some important elements of the promoter of YLL036C are
located in the coding region of YLL035W, the promoters of YLL035W and
YLL036C were changed to pCYC1 and the terminator of YLL035W was changed
to tCYC1^[299]20. Segments 1–4 were amplified using the gDNA of BY4742
containing ptWT10 as template. The coordinates for these segments in
the ptWT10 reference sequence are: segment 1, encoding YLL050C and
YLL036C, 2532–5168; segment 2, encoding YLL035W, 5492–7390; segment 3,
encoding YLL034C, YLL031C and YLL018C, 7545–17,189; segment 4, encoding
YLL011W, YLL008W, YLL004W and YLL003W, 17,190–27,887. The same fused
linker in ptWT10 was also used.
(C) The ptWT12 was constructed by integrating a segment containing
YLL002W, YLL006W and KanMX4 at the right arm of ptWT10. The coordinates
for YLL002W and YLL006W in chrXII ([300]NC_001144.5) are: YLL002W,
146,041–147,802; YLL006W, 136,300–137,939.
(D) The ptWT18/25/27/31 were constructed similarly as ptWT12. Segment 1
containing eight nonessential genes and a KanMX4 selection marker was
integrated between URR3 and URR4 of ptWT10 firstly (ptWT18), then
segment 2, containing 7 nonessential genes and a NatNT2 selection
marker, was integrated at the middle of URR4 (ptWT25). The coordinates
for these 15 nonessential genes in chrXII ([301]NC_001144.5) are:
YLL002W, 146,041–147,802; YLL006W, 136,300–137,939; YLL009C,
131,005–131,728; YLL039W, 63,593–65,707; YLL040C, 54,011–63,925;
YLL043W, 49,438-52,087; YLL045C, 47,659–49,129; YLL049W, 40,179–41,381;
YLL018C-A, 108,476–109,472; YLL027W, 86,903–88,356; YLL028W,
84,304–86,765; YLL030C, 80,156–81,197; YLL033C, 72,909–74,302; YLL038C,
65,575–67018; YLL042C, 51,877–53,090. For ptWT31, four segments
containing six non-essential genes were integrated to replace the
NatNT2 selection marker of ptWT25. The coordinates for the 6
nonessential genes in chrXII ([302]NC_001144.5) are: YLL032C,
74,070–77,247; YLL029W, 80,961–83,910; YLL019C, 105,486-108,399;
YLL014W and YLL013C, 120,822–125,214; YLL001W, 147,390-150,363. For
ptWT27, the segment containing 2 nonessential genes and URA3 was
integrated into ptWT25 to replace KanMX4. The coordinates for the 2
nonessential genes in chrXII ([303]NC_001144.5) are: YLL013C,
121,837–125,222; YLL041C, 52,931–54,211.
(E) In total, seven segments were used for assembling the ptSYN10
neochromosome. Segments L, R, and M were prepared using the same method
as ptWT10 and ptWT10U. Segments 1–3 were released from the synthesized
plasmids using NotI digestion. The same fused linker in ptWT10 was also
used. The coordinates for these segments in the ptSYN10 reference
sequence are: segment 1, encoding syn035w and syn031c, 2536–8907;
segment 2, encoding syn003w, syn050c, syn036c, and syn034c,
8097–17,510; segment 3, encoding syn018c, syn011w, syn008w and
syn004w,17,5111–25,503.
(F) 7 segments were used for assembling the ptSYN12 neochromosome.
Segment L, R and M were prepared using the same method as ptWT10 and
ptWT10U. Segments 1–3 were prepared using the same method as ptSYN10.
Segment 4 was the fused product, including URR3, URR4, and the DNA
encoding syn002w and syn006w. The coordinates for these segments in the
ptSYN12 reference sequence are: segment 1, encoding syn035w and
syn031c, 2536–8907; segment 2, encoding syn003w, syn050c, syn036c and
syn034c, 8097–17,510; segment 3, encoding syn018c, syn011w, syn008w,
and syn004w, 17,511–25,503; segment 4, encoding syn006w and syn002w,
29,363-32,365.
(G) There were two steps to construct the ptSYN24 neochromosome based
on ptSYN10. The first step integrated a segment containing KanMX4
selection marker franked with two I-SceI sites between URR3 and URR4.
Six segments for assembling the ptSYN24 neochromosome were transformed
into the first-step strain expressing I-SceI restriction enzyme.
Segment 1, 2, and 4 were released from the plasmids containing
synthetic genes by BsaI digestion. Segment 3 was amplified using the
plasmid containing syn040c. Segment 5 and 6 were amplified by
overlapping PCR. The coordinates for these segments in the ptSYN24
reference sequence are: segment 1, encoding syn002w, syn006w, syn009c,
syn018c, and syn027w, 28,813–34,513; Segment 2, encoding syn028w,
syn030c, syn033w and syn038c, 34,474–39,327; Segment 3, encoding
syn040c, 39,284–49,041; Segment 4, encoding syn042c, syn043w, syn045c,
and syn049w, 48,764–54,652; segment 5, 34,194–34,793; segment 6,
39,003–39,602.
Yeast transformation
the protocol described in our previous paper^[304]29 was used for yeast
transformation. Briefly, 5 mL log-phase cells with OD[600] ~ 0.4–0.6
were harvested and washed with ddH[2]O and 0.1 M LiOAc/TE,
respectively. And 100 μL 0.1 M LiOAc/TE were used to resuspend the
cells. DNA fragments or plasmids were added to the cells. The mixture,
including 312 μL PEG3350, 41 μL 1 M LiOAc and 25 μL ssDNA (which were
boiled at 100 °C for 10 min and cooled on the ice for 5 min before
used), were added and mixed. After a 30 min incubation at 30 °C, 50 μL
DMSO were added and mixed. Then the tube was subjected to the
heat-shock for 15 min at 42 °C. Cells were washed once with 5 mM
CaCl[2]and plated onto the appropriate plates. The plates were cultured
at 30 °C for the appropriate time. The linear neochromosomes were
usually assembled in BY4742. Then these strains were used to mate with
MATa strains containing either multi-gene deletion or truncated chrXIIL
to construct the diploid strains which could sporulate to generate the
haploid strains for functional test of the neochromosomes, such as
ptWT10-10KO and ySYN24.
PCR identification
primers for synthetic sequence identification (PCRtags) and junction
verification were designed and listed in Supplementary Data [305]5. The
specificity of PCRtags was verified using the genomic DNA of strains
without the corresponding neochromosome as templates. PCR were
performed the same as our previous report^[306]29. Only colonies
positive for all junctions and PCRtags were subjected to further
sequencing analysis.
Nanopore sequencing
Total DNA was extracted using the QIAGEN Genomic-tips 100/G with
Genomic DNA buffer Set following the manufacturer’s instruction. DNA
quality was assessed by NanoDrop, gel-electrophoresis and Qubit.
Libraries were prepared using the Ligation Sequencing Kit (SQK-LSK109)
with the barcoding kits Native Barcoding Expansion 1–12 (PCR-free,
EXP-NDB104) and Native Barcoding Expansion 13–24 (PCR-free,
EXP-NDB104). Sequencing was performed using the MinlON platform with
FLO-MIN106D for a 72-h run. Base calling, adapter removal, and
low-quality base filtering based on fast5 files were done by ONT
software MinKNOW. The FastQ files were filtered by NanoFilt^[307]53 to
remove short (read length < 500) and low-quality (average read quality
score <7) reads. The remaining reads were mapped to reference genome by
NGMLR^[308]54 and assembled into contigs using Canu 2.1.1^[309]55.
Alignments between contigs and reference genomes were generated by
MUMmer-nucmer 3.23^[310]56 with default parameters and were used to
create collinearity dot plot by Dot
([311]https://github.com/dnanexus/dot). We divided the reads into two
parts: the linear neochromosome and native chromosomes. The mean
read-depth of a 1 kb-sized bin for all native chromosomes was
normalized to 1 and the ratio of the mean depth of the neochromosome to
that of all native chromosomes was calculated as the average copy
number of the neochromosome per haploid genome. The reads containing
full-length neochromosome were extracted and aligned to reference
sequence to infer the actual length of telomere TG repeats (TG repeats
to the extremity).
Pulsed-field gel electrophoresis and Southern blotting
About 2 × 10^8 cells at stationary phase were fixed in the 0.6% low
melting point agarose for each plug and genomic DNA was prepared as
described before^[312]29. Plug samples were resolved on a 1% agarose
gel in 0.5 X TBE for 16 h at 14 °C on a BioRad CHEF Mapper XA Pulsed
Field Electrophoresis System. The voltage was 6 V/cm, at an angle of
120° and switch time from initial 0.5 s to 1.5 s. The gels after PFGE
were washed with ddH[2]O twice and transferred onto Hybond-N+ membrane
(Amersham). The samples were UV crosslinked and hybridized with
DIG-labeled probes. Five DNA probes were amplified from corresponding
linear chromosomes for probe labeling and mixed together for
hybridization. Probe preparation, hybridization and detection were
conducted with DIG-High Prime DNA Labeling and Detection Starter Kit
(Roche, 11585614910). The primers for probes amplification are listed
in Supplementary Data [313]5.
Stability analysis of assembled chromosomes
Two independent colonies of the targeted strain were inoculated into
5 mL SC-His medium and cultured at 30 °C with shaking at 220 rpm for 24
hrs. Then 5 μL of the culture was added into 5 mL fresh SC-His medium
and grew for another 24 h. The cells after 10 days’ passages were
plated onto SC-His plates to isolate single colonies. Ten colonies of
each parent were picked and cultured separately for gDNA preparation
and PCRtag analysis to see whether some deletion events have happened
during about 125 generations.
Three out of the twenty colonies were further analyzed by whole genome
sequencing using the Miseq platform. Because the neochromosomes are
non-essential for BY4742, SC-His medium was used to select the
neochromosome. The indicated strain was inoculated into 5 mL SC-His
liquid medium and incubated at 30 °C for 12 h with shaking at 220 rpm.
5 × 10^7 yeast cells were harvested for total DNA extraction using the
Monarch® Genomic DNA Purification Kit based on the manufacturer’s
instructions. 300-1000 bp libraries were prepared using the Nextera DNA
Flex Library Prep Kit for purified genomic DNA and then sequenced on
the Illumina Miseq platform with PE250 strategy.
After adapter removal, the low-quality reads were trimmed by
Trimmomatic^[314]57 with parameters “SLIDINGWINDOW:5:20 LEADING:5
TRAILING:5 MINLEN:50”, and the remaining reads were mapped to reference
genome with BWA-mem^[315]58. After marking PCR repeat using
GATK-MarkDuplicates, the SNPs were called by GATK- HaplotypeCaller and
GATK- GenotypeGVCFs. Structural variations were called using
DELLY^[316]59.
Serial dilution
The serial dilution was performed as previously mentioned^[317]29. In
short, a single colony was inoculated into 3 mL YPD medium and
incubated at 30 °C with shaking at 220 rpm for 24 h. The OD[600] was
measured, and the overnight cultures were diluted by sterile water to
OD[600] = 0.2. After four tenfold gradient dilutions, well-mixed cells
were dropped onto indicated plates. The plates were cultured at 30 °C
for appropriate time unless specifically mentioned.
Doubling time assay
The measurements of growth curve were performed as before^[318]60.
Briefly, the log-phase cells were diluted with fresh medium to the same
density, and 100 µL of the diluted cells were added to each well of the
Costar clear polystyrene 96-well plates. For each culture, three or
four technical replicates were examined. The 96-well plate sealed with
Breathe-Easy membrane (Sigma, MKBZ0331) was cultivated in an Epoch2
microplate photometer (BioTek) at 30 °C for 36–48 h in a selected
medium. The OD[600] of each well was recorded every 10 min. The
doubling time (D) of each strain were acquired using the GraphPad Prism
8 software.
To analyze the resistance phenotype of yWT25, strain fitness was
measured using logarithmic strain coefficients (LSC) and logarithmic
phenotype index (LPI)^[319]61,[320]62. The growth of cells from three
colonies of BY4742 or yWT25 in YPD with or without MMS were analyzed.
Doubling times were used to generate LSC and LPI based on the following
formula:
[MATH: LSCi−j=lnDWT−<
mi>jDi−j<
/mrow> :MATH]
and
[MATH: LPIi−j=LSCi−j−LSCi−YPD :MATH]
where D[WT-j] represents the average doubling time of the three BY4742
strains under condition j, D[i-j] represents the doubling time of
strain i under condition j. The LPI[MMS] significantly greater than
zero indicates the resistance phenotype of corresponding strain.
Differential fitness scoring
To derive quantitative estimates of strain fitness, the same batch of
original images of agar plates after 3-day culture at 30 °C captured by
the same camera in serial dilution assay were used for scoring. These
original images were in the format of.JPG with a resolution of
3168 pixel × 4752 pixel. To meet the resolution restriction of
CellProfiler^[321]63, the original images were unified compressed by
50% before the modified pipeline from the example on the website
([322]https://cellprofiler.org/examples). To identify colonies
effectively, we set the “Typical diameter of objects” parameter in the
“Identify Primary Objects” step as 2 to 150^[323]64, and manually
checked the positions of the forced spots and the natural spots to
avoid misidentification. The “Mean Intensity” value of the spots in the
fourth gradient, where the wild-type strain did not reach the up limit
of the “Mean Intensity” value, was used as a measure of cell growth for
corresponding strains^[324]63,[325]65. To estimate strain fitness in
each condition, the average “Mean Intensity” for the four replicates of
wild type cells at different positions in the same plate in each
condition were normalized to be 1, and the normalized “Mean Intensity”
for strain i in condition j was defined as the fitness score in
condition j (f[ij]). The average fitness of the three replicates for
strain i on YPD plates was defined as the reference fitness (f[ir]).
Similar to previous study^[326]66, we calculated the differential
fitness score for each strain by measuring the difference between the
f[ir] and the f[ij]. The 2way ANOVA analysis was used to calculate the
difference significance. The stress conditions included MMS, benomyl,
H[2]O[2] and nocodazole.
Transcriptome analysis
The non-stranded RNA sequencing libraries were prepared and sequenced
by Beijing Novogene Bioinformatics Technology Co., Ltd. using Next®
Ultra^TM RNA Library Prep Kit for Illumina®. Because of the deletion of
a few genes in our strains, we first generated the diminished reference
genomes according to our design. We used Cutadapter software^[327]67 to
remove adapters in raw data. HISAT2^[328]68 and Picard were then used
to accomplish the alignments of cleaned reads and remove PCR
duplicates. Hereafter, Htseq-count software was employed to calculate
read counts of each gene while intersection-nonempty option was set.
The downstream statistical analysis was achieved by the DEseq2^[329]69
package in R. Considering that the remaining fragment of his3Δ1 in
chrXV can still be transcribed, we only use the deleted part in his3Δ1
to quantify HIS3. Differential expression in this paper was defined as
|log[2] FC| > 1 and −log[10] (Adjusted p value) > 4.
The transcriptome-wide disturbance was inferred according to
|log[2]FC|. For SAFE analysis, the SAFE add-on with default parameters
in cytoscape was used^[330]70. The original network subjected to SAFE
was derived from the global genetic interaction similarity network
published^[331]40. The GO biological process annotation was also
provided by the SAFE add-on. The transparency of the differentially
expressed genes in ySYN24 is set to 255 while the other genes are set
to 0.
Metabolome analysis
Single colonies were inoculated into 5 mL of SC medium for overnight at
30 °C. Each culture was diluted to OD[600] = 0.1 in fresh SC medium.
Cells were collected from 25 mL culture at different time points (8 h,
24 h and 48 h) and extracted for untargeted metabolomics according to
the previous methods with modifications^[332]71. A 1 mL solution
(methanol: water = 4:1, V/V) was added into the sample, which were
further placed in liquid nitrogen for 5 min and on the dry ice for
5 min, and then were thawed on ice and vortexed for 2 min. This above
freeze-thaw circle was repeated three times. The sample was centrifuged
at 13.400 g for 10 min and the supernatant was transferred and placed
in −20 °C for further analysis. Ultraperformance LC/QTOF MS
(UPLC-QTOF-MS/MS) acquisition was applied for untargeted metabolomic
analysis. Chromatographic separation of the metabolome was performed on
an ACQUITY UPLC BEH C18 (1.8 µm, 2.1 mm * 100 mm; Waters, Milford, MA,
USA) with column temperature maintained at 40 °C. Binary mobile phases
with phase A of water containing 0.1% formic acid (v/v) and phase B of
acetonitrile (0.1 % formic acid) were used for elution, respectively.
The column was eluted with the following linear gradient program:
0 min, 5% B; 11 min, 90% B; 12 min, 90%B; 12.1 min, 5% B; and 15 min,
5% B. The flow rate was set at 0.4 ml/min, and the injection volume was
2 μL. The MS data acquisition was operated using the
information-dependent acquisition mode using Analyst TF 1.7.1 Software
(Sciex, Concord, ON, Canada).
Raw metabolome data was converted into mzML format by ProteoWizard
software^[333]72. Peak extraction, peak alignment and retention time
correction were respectively performed by XCMS program. The “SVR”
method was used to correct the peak area. The peaks with detetion rate
lower than 50% in each group of samples were discarded. After that,
metabolic identification information was obtained by searching the
laboratory’s self-built database, integrated public database, AI
database and metDNA. Data were loaded in R ([334]www.r-project.org) and
unsupervised PCA was performed by statistics function prcomp. The
hierarchical cluster analysis (HCA) results of samples and metabolites
were presented as heatmaps with dendrograms, while pearson correlation
coefficients (PCC) between samples were caculated by the cor function
in R and presented as only heatmaps. Both HCA and PCC were carried out
by R package ComplexHeatmap. Differential metabolites were determined
by |log[2]FC| > 1 and P value (P value < 0.05, Student’s t test for
two-group analysis and ANOVA for multi-group analysis).
Identified metabolites were annotated using KEGG Compound database
([335]http://www.kegg.jp/kegg/compound/) and annotated metabolites were
then mapped to KEGG Pathway database
([336]http://www.kegg.jp/kegg/pathway.html). Significantly enriched
pathways are identified with a hypergeometric test’s P value for a
given list of metabolites^[337]73. For visualization of DMs in the
metabolic network, the KEGG compound IDs of DMs (8 h) are mapped to
iPATH3^[338]37 yeast metabolism pathway map
([339]https://pathways.embl.de/, species filter set to “sce”) to
generate dots. The uniprot ID of differentially expressed genes from
transcriptomic data are also mapped to the identical map to generate
lines.
Full-length transcriptome analysis
The transcriptomes of indicated strains were sequenced using the PacBio
platform at Grandomics Company. Circular Consensus Sequencing (CCS)
reads were generated using SMRT-Link (version 8.0.0.80529), with the
following modified parameters: “--min-passes 0 --min-length 50
--max-length 21000 --min-rq 0.75”. We used Lima (version 1.10.0, commit
SL-release-8.0.0) for Single Cell Full-Length Non-Concatemer (FLNC)
reads detection. Lima is integrated into the PacBio official SMRT-Link
(version 8.0.0.80529) software package. Lima map 5′ and 3′ primers to
CCS reads first, then parse standard pair of 5′ and 3′ primers CCS as
the full-length isoform, next trim the primer sequence and polyA tail
in each full-length isoform. Here, each isoform was oriented and
correspond to cDNA orientation from 5′ to 3′ end.
After FLNC detection, primer and polyA tail trimming, the remaining
fraction of each isoform FLNC read was split into wild-type genome
derived reads and neochromosome derived reads according to PCRtags
using LAST software ([340]https://gitlab.com/mcfrith/last), except the
ptSYN10 libraries (alignment methods can distinguish the completely
recoded genes on ptSYN10 from the wild-type ones). The neochromosome
derived reads or all reads from ptSYN10 libraries were aligned to the
designate reference genome with minimap2^[341]74 (version
2.17-r974-dirty) in spliced alignment mode with parameters: “-ax splice
-uf --secondary=no -C5”. Reads with more than 95% identity and 30%
coverage of a certain CDS are directly counted from paf files.
To ensure the generation of transcripts with high accuracy, we use
cDNA_Cupcake ([342]https://github.com/Magdoll/cDNA_Cupcake) python
script “collapse_isoforms_by_sam.py” to collapse redundant isoforms.
The “--flnc-coverage” for minimum collapsed reads is set to 5 and the
other parameters are set to default. After redundant isoforms
collapsing, unique isoforms can be reported as GFF file. A homemade R
script is used to illustrate the TSS, TES and transcription direction
based on gggenes ([343]https://github.com/wilkox/gggenes).
Truncation of chrXIIL
The four homologous fragments were amplified from the synXIIL and
respectively cloned into the plasmid with TeSS sequences using the
restriction sites, XmaI and SalI. The TeSS-Marker-HR fragments were
released by BsaI digestion and transformed into the heterozygous
diploid cells (chrXII X synXIIL). The chromosome arm capped strains
were screened with the PCRtags which have been reported to distinguish
chrXIIL(1.0) and synXIIL(2.0)^[344]29. The colonies which were positive
for 1.0 PCRtags but negative for 2.0 PCRtags indicated the specific
loss of sequences on synXIIL.
Tetrad analysis
Diploid strains were cultured in selective medium at 30 °C overnight.
About 8 × 10^7 cells were harvested and washed with ddH[2]O twice.
Cells were resuspended with 50 μL 1 × Sporulation medium (10 g/L
potassium acetate, 0.05 g/L zinc acetate dehydrate) and transferred
into 2 ml 1 × Sporulation medium. The tubes were incubated at 25 °C for
3–10 days. Then cells were harvested and resuspended in 30 μL
Zymolyase-100T (0.5 mg/ml Zymolyase-100T in 1 M sorbitol) for about
4–6 min at RT. 300 μL pre-cold ddH[2]O was added to stop digestion and
20 μL suspension was gently spread on YPD plates for further dissection
under the microscope. These plates were cultured at 30 °C for
appropriate days before imaging and replicated onto various selective
media to identify their auxotroph and mating type.
Analysis of protein length, genetic interaction, and gene ontology (GO)-terms
The theoretical protein length and genetic interaction information of
each gene are queried through SGD YeastMine service
([345]https://yeastmine.yeastgenome.org/yeastmine). Self-interactions
of a gene were eliminated before calculation of GGI. The synthetic
lethality is simultaneously inferred from the queries. The GO
information of each gene is collected through R package org.Sc.sgd.db.
Only the directly evidenced terms of each gene are represented in this
package. The GO terms of all protein coding genes are inferred and
summarized. The average number of GO terms for the genes on all
chromosome arms are subsequently calculated.
Assembly and screening of functional non-essential TUs
The pMV-AmpR plasmids, ORF plasmids and HCKan_terminator plasmids,
hosting promoter pool (P), ORF (O) and terminator pool (T) were
prepared for YeastFab assembly. The promoter fragments were amplified
by ExTaq DNA polymerase (TaKaRa). The YeastFab assembly is performed
according to the reaction system below: 1 μL 10X Buffer for T4 DNA
Ligase (NEB), 0.1 μL Purified BSA 100X (Thermo), 0.2 μL T4 DNA Ligase
(Thermo), 0.5 μL Esp3I (Thermo), 2 μL purified promoter PCR product,
2 μL ORF plasmid (20 ng/μL), 2 μL HCKan_terminator plasmid (20 ng/μL),
2 μL vector plasmid (20 ng/μL) and 0.2 μL ddH[2]O. Then carry out the
following program in a thermal cycler: 37 °C for 2 h, 55 °C for 15 min,
80 °C for 15 min. The reaction products were transformed into E. coli
DH5α and 5 mL LB liquid medium containing carbenicillin disodium
(100 μg/mL) was added into the bacteria mix, shaking at 37 °C
overnight. Plasmids were extracted from the bacteria mixture,
transformed into yeast, and plated on selective plates.
Six individual colonies were randomly picked and cultured in selective
liquid media overnight at 30 °C. The OD[600] was measured, and the
overnight cultures were diluted in sterile water to OD[600] = 1. After
four 10-fold gradient dilution, well-mixed cells were dropped onto
indicated plates. The plates were cultured at appropriate temperature.
The yeast strains with recovered phenotypes were selected. The plasmids
carrying the synthetic TUs were isolated and identified by sanger
sequencing.
Flow cytometry analysis
Samples were selected at corresponding time points and cells were fixed
with 70% ethanol overnight at 4 °C. Cells were resuspended in 50 mM
sodium citrate (pH 7.0) and briefly sonicated on ice. Cells were
resuspended in 50 mM sodium citrate (pH 7.0) and added with RnaseA
(0.25 mg/mL) for incubation at 37 °C. Cells were washed with 50 mM
sodium citrate (pH 7.0) and resuspended in 50 mM sodium citrate (pH
7.0) containing propidium iodide (16 µg/mL). The cells were incubated
at room temperature for at least 1 h. Samples were proceeded with BD
FACS Celesta for measurement. The software FlowJo was used for analysis
and the fraction of cells at different stages was calculated with the
Dean-Jett-Fox model.
Microscope imaging
Cells were cultured in YPD overnight and subcultured into fresh YPD for
several hours to get cells at log phase. Cells were harvested gently
and washed once with ddH[2]O. Then cells were dropped on slides for
further imaging with a Nikon A1 confocal microscope under 60×
objective. To calculate cell size of strains at different stages with
Image J, the cells of each strain were defined into two groups: G1
cells (not budding) and dividing cells (budding).
Statistics and reproducibility
Error bars in this study represent SD. Unless specially noted,
two-tailed t tests were used to compare different groups in this paper.
All experiments were repeated independently at least three times.
Differences were considered as statistically significant at p
value < 0.05. * indicates p < 0.05, ** indicates p < 0.01, ***
indicates p < 0.001, **** indicates p < 0.0001.
Reporting summary
Further information on research design is available in the [346]Nature
Portfolio Reporting Summary linked to this article.
Supplementary information
[347]Supplementary information^ (2.7MB, pdf)
[348]41467_2023_43531_MOESM2_ESM.pdf^ (28.8KB, pdf)
Description of Additional Supplementary Files
[349]Supplementary Data 1^ (157.5KB, xlsx)
[350]Supplementary Data 2^ (195.2KB, xlsx)
[351]Supplementary Data 3^ (34.4KB, xlsx)
[352]Supplementary Data 4^ (13.1KB, xlsx)
[353]Supplementary Data 5^ (18.7KB, xlsx)
[354]Reporting Summary^ (356.1KB, pdf)
[355]Peer Review File^ (3.2MB, pdf)
Source data
[356]Source data^ (1.8MB, xlsx)
Acknowledgements