Pathway In Saccharomyces Cerevisiae Research Articles

Efficient biosynthesis of resveratrol via combining phenylalanine and tyrosine pathways in Saccharomyces cerevisiae

BackgroundResveratrol is a commercially available stilbenoid widely used as dietary supplements, functional food ingredients, and cosmetic ingredients due to its diverse physiological activities. The production of resveratrol in microorganisms provides an ideal source that reduces the cost of resveratrol, but the titer in Saccharomyces cerevisiae was still much lower than that in other hosts.ResultsTo achieve enhanced production of resveratrol in S. cerevisiae, we constructed a biosynthetic pathway via combining phenylalanine and tyrosine pathways by introducing a bi-functional phenylalanine/tyrosine ammonia lyase from Rhodotorula toruloides. The combination of phenylalanine pathway with tyrosine pathway led to a 462% improvement of resveratrol production in yeast extract peptone dextrose (YPD) medium with 4% glucose, suggesting an alternative strategy for producing p-coumaric acid-derived compounds. Then the strains were further modified by integrating multi-copy biosynthetic pathway genes, improving metabolic flux to aromatic amino acids and malonyl-CoA, and deleting by-pathway genes, which resulted in 1155.0 mg/L resveratrol in shake flasks when cultured in YPD medium. Finally, a non-auxotrophic strain was tailored for resveratrol production in minimal medium without exogenous amino acid addition, and the highest resveratrol titer (4.1 g/L) ever reported was achieved in S. cerevisiae to our knowledge.ConclusionsThis study demonstrates the advantage of employing a bi-functional phenylalanine/tyrosine ammonia lyase in the biosynthetic pathway of resveratrol, suggesting an effective alternative in the production of p-coumaric acid-derived compounds. Moreover, the enhanced production of resveratrol in S. cerevisiae lays a foundation for constructing cell factories for various stilbenoids.Graphical

Assembly of nitrogenase biosynthetic pathway in Saccharomyces cerevisiae by using polyprotein strategy.

Nitrogenase in some bacteria and archaea catalyzes conversion of N2 to ammonia. To reconstitute a nitrogenase biosynthetic pathway in a eukaryotic host is still a challenge, since synthesis of nitrogenase requires a large number of nif (nitrogen fixation) genes. Viral 2A peptide mediated "cleavage" of polyprotein is one of strategies for multigene co-expression. Here, we show that cleavage efficiency of NifB-2A-NifH polyprotein linked by four different 2A peptides (P2A, T2A, E2A, and F2A) in Saccharomyces cerevisiae ranges from ~50% to ~90%. The presence of a 2A tail in NifB, NifH, and NifD does not affect their activity. Western blotting shows that 9 Nif proteins (NifB, NifH, NifD, NifK, NifE, NifN, NifX, HesA, and NifV) from Paenibacillus polymyxa that are fused into two polyproteins via 2A peptides are co-expressed in S. cerevisiae. Expressed NifH from Klebsiella oxytoca NifU and NifS and P. polymyxa NifH fusion linked via 2A in S. cerevisiae exhibits Fe protein activity.

New Light on the Varietal Thiols Pathway during Alcoholic Fermentation: Role of 3-S-(N-Acetyl-cysteinyl)-hexan-1-ol (NAC3SH).

For many years, knowledge on thiol precursors has been limited to S-conjugates of glutathione (G3SH), cysteine (Cys3SH), and later on the dipeptides γ-GluCys and CysGly. In this work, we took the parallel between precursor degradation and the glutathione-mediated detoxification pathway a step further by considering a new type of derivative, 3-S-(N-acetyl-l-cysteinyl)hexanol (NAC3SH). This compound was synthesized and then added to the existing liquid chromatography with tandem mass spectrometry (LC-MS/MS) method of thiol precursors. This intermediate was only identified during alcoholic fermentation in synthetic must spiked with G3SH (1 mg/L or 2.45 μmol/L) in the presence of copper with concentration above 1.25 mg/L, which demonstrates for the first time the existence of this new derivative (until 126 μg/L or 0.48 μmol/L) and the capacity of the yeast to produce such a compound. Its status as a precursor was also studied during fermentation, in which a release of 3-sulfanylhexanol was noted corresponding to a conversion yield close to 0.6%. This work completed the thiol precursor's degradation pathway in Saccharomyces cerevisiae in synthetic conditions with a new intermediate, confirming its connection with the xenobiotic detoxification pathway and giving new insights on the precursor's fate.

TrLys9 participates in fungal development and lysine biosynthesis in Trichoderma reesei.

Fungi uniquely synthesize lysine through the α-aminoadipate pathway. The saccharopine reductase ScLys9 catalyzes the formation of saccharopine from ɑ-aminoadipate 6-semialdehyde, the seventh step in the lysine biosynthesis pathway in Saccharomyces cerevisiae. Here, we characterized the functions of TrLys9, an ortholog of S. cerevisiae ScLys9 in the industrial filamentous fungus Trichoderma reesei. Transcriptional level analysis indicated that TrLYS9 expression was higher in the conidial stage than in other stages. Disruption of TrLYS9 led to lysine auxotrophy. Phenotype analysis of the ΔTrlys9 mutant showed that TrLYS9 was involved in fungal development including vegetative growth, conidiation, and conidial germination and lysine biosynthesis. Cellulase production was also impaired in the ΔTrlys9 mutant due to the failure of conidial germination in liquid cellulase-inducing medium. Defects in radial growth and asexual development of the ΔTrlys9 mutant were fully recovered when exogenous lysine was added to the medium. These results imply that TrLys9 is involved in fungal development and lysine biosynthesis in T. reesei.

Comparison of Genome and Plasmid-Based Engineering of Multigene Benzylglucosinolate Pathway in Saccharomyces cerevisiae.

Intake of brassicaceous vegetables such as cabbage is associated with numerous health benefits. The major defense compounds in the Brassicales order are the amino acid-derived glucosinolates that have been associated with the health-promoting effects. This has primed a desire to build glucosinolate-producing microbial cell factories as a stable and reliable source. Here, we established-for the first time-production of the phenylalanine-derived benzylglucosinolate (BGLS) in Saccharomyces cerevisiae using two different engineering strategies: stable genome integration versus plasmid-based introduction of the biosynthetic genes. Although the plasmid-engineered strain showed a tendency to generate higher expression level of each gene (except CYP83B1) in the biosynthetic pathway, the genome-engineered strain produced 8.4-fold higher BGLS yield compared to the plasmid-engineered strain. Additionally, we optimized the genome-engineered strain by overexpressing the entry point genes CYP79A2 and CYP83B1, resulting in a 2-fold increase in BGLS production but also a 4.8-fold increase in the level of the last intermediate desulfo-benzylglucosinolate (dsBGLS). We applied several approaches to alleviate the metabolic bottleneck in the step where dsBGLS is converted to BGLS by sulfotransferase, SOT16 dependent on 3'-phosphoadenosine-5'-phosphosulfate (PAPS). BGLS production increased 1.7-fold by overexpressing SOT16 and 1.7-fold by introducing APS kinase, APK1, from Arabidopsis thaliana involved in the PAPS regeneration cycle. Modulating the endogenous sulfur assimilatory pathway through overexpression of MET3 and MET14 resulted in 2.4-fold to 12.81 μmol/L (=5.2 mg/L) for BGLS production. IMPORTANCE Intake of brassicaceous vegetables such as cabbage is associated with numerous health benefits. The major defense compounds in the Brassicales order are the amino acid-derived glucosinolates that have been associated with the health-promoting effects. This has primed a desire to build glucosinolate-producing microbial cell factories as a stable and reliable source. In this study, we engineered for the first time the production of phenylalanine-derived benzylglucosinolate in Saccharomyces cerevisiae with two engineering strategies: stable genome integration versus plasmid-based introduction of the biosynthetic genes. Although the plasmid-engineered strain generally showed higher expression level of each gene (except CYP83B1) in the biosynthetic pathway, the genome-engineered strain produced higher production level of benzylglucosinolate. Based on the genome-engineered strain, the benzylglucosinolate level was improved by optimization. Our study compared different approaches to engineer a multigene pathway for production of the plant natural product benzylglucosinolate. This may provide potential application in industrial biotechnology.

An accessory enzymatic system of cellulase for simultaneous saccharification and co-fermentation

The enhanced hydrolysis of xylan-type hemicellulose is important to maximize ethanol production yield and substrate utilization rate in lignocellulose-based simultaneous saccharification and co-fermentation system. In this study, we conduct δ-integration CRISPR Cas9 to achieve multicopy chromosomal integration with high efficiency of reductase–xylitol dehydrogenase pathway in Saccharomyces cerevisiae. Subsequently, we devise a consolidated bioprocessing-enabling S. cerevisiae consortium, in which every engineered yeast strain could secrete or display different assembly components to be adaptively assembled on the surface of scaffoldin-displaying yeast cell for synergistic catalysis and co-fermentation from steam-exploded Pennisetum purpureum. Despite the accumulation of xylitol, the maximum ethanol titer of the genetically engineered yeast strain reached 12.88 g/l with the cellulose conversion of 91.21% and hemicellulose conversion of 55.25% under 30 ºC after 96 h with the addition of commercial cellulase. The elaborated cellulosomal organization toward genetic engineering of an industrially important microorganism presents a designed approach for advanced lignocellulolytic potential and improved capability of biofuel processing.Graphical

A High-Copy Suppressor Screen Reveals a Broad Role of Prefoldin-like Bud27 in the TOR Signaling Pathway in Saccharomyces cerevisiae.

Bud27 is a prefoldin-like, a member of the family of ATP-independent molecular chaperones that associates with RNA polymerases I, II, and III in Saccharomyces cerevisiae. Bud27 and its human ortholog URI perform several functions in the cytoplasm and the nucleus. Both proteins participate in the TOR signaling cascade by coordinating nutrient availability with gene expression, and lack of Bud27 partially mimics TOR pathway inactivation. Bud27 regulates the transcription of the three RNA polymerases to mediate the synthesis of ribosomal components for ribosome biogenesis through the TOR cascade. This work presents a high-copy suppression screening of the temperature sensitivity of the bud27Δ mutant. It shows that Bud27 influences different TOR-dependent processes. Our data also suggest that Bud27 can impact some of these TOR-dependent processes: cell wall integrity and autophagy induction.

Deletion of autophagy related, ATG1 and F-box motif encoding YDR131C, together, lead to synthetic growth defects and flocculation behavior in Saccharomyces cerevisiae.

Ubiquitin proteasome system (UPS) and autophagy both pathways are involved in clearing the nonessential cellular components and also crosstalk during cellular response to normal and stress conditions. The F-box motif proteins constitute the SCF-E3 ligase complex of the UPS pathway in Saccharomyces cerevisiae and are involved in the substrate recruitment for ubiquitination. The ATG1 encoded Atg1p, a conserved serine-threonine kinase is crucial for the autophagy process. Here in this study, we report that loss of F-box motif encoding YDR131C and ATG1 together results in growth defects, floc formation, sensitivity to hydroxyurea, methyl methanesulfonate, and hydrogen peroxide. Both the genes also interact with the flocculation-related genes (FLO) and associate with gene ontology terms "ubiquitin-protein transferase activity" and "cellular catabolic process."Based on in silico analysis and experimental evidence we conclude that YDR131C and ATG1 function in parallel pathways to regulate the growth, flocculation, and stress response.

Curcumin biosynthesis from ferulic acid by engineered Saccharomyces cerevisiae.

Curcumin is a polyphenolic compound found in Curcuma longa. This bioactive molecule has several reported health-benefit effects, being the anticarcinogenic activity among the most promising ones. However, curcumin extraction from natural sources is hampered by impure products obtained from harsh chemicals and limited by plant seasonality and high prices. Therefore, curcumin heterologous production emerged as an interesting alternative. Escherichia coli has been explored as chassis but the implementation of the pathway in Saccharomyces cerevisiae can have several advantages, including its generally regarded as safe status. Hence, S. cerevisiae was engineered for the first time to produce curcumin from its precursor ferulic acid. The enzymes 4-coumarate-CoA ligase (4CL1) from Arabidopsis thaliana or feruloyl-CoA synthetase (FerA) from Pseudomonas paucimobilis and type III polyketide synthases (PKSs) from Oryza sativa or C. longa were expressed in BY4741 strain. To avoid ferulic acid deviation, the gene FDC1 coding a ferulic acid decarboxylase was deleted. The maximum curcumin titer was obtained with FerA combined with C. longa PKSs (2.7mg L-1 ). Up to our knowledge, this is the first work reporting the expression of a feruloyl-CoA synthase and also curcuminoid biosynthetic enzymes in S. cerevisiae, and consequently, curcumin production.

The CWI Pathway: A Versatile Toolbox to Arrest Cell-Cycle Progression.

Cell-signaling pathways are essential for cells to respond and adapt to changes in their environmental conditions. The cell-wall integrity (CWI) pathway of Saccharomyces cerevisiae is activated by environmental stresses, compounds, and morphogenetic processes that compromise the cell wall, orchestrating the appropriate cellular response to cope with these adverse conditions. During cell-cycle progression, the CWI pathway is activated in periods of polarized growth, such as budding or cytokinesis, regulating cell-wall biosynthesis and the actin cytoskeleton. Importantly, accumulated evidence has indicated a reciprocal regulation of the cell-cycle regulatory system by the CWI pathway. In this paper, we describe how the CWI pathway regulates the main cell-cycle transitions in response to cell-surface perturbance to delay cell-cycle progression. In particular, it affects the Start transcriptional program and the initiation of DNA replication at the G1/S transition, and entry and progression through mitosis. We also describe the involvement of the CWI pathway in the response to genotoxic stress and its connection with the DNA integrity checkpoint, the mechanism that ensures the correct transmission of genetic material and cell survival. Thus, the CWI pathway emerges as a master brake that stops cell-cycle progression when cells are coping with distinct unfavorable conditions.

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways.

Endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs compromise genome integrity and are eliminated by multiple repair pathways. Aberrant Top1-DNA crosslinks, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae. It remains obscure how cells choose between diverse mechanisms of DPC repair. Here, we show that several SUMO biogenesis factors (Ulp1, Siz2, Slx5, and Slx8) control repair of Top1cc or an analogous DPC lesion. Genetic analysis reveals that SUMO promotes Top1cc processing in the absence of Tdp1 but has an inhibitory role if cells additionally lack Wss1. In the tdp1Δ wss1Δ mutant, the E3 SUMO ligase Siz2 stimulates sumoylation in the vicinity of the DPC, but not SUMO conjugation to Top1. This Siz2-dependent sumoylation inhibits alternative DPC repair mechanisms, including Ddi1. Our findings suggest that SUMO tunes available repair pathways to facilitate faithful DPC repair.

Engineering of Saccharomyces cerevisiae for sensing sweetness

With the rapidly increasing demand for sweeteners, it is necessary to establish an effective sweetness evaluation system for exploring new sweeteners. In this study, a gene circuit was constructed to couple with the inherent mitogen-activated protein kinase (MAPK) pathway in Saccharomyces cerevisiae to sense sweeteners. The Sst2, Far1, and Ste2 genes were knocked out to amplify MAPK pathway signaling. Then, the C-terminus of the inherent Gpa1 of S. cerevisiae was replaced with the five residues of human taste converting protein so that Gpa1 could interact with T1R2/T1R3 for sweetness signaling. Finally, the human sweetness receptor T1R2/T1R3 was heterologously expressed in engineered S. cerevisiae by N-terminal signal peptide engineering, creating strain D3Gm, which showed the ability to sense sweeteners. This study provided proof of the concept that S. cerevisiae can be engineered to sense sweetness.

Variation in the modality of a yeast signaling pathway is mediated by a single regulator.

Bimodal gene expression by genetically identical cells is a pervasive feature of signaling networks and has been suggested to allow organisms to hedge their 'bets' in uncertain conditions. In the galactose-utilization (GAL) pathway of Saccharomyces cerevisiae, gene induction is unimodal or bimodal depending on natural genetic variation and pre-induction conditions. Here, we find that this variation in modality arises from regulation of two features of the pathway response: the fraction of cells that show induction and their level of expression. GAL3, the galactose sensor, controls the fraction of induced cells, and titrating its expression is sufficient to control modality; moreover, all the observed differences in modality between different pre-induction conditions and among natural isolates can be explained by changes in GAL3's regulation and activity. The ability to switch modality by tuning the activity of a single protein may allow rapid adaptation of bet hedging to maximize fitness in complex environments.

Distinct role of HAMP and HAMP-like linker domains in regulating the activity of Hik1p, a hybrid histidine kinase 3 from Magnaporthe oryzae.

Nik1 orthologs or group III hybrid histidine kinases (HHK3) represent a unique cytoplasmic osmosensor that act upstream of HOG/p38 MAPK pathway in fungi. It is an important molecular target for developing new antifungal agents against human pathogens. HHK3 orthologs contain a linear array of alternative HAMP and HAMP-like linker domains (poly-HAMP) in the N-terminal region. HAMP domains are quite common in prokaryotic histidine kinases where it mostly functions as signal transducer mediating conformational changes in the kinase domains. In contrast, poly-HAMP in HHK3 acts as a sensor and signal transducer to regulate histidine kinase activity. However, the mechanistic detail of this is poorly understood. Interestingly, recent studies indicate that the poly-HAMP-mediated regulation of the kinase activity varies among the orthologs. Hik1 is an important HHK3 ortholog from fungus Magnaporthe oryzae. In this paper, we aimed to decipher the role HAMP and HAMP-like linker domains in regulating the activity of Hik1p. We show that Hik1p acts as a bona fide osmosensor and negatively regulates the downstream HOG/p38 MAPK pathway in Saccharomyces cerevisiae. Our data suggest a differential role of the HAMP domains in the functionality of Hik1p. Most interestingly, the deletion of individual domains in poly-HAMP resulted in distinct active forms of Hik1p and thereby indicating that the poly-HAMP domain, instead of acting as on-off switch, regulates the histidine kinase activity by transition through multiple conformational states.

Abstract P-12:Three-dimensional Structure of the Yeast Transmembrane Mechanosensor Wsc1

Background: Wsc1 is the best studied of the five mechanosensors of the cell wall integrity (CWI) signal transduction pathway in Saccharomyces cerevisiae. In genetic and biophysical studies, Wsc1 functions were assessed either in living yeast cells or in crude cell extracts. So far, no attempts to purify the sensor and determine its structure have been reported. Methods: The Wsc1-green fluorescent protein (GFP) fusion was expressed in S. cerevisiae following standard protocols. For solubilization, a 5% (w/v) solution of styrene-maleic acid (SMA) copolymer was added dropwise to the membrane suspension to get a final cell-to-SMA weight ratio of 1:2.5. The suspension was incubated for 30 min at room temperature (RT) and then for 16 hours at 4 °C, followed by centrifugation for 20 min at 134000 g at 4 °C. The supernatant was subsequently purified on affinity resin. Protein samples (3 µL) were placed onto the glow-discharged grid, stained twice, using 1% aquatic uranyl acetate solution for 30 s at RT, and air-dried. Micrographs were acquired using a transmission electron microscope Jem-2100 (Jeol, Japan) equipped with a 2K x 2K CCD camera Ultrascan 1000XP (Gatan, USA). The microscope operated at 200 kV in a low dose mode, with a magnification x40000 (2.5 Å/pix) and defocus 0.5-1.9 µm. Images were acquired automatically with SerialEM software. Results: We applied the 3:1 SMA copolymer to isolate Wsc1-GFP fusions into SMA/lipid particles (SMALPs) directly from native yeast membranes. We purified Wsc1-GFP-containing SMALPs by affinity chromatography; characterization by dynamic light scattering confirmed the presence of monodisperse nano-sized particles. The intensity-weighted diameter was estimated to be in the 10-nm range. The purified Wsc1-GFP-containing SMALPs were further analyzed by transmission electron microscopy (TEM). 46500 single particles were used to generate the 3D reconstructions of Wsc1-GFP. The resulting model showed the three functional parts of the Wsc1 sensor: the extracellular part, which has a clasp-like appearance, the transmembrane domain, and the short cytosolic part of Wsc1 fused to the GFP beta-barrel. Conclusion: The 3:1 SMA copolymer enabled isolation of Wsc1-GFP fusion complexes into SMALPs directly from native yeast membranes. Image analysis of the TEM data showed the Wsc1 in its contracted conformation. We propose a model of conformational changes in Wsc1 in response to mechanical stress.

TORC1 regulates the transcriptional response to glucose and developmental cycle via the Tap42-Sit4-Rrd1/2 pathway in Saccharomyces cerevisiae

BackgroundTarget of Rapamycin Complex 1 (TORC1) is a highly conserved eukaryotic protein complex that couples the presence of growth factors and nutrients in the environment with cellular proliferation. TORC1 is primarily implicated in linking amino acid levels with cellular growth in yeast and mammals. Although glucose deprivation has been shown to cause TORC1 inactivation in yeast, the precise role of TORC1 in glucose signaling and the underlying mechanisms remain unclear.ResultsWe demonstrate that the presence of glucose in the growth medium is both necessary and sufficient for TORC1 activation. TORC1 activity increases upon addition of glucose to yeast cells growing in a non-fermentable carbon source. Conversely, shifting yeast cells from glucose to a non-fermentable carbon source reduces TORC1 activity. Analysis of transcriptomic data revealed that glucose and TORC1 co-regulate about 27% (1668/6004) of yeast genes. We demonstrate that TORC1 orchestrates the expression of glucose-responsive genes mainly via the Tap42-Sit4-Rrd1/2 pathway. To confirm TORC1’s function in glucose signaling, we tested its role in spore germination, a glucose-dependent developmental state transition in yeast. TORC1 regulates the glucose-responsive genes during spore germination and inhibition of TORC1 blocks spore germination.ConclusionsOur studies indicate that a regulatory loop that involves activation of TORC1 by glucose and regulation of glucose-responsive genes by TORC1, mediates nutritional control of growth and development in yeast.

Sensing, Uptake and Catabolism of L-Phenylalanine During 2-Phenylethanol Biosynthesis via the Ehrlich Pathway in Saccharomyces cerevisiae.

2-Phenylethanol (2-PE) is an important flavouring ingredient with a persistent rose-like odour, and it has been widely utilized in food, perfume, beverages, and medicine. Due to the potential existence of toxic byproducts in 2-PE resulting from chemical synthesis, the demand for “natural” 2-PE through biotransformation is increasing. L-Phenylalanine (L-Phe) is used as the precursor for the biosynthesis of 2-PE through the Ehrlich pathway by Saccharomyces cerevisiae. The regulation of L-Phe metabolism in S. cerevisiae is complicated and elaborate. We reviewed current progress on the signal transduction pathways of L-Phe sensing, uptake of extracellular L-Phe and 2-PE synthesis from L-Phe through the Ehrlich pathway. Moreover, the anticipated bottlenecks and future research directions for S. cerevisiae biosynthesis of 2-PE are discussed.

Bornyl Diphosphate Synthase From Cinnamomum burmanni and Its Application for (+)-Borneol Biosynthesis in Yeast.

(+)-Borneol is a desirable monoterpenoid with effective anti-inflammatory and analgesic effects that is known as soft gold. (+)-bornyl diphosphate synthase is the key enzyme in the (+)-borneol biosynthesis pathway. Despite several reported (+)-bornyl diphosphate synthase genes, relatively low (+)-borneol production hinders the attempts to synthesize it using microbial fermentation. Here, we identified the highly specific (+)-bornyl diphosphate synthase CbTPS1 from Cinnamomum burmanni. An in vitro assay showed that (+)-borneol was the main product of CbTPS1 (88.70% of the total products), and the Km value was 5.11 ± 1.70 μM with a kcat value of 0.01 s–1. Further, we reconstituted the (+)-borneol biosynthetic pathway in Saccharomyces cerevisiae. After tailored truncation and adding Kozak sequences, the (+)-borneol yield was improved by 96.33-fold to 2.89 mg⋅L–1 compared with the initial strain in shake flasks. This work is the first reported attempt to produce (+)-borneol by microbial fermentation. It lays a foundation for further pathway reconstruction and metabolic engineering production of this valuable natural monoterpenoid.

Improved aromatic alcohol production by strengthening the shikimate pathway in Saccharomyces cerevisiae

Aromatic alcohols, such as tyrosol and 2-phenylethanol, are widely used in various industries owing to their superior flavors and properties. In this study, a yeast metabolic modification platform for the de novo synthesis of tyrosol and 2-phenylethanol was established. Alleviation of the feedback inhibition and overexpression of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase and chorismate mutase were achieved by introducing aroGD146N and pheAfbr genes from Escherichia coli. Shikimate kinase (aroL) and shikimate dehydrogenase (ydiB), analogues of the penta-functional enzyme Aro1p, were overexpressed to release the metabolic bottlenecks. Furthermore, in addition to ARO10-overexpression, ARO9 was disrupted to reduce the by-product generation. Finally, by introducing EcaroGD146N, EcpheAfbr, EcydiB and EcaroL in the ARO9-deleted strain, together with overexpression of transketolase (TKL1) and phenylpyruvate decarboxylase (ARO10), the engineered strain JM26 was constructed. Using glucose as the sole carbon source, JM26 achieved 541 mg/L (73.5 mg/g DCW) of tyrosol and 643 mg/L (92.0 mg/g DCW) of 2-phenylethanol, which is 5.7-fold and 6.1-fold higher than the control strain, respectively. These results demonstrate that the strategies applied improved de novo production of tyrosol and 2-phenylethanol, and showed potential for application as a metabolic chassis for other aromatic compounds in Saccharomyces cerevisiae and the exploration of their biological functions.

Ancient balancing selection maintains incompatible versions of the galactose pathway in yeast.

Metabolic pathways differ across species but are expected to be similar within a species. We discovered two functional, incompatible versions of the galactose pathway in Saccharomyces cerevisiae We identified a three-locus genetic interaction for growth in galactose, and used precisely engineered alleles to show that it arises from variation in the galactose utilization genes GAL2, GAL1/10/7, and phosphoglucomutase (PGM1), and that the reference allele of PGM1 is incompatible with the alternative alleles of the other genes. Multiloci balancing selection has maintained the two incompatible versions of the pathway for millions of years. Strains with alternative alleles are found primarily in galactose-rich dairy environments, and they grow faster in galactose but slower in glucose, revealing a trade-off on which balancing selection may have acted.