Global rewiring of cellular metabolism renders Saccharomyces cerevisiae Crabtree negative

Saccharomyces cerevisiae is a Crabtree-positive eukaryal model organism. It is believed that the Crabtree effect has evolved as a competition mechanism by allowing for rapid growth and production of ethanol at aerobic glucose excess conditions. This inherent property of yeast metabolism and the multiple mechanisms underlying it require a global rewiring of the entire metabolic network to abolish the Crabtree effect. Through rational engineering of pyruvate metabolism combined with adaptive laboratory evolution (ALE), we demonstrate that it is possible to obtain such a global rewiring and hereby turn S. cerevisiae into a Crabtree-negative yeast. Using integrated systems biology analysis, we identify that the global rewiring of cellular metabolism is accomplished through a mutation in the RNA polymerase II mediator complex, which is also observed in cancer cells expressing the Warburg effect.

T he yeast Saccharomyces cerevisiae is a widely used model organism for studying the biology of eukaryal cells as well as it is extensively used as a cell factory for the production of pharmaceuticals, chemicals, and biofuels 1,2 . Its metabolism has evolved to have oxidative fermentation, meaning that even in the presence of oxygen, the yeast uses fermentative metabolism when glucose is in excess, a metabolic feature that is generally referred to as the Crabtree effect 3,4 . This million-year-old evolution feature ensures the advant.age in its ecological niche due to the ability to rapidly consume glucose and produce ethanol that has antiseptic properties. However, it generally results in reduced yields when this yeast is used as a cell factory. There is therefore much interest in rewiring the central carbon metabolism to abolish the Crabtree effect.
Eliminating pyruvate decarboxylase activity in yeast completely abolishes the Crabtree effect, but the growth deficiency of pyruvate decarboxylase minus (Pdc − ) strains in excess glucose conditions 5 limits their application for biotechnology. Even though Pdc − strains have been studied for last 25 years, only one strategy has so far enabled successful restoration of the growth of Pdc − strains in a minimal medium with excess glucose. This strategy involves introducing MTH1 mutations, which were originally identified from Pdc − strains evolved to grow in excess glucose 6 . However, the specific growth rate of this strain was only at 0.1 h −1 , and acetyl-CoA generation in the cytosol relies on acetate supplementation and the native ATP-dependent acetyl-CoA synthetase 7 .
To overcome this challenge, we create an alternative pyruvate dehydrogenase (PDH) bypass in S. cerevisiae with an ATPindependent acetyl-CoA synthesis pathway. With this, growth of a Pdc − strain is successfully restored in minimal media with excess glucose. Combining rational design, adaptive laboratory evolution (ALE), and reverse engineering, the specific growth rate of the best strain reaches 0.218 h −1 , which is close to the maximum growth of S. cerevisiae with purely respiratory metabolism 8 and the maximum specific growth rate of most Crabtree-negative yeasts 9 . We find that, to unlock the millions of years of evolution that has determined metabolic features of S. cerevisiae, many different metabolic parts need to be engineered, and an important element is enabling global transcriptional alteration by having a mutation in the mediator complex that supports rewiring of cellular metabolism.
Increasing growth of the Crabtree-negative S. cerevisiae. Although the specific growth rate of this S. cerevisiae strain is similar to that of some natural Crabtree-negative yeasts, such as Kluyveromyces nonfermentans (0.101 h −1 ) and Eremothecium sinecaudum (0.117-0.122 h −1 ), it is still much lower than for most natural Crabtree-negative yeasts (0.249-0.429 h −1 ) (Supplementary Table 2).
We therefore established three independent yeast populations based on exposing sZJD-24 (prototrophic strain based on sZJD-23) (Supplementary Table 1) to ALE for 40 days, which is a duration compromising the selection of clones with improved fitness and not accumulating too many mutations. All of the three evolved populations have a higher specific growth rate compared with starting strain sZJD-24. (Supplementary Figure 4). The maximum specific growth rate of clones picked from each of these three populations reached 0.217 h −1 , 0.221 h −1 , and 0.209 h −1 , respectively (Fig. 2a). Through genome sequencing of seven clones (two each from sZJD-24A and sZJD-24B, three from sZJD-24C), we found a total of 19 single nucleotide variations (SNVs) in 18 genes (Supplementary Table 3). Although there were no shared mutations among all seven clones (Fig. 2b), a nonstop mutation in MED2 was identified in five clones derived from lines sZJD-24A and sZJD-24C. The two clones of line sZJD-24B shared a mutation in MED3. Both Med2 and Med3 are components of the tail module of the RNA polymerase II mediator complex 10 . These results indicated that the mediator complex may play a key role in regulating cell growth. Only three SNVs were identified in the clones from line sZJD-24C, which shared a nonsense mutation in GPD1 encoding a NADH-dependent glycerol-3-phosphate dehydrogenase 11 . For all clones in line sZJD-24B, besides MED3, we found shared mutations in SIW14 and MHO1. Siw14 is tyrosine phosphatase involved in actin organization and endocytosis 12 , Mho1 is a protein of unknown function.
Therefore, MED2, MED3, GPD1, HXK2, and SIW14 were chosen as reverse engineering targets to evaluate if mutations in these gene were causal. We successfully obtained the GPD1 W71* and MED2 *432Y single mutant strains sZJD-26 and sZJD-27, respectively, using the Cas9-expressing strain sZJD-25. The The genes with mutations identified in all of the isolated strains. sZJD-24A1 and A2 were from lines sZJD-24A, sZJD-24B1; B2 was from lines sZJD-24B, sZJD24C1; C2 and C3 were from line sZJD-24C. c Determination of the specific growth rate of reverse engineered strains based on sZJD-23 in shake flasks. d Growth and metabolite profiles of sZJD-28 in 20 g l −1 glucose minimal medium. All data represent the mean ± s.d. of biological triplicates specific growth rate of these two strains increased by 31.5 and 47.0% compared with the starting strain sZJD-25, reaching 0.139 h −1 , and 0.156 h −1 respectively. A MED2 *432Y and GPD1 W71* double-mutant strain sZJD-28 reached an even higher specific growth rate of 0.205 h −1 , which is 98% of the specific growth rate of the evolved line sZJD-24C (Fig. 2c), showing a clear causal effect of these two mutations. This is consistent with the finding that these two mutations were the only two SNVs found in the evolved strains sZJD-24C2 and sZJD-24C3 (Fig. 2b). sZJD-28 consumed glucose with faster rate and reached higher OD 600 value compared with starting strain sZJD-23. The extracellular metabolites were also lower than those of sZJD-23 (Figs. 2d, 1e). Compared with wild-type strain CEN.PK113-11c, sZJD-28 had much higher biomass yield and lower RQ value, which is close to 1. The maximal specific growth rate of sZJD-28 reached 0.218 h −1 (Table 1). Thus, through identification of targets using ALE, we managed to engineer a better and faster growing Crabtree-negative S. cerevisiae with reduced carbon loss.
Transcriptional profiles of the Crabtree-negative strain. To understand the underlying mechanism of how the mutations results in faster growth rate of the reverse engineered strains, we used RNA-Seq to perform transcriptome analysis of the parental strain sZJD-25 and the three strains sZJD-26, sZJD-27, and sZJD-28. Transcriptome analysis showed that in sZJD-28, totally, 2096 genes, about 33% of all genes of S. cerevisiae, were significantly (padj < 0.01) differentially expressed and 1562 genes in sZJD-27 compared with sZJD-25 (Fig. 3a). This indicated that the nonstop mutation of MED2 resulted in a global impact on the metabolic network. Med2 is one of the subunits of the tail module 10 , which is one of the four parts of the mediator complex, and it is required for the regulated transcription of nearly all RNA polymerase IIdependent genes in S. cerevisiae [13][14][15] . The tail module mediates mediator complex-associated transcriptional regulation on SAGA-regulated, TATA-containing genes which account for about 15% of all the genes in yeast 16,17 (Supplementary Figure 5). Indeed, almost half of all TATA-containing genes had significantly altered expression in the MED2 mutant strains sZJD-28 (7.5%) and sZJD-27 (6.8%) (Fig. 3b). It indicated a clear causal effect of this mutation. Additionally, the expession level of TATA-containing genes in the MED2 mutant strain were predominantly downregulated, especially in the double-mutant strain sZJD-28 (Fig. 3c), which is clearly seen in the Volcano plot of sZJD-28 (Supplementary Figure 6). GO Slim Mapper analysis on the 114 shared TATA-containing genes showed that GO terms related to response to cellular amino acid metabolic processes, carbon metabolism, and chemical and oxidative stress were enriched (Supplementary Data 1). It indicated that altering the expression of genes with these GO terms may play an indispensable role in improving the cell growth rate.
The reporter GO term analysis showed that genes associated with GO terms related to translation are upregulated (upper part of heat map in Fig. 3d), whereas genes associated with GO terms related to carbon metabolism are downregulated (lower part of heat map in Fig. 3d). Protein synthesis is required for cell growth and needs ribosomes to polymerize amino acids into polypeptide chains. The cellular growth rate is linearly correlated with ribosome abundance, and the expression of ribosome-associated genes, including genes coding for ribosomal proteins and rRNA biogenesis, therefore affects the growth rate 18,19 . Indeed, in sZJD-28 and sZJD-27, protein synthesis-associated genes were significantly upregulated ( Fig. 3d and Supplementary Figure 7). In addition, reporter TFs analysis showed that genes controlled by chromatin remodeling-related TFs such as Snf2, Snf6, Sin3, Sas3, and Rsc1 were significantly changed in sZJD-27 and sZJD-28, in contrast to sZJD-25 (Supplementary Figure 8-9). It suggested that gene transcription may have become more active, supporting increased protein synthesis in these two strains. However, increasing the ribosomal protein fraction would reduce the fraction of metabolic proteins 20 . Indeed, GO terms related to carbon metabolism-contained genes were significantly downregulated in sZJD-27 and sZJD-28 ( Fig. 3d and Supplementary  Figure 7). Downregulating of these metabolic genes would save the resource for ribosomal proteins synthesis, as glycolytic enzymes account for a major fraction of the cellular proteome. These results suggested that the introduced mutations led to redistributed and active protein synthesis, which may support a faster cell growth rate.

Disscusion
Pdc − S. cerevisiae strains cannot grow in batch cultures on synthetic glucose medium. Two reasons are lacking of cytosolic acetyl-CoA supply and limited capacity of reoxidation of cytosolic NADH 21 . In sZJD-25, the PO/PTA pathway can produce acetyl-CoA in the cytosol, which supported the growth of Pdc − S. cerevisiae strains in an excess glucose medium. However, the reoxidation of cytosolic NADH still mainly relies on the mitochondrial respiratory chain due to the absence of alcoholic fermentation. In sZJD-25, the unrestricted glucose uptake and high glycolytic activity would particularly cause problems with recycling of cytosolic NADH to NAD + . Compared with sZJD-25, the high-affinity glucose transporter genes HXT2, HXT4, HXT6, HXT7, HXT10, and HXT14 were upregulated and the low/medium-affinity glucose transporter genes HXT1, HXT3, HXT5, HXT9, and HXT11 were downregulated in sZJD-28. (Fig. 3e). The fold changes of high-affinity glucose transporter genes were higher than that of the low-affinity ones (Supplementary  Figure 10). Expression of high-affinity glucose transporters were repressed by high levels of glucose and induced by low levels of glucose 22 . Transriptional changes in these glucose transporters indicated that glucose uptake was restricted in sZJD-28 compared with sZJD-25. Additonally, almost all of the genes in glycolysis were downregulated in sZJD-28 (Fig. 3e). Restricted glucose uptake and activity of glycolysis would release the burden on the respiration chain, which is consistent with the the expression profiles of oxidative phosphorylation (OXPHOS) genes, especially in sZJD-28 NADH dehydrogenase genes (NDE1 and NDE2) and ubiquinol-cytochrome c oxidoreductase genes (complex III), which were significantly downregulated compared with sZJD-25 (Supplementary Figure 11). These results indicate that the limited glucose consumption capacity of sZJD-25, which was enhanced by the introduced mutations led to higher glycolytic flux in sZJD-28, but now with balancing of NADH formation and oxidation back to NAD+. The faster glucose consumption rate, higher biomass yield and lower RQ of sZJD-28 compared with that of sZJD-25 (Table 1) further confirmed the higher glycolytic flux and respiration rate in the reverse engineered sZJD-28. The balance between fermentation and respiration may lead to efficient carbon and electron flux in the cell, which can support the faster growth rate. Taken together, we demonstrated that the Crabtree-positive S. cerevisiae can be turned into a Crabtree-negative yeast by systematic engineering, which included rational pathway design and   system biology analysis. The growth rate of this engineered S. cerevisiae reached 0.218 h −1 , which was two folds of previous MTH1 reverse engineered strains and almost reached the level of many natural Crabtree-negative yeasts. By systems biology analysis, the mediator complex was identified as a global regulator involved in rewiring the central carbon metabolism and allocating protein synthesis in a way that favors faster growth of this Crabtree-negative S. cerevisiae. We believe that the derived yeast strain represents a possible platform strain for use in biotechnology as well as global rewiring of yeast metabolism through engineering; the mediator complex may be used as a strategy in metabolic engineering of yeast. Additionally, our finding on restricting glucose flux by modulation of the conservative mediator complex may give an insight into cancer metabolism due to the similarity between Crabtree effect and Warburg effect in cancer cells 23 . Thus, many cancer cells have altered pyruvate metabolism 24 and mutations in the mediator complex 25,26 .   Germany). The oxygen uptake rate at each time point was calculated by correcting for the dilution caused by CO 2 production according to the Eq. 1:

Methods
where, F is the gas flow existing the bioreactor, O 2 in is the fractional concentration of O 2 in the air entering the bioreactor, O 2 out is the fractional concentration of O 2 in the gas existing the bioreactor, N 2 in is the combined fractional concentration of nitrogen and argon in the air entering the bioreactor (0.791) and N 2 out is the combined fractional concentration of nitrogen and argon existing the bioreactor (1-O 2 out-CO 2 out). The total mols of oxygen consumed until each time point were calculated by integrating the OUR curve against time, and the resulting values were against the biomass concentration in grams of cell dry weight. The specific oxygen consumption rate was calculated by taking the slope of the linear trendline and multiplying by the growth rate. For CO 2 calculation, first the release rate was calculated by Eq. 2: and then, it is the same procedure as for O 2 .
Genetic manipulation. For plasmid construction, standard procedures of restriction enzyme digestion and ligation were used 29 . For gene deletion, integration, and point mutation, two strategies were used in this study. One way is seamless gene deletion or integration by using Kluyveromyces lactis URA3 (KlURA3) as a selection marker 30 . The KlURA3 marker was flanked by two homologous sequences, which were used for looping out the KlURA3 marker after gene deletion and integrating via yeast homologous recombination system. Removing the KlURA3 marker from the strain can be performed by using selective SCD+ 5'-FOA or SCE+ 5'-FOA plates. The other way for gene deletion, integration, and point mutation is CRISPR-Cas9-based strategy 31 . The Cas9 expression cassette was first fused with a KanMX cassette and integrated into X-2 site of the yeast genome for utilization of CRISPR-Cas9 system. For ALE, URA3 and HIS3 were integrated into XI-5 and X-2 sites of the starting strain sZJD-23, respectively, resulting in strain sZJD-24. All of the plasmids and gene deletion or integration cassettes were transformed into yeast strains by using the LiAc/SS DNA/PEG method 32 .
Metabolite extract and analysis. For HPLC analysis, 1 ml of the fermentation broth was centrifuged at 18,000 g for 5 min at 4°C. The supernatant was filtered through a 0.2 μm filter (VWD) then analyzed on an Aminex HPX-87H column (Bio-rad) in an Ultimate 3000 HPLC system (ThermoFisher Scientific). To detect extracellular glucose, ethanol, glycerol, acetate, pyruvate, and succinate, the column was set at 45°C and eluted with 5 mM H 2 SO 4 at a flow rate of 0.6 ml min −1 . were used with helium as carrier gas at a flow rate of 1 ml min −1 . Initial oven temperature was set at 50°C for 1.5 min, increased up to 170°C (30°C min −1 ) and held for 1.5 min. The temperature was then increased to 300°C (15°C min −1 ) and maintained for 3 min.
Enzyme activity assay. For cell extract preparation, yeast cells were harvested during the exponential phase and washed with cold enzyme activity detection buffer (1 M potassium phosphate, pH 6.7 and 100 mM Tris HCl, pH 7.4 for pyruvate oxidase and phosphotransacetylase, respectively). Then, the cell suspension was transferred into 2 ml lysing matrix tubes containing beads (MP Biomedicals). Cells were lysed by the MP FastPrep-24 instrument with four 20 sec cycles at the speed of 4.0 m s −1 ; the cell lysate was cooled down in ice for 5 min between cycles. Cell debris was removed from the cell lysate by centrifugation at 18,000 g for 5 min at 4°C, and the supernatant was used for enzyme activity assays. Protein concentration was determined using the Quick Start Bradford Protein Assay (Bio-Rad). For pyruvate oxidase and phosphotransacetylase activity analysis, previously described methods were used 35,36 . Specifically, for pyruvate oxidase, spectrophotometric stop rate determination method was used. The assay condition was: 37°C, pH = 6.7, 565 nm, and light path = 1 cm. Adaptive laboratory evolution. Adaptive laboratory evolution experiments with S. cerevisiae sZJD-24 were carried out by serial dilutions in shake flasks. The strains were cultured at 30°C, 200 r.p.m. in minimal medium with 20 g l −1 glucose. Cells from three independent colonies were used for three independent evolution series. Serial transfer was performed every day or every second day. For every transfer, the cell culture was diluted by a factor ranging from 1:6 to 1:10 into minimal glucose medium to give an initial OD 600 of about 0.1. After 40 days, the three populations were spread on YPD plates, and three clones were randomly picked from each line of evolution: sZJD-24-1A, sZJD-24-1B, and sZJD-24-1C from the first line, sZJD-24-2A, sZJD-24-2B, and sZJD-24-2C from the second line, and sZJD-24-3A, sZJD-24-3B, and sZJD-24-3C from the third line. The specific growth rates of these nine strains were determined in shake flasks with 20 g l −1 minimal glucose medium.
Genome sequencing. The genomic DNA of the ALE strains was extracted by using the Blood & Cell culture DNA Kit. The quality of the genomic DNA was assessed by the Agilent 2100 Bioanalyzer according to the manufacturer's instructions. The genomic DNA of sZJD-24-1C and sZJD-24-2C failed to meet the sequencing quality requirement and was discarded. DNA from strains ZJD-24-1A, sZJD-24-1B, sZJD-24-2A, sZJD-24-2B, sZJD-24-3A, sZJD-24-3B, sZJD-24-3C, and parental strain sZJD-24 was sent for sequencing. The Illumina TruSeq Nano HT 96 was used for preparing libraries for genome sequencing, and whole-genome sequencing was performed on the Illumina NextSeq by using the 2 × 150 bp pairedend method according to manufacturer's manual. S. cerevisiae CEN.PK 113-7D was used as the reference genome (cenpk.tudelft.nl) for mapping the reads. Breseq-0.28.1 was used for detecting single nucleotide variants, insertions, and small deletions 37 .
Transcriptome analysis. Cells were collected at OD 600 ≈ 1 and stored at −80°C before processing for RNA extraction. Total RNA was extracted by using the RNeasy Mini Kit. The quality of RNA samples was assessed by using the Agilent 2100 Bioanalyzer according to the manufacturer's instructions. The RNA samples were prepared by using the TruSeq RNA Stranded HT Sample Prep Kit (Illumina) and sequenced by NextSeq Series Mid-Output Kit (2 × 75) (Illumina). Reads were aligned on the yeast genome by using Bowtie2 38 and then further processed by SAMTools 39 and BEDTools 40 to count the number of reads aligning to each gene. Differential gene expression was analyzed by using the DEseq package in the R programming language 41 . Reporter analysis on GO terms and transcription factors was performed by using the Platform for Integrative Analysis of Omics (PIANO) R package 42 . Differential expression levels and p-adj values were used as input in reporter analysis. GO slim mapper analysis was conducted with the online tool at the SGD website (http://www.yeastgenome.org/cgi-bin/GO/goSlimMapper.pl).
Data availability. The RNA-Seq raw data of the reverse engineered strains and the control strain can be downloaded from the European Nucleotide Archive with the access number PRJEB23677 (https://www.ebi.ac.uk/ena/data/search?query = + PRJEB23677). The data that support the findings of this study are available within the article and its Supplementary Information file or available from the corresponding author upon reasonable request.