KR102670084B1 - Novel kinase for treatment and prevention of fungal infection and the use thereof - Google Patents

Abstract

The present invention relates to the use of kinases for the treatment and prevention of fungal meningoencephalitis caused by pathogenic fungi of the genus Cryptococcus. Specifically, the present invention provides an antifungal agent screening method comprising measuring the amount or activity of a virulence-regulating kinase protein of Cryptococcus neoformans, or the expression level of a gene encoding the protein; and an antifungal pharmaceutical composition comprising an inhibitor for the pathogenicity-regulating kinase protein of Cryptococcus neoformans or the gene encoding the same, which is an antifungal agent for treating meningoencephalitis, etc. using the antifungal agent screening method according to the present invention. can be effectively screened, and meningoencephalitis, etc. can be effectively treated using the antifungal pharmaceutical composition according to the present invention, and can be widely used in related industrial fields such as the pharmaceutical field and life science field.

Description

NOVEL KINASE FOR TREATMENT AND PREVENTION OF FUNGAL INFECTION AND THE USE THEREOF}

The present invention relates to novel kinases and their use for the prevention and treatment of fungal infections caused by pathogenic fungi. In addition, the present invention provides an antifungal agent screening method comprising measuring the amount or activity of a virulence-regulating kinase protein of Cryptococcus neoformans , or the expression level of a gene encoding the protein; and an antifungal pharmaceutical composition comprising an inhibitor for the virulence-regulating kinase protein of Cryptococcus neoformans or the gene encoding it.

Cryptococcus neoformans is a fungal pathogen that is distributed in a variety of natural environments, including soil, trees, and bird guano, has a variety of infectious agents, and uses a variety of hosts ranging from lower eukaryotic cells to aquatic and terrestrial animals (Lin, & Heitman, J. The biology of the Cryptococcus neoformans species Rev. 60, 69-105, 2006). Cryptococcus neoformans is a major cause of death due to fungal meningitis, and is known to cause approximately one million new infections and approximately 600,000 deaths worldwide each year (Park, BJ et al. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23, 525-530, doi:10.1097/QAD.0b013e328322ffac, 2009). However, only limited treatments are used to treat systemic Cryptococcus's (Perfect, JR et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america. Clin Infect Dis 50, 291-322, doi:10.1086/649858, 2010). Meanwhile, C. neoformans is considered an ideal fungal model system for basidiomycetes, with a fully sequenced and well-described genome database, precise genetic analysis methods through sexual differentiation, and efficient reverse and forward genetic methods. , and the availability of various heterologous host model systems (Idnurm, A. et al. Deciphering the model pathogenic fungus Cryptococcus neoformans. Nat. Rev. Microbiol. 3, 753-764, 2005).

To understand the pathogenic mechanisms of C. neoformans, extensive research has been conducted over the past decades. In addition to efforts to analyze the function of individual genes/proteins, recent large-scale functional genetic analyzes have led to a greater understanding of the overall biological circuitry of C. neoformans. However, the general biological characteristics and signaling and metabolic pathways governing pathogenicity of C. neoformans are not yet fully understood. The main reason for this is that the functions of kinases, which play an important role in signaling pathways and control the activation or expression of transcription factors (TFs), have not been sufficiently characterized on a genome scale. In general, kinases affect approximately 30% of cellular proteins through phosphorylation and play key roles in growth, cell cycle regulation, differentiation, development, stress response, and many other cellular functions (Cohen, P. The regulation of protein function by multisite phosphorylation-a 25 year update. Trends Biochem Sci 25, 596-601, 2000). In addition, kinases are considered a protein family that is a major target for drug development because their activity can be easily inhibited by small molecules such as compounds or antibodies (Rask-Andersen, M., Masuram, S. & Schioth, HB The druggable genome: Evaluation of drug targets in clinical trials suggests major shifts in molecular class and indication. Annu Rev Pharmacol Toxicol 54, 9-26, doi:10.1146/annurev-pharmtox-011613-135943, 2014). Therefore, systematic and functional profiling of fungal kinases in human fungal pathogens is needed more than ever before to identify virulence-related kinases that can be developed as antifungal drugs.

Accordingly, the present inventors constructed a library of 226 high-quality marker gene deletion strains through homologous recombination of 114 putative kinases, and developed growth, differentiation, stress response, antifungal resistance, and virulence factor production (capsule, melanin, and urease). Systematic functional profiling of the kinome network in C. neoformans as well as Basidiomycota was performed by examining their phenotypes under 30 different in vitro growth conditions, including . Additionally, the present inventors investigated their potential pathogenicity and infectivity in insect and murine host models.

The object of the present invention relates to novel kinases and their use for the prevention and treatment of fungal infections caused by pathogenic fungi. Additionally, the present invention provides screening of antifungal agents by measuring the amount or activity of the virulence-regulating kinase protein of Cryptococcus neoformans , or the expression level of the gene encoding the protein. In addition, the present invention provides an antifungal pharmaceutical composition containing an inhibitor and/or activator for the virulence-regulating kinase protein of Cryptococcus neoformans or the gene encoding it. Additionally, the present invention provides a method for screening drug candidates for the treatment and prevention of cryptococcosis or meningitis. Additionally, the present invention provides a pharmaceutical composition for the treatment and prevention of cryptococcosis or meningitis. Additionally, the present invention provides a method for diagnosing fungal infections.

In order to achieve the above object, the present invention provides a novel pathogenicity regulatory kinase protein. Specifically, the novel pathogenicity regulatory kinases according to the present invention are Fpk1, Bck1, Gal83, Kic1, Vps15, Ipk1, Mec1, Urk1, Yak1, Pos5, Irk1, Hsl101, Irk2, Mps1, Sat4, Irk3, Cdc7, Irk4, Swe102. , Vrk1, Fbp26, Psk201, Ypk101, Pan3, Ssk2, Utr1, Pho85, Bud32, Tco6, Arg5,6, Ssn3, Irk6, Dak2, Rim15, Dak202a, Snf101, Mpk2, Cmk1, Irk7, Cbk1, Kic102, Mkk2, Cka1 , Bub1, but is not limited thereto.

In addition, the present invention includes the steps of (a) contacting a sample to be analyzed with cells containing a pathogenicity-regulating kinase protein; (b) measuring the amount or activity of the protein; and (c) determining that the sample is an antifungal agent when the amount or activity of the protein is measured to be down or up regulated.

In addition, the present invention includes the steps of (a) contacting a sample to be analyzed with a cell containing a gene encoding a pathogenicity regulatory kinase protein; (b) measuring the expression level of the gene; and (c) determining that the sample is an antifungal agent when the expression level of the gene is measured to be down or up regulated.

In the present invention, the cells used for antifungal agent screening may be fungi, for example, Cryptococcus neoformans.

In the present invention, the antifungal agent may be used to treat and prevent meningoencephalitis or cryptococcosis, but is not limited thereto.

In the present invention, the term "sample" refers to an unknown candidate substance used in screening to test whether it affects the expression level of a gene or affects the amount or activity of a protein, such as a chemical substance or nucleotide. , antisense-RNA, siRNA (small interference RNA), and natural product extracts, but are not limited thereto.

In the present invention, the term "antifungal agent" refers to an agent that inhibits the growth of bacteria and/or fungi, and includes inorganic antifungal agents, organic natural product extract antifungal agents, organic aliphatic compound antifungal agents, and organic aromatic compound antifungal agents. Inorganic antifungal agents include chlorine compounds represented by sodium hypochlorite, boric acid peroxide represented by hydrogen peroxide, boric acid compounds represented by sodium borate, copper compounds represented by copper sulfate, zinc compounds represented by zinc sulfate and zinc chloride, sulfur, and polysulfuric acid. There are sulfur-based substances represented by lime and hydrated sulfur, calcium compounds represented by calcium oxide, sodium thiosulfate, silver complexes, and silver compounds represented by silver nitrate, as well as oxo and sodium silicofluoride. Examples of antifungal agents derived from organic natural products include , hinokitiol, Maengjong porridge extract, creosote oil, etc., but are not limited thereto.

In the present invention, measurement of changes in gene expression level can be performed through various known methods. For example, RTPCR (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press, 2001), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102- 108, CRCpress), hybridization reaction using a cDNA microarray (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press, 2001), or in situ hybridization reaction (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press, 2001). When performed according to the RT-PCR protocol, total RNA is first isolated from the cells treated with the sample, and then single-stranded cDNA is prepared using oligo dT primer and reverse transcriptase. Subsequently, a PCR reaction is performed using single-stranded cDNA as a template and a gene-specific primer set. Gene-specific primer sets used in the present invention are shown in Tables 2 and 3 below. Next, the PCR amplification product is electrophoresed, and the formed band is analyzed to measure changes in gene expression.

In the present invention, changes in the amount or activity of a protein can be performed through various known immunoassay methods. Examples include, but are not limited to, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, and sandwich assay. The method of the immunoassay or immunostaining is described in various literature (Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999. For example, when using a radioimmunoassay, protein-specific antibodies labeled with radioisotopes (e.g., C14, I125, P32, and S35) can be used.

When using ELISA in one embodiment of the present invention, (i) coating the extract of the sample-treated cells on the surface of a solid substrate, (ii) the protein of the kinase itself or a labeled protein-specific antibody as a primary antibody. and reacting the cell extract, (iii) reacting the result of step (ii) with a secondary antibody to which the enzyme is bound, and (iv) measuring the activity of the enzyme. Suitable solid substrates are hydrocarbon polymers (eg polystyrene and polypropylene), glass, metal or gel, most preferably microtiter plates. Enzymes bound to the secondary antibody include, but are not limited to, enzymes that catalyze color reaction, fluorescence reaction, luminescence reaction, or infrared reaction, for example, alkaline phosphatase, β-galactosidase , horseradish peroxidase, luciferase and cytochrome P450. When alkaline phosphatase is used as an enzyme that binds to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitroblue tetrazolium (NBT), and naphthol-ASB1-phosphate (naphthol-AS-B1) are used as substrates. -phosphate) and ECF (enhanced chemifluorescence) can be used, and when horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, and lucigenin (bis- N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), TMB (3,3,5,5-tetramethylbenzidine), Substrates such as ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]) and o -phenylenediamine (OPD) can be used. In the ELISA method, the final enzyme activity measurement or signal measurement can be performed according to various generally known methods. When biotin is used as a label, the signal can be easily detected with streptavidin, and when luciferase is used as a label, the signal can be easily detected with luciferin.

In addition, one embodiment of the present invention provides an antifungal pharmaceutical composition containing an agent (inhibitor or activator) for a fungal pathogenicity-regulating kinase protein. In another embodiment, the fungus is Cryptococcus neoformans.

In addition, the present invention provides, in one embodiment, an antifungal pharmaceutical composition containing an agonist (inhibitor or activator) for a gene encoding a fungal pathogenicity-regulating kinase protein. In one embodiment, the fungus is Cryptococcus neoformans.

In the present invention, the pharmaceutical composition may be used to treat meningoencephalitis or cryptococcosis, but is not limited thereto.

In the present invention, the agent may be an antibody. In one embodiment, the inhibitor may be an inhibitor that blocks signal transduction by binding to a protein and inhibiting the activity of the protein. For example, it may be a peptide or compound that binds to a protein, and these peptides or compounds may be selected by a screening method including protein structure analysis, etc., and may be designed using generally known methods. Additionally, in the case of a polyclonal or monoclonal antibody against a protein, it can be produced using generally known antibody production methods.

In the present invention, the term "antibody" refers to synthetic antibodies, monoclonal antibodies, polyclonal antibodies, recombinantly produced antibodies, intrabodies, multispecific antibodies (including dual-specific antibodies), human antibodies, Humanized antibody, chimeric antibody, single-chain Fv (scFv) (including dual-specific scFv), BiTE molecule, single chain antibody, Fab fragment, F(ab') fragment, disulfide-linked Fv (sdFv), or one of the above It may be an epitope-binding fragment of any of the following. In the present invention, the antibody may be any of the immunoglobulin molecules or immunologically active portions of immunoglobulin molecules, and may be of any isotype. Additionally, in the present invention, the antibody may be a full-length antibody containing variable and constant portions, a single-chain antibody, or an antigen-binding fragment thereof such as a Fab or Fab'2 fragment. In the present invention, antibodies may be conjugated or linked to therapeutic agents such as cytotoxins or radioactive isotopes.

In the present invention, the agent may be an anti-sense oligonucleotide for a gene, siRNA, shRNA, miRNA, or a vector containing these, but is not limited thereto.

In the present invention, the inhibitor may be an inhibitor that blocks signal transduction by inhibiting the expression of a gene, interferes with transcription by binding to a gene, or interferes with translation of mRNA by binding to an mRNA transcribed from a gene. In one embodiment, the inhibitor may be, for example, a peptide, nucleic acid, or compound that binds to a gene, may be selected through methods such as cell-based screening, and may be designed using generally known methods. For example, it may be an antisense oligonucleotide for a gene, siRNA, shRNA, miRNA, or a vector containing these, and these can be produced by generally known methods.

In the present invention, the term "antisense oligonucleotide" refers to DNA, RNA, or a derivative thereof having a nucleic acid sequence complementary to the sequence of a specific mRNA, and binds to the complementary sequence in the mRNA and inhibits the translation of the mRNA into a protein. It works. In one embodiment, the antisense oligonucleotide is 6 to 100 bases in length, preferably 8 to 60 bases, and even more preferably 10 to 40 bases. In one embodiment of the present invention, the antisense oligonucleotide may be modified at one or more base, sugar or backbone positions to improve efficacy (De Mesmaeker et al., Curr Opin Struct Biol., 5(3) ):343-55, 1995). The nucleic acid backbone can be modified with phosphorothioate, phosphotriester, methylphosphonate, short-chain alkyl, cycloalkyl, short-chain heteroatomic, heterocyclic intersaccharide linkages, etc. Additionally, antisense oligonucleotides may contain one or more substituted sugar moieties. Antisense oligonucleotides may contain modified bases. Modified bases include hypoxanthine, 6-methyladenine, 5-Me pyrimidine (especially 5-methylcytosine), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosyl HMC, 2- Aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, 2,6- Diaminopurine, etc. Additionally, the antisense oligonucleotide of the present invention may be chemically combined with one or more moieties or conjugates that improve its activity and cell adsorption. In one embodiment of the invention, the moiety includes cholesterol moiety, cholesteryl moiety, cholic acid, thioether, thiocholesterol, fatty chain, phospholipid, polyamine, polyethylene glycol chain, adamantane acetic acid, palmityl moiety, It may be a fat-soluble moiety such as octadecylamine or hexylamino-carbonyl-oxycholesterol moiety, but is not limited thereto. Oligonucleotides containing lipid-soluble moieties and methods for their preparation are well known in the art (see U.S. Patents Nos. 5,138,045, 5,218,105 and 5,459,255). In one embodiment of the present invention, the modified nucleic acid can increase stability against nucleases and increase the binding affinity between the antisense nucleic acid and the target mRNA. In one embodiment, antisense oligonucleotides may be synthesized in a test tube and administered in vivo by a generally well-known method, or may be synthesized in vivo. One example of synthesizing antisense oligonucleotides in a test tube is using RNA polymerase I. An example of synthesizing antisense RNA in vivo is to transcribe antisense RNA using a vector with the origin of the recognition site (MCS) in the opposite direction, and such antisense RNA is made into a peptide by inserting a translation stop codon in the sequence. It is desirable to avoid translation into sequences.

In the present invention, the term “siRNA” refers to a nucleic acid molecule capable of mediating RNA interference or gene silencing (see: WO00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409 and WO 00/44914). Since siRNA suppresses the expression of target genes, it can be provided as an efficient gene knock-down method or gene therapy method. In the present invention, the siRNA molecule may have a double-stranded structure in which the sense strand (sequence corresponding to the mRNA sequence) and the antisense strand (sequence complementary to the mRNA sequence) are located on opposite sides of each other. In the present invention, siRNA molecules may have a single-stranded structure with self-complementary sense and antisense strands. In one embodiment of the present invention, siRNA is not limited to complete pairing of double-stranded RNA portions of RNA, but also includes mismatches (corresponding bases are not complementary), bulges (one side of the chain), and Unpaired parts may be included due to reasons such as (there is no corresponding base). In one embodiment of the present invention, the total length of siRNA may be 10 to 100 bases, preferably 15 to 80 bases, and more preferably 20 to 70 bases. In addition, in the present invention, the siRNA terminal structure can be either a blunt end or a cohesive end as long as it can inhibit gene expression, and the cohesive end structure can be either a 3'-end protrusion structure or a 5'-end protrusion structure. possible. In the present invention, the siRNA molecule may have a short nucleotide sequence (e.g., about 5 to 15 nucleotides) inserted between the self-complementary sense and antisense strands, in which case the siRNA molecule formed by expression of the nucleotide sequence is a molecule. By internal hybridization, a hairpin structure is formed, and an overall stem-and-loop structure is formed.

In the present invention, the term "shRNA" refers to an oligoDNA that connects a 3 to 10 base linker between the sense and complementary nonsense of the target gene siRNA base sequence, and then added to the plasmid vector. When cloned or expressed by inserting shRNA into retroviruses such as lentivirus and adenovirus, shRNA (short hairpin RNA) with a looped hairpin structure is generated and converted to siRNA by intracellular Dicer. This means that it exhibits an RNAi effect. The shRNA exhibits a relatively long-term RNAi effect compared to siRNA.

In the present invention, the term "miRNA (microRNA)" refers to a single-stranded RNA molecule of 18 to 25 nucleotides (nt), and is a regulator that controls gene expression in eukaryotes. miRNAs are known to bind complementary to target mRNAs and act as posttranscriptional gene suppressors, inducing translational repression and mRNA destabilization.

In the present invention, the term "vector" refers to a genetic construct containing foreign DNA inserted into a genome encoding a polypeptide, such as a DNA vector, plasmid vector, cosmid vector, bacteriophage vector, yeast vector, or viral vector. Includes.

In one embodiment of the present invention, the pharmaceutical composition includes at least one azole antifungal agent selected from the group consisting of fluconazole, itraconazole, voriconazole, and ketoconazole; or one or more selected from the group consisting of amphotericin B, natamycin, rimocidin, nystatin, flucytosine and fludioxonil. It may be administered in combination with non-azole antifungal agents.

In the present invention, the antifungal pharmaceutical composition may include pharmaceutically suitable and physiologically acceptable adjuvants in addition to the active ingredient, and these adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, and glidants. Or it may be a solubilizer such as a flavoring agent.

The antifungal pharmaceutical composition of the present invention may include one or more pharmaceutically acceptable carriers in addition to the active ingredient. In one embodiment, the carrier used in the pharmaceutical composition formulated as a liquid solution is sterile and biocompatible, and includes saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, and ethanol. And one or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, or bacteriostatic agents can be added as needed.

In addition, in one embodiment of the present invention, the antifungal pharmaceutical composition further includes a diluent, dispersant, surfactant, binder or lubricant and is formulated into an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. It can be formulated. Furthermore, it can be preferably formulated according to each disease or ingredient by using a method disclosed in the literature (Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA) as an appropriate method in the field. In one embodiment of the present invention, the pharmaceutical preparation form of the pharmaceutical composition is granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release of the active compound. It can be a brother, sister, etc. The pharmaceutical composition of the present invention can be administered in any conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, transdermal, intranasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. It can be administered.

In the present invention, an effective amount of an active ingredient in a pharmaceutical composition refers to the amount required for the prevention or treatment of a disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form and the patient's age, weight, general health condition, gender and diet, administration time, administration route and composition. It can be adjusted depending on a variety of factors, including secretion rate, duration of treatment, and concurrent medications.

Using the kinase according to the present invention, novel antifungal drug candidates can be effectively screened. In addition, fungal infections can be effectively prevented, treated, and/or diagnosed using the antifungal pharmaceutical composition containing an agonist (inhibitor or suppressor) against the kinase of the present invention.

In Figure 1, a shows the phylogenetic correlation between protein kinases in Cryptococcus neoformans, and b shows a comparison of major kinases in Cryptococcus neoformans, C. albicans , and A. fumigatus. . Figure 1a shows protein sequence-based alignment performed using ClustalX2 (University College Dublin). Alignment data were used, and a phylogenetic tree was created using Interactive Tree Of Life (http://itol.embl.de) (Letunic, I. & Bork, P. Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acids Res 39, W475-478, doi:10.1093/nar/gkr201 (2011). From the 183 kinases of C. neoformans, the present inventors constructed 114 gene-deleted kinases and named them based on the gene naming of the S. cerevisiae orthologs. Colored lines refer to the kinase classes predicted by Kinomer1.0 (http://www.compbio.dundee.ac.uk/kinomer) (Martin, DM, Miranda-Saavedra, D. & Barton, GJ Kinomer v. 1.0: a database of systematically classified eukaryotic protein kinases. Nucleic Acids Res 37, D244-250, doi:10.1093/nar/gkn834 (2009). Red letters show the 60 pathogenicity-related kinases and the distribution of these kinases across all kinases and various classes. Figure 1b shows the distribution of kinase classes in the human infectious fungal pathogens C. neoformans, C. albicans, and A. fumigatus, and is a pie chart for the kinase classes predicted by Kinomer1.0. .
Figure 2 shows the phenotypic clustering of protein kinases in C. neoformans. Phenotypes were scored on a 7 scale (-3: strong susceptibility/reduction, -2: moderate susceptibility/reduction, -1: weak susceptibility/reduction, 0: similar to wild type, +1: weak resistance/intermediate valence, +2 : Medium resistance/increase, +3: Strong resistance/increase). An Excel file containing the phenotypic score of each kinase variant was loaded into Gene-E software (http://www.broadinstitute.org/cancer/software/GENE-E/), and kinase phenome clustering was performed using One Minus Pearson. It can be derived using correlation (One minus Pearson correlation). Abbreviations used in Figure 2 have the following meanings: [T25: 25 °C, T30: 30 °C, T37: 37 °C, T39: 39 °C, CAP: capsule production, MEL: melanin production, URE: urease production, MAT: mating filamentation, HPX: hydrogen peroxide, TBH: tert-butyl hydroperoxide, MD: menadione, DIA: diamide ( diamide), MMS: methyl methanesulfonate, HU: hydroxyurea, 5FC: 5-flucytosine, AMB: amphotericin B, FCZ: fluconazole (fluconazole), FDX: fludioxonil, TM: tunicamycin, DTT: dithiothreitol, CDS: cadmium sulfate, SDS: sodium dodecyl sulfate dodecyl sulfate), CR: congo red, CFW: calcofluor white, KCR: YPD + KCl, NCR: YPD + NaCl, SBR: YPD + sorbitol, KCS: YP + KCl , NCS: YP + NaCl, SBS: YP + sorbitol]
Figure 3 shows the results of observing the phenotypes of the gal83△ mutant and the snf1△ mutant. Figure 3(a) is the result of comparing phenotypic traits between the wild-type strain, snf1△ , and gal83△ mutants under various stress conditions. Under 1 ㎍/㎖ fludioxonil (FDX) conditions, snf1△ , gal83△ mutants were superior to the wild-type strain. The results showed increased susceptibility, and at 0.65 mM 4-butyl hydroperoxide (tBOOH), the snf1△ and gal83△ mutants showed increased resistance compared to the wild type strain. Figure 3b shows the results of comparing the carbon source usage abilities of the wild type strain, snf1△ , and gal83△ mutants. 2% glucose, 2% galactose, 3% glycerol, 3% ethanol, 2% maltose, 2% sucrose, 2% sodium acetate , experiments were performed under 1% potassium acetate conditions and the results showed that snf1△ and gal83△ mutants were required to use ethanol, sodium acetate, and potassium acetate as carbon sources.
Figure 4 shows that Fpk1 is C. This is the result of an experiment to check whether Neoformance regulates Ypk1-dependent phenotypic characteristics. (a) This is an experiment to create an overexpression strain by replacing the FPK1 promoter with the histone H3 promoter. (b) The FPK1 overexpressing strain was confirmed through Southern blot analysis, and YSB3986 and YSB3981 strains were generated by overexpressing FPK1 using the ypk1△ mutant as the parent. (c) Overexpression of FPK1 was confirmed through Northern blot quantification. The rRNA amount was used as a control. (d) Wild-type H99S strain, ypk1△ (YSB1736) mutant, and FPK1 overexpression strain (YSB3986 and YSB3981) were cultured in YPD liquid medium for 16 hours and then placed on solid medium to observe the degree of growth at the indicated temperature. (e and f) 1.5 M NaCl and 0.04% sodium dodecyl sulphate, 1 μg/ml fludioxonil, 1 μg/ml amphotericin B, 3 mM hydrogen peroxide, 3 mg/ml YPD containing ml calcofluor white, 100 mM hydroxyurea, 2 mM diamide, 300 μg/ml flucytosine and 5 mg/ml fluconazole. Tested on medium. Cells were cultured for an additional 3 days and then photographed. (g) This is the regulation model of Ypk1 and Fpk1 kinases in C. neoformans, which can be expected from the experimental results.
Figure 5 shows the results of a pathogenic kinase experiment through insect death analysis. Each mutant was cultured in YPD liquid medium for 16 hours, washed three times with PBS buffer, and then injected into G. mellonella larvae. At this time, 15 larvae are used for each mutant, and 4,000 mutant cells are injected per larva. Afterwards, the larvae were cultured at 37°C and their survival was checked daily. Statistical analysis of the experimental results was performed using the log-rank Mantel-Cox test. Figure 5a shows the results of an experiment performed using two independent mutants for each kinase. Figure 5b shows the results of two repeated experiments on a kinase in which only one mutant was created.
Figure 6 shows the results of the marker mutation (STM)-based mouse model infectivity test. The ste50△ and hxl1△ strains were used to control pathogenicity and non-pathogenicity for STM studies. STM quantitative values were quantified by analyzing biological triplicate experiments through qPCR using STM number-specific primers labeled for the strain as shown in Table 2. (ad) All kinase variants were divided into four groups and tested. Each set of genes consisted of variants from two independent strains, and when one variant was present, two experiments were performed.
Figure 7 is a diagram summarizing the kinases involved in the pathogenicity of C. neoformans. The STM score was calculated using quantitative PCR method and distinguished using numbers and colors (Figure 7a). Red letters indicate infectiousness-related kinases, which were newly identified in the present invention. Twenty-five kinases were jointly identified in the analysis of both insect and mouse models, and the gene names of these 25 kinases were written on the STM zero line. P values between control and mutant strains were calculated by one-way ANOVA using three mice per STM set and Bonferroni correlation. Each set was repeated twice using independent strains. For single strain variants, two independent experiments were repeated with each single strain. In the STM study, the role of a total of 54 kinases in the infectivity of C. neoformans was analyzed. Referring to Figures 4 and 5, six kinases were not confirmed to have a pathogenicity-regulating function in the mouse model infection test, but were confirmed as pathogenicity-related kinases in a wax moth killing assay (Figure 7b). For single mutant strains of bub1 and kin4, two replicates were performed.
Figure 8 shows the phylogenetic relationships between pathogenicity-related kinases in C. neoformans and other eukaryotic protein kinases. Figure 8a shows a BLAST matrix using the CFGP (Comparative Fungal Genomics Platform, http://cfgp.riceblast.snu.ac.kr) database for 60 pathogenicity-related kinases, and the protein sequences of 60 pathogenicity-related kinases are As a result of the search, orthologue proteins were matched in 35 genome databases. Figure 8b shows the correlation of pathogenicity-related kinases in fungal pathogens. To identify orthologous proteins, each protein sequence was subjected to BLAST and reverse analysis using genome databases (CGD; Candida Genome Database for C. albicans, Broad Institute Database for F. graminearum and C. neoformans). Analyzed by BLAST. Twenty-one kinases were associated with pathogenicity in both F. graminearum and C. neoformans. Thirteen kinases have been implicated in the pathogenicity of C. neoformans and C. albicans. Among these, five kinases, including Sch9, Snf1, Pka1, Hog1 and Swe1, were involved in the virulence of all three fungal pathogens and are shown in red.
Figure 9 shows the pleiotropic role of Ipk1 in Cryptococcus neoformans. Various experiments were performed using WT (wild type) and ipk1Δ mutants (YSB2157 and YSB2158). In Figure 9A, the ipk1△ mutant showed reduced virulence in an insect-based in vivo virulence assay. In this assay, the control group was injected with only WT and PBS. In Figure 9b, the ipk1△ mutant induced capsule. Cells cultured overnight were placed on DME plates at 37°C for 2 days. 50 μl of ^1.5 After 3 days of cells falling by gravity, packed cell volume was calculated from the total volume and normalized to WT. The P value for each strain was less than 0.05. (*) Error lines represent SEM. In Figure 9c , the ipk1△ mutant shows a melanin-deficient phenotype. Melanin production was confirmed after 3 days on Niser seed plates containing 0.2% glucose. In Figure 9D, the ipk1△ deletion mutant was defective in urease production. Urease production was confirmed after 2 days on Christensen's agar medium at 30°C. In Figure 9e, the ipk1△ mutant showed severe defects in hyphal growth. Mating analysis was confirmed after 9 days on a V8 medium (pH 5, 1L standard: 50 ml of V8 juice (Campbell), 0.5 g of KH2PO4 (Bioshop, PPM302), 40 g of Agar (Bioshop, AGR001.500)). Figure 9f, g are micrographs obtained from spot analysis diluted 10 times (10 ² to 10 ⁵ times diluted). Growth rates were measured under various growth conditions shown in the photos. For chemical susceptibility analysis, the following chemicals were treated on YPD medium: HU; hydroxyurea 100 mM, TM as a DNA damaging reagent; As an ER stress (endoplasmic reticulum stress) inducing reagent, tunicamycin 0.3 μg/ml, CFW; Calcofluor white 3 mg/ml, SDS as a cell wall damage reagent; Sodium dodecyl sulfate 0.03%, CDS for membrane stability testing; CdSO4 30 M, HPX as heavy metal stress reagent; As an oxidizing reagent, hydrogen peroxide (Hydrogen peroxide) 3 mM, 1M NaCl for osmotic shock, and AmpB (amphoteracin B) 0.9 mg/ml, FCZ (fluconazole) 14 μg/ml, 5-FC ( Flucytosine) 300 μg/ml, and FDX (fludioxonil) 1 μg/ml.
Figure 10 shows protein kinases involved in the growth and pathogenicity of Cryptococcus neoformans. Functional network analysis of 60 virulence-regulating kinases of C. neoformans by previously reported CryptoNet (http://www.inetbio.org/cryptonet) (Kim, H. et al. Network-assisted genetic dissection of pathogenicity and drug resistance in the opportunistic human pathogenic fungus Cryptococcus neoformans . Scientific reports 5, 8767, doi:10.1038/srep08767 (2015). The network was created based on 100 candidate genes predicted by CryptoNet to be functionally related to 60 pathogenicity-regulated kinases. In this pathogenic network, genes were classified according to their predicted biological functions described in Gene Ontology (GO) terms. Six kinases (Arg5/6, Ipk1, Irk2, Irk4, Irk6, and vrk1) in CryptoNet had no functionally relevant genes.
Figure 11 shows the results of experiments using cdc7△, cbk1△, and kic1△ mutants. (ac) cdc7△ mutants (YSB2911, YSB2912), met1△ mutants (YSB3063, YSB3611), and cka1 (YSB3051, YSB3052) were grown overnight in YPD medium and serially diluted 10-fold to solid YPD medium and 100 mM hydroxyurea (HU), 0.06 YPD medium containing % methyl methanesulphonate (MMS), 1 ㎍/㎖ amphotericin B (AmpB), 1 ㎍/㎖ fludioxonil (FDX), 3 mM hydrogen peroxide (HPX), and 300 ㎍/㎖ flucytosine (5-FC). Spotted. Spotted cells were cultured for an additional 3 days at 30°C or the indicated temperature and then photographed. (d) Wild type and the following mutants [ kic1△ (YSB2915, YSB2916), cbk1△ (YSB2941, YSB2942), cka1△ (YSB3051, YSB3052)] were cultured in YPD medium for more than 16 hours and then incubated with 10% paraformaldehyde for 15 minutes. It was fixed and washed twice with PBS solution. The fixed cells were stained with 10 μg/ml Hoechst solution (Hoechst 33342, Invitrogen) for 30 minutes and then observed under a fluorescence microscope (Nikon eclipse Ti microscope).
Figure 12 shows the results of an experiment on the bud32Δ mutant. (a) Wild type and bud32Δ mutants (YSB1968, YSB1969) were cultured overnight in YPD medium, serially diluted 10-fold, and then spotted on YPD medium containing the following chemicals to observe growth rates under various growth conditions. [1.5 M NaCl, 1.5 M KCl, 2 M Sorbitol, 1 ㎍/㎖ amphotericin B (AmpB), 14 ㎍/㎖ fluconazole (FCZ), 1 ㎍/㎖ fludioxonil (FDX), 300 ㎍/㎖ flucytosine, 100 mM hydroxyurea (HU), 0.04% methyl methanesulphonate (MMS), 3mM hydrogen peroxide (HPX), 0.7mM tert-butyl hydroperoxide (tBOOH), 2mM diamide (DIA), 0.02mM menadione (MD), 0.03% sodium dodecyl sulphate ( SDS)] Cells spotted on YPD medium containing these chemicals were further cultured at 30°C and then photographed. (b) The melanin production ability of the wild type and bud32Δ mutant was confirmed on a Niser seed plate containing 0.1% glucose, and the urease production was confirmed after culturing on Christensen's agar medium at 30°C. To confirm capsule production ability, cells cultured overnight were placed on a DME plate at 37°C for 2 days. ⁵⁰ μl of 1.5 The results were calculated using one-way ANOVA using the Bonferroni correlation method and were repeated three times. (c) To compare the mating efficacy of the wild type and the bud32Δ mutant, they were spotted on V8 medium and cultured in the dark for 9 days. (d) After culturing the wild type and bud32Δ mutant at 30°C to logarithmic growth phase, the culture was divided into two samples, one was treated with fluconazole (FCZ) for 90 minutes and the other was not. Total RNA was extracted from each sample, and the expression level of ERG11 was confirmed through Northern analysis.
Figure 13 shows experimental results for arg5,6Δ mutant and met3Δ . (a, b) Wild type (H99S), arg5,6Δ mutants (YSB2408, YSB2409, YSB2410), and met3Δ mutants (YSB3329, YSB3330) were cultured overnight in YPD medium and then washed with PBS. Washed cells were serially diluted 10-fold and spotted on solid synthetic complete medium. [SC; yeast nitrogen base without amino acids (Difco) supplemented with the indicated concentration of the following amino acids and nucleotides: 30 mg/l L-isoleucine, 0.15 g/l L-valine, 20 mg/l adenine sulphate, 20 mg/l L -histidine-HCl, 0.1 g/l L-leucine, 30 mg/l L-lysine, 50 mg/l L-phenylalanine, 20 mg/l L-tryptophan, 30 mg/l uracil, 0.4 g/l L-serine , 0.1 g/l glutamic acid, 0.2 g/l L-threonine, 0.1 g/l L-aspartate, 20 mg/l L-arginine, 20 mg/l L-cysteine, and 20 mg/l L-methionine]. SC-arg (a), SC-met and SC-met-cys (b) media indicate the SC medium lacking arginine, methionine and/or cysteine supplements. (b) Schematic diagram of the biosynthetic pathway of methionine and cysteine. (c) The wild type, arg5,6Δ mutant, and met3Δ mutant were cultured overnight in YPD medium, serially diluted 10-fold, and then spotted on YPD medium containing the following chemicals to observe growth rates under various growth conditions. [1 ㎍/㎖ amphotericin B (AmpB), 14 ㎍/㎖ fluconazole (FCZ), 1 ㎍/㎖ fludioxonil (FDX), 3 mM hydrogen peroxide (HPX)]. Spotted cells were photographed after culturing for 3 days at 30°C or the indicated temperature.
Figure 14 shows retrograde vacuole trafficking that regulates the pathogenicity of Cryptococcus neoformans. Retrograde vacuolar transport regulates the pathogenicity of C. neoformans. Various experiments were performed using WT and vps15Δ mutants [YSB1500, YSB1501]. In Figure 14A, Vps15 is required for virulence of C. neoformans. WT and PBS buffer were used as positive and negative controls, respectively. In Figure 14b, the vps15Δ mutant shows increased vacuole production. The scale represents 10 μl. In Figure 14c, the vps15Δ mutant showed severe growth defects under ER stress. Cells cultured overnight were spotted on YPD medium containing 15 mM dithiothreitol (DTT) or 0.3 μg/ml tunicamycin (TM) and cultured for an additional 3 days at 30°C. After doing so, a photo was obtained. In Figure 14D, the vps15Δ mutant showed severe growth defects in response to high temperature, cell membrane stress, and cell wall stress. Cells from overnight culture were spotted on YPD medium and further incubated at the indicated temperature, or spotted on YPD medium containing 0.03% SDS or 5 mg/ml Calcofluor White (CFW) and incubated at 30°C. Additional culture was performed. The plate was photographed 3 days later. In Figure 14e, Vps15 was not involved in the calcineurin pathway in C. neoformans. For quantitative RT-RCR (qRT-PCR), RNAs were obtained from three replicates of WT and vps15Δ mutants. CNA1, CNB1, CRZ1, and UTR2 expression levels were normalized to the control ACT1 expression level. Data were collected from three replicates. The error line represents the standard error of means (sem). In Figure 14f, Vps15 negatively regulates Hxl1 splicing. For RT-PCR, RNAs were obtained from WT and vps15Δ mutants and cDNAs were synthesized. For RT-PCR, HXL1 and ACT1-specific primer pairs were used (Table 3). The experiment was repeated twice, and one experiment is shown as a representative example.
Figure 15 shows the results of an experiment on the vrk1Δ mutant. Figure 15A shows WT and vrk1Δ strains on YPD medium and YPD containing 2.5 mM hydrogen peroxide (HPX), 600 μg/ml flucytosine (5-FC), or 1 μg/ml fludioxonil (FDX). Indicates spotting on the medium. The strain was cultured for 3 days at 30 °C and photographed. Figure 15b shows the relative quantification of the packed cell volume. Three independent measurements show significant differences between WT and vrk1Δ strains (***; 0.0004 and **; 0.0038, sem). Figure 15c shows relative quantification of Vrk1-mediated phosphorylation. Peptide samples were analyzed on average three times in independent experiments, and peptides were obtained in two independent experiments. Results are the mean ± sem of two independent experiments. Student's unpaired t-test was applied to examine statistical significance. ***P<0.001, **P<0.01, *P<0.05. PSMs represent peptide spectral matching.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Animal care and all experiments were performed in accordance with the ethical guidelines of Yonsei University's Institutional Animal Care and Use Committee (IACUC). Yonsei University IACUC approved all vertebrate animal studies.

Example

Example 1: Cryptococcus neoformans ( Cryptococcus neoformans ) Identification of protein kinases within

To select putative kinase genes in the genome of C. neoformans var. grubii (strain H99), two approaches were used. First, Kinomer v. 1.0 database (Kinome v. 1.0 database; www.compbio.dundee.ac.uk/kinomer/) was used, which is a highly sensitive and accurate hidden Markov model (HMM)-based method. It is intended to systematically predict and classify eukaryotic protein kinases based on (Martin, DM, Miranda-Saavedra, D. & Barton, GJ Kinomer v. 1.0: a database of systematically classified eukaryotic protein kinases. Nucleic Acids Res 37, D244- 250, doi:10.1093/nar/gkn834, 2009). With the kinomer database, 97 putative kinases were predicted in the genome of serotype D C. neoformans (strain JEC21). Next, each JEC21 kinase gene ID was mapped to the H99 strain based on the most recent genome annotation (version 7), and 95 putative kinases were searched. However, these kinomer lists were found to be incomplete as they did not represent all histidine kinases and some known kinases such as Hog1. Therefore, we further investigated the annotations produced in the H99 genome database provided by the Broad Institute (www.broadinstitute.org/annotation/genome/cryptococcus_neoformans) and the JEC21 genome database by the National Center for Biotechnology Information (NCBI). The approach was used. For each gene with a kinase-related annotation, Pfam (http://pfam.xfam.org/ ) was used to perform protein domain analysis. Through this analysis, an additional 88 putative kinase genes were retrieved. As a result, a total of 183 putative kinase genes were identified in C. neoformans. Their phylogenetic relationship is shown in Figure 1.

The eukaryotic protein kinase superfamily is broadly divided into two groups: six conventional protein kinase groups (ePKs) and three atypical groups (aPKs) (Miranda-Saavedra, D. & Barton, G.J. Classification and functional annotation of eukaryotic protein kinases. Proteins 68, 893-914, doi:10.1002/prot.21444, 2007). ePKs include the AGC group (including cyclic nucleotide and calcium-phospholipid-dependent kinases, ribosomal S6-phosphorylated kinases, G protein-coupled kinases, and all similar homologues of this set), CAMKs (calmodulin-regulated kinases) ; CK1 group (casein kinase 1, and similar homologs), CMGC group (including cyclin-dependent kinases, mitogen-activated protein kinases, glycogen synthase kinases, and CDK-like kinases), RGC group (receptor guanylate cyclase) ), STEs (which contain many kinase functions in the MAP kinase cascade), TKs (tyrosine kinases), and TKLs (tyrosine kinase-like kinases) (Figure 1). aPKs include the alpha-kinase group, PIKK (phosphatidylinositol 3-kinase-related kinase), RIO, and PHDK (pyruvate dehydrogenase kinase). To classify the 183 C. neoformans kinases according to these criteria, their amino acid sequences were searched in the kinomer database. Several previously classified kinases (Martin, DM, Miranda-Saavedra, D. & Barton, GJ Kinomer v. 1.0: a database of systematically classified eukaryotic protein kinases. Nucleic Acids Res 37, D244-250, doi:10.1093/ nar/gkn834, 2009) were classified differently (14 out of 95), probably due to sequence differences between JEC21 and H99. Most of the other kinases identified by annotation search did not correspond to existing categories (82 out of 88) and were classified as “others.” Therefore, the C. neoformans kinome consists of 89 ePKs (18 AGC, 22 CAMK, 2 CK1, 24 CMGC, 2 PDHK, 18 STE, 3 TKL), 10 aPKs (2 PDHK, 6 PIKK, 2 RIO), and It was identified as consisting of 84 “other” groups (Figure 1). The other group includes seven histidine kinases (Figure 1). Superfamily (version 1.73) (Wilson, D. et al. SUPERFAMILY--sophisticated comparative genomics, data mining, visualization and phylogeny. Nucleic Acids Res 37, D380-386, doi:10.1093/nar/gkn762 (2009)) (Supplementary Based on predictions by the HMMER sequence profile in Data 2 and Fig. 1b), two human fungal pathogens, C. albicans and A. fumagatus, were identified to have 188 and 269 protein kinases, respectively. Among pathogenic fungal kinases, CMGC (12-13%), CAMK (12-18%), STE (6-10%), and AGC (6-10%) appear to be the most common clades (Figure 1).

Considering that most eukaryotic genomes are predicted to contain kinases at a rate of approximately 1–2% of the genome, the proportion of protein kinases in C. neoformans (~2.6%) was higher than expected. This indicates that C. neoformans has both saprobic and parasitic life cycles, in which the pathogenic yeast comes into contact with a wider variety of environmental and host signals. Nevertheless, whether all of these predicted kinases have biologically meaningful kinase activity remains to be elucidated. Phylogenetic comparison of 183 putative C. neoformans kinases with other fungi and higher eukaryotes suggests that kinases are conserved to a much higher degree among fungi and other eukaryotes than transcription factors (TFs). In conclusion, the kinome network appears to be evolutionarily conserved among fungi, at least in terms of sequence similarity, which contrasts sharply with the evolutionarily dispersed nature of the TF network.

Example 2: C. Construction of a library of kinase gene-deletion variants in Neoformans

To understand the biological function of the Cryptococcal kinome network and its complexity, gene-deletion mutants for each kinase were constructed and their functional properties were confirmed. Among the analyzed kinases, there were variants for 22 kinases ( TCO1, TCO2, TCO3, TCO4, TCO5, TCO7, SSK2, PBS2, HOG1, BCK1, MKK1/2, MPK1, STE11, STE7, CPK1, PKA1, PKA2, HRK1, PKP1, IRE1, SCH9, and YPK1 ) have already been partially characterized by the present inventors. (Bahn, YS, Geunes-Boyer, S. & Heitman, J. Ssk2 mitogen-activated protein kinase kinase kinase governs divergent patterns of the stress-activated Hog1 signaling pathway in Cryptococcus neoformans. Eukaryot. Cell 6, 2278-2289 (2007) ; Bahn, YS, Hicks, JK, Giles, SS, Cox, GM & Heitman, J. Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein kinase A cascade. -1491 (2004); Bahn, YS, Kojima, K., Cox, GM & Heitman, J. Specialization of the HOG pathway and its impact on differentiation and virulence of Cryptococcus neoformans Cell 16, 2285-2300. 2005); Cox, GM & Heitman, J. A unique fungal two-component system regulates stress responses, drug sensitivity, and virulence of Cryptococcus neoformans. , 3122-3135 (2006); Network-assisted genetic dissection of pathogenicity and drug resistance in the opportunistic human pathogenic fungus Cryptococcus neoformans. Scientific reports 5, 8767, doi:10.1038/srep08767 (2015); Kim, MS, Kim, SY, Yoon, JK, Lee, YW & Bahn, YS An efficient gene-disruption method in Cryptococcus neoformans by double-joint PCR with NAT-split markers. Biochem. Biophys. Res. Commun. 390, 983-988, doi:S0006-291X(09)02080-4 [pii]10.1016/j.bbrc.2009.10.089 (2009); Kim, S. Y. et al. Hrk1 plays both Hog1-dependent and -independent roles in controlling stress response and antifungal drug resistance in Cryptococcus neoformans. PLoS One 6, e18769, doi:doi:10.1371/journal.pone.0018769 (2011); Kojima, K., Bahn, YS & Heitman, J. Calcineurin, Mpk1 and Hog1 MAPK pathways independently control fludioxonil antifungal sensitivity in Cryptococcus neoformans. Microbiology 152, 591-604 (2006); Maeng, S. et al. Comparative transcriptome analysis reveals novel roles of the Ras and cyclic AMP signaling pathways in environmental stress response and antifungal drug sensitivity in Cryptococcus neoformans. Eukaryot. Cell 9, 360-378, doi:EC.00309-09 [pii];10.1128/EC.00309-09 (2010); Cheon, S. A. et al. Unique evolution of the UPR pathway with a novel bZIP transcription factor, Hxl1, for controlling pathogenicity of Cryptococcus neoformans. PLoS Pathog. 7, e1002177, doi:10.1371/journal.ppat.1002177 (2011)).

For the other 161 kinases, large-scale homologous recombination and in vitro and in vivo phenotypic analysis were used to construct gene-deletion variants and deposit them (Accession Number: KCCM 51297). To perform large-scale infectivity studies in mouse hosts, a dominant nourseothricin resistance marker (NAT) is used, which contains a signature tag of a series of distinct oligonucleotide sequences (Table 1). did. To verify the exact gene deletion and absence of ectopic integration of each gene-disruption cassette, Southern blot analysis was performed.

26 kinase gene deletion strains CNAG_Num. gene name Experiment Serial Number (YSB #) genotype CNAG_00047 PKP1 558, 608 MAT α pkp1 Δ ::NAT-STM#224 CNAG_00106 TCO5 286, 287 MAT α tco5 Δ ::NAT-STM#125 CNAG_00130 HRK1 270, 271 MAT α hrk1 Δ ::NAT-STM#58 CNAG_00363 TCO6 2469, 2554 MAT α tco6 Δ ::NAT-STM#58 CNAG_00396 PKA1 188, 189 MAT α pka1 Δ ::NAT-STM#191 CNAG_00405 KIC1 2915, 2916 MAT α kic1 Δ ::NAT-STM#201 CNAG_00415 CDC2801 2370, 3699 MAT α cdc2801 Δ ::NAT-STM#191 CNAG_00636 CDC7 2911, 2912 MAT α cdc7 Δ ::NAT-STM#213 CNAG_00745 HRK1/NPH1 1438, 1439 MAT α hrk1/mph1 Δ ::NAT-STM#210 CNAG_00769 PBS2 123, 124 MAT α pbs2 Δ ::NAT-STM#213 CNAG_00782 SPS1 3229, 3325 MATα sps1Δ ::NAT-STM#288 CNAG_00826 DAK2 1912, 1913 MAT α dak2 Δ ::NAT-STM#282 CNAG_01062 PSK201 1989, 1990 MAT α psk201 Δ ::NAT-STM#191 CNAG_01155 GUT1 1241, 2761 MAT α gut1 Δ ::NAT-STM#242 CNAG_01162 MAK322 3824, 3825 MAT α mak322 Δ ::NAT-STM#159 CNAG_01165 LCB5 3789, 3790 MAT α lcb5 Δ ::NAT-STM#213 CNAG_01209 FAB1 3172 MAT α fab1 Δ ::NAT-STM#169 CNAG_01294 IPK1 2157, 2158 MAT α ipk1 Δ ::NAT-STM#184 CNAG_01333 ALK1 1571, 1573 MAT α alk1 Δ ::NAT-STM#122 CNAG_01523 HOG1 64, 65 MAT α hog1 Δ ::NAT-STM#177 CNAG_01612 PSK202 3922, 3924 MAT α psk202 Δ ::NAT-STM#208 CNAG_01704 IRK6 3830, 3831 MAT α irk6 Δ ::NAT-STM#5 CNAG_01730 STE7 342, 343 MAT α ste7 Δ ::NAT-STM#225 CNAG_01850 TCO1 278, 279 MAT α yco1 Δ ::NAT-STM#102 CNAG_01905 KSP1 1807, 1808, 1809 MAT α ksp1 Δ ::NAT-STM#159 CNAG_01938 KIN1 3930, 3931 MAT α kin1 Δ ::NAT-STM#6 CNAG_01988 TCO3 284, 285 MAT α tco3 Δ ::NAT-STM#119 CNAG_02233 MEC1 3063, 3611 MAT α mec1 Δ ::NAT-STM#204 CNAG_02296 RBK1 1510, 1511 MAT α rbk1 Δ ::NAT-STM#219 CNAG_02357 MKK2 330, 331 MAT α mkk2 Δ ::NAT-STM#224 CNAG_02389 YPK101 1885, 1886 MAT α ypk101 Δ ::NAT-STM#242 CNAG_02511 CPK1 127, 128 MAT α cpk1 Δ ::NAT-STM#184 CNAG_02531 CPK2 373, 374 MAT α cpk2 Δ ::NAT-STM#122 CNAG_02542 IRK2 1904, 1905 MAT α irk2 Δ ::NAT-STM#232 CNAG_02551 DAK3 1940, 1941 MAT α dak3Δ ::NAT-STM#295 CNAG_02675 HSL101 1800, 1801 MAT α hsl101 Δ ::NAT-STM#146 CNAG_02680 VPS15 1500, 1501 MAT α vps15 Δ ::NAT-STM#123 CNAG_02712 BUD32 1968, 1969 MAT α bud32Δ ::NAT-STM#295 CNAG_02799 DAK202A 2487, 2489 MAT α dak202a Δ ::NAT-STM#119 CNAG_02802 ARG2 1503, 1504 MAT α arg2 Δ ::NAT-STM#125 CNAG_02820 PKH201 1234, 1235, 1236 MAT α pkh201 Δ ::NAT-STM#219 CNAG_02859 POS5 3714, 3715 MAT α pos5 Δ ::NAT-STM#58 CNAG_02947 SCY1 2793, 2794 MAT α scy1 Δ ::NAT-STM#150 CNAG_03024 RIM15 1216, 1217 MAT α rim15 Δ ::NAT-STM#191 CNAG_03048 IRK3 1486, 1487 MAT α irk3 Δ ::NAT-STM#273 CNAG_03167 CHK1 1825, 1828 MAT α chk1 Δ ::NAT-STM#205 CNAG_03184 BUB1 3398 MAT α bub1 Δ ::NAT-STM#201 CNAG_03216 SNF101 1575, 1576 MAT α snf101 Δ ::NAT-STM#146 CNAG_03258 TPK202A 2443, 2444 MAT α psk202a Δ ::NAT-STM#208 CNAG_03290 KIC102 3211, 3212 MAT α kic102 Δ ::NAT-STM#201 CNAG_03355 TCO4 417, 418 MAT α tco4 Δ ::NAT-STM#123 CNAG_03367 URK1 1266, 1267 MAT α urk1 Δ ::NAT-STM#43 CNAG_03369 SWE102 1564, 1565 MAT α swe102 Δ ::NAT-STM#169 CNAG_03567 CBK1 2941, 2942 MAT α cbk1 Δ ::NAT-STM#232 CNAG_03592 THI20 3219, 3220 MAT α THI20 Δ ::NAT-STM#231 CNAG_03670 IRE1 552, 554 MAT α ire1 Δ ::NAT-STM#224 CNAG_03811 IRK5 2952, 2953 MAT α irk5 Δ ::NAT-STM#213 CNAG_03843 ARK1 1725, 1726 MAT α ark1 Δ ::NAT-STM#43 CNAG_03946 GAL302 2852, 2853 MAT α gal302 Δ ::NAT-STM#218 CNAG_04040 FPK1 2948, 2949 MAT α fpk1 Δ ::NAT-STM#211 CNAG_04108 PKP2 2439, 2440 MATα pkp2Δ ::NAT-STM#295 CNAG_04162 PKA2 194, 195 MAT α pka2 Δ ::NAT-STM#205 CNAG_04197 YAK1 2040, 2096, 4139 MAT α yak1 Δ ::NAT-STM#184 CNAG_04215 MET3 3329, 3330 MAT α met3 Δ ::NAT-STM#205 CNAG_04221 FBP26 3669 MAT α fbp26 Δ ::NAT-STM#146 CNAG_04230 THI6 1468, 1469 MAT α thi6Δ ::NAT-STM#290 CNAG_04282 MPK2 3236, 3238 MAT α mpk2 Δ ::NAT-STM#102 CNAG_04316 UTR1 2892, 2893 MAT α utr1 Δ ::NAT-STM#5 CNAG_04408 CKI1 1804, 1805 MAT α cki1 Δ ::NAT-STM#218 CNAG_04433 YAK103 3736, 3737 MAT α YAK103 Δ ::NAT-STM#231 CNAG_04514 MPK1 3814, 3816 MAT α mpk1 Δ ::NAT-STM#240 CNAG_04631 RIK1 1579, 1580 MAT α CNAG_04631 Δ ::NAT-STM#150 CNAG_04678 YPK1 1736, 1737 MAT α ypk1 Δ ::NAT-STM#58 CNAG_04755 BCK1 273, 274 MAT α bck1 Δ ::NAT-STM#43 CNAG_04821 PAN3 2809, 2810 MAT α pan3 Δ ::NAT-STM#204 CNAG_04927 YFH702 2826, 3716 MAT α yfh702 Δ ::NAT-STM#220 CNAG_05005 ATG1 1935, 1936 MAT α atg1Δ ::NAT-STM#288 CNAG_05063 SSK2 264, 265 MAT α ssk2 Δ ::NAT-STM#210 CNAG_05097 CKY1 1245, 1246 MAT α CNAG_05097 Δ ::NAT-STM#282 CNAG_05216 RAD53 3785, 3786 MAT α rad53 Δ ::NAT-STM#184 CNAG_05220 TLK1 3153, 3188 MAT α tlk1 Δ ::NAT-STM#116 26 kinase gene deletion strains CNAG_Num. gene name Experiment Serial Number (YSB #) genotype CNAG_05243 XKS1 2851 MAT α xks1 Δ ::NAT-STM#125 CNAG_05439 CMK1 1883, 1901, 1902 MAT α cmk1 Δ ::NAT-STM#227 CNAG_05558 KIN4 2955 MAT α kin4 Δ ::NAT-STM#225 CNAG_05590 TCO2 281, 282 MAT α tco2 Δ ::NAT-STM#116 CNAG_05600 IGI1 1514, 1515 MAT α CNAG_05600 Δ ::NAT-STM#230 CNAG_05694 CKA1 3051, 3052, 3053 MAT α cka1 Δ ::NAT-STM#6 CNAG_05753 ARG5,6 2408, 2409, 2410 MAT α arg5/6 Δ ::NAT-STM#220 CNAG_05771 TEL1 3844, 3845 MAT α tel1 Δ ::NAT-STM#225 CNAG_05965 IRK4 2806, 2808 MAT α irk4 Δ ::NAT-STM#211 CNAG_06033 MAK32　 3240, 3241 MAT α mak32 Δ ::NAT-STM#169 CNAG_06051 GAL1 2829, 2830 MAT α gal1 Δ ::NAT-STM#224 CNAG_06086 SSN3 3038, 3039 MAT α ssn3 Δ ::NAT-STM#219 CNAG_06161 VRK1 2216, 2217 MAT α vrk1 Δ ::NAT-STM#123 CNAG_06193 CRK1 1709, 1710 MAT α crk1 Δ ::NAT-STM#43 CNAG_06278 TCO7 348 MAT α tco7 Δ ::NAT-STM#209 CNAG_06301 SCH9 619, 620 MAT α sch9 Δ ::NAT-STM#169 CNAG_06310 IRK7 2136, 2137 MAT α irk7 Δ ::NAT-STM#208 CNAG_06366 HRR2502 2053 MAT α hrr2502 Δ ::NAT-STM#125 CNAG_06552 SNF1 2372, 2373 MAT α snf1 Δ ::NAT-STM#204 CNAG_06553 GAL83 2415, 2416 MAT α gal83Δ ::NAT-STM#288 CNAG_06568 SKS1 1410, 1411 MAT α sks1 Δ ::NAT-STM#211 CNAG_06632 ABC1 2072, 2797 MAT α CNAG_06632 Δ ::NAT-STM#119 CNAG_06671 YKL1 3926, 3927 MAT α CNAG_06671 Δ ::NAT-STM#122 CNAG_06697 MPS1 3632, 3633 MAT α mps1 Δ ::NAT-STM#116 CNAG_06730 GSK3 2038, 2039 MAT α gsk3 Δ ::NAT-STM#123 CNAG_06809 IKS1 1310, 2119 MAT α iks1 Δ ::NAT-STM#116 CNAG_06980 STE11 313, 314 MAT α ste11 Δ ::NAT-STM#242 CNAG_07359 IRK1 1950, 1951 MAT α irk1 Δ ::NAT-STM#5 CNAG_07580 TRM7 3056, 3057 MAT α trm7 Δ ::NAT-STM#102 CNAG_07667 SAT4 3612 MAT α sat4 Δ ::NAT-STM#212 CNAG_07744 PIK1 1493, 1494 MAT α pik 1 Δ ::NAT-STM#227 CNAG_07779 TDA10 2663, 3223 MAT α tda10 Δ ::NAT-STM#102 CNAG_08022 PHO85 3702, 3703 MAT α pho85 Δ ::NAT-STM#218

*CNAG: Abbreviation for Cryptococcus neoformans serotype A genome database, which is the H99 genome database gene number provided by the Broad Institute. For gene deletion through homologous recombination, norseothricin-resistance (NAT; A gene-disruption cassette containing the marker (nourseothricin acetyl transferase) was produced by conventional overlap PCR or NAT split marker/double-joint (DJ) PCR method (Davidson, RC et al. A PCR- based strategy to generate integrative targeting alleles with large regions of homology. Microbiology 148, 2607-2615 (2002); Kim, MS, Kim, SY, Jung, KW & Bahn, YS Targeted gene disruption in Cryptococcus neoformans using double-joint PCR with split dominant selectable markers. Methods Mol Biol 845, 67-84, doi:10.1007/978-1-61779-539-8_5 (2012) (Table 1 above) to evaluate variant phenotypes and exclude irrelevant mutation effects. , more than two independent deletion strains were prepared for each kinase variant (see Table 1). If two independent kinase variants showed different phenotypes (inter-isolate inconsistency), three or more variants were prepared. As a result, 220 gene deletion variants corresponding to 114 kinases (along with those previously reported) were successfully prepared (Table 1 above), and at least two independent variants were prepared for 106 kinases. Several kinases were independently deleted with unique marker markers to perform in vitro and in vivo phenotypic analyses. In cases where two independent kinase variants displayed inconsistent phenotypes (known as inter-isolated mismatches), three or more variants were prepared. For the remaining 69 kinases, despite repeated attempts by the inventors, no variants were identified. couldn't even make it. Among these, in many cases, viable transformants could not be obtained at all, or it was observed that the wild-type allele was maintained in addition to the collapsed allele. The variant creation rate for kinases (114 out of 183 [62%]) was lower than that of previously reported TFs (155 out of 178 [87%]) (Jung, KW et al. Systematic functional profiling of transcription factors networks in Cryptococcus neoformans. Nat Comms 6, 6757, doi:10.1038/ncomms7757, 2015). This may be because, in general, kinases are much more evolutionarily conserved among fungi than TFs and contain a greater number of essential genes. In fact, 24 (35%) of the kinases are orthologous to kinases essential for S. cerevisiae growth. In particular, eight of them (RAD53, CDC28, CDC7, CBK1, UTR1, MPS1, PIK1, and TOR2), for which deletion mutants could be created in C. neoformans, are known to be essential in S. cerevisiae, which are We suggest that there is functional differentiation in several protein kinases between fungi and basidiomycete fungi. In the first round of PCR, the 5'- and 3'-flanking regions for the target kinase gene were amplified using H99S genomic DNA as a template and primer pairs L1/L2 and R1/R2, respectively. For overlap PCR, primers M13Fe (extended forward M13) and M13Re were used, using plasmid pNATSTM (obtained from Joeseph Heitman's laboratory at Duke University, USA) containing the NAT gene, each with a unique marker sequence, for the entire NAT marker. (extended reverse M13). For split marker/DJ-PCR, the split 5'- and 3'-regions of the NAT marker were amplified with pNATSTM using primer pairs M13Fe/NSL and M13Re/NSR, respectively. In the second round of PCR for overlap PCR, the kinase gene-disruption cassette was amplified with primers L1 and R2, using the combined first round PCR product as a template. In the second round of PCR for cleavage marker/DJ-PCR, the NAT-cleavage gene-disruption cassette was used as a template with the corresponding first round PCR product combined, 5'- and 3' of the kinase gene and NAT marker. The '-region was amplified with primer pairs L1/NSL and R2/NSR2, respectively. For transformation, the H99S strain (obtained from Joeseph Heitman's laboratory, Duke University, USA) was cultured in 30 to 50 ml of yeast extract-peptone-dextrose (YPD) medium [Yeast extract (Becton, Dickison and company #212750), Peptone (Becton, Dickison and company #211677), Glucose (Duchefa, #G0802)], cultured overnight, pelleted, and resuspended in 5 ml of distilled water. Approximately 200 μl of the cell suspension was spread on YPD solid medium containing 1 M sorbitol and further incubated at 30 °C for 3 hours. PCR-amplified gene disruption cassettes were coated on 600 μg of 0.6 μm gold microcarrier beads (PDS-100, BioRad) and biologically injected into cells with a particle delivery system (PDS-100, BioRad). Transformed cells were cultured at 30 °C and scraped after 3 hours to restore cell membrane integrity. The scraped cells were transferred to selective medium (YPD solid plate containing 100 μg/ml norceothricin; YPD+NAT). Stable norseothricin-resistant (NATr) transformants were selected through two or more passages on YPD+NAT plates. All NAT ^r strains were confirmed by PCR using each screening primer listed in Table 2, and the correct genotype was finally confirmed by Southern blot analysis (Jung, KW, Kim, SY, Okagaki, LH, Nielsen, K. & Bahn , YS Ste50 adapter protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway. Fungal Genet 48, 154-165, doi:S1087-1845(10)00191-X [pii] 10.1016/j.fgb. .2010.10.006 (2011).

List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') One CNAG_00047 PKP1 L1 CNAG_00047 5' flanking region primer 1 AATGAAGTTCCTGCGACAG L2 CNAG_00047 5' flanking region primer 2 GCTCACTGGCCGTCGTTTTACAATGGGATGAGAACGCAC R1 CNAG_00047 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAGCATTTTCCAGCATCAGC R2 CNAG_00047 3' flanking region primer 2 GGTGTGGAACATCTTTTGAG SO CNAG_00047 diagnostic screening primer, pairing with B79 CCTCTGACAGCCACATACTG PO1 CNAG_00047 Southern blot probe primer 1 CTGGTTCATCTTGGGTGTC PO2 CNAG_00047 Southern blot probe primer 2 TCTGAGCATACCACTCCTTTAC STM NAT#224 STM primer AACCTTTAAATGGGTAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 2 CNAG_00106 TCO5 L1 CNAG_00106 5' flanking region primer 1 TACACGAGATTGGCTGGCAACC L2 CNAG_00106 5' flanking region primer 2 CTGGCCGTCGTTTTACAAGTGAACGCCACACCGATGAG R1 CNAG_00106 3' flanking region primer 1 GTCATAGCTGTTTCCTGTCTCCCGAGGATGTCTTAG R2 CNAG_00106 3' flanking region primer 2 TGCCAAAGCGTGTAAGTG SO CNAG_00106 diagnostic screening primer, pairing with B79 ATGGGAAAGGTCAGTAGCACCG PO1 CNAG_00106 Southern blot probe primer 1 TCGTCTTTTCTTGGTCCAG PO2 CNAG_00106 Southern blot probe primer 2 TGAGGGCGTAGTTGATAATG STM NAT#125 STM primer CGCTACAGCCAGCGCGCGCAAGCG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 3 CNAG_00130 HRK1 L1 CNAG_00130 5' flanking region primer 1 TTCCAGTCAACCGAGTAGC L2 CNAG_00130 5' flanking region primer 2 CTGGCCGTCGTTTTACCTGTATTCATCATTGCGGC R1 CNAG_00130 3' flanking region primer 1 GTCATAGCTGTTTCCTGCGTCAAATCCAAGAACATCGTG R2 CNAG_00130 3' flanking region primer 2 GCCTTCATCGTCGTTAGAC SO CNAG_00130 diagnostic screening primer, pairing with B79 AAGACGACCACATCTCAGAG PO1 CNAG_00130 Southern blot probe primer 1 AGGACTCTGCTCCATCAAG PO2 CNAG_00130 Southern blot probe primer 2 GAAAGAGCCTCAGAAAAGTAGG STM NAT#58 STM primer CGCAAAATCACTAGCCCTATAGCG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 4 CNAG_00266 L1 CNAG_00266 5' flanking region primer 1 GGTCGTATTCTCTCTTTTCAAGC L2 CNAG_00266 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTGACGAGTTGTTCAGGGG R1 CNAG_00266 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGATGTGGATGAGAAGGTAGC R2 CNAG_00266 3' flanking region primer 2 GTGCCGACGAGAAGATAAC SO CNAG_00266 diagnostic screening primer, pairing with B79 AAGGGATAATGGATGACCAC P.O. CNAG_00266 Southern blot probe primer TCAGTGAGATTCAAGGATGC STM NAT#213 STM primer CTGGGGATTTTGATGTGTCTATGT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 5 CNAG_00363 TCO6 L1 CNAG_00363 5' flanking region primer 1 GAGAGAATAACAAAAGGGCG L2 CNAG_00363 5' flanking region primer 2 TCACTGGCCGTCGTTTTACACACGAGGGTTAGAGTTGG R1 CNAG_00363 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAGCGTCTTTGTAACCCG R2 CNAG_00363 3' flanking region primer 2 GCAGGTATCTTACACTCCGTTG SO CNAG_00363 diagnostic screening primer, pairing with B79 ATTAGACACACGACCTGGG P.O. CNAG_00363 Southern blot probe primer TGAGGATACTGGTTGACGC STM NAT#58 STM primer CGCAAAATCACTAGCCCTATAGCG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 6 CNAG_00388 L1 CNAG_00388 5' flanking region primer 1 TTTTGAGCGGGGAAACAC L2 CNAG_00388 5' flanking region primer 2 TCACTGGCCGTTCGTTTTACGGGTCTCGTCTGTATTTTCG R1 CNAG_00388 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGATACCCAGGATTCCACTG R2 CNAG_00388 3' flanking region primer 2 ACCATTATCGTCGCCTTCG SO CNAG_00388 diagnostic screening primer, pairing with B79 CAATCCCAATGGCTTTCAG P.O. CNAG_00388 Southern blot probe primer CGGGTCAAGATGAAAATGTTCGTC STM NAT#208 STM primer TGGTCGCGGGAGATCGTGGTTT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 7 CNAG_00396 PKA1 L1 CNAG_00396 5' flanking region primer 1 AAACGACTGTGTAATGCGAG L2 CNAG_00396 5' flanking region primer 2 CTGGCCGTCGTTTTACGGAGCCAGAATAAAGGAGTTG R1 CNAG_00396 3' flanking region primer 1 GTCATAGCTGTTTCCTGGCACTAAATGGGTGAGACAC R2 CNAG_00396 3' flanking region primer 2 CGATTTGTCCAGTGATTCAGTGAC SO CNAG_00396 diagnostic screening primer, pairing with B79 GTTGGAAGTAGCAGTGTCTTG P.O. CNAG_00396 Southern blot probe primer TGTCGGAGGAAATGAACG STM NAT#191 STM primer ATATGGATGTTTTTAGCGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 8 CNAG_00405 KIC1 L1 CNAG_00405 5' flanking region primer 1 AAGATGAGCGTTGCGAAG L2 CNAG_00405 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGCGTGGTGCTAAGAACAAC R1 CNAG_00405 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGAGGTAGACTCCCAGAATGC R2 CNAG_00405 3' flanking region primer 2 TAATGTGTCAACTGCCGC SO CNAG_00405 diagnostic screening primer, pairing with B79 TTGGTTTCAAGGGGGAAC P.O. CNAG_00405 Southern blot probe primer AAAGTGGACCGTTTGGAG STM NAT#201 STM primer CACCCTCTATCTCGAGAAAGCTCC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 9 CNAG_00415 CDC2801 L1 CNAG_00415 5' flanking region primer 1 CGCATTCTGGACAAAAGC L2 CNAG_00415 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTTGCCGTATCTTTCCTGG R1 CNAG_00415 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGATGTATCTAATCCCTCCG R2 CNAG_00415 3' flanking region primer 2 AGATTCGGTGCTTTGTGTC SO CNAG_00415 diagnostic screening primer, pairing with B79 TTGGTCTGGGAACCTTTAC P.O. CNAG_00415 Southern blot probe primer AATGTGCTACTGCCGACAG STM NAT#191 STM primer ATATGGATGTTTTTAGCGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 10 CNAG_00556 L1 CNAG_00556 5' flanking region primer 1 GAACCGAAAAGGGCATTC L2 CNAG_00556 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGGAGCAGGTGGTTCTAAG R1 CNAG_00556 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCCAGGAGAGAGGAATGAAAC R2 CNAG_00556 3' flanking region primer 2 CCACCGTCCATTACTTACTG SO CNAG_00556 diagnostic screening primer, pairing with B79 TGTCAACCCGCTCAAACAC P.O. CNAG_00556 Southern blot probe primer AGAGAAGTCCTTGCGATTG STM NAT#290 STM primer ACCGACAGCTCGAACAAGCAAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 11 CNAG_00636 CDC7 L1 CNAG_00636 5' flanking region primer 1 GCTGGAAGCGTGATGATAC L2 CNAG_00636 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGTGTAGGAGGGGAGATGAG R1 CNAG_00636 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAGGACATCCACCAGAGAGG R2 CNAG_00636 3' flanking region primer 2 CAAATGGGTGTCTCAGAGC SO CNAG_00636 diagnostic screening primer, pairing with B79 TGAGTGATGCCTTACGCTG P.O. CNAG_00636 Southern blot probe primer CCCTGTAGACTTACCTTCCC STM NAT#213 STM primer CTGGGGATTTTGATGTGTCTATGT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 12 CNAG_00683 L1 CNAG_00683 5' flanking region primer 1 GAAAACGAGTCCTGGATAGTTC L2 CNAG_00683 5' flanking region primer 2 TCACTGGCCGTCGTTTTACATGGTTGGATGGGTAGGAG R1 CNAG_00683 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCCTGCCAACAGACATCAAC R2 CNAG_00683 3' flanking region primer 2 AGAAAAACTCGGACACCTG SO CNAG_00683 diagnostic screening primer, pairing with B79 TGTAAAAAACAGAGGAGCCC P.O. CNAG_00683 Southern blot probe primer TTCAGAGTCATCCCACGTG STM NAT#273 STM primer GAGATCTTTCGGGAGGTCTGGATT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 13 CNAG_00745 HRK1/NPH1 L1 CNAG_00745 5' flanking region primer 1 GCAAAAATGGGGAAGATAGG L2 CNAG_00745 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTCCCCAAAATCACTCCC R1 CNAG_00745 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGGAGATGAGTGGGTGAAG R2 CNAG_00745 3' flanking region primer 2 TGTGTCAGACCTGTTATCGTTC SO CNAG_00745 diagnostic screening primer, pairing with CTCAACCACTCTCTTACGGA P.O. CNAG_00745 Southern blot probe primer CGAGGTTAGGAGGGAAAGGTC STM NAT#210 STM primer CTAGAGCCCGCCACAACGCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 14 CNAG_00769 PBS2 L1 CNAG_00769 5' flanking region primer 1 AGGAAGTGGAGTGTTGTG L2 CNAG_00769 5' flanking region primer 2 CTGGCCGTTCGTTTTACATGCGAGGAAGAAGAAAGGTCG R1 CNAG_00769 3' flanking region primer 1 GTCATAGCTGTTTCCTGAACCGACGACCGACTTATGC R2 CNAG_00769 3' flanking region primer 2 GTAAGGTAGTCGCAACAACG SO CNAG_00769 diagnostic screening primer, pairing with B79 CGATACCCTTCTTGCCTGTAG PO1 CNAG_00769 Southern blot probe primer 1 AACACGACAGGAAATCCG PO2 CNAG_00769 Southern blot probe primer 2 TGGAAGGTTACAAGCCGAC STM NAT#213 STM primer CTGGGGATTTTGATGTGTCTATGT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 15 CNAG_00782 SPS1 L1 CNAG_00782 5' flanking region primer 1 CCCGATGAAAGTAATGGC L2 CNAG_00782 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAATGTCCTCTCTTCTGCTCTC R1 CNAG_00782 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGATGACTGCGAAGAAAGGC R2 CNAG_00782 3' flanking region primer 2 CTTACATCCAGACATCCCAC SO CNAG_00782 diagnostic screening primer, pairing with B79 GGGTGAGCAACAAGAAATG P.O. CNAG_00782 Southern blot probe primer CTCCTCCTTTCTTTTATGCC STM NAT#288 STM primer CTATCCAACTAGACCTCTAGCTAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 16 CNAG_00826 DAK2 L1 CNAG_00826 5' flanking region primer 1 AGTTTGAATGAAGGGGCG L2 CNAG_00826 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGGAAGATGTGTCGGTCTGTC R1 CNAG_00826 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCGGAAGGTATTCTCAAGGC R2 CNAG_00826 3' flanking region primer 2 GCTGTTCAGTTTCCTCTCTATG SO CNAG_00826 diagnostic screening primer, pairing with B79 ACAGCGATGTGGGGATAAG P.O. CNAG_00826 Southern blot probe primer CATACTTTCCTCGGGATTTC STM NAT#282 STM primer TCTCTATAGCAAAACCAATC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 17 CNAG_00877 L1 CNAG_00877 5' flanking region primer 1 TCCACACACGAATGGTATC L2 CNAG_00877 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTGTCAGCAAGGGAATGGGCAGTG R1 CNAG_00877 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCGGATGATTTGAGGGATAG R2 CNAG_00877 3' flanking region primer 2 ATTGAAAACTACCAGTGGCACCCCG SO CNAG_00877 diagnostic screening primer, pairing with B79 CCAATACGGTGCTTATTGTGAC P.O. CNAG_00877 Southern blot probe primer CGCAGAGTAGGTTGTGTTG STM NAT#204 STM primer GATCTCTCGCGCTTGGGGGA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 18 CNAG_01061 L1 CNAG_01061 5' flanking region primer 1 AAAAGGGGTGGGTCAAAAG L2 CNAG_01061 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGGGTATTGGGTTTCCTCTG R1 CNAG_01061 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCCATTAGCATTCGGAGAG R2 CNAG_01061 3' flanking region primer 2 GAAGTATCAGAGGAGTCCCG SO CNAG_01061 diagnostic screening primer, pairing with B79 CGTGGTCACTTATGTCCTTC P.O. CNAG_01061 Southern blot probe primer AAAAGTGCGAAGGGGAGGTC STM NAT#220 STM primer CAGATCTCGAACGATACCCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 19 CNAG_01062 PSK201 L1 CNAG_01062 5' flanking region primer 1 GTCCACTTTATTTTTCGGGC L2 CNAG_01062 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGAGGAGTAATGACCGTGACC R1 CNAG_01062 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGGTAAAAAGGGGGTGGGTC R2 CNAG_01062 3' flanking region primer 2 GGTATTGGGTTTCCTCTGTG SO CNAG_01062 diagnostic screening primer, pairing with B79 GATTAGTATTCCTGTGCCACC P.O. CNAG_01062 Southern blot probe primer GGAAATGTAGGGGGTAGACG STM NAT#191 STM primer ATATGGATGTTTTTAGCGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 20 CNAG_01155 GUT1 L1 CNAG_01155 5' flanking region primer 1 AATCGTCCCTTCCTAAGC L2 CNAG_01155 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAAACCGAGACCTCTGAAGG R1 CNAG_01155 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGGAGAAAGCCAGACTGAAG R2 CNAG_01155 3' flanking region primer 2 ATGGTAGTTTTGCGGGTG SO CNAG_01155 diagnostic screening primer, pairing with B79 CAGAGAAGTTGACTGGGATG P.O. CNAG_01155 Southern blot probe primer GTTCATCGGCTTCAACCAG STM NAT#242 STM primer GTAGCGATAGGGGTGTCGCTTTAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 21 CNAG_01162 MAK322 L1 CNAG_01162 5' flanking region primer 1 GACCGCAGTAGAACTTACACC L2 CNAG_01162 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGAGGAAATGTTGAAGGTGTG R1 CNAG_01162 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCGGAAGGAAAGAGTTTAGACG R2 CNAG_01162 3' flanking region primer 2 ATCAGGCAACCGCATAAC SO CNAG_01162 diagnostic screening primer, pairing with B79 ATGCTGCCAGAACACTTG P.O. CNAG_01162 Southern blot probe primer TCCTCCCAAATAAGTGCC STM NAT#159 STM primer ACGCACCAGACACACAACCAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 22 CNAG_01165 LCB5 L1 CNAG_01165 5' flanking region primer 1 CCCAAATCTCGTTCGTTG L2 CNAG_01165 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTGTGTGGCTGTAGAGGTG R1 CNAG_01165 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCCATCGCACATAACTTTC R2 CNAG_01165 3' flanking region primer 2 ATTCTGAAGGCGTAAGTCG SO CNAG_01165 diagnostic screening primer, pairing with B79 AAAAGGGTCGTAAGATGGG P.O. CNAG_01165 Southern blot probe primer ACGCCGAATAGGTTTGTG STM NAT#213 STM primer CTGGGGATTTTGATGTGTCTATGT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 23 CNAG_01209 FAB1 L1 CNAG_01209 5' flanking region primer 1 TTTCTGATGGGAGGGAGTG L2 CNAG_01209 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGCGTGGTATGGATAGACAAG R1 CNAG_01209 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAAAGATTTGGGGGCTGG R2 CNAG_01209 3' flanking region primer 2 GCTGAAGGTGAGCGATAAG SO CNAG_01209 diagnostic screening primer, pairing with B79 AGTCAGTGTCCAAACTTCTGTC P.O. CNAG_01209 Southern blot probe primer AAAGGGAATCCAGGAACG STM NAT#169 STM primer ACATCTATATCACTATCCCGAACC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 24 CNAG_01250 L1 CNAG_01250 5' flanking region primer 1 GCTTTTCGTTGGAGGTG L2 CNAG_01250 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGCTCTGTCATCTTCCAGC R1 CNAG_01250 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGATAGCGTGTTACCACAGGC R2 CNAG_01250 3' flanking region primer 2 CGTCCTCAAAATACAACTCG SO CNAG_01250 diagnostic screening primer, pairing with B79 TGGTAAATCTCTCGTGCTG P.O. CNAG_01250 Southern blot probe primer GCGAAAGTAACCCAGATGC STM NAT#227 STM primer TCGTGGTTTAGAGGGAGCGC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 25 CNAG_01285 L1 CNAG_01285 5' flanking region primer 1 CAATAACCCATTACCACTGC L2 CNAG_01285 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTGTTGGCAAGACCACTG R1 CNAG_01285 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGTTTCTCCTGAAGCCACTG R2 CNAG_01285 3' flanking region primer 2 TTAGAGGGCGGTAGTTACGG SO CNAG_01282 diagnostic screening primer, pairing with B79 TTACGATACTTGGCTGAAGC P.O. CNAG_01285 Southern blot probe primer AGCATTTTGGCTGTAGGC STM NAT#240 STM primer GGTGTTGGATCGGGGTGGAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 26 CNAG_01294 IPK1 L1 CNAG_01294 5' flanking region primer 1 GGAAAGAGAAGAGCACGG L2 CNAG_01294 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCATCAACCATAGCAAGCAAC R1 CNAG_01294 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGGCTGGTCAAAGAATGGAC R2 CNAG_01294 3' flanking region primer 2 TGGTAGGATGTGTTGTGGAG SO CNAG_01294 diagnostic screening primer, pairing with B79 TTTGCTCTCTTCGCCAAC P.O. CNAG_01294 Southern blot probe primer CGCATTCTCATCTTATCCC STM NAT#184 STM primer ATATATGGCTCGAGCTAGATAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 27 CNAG_01333 ALK1 L1 CNAG_01333 5' flanking region primer 1 GCATTTTCATTGCTGGTCAC L2 CNAG_01333 5' flanking region primer 2 TCACTGGCCGTCGTTTTACACGGAAGGAGGAGATAACTAAC R1 CNAG_01333 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGAGTTGTATGGCGAGGATG R2 CNAG_01333 3' flanking region primer 2 GTCCTGTGAATCGGGAGAT SO CNAG_01333 diagnostic screening primer, pairing with B79 TGTTTCACCAGAGTCAGCC P.O. CNAG_01333 Southern blot probe primer ACGGGAGTGTTGTATGAGC STM NAT#122 STM primer ACAGCTCCAAACCTCGCTAAACAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 28 CNAG_01364 L1 CNAG_01364 5' flanking region primer 1 TCGCTCGCCTTGATTTGAC L2 CNAG_01364 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAAGTGGCTGTTGTGGAGGTCTG R1 CNAG_01364 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTGCGGTGATACCTTGCCAG R2 CNAG_01364 3' flanking region primer 2 TCCCCCGTTACCTTTATG SO CNAG_01364 diagnostic screening primer, pairing with B79 CAGCCAATCTTTTCCCTG P.O. CNAG_01364 Southern blot probe primer TTTTCGCCAGCCACCTTCAG STM NAT#5 STM primer TGCTAGAGGGCGGGAGAGTT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 29 CNAG_01523 HOG1 L1 CNAG_01523 5' flanking region primer 1 TGTGGTAGGTGCGTTATCG L2 CNAG_01523 5' flanking region primer 2 CTGGCCGTCGTTTTACAGAAAGCCCATCCATCAG R1 CNAG_01523 3' flanking region primer 1 GTCATAGCTGTTTCCTGTCTTGGTAAGTCTCTGTGCC R2 CNAG_01523 3' flanking region primer 2 TACTCAACCCCATACTCACTCCCG SO CNAG_01523 diagnostic screening primer, pairing with B79 TGAAGACAAAGGCGTGGG PO1 CNAG_01523 Southern blot probe primer 1 TCACAGAGCGTTGATTACG PO2 CNAG_01523 Southern blot probe primer 2 CAGGCTCATCGGTAGGATCA STM NAT#177 STM primer CACCAACTCCCCATCTCCAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 30 CNAG_01612 PSK202 L1 CNAG_01612 5' flanking region primer 1 ACGCTTGTTTCTTCGTCC L2 CNAG_01612 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTCGGGATGATAAGTGAGG R1 CNAG_01612 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTCTTCCCCTTTCTGATGG R2 CNAG_01612 3' flanking region primer 2 CCGACCAAAAACAGGTTC SO CNAG_01612 diagnostic screening primer, pairing with B79 AACTGGCATTGAAGGTGTC P.O. CNAG_01612 Southern blot probe primer GACAAGCATTGGGAAACC STM NAT#208 STM primer TGGTCGCGGGAGATCGTGGTTT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 31 CNAG_01664 L1 CNAG_01664 5' flanking region primer 1 CCTACATCCAGGACAAACG L2 CNAG_01664 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCACCTTCTCCGACCTTTTC R1 CNAG_01664 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCCGCATAAAGAAAAGCC R2 CNAG_01664 3' flanking region primer 2 AAAGCGAGGTTGAAGAGGG SO CNAG_01664 diagnostic screening primer, pairing with B79 CGTCGTAGTGGGTGTAGATG P.O. CNAG_01664 Southern blot probe primer AGGACAACAAGTCTGGATAGC STM NAT#218 STM primer CTCCACATCCATCGCTCCAA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 32 CNAG_01687 L1 CNAG_01687 5' flanking region primer 1 GCTCCTAAATACCTGCCACTC L2 CNAG_01687 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCTCATCCGCAGAAATGTATC R1 CNAG_01687 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGTTCGCTTATGGTCTATGG R2 CNAG_01687 3' flanking region primer 2 TTGCGACCTTTTTCTCGG SO CNAG_01687 diagnostic screening primer, pairing with B79 TGTTAGAAAAGCCTGTGACG P.O. CNAG_01687 Southern blot probe primer CCCAAGATAGTCTCGTTTGC STM NAT#290 STM primer ACCGACAGCTCGAACAAGCAAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 33 CNAG_01704 IRK6 L1 CNAG_01704 5' flanking region primer 1 GGTCAACTTTCCCTTGTCG L2 CNAG_01704 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTGAGAGAGCGTGATAAAGC R1 CNAG_01704 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCACATTGACCTTCCTGTAAC R2 CNAG_01704 3' flanking region primer 2 GCCCTAAACAAACTAACTCTGTCC SO CNAG_01704 diagnostic screening primer, pairing with B79 AGCCTCCTCTTTCCTTACAG P.O. CNAG_01704 Southern blot probe primer GCTGGTGCCTCTTTGATTC STM NAT#5 STM primer TGCTAGAGGGCGGGAGAGTT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 34 CNAG_01730 STE7 L1 CNAG_01730 5' flanking region primer 1 TTGTAAGGCTCTCATTCGC L2 CNAG_01730 5' flanking region primer 2 CTGGCCGTCGTTTTACTGAAGGCAAAACTGGTGC R1 CNAG_01730 3' flanking region primer 1 GTCATAGCTGTTTCCTGCCTTACCGTGCTTTTCTGC R2 CNAG_01730 3' flanking region primer 2 TTACTTCCGCCCAACGACAC SO CNAG_01730 diagnostic screening primer, pairing with B79 TCCTCGCTCACAAAATGGGC PO1 CNAG_01730 Southern blot probe primer 1 CCAATAGACATCAAGCCGTC PO2 CNAG_01730 Southern blot probe primer 2 AAACAGAGAAGAGAAGGGACC STM NAT#225 STM primer CCATAGAACTAGCTAAAGCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 35 CNAG_01820 L1 CNAG_01820 5' flanking region primer 1 TCAGAGCAGACAAGGCGTC L2 CNAG_01820 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTTTGGGGAGGAAAGTGCTGAGG R1 CNAG_01820 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGTTGGTCATTTGTGCGAC R2 CNAG_01820 3' flanking region primer 2 GGCATTATGAGCAAATCGG SO CNAG_01820 diagnostic screening primer, pairing with B79 TAGCAGAAGGAGGAGGACGGTTC P.O. CNAG_01820 Southern blot probe primer CCTTGACGATGTTGGTCTG STM NAT#6 STM primer ATAGCTACCACACGATAGCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 36 CNAG_01845 L1 CNAG_01845 5' flanking region primer 1 GAATAATCAGCAGGCGGTG L2 CNAG_01845 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGTTCGTTGTTGGTTGTCG R1 CNAG_01845 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGGGAGCCAATAATGTGGAG R2 CNAG_01845 3' flanking region primer 2 TCTTCATCCTTCCCTTGC SO CNAG_01845 diagnostic screening primer, pairing with B79 TAAGGGCAAAAGGGTCAG P.O. CNAG_01845 Southern blot probe primer TTTTTAGCGTCCGTCTCG STM NAT#205 STM primer TATCCCCCTCTCCGCTCTCTAGCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 37 CNAG_01850 TCO1 L1 CNAG_01850 5' flanking region primer 1 GTTTCTGCTTCCACCTCAC L2 CNAG_01850 5' flanking region primer 2 CTGGCCGTCGTTTTACTTTACACACACGGGCGATGTCCTG R1 CNAG_01850 3' flanking region primer 1 GTCATAGCTGTTTCCTGACTGAGCAAATCGGCGTAGG R2 CNAG_01850 3' flanking region primer 2 AAGTGAGGGGCATTACAGG SO CNAG_01850 diagnostic screening primer, pairing with B79 CGACACAATACTCTAACTGCG PO1 CNAG_01850 Southern blot probe primer 1 CTTTCGTCTTTGCCACAC PO2 CNAG_01850 Southern blot probe primer 2 AATCACCCTTTGCTACGG STM NAT#102 STM primer CCATAGCGATATCTACCCCAATCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 38 CNAG_01905 KSP1 L1 CNAG_01905 5' flanking region primer 1 CGATTTTGTCTGGGCTCTC L2 CNAG_01905 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAAGATGATTCGGGCACAG R1 CNAG_01905 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCCCTCTTTCTCAATCATCG R2 CNAG_01905 3' flanking region primer 2 ACAACATCTTCGCCAACG SO CNAG_01905 diagnostic screening primer, pairing with B79 TACCGACTCGCAATACACC P.O. CNAG_01905 Southern blot probe primer ATACCTTTGTGGCTTCGC STM NAT#159 STM primer ACGCACCAGACACACAACCAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 39 CNAG_01907 L1 CNAG_01907 5' flanking region primer 1 GCATTCTCTCAACTCGCTC L2 CNAG_01907 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTCGTAGCTCTGTCTCTATCCC R1 CNAG_01907 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAGTTTTCAGCCAATACCAGG R2 CNAG_01907 3' flanking region primer 2 TGAACCCCTTTGACCCATCC SO CNAG_01907 diagnostic screening primer, pairing with B79 CCTCTTCTGTATGCTGCGAG P.O. CNAG_01907 Southern blot probe primer TCTGGAATGGAGGCTTTC STM NAT#282 STM primer TCTCTATAGCAAAACCAATC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 40 CNAG_01938 KIN1 L1 CNAG_01938 5' flanking region primer 1 AGAGACAAAGGTGAGGTCG L2 CNAG_01938 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCACGGGATAATGTTGACG R1 CNAG_01938 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCAGTATCAAATGCTGGC R2 CNAG_01938 3' flanking region primer 2 AGATAATAAGGGTGCGGC SO CNAG_01938 diagnostic screening primer, pairing with B79 TGAGGTGGAGGCTTGTCTAC P.O. CNAG_01938 Southern blot probe primer GGACTTCTTTGGTTGGGAG STM NAT#6 STM primer ATAGCTACCACACGATAGCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 41 CNAG_01988 TCO3 L1 CNAG_01988 5' flanking region primer 1 CCCAGAAAAGAAGGTTGG L2 CNAG_01988 5' flanking region primer 2 CTGGCCGTCGTTTTACTTGTGGTTTTGTGGGTAGCGTGG R1 CNAG_01988 3' flanking region primer 1 GTCATAGCTGTTTCCTGGGCATCATTGCTCATTCTTGTG R2 CNAG_01988 3' flanking region primer 2 AAAAGGTGAAATAGGGGGCGGCG SO CNAG_01988 diagnostic screening primer, pairing with B79 TGTTCCTCAATGAAGTGTCC P.O. CNAG_01988 Southern blot probe primer 1 ATGGGGAGGTCTATGCGTTAGC PO2 CNAG_01988 Southern blot probe primer 2 TAAAGTCTTCTTCCCCACCCG STM NAT#119 STM primer CTCCCCACATAAAGAGAGCTAAAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 42 CNAG_02007 L1 CNAG_02007 5' flanking region primer 1 GAGCAGCGAAATAACCAAAG L2 CNAG_02007 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCAGTAGCGAGGTGACAGATG R1 CNAG_02007 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCGATTGGACACTTACCAC R2 CNAG_02007 3' flanking region primer 2 AGCCCGAGTTCTTTTTAGAC SO CNAG_02007 diagnostic screening primer, pairing with B79 AGAAATAGCGTTGCCACC P.O. CNAG_02007 Southern blot probe primer GCTTGTTTGGTAGATAGTCAGC STM NAT#232 STM primer CTTTAAAGGTGGTTTTGTG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 43 CNAG_02028 L1 CNAG_02028 5' flanking region primer 1 AAATCCGCAGGGGAAAAC L2 CNAG_02028 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGGGAAAAGGATGGACAGG R1 CNAG_02028 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCCTCCGTCCTCAAAGAAAAATACC R2 CNAG_02028 3' flanking region primer 2 TTCCGTTCCAATCGCAAG SO CNAG_02028 diagnostic screening primer, pairing with B79 TTTTGCCCTTGCCCTGTTG P.O. CNAG_02028 Southern blot probe primer ATCTTGCTCATACCGAACC STM NAT#225 STM primer CCATAGAACTAGCTAAAGCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 44 CNAG_02194 L1 CNAG_02194 5' flanking region primer 1 TTGGTCCTCTGCGAAAAC L2 CNAG_02194 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGCTGTTGCTGAGAGTTTGTG R1 CNAG_02194 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTCAAACCCGAAGGTGAAG R2 CNAG_02194 3' flanking region primer 2 ACGACTTATTCCCCATCCC SO CNAG_02194 diagnostic screening primer, pairing with B79 CACCTCGTTGATGAATGC P.O. CNAG_02194 Southern blot probe primer CTTCTCTCCTTCTCGTATCTGG STM NAT#273 STM primer GAGATCTTTCGGGAGGTCTGGATT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 45 CNAG_02202 L1 CNAG_02202 5' flanking region primer 1 AACAACCGAAACCAGCGAC L2 CNAG_02202 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGGAAGGTGATGTTTGTGGC R1 CNAG_02202 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCGCCGACAATGGTCTTATC R2 CNAG_02202 3' flanking region primer 2 TCCTGGTCATCGTGCTAACC SO CNAG_02202 diagnostic screening primer, pairing with B79 CTTATGCCACTCCTAACCG P.O. CNAG_02202 Southern blot probe primer GCCGAGATACCTGTAAAGTCC STM NAT#6 STM primer ATAGCTACCACACGATAGCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 46 CNAG_02233 MEC1 L1 CNAG_02233 5' flanking region primer 1 TTCCTCATCCACGATACTTC L2 CNAG_02233 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGACAGAGGTTTTGAGGATGC R1 CNAG_02233 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTTTGTCCACGACCCTCTC R2 CNAG_02233 3' flanking region primer 2 TCATTGCCACCTCCCACCAAG SO CNAG_02233 diagnostic screening primer, pairing with B79 CTGATTGAAGGAACTTACCTCG P.O. CNAG_02233 Southern blot probe primer GGAGAAGTTCACGAAGGTCTG STM NAT#204 STM primer GATCTCTCGCGCTTGGGGGA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 47 CNAG_02285 L1 CNAG_02285 5' flanking region primer 1 TCCTCTGTTCTTGTCGTGG L2 CNAG_02285 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCTGCTCAGTGGTAGACATTTTG R1 CNAG_02285 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTCTCAGGCTTGGCTCTAC R2 CNAG_02285 3' flanking region primer 2 CGCCCTGTGATGATAATAACCTTC SO CNAG_02285 diagnostic screening primer, pairing with B79 TGGACAAAGGGACACTTACC P.O. CNAG_02285 Southern blot probe primer TGACAACACCAACGATGG STM NAT#150 STM primer ACATACACCCCCATCCCCCC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 48 CNAG_02296 RBK1 L1 CNAG_02296 5' flanking region primer 1 TCACTCATCACCAGGTAACG L2 CNAG_02296 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAGAAACTGGAAAGCAGACG R1 CNAG_02296 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCTTGCTTAGGAAAATCACCC R2 CNAG_02296 3' flanking region primer 2 GCACAAGAAAACCAGTCCAG SO CNAG_02296 diagnostic screening primer, pairing with B79 GCTCGGTATGTTTATCACCTG P.O. CNAG_02296 Southern blot probe primer GAGTGTGGAAGAGAGAGGAAC STM NAT#219 STM primer CCCTAAACCCTACAGCAAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 49 CNAG_02357 MKK2 L1 CNAG_02357 5' flanking region primer 1 GCGTCATTTCCCAATCAC L2 CNAG_02357 5' flanking region primer 2 CTGGCCGTCGTTTTACTCGGTGCTTCAGTTCAGAG R1 CNAG_02357 3' flanking region primer 1 GTCATAGCTGTTTCCTGACCCTACCCTTGGCAACTAC R2 CNAG_02357 3' flanking region primer 2 CCCTTTGTTTGTTGCTGAC SO CNAG_02357 diagnostic screening primer, pairing with B79 TTTTGCCCACTCCCCCTTTTACCAC PO1 CNAG_02357 Southern blot probe primer 1 GCAAAGTCACATACACGGC PO2 CNAG_02357 Southern blot probe primer 2 GATGTCCGAGTGATAACCTG STM NAT#224 STM primer AACCTTTAAATGGGTAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 50 CNAG_02389 YPK101 L1 CNAG_02389 5' flanking region primer 1 TACCTGCCGACAAATGAC L2 CNAG_02389 5' flanking region primer 2 TCACTGGCCGTCGTTTTACACATAGCGGCTGCTTTTC R1 CNAG_02389 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGGGGGTTCTAAAAGACG R2 CNAG_02389 3' flanking region primer 2 ACCATCATCTCTGCGTTG SO CNAG_02389 diagnostic screening primer, pairing with B79 AACCGCAAGTAGGGCATAC P.O. CNAG_02389 Southern blot probe primer TGAGCAAAAAAGGCGAGC STM NAT#242 STM primer GTAGCGATAGGGGTGTCGCTTTAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 51 CNAG_02459 L1 CNAG_02459 5' flanking region primer 1 TCTCGGGGTCTTCAATCTC L2 CNAG_02459 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGTGCGGATTCGTTATTTGG R1 CNAG_02459 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAAGAGGGTTAGGTTTGGC R2 CNAG_02459 3' flanking region primer 2 GCCACTTCCGTATCAAAAG SO CNAG_02459 diagnostic screening primer, pairing with B79 GCACTGCTGCTTGAAATC P.O. CNAG_02459 Southern blot probe primer ATAGATTCTGATGGCGGCG STM NAT#122 STM primer ACAGCTCCAAACCTCGCTAAACAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 52 CNAG_02511 CPK1 L1 CNAG_02511 5' flanking region primer 1 CTGTAGAAGATTGTGAGTTTGGG L2 CNAG_02511 5' flanking region primer 2 CTGGCCGTCGTTTTACTGATTGATGAGAGATACGGG R1 CNAG_02511 3' flanking region primer 1 GTCATAGCTGTTTCCTGGGCGGAGAAATAGAGGTTG R2 CNAG_02511 3' flanking region primer 2 CGCACAAGAAGTAAGAGGGTG SO CNAG_02511 diagnostic screening primer, pairing with B79 GGCTATGACCGTATTCAC PO1 CNAG_02511 Southern blot probe primer 1 TATCTCACAAGCCACTCCC PO2 CNAG_02511 Southern blot probe primer 2 ATGCTGCTCACCGTTAGTC STM NAT#184 STM primer ATATATGGCTCGAGCTAGATAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 53 CNAG_02531 CPK2 L1 CNAG_02531 5' flanking region primer 1 ATGTGCTTGGTTTGCCCGAG L2 CNAG_02531 5' flanking region primer 2 CTGGCCGTCGTTTTACAACCTGACTTTGCGAGGAGC R1 CNAG_02531 3' flanking region primer 1 GTCATAGCTGTTTCCTGGGAAGAGTTGAAGAGGCTG R2 CNAG_02531 3' flanking region primer 2 ACTGTGGCTGTTGTTCAGGC SO CNAG_02531 diagnostic screening primer, pairing with B79 CCAAGGGAAGTCTACCAATAC PO1 CNAG_02531 Southern blot probe primer1 GGGGAAAGATTAGTGCGTC PO2 CNAG_02531 Southern blot probe primer2 GTGCGTAGATGAACGAGTG STM NAT#122 STM primer ACAGCTCCAAACCTCGCTAAACAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 54 CNAG_02542 IRK2 L1 CNAG_02542 5' flanking region primer 1 TGTGCTGGTATCTGATGAGC L2 CNAG_02542 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGTGAGCGGCTTTGAAAATG R1 CNAG_02542 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCGGCTATCTTTGTGTATGC R2 CNAG_02542 3' flanking region primer 2 CCCTTTGCTCACTTTCATACC SO CNAG_02542 diagnostic screening primer, pairing with B79 TTTTTCGGGTCTGACGAC P.O. CNAG_02542 Southern blot probe primer CTGTTCACCAAGTTCCCTAATC STM NAT#232 STM primer CTTTAAAGGTGGTTTTGTG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 55 CNAG_02551 DAK3 L1 CNAG_02551 5' flanking region primer 1 ATCTAATCCTCCCTGTCCAC L2 CNAG_02551 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGCGTGATTTCAGGTTCAG R1 CNAG_02551 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAGCGTGGTTTCCTGTAAG R2 CNAG_02551 3' flanking region primer 2 GGTCATAACTCAGAGGGGTC SO CNAG_02551 diagnostic screening primer, pairing with B79 GAGAGCGAAGCAATAGGAAG P.O. CNAG_02551 Southern blot probe primer AAGCAATCTCCAGACTCCC STM NAT#295 STM primer ACACCTACATCAAACCCTCCC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 56 CNAG_02675 HSL101 L1 CNAG_02675 5' flanking region primer 1 CAATGCCGTCATCATCAAAC L2 CNAG_02675 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAAGGGCGAACAGGATAATAC R1 CNAG_02675 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCCTAATGTGAGAGCAGCAATAC R2 CNAG_02675 3' flanking region primer 2 TATTGTGCAGAAAACCGTG SO CNAG_02675 diagnostic screening primer, pairing with B79 GCTGTCTTGTTTGCGTTG P.O. CNAG_02675 Southern blot probe primer AGGAGTAGTATCACTTCGGG STM NAT#146 STM primer ACTAGCCCCCCCTCACCACCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 57 CNAG_02680 VPS15 L1 CNAG_02680 5' flanking region primer 1 AGGACCTTCATCAGGACGAC L2 CNAG_02680 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAAACTACCTCCCCCGTTAC R1 CNAG_02680 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCCAAATGTATGGATTCGCC R2 CNAG_02680 3' flanking region primer 2 CTGCGAATCTCGTCTAAGG SO CNAG_02680 diagnostic screening primer, pairing with B79 TTGAAAGGTCCCACCAGAC P.O. CNAG_02680 Southern blot probe primer GGGAGGAAGTGAGGAGTATG STM NAT#123 STM primer CTATCGACCAACCAACACAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 58 CNAG_02686 L1 CNAG_02686 5' flanking region primer 1 CACACTTTGCTCTTGTTCTGAG L2 CNAG_02686 5' flanking region primer 2 TCACTGGCCGTCGTTTTACATGGAGATGCGATAAGCG R1 CNAG_02686 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGAATCCTCCCTCCAACGAG R2 CNAG_02686 3' flanking region primer 2 AAAGACGACGCCTACTCTGC SO CNAG_02686 diagnostic screening primer, pairing with B79 TGTTCCTCTTCCCTGACAG P.O. CNAG_02686 Southern blot probe primer CACAATCAAAGCGTTAGGG STM NAT#191 STM primer ATATGGATGTTTTTAGCGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 59 CNAG_02712 BUD32 L1 CNAG_02712 5' flanking region primer 1 ATAGGGGATGACCTTGGAG L2 CNAG_02712 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGATGCCAAAGACCAGTG R1 CNAG_02712 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGAGAAGAGGAAGGAAGAGAGAC R2 CNAG_02712 3' flanking region primer 2 GAGCGATAATAGCCACCAC SO CNAG_02712 diagnostic screening primer, pairing with B79 GGGCAATCTTTCTTCGTC P.O. CNAG_02712 Southern blot probe primer CTCGTTCTCTGGTTCTTCTG STM NAT#296 STM primer CGCCCGCCCTCACTATCCAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 60 CNAG_02787 L1 CNAG_02787 5' flanking region primer 1 AACCCCTTGTGTCCCCAAAC L2 CNAG_02787 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGAGCAGGCGGATACGATAC R1 CNAG_02787 3' flanking region primer 1 ATGGTCATAGCTGTTTCCTGGCAAAAAGGACAGAAGAAGAGG R2 CNAG_02787 3' flanking region primer 2 TTCTCCCATTTCTCCACCC SO CNAG_02787 diagnostic screening primer, pairing with B79 AGCAGAGCCAGATGGTAGAG P.O. CNAG_02787 Southern blot probe primer TTCCACTTGGCAACTGTCC STM NAT#227 STM primer TCGTGGTTTAGAGGGAGCGC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 61 CNAG_02799 DAK202A L1 CNAG_02799 5' flanking region primer 1 TTGATACTTTGGGTCTGGG L2 CNAG_02799 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCGGGAGCCATTATTGGTAAG R1 CNAG_02799 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTTTGGATGGCTTGCGAGGG R2 CNAG_02799 3' flanking region primer 2 CCATACAATGACCTGGGAC SO CNAG_02799 diagnostic screening primer, pairing with B79 AACCATCAACTGCCCTCAC P.O. CNAG_02799 Southern blot probe primer GGTAGTATCGGTGATTTGAGTGAG STM NAT#119 STM primer CTCCCCACATAAAGAGAGCTAAAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 62 CNAG_02802 ARG2 L1 CNAG_02802 5' flanking region primer 1 CCAGCAGTTAGGGATTCAG L2 CNAG_02802 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCATCGTAGAGTCGTTATTACCG R1 CNAG_02802 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGATTTGGAGTCCTATCGCC R2 CNAG_02802 3' flanking region primer 2 ATGTCAATGGTAGCCCACC SO CNAG_02802 diagnostic screening primer, pairing with B79 TTTGTTGTTGCCTGACCC P.O. CNAG_02802 Southern blot probe primer GTCGCTCAAAGTGTCTTCTC STM NAT#125 STM primer CGCTACAGCCAGCGCGCGCAAGCG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 63 CNAG_02820 PKH201 L1 CNAG_02820 5' flanking region primer 1 CCCTCGCCAGAATCAATAC L2 CNAG_02820 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGAGAGGATGTTGAGGTTGC R1 CNAG_02820 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTGGGATTAGGGCGTATC R2 CNAG_02820 3' flanking region primer 2 TCTGCCTCTACAAAACCACTG SO CNAG_02820 diagnostic screening primer, pairing with B79 GGAGAGACAGGGGATAAAGC P.O. CNAG_02820 Southern blot probe primer ATACCTCCCTTCTCCCAAC STM NAT#219 STM primer CCCTAAACCCTACAGCAAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 64 CNAG_02847 L1 CNAG_02847 5' flanking region primer 1 AGACCGATAAAAACAGGACC L2 CNAG_02847 5' flanking region primer 2 TCACTGGCCGTTCGTTTTACAACAATGAAGGCACCTCG R1 CNAG_02847 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGGAACATTCAAACGGAGAC R2 CNAG_02847 3' flanking region primer 2 ACCAGTTGACAAAGGTATCG SO CNAG_02847 diagnostic screening primer, pairing with B79 AAGAATACTCCAGAAGGGACC P.O. CNAG_02847 Southern blot probe primer GCTTCGGGGATAAGGTGAG STM NAT#296 STM primer CGCCCGCCCTCACTATCCAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 65 CNAG_02859 POS5 L1 CNAG_02859 5' flanking region primer 1 TACACGACAGTAACTCCCTCCG L2 CNAG_02859 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGAAATAACACACACGCTGC R1 CNAG_02859 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGAAAGTGGCTGGGTGAAG R2 CNAG_02859 3' flanking region primer 2 AAAGAACTTGAGAAGACCCG SO CNAG_02859 diagnostic screening primer, pairing with B79 AGCAACGAGTCCACATACC P.O. CNAG_02859 Southern blot probe primer TACACACCTCCAGTTTGACCTCGC STM NAT#58 STM primer CGCAAAATCACTAGCCCTATAGCG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 66 CNAG_02866 L1 CNAG_02866 5' flanking region primer 1 GAAGATAGTCAATCCGCAAG L2 CNAG_02866 5' flanking region primer 2 TCACTGGCCGTCGTTTTACATCTACCACTATTCTCCTGGC R1 CNAG_02866 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCTGATTGTTCTTGACATTCCG R2 CNAG_02866 3' flanking region primer 2 AAGGAGGATGAAGGAAGGC SO CNAG_02866 diagnostic screening primer, pairing with B79 ACAGGAACCTCCGTAACAG P.O. CNAG_02866 Southern blot probe primer ATTGGTGAAGGTCTGGGCAGTTCG STM NAT#102 STM primer CCATAGCGATATCTACCCCAATCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 67 CNAG_02897 L1 CNAG_02897 5' flanking region primer 1 GATGTAGCGGATTGTTTGAC L2 CNAG_02897 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTCCTTCTGCCTGGTGTTTC R1 CNAG_02897 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGATTTGGTGTTTGCTAACGG R2 CNAG_02897 3' flanking region primer 2 CTCCATCCAGCAACTCTATG SO CNAG_02897 diagnostic screening primer, pairing with B79 AGGAAGCAACGCTGACTGTC P.O. CNAG_02897 Southern blot probe primer TGGTTGTAATGGCACCGTC STM NAT#122 STM primer ACAGCTCCAAACCTCGCTAAACAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 68 CNAG_02915 PKH202 L1 CNAG_02915 5' flanking region primer 1 TGGTGGAAATGGACTGTG L2 CNAG_02915 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCAGCCTCGGGTTTTTTTG R1 CNAG_02915 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAGCACGAAAAGCACGAAG R2 CNAG_02915 3' flanking region primer 2 TCCTTGGACAACTGGTAGC SO CNAG_02915 diagnostic screening primer, pairing with B79 AGGTGGGATTGCTCAAAC P.O. CNAG_02915 Southern blot probe primer TGAAGGCGTGCTCAAATG STM NAT#177 STM primer CACCAACTCCCCATCTCCAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 69 CNAG_02947 SCY1 L1 CNAG_02947 5' flanking region primer 1 CGTCACCAACAAGTCACAG L2 CNAG_02947 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGAGAAGAGGTTTGAGGCTG R1 CNAG_02947 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAACCTGTCTGGGAGAAGAGC R2 CNAG_02947 3' flanking region primer 2 TTCCAAGACTTCCCCCAAC SO CNAG_02947 diagnostic screening primer, pairing with B79 CCATTACCTTTATGTCCCCAC P.O. CNAG_02947 Southern blot probe primer TTGCCCATTCCTGTCTTAG STM NAT#150 STM primer ACATACACCCCCATCCCCCC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 70 CNAG_02962 L1 CNAG_02962 5' flanking region primer 1 CAAGGGCTTTCTTCTTTGG L2 CNAG_02962 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGTCGTGATAATGGCGTTTG R1 CNAG_02962 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCTAAAAGATTGACTCCGAGG R2 CNAG_02962 3' flanking region primer 2 GAATAGGTCGTGAATGGATGTC SO CNAG_02962 diagnostic screening primer, pairing with B79 CTGATAAAAGAGCAGAGAGGG P.O. CNAG_02962 Southern blot probe primer GGTGGCTATCAAAGTTGTTAGG STM NAT#242 STM primer GTAGCGATAGGGGTGTCGCTTTAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') > 71 CNAG_02976 L1 CNAG_02976 5' flanking region primer 1 GCAAAGTGAAGAAGGCGAG L2 CNAG_02976 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTGGTGACGGGTCCCTTCAAG R1 CNAG_02976 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAATCCTTGCTGGGGGAAGC R2 CNAG_02976 3' flanking region primer 2 CGATTCATCTCCATAACCAGTG SO CNAG_02976 diagnostic screening primer, pairing with B79 GGCATAATGAAACCAGGG P.O. CNAG_02976 Southern blot probe primer CGCAAAAACTCGTCATAGG STM NAT#169 STM primer ACATCTATATCACTATCCCGAACC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 72 CNAG_03024 RIM15 L1 CNAG_03024 5' flanking region primer 1 CTGAGTGCGATGATTGTTTG L2 CNAG_03024 5' flanking region primer 2 GCTCACTGGCCGTCGTTTTACTTTCCTGACTTTGGGTGC R1 CNAG_03024 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTGAGGACAGATTCTATGGC R2 CNAG_03024 3' flanking region primer 2 CAGAGAATAAGGTCCCCTCC SO CNAG_03024 diagnostic screening primer, pairing with B79 TCAAGGGATAGAAGTTCGC P.O. CNAG_03024 Southern blot probe primer GAGATAAACAGAGCCAAACG STM NAT#191 STM primer ATATGGATGTTTTTAGCGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 73 CNAG_03048 IRK3 L1 CNAG_03048 5' flanking region primer 1 GATTGAGTTTCGGTTGGG L2 CNAG_03048 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCTAAAAACGGAGCGGAAG R1 CNAG_03048 3' flanking region primer 1 ATGGTCATAGCTGTTTCCTGCGAACTTCTCAAGCAACG R2 CNAG_03048 3' flanking region primer 2 ATACAACCCCCATACTCCC SO CNAG_03048 diagnostic screening primer, pairing with B79 AAAGGGATTCGGGGCTTAC P.O. CNAG_03048 Southern blot probe primer CCAGGGGTTTGATGTCATAC STM NAT#273 STM primer GAGATCTTTCGGGAGGTCTGGATT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 74 CNAG_03137 L1 CNAG_03137 5' flanking region primer 1 CAAGGAGGTCAACCCTACAG L2 CNAG_03137 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAGGCGTCTTCTGTCCATAG R1 CNAG_03137 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAGTCGTCCTCTTTTTGTGC R2 CNAG_03137 3' flanking region primer 2 AGGACTTGTCGGTCTTCAG SO CNAG_03137 diagnostic screening primer, pairing with B79 GGTAAGTTGCTTTATCCCCC P.O. CNAG_03137 Southern blot probe primer GCTTGAGCAGTTGATACG STM NAT#211 STM primer GCGGTCGCTTTATAGCGATT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 75 CNAG_03167 CHK1 L1 CNAG_03167 5' flanking region primer 1 GTATTCCCATCCCACACATC L2 CNAG_03167 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTGACAGAGAGGGGCTTAC R1 CNAG_03167 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTACATTGGAGGGCGTTG R2 CNAG_03167 3' flanking region primer 2 CTGACAACAAGCAGCCTATC SO CNAG_03167 diagnostic screening primer, pairing with B79 ATACCACCACAAAACGCCTC P.O. CNAG_03167 Southern blot probe primer GGACTACTTTCCGAAGGTTC STM NAT#205 STM primer TATCCCCCTCTCCGCTCTCTAGCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 76 CNAG_03171 L1 CNAG_03171 5' flanking region primer 1 CGTCCAACCATCAATCAC L2 CNAG_03171 5' flanking region primer 2 TCACTGGCCGTCGTTTTACACCTTGGTAGGAGTGTGGAG R1 CNAG_03171 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTAGTTGCGATTCTGTGGG R2 CNAG_03171 3' flanking region primer 2 TAGGGACGAGTATCAGGAGCAG SO CNAG_03171 diagnostic screening primer, pairing with B79 TCCTCTGTTCTTGTCGTGG P.O. CNAG_03171 Southern blot probe primer TAAGCCTCGTAGAGCCAAG STM NAT#159 STM primer ACGCACCAGACACACAACCAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 77 CNAG_03184 BUB1 L1 CNAG_03184 5' flanking region primer 1 CAACGCCATTGAGGAAAG L2 CNAG_03184 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGCCTGATGTTCTCTTTCTGAG R1 CNAG_03184 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAGCGACTTTGAGGGATGGC R2 CNAG_03184 3' flanking region primer 2 ATCCCAGAACAGTGGCAGAC SO CNAG_03184 diagnostic screening primer, pairing with B79 GGAGGATACATCAGGTGAGC P.O. CNAG_03184 Southern blot probe primer AACAGCACTTTGGGGTAAC STM NAT#201 STM primer CACCCTCTATCTCGAGAAAGCTCC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 78 CNAG_03216 SNF101 L1 CNAG_03216 5' flanking region primer 1 GGAGATGAAGGGGAATGAGTC L2 CNAG_03216 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCGACGCAAGAGGATAACAAC R1 CNAG_03216 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGGCAGGGAGATGAGGGATAG R2 CNAG_03216 3' flanking region primer 2 CTGCTCTTGTTTAGCCACC SO CNAG_03216 diagnostic screening primer, pairing with B79 TCCGACTCTGATAACGACTG P.O. CNAG_03216 Southern blot probe primer AAAGCCTCCTCTTCCAACC STM NAT#146 STM primer ACTAGCCCCCCCTCACCACCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 79 CNAG_03258 TPK202A L1 CNAG_03258 5' flanking region primer 1 AGGGACTGAATCCAAAGGG L2 CNAG_03258 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTCTCGTCTTCGGCAAGGCAAGTG R1 CNAG_03258 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAGGACAAGGGCTAATGG R2 CNAG_03258 3' flanking region primer 2 AAGGCTGGACTTTGTTGGGGAC SO CNAG_03258 diagnostic screening primer, pairing with B79 GATTGCGAAGATGTGAACTC P.O. CNAG_03258 Southern blot probe primer TTTCCCTGTTGCCATCTC STM NAT#208 STM primer TGGTCGCGGGAGATCGTGGTTT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 80 CNAG_03290 KIC102 L1 CNAG_03290 5' flanking region primer 1 CGCTGACTTGGAGTATGTG L2 CNAG_03290 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAAGTCTGCGGAAAGGTTC R1 CNAG_03290 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTCACCTCTGCTTTTGTCTTG R2 CNAG_03290 3' flanking region primer 2 CCGACAAGGATGAAAACAAAGATGG SO CNAG_03290 diagnostic screening primer, pairing with B79 TGGATGTCTTAGAAGGGAGC P.O. CNAG_03290 Southern blot probe primer GGAAGACAAGAACAAACGG STM NAT#201 STM primer CACCCTCTATCTCGAGAAAGCTCC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 81 CNAG_03355 TCO4 L1 CNAG_03355 5' flanking region primer 1 AATGCCATAGGACACCTCTGACCC L2 CNAG_03355 5' flanking region primer 2 CTGGCCGTCGTTTTACTGTGACTATGGTAAGCACCG R1 CNAG_03355 3' flanking region primer 1 GTCATAGCTGTTTCCTGAATGCCATAGGACACCTCTGACCC R2 CNAG_03355 3' flanking region primer 2 TGTGACTATGGTAAGCACCG SO CNAG_03355 diagnostic screening primer, pairing with B79 GTTGCTTGGTTTTTCTTCGG PO1 CNAG_03355 Southern blot probe primer 1 AAACGCAGCATTGACTAC PO2 CNAG_03355 Southern blot probe primer 2 TATGTAAGCAGCCTGTTCG STM NAT#123 STM primer CTATCGACCAACCAACACAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 82 CNAG_03358 L1 CNAG_03358 5' flanking region primer 1 GCAGAATCGTGAAACATTACCC L2 CNAG_03358 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTCATTGAGGAGGTAGGGAGG R1 CNAG_03358 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGAAAGGTGTCGGGGATAG R2 CNAG_03358 3' flanking region primer 2 ACGGAGAAGCAGGAACATC SO CNAG_03358 diagnostic screening primer, pairing with B79 CAGACAATCGCAGAGTGAG P.O. CNAG_03358 Southern blot probe primer CTCTCGGAACTTCTTGACG STM NAT#230 STM primer ATGTAGGTAGGGTGATAGGT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 83 CNAG_03367 URK1 L1 CNAG_03367 5' flanking region primer 1 ACCCTTCTTTTTGGTCCC L2 CNAG_03367 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTGGTTTTTGCTCTGCGGC R1 CNAG_03367 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGTTTGCTGTTTGGATTCGC R2 CNAG_03367 3' flanking region primer 2 ATTTCCCCGCATTTGCCAC SO CNAG_03367 diagnostic screening primer, pairing with B79 TCGCACATTCTTGTCAGAG P.O. CNAG_03367 Southern blot probe primer GATGATGGAAAGAGTAGACCG STM NAT#43 STM primer CCAGCTACCAATCACGCTAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 84 CNAG_03369 SWE102 L1 CNAG_03369 5' flanking region primer 1 TGCTACGCTAAGACTGGACTAC L2 CNAG_03369 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGGAGGCTGGTTGAAAGAAC R1 CNAG_03369 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGACGAACTTGTGCTCTCTGC R2 CNAG_03369 3' flanking region primer 2 ACAGTTTCCTGACGAGAATG SO CNAG_03369 diagnostic screening primer, pairing with B79 GCCGATACATTTTGGGTAG P.O. CNAG_03369 Southern blot probe primer TGGATGGTGAGGAGTTGAG STM NAT#169 STM primer ACATCTATATCACTATCCCGAACC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 85 CNAG_03567 CBK1 L1 CNAG_03567 5' flanking region primer 1 CAACCGATTTGCCAAGAG L2 CNAG_03567 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTGTTGTCCCTGGATTGG R1 CNAG_03567 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTAAGGAGTGCGATGGATG R2 CNAG_03567 3' flanking region primer 2 CGTTTTTCATCCTGCGAG SO CNAG_03567 diagnostic screening primer, pairing with B79 TCATTCCCACCATTCACG P.O. CNAG_03567 Southern blot probe primer TCTGACTTCACCGAATGC STM NAT#232 STM primer CTTTAAAGGTGGTTTTGTG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 86 CNAG_03592 THI20 L1 CNAG_03592 5' flanking region primer 1 TTGTGAGCAGGTTTCCGTG L2 CNAG_03592 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTACCTGAATACCAGCACCACCG R1 CNAG_03592 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAGATAGTGGCAGGACCTTGC R2 CNAG_03592 3' flanking region primer 2 TTACATCGCCGCTGTTTTCC SO CNAG_03592 diagnostic screening primer, pairing with B79 TGTCTCTGGTGTCTGGTTG P.O. CNAG_03592 Southern blot probe primer GAAAGCAGTAGCGATAGCAG STM NAT#231 STM primer GAGAGATCCCAACATCACGC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 87 CNAG_03670 IRE1 L1 CNAG_03670 5' flanking region primer 1 GCCCCATCATCATAATCAC L2 CNAG_03670 5' flanking region primer 2 GCTCACTGGCCGTCGTTTTACACTATGTGTCCATCTGAGGC R1 CNAG_03670 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAGTGAGTTGAGGGGAGGAAAG R2 CNAG_03670 3' flanking region primer 2 GAAGAAGAGCGTCAAGAAGG SO CNAG_03670 diagnostic screening primer, pairing with B79 AGGAATACGAGGGTTTATCGG P.O. CNAG_03670 Southern blot probe primer AGCATTAGGGGTGTAGGTG STM NAT#224 STM primer AACCTTTAAATGGGTAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 88 CNAG_03701 L1 CNAG_03701 5' flanking region primer 1 AGCGTATTCTTCAGGGGCTC L2 CNAG_03701 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAAGAAGGGAGAGTGGTTGTGACGG R1 CNAG_03701 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGAAGTGTTTTCCCGTCCC R2 CNAG_03701 3' flanking region primer 2 TAAAGGAGTGTTGGACCCC SO CNAG_03701 diagnostic screening primer, pairing with B79 ACAAACCTCACTGTGCCTC P.O. CNAG_03701 Southern blot probe primer CAATACCGACTGAGACACACTC STM NAT#125 STM primer CGCTACAGCCAGCGCGCGCAAGCG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 89 CNAG_03791 L1 CNAG_03791 5' flanking region primer 1 GAAGCATCCTCAAAAGGG L2 CNAG_03791 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGGCTGGAGATTTGAAAGAG R1 CNAG_03791 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCTTTTGGAAGTAAACGGGG R2 CNAG_03791 3' flanking region primer 2 GCAACTCGTCAAAGACCTG SO CNAG_03791 diagnostic screening primer, pairing with B79 CGACTTCTTCAGCAATGG P.O. CNAG_03791 Southern blot probe primer TATTCCAGTCCGAGTAGCG STM NAT#210 STM primer CTAGAGCCCGCCACAACGCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 90 CNAG_03796 L1 CNAG_03796 5' flanking region primer 1 AGGTCGGAAGATTTTGCG L2 CNAG_03796 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTAGGGTCGTTTTGTGTTATCC R1 CNAG_03796 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCTTTTGGCTTTGGGTCAG R2 CNAG_03796 3' flanking region primer 2 TGAGCAGTAGTGTATTGGGTG SO CNAG_03796 diagnostic screening primer, pairing with B79 AATCTCCTCTTGGGCTCAG P.O. CNAG_03796 Southern blot probe primer ATACCACAGCACCCACAAG STM NAT#240 STM primer GGTGTTGGATCGGGGTGGAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 91 CNAG_03811 IRK5 L1 CNAG_03811 5' flanking region primer 1 TCTTTAGCGTTTGACCCTG L2 CNAG_03811 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTCCAACACTCCGTAGCAG R1 CNAG_03811 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCTGATGGAAGATGTTGAAGC R2 CNAG_03811 3' flanking region primer 2 GTCGCATCTTTTTTGCTGG SO CNAG_03811 diagnostic screening primer, pairing with B79 TCACAATCATTCTGACCAGG P.O. CNAG_03811 Southern blot probe primer CCGCAAAGGTAAAGTTCG STM NAT#213 STM primer CTGGGGATTTTGATGTGTCTATGT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 92 CNAG_03821 L1 CNAG_03821 5' flanking region primer 1 GGGTCATTTTCACCGAATC L2 CNAG_03821 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCTTTGTGTGCCGTTCTAAAC R1 CNAG_03821 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCCAGATGGTCATTTCTTC R2 CNAG_03821 3' flanking region primer 2 GGAAATAGAAACAGCGGTG SO CNAG_03821 diagnostic screening primer, pairing with B79 ACCAGGTCTTCCTCCATTG P.O. CNAG_03821 Southern blot probe primer TGAGAGATTCTTGTTCCGAG STM NAT#177 STM primer CACCAACTCCCCATCTCCAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 93 CNAG_03843 ARK1 L1 CNAG_03843 5' flanking region primer 1 CAATAGGCGTGAACAAGC L2 CNAG_03843 5' flanking region primer 2 TCACTGGCCGTTCGTTTTACGGGATACTGGTGTTTTTTGG R1 CNAG_03843 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAGGTCAAACAATGCGTCAG R2 CNAG_03843 3' flanking region primer 2 GAAAGGAAGGAGCGAAAG SO CNAG_03843 diagnostic screening primer, pairing with B79 ATAGAGCGGGAGGAAATG P.O. CNAG_03843 Southern blot probe primer TGGGTGGGAGTGATTTCTG STM NAT#43 STM primer CCAGCTACCAATCACGCTAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 94 CNAG_03946 GAL302 L1 CNAG_03946 5' flanking region primer 1 AAAACTCACATCCGCTGC L2 CNAG_03946 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGCAGAGAGTTGAAGACGGTG R1 CNAG_03946 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCTGGAGGTGAGTTCTGTAATC R2 CNAG_03946 3' flanking region primer 2 CCCTATTCCTTTCTCTTGTTC SO CNAG_03946 diagnostic screening primer, pairing with B79 AGACCAATGTAGACCCTATGTG P.O. CNAG_03946 Southern blot probe primer ACAAGCACATCCATTCCTAC STM NAT#218 STM primer CTCCACATCCATCGCTCCAA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 95 CNAG_04040 FPK1 L1 CNAG_04040 5' flanking region primer 1 ATCGTCTCAGCTCAACAG L2 CNAG_04040 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTCTTCCACTTTGACGGTG R1 CNAG_04040 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTCCGTTTGGGGAGTTTAG R2 CNAG_04040 3' flanking region primer 2 GGCTATTCTTCTTGGCTTGC SO CNAG_04040 diagnostic screening primer, pairing with B79 CCTTTGGGTTTTTGGGAC P.O. CNAG_04040 Southern blot probe primer ATTAGTCTGCCCAAACGG STM NAT#211 STM primer GCGGTCGCTTTATAGCGATT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG OEL2 CNAG_04040 5' flaking region for overexpression construction CACTCGAATCCTGCATGCGGGATGTTTGTGTGACTGAG OER1 CNAG_04040 5' coding region for overexpression construction CCACAACACATCTATCACATGTCGTCTCTCGCGTCACC NP1 CNAG_04040 Northern blot probe primer TTCAAACTCGGGAGGACAG 96 CNAG_04083 L1 CNAG_04083 5' flanking region primer 1 TTCCTCCATCTTCGCATC L2 CNAG_04083 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTCGTGCCCTTTTTGGTAG R1 CNAG_04083 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAAGAAAGAACACCCCTCC R2 CNAG_04083 3' flanking region primer 2 AACAGGTTGCGATTGTGC SO CNAG_04083 diagnostic screening primer, pairing with B79 GCCGTTATGGGTGAAAGAG P.O. CNAG_04083 Southern blot probe primer GAAAGGGAGAAGAGTGAAGG STM NAT#210 STM primer CTAGAGCCCGCCACAACGCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 97 CNAG_04108 PKP2 L1 CNAG_04108 5' flanking region primer 1 AAAAGAGGAGGGAGAAGGG L2 CNAG_04108 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGAAGTATCCACACACCCC R1 CNAG_04108 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCGTCTTTGAGTTAGGTGCTG R2 CNAG_04108 3' flanking region primer 2 TGATTGGGGAAGCGTTAG SO CNAG_04108 diagnostic screening primer, pairing with B79 TGTCGGTTTTGTGGTTCC P.O. CNAG_04108 Southern blot probe primer TTAGCCTCTTGCCAACTCC STM NAT#295 STM primer ACACCTACATCAAACCCTCCC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 98 CNAG_04118 L1 CNAG_04118 5' flanking region primer 1 TCAGGCGAGATGATAGGTCG L2 CNAG_04118 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCCGCTATCTCTATCTCTGTCC R1 CNAG_04118 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGACAAGATAAAGATTGGCGG R2 CNAG_04118 3' flanking region primer 2 CGCCATCTCCTTTCTATCG SO CNAG_04118 diagnostic screening primer, pairing with B79 CAAAAGAGAATCCTGGAGACC P.O. CNAG_04118 Southern blot probe primer GGAGAATGAGTCAAATGCTG STM NAT#212 STM primer AGAGCGATCGCGTTATAGAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 99 CNAG_04148 L1 CNAG_04148 5' flanking region primer 1 GAAGCCCTTGGTATTTTCC L2 CNAG_04148 5' flanking region primer 2 TCACTGGCCGTTCGTTTTACCCTCGTAGCCCAAGAAATG R1 CNAG_04148 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTCGTATTGGGTGAATGGC R2 CNAG_04148 3' flanking region primer 2 TGCTGATACCCTGTTTCG SO CNAG_04148 diagnostic screening primer, pairing with B79 CGATGATAGGTCCGAAATC P.O. CNAG_04148 Southern blot probe primer AGACCAAACATCCCAAGC STM NAT#224 STM primer AACCTTTAAATGGGTAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 100 CNAG_04156 L1 CNAG_04156 5' flanking region primer 1 TTCTCCTCCTTCTTTATGCC L2 CNAG_04156 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAGACAAGAGGGTTTACCTGC R1 CNAG_04156 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGATTACTGAGGCTGCGTTCC R2 CNAG_04156 3' flanking region primer 2 GCGGATAGAAGCACTGAAAC SO CNAG_04156 diagnostic screening primer, pairing with B79 GTCCATCGGTAACAAGTCC P.O. CNAG_04156 Southern blot probe primer GTGGTAAGCACGGCTAATC STM NAT#177 STM primer CACCAACTCCCCATCTCCAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 101 CNAG_04162 PKA2 L1 CNAG_04162 5' flanking region primer 1 AATAACACACCAGCCGCTCTGACC L2 CNAG_04162 5' flanking region primer 2 CTGGCCGTCGTTTTACTGATGTGATGGATGTGC R1 CNAG_04162 3' flanking region primer 1 GTCATAGCTGTTTCCTGCGGCAGTAGAGATAGCACAG R2 CNAG_04162 3' flanking region primer 2 GGAGGTGGTGGAGAATGTTC SO CNAG_04162 diagnostic screening primer, pairing with B79 TACCTGCTGCTATGACCCTACG P.O. CNAG_04162 Southern blot probe primer CCACTTGCTTCCAACCTCAC STM NAT#205 STM primer TATCCCCCTCTCCGCTCTCTAGCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 102 CNAG_04191 L1 CNAG_04191 5' flanking region primer 1 CAAGTGGTGTCGCATTTC L2 CNAG_04191 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCGCAACCTGTTTAGTCAGAC R1 CNAG_04191 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCAAAAGAAGAGCAAGGC R2 CNAG_04191 3' flanking region primer 2 GGGCTAAGAAGTTTGATGTTCC SO CNAG_04191 diagnostic screening primer, pairing with B79 ATGAGGGTTTTCAGCACC P.O. CNAG_04191 Southern blot probe primer GGGAAGGAGTGACAAAGATAG STM NAT#159 STM primer ACGCACCAGACACACAACCAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 103 CNAG_04197 YAK1 L1 CNAG_04197 5' flanking region primer 1 GTGTGTCATTGGGTTTTGC L2 CNAG_04197 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAATGAATCTGCGGGAGTC R1 CNAG_04197 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAGAAGTTGACTCGGCATCG R2 CNAG_04197 3' flanking region primer 2 GCTTCGTCATCAAACAGTTC SO CNAG_04197 diagnostic screening primer, pairing with GGTGATTTTTCATCGCCC P.O. CNAG_04197 Southern blot probe primer CAGCAGATGGCTCCTCTATC STM NAT#184 STM primer ATATATGGCTCGAGCTAGATAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 104 CNAG_04215 MET3 L1 CNAG_04215 5' flanking region primer 1 CTCAAAATGAAAGCAGCAG L2 CNAG_04215 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGAGAAGAGAATCGTGAAGAGC R1 CNAG_04215 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCTTGTAGCGTTGTAGATGG R2 CNAG_04215 3' flanking region primer 2 GCGTTGTTTATTCACAGGAG SO CNAG_04215 diagnostic screening primer, pairing with B79 CTGTTCTTTGTGTCTTTGCG P.O. CNAG_04215 Southern blot probe primer TCTTTCGGATAACGGCGTG STM NAT#205 STM primer TATCCCCCTCTCCGCTCTCTAGCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 105 CNAG_04221 FBP26 L1 CNAG_04221 5' flanking region primer 1 TGGAGGTCAGTAATCGGTCG L2 CNAG_04221 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGGATTGGATGGATGTGAAC R1 CNAG_04221 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTCCGATGTATGCTCTGGTC R2 CNAG_04221 3' flanking region primer 2 TGTTTCTCCCCTTGTCACC SO CNAG_04221 diagnostic screening primer, pairing with B79 TGGAAATGAGTTCTCTTGGG P.O. CNAG_04221 Southern blot probe primer TCCTAAAATCCCGCTCTGC STM NAT#146 STM primer ACTAGCCCCCCCTCACCACCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 106 CNAG_04230 THI6 L1 CNAG_04230 5' flanking region primer 1 TCATCACCAGTAACGAAAGG L2 CNAG_04230 5' flanking region primer 2 TCACTGGCCGTTCGTTTTACAGGCTCAACAAAACCGAG R1 CNAG_04230 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAGACTCGGACCCATTCAG R2 CNAG_04230 3' flanking region primer 2 TGGTGAGGTCTTTGCGAAG SO CNAG_04230 diagnostic screening primer, pairing with B79 TGACCCGAGGTAGAGAATC P.O. CNAG_04230 Southern blot probe primer ATCAAGAATTCTCGCCCAC STM NAT#290 STM primer ACCGACAGCTCGAACAAGCAAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 107 CNAG_04272 L1 CNAG_04272 5' flanking region primer 1 GCCTGAAAAGAAGGAAACC L2 CNAG_04272 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCCTTCCTAATGTCTTTCCAGTC R1 CNAG_04272 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAGGAAGTGGAAGGCGTTC R2 CNAG_04272 3' flanking region primer 2 TCGTCTTCGCCAAACTCTGC SO CNAG_04272 diagnostic screening primer, pairing with B79 GAACGCCGAAACAAAACC P.O. CNAG_04272 Southern blot probe primer CTGGGAGGAAAATCAGC STM NAT#212 STM primer AGAGCGATCGCGTTATAGAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 108 CNAG_04282 MPK2 L1 CNAG_04282 5' flanking region primer 1 ATGGCAGCAAGCGTAACTC L2 CNAG_04282 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGTTTTATGCCCGTTGTGTTG R1 CNAG_04282 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCCCAAAGTCAGTCTGGTAACC R2 CNAG_04282 3' flanking region primer 2 ATACATCTTCGTAGCCCCG SO CNAG_04282 diagnostic screening primer, pairing with B79 TCCAAATAGACCAAGCCC P.O. CNAG_04282 Southern blot probe primer CGTTGAGTGTTTGGTAGCC STM NAT#102 STM primer CCATAGCGATATCTACCCCAATCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 109 CNAG_04314 L1 CNAG_04314 5' flanking region primer 1 CCATTCGTAGCCCTTATCTG L2 CNAG_04314 5' flanking region primer 2 TCACTGGCCGTCGTTTTACACGGAGTCTGGTTTTCAGG R1 CNAG_04314 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTTGATGGAAGGAGTCGC R2 CNAG_04314 3' flanking region primer 2 AAGAGGGCATCACTAAGGC SO CNAG_04314 diagnostic screening primer, pairing with B79 ATTGGACTGGACCATAGCC P.O. CNAG_04314 Southern blot probe primer GATAAAAGACAGAACTCAGCACC STM NAT#231 STM primer GAGAGATCCCAACATCACGC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 110 CNAG_04316 UTR1 L1 CNAG_04316 5' flanking region primer 1 GGTGATTGCCTTGTTGTTG L2 CNAG_04316 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAGACGAAGGAGGAGGAGTAG R1 CNAG_04316 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCAGTGGTTCAGAGGAATAAG R2 CNAG_04316 3' flanking region primer 2 ACTTGCCCATACTGGAGGTC SO CNAG_04316 diagnostic screening primer, pairing with B79 CAGGATGTAGTGGAGACTGC P.O. CNAG_04316 Southern blot probe primer CCAGTAACCCATCACCTATTAG STM NAT#5 STM primer TGCTAGAGGGCGGGAGAGTT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 111 CNAG_04335 L1 CNAG_04335 5' flanking region primer 1 CGATAGAGTAGTAGTTTTAGGGGG L2 CNAG_04335 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCTTACGAGTCCATCTTCGC R1 CNAG_04335 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAACCGATTCCAGTTACAGC R2 CNAG_04335 3' flanking region primer 2 AGATGGACGAGGTGGTGATG SO CNAG_04335 diagnostic screening primer, pairing with B79 TGATGTGCTACTGGAAGCC P.O. CNAG_04335 Southern blot probe primer TCATCAATGTCAGGCTGGG STM NAT#146 STM primer ACTAGCCCCCCCTCACCACCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 112 CNAG_04347 L1 CNAG_04347 5' flanking region primer 1 GAGTTTGAGCGGTCATTG L2 CNAG_04347 5' flanking region primer 2 TCACTGGCCGTTCGTTTTACAGGTCCTCAAGGTATGGAGC R1 CNAG_04347 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCCCTCAATGTTATCCACG R2 CNAG_04347 3' flanking region primer 2 GTAGCGAGAGCGATTCATC SO CNAG_04347 diagnostic screening primer, pairing with B79 TCCAGGGAACAGTGAGTAAC P.O. CNAG_04347 Southern blot probe primer TTCAATGATGCCCGAGCAG STM NAT#210 STM primer CTAGAGCCCGCCACAACGCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 113 CNAG_04408 CKI1 L1 CNAG_04408 5' flanking region primer 1 CGTCATTTCTGGGATAGACTG L2 CNAG_04408 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTCCTTCTATGCCTGGGGTAGC R1 CNAG_04408 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAACGCAAGGATGTCCCAGCAG R2 CNAG_04408 3' flanking region primer 2 TGCTTGTAGGCAAATGGCTGG SO CNAG_04408 diagnostic screening primer, pairing with B79 GATTTCATCCGCCTGTTG P.O. CNAG_04408 Southern blot probe primer ATCTTCCGCTGCTTCAGAC STM NAT#218 STM primer CTCCACATCCATCGCTCCAA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 114 CNAG_04433 YAK103 L1 CNAG_04433 5' flanking region primer 1 AGCCTGTGAGTTGTGCGTTG L2 CNAG_04433 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGGTTTTCCTGCTATCACGC R1 CNAG_04433 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGACCTCAAAACTCAGCATTG R2 CNAG_04433 3' flanking region primer 2 AAGAAACCTCTCCATTCCC SO CNAG_04433 diagnostic screening primer, pairing with B79 AATACCTTGTTGGCGAGAC P.O. CNAG_04433 Southern blot probe primer CATCAGGAGGGTTTACCACC STM NAT#231 STM primer GAGAGATCCCAACATCACGC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 115 CNAG_04514 MPK1 L1 CNAG_04514 5' flanking region primer 1 TTTGCTTGCTCCTCTTCTC L2 CNAG_04514 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGAGAAGTAGAGGCAGTGACG R1 CNAG_04514 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTGGAGAAACAGTTGGAGAG R2 CNAG_04514 3' flanking region primer 2 TTCAGCAGGTCAATCAGG SO CNAG_04514 diagnostic screening primer, pairing with B79 CGACTCACGATGTAACTTCC P.O. CNAG_04514 Southern blot probe primer ACCTCAACTCTCTCAGACACC STM NAT#240 STM primer GGTGTTGGATCGGGGTGGAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 116 CNAG_04577 L1 CNAG_04577 5' flanking region primer 1 AGGTTTGAGCCATCTGAAC L2 CNAG_04577 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAAAGGGCATAACCAGTGAC R1 CNAG_04577 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGTTGGAGTATGGGAGATGC R2 CNAG_04577 3' flanking region primer 2 GTCTTTTCTTTCCCACTTGG SO CNAG_04577 diagnostic screening primer, pairing with B79 GAGATGGGTAATGGTGATGAG P.O. CNAG_04577 Southern blot probe primer GCTTGTAACCAGCGCTCTATC STM NAT#282 STM primer TCTCTATAGCAAAACCAATC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 117 CNAG_04631 RIK1 L1 CNAG_04631 5' flanking region primer 1 TCATCAGTTTCGTCCAGC L2 CNAG_04631 5' flanking region primer 2 TCACTGGCCGTCGTTTTACATAACGGGATTGGGGTTG R1 CNAG_04631 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTGCTGATGAGGTCAAGG R2 CNAG_04631 3' flanking region primer 2 ATCTCACTGCCCTATTCCC SO CNAG_04631 diagnostic screening primer, pairing with B79 TTCCACTCCTTTCTCCCTCTG P.O. CNAG_04631 Southern blot probe primer CAGGAAGGCTAAACCACAG STM NAT#150 STM primer ACATACACCCCCATCCCCCC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 118 CNAG_04678 YPK1 L1 CNAG_04678 5' flanking region primer 1 CGACTATGGGTTCGTTACTGG L2 CNAG_04678 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGTCTATGCGTTTTCCGAC R1 CNAG_04678 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGGTGTAGAATGGCAGAGC R2 CNAG_04678 3' flanking region primer 2 GCACCGTGGAGGTAGTAATG SO CNAG_04678 diagnostic screening primer, pairing with B79 TACCCATCATTCCCTGCTC P.O. CNAG_04678 Southern blot probe primer ACACCGTATCAGCACAAGC STM NAT#58 STM primer CGCAAAATCACTAGCCCTATAGCG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 119 CNAG_04755 BCK1 L1 CNAG_04755 5' flanking region primer 1 GCTGTTGGTTCTCTCTTGC L2 CNAG_04755 5' flanking region primer 2 CTGGCCGTCGTTTTACGGTTTGCGATGAATAGTCC R1 CNAG_04755 3' flanking region primer 1 GTCATAGCTGTTTCCTGTTCCGAACGCTCATACTCC R2 CNAG_04755 3' flanking region primer 2 TTCCTTCGTTTGTCCGTCG SO CNAG_04755 diagnostic screening primer, pairing with B79 CAGGCTTTTTTTCTGGCTAC PO1 CNAG_04755 Southern blot probe primer 1 TACCTCCTTCATTCCTGCCGTC PO2 CNAG_04755 Southern blot probe primer 2 GCTTCGTTATCAGTCGTCAC STM NAT#43 STM primer CCAGCTACCAATCACGCTAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 120 CNAG_04821 PAN3 L1 CNAG_04821 5' flanking region primer 1 CTCTTACAGACGGTTCTTTAGG L2 CNAG_04821 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTCTCCTTTGCCTTTCTCCGAG R1 CNAG_04821 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAGAATGCGGGCAATAACC R2 CNAG_04821 3' flanking region primer 2 GCCAAAAAGCAAAAAGTGGAGC SO CNAG_04821 diagnostic screening primer, pairing with B79 GCAGGAAGAACAAGGTGTC P.O. CNAG_04821 Southern blot probe primer GGAACGAGAGAGTGATACACG STM NAT#204 STM primer GATCTCTCGCGCTTGGGGGA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 121 CNAG_04843 L1 CNAG_04843 5' flanking region primer 1 CAATCAAACAAGCGACCTC L2 CNAG_04843 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGAAGATTTCTCAACAAGCGG R1 CNAG_04843 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGACAGCATAGAGAGGGTGTGTG R2 CNAG_04843 3' flanking region primer 2 TCCTCCACCATTTCAGACG SO CNAG_04843 diagnostic screening primer, pairing with B79 GGGGAGCAAACTCTTGAAC P.O. CNAG_04843 Southern blot probe primer CATCTCATCCGTTTCCTGC STM NAT#116 STM primer GCACCCAAGAGCTCCATCTC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 122 CNAG_04927 YFH702 L1 CNAG_04927 5' flanking region primer 1 GGCATAACTTTCAACGGC L2 CNAG_04927 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAGTCTCCACGACATCTTCTG R1 CNAG_04927 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTATGCCAGTGGTCAGGTTC R2 CNAG_04927 3' flanking region primer 2 TCGTATTTGACTTCCCTGG SO CNAG_04927 diagnostic screening primer, pairing with B79 TGTTTTGAGAGTCCTTCGG P.O. CNAG_04927 Southern blot probe primer TGTCTTTGTGCGTTATGGG STM NAT#220 STM primer CAGATCTCGAACGATACCCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 123 CNAG_05005 ATG1 L1 CNAG_05005 5' flanking region primer 1 CGCAGAACAGTCCTACACAAC L2 CNAG_05005 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCTCCTTGCGAGTTTGAGTC R1 CNAG_05005 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCCCTGAGAAAAAAGTTGGC R2 CNAG_05005 3' flanking region primer 2 CGGGAGGAAAACTTGTTC SO CNAG_05005 diagnostic screening primer, pairing with B79 GATTCACACAAGAGAGCGG P.O. CNAG_05005 Southern blot probe primer TTCCCCTCCTCATTTGTC STM NAT#288 STM primer CTATCCAACTAGACCTCTAGCTAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 124 CNAG_05063 SSK2 L1 CNAG_05063 5' flanking region primer 1 CCTATCTTATTTTTTGCGGGG L2 CNAG_05063 5' flanking region primer 2 CTGGCCGTCGTTTTACTCCTCTTTGTGCCGTATTC R1 CNAG_05063 5' flanking region primer 2 GTCATAGCTGTTTCCTGATGTTGGAGCAGATGGTG R2 CNAG_05063 3' flanking region primer 2 CGACTCGTCCAACCAAGTTAC SO CNAG_5063 diagnostic screening primer, pairing with B79 CTAAGGATAGGATGTGGAAGG PO1 CNAG_05063 Southern blot probe primer 1 AAGGACGACGAGAGTGAGTAG PO2 CNAG_05063 Southern blot probe primer 2 TCCAAACGAACCTTGACAG STM NAT#210 STM primer CTAGAGCCCGCCACAACGCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 125 CNAG_05097 CKY1 L1 CNAG_05097 5' flanking region primer 1 TGTTCTTCCTTGATGCTCTC L2 CNAG_05097 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGCAGATACGGAGAAGTCAGAC R1 CNAG_05097 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAGAACATCCCTGTCCTTGC R2 CNAG_05097 3' flanking region primer 2 ATTATGGGAGAGGGCGATG SO CNAG_05097 diagnostic screening primer, pairing with B79 ATCTTTGTCGGTGTCAGCC P.O. CNAG_05097 Southern blot probe primer AGTCCATCACTACCTTCGG STM NAT#282 STM primer TCTCTATAGCAAAACCAATC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 126 CNAG_05104 L1 CNAG_05104 5' flanking region primer 1 GCTTTTTGACGAGACAACTG L2 CNAG_05104 5' flanking region primer 2 TCACTGGCCGTTCGTTTTACGATAAAACCCGAGGACATTC R1 CNAG_05104 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCGTTGCTTCCGTATCTGTTG R2 CNAG_05104 3' flanking region primer 2 AGCAAGTGAAAAGAAGGGC SO CNAG_05104 diagnostic screening primer, pairing with B79 TATCAGGGCTTGGGTGTAG P.O. CNAG_05104 Southern blot probe primer TCTGATAGGGAGCCATACG STM NAT#208 STM primer TGGTCGCGGGAGATCGTGGTTT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 127 CNAG_05125 L1 CNAG_05125 5' flanking region primer 1 TGGTTTGGCTGCTTCTG L2 CNAG_05125 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGTGAGCAGGTGTTAGAGTGC R1 CNAG_05125 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGAGGACAGTTTATTGGGGG R2 CNAG_05125 3' flanking region primer 2 CACCCAGTAAATACCATCCTG SO CNAG_05125 diagnostic screening primer, pairing with B79 AGGTTCAAGCGTGATGTG P.O. CNAG_05125 Southern blot probe primer CGCTGACAACACAGATAAGAG STM NAT#219 STM primer CCCTAAACCCTACAGCAAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 128 CNAG_05200 L1 CNAG_05200 5' flanking region primer 1 TCCGACAACGAGATTGAAC L2 CNAG_05200 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTCTCCATCTTGACACATTCC R1 CNAG_05200 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGTTTACACCTTACCTCCCAC R2 CNAG_05200 3' flanking region primer 2 GGAATGGGCAAATGCTAC SO CNAG_05200 diagnostic screening primer, pairing with B79 TATCCCCACCAAGAAGTCC P.O. CNAG_05200 Southern blot probe primer ACAGACCCGTTCCAATGTC STM NAT#224 STM primer AACCTTTAAATGGGTAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 129 CNAG_05216 RAD53 L1 CNAG_05216 5' flanking region primer 1 CCTTGCTGACACTTTACC L2 CNAG_05216 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCTGTGTGTTTTGGGTTTGG R1 CNAG_05216 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTCCATTATGAAGGAGTCGG R2 CNAG_05216 3' flanking region primer 2 GTAGACCCTCTTCTTCCTCG SO CNAG_05216 diagnostic screening primer, pairing with B79 TAGGAGCGATTGCTGAAG P.O. CNAG_05216 Southern blot probe primer ACCAATCAATCAGCCGAC STM NAT#184 STM primer ATATATGGCTCGAGCTAGATAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 130 CNAG_05220 TLK1 L1 CNAG_05220 5' flanking region primer 1 ATCGCTTCTCGTTTGACC L2 CNAG_05220 5' flanking region primer 2 TCACTGGCCGTCGTTTTACATCAACGACCATCTGGGAC R1 CNAG_05220 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGGCTACTGCTGTGTATTGC R2 CNAG_05220 3' flanking region primer 2 GCGGTAAAGGTGGAAAGTC SO CNAG_05220 diagnostic screening primer, pairing with B79 CTTTGAAACCGACCATAGG P.O. CNAG_05220 Southern blot probe primer GGACCGAGACACTACTCACAAC STM NAT#116 STM primer GCACCCAAGAGCTCCATCTC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 131 CNAG_05243 XKS1 L1 CNAG_05243 5' flanking region primer 1 GCACGAATAAAATGCTGC L2 CNAG_05243 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCTGAGCAAAGGACTTACCTG R1 CNAG_05243 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCGGATTGGAATGCCTGTAG R2 CNAG_05243 3' flanking region primer 2 GGAGAGGTGTTGGAATACGGTAG SO CNAG_05243 diagnostic screening primer, pairing with B79 AGCCGAAGCCATTTTGAG P.O. CNAG_05243 Southern blot probe primer CATCATCACCAGCGATTG STM NAT#125 STM primer CGCTACAGCCAGCGCGCGCAAGCG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 132 CNAG_05274 L1 CNAG_05274 5' flanking region primer 1 ATGCTGTTTTGTGGGGGTAGGC L2 CNAG_05274 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGCTTCTCCGTTTGTTTCG R1 CNAG_05274 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTATCACAGGGCTTGACGGACTGAG R2 CNAG_05274 3' flanking region primer 2 CACTTTTCTTTCTGTCTCTCCC SO CNAG_05274 diagnostic screening primer, pairing with B79 CAACAACGCCAAGAAAGC P.O. CNAG_05274 Southern blot probe primer TTGGCGGAACGGATGAATCG STM NAT#58 STM primer CGCAAAATCACTAGCCCTATAGCG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 133 CNAG_05386 L1 CNAG_05386 5' flanking region primer 1 TTGCGGAATAAGAAGGGG L2 CNAG_05386 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGTGCTTTATGTGGATTTGGG R1 CNAG_05386 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCCAATCCAAATGAGTGACG R2 CNAG_05386 3' flanking region primer 2 ACAGGAAGACAGCAGGAG SO CNAG_05386 diagnostic screening primer, pairing with B79 GCTATGGGATTTTTCCG P.O. CNAG_05386 Southern blot probe primer GCAAATGGGCGTTATTCC STM NAT#177 STM primer CACCAACTCCCCATCTCCAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 134 CNAG_05439 CMK1 L1 CNAG_05439 5' flanking region primer 1 GGATTGTTAGGTAGGTAGGGG L2 CNAG_05439 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAAGAAGGCGGCTGGATAAG R1 CNAG_05439 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGAAGCCCACAATCAAAGTC R2 CNAG_05439 3' flanking region primer 2 GTGTCATCGTAGGGGTTTC SO CNAG_05439 diagnostic screening primer, pairing with B79 ATTGCCTATCTGCCTGTGC P.O. CNAG_05439 Southern blot probe primer TCAATGAAACGGGTGTG STM NAT#227 STM primer TCGTGGTTTAGAGGGAGCGC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 135 CNAG_05484 L1 CNAG_05484 5' flanking region primer 1 CCAACACCGCCTATTTATC L2 CNAG_05484 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGTGAGTGCCGAGAAAAAATG R1 CNAG_05484 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCTGTGTTGTATGGGACGAG R2 CNAG_05484 3' flanking region primer 2 TCTCACTCATTCTCAAAACGC SO CNAG_05484 diagnostic screening primer, pairing with B79 TGCTGTTTTAGCCCTTGC P.O. CNAG_05484 Southern blot probe primer AGAGATTGGTGATGGAGCC STM NAT#205 STM primer TATCCCCCTCTCCGCTCTCTAGCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 136 CNAG_05549 L1 CNAG_05549 5' flanking region primer 1 GGAAGCAGAGGAAGTCTTTAG L2 CNAG_05549 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAGGGTTTTTCCAGACAGC R1 CNAG_05549 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAGAGACCTCCTTCCGACAG R2 CNAG_05549 3' flanking region primer 2 GATTCGTCCACAACAAAGAC SO CNAG_05549 diagnostic screening primer, pairing with B79 GACGGCATCAAGGAAAATG P.O. CNAG_05549 Southern blot probe primer GAGGTGGTGATGTAGAAATAGG STM NAT#230 STM primer ATGTAGGTAGGGTGATAGGT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 137 CNAG_05558 KIN4 L1 CNAG_05558 5' flanking region primer 1 ATTCAATGGAGCGGGGAGTG L2 CNAG_05558 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCGAATAAGAATGATGGTGAGCG R1 CNAG_05558 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGATTGAGTAAGTTCCGCCCC R2 CNAG_05558 3' flanking region primer 2 AAGGCTGAGGACTGCTACTAC SO CNAG_05558 diagnostic screening primer, pairing with B79 ATTCTGGTATGAAGCCTCGCAGCC P.O. CNAG_05558 Southern blot probe primer TTCCAACTTCAGGTCACG STM NAT#225 STM primer CCATAGAACTAGCTAAAGCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 138 CNAG_05590 TCO2 L1 CNAG_05590 5' flanking region primer 1 CAAAACTGGAAAGAAGCGAAG L2 CNAG_05590 5' flanking region primer 2 CTGGCCGTCGTTTTACTTGCCAGATGAAGAGTCACGCC R1 CNAG_05590 3' flanking region primer 1 GTCATAGCTGTTTCCTGTCCCATCCTCTGTGATTCCC R2 CNAG_05590 3' flanking region primer 2 ATTGTGGAGTGGTGGAGTGGAC SO CNAG_05590 diagnostic screening primer, pairing with B79 TGAGGAGGGAAAGTTTTAGCG PO1 CNAG_05590 Southern blot probe primer 1 GTTACCGATTCTGGACCTG PO2 CNAG_05590 Southern blot probe primer 2 TGCTTCACCCTTTCAGTCTC STM NAT#116 STM primer GCACCCAAGAGCTCCATCTC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 139 CNAG_05600 IGI1 L1 CNAG_05600 5' flanking region primer 1 TTCTTCTCCTCTATCCCCG L2 CNAG_05600 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGATGATAGCGATGGTAGCC R1 CNAG_05600 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGGAAAGAAGTTTGGGTTCG R2 CNAG_05600 3' flanking region primer 2 TGGGGAAGAACCAGAAGTAG SO CNAG_05600 diagnostic screening primer, pairing with B79 TCCCTGTAAGATTCGCCAG P.O. CNAG_05600 Southern blot probe primer TTCTCCATAGGTAGCCACG STM NAT#230 STM primer ATGTAGGTAGGGTGATAGGT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 140 CNAG_05694 CKA1 L1 CNAG_05694 5' flanking region primer 1 TGTCAAAAGCACACTCAGG L2 CNAG_05694 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGCGAATAGTTGCTGCTC R1 CNAG_05694 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTGACCTGCCGTGTATTTAG R2 CNAG_05694 3' flanking region primer 2 AAACATCACTCACCGTTCC SO CNAG_05694 diagnostic screening primer, pairing with B79 CGACAAGTTGCTGAAGTTTC P.O. CNAG_05694 Southern blot probe primer ACATTTGGAGTCGGTTGG STM NAT#6 STM primer ATAGCTACCACACGATAGCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 141 CNAG_05753 ARG5,6 L1 CNAG_05753 5' flanking region primer 1 ATTTTCCAGTCGTCCGTC L2 CNAG_05753 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTAATACTGAGGGCAGAGCG R1 CNAG_05753 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGATCCTTTGACCATCCAGGG R2 CNAG_05753 3' flanking region primer 2 TTGATGTTTCGCAGCACC SO CNAG_05753 diagnostic screening primer, pairing with JOHE12579 ACCAGTCAGCAACGAAACG P.O. CNAG_05753 Southern blot probe primer CGACAGCAAGGGTTTTTTG STM NAT#220 STM primer CAGATCTCGAACGATACCCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 142 CNAG_05771 TEL1 L1 CNAG_05771 5' flanking region primer 1 ACCCTCCATACATCCTTCC L2 CNAG_05771 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGGCTATCGTTTCGGTAAGG R1 CNAG_05771 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCAGTATGGATGGGGAGTAATAG R2 CNAG_05771 3' flanking region primer 2 AACTCCCAAAGATGAGCC SO CNAG_05771 diagnostic screening primer, pairing with B79 TAGCAGCAAAAGTGAGCG P.O. CNAG_05771 Southern blot probe primer GAAATCGTCAAACTCGTTCC STM NAT#225 STM primer CCATAGAACTAGCTAAAGCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 143 CNAG_05935 L1 CNAG_05935 5' flanking region primer 1 GGTCAATCCAGATGCTATCAG L2 CNAG_05935 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTTGGGGTTTGGGTTTGGGCAGC R1 CNAG_05935 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCCCGTGTTGTTCTTTCGTAG R2 CNAG_05935 3' flanking region primer 2 CAAGGTGTTGGTATCTACG SO CNAG_05935 diagnostic screening primer, pairing with B79 CGGAAGATTACATCCTGGG P.O. CNAG_05935 Southern blot probe primer TTACTCATACCGCAGGACCC STM NAT#220 STM primer CAGATCTCGAACGATACCCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 144 CNAG_05965 IRK4 L1 CNAG_05965 5' flanking region primer 1 TCATAGACGATGTTGCCG L2 CNAG_05965 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCAAGATGGAAGCCAGACTTAC R1 CNAG_05965 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCCATCTTCCTTCTCCGAAC R2 CNAG_05965 3' flanking region primer 2 TTTCGGGAGAGTTTTGCG SO CNAG_05965 diagnostic screening primer, pairing with B79 GCTGTTGTTTCTCACTGTAACC P.O. CNAG_05965 Southern blot probe primer GATGTATCTGGCAAAGGGTC STM NAT#211 STM primer GCGGTCGCTTTATAGCGATT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 145 CNAG_05970 L1 CNAG_05970 5' flanking region primer 1 TGAAGGCGTGAGTGTAAACG L2 CNAG_05970 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGGGCAAAGGAATGTGATG R1 CNAG_05970 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCTCATTCTTGGATTTCCCTG R2 CNAG_05970 3' flanking region primer 2 ACAGAAAGGGGTGAAACG SO CNAG_09570 diagnostic screening primer, pairing with B79 AGACTTGCCCGATTTTGG P.O. CNAG_05970 Southern blot probe primer TGGCGGTTTATCCTTTCC STM NAT#212 STM primer AGAGCGATCGCGTTATAGAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 146 CNAG_06001 L1 CNAG_06001 5' flanking region primer 1 ATCTCCACCTCTTCGCCAACTTCC L2 CNAG_06001 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCGTCATTTTTTTGGGATACGCC R1 CNAG_06001 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAGAAGAAGTTGCGGAAGTC R2 CNAG_06001 3' flanking region primer 2 GGAAGAAAGCGATTTACGG SO CNAG_06001 diagnostic screening primer, pairing with B79 TTCCTTGCCCTTCCAATCC P.O. CNAG_06001 Southern blot probe primer GGATAAAAGCCTGTCAGTCG STM NAT#119 STM primer CTCCCCACATAAAGAGAGCTAAAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 147 CNAG_06033 MAK32 L1 CNAG_06033 5' flanking region primer 1 CAAACAACAGATTCCGCC L2 CNAG_06033 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTCGGATGGACGGATGTAG R1 CNAG_06033 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGGACATTTCTCTGCCATCC R2 CNAG_06033 3' flanking region primer 2 AAGCCTGGAAAACTACC SO CNAG_06033 diagnostic screening primer, pairing with B79 CAGCGTGAAAGTAGCATTG P.O. CNAG_06033 Southern blot probe primer GCTCTTGTCATTCTCGTTCC STM NAT#169 STM primer ACATCTATATCACTATCCCGAACC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 148 CNAG_06051 GAL1 L1 CNAG_06051 5' flanking region primer 1 GCGGTTGAGTTGTTATTG L2 CNAG_06051 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGCTCCCCTAACACATTGACTC R1 CNAG_06051 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGTCCTGACGCTCTGATACTG R2 CNAG_06051 3' flanking region primer 2 GCTATGGGTATGAATCGCC SO CNAG_06051 diagnostic screening primer, pairing with B79 AGAGACCAGAAGTGAGGAGGAC P.O. CNAG_06051 Southern blot probe primer GACGCTGACAACAAAAGC STM NAT#224 STM primer AACCTTTAAATGGGTAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 149 CNAG_06086 SSN3 L1 CNAG_06086 5' flanking region primer 1 CGGAGTCTACATTGCTCAGAG L2 CNAG_06086 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAGTAATCGGTTATCCCACG R1 CNAG_06086 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGAGGATAACGGTGATGCTAAG R2 CNAG_06086 3' flanking region primer 2 CCACTTGTTTTGCTTGTGC SO CNAG_06086 diagnostic screening primer, pairing with B79 AGGCACGGGGATTTTTAG P.O. CNAG_06086 Southern blot probe primer ATTTGAACCCACCGACAG STM NAT#219 STM primer CCCTAAACCCTACAGCAAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 150 CNAG_06161 VRK1 L1 CNAG_06161 5' flanking region primer 1 TATCGGCAGCGACTCTACTC L2 CNAG_06161 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCGCAACCATCAACCTAAGC R1 CNAG_06161 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGATAGACGCCAAACGCATC R2 CNAG_06161 3' flanking region primer 2 CCAACCCAACTACTACATACTGC SO CNAG_06161 diagnostic screening primer, pairing with B79 GAAGAACTGGAAGCATTGG P.O. CNAG_06161 Southern blot probe primer CGAGAAGAGTGAGAAATGGG STM NAT#123 STM primer CTATCGACCAACCAACACAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 151 CNAG_06174 L1 CNAG_06174 5' flanking region primer 1 GCTCACATCGTAACGGTTG L2 CNAG_06174 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAATGAGCCGAGAACTTACG R1 CNAG_06174 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTGGAGGGCTTTGTTAGC R2 CNAG_06174 3' flanking region primer 2 GCTCAACAACAACACAGCAAGAG SO CNAG_06174 diagnostic screening primer, pairing with B79 TCCGATGCTCACGAATAC P.O. CNAG_06174 Southern blot probe primer GTCTCGCACTGTATCAATAAGC STM NAT#119 STM primer CTCCCCACATAAAGAGAGCTAAAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 152 CNAG_06193 CRK1 L1 CNAG_06193 5' flanking region primer 1 TCCCCTGCTGTATTCATTG L2 CNAG_06193 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCTTGTGCTAATGTTGTCACG R1 CNAG_06193 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTAACCAGTCTCATCCTCCCAC R2 CNAG_06193 3' flanking region primer 2 TATTCCAGAGGTAGCGGGCGTCAAG SO CNAG_06193 diagnostic screening primer, pairing with B79 ATAAGGGGGAAAGACCGAG P.O. CNAG_06193 Southern blot probe primer GGTTGCCTTCCATACACTC STM NAT#43 STM primer CCAGCTACCAATCACGCTAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 153 CNAG_06278 TCO7 L1 CNAG_06278 5' flanking region primer 1 CCACCTTTCTCATTCGTATG L2 CNAG_06278 5' flanking region primer 2 CTGGCCGTCGTTTTACTCTTCTTCAGATGGTTCCC R1 CNAG_06278 3' flanking region primer 1 GTCATAGCTGTTTCCTGCACACTCACTCAACGCATC R2 CNAG_06278 3' flanking region primer 2 CTCCATTTGTTCCATTAGCC SO CNAG_06278 diagnostic screening primer, pairing with B79 TAAGCCTCGGAAACACTC P.O. CNAG_06278 Southern blot probe primer CCTTTCTCATTCGTATGGTGTG STM NAT#209 STM primer AGCACAATCTCGCTCTACCCATAA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 154 CNAG_06301 SCH9 L1 CNAG_06301 5' flanking region primer 1 TTCTTCGTGCTGAGAGGAG L2 CNAG_06301 5' flanking region primer 2 GCTCACTGGCCGTCGTTTTACAGATGTGGCGTAGTCAGCAC R1 CNAG_06301 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAATGAGAATGCGGTGGAC R2 CNAG_06301 3' flanking region primer 2 GGATGGATGGATGCTCAT SO CNAG_06301 diagnostic screening primer, pairing with B79 TTCTTCGTGCTGAGAGGAG P.O. CNAG_06301 Southern blot probe primer AACCGAAACCTCGAGAACC STM NAT#169 STM primer ACATCTATATCACTATCCCGAACC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 155 CNAG_06310 IRK7 L1 CNAG_06310 5' flanking region primer 1 GGTGCTAAAGGATGGTATGG L2 CNAG_06310 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGTTGCTGTTGTTTCTGTAGGTC R1 CNAG_06310 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTGGTTATCCGCTTACGAC R2 CNAG_06310 3' flanking region primer 2 GTATGGCTATCAACCTGCTG SO CNAG_06310 diagnostic screening primer, pairing with B79 CCGACCAAGATGAAAAGC P.O. CNAG_06310 Southern blot probe primer GATAGCAACTTTACCCCCC STM NAT#208 STM primer TGGTCGCGGGAGATCGTGGTTT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 156 CNAG_06366 HRR2502 L1 CNAG_06366 5' flanking region primer 1 TTCTCGTCTTCGCTTTCG L2 CNAG_06366 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGGAGAAGGCATTGCTAAAC R1 CNAG_06366 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGATTGTGCCCTCGTAATGG R2 CNAG_06366 3' flanking region primer 2 TTCGCTGACTTGCTTGAG SO CNAG_06366 diagnostic screening primer, pairing with B79 TTCCTCGCTTTCAACTCC P.O. CNAG_06366 Southern blot probe primer GTTTCCTTCTTCACCCTACC STM NAT#125 STM primer CGCTACAGCCAGCGCGCGCAAGCG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 157 CNAG_06432 L1 CNAG_06432 5' flanking region primer 1 CGTCACACAACACTGCTACAG L2 CNAG_06432 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTGATTGACGAGGAACCG R1 CNAG_06432 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCGAACTTAGTGGGTCTTGACG R2 CNAG_06432 3' flanking region primer 2 GCGGTGATGGTTGTTATC SO CNAG_06432 diagnostic screening primer, pairing with B79 ACTTGGCGGTAGTCTGAAG P.O. CNAG_06432 Southern blot probe primer ATACCTGGCGGCTAATCAG STM NAT#224 STM primer AACCTTTAAATGGGTAGAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 158 CNAG_06445 L1 CNAG_06445 5' flanking region primer 1 GCGATAGGTCAGTAGATTGGG L2 CNAG_06445 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGCTTACATCTGTTGGCACG R1 CNAG_06445 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCGCCTCACAAGAGTCAAAG R2 CNAG_06445 3' flanking region primer 2 CAATCAGGACAATCATACGC SO CNAG_06445 diagnostic screening primer, pairing with B79 GAAGAGGAAATGTCAGGGTC P.O. CNAG_06445 Southern blot probe primer CAGAAAGGAACTCACAGGC STM NAT#122 STM primer ACAGCTCCAAACCTCGCTAAACAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 159 CNAG_06454 L1 CNAG_06454 5' flanking region primer 1 AAAAAACCGCTGGCAACACCC L2 CNAG_06454 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTCCAGAGTCTTCTTCAGGCG R1 CNAG_06454 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGACCAAGATGCCAAAAGC R2 CNAG_06454 3' flanking region primer 2 AATGGTTGACAAGCGTGCC SO CNAG_06454 diagnostic screening primer, pairing with B79 ACCCCTTACTGGCGAAAAC P.O. CNAG_06454 Southern blot probe primer GGCAAAACTTACACCTCGC STM NAT#232 STM primer CTTTAAAGGTGGTTTTGTG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 160 CNAG_06489 L1 CNAG_06489 5' flanking region primer 1 TTCTGGAGACCCATCGTCAG L2 CNAG_06489 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCAACGCCCTGTTATTTCTTC R1 CNAG_06489 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTGGTCAGATGTGTGTCGG R2 CNAG_06489 3' flanking region primer 2 CTACTTTGCCCGAGTCTCAAG SO CNAG_06489 diagnostic screening primer, pairing with B79 CAGGACTTGCGTAGCCTATC P.O. CNAG_06489 Southern blot probe primer TGGGTGATGACGATGAGAC STM NAT#125 STM primer CGCTACAGCCAGCGCGCGCAAGCG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 161 CNAG_06490 L1 CNAG_06490 5' flanking region primer 1 GGAGGGGTTTTTGAGGTC L2 CNAG_06490 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGGGGACTTTTTTGATGGC R1 CNAG_06490 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGAAGAGGAAGAGGAAGATGAAG R2 CNAG_06490 3' flanking region primer 2 TCGTTCTGGTTGTCTGCTC SO CNAG_06490 diagnostic screening primer, pairing with B79 GGTGAGAAAGTAGCCTTCG P.O. CNAG_06490 Southern blot probe primer CAGGACTTGCGTAGCCTATC STM NAT#231 STM primer GAGAGATCCCAACATCACGC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 162 CNAG_06500 L1 CNAG_06500 5' flanking region primer 1 GATACAGCGGGCAAAAG L2 CNAG_06500 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAGAATGGGATGTGGTCGTC R1 CNAG_06500 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGAACGGGGTTGTGTTTG R2 CNAG_06500 3' flanking region primer 2 ATACAGACACTCCGATGCG SO CNAG_06500 diagnostic screening primer, pairing with B79 ATAAAGAGGTTTGGGGC P.O. CNAG_06500 Southern blot probe primer ATCGCATTTCAAGGGTGG STM NAT#225 STM primer CCATAGAACTAGCTAAAGCA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 163 CNAG_06552 SNF1 L1 CNAG_06552 5' flanking region primer 1 CCATCATCCTTTCGGTTTTTC L2 CNAG_06552 5' flanking region primer 2 TCACTGGCCGTTCGTTTTACAGTTGTTATTGCCAGCGG R1 CNAG_06552 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCTTTTTGGAGATGGCTTGC R2 CNAG_06552 3' flanking region primer 2 ATACCACGGAAAGGCGTTC SO CNAG_06552 diagnostic screening primer, pairing with B79 GGATTGTGGTGTTGAAGTCG P.O. CNAG_06552 Southern blot probe primer ATGCTTGCCTTTCTGGAC STM NAT#204 STM primer GATCTCTCGCGCTTGGGGGA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 164 CNAG_06553 GAL83 L1 CNAG_06553 5' flanking region primer 1 TGAGCACTTTGAGGTATTGG L2 CNAG_06553 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGTGTGATGTATGGGTGTGTG R1 CNAG_06553 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCATCTGCTGTGAAACATTGG R2 CNAG_06553 3' flanking region primer 2 GGAAAGGGGTGAAAATGG SO CNAG_06553 diagnostic screening primer, pairing with B79 ATGCTTGCCTTTCTGGAC P.O. CNAG_06553 Southern blot probe primer TATTGACCAGGAGGAAGGC STM NAT#288 STM primer CTATCCAACTAGACCTCTAGCTAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 165 CNAG_06568 SKS1 L1 CNAG_06568 5' flanking region primer 1 AATAAGGTTCCCAGCCTCG L2 CNAG_06568 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCCACCATCAATGAACTGC R1 CNAG_06568 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAACGACCTGTTGATGACG R2 CNAG_06568 3' flanking region primer 2 CAAGTTGAATGCTGGGAG SO CNAG_06568 diagnostic screening primer, pairing with B79 AGCAAGTGGGCAAAGAAGC P.O. CNAG_06568 Southern blot probe primer AACCGAAGTCACAGAGGCG STM NAT#211 STM primer GCGGTCGCTTTATAGCGATT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 166 CNAG_06632 ABC1 L1 CNAG_06632 5' flanking region primer 1 ACGACCTGGTAAAGAGTGTG L2 CNAG_06632 5' flanking region primer 2 TCACTGGCCGTCGTTTTACAGATGGGCGAAATGTCTC R1 CNAG_06632 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCACCTCTTATCACCTCAATGAC R2 CNAG_06632 3' flanking region primer 2 ACCTTCACGACCAAGTGTC SO CNAG_06632 diagnostic screening primer, pairing with B79 CTATCGCAGAAGAGGATGAG P.O. CNAG_06632 Southern blot probe primer AATACCCCTACAACCTCGTC STM NAT#119 STM primer CTCCCCACATAAAGAGAGCTAAAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 167 CNAG_06642 L1 CNAG_06642 5' flanking region primer 1 CCTTTTCCTTTTACCTGGC L2 CNAG_06642 5' flanking region primer 2 TCACTGGCCGTTCGTTTTACCGCTGAAAGATGTTGTCG R1 CNAG_06642 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGGATTGACTGGACGAAAC R2 CNAG_06642 3' flanking region primer 2 CTGGTATGGCGTAAAGACTTGAC SO CNAG_06642 diagnostic screening primer, pairing with B79 CCTGCTGAACGGATGATAG P.O. CNAG_06642 Southern blot probe primer GAAGGTTAGTTCGCAAATGG STM NAT#43 STM primer CCAGCTACCAATCACGCTAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 168 CNAG_06671 YKL1 L1 CNAG_06671 5' flanking region primer 1 CCGACCTACTGATTCGTCTAC L2 CNAG_06671 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCTCGCCCCTTTTCATAATG R1 CNAG_06671 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGTCCAATCAACAACAGCG R2 CNAG_06671 3' flanking region primer 2 TGCGGAGGAGATTACCATAC SO CNAG_06671 diagnostic screening primer, pairing with B79 TTCGCCTTTGAAGTTCCC P.O. CNAG_06671 Southern blot probe primer GGAAAGTGTAGATTGTCGGC STM NAT#123 STM primer CTATCGACCAACCAACACAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 169 CNAG_06697 MPS1 L1 CNAG_06697 5' flanking region primer 1 GCGATAACTTTTCATCCCC L2 CNAG_06697 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGGTTTTTCCTTTCTCCAGTC R1 CNAG_06697 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCGGAACTGTCAGATGGTAATC R2 CNAG_06697 3' flanking region primer 2 CCTTTCTTCACCCTACTCTGG SO CNAG_06697 diagnostic screening primer, pairing with B79 CCAATCTCGCATTTACACC P.O. CNAG_06697 Southern blot probe primer TCCTTAGTTATCCTATCCCAGC STM NAT#116 STM primer GCACCCAAGAGCTCCATCTC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 170 CNAG_06730 GSK3 L1 CNAG_06730 5' flanking region primer 1 GTGAGTCTATCCTTCGTTTCTGTC L2 CNAG_06730 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCGGCTTCCAAAAAAGTCAG R1 CNAG_06730 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCTGAACAACTGCGTGTCAC R2 CNAG_06730 3' flanking region primer 2 CTTGAAAGATGACGCTCG SO CNAG_06730 diagnostic screening primer, pairing with B79 ACATCCTTTGTCTCCCCCAC PO1 CNAG_06730 Southern blot probe primer 1 CGGAAGACTTTGGTGAAGG STM NAT#123 STM primer CTATCGACCAACCAACACAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 171 CNAG_06809 IKS1 L1 CNAG_06809 5' flanking region primer 1 TGGAAGAGGATGAAAGACC L2 CNAG_06809 5' flanking region primer 2 TCACTGGCCGTCGTTTTACACAACTAAAGGCACAAGGG R1 CNAG_06809 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGATGAGCGAGCAATGACCTGC R2 CNAG_06809 3' flanking region primer 2 CAGAACGGTCTTTTTGCTTC SO CNAG_06809 diagnostic screening primer, pairing with B79 TACAGTATCGCTGGTTGCC P.O. CNAG_06809 Southern blot probe primer AGCGAGACTGGAAATGTGGAG STM NAT#116 STM primer GCACCCAAGAGCTCCATCTC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 172 CNAG_06845 L1 CNAG_06845 5' flanking region primer 1 GTTATTTGGATGCCAGAGC L2 CNAG_06845 5' flanking region primer 2 TCACTGGCCGTTCGTTTTACATGCGGTTACCTCATTCG R1 CNAG_06845 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAGGGAGAAGTAGTTTCGGG R2 CNAG_06845 3' flanking region primer 2 TGGAGGTTTCGGGTATCAC SO CNAG_06845 diagnostic screening primer, pairing with B79 GCAAAAAACCGAGACTGTG P.O. CNAG_06845 Southern blot probe primer TTGAGGGGTTATGCCTTC STM NAT#201 STM primer CACCCTCTATCTCGAGAAAGCTCC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG List of primers used to construct the azyme variant library No. H99 locus tag (Broad ID) Cn gene name Primer name Primer description Primer sequence (5'-3') 173 CNAG_06980 STE11 L1 CNAG_06980 5' flanking region primer 1 TCTCAGCCACATCAGTTAGC L2 CNAG_06980 5' flanking region primer 2 CTGGCCGTCGTTTTACGGGTGCTCTAAATCTCCTTG R1 CNAG_06980 3' flanking region primer 1 GTCATAGCTGTTTCCTGCCATTTTCCGAGTCAGTAGG R2 CNAG_06980 3' flanking region primer 2 ATCCTGATGCCAGATTCG SO CNAG_06980 diagnostic screening primer, pairing with B79 TCATCTGTCTCACCAACTGC PO1 CNAG_06980 Southern blot probe primer 1 GGACGCACAGTCTGGTTTAC PO2 CNAG_06980 Southern blot probe primer 2 TGGGTCAAGTTTAGGGATG STM NAT#242 STM primer GTAGCGATAGGGGTGTCGCTTTAG STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 174 CNAG_07359 IRK1 L1 CNAG_07359 5' flanking region primer 1 CGCATTTGGTGTATGATGAC L2 CNAG_07359 5' flanking region primer 2 TCACTGGCCGTCGTTTTACGGAGGAAGAAGGAGATGAAG R1 CNAG_07359 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGCTTCGCCTTGATTGTC R2 CNAG_07359 3' flanking region primer 2 TGCTGAAGATTTCGGAGG SO CNAG_07359 diagnostic screening primer, pairing with B79 TGATGGTAGAAATGGCGG PO1 CNAG_07359 Southern blot probe primer 1 GCATTCGGAGGTAGTTGAAG STM NAT#5 STM primer TGCTAGAGGGCGGGAGAGTT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 175 CNAG_07372 L1 CNAG_07372 5' flanking region primer 1 CCAAACGGTGTGAAAAGG L2 CNAG_07372 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGTAGTCGCCGATGGAGTAG R1 CNAG_07372 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGGCAAGACGAGAAGTAGAGC R2 CNAG_07372 3' flanking region primer 2 GAACCTGAACCTGAACCAG SO CNAG_07372 diagnostic screening primer, pairing with B79 TTTGTAGTTGGGTGTGGTG P.O. CNAG_07372 Southern blot probe primer CTTCGCCTTTTGCCTTTC STM NAT#295 STM primer ACACCTACATCAAACCCTCCC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 176 CNAG_07377 L1 CNAG_07377 5' flanking region primer 1 CGATAACGCAACTTACGG L2 CNAG_07377 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTTTGGCTTGATTCTCCGC R1 CNAG_07377 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCTCTCAATCTCGCTCAAATG R2 CNAG_07377 3' flanking region primer 2 CTGAGCCGATAGAGTTCAAC SO CNAG_07377 diagnostic screening primer, pairing with B79 ACCAACCGCACATCTACCTC P.O. CNAG_07377 Southern blot probe primer TTATTCTACCGAAGTTGGCTG STM NAT#296 STM primer CGCCCGCCCTCACTATCCAC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 177 CNAG_07408 L1 CNAG_07408 5' flanking region primer 1 GCTGGCATAAAACCGTTC L2 CNAG_07408 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCTCTTACTCCACATAAATGCCC R1 CNAG_07408 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTTGAAGTCACCCGAGAAAC R2 CNAG_07408 3' flanking region primer 2 ACACTGCGGATTACGAAGC SO CNAG_07408 diagnostic screening primer, pairing with B79 TGTGGCTGAGATGAGGTAGG P.O. CNAG_07408 Southern blot probe primer TCTGGCTGAAGTCTACTAAAC STM NAT#6 STM primer ATAGCTACCACACGATAGCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 178 CNAG_07427 CCK2 L1 CNAG_07427 5' flanking region primer 1 AGATTCACTCGTCATCGCC L2 CNAG_07427 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTAAGATGCGATAGGTGGGCG R1 CNAG_07427 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGCAGACTAAAGCCAGGGACAC R2 CNAG_07427 3' flanking region primer 2 GGAAGGTCAAGCCATTAGC SO CNAG_07427 diagnostic screening primer, pairing with B79 TCAAGGCTTTCATCCCGAC P.O. CNAG_07427 Southern blot probe primer CGAGACCAGTTATGTTTGAGAG STM NAT#230 STM primer ATGTAGGTAGGGTGATAGGT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 179 CNAG_07580 TRM7 L1 CNAG_07580 5' flanking region primer 1 GGTGGAGAGATGTTTATGGC L2 CNAG_07580 5' flanking region primer 2 TCACTGGCCGTCGTTTTACATAGAGGACTTGGAGGTGGG R1 CNAG_07580 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGCAATGCTGTGAATCTTGTG R2 CNAG_07580 3' flanking region primer 2 AGAGTAGGGCTGAGCAAGAC SO CNAG_07580 diagnostic screening primer, pairing with B79 TGGAAAGACCTGTTGCGAC P.O. CNAG_07580 Southern blot probe primer TTCTTCGGGAAATGGACTG STM NAT#102 STM primer CCATAGCGATATCTACCCCAATCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 180 CNAG_07667 SAT4 L1 CNAG_07667 5' flanking region primer 1 GATTTTGTGGCTGTTGTGC L2 CNAG_07667 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGCTTCAAAACCTGGGCTCC R1 CNAG_07667 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGGTGTAGATTGTTCAGGATGACG R2 CNAG_07667 3' flanking region primer 2 AGATAGGGCGTGCTACCGATG SO CNAG_07667 diagnostic screening primer, pairing with ATCGGCTTACCATTCTGG P.O. CNAG_07667 Southern blot probe primer TCGGTCCCATAATAGACGG STM NAT#212 STM primer AGAGCGATCGCGTTATAGAT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 181 CNAG_07744 PIK1 L1 CNAG_07744 5' flanking region primer 1 TGGTAGTATGCCAAGAGGTG L2 CNAG_07744 5' flanking region primer 2 TCACTGGCCGTCGTTTTACTGGGATACTCTCTCTCTCTGAG R1 CNAG_07744 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGAAAGGGCAAAGGCAGAAG R2 CNAG_07744 3' flanking region primer 2 GGAGATGAAGTCAAGATGCG SO CNAG_07744 diagnostic screening primer, pairing with B79 TCATCTTCATTGTCCTCCC P.O. CNAG_07744 Southern blot probe primer TAAAGAGCGGTAAGGCGAG STM NAT#227 STM primer TCGTGGTTTAGAGGGAGCGC STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 182 CNAG_07779 TDA10 L1 CNAG_07779 5' flanking region primer 1 TGGGAAGCGTTACTTATGC L2 CNAG_07779 5' flanking region primer 2 TCACTGGCCGTCGTTTTACCTGTAGCAGTCATAATGGCTTG R1 CNAG_07779 3' flanking region primer 1 CATGGTCATAGCTGTTTCCTGTGAGCAGGGTCCGACATTTC R2 CNAG_07779 3' flanking region primer 2 CATCGCTCTTTCCTACTCG SO CNAG_07779 diagnostic screening primer, pairing with B79 TTTGGAGCCAGTTTAGGG P.O. CNAG_07779 Southern blot probe primer AAAACGAAGCCTTTGCCCC STM NAT#102 STM primer CCATAGCGATATCTACCCCAATCT STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG 183 CNAG_08022 PHO85 L1 CNAG_08022 5' flanking region primer 1 CCTTGCTTTTTGAGCGAG L2 CNAG_08022 5' flanking region primer 2 CTGGCCGTTCGTTTTACCCTTCACCAAGTTTCTCAAG R1 CNAG_08022 3' flanking region primer 1 GTCATAGCTGTTTCCTGCAAATGGCTCAACAAGGG R2 CNAG_08022 3' flanking region primer 2 CCACAGTCGTCTTTTTATC SO CNAG_08022 diagnostic screening primer, pairing with B79 ATAGGGGTGATTATCGGGGC P.O. CNAG_08022 Southern blot probe primer TCGGCATTATCTCTTCCTC STM NAT#218 STM primer CTCCACATCCATCGCTCCAA STM common STM common primer GCATGCCCTGCCCCTAAGAATTCG

Primers used to produce Naase variant library and identify function Primer name Primer description Primer sequence (5'-3') B1026 M13 Forward extended GTAAAACGACGGCCAGTGAGC B1027 M13 Reverse extension CAGGAAACAGCTATGACCATG B1454 NAT split marker primer (NSR) AAGGTGTTCCCCGACGACGAATCG B1455 NAT split marker primer (NSL) AACTCCGTCGCGAGCCCCATCAAC B1886 NEO split marker primer (GSR) TGGAAGAGATGGATGTGC B1887 NEO split marker primer (GSL) ATTGTCTGTTGTGCCCAG B4017 Primer 1 for overexpression promoter with NEO marker GCATGCAGGATTCGAGTG B4018 Primer 2 for overexpression promoter with NEO marker GTGATAGATGTGTTGTGGTG B678 Northern probe primer1 for ERG11 TTCAGGGAACTTGGGAACAGC B1598 Northern probe primer2 for ERG11 CAGGAGCAGAAAACAAAGC B3294 Northern probe primer1 for ACT1 GCACCATACCTTCTACAATGAG B3295 Northern probe primer2 for ACT1 ACTTTCGTGGACGATTG B5251 RT-PCR primer for HXL1 of H99 CACTCCATTCCTTTTCTGC B5252 RT-PCR primer for HXL1 of H99 CGTAACTCCACTGTGTCC B7030 qRT-PCR primer for CNA1 of H99 AGACTGTTTACAATGCCTGC B7031 qRT-PCR primer for CNA1 of H99 TCTGGCGACAAGCCCACCATG B7032 qRT-PCR primer for CNB1 of H99 AAGATGGAAGTGGAACGG B7033 qRT-PCR primer for CNB1 of H99 TTGAAAGCGAATCTCAGCTT B7034 qRT-PCR primer for CRZ1 of H99 ACCACGGACATTATCTTCAG B7035 qRT-PCR primer for CRZ1 of H99 AGCCCAGCCTTGCTGTTCGT B7036 qRT-PCR primer for UTR2 of H99 TTTCTATGCCCATCTACAGC B7037 qRT-PCR primer for UTR2 of H99 CTTCGTGGGAGTACAGTGGC B679 qRT-PCR primer for ACT1 of H99 CGCCCTTGCTCCTTCTTCTATG B680 qRT-PCR primer for ACT1 of H99 GACTCGTCGTATTCGCTCTTCG

Example 3: Systematic phenotypic profiling and clustering of the Cryptococcal kinome network

With the kinase variant library constructed in the above example, a series of in vitro phenotypic analyzes were performed on the following six major phenotypic classes: growth, differentiation, stress response and adaptation, antifungal drug resistance, and virulence factor production (a total of 30 phenotypic traits). was performed, and more than 6,600 phenotypic data were created. This comprehensive kinase phenome data can be searched in the C. neoformans kinome database (http://kinase.cryptococcus.org). In order to reveal the connection in function and regulation between the kinases, the kinases were grouped through trait analysis through Pearson Correlation analysis (see Figure 2). The rationale for this analysis is that groups of kinases within any signaling pathway tend to cluster together based on shared phenotypic characteristics. For example, variants of the three-stage mitogen-activated protein kinase (MAPK) cascade should be clustered together because they exhibit nearly identical phenotypes. Indeed, cell wall integrity MAPKs ( bck1△, mkk1△, mpk1△ ) and high osmolality glycerol-responsive (HOG) MAPKs ( ssk2△, pbs2△, hog1△ ), and pheromone-responsive MAPKs ( ste11△, ste7△, The three sets of kinases in the cpk1Δ ) pathway were each clustered together based on their shared functions (Figure 2). Therefore, it is highly likely that a group of kinases clustered together by this analysis function in the same or related signaling cascades. The present inventors identified several novel, unknown kinases that are functionally associated with known signaling pathways. We identified CNAG_06553, which encodes a protein orthologous to yeast Gal83, one of three possible β-subunits of the Snf1 kinase complex in S. cerevisiae. The yeast Snf1 kinase complex is composed of Snf1, a catalytic α-subunit, Snf4, a regulatory γ subunit, and one of three possible β subunits, Gal83, Sip1 or Sip2, and regulates transcriptional changes under glucose depression. (Jiang, R. & Carlson, M. The Snf1 protein kinase and its activating subunit, Snf4, interact with distinct domains of the Sip1/Sip2/Gal83 component in the kinase complex. Mol Cell Biol 17, 2099-2106, 1997; Schuller , HJ Transcriptional control of nonfermentative metabolism in the yeast Saccharomyces cerevisiae. Curr Genet 43, 139-160, doi:10.1007/s00294-003-0381-8, 2003). The function of Snf1 in C. neoformans has already been reported (Hu, G., Cheng, PY, Sham, A., Perfect, JR & Kronstad, JW Metabolic adaptation in Cryptococcus neoformans during early murine pulmonary infection. Molecular microbiology 69, 1456-1475, doi:10.1111/j.1365-2958.2008.06374.x, 2008). Several experimental evidences have demonstrated that Gal83 functions in association with Snf1 in C. neoformans. First, the in vitro (in vitro) phenotype of the gal83△ mutant is almost identical to that of the snf1△ mutant (Figure 3). Both gal83Δ and snf1Δ mutants showed increased susceptibility to fludioxonil and increased resistance to organic peroxide (tert-butyl hydroperoxide). Second, the growth defect of the snf1△ mutant on other carbon sources (e.g., potassium acetate, sodium acetate, ethanol) was also observed in the gal83△ mutant (Figure 3). Therefore, Gal83 may be one of the possible β-subunit complexes of the Snf1 kinase complex in C. neoformans.

Additionally, we identified several kinases with potential functions upstream or downstream of the TOR kinase complex. Because the Tor1 kinase is essential for C. neoformans, we could not knock it down, but three kinases (Ipk1, Ypk1, and Gsk3), which are found clustered in most eukaryotes, are responsible for the Tor1-dependent signaling cascade. It was found that ids can cluster in C. neoformans. Recently, Lev et al. proposed that S. cerevisiae Ipk1 may be involved in the production of inositol hexaphosphate (IP6) based on limited sequence homology (Lev, S. et al. Fungal Inositol Pyrophosphate IP7 Is Crucial for Metabolic Adaptation to the Host Environment and Pathogenicity. MBio 6, e00531-00515, doi:10.1128/mBio.00531-15 (2015). IPMK (inositol polyphosphate multikinase), identified as Arg82 in yeast, and, in mammals, IP6, the precursor of 5-IP7, inhibit Akt activity and thereby reduce mTORC1-mediated protein translation and GSK3-mediated glucose homeostasis. Increase adipogenesis, and behavior (Chakraborty, A., Kim, S. & Snyder, SH Inositol pyrophosphates as mammalian cell signals. Sci Signal 4, re1, doi:10.1126/scisignal.2001958 (2011)) I order it. In S. cerevisiae , Ypk1 was reported to be a direct target of TORC2 by promoting autophagy during amino acid starvation (Vlahakis, A. & Powers, T. A role for TOR complex 2 signaling in promoting autophagy. Autophagy 10, 2085 -2086, doi:10.4161/auto.36262 (2014)). In C. neoformans, Ypk1 , a potential downstream target of Tor1, is involved in sphingolipid synthesis, and deletion of YPK1 results in a significant reduction in virulence (Lee, H., Khanal Lamichhane, A., Garraffo , HM, Kwon-Chung, KJ & Chang, YC Involvement of PDK1, PKC and TOR signaling pathways in basal fluconazole tolerance in Cryptococcus neoformans Mol. .x (2012)). Reflecting the essential role of Tor1, ipk1Δ, ypk1Δ , and gsk3Δ mutants all exhibited growth factor deficiencies, especially at high temperatures.

There are two major limitations in this phenotypic clustering analysis. First, kinases that are oppositely regulated in the same pathway cannot be clustered. Second, a kinase that regulates a subset of phenotypes regulated in a signaling pathway cannot be clustered with its upstream kinases. These examples include Hrk1 (CNAG_00130; Hog1-regulated kinase 1). Although we have demonstrated that Hrk1 is regulated by Hog1, Hrk1 and Hog1 cannot be clustered together because Hrk1 regulates only a subset of Hog1-dependent phenotypes. Fpk1, a phospholipid flippase kinase, is another example. In S. cerevisiae, the activity of Fpk1 is inhibited by direct phosphorylation by Ypk136. As expected, Fpk1 and Ypk1 clustered together. To determine whether Fpk1 regulates C. neoformans Ypk1-dependent phenotypic characteristics, we performed epistatic analyzes constructing FPK1 overexpression strains constructed in ypk1Δ and wild-type strain backgrounds. . As expected, overexpression of FPK1 partially restored normal growth, and stress (osmotic, oxidative, genotoxic, and cell wall/membrane stress) resistance and antifungal drug (amphotericin B) resistance in ypk1 mutants (Figure 4) . However, the azole sensitivity of the ypk1Δ mutant could not be restored by FPK1 overexpression (see Figure 4). These results suggest that Fpk1 may be one of the downstream targets of Ypk1 and may be positively regulated by Ypk1.

Example 4: C. Pathogenic kinome networks within neoformance.

To identify the kinases that regulate pathogenicity, which are governed by both infectivity and virulence, we performed two large-scale in vivo animal experiments: a virulence assay and an STM-based mouse infectivity assay. In both assays, two independent variants for each kinase were monitored, except for a few kinases that had a single variant. As a result, 31 virulence-regulating kinases were discovered in the insect killing assay (Fig. 5a and b) and 54 infectivity-regulating kinases were discovered in the STM-based mouse infectivity assay (Fig. 6a, b, c and d). Among these, 25 kinases were commonly identified in both assays (Figure 7a), indicating that virulence in insect hosts and infectivity in mouse hosts are closely related, as previously reported (Figure 7A). Jung, KW et al. Systematic functional profiling of transcription factor networks in Cryptococcus neoformans. Nat Comms 6, 6757, doi:10.1038/ncomms7757, 2015). Only six kinase variants were discovered by insect kill assay (Figure 7b). The present inventors discovered a total of 60 kinase variants associated with the pathogenicity of C. neoformans.

Additionally, a large number of known virulence-regulating kinases (a total of 15 kinases) were reidentified in the present invention (kinase shown in black in Figure 7 a). These are Mpk1 MAPK (Gerik, KJ, Bhimireddy, SR, Ryerse, JS, Specht, CA & Lodge, JK PKC1 is essential for protection against both oxidative and nitrosative stresses, cell integrity, and normal manifestation of virulence factors in the pathogenic fungus Cryptococcus neoformans Eukaryot Cell 7, 1685-1698, 2008; Kraus, PR, Fox, DS, Cox, GM & Heitman, J. The Cryptococcus neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal drugs and loss of calcineurin function. Microbiol 48, 1377-1387, 2003); Ssk2 in the high osmolarity glycerol response (HOG) pathway (Bahn, YS, Geunes-Boyer, S. & Heitman, J. Ssk2 mitogen-activated protein kinase kinase kinase governs divergent patterns of the stress-activated Hog1 signaling pathway in Cryptococcus neoformans Eukaryot. Cell 6, 2278-2289, 2007), the main catalytic subunit of protein kinase A (Pka1) in the cAMP pathway (D'Souza, CA et al. Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans. Mol. Cell. 21, 3179-3191, Ire1 kinase/endoribonuclease in the unfolded protein response (UPR) pathway (Cheon, SA et al. Unique evolution of the UPR pathway with a novel bZIP transcription factor, Hxl1, for controlling pathogenicity of Cryptococcus neoformans. PLoS Pathog. 7 , e1002177, doi:10.1371/journal.ppat.1002177, 2011); Ypk1 (Kim, H. et al. Network-assisted genetic dissection of pathogenicity and drug resistance in the opportunistic human pathogenic fungus Cryptococcus neoformans. Scientific reports 5, 8767, doi:10.1038/srep08767, 2015; Lee, H., Khanal Lamichhane, A., Garraffo, HM, Kwon-Chung, KJ & Chang, Y.C. Involvement of PDK1, PKC and TOR signaling pathways in basal fluconazole tolerance in Cryptococcus neoformans Mol 84, 130-146, doi:10.1111/j.1365. -2958.2012.08016.x, 2012); and Snf1 (Hu, G., Cheng, PY, Sham, A., Perfect, JR & Kronstad, JW Metabolic adaptation in Cryptococcus neoformans during early murine pulmonary infection. Molecular microbiology 69, 1456-1475, doi:10.1111/j.1365 -2958.2008.06374.x, 2008). By examining the function of Gsk3 in serotype D (B3501A), it has been demonstrated that Gsk3 is required for survival of C. neoformans at low oxygen tension (1%) and for virulence in a mouse model system (Chang, YC, Ingavale, SS, Bien, C., Espenshade, P. & Kwon-Chung, K.J. Conservation of the sterol regulatory element-binding protein pathway and its pathobiological importance in Eukaryot Cell 8, 1770-1779, doi:10.1128. /EC.00207-09, 2009). We found that Gsk3 is also required for serotype A C. neoformans (H99S). Although not previously reported, we also found that deletion variants of known virulence-regulating kinases and functionally related kinases attenuate virulence or infectivity. In the present invention, bck1△ and mkk1/2△ mutants (related to Mpk1) and gal83△ mutants (related to Snf1) are included. In particular, for 44 of these kinases, their role in fungal pathogenicity was confirmed for the first time.

We analyzed the phenotypic relationship of 60 pathogenicity-related kinases in C. neoformans to their possible orthologs in fungal species and other eukaryotic kingdoms, animals and plants ( Figure 8 a). For broad-spectrum inhibition of fungal pathogens, it is ideal to target kinases that are not present in humans and are required by many fungal pathogens (broad-spectrum antifungal targets). These large-scale virulence data from C. neoformans were compared to other fungal pathogens. In the wheat scab plant pathogenic fungus Fusarium graminearum, a large-scale kinome analysis was performed and 42 protein kinases involved in virulence were discovered (Wang, C. et al. Functional analysis of the kinome of the wheat scab fungus Fusarium graminearum . PLoS Pathog 7, e1002460, doi:10.1371/journal.ppat.1002460, 2011). Of these, 21 were implicated in the pathogenicity of both fungi and were therefore considered broad-spectrum antifungal targets: BUD32 (Fg10037), ATG1 (Fg05547), CDC28 (Fg08468), KIC1 (Fg05734), MEC1 (Fg13318), KIN4 ( Fg11812), MKK1/2 (Fg07295), BCK1 (Fb06326), SNF1 (Fg09897), SSK2 (Fg00408), PKA1 (Fg07251), GSK3 (Fg07329), CBK1 (Fg01188), KIN1 (Fg09274), SCH9 (Fg00) 472), RIM15 (Fg01312), HOG1 (Fg09612), and YAK1 (Fg05418). In another human fungal pathogen, Candida albicans, large-scale pathogenicity kinome analyzes have not been performed. According to information obtained from the constructed Candida genome database (http://www.candidagenome.org/), 33 kinases are known to be involved in the pathogenicity of Candida albicans. Of these, 13 were confirmed to be related to the pathogenicity of C. neoformans and C. candida (Figure 8b). In particular, five kinases (Sch9, Snf1, Pka1, Hog1, and Swe1) were found to be important pathogenic kinases as they were involved in the pathogenicity of all three fungi.

Conversely, to selectively inhibit C. neoformans, it is ideal to target virulence-related kinases present in C. neoformans but not in other fungi or humans (narrow-spectrum anticryptococcal agents). increase target). Among these, there is limited orthology to inositol 1,3,4,5,6-pentakisphosphate 2-kinase (inositol 1,3,4,5,6-pentakisphosphate 2-kinase) present in plants, but ascomycetes CNAG_01294 (named IPK1 ), which encodes a protein that is absent or distantly related to humans, was considered a good anti-cryptococcussis target. The ipk1Δ mutant exhibited a lack of virulence and a pleiotropic phenotype (Figure 9). Deletion of IPK1 slightly increased capsule production (Figure 8b), but did not produce melanin and urease (Figure 8c and d), made it defective in componentization (Figure 8e), and caused cells to This made them highly sensitive to high temperatures (Figure 8f) and various stresses (Figure 8g). In particular, Ipk1 can be a useful target for combination therapy because its deletion significantly increases susceptibility to various types of antifungal drugs (Figure 8g). As such, the present inventors revealed a narrow- and broad-spectrum of anticryptococcal and antifungal drug targets through analysis of the pathogenic kinome of C. neoformans.

Example 5: C. Biological functions of the kinases that govern the pathogenicity of Neoformans

To further clarify the functional network of pathogenicity-related kinases, we used CryptoNet (www.inetbio.org/cryptonet), a genome-scale co-functional network for C. neoformans that we recently constructed. (Kim, H. et al. Network-assisted genetic dissection of pathogenicity and drug resistance in the opportunistic human pathogenic fungus Cryptococcus neoformans. Scientific reports 5, 8767, doi:10.1038/srep08767 (2015)). By searching for any proteins functionally linked to pathogenicity-related kinases, Gene Ontology (GO) terms of previously reported information and corresponding kinase orthologs in C. neoformans and S. Interacting proteins were searched for in cerevisiae and other fungi (Figure 10). This analysis shows that the biological functions of pathogenicity-related kinases include cell cycle regulation, metabolic processes, cell wall biogenesis and organization, DNA damage repair, histone modification, including transmembrane transport and vacuole trafficking, tRNA processing, cytoskeleton organization, stress response and signal transduction, protein folding, mRNA processing, and transcription regulation. revealed that a variety of different biological and physiological functions influence the virulence of C. neoformans (Figure 10). Among the pathogenicity-related kinases, those involved in cell cycle and growth regulation were most frequently identified. These include CDC7, SSN3, CKA1, and MEC1 . In particular, in S. cerevisiae, Cdc7 is an essential catalytic subunit of the Dbf4-dependent protein kinase, and Cdc7-Dbf4 is essential for replication origin operation through S phase in S. cerevisiae (Diffley, JF) , Cocker, JH, Dowell, SJ, Harwood, J. & Rowley, A. Stepwise assembly of initiation complexes at budding yeast replication origins during the cell cycle. J Cell Sci Suppl 19, 67-72, 1995). Although not essential at ambient temperature, the cdc7Δ mutant exhibits severe growth defects at high temperatures (Figure 11a), which is likely to affect the virulence of C. neoformans. The highly sensitive nature of the cdc7△ mutant in C. neoformans to genotoxic agents such as methyl methanesulfonate (MMS) and hydroxyurea (HU) suggests that Cdc7 may be responsible for DNA replication and repair (Figure 11 a). Mec1 is required for cell cycle checkpoints, telomere maintenance and silencing, and DNA damage repair in S. cerevisiae (Mills, KD, Sinclair, DA & Guarente, L. MEC1-dependent redistribution of the Sir3 silencing protein from telomeres to DNA double-strand breaks. Cell 97, 609-620, 1999). Reflecting this role, deletion of MEC1 increased cellular susceptibility to genotoxic agents in C. neoformans (Figure 11b), suggesting that Mec1's role in maintaining chromosome integrity may be preserved. It indicates that it is possible. Similar to the case of C. neoformans, deletion of MEC1 did not cause any lethality or growth defects in C. albicans (Legrand, M., Chan, CL, Jauert, PA & Kirkpatrick, DT The contribution of the S-phase checkpoint genes MEC1 and SGS1 to genome stability maintenance in Candida albicans. Fungal Genet Biol 48, 823-830, doi:10.1016/j.fgb.2011.04.005, 2011). Cka1 and Cka2 are the catalytic α-subunits of the protein kinase CK2 and are essential for growth and reproduction of S. cerevisiae, and deletion of both kinases causes lethality (Padmanabha, R., Chen-Wu, JL, Hanna , DE & Glover, CV Isolation, sequencing, and disruption of the yeast CKA2 gene: casein kinase II is essential for viability in Saccharomyces cerevisiae. Mol Cell Biol 10, 4089-4099, 1990). Interestingly, C. neoformans has been shown to contain a single protein (CKA1) that is orthologous to both Cka1 and Cka2. Deletion of CKA1, although not essential, was found to have a significant impact on growth in C. neoformans (Figure 11c). In particular, the cka1△ mutant showed an elongated and deformed cell shape (Figure 11d), which was comparable to two kinase mutants ( cbk1△ and kic1△ ) in the RAM pathway. Cbk1 and Kic1 are known to regulate cell polarity and morphology in C. neoformans, but their relationship to virulence is unknown (Walton, FJ, Heitman, J. & Idnurm, A. Conserved Elements of the RAM Signaling Pathway Establish Cell Polarity in the Basidiomycete Cryptococcus neoformans in a Divergent Fashion from Other Biol Cell, 2006). The present inventors revealed that cell polarity and morphology are related to virulence in C. neoformans.

Bud32 is also believed to be potentially essential for growth through its involvement in tRNA modification. Bud32 belongs to the piD261 family of atypical protein kinases conserved in bacteria, archaea, and eukaryotes, and unlike other eukaryotic protein kinases that recognize basic substances, it recognizes acidic substances (Stocchetto, S., Marin, O. , Carignani, G. & Pinna, LA Biochemical evidence that Saccharomyces cerevisiae YGR262c gene, required for normal growth, encodes a novel Ser/Thr-specific protein kinase. FEBS Lett 414, 171-175, 1997). In S. cerevisiae, Bud32 is an EKC (Endopetidase-like and Kinase-associated to transcribed Chromatin)/KEOPS (Kinase, Putative Endopeptidase, and Other Proteins of Small Size) Kinase, putative endopetidase and other proteins of small size) complex. This complex is required for N6-threonylcarbamoyladenosine (t6A) tRNA modification, which is important for maintaining codon-anticodon interactions for all tRNAs. Therefore, cells damaged in the EKC/KEOPS complex tend to have increased frameshift mutation rates and low growth rates (Srinivasan, M. et al. The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification , t6A. EMBO J 30, 873-881, doi:10.1038/emboj.2010.343, 2011). As expected, this defect in tRNA modification was shown to have dramatic effects on various biological aspects of C. neoformans, which in turn affects virulence. The bud32Δ mutant not only displayed highly defective growth under both normal and most stress conditions (Figure 12a), but also produced much lower amounts of capsule, melanin, and urease (Figure 12b). Additionally, the bud32 mutant showed a significant defect in sexual reproduction (Figure 12c). One exception was fluconazole resistance (Figure 12a). Interestingly, the present inventors confirmed that BUD32 deletion impaired ERG11 induction that occurred upon sterol deletion by fluconazole treatment (Figure 12d), indicating that Bud32 is involved in ergosterol gene expression and sterol biosynthesis in C. neoformans. suggests that it has a potential role.

Kinases involved in nutrient metabolism are also involved in the pathogenicity of C. neoformans. In S. cerevisiae, Arg5,6p is synthesized as a single protein, which is then processed within the mitochondrial matrix by two distinct enzymes, acetylglutamate kinase and N -acetyl-γ-glutamyl-phosphate reductase (Boonchird, C., Messenguy, F. & Dubois, E. Determination of amino acid sequences involved in the processing of the ARG5/ARG6 precursor in Saccharomyces cerevisiae. Eur J Biochem 199, 325-335, 1991). These enzymes catalyze ornithine biosynthesis, an intermediate in arginine biosynthesis. In support of this, the present inventors confirmed that the arg5,6p△ mutant is arginine-auxotrophic (Figure 13a). In S. cerevisiae, MET3, encoding ATP sulfurylase, catalyzes the initial step of the sulfur assimilation pathway, which generates hydrogen sulfide, the precursor for the biosynthesis of homocysteine, cysteine, and methionine (Cherest, H., Nguyen, NT & Surdin-Kerjan, Y. Transcriptional regulation of the MET3 gene of Saccharomyces cerevisiae Gene 34, 269-281, 1985; Ullrich, TC, Blaesse, M. & Huber, R. Crystal structure of ATP sulfurylase. from Saccharomyces cerevisiae, a key enzyme in sulfate activation. EMBO J 20, 316-329, doi:10.1093/emboj/20.3.316, 2001). In fact, the met3Δ mutant was found to be auxotrophic for both methionine and cysteine (Figure 13b). In particular, both arg5,6p△ and met3△ mutants did not show any growth defects in nutrient-rich medium (YPD), but showed severe growth defects under various stress conditions (Figure 13c), which is similar to arg5,6p△ and met3. △ may collectively be responsible for the virulence defects observed in the mutants.

Example 6: C. Retrograde vacuolar transport dominates the pathogenicity of neoformans.

A notable unknown biological function responsible for the pathogenicity of C. neoformans is retrograde vacuolar transport. It has already been reported that in C. neoformans, an ESCRT complex-mediated unusual vacuolar sorting process is involved in the virulence process, since several virulence factors, capsule and melanin, must be secreted extracellularly. (Godinho, RM et al. The vacuolar-sorting protein Snf7 is required for export of virulence determinants in members of the Cryptococcus neoformans complex. Scientific reports 4, 6198, doi:10.1038/srep06198, 2014; Hu, G. et al. Cryptococcus neoformans requires the ESCRT protein Vps23 for iron acquisition from heme, for capsule formation, and for virulence. Immun 81, 292-302, doi:10.1128/IAI.01037-12, 2013). Nevertheless, the role of endosome-to-Golgi retrograde transport in the virulence of C. neoformans is unknown. In the present invention, the present inventors found that deletion of CNAG_02680, which encodes a VPS15 ortholog involved in the vacuolar sorting process, significantly reduces virulence (Figure 14a). These results are consistent with the finding that mutation of VPS15 suppresses the virulence of C. albicans (Liu, Y. et al. Role of retrograde trafficking in stress response, host cell interactions, and virulence of Candida albicans. Eukaryot Cell 13, 279-287, doi:10.1128/EC.00295-13, 2014), strongly suggesting that the role of Vps15 in fungal virulence is evolutionarily conserved. In S. cerevisiae, Vps15 constitutes a vacuolar proteotypic complex (Vps15/30/34/38) that mediates endosome-to-Golgi retrograde protein transport (Stack, JH, Horazdovsky, B. & Emr, SD Receptor-mediated protein sorting to the vacuole in yeast: roles for a protein kinase, a lipid kinase and GTP-binding proteins. Annu Rev Cell Dev Biol 11, 1-33, doi:10.1146/annurev.cb.11.110195.000245, 1995. ).

To confirm the role of Vps15 in vacuole sorting and retrograde protein transport, the vacuole morphology of the vps15△ mutant was examined compared with that of the wild-type strain. Similar to the vps15△ null mutant of C. albicans, the C. neoformans vps15△ mutant also showed a highly expanded vacuole form (Figure 14b). Defects in retrograde vacuolar transport are known to cause extracellular secretion of the ER-intrinsic chaperone protein, Kar2 (Liu, Y. et al. Role of retrograde trafficking in stress response, host cell interactions, and virulence of Candida albicans. Eukaryot Cell 13, 279-287, doi:10.1128/EC.00295-13 (2014)). In support of this, the present inventors confirmed that the vps15Δ mutant is very sensitive to ER stress agents, such as dithiothreitol (DTT) and tunicamycin (TM) (Figure 14c) ). The main cause of the greatly weakened virulence of the vps15Δ mutant was its growth defect at 37°C (Figure 14d). This may result from increased cell wall and membrane instability in the vps15Δ mutant. In C. albicans, impaired retrograde transport in the vps15Δ mutant causes cell wall stress, which activates the calcineurin signaling pathway by transcriptionally upregulating CRZ1, CHR1 , and UTR2 (Liu, Y. et al. Role of retrograde) trafficking in stress response, host cell interactions, and virulence of Candida albicans Cell 13, 279-287, doi:10.1128/EC.00295-13, 2014). However, in C. neoformans, activation of the calcineurin pathway was not observed in this vps15Δ mutant (Figure 14e). The expression levels of CRZ1, CHR1 , and UTR2 in the vps15△ mutant were equivalent to those of the wild type strain. In C. neoformans, cell wall integrity is also controlled by the unfolded protein response (UPR) pathway (Cheon, SA et al. Unique evolution of the UPR pathway with a novel bZIP transcription factor, Hxl1, for controlling pathogenicity of Cryptococcus neoformans PLoS Pathog. 7, e1002177, doi:10.1371/journal.ppat.1002177 (2011). In previous studies, the present inventors have shown that activation of the UPR pathway through Ire1 kinase results in unusual splicing in HXL1 mRNA, thereby triggering an ER stress response (Cheon, SA et al. Unique evolution of the UPR pathway with a novel bZIP transcription factor, Hxl1, for controlling pathogenicity of Cryptococcus neoformans. PLoS Pathog 7, e1002177 (2011). In fact, we confirmed that VPS15 -deleted cells contained more spliced HXL1 mRNA (HXL1s) compared to the wild-type strain under basal conditions, which was expressed in retrograde vacuoles in C. neoformans. This suggests that when transport is perturbed, the UPR pathway may be activated instead of the calcineurin pathway.

Example 7: C. A novel virulence and infectivity regulatory kinase related to neoformance.

Among the 60 virulence-related kinases, 8 did not appear to have clear orthologs in model yeast and were accordingly named virulence-regulated kinase (Vrk1) or infectivity-regulated kinase 1-7 (irk1-7). In particular, deletion of Vrk1 (CNAG_06161) reduced the virulence of C. neoformans in the insect host model (Figure 5) and attenuated infectivity in the mouse host model (Figure 6), so we focused on Vrk1 (Figure 15). . Its closest yeast ortholog is Fab1 (score: 140.9, e-value: 3.2E-34), but the closest Fab1 ortholog in C. neoformans is CNAG_01209 (score: 349.7, e-value: 0.0) . Interestingly, VRK1 deletion increases cellular resistance to hydrogen peroxide and capsule formation (Figure 15a and b). In addition, cell resistance to 5-flucytosine increased and sensitivity to fludioxonil (Figure 15a). Based on our kinase variant phenotypic clusters (Figure 2), Vrk1 was not clearly grouped with other kinases. To further understand the regulatory mechanism of Vrk1, the present inventors performed comparative phosphoproteomics analysis on wild type and vrk1△ and identified Vrk1-specific phospho-target proteins. TiO ₂ -enriched based phosphoproteomics analysis suggested eight potential Vrk1 substrates: CNAG_04190 ( TOP1 , topoisomerase I), CNAG_01744 ( GPP2 , a DL-glycerol-3-phosphate phosphatase), CNAG_05661 (POB3). Heterodimer Fact Complex Sub Unit), CNAG_01972, CNAG_07381, CNAG_00055, CNAG_02943 ( SLM1 , A Force Deal Innositol-4,5 PHATE BINDING Protein), and CNAG_07878 ( NOC2 a nucleolar complex associated protein). CNAG_01972, 07381 and 00055 had no clear fungal orthology. Although it is not clear whether the candidate proteins are directly or indirectly phosphorylated by Vrk1, we found that five candidate proteins (TOP1, GPP2, POB3, CNAG_01972, and CNAG_07381) were damaged in the vrk1Δ mutant (Figure 15c), which suggests that they are dependent on Vrk1. It is suggested that it can be directly phosphorylated by To further understand the Vrk1-dependent functional network, the present inventors searched for Vrk1-regulated target proteins and any proteins functionally linked to Vrk1 itself using CryptoNet, and were able to construct their functional networks. CNAG_01972 and 00055 had no significant relationship to any known protein. Among the various potential biological functions associated with Vrk1 and its substrates, rRNA processing was the most overrepresented, suggesting that Vrk1 may be directly or indirectly involved in ribosome biogenesis and transport (Figure 15D).

Example 8: C. Kinase analysis related to antifungal drug resistance in Neoformans

Based on antifungal drug analysis of the kinase variant library, a total of 43, 38, and 42 kinases were identified as clinically used antifungal drugs amphotericin B (a polyene), fluconazole (an azole), and Increased or decreased susceptibility to flucytosine (a nucleotide analog) was shown, respectively (Table 4). For keykinases with deletions that increased susceptibility to these drugs, we identified 39 (for amphotericin B), 24 (for fluconazole), and 28 (for flucytosine) kinases. It was confirmed, and this could be developed as a parallel treatment target for each drug.

Kinases involved in antifungal drug resistance in Opomans Antifungal agents Kinase mutants showing
increased resistance Kinase mutants showing
increased susceptibility Polyene
(Amphotericin B) HRK1/NPH1, SPS1, SWE102 , TCO4 YPK1, VPS15, CBK1, HOG1, SSK2 ,PBS2, ARG5,6, GAL83, SNF1, MKK2, MPK1, BUD32, CKA1, IPK1, IRE1, CDC7, KIC1, PKA1 , CRK1, BCK1 , TCO2, IRK5 ,IGI1, GSK3, UTR1, MEC1, MET3, PAN3, MPS1 , PKH201, PIK1 ,HRK1, KIC102 , ALK1, TLK1, ARK1, IRK3, KIN1, POS5 Azole
(Fluconazole) GAL83, PAN3 , ALK1, TCO1, STE11, TCO2, SCH9, SSK2 ,PBS2, HOG1 , BUD32,PKA1 ,CHK1, YAK1 YPK1, VPS15, CBK1, MKK2, MPK1, IPK1, IRE1, BCK1 ,IGI1, GSK3, UTR1, PIK1 , HRK1/NPH1, CDC7 ,HRK1, PSK201, MPK2 , RAD53, ARG5,6, KIC1, KIC102 ,SPS1, IRK6 , MAK322 5-flucyotosine BCK1, PSK201, ARG5,6, GAL83 , TCO2, SNF1, IRK5 , PKH201, VRK1 , CKI1, TCO5, STE7, IGI1 , URK1 YPK1, VPS15, GSK3, UTR1 , HRK1/NPH1, SCH9, BUD32, CKA1, MEC1, FBP26, CBK1 , HOG1, IPK1, IRE1, SSK2, PBS2, MET3 , CDC7, KIC1, PAN3 ,TCO1, PKA1 , CHK1, CRK1, MPS1, CDC2801, TCO6, BUB1

* The underlined kinase was first identified in the present invention.

Example 9: Growth and chemical susceptibility testing

To analyze the growth and chemical sensitivity of the kinase variant library, C. neoformans cells grown overnight at 30°C were serially diluted 10-fold (from 0 to 10 ⁴ ) and spotted on YPD medium containing the following chemical reagents. were administered: 2 M sorbitol for osmotic stress and 1 to 1.5 M NaCl and KCl for cation/salt stress, under glucose-rich (YPD) or glucose-starved (YPD without dextrose; YP) conditions; Hydrogen peroxide (H ₂ O ₂ ), tert-butyl hydroperoxide (tan organic peroxide), menadione (superoxide anion generator), diamide (thiol-specific oxidizing agent) for oxidative stress. ; CdSO ₄ for toxic heavy metal stress; methyl methanesulfonate and hydroxyurea for genotoxic stress; Sodium dodecyl sulfate (SDS) for membrane destabilizing stress; calcofluor white and Congo red for cell wall destabilizing stress; tunicamycin and dithiothreitol (DTT) for ER stress and stress reduction; Fludioxonil, fluconazole, amphotericin B, and flucytosine for antifungal drug susceptibility. Cells were cultured at 30°C and photographed after 2 to 5 days of treatment. To test the growth rate of each variant at different temperatures, YPD plates spotted with serially diluted cells were incubated at 25°C, 30°C, 37°C, and 39°C and photographed after 2-4 days.

Example 10: Mating analysis

To analyze the mating phenotype of each kinase mutant, the MATα kinase mutants shown in Table 1 above were co-cultured with the mating partner, the serotype A MATa wild-type KN99a strain. Each kinase mutant MATα strain and MATa WT KN99a strain (obtained from Joeseph Heitman's laboratory, Duke University, USA) were cultured in YPD medium for 30 and 16 hours, pelleted, washed, and resuspended in distilled water. The resuspended α and a cells were mixed at the same concentration (10 ⁷ cells/ml), and 5 μl of them were spotted on V8 mating medium (pH 5). The mated plates were cultured in the dark at room temperature for 7 to 14 days and then observed weekly.

Example 11: In vitro virulence-factor production analysis

As a virulence-factor assay, capsule, melanin and urease production were examined for each kinase variant. Capsule formation was qualitatively analyzed by India Ink staining (Bahn, YS, Hicks, JK, Giles, SS, Cox, GM & Heitman, J. Adenylyl cyclase-associated protein Aca1 regulates virulence and differentiation of Cryptococcus neoformans via the cyclic AMP-protein Kinase A cascade. Cell 3, 1476-1491 (2004), each kinase mutant was grown overnight at 30°C in YPD medium and subjected to quantitative analysis using Cryptocrit. After spotting on solid medium (Eagle's), cells were cultured at 37°C for 2 days, washed with PBS, fixed with 10% formalin solution, and washed again with PBS. For the mutants, the cell concentration was adjusted to ³ The relative packed cell volume ratio was measured by calculating the packed cell volume ratio as the ratio of the length of the packed cell phase to the total phase (cell phase and liquid phase). The statistical difference in the relative packed cell volume ratio was confirmed by one-way ANOVA analysis with Bonferroni correction using Prism 6. (GraphPad software).

To test melanin production, each kinase variant was grown overnight in YPD medium at 30 °C, and 5 μl of each culture was spotted on Niger seed medium containing 0.1% or 0.2% glucose. The niger seed plates were incubated at 37°C and photographed after 3-4 days. For kinase variants showing growth defects at 37°C, melanin and capsule production were assessed at 30°C. To test urease production, each kinase variant was grown overnight in YPD medium at 30°C, washed with distilled water, and equal numbers of cells ( ⁵ Plates were incubated at 30°C for 2-3 days and photographs were taken.

Example 12: Insect-based in vivo virulence assay

For each C. neoformans strain tested, 15 Galleria mellonella larvae in the final instar larval stage weighing 200 to 300 mg were randomly selected as one group. For these larvae, larvae that arrived within 7 days from the date of transport were used (Vanderhorst Inc. St Marys, OH, USA). Each C. neoformans strain was grown in YPD liquid medium at 30°C overnight, washed three times, and then resuspended in PBS at the same concentration (10 ⁶ cells/ml). A total of 4,000 C. neoformans cells per 4-μl volume of larvae were inoculated through the second to last antrum using a 100 μl Hamilton syringe equipped with a 10 μl-size needle and a continuous dispenser (PB600-1, Hamilton). . An equal volume (4 μl) of PBS was used as a non-infected control. Infected larvae were placed in a Petri dish in a humidifying chamber, cultured at 37°C, and observed daily. Larvae that did not show movement when contacted were considered dead. Larvae that pupated during the experiment were examined for statistical analysis. Survival curves were displayed using Prism 6 (GraphPad). The log-rank (Mantel-Cox) test was used for statistical analysis. For each kinase variant, two independent variant strains were tested. For kinase variants with a single strain, the experiment was repeated twice.

Example 13: Marker mutagenesis-based mouse infectivity assay

For high-through murine infectivity analysis, a group of kinase variants with a NAT selection marker containing 45 different markers was pooled (a total of 4 groups). ste50△ and hxl1△ mutants were used as virulent and non-virulent control strains, respectively (Cheon, SA et al. Unique evolution of the UPR pathway with a novel bZIP transcription factor, Hxl1, for controlling pathogenicity of Cryptococcus neoformans. PLoS Pathog. 7 , e1002177, doi:10.1371/journal.ppat.1002177 (2011), Jung, KW, Kim, SY, Okagaki, LH, Nielsen, K. & Bahn, YS Ste50 adapter protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway. Biol 48, 154-165, doi:S1087-1845(10)00191-X [pii]10.1016/j.fgb.2010.10.006). The kinase variant library of each group was grown separately in YPD medium at 30°C for 16 hours and washed three times with PBS buffer. The concentration of each variant was adjusted to 10 ⁷ cells/ml, and 50 μl of each sample was placed in a tube. To prepare the input genomic DNA of each kinase variant pool, 200 μl of the variant pool was spread on a YPD plate, incubated at 30°C for 2 days, and then scraped. For preparation of output genomic DNA samples, 50 μl of variant pool (5 x 10 ⁵ cells per mouse) was infected into 7-week-old female A/J mice (Jackson Laboratory) via intranasal inhalation. Infected lungs were obtained from mice sacrificed by Avertin overdose 15 days after infection, homogenized in 4 ml PBS and spread on YPD plates containing 100 μg/ml chloramphenicol, and incubated at 30°C. It was cultured for 2 days and then scraped. Total genomic DNA was extracted from input and production cells by the CTAB method (Jung, KW, Kim, SY, Okagaki, LH, Nielsen, K. & Bahn, YS Ste50 adapter protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway. Biol 48, 154-165, doi:S1087-1845(10)00191-X [pii]10.1016/j.fgb.2010.10.006). Quantitative PCR was performed with the MyiQ2 real-time PCR detection system (BioRad) using the tag-specific primers listed in Tables 2 and 3. STM scores were calculated (Jung, KW et al. Systematic functional profiling of transcription factor networks in Cryptococcus neoformans. Nat Comms 6, 6757, doi:10.1038/ncomms7757 (2015)). To determine the STM score, the relative change in the amount of genomic DNA was measured using the 2 ^-△△CT method (Choi, J. et al. CFGP 2.0: a versatile web-based platform for supporting comparative and evolutionary genomics of fungi and Oomycetes. Nucleic Acids Res 41, D714-719, doi:10.1093/nar/gks1163 (2013)). The average fold change of input versus production samples was calculated as a log score (Log ₂ 2 ^{-(Ct,Target - Ct,Actin)output - (Ct,Target - Ct,Actin)input} ).

Example 14: Vacuole staining

To visualize vacuole morphology, the wild-type H99S strain and vsp15Δ strains (YSB1500 and YSB1501) (obtained from Joeseph Heitman's laboratory, Duke University, USA) were cultured in liquid YPD medium for 30 to 16 hours. FM4-64 dye (Life Technologies) was added to each culture at a final concentration of 10 μM and incubated at 30°C for 30 minutes. Cells were pelleted by centrifugation, resuspended in fresh liquid YPD medium, and further incubated at 30°C for 30 minutes. The cells were pelleted again, washed three times with PBS buffer, and then resuspended in 1 ml of PBS. 10 ml of cells and 10 ml of mounting solution (Biomeda) were mixed and spotted on a glass slide. The slide glass was observed with a confocal microscope (Olympus BX51 microscope).

Example 15: TiO ₂ -Enrichment-based phosphoproteomics

To identify the phosphorylated targets of Vrk1 at a genomic scale, H99S and vrk1△ mutant strains were cultured in YPD broth for 16 h at 30 °C, subcultured into 1 liter of fresh YPD broth, and then incubated at 600 nm (OD ₆₀₀ ) and were further incubated at 30°C until an OD of approximately 0.9 was reached. Each of the total cell lysates was incubated with 50 mM Tris-Cl (pH 7.5), 1% sodium deoxycholate, 5 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, and 50 mM. It was prepared with lysis buffer (Calbiochem) containing NaF, 0.1% SDS, 1% Triton . The protein concentration of each cell lysate was measured using the Pierce BCA protein kit (Life Technology). Reduce sulfhydryl bonds between cysteine residues in the protein lysate by incubating 10 mg of total protein lysate with 10 mM DTT for 1 hour at room temperature, followed by alkyl incubation with 50 mM iodoacetamide for 1 hour at room temperature in the dark. Rated. These samples were again treated with 40 mM DTT for 30 minutes at room temperature and then digested with trypsin (Sequencing grade trypsin, Promega) at a substrate ratio of 1:50 (w/w) by incubation at 37°C overnight. Next, the trypsin-digested protein lysate was purified using a Sep-Pak C18 column (Waters corporation, Milford, MA), lyophilized, and stored at -80°C. Phosphopeptides were incubated with TiO2MagSepharose beads (GE Healthcare) and then lyophilized for LC-MS/MS analysis. Q Exactive Hybrid Dionex U 3000 RSLC nano HPLC system equipped with nano-electrospray ionization source and Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, MA, USA) fitted with fused silica emitter tips (New Objective, Wobum, MA). Mass spectrometry was performed using . All phosphopeptide samples were reconstituted in solution A (water/acetonitrile (98:2, v/v), 0.1% formic acid) and injected into the LC-Nano ESI-MS/MS system. Samples were first trapped on an Acclaim PepMap 100 trap column (100 ㎛ id After washing with Solution A, the cells were separated on an Acclaim PepMap 100 capillary column (75 ㎛ id x 15 cm, nanoViper C18, 3 ㎛ particle size, 100 Å pore size, Thermo Scientific) at a flow rate of 400 nl/min. Peptides were added in a 2% to 35% solution B (water/acetonitrile (2:98, v/v), 0.1% formic acid) gradient over 90 minutes, in a 35% to 90% gradient. The analysis was performed for 10 minutes, then at 90% for 5 minutes, and finally at 5% for 15 minutes. The resulting peptide was electrosprayed with a coated silica tip (PicoTip emitter, New Objective, MA, USA) at an ion spray voltage of 2,000 eV. To assign peptides, MS/MS spectra of C. neoformans var. grubii H99 protein database (www.uniprot.org). The following search parameters were used: cysteine carbamidomethylation as a fixed modification, methionine oxidation and serine/threonine/tyrosine phosphorylation as variable modifications. Peptide identification was possible by two miss cleavages. Peptide identification was filtered with a 1% FDR cutoff. The relative excess amount of phosphopeptides of wild type and mutant was predicted by spectral counts. To evaluate differences between samples, Student's t-test was used.

Example 16: ER Stress Assay

To monitor ER stress-mediated UPR induction, H99S and vps15Δ mutant strains were cultured in YPD at 30°C for 16 h, subcultured into fresh YPD liquid medium, and then reached early logarithmic growth phase (OD600 = 0.6). Further culture was performed at 30°C. Cells were treated with 0.3 μg/ml tunicamycin (TM) for 1 hour. The cell pellet was immediately frozen in liquid nitrogen and lyophilized. Total RNAs were extracted with easy-BLUE (total RNA extraction kit, iNtRON Biotechnology), and cDNA was synthesized with MMLV reverse transcriptase (Invitrogen). HXL1 splicing patterns (UPR-induced spliced form of HXL1 [ HXL1S ] and unspliced form of HXL1 [ HXL1U ]) were analyzed by PCR using cDNA samples from each strain and primers (B5251 and B5252). analyzed (Table 3).

Example 17: Expression analysis

To analyze the expression of ERG11 , the H99S strain and the bud32Δ mutant were cultured in liquid YPD medium at 30°C for 16 hours in YPD, and then sub-cultured with fresh liquid YPD. When cells reached early logarithmic growth phase (OD ₆₀₀ = 0.6), the culture was divided into two samples: one treated with fluconazole (FCZ) for 90 min and one untreated. The cell pellet was immediately frozen in liquid nitrogen and lyophilized. Total RNA was extracted, and Northern analysis was performed on the total RNA samples for each strain as previously reported (Jung, KW, Kim, SY, Okagaki, LH, Nielsen, K. & Bahn, YS Ste50 adapter protein governs sexual differentiation of Cryptococcus neoformans via the pheromone-response MAPK signaling pathway. Fungal Genet 48, 154-165, doi:S1087-1845(10)00191- 2011)). For quantitative reverse transcription-PCR (qRT-PCR) analysis of genes involved in the calcineurin pathway, the H99S strain and the vps15Δ mutant were cultured in liquid YPD at 30°C for 16 h and grown to early logarithmic growth phase (OD ₆₀₀₎ in fresh liquid YPD medium. = 0.8) was secondary cultured. Next, the cells were pelleted by centrifugation, immediately frozen in liquid nitrogen, and then lyophilized. After extracting total RNAs, cDNA was synthesized using RTase (Thermo Scientific). CNA1, CNB1, CRZ1, UTR2 and ACT1 -specific primer pairs (B7030 and B7031, B7032 and B7033, B7034 and B7035, B7036 and B7037, B679 and B680, respectively) (Table 3) were used for qRT-PCR.

Example 18: Construction of FPK1 overexpression strain

To create a strain overexpressing FPK1, the promoter of FPK1 was replaced with the promoter of histone H3 using an amplified homologous recombination cassette (Figure 4a). In the first round PCR, primer pair L1/OEL2 was used for the 5'-flanking region of FPK1 , and primer pair OER1/PO was used for the 5'-coding region of FPK1 . The NEO-H3 promoter was amplified with primer pair B4017/B4018. P _H3 : For the second round PCR of the 5' or 3' region of the FPK1 cassette, the first round result was over-processed by DJ-PCR with the primer pair L1/GSL or GSR/PO (primers in Tables 2 and 3). It was made to be wrapped. Next, the PH3:FPK1 cassette was introduced into the wild-type strain H99S (obtained from the Joeseph Heitman laboratory at Duke University, USA) and the ypk1Δ mutant strain (YSB1736) by biolistic transformation. Stable transformants were selected on YPD medium containing G418 and screened by diagnostic PCR using the primer pair (SO/B79). The correct genotype was confirmed by Southern blot using a specific probe amplified by PCR using primers L1/PO. Overexpression of FPK1 was confirmed by a specific Northern blot probe amplified by PCR using primers NP1 and PO (Figure 4b, c).

Example 19: Kinase phenome clustering

The in vitro phenotypic characteristics of each kinase variant were scored using the following quantitative criteria: -3 (strongly susceptible or defective), -2 (moderately susceptible or defective), -1 (weakly susceptible or defective), 0 (like wild type). , +1 (mild resistance or enhancement), +2 (medium resistance or enhancement), and +3 (high resistance or enhancement). Excel files containing phenotypic scores for each variant were uploaded to Gene-E software (http://www.broadinstitute.org/cancer/software/GENE-E/), and kinase phenome clustering was performed using one-minus Pearson correlation ( It can be derived as One minus Pearson correlation).

Example 20: Cryptococcus Kinome Web-Database

In order to provide public access to phenomic and genomic data for the C. neoformans kinase variant library constructed by the present inventors, a Cryptococcus Kinase Database was developed (http://kinase.cryptococcus.org/). C. neoformans var. grubii H99 was downloaded from the Broad Institute (https://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.html) and analyzed by the Comparative Fungal Genomics Platform (CFGP). 2.0; http://cfgp.snu.ac.kr/) (Choi, J. et al. CFGP 2.0: a versatile web-based platform for supporting comparative and evolutionary genomics of fungi and Oomycetes) Nucleic Acids Res 41, D714-719, doi:10.1093/nar/gks1163 (2013). Protein kinase classification was performed using the HMMER sequence profile of SUPERFAMILY (version 1.73) (Wilson, D. et al. SUPERFAMILY--sophisticated comparative genomics, data mining, visualization and phylogeny. Nucleic Acids Res 37, D380-386, doi :10.1093/nar/gkn762 (2009)). A total of 64 families belonging to 38 superfamilies were used for potential kinase prediction. Also, Kinomer (version 1.0) (Martin, DM, Miranda-Saavedra, D. & Barton, GJ Kinomer v. 1.0: a database of systematically classified eukaryotic protein kinases. Nucleic Acids Res 37, D244-250, doi:10.1093/nar /gkn834 (2009); Miranda-Saavedra, D. & Barton, G.J. Classification and functional annotation of eukaryotic protein kinases, doi:10.1002/prot.21444 (2007)) Sequence Profile and Microbial Kinome (Kannan) , N., Taylor, SS, Zhai, Y., Venter, JC & Manning, G. Structural and functional diversity of the microbial kinome. PLoS Biol 5, e17, doi:10.1371/journal.pbio.0050017 (2007). Kinase prediction was supplemented using . C. neoformans var. predicted by InterProScan 62. grubii H99 and the genome annotation of protein domains were adopted to find the maximum coverage of likely kinase-encoding genes. For each gene, results from eight bioinformatics programs were provided to provide clues for gene annotation. Additionally, results from SUPERFAMILY, Kinomer, and Microbial Kinome are presented to support the robustness of the prediction. The gene is C. neoformans var. In case of orthologs in neoformans JEC21, a link to the KEGG database was also provided. Seoul National University Genome Browser (SNUGB; http://genomebrowser.snu.ac.kr/) to view genome context with important biological properties (Jung, K. et al. SNUGB: a versatile genome browser supporting comparative and functional fungal) genomics. BMC Genomics 9, 586, doi:10.1186/1471-2164-9-586 (2008)) is provided in the Cryptococcus Kinase Phenome Database. In the Kinase Browser, a direct link to the SNUGB module is provided for each gene. The Cryptococcal Kinase Phenome Database is provided with MySQL 5.0.81 (source code distribution) for database management and PHP 5.2.6 for web interface. The web-based user interface is provided through the Apache 2.2.9 web server.

Korea Center for Microbial Conservation (KCCM) KCCM51297 20151106

Claims (3)

In determining an antifungal agent, a method of screening an antifungal agent to determine virulence control of Cryptococcus neoformans comprising the following steps:
(a) contacting the sample to be analyzed with Cryptococcus neoformans containing IRK4, YAK1, MEC1, VPS15 or VRK1 as a pathogenicity-regulating serine/threonine protein kinase ;
(b) measuring the amount or activity of the protein; and
(c) determining that the sample is an antifungal agent when the amount or activity of the protein is measured to be down regulated.
In determining an antifungal agent, a method of screening an antifungal agent to determine virulence control of Cryptococcus neoformans comprising the following steps:
(a) contacting the sample to be analyzed with Cryptococcus neoformans containing genes encoding IRK4, YAK1, MEC1, VPS15 or VRK1 as virulence-regulated serine/threonine protein kinases;
(b) measuring the expression level of the gene; and
(c) determining that the sample is an antifungal agent when the expression level of the gene is measured to be down-regulated.
The antifungal agent screening method according to claim 1 or 2, wherein the antifungal agent is used to treat meningoencephalitis or cryptococcosis.