Use of pyridoxatin and related analogs for control of candida auris

Abstract

Fungi are prolific producers of secondary metabolites and represent a rich and relatively untapped source for novel drug leads. Numerous successful therapeutics have derived from fungi, including penicillin, cephalosporin, statins, and cyclosporin. However, many promising classes of natural product are never developed into drugs because their chemical synthesis is complex or because the native organisms that naturally produce them, do so at impractically low levels. Pyridoxatins are a class of promising anti-fungal secondary metabolites produced by several naturally occurring strains filamentous fungi. The molecules have complex structures that impede straightforward chemical synthesis or analoging to increase efficacy. The disclosure herein details novel a novel use of pyridoxatin class molecules for the treatment of Candida auris and novel methods for the biosynthesis of pyridoxatins through fermentation of engineered microbes.

Description

[0001] USE OF PYRIDOXATIN AND RELATED ANALOGS FOR CONTROL OF CANDIDA AURIS

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the synthesis of biochemical molecules of the pyridoxatin class and methods of use of pyridoxatin and its functional chemical analogs for the control of Candida species.

[0004] BACKGROUND

[0005] In general, fungal pathogens pose a significant but often overlooked problem for human health, causing a variety of diseases including infections of the skin, respiratory' tract, and systemic infections. According to the World Health Organization, approximately 1.5 million people die from fungal infections each year (WHO, Fungal diseases factsheet. (2019) www.who.int / news-room / fact-sheets / detail / fungal-diseases) These diseases can be difficult to treat, as there has been a general underinvestment in antimicrobial medicine development; existing antifungal drugs often have toxic side effects and many fungal pathogens have now evolved resistance to available drugs.

[0006] First reported in the United States in 2016, the fungal pathogen Candida auris has spread at an alarming rate. C. auris is a yeast that can cause severe illness and spreads easily among patients in healthcare facilities. The pathogen is characterized by high rate of resistance with reduced susceptibility to azoles, polyenes, and echinocandins. In particular, higher resistance to fluconazole in a Candida non-albicans species has become one of the distinguishing characteristics indicative of a potential C. auris infection (Ademe and Girma. 2020. Infect Drug Resist.; 13: 1287-1294. doi: 10.2147 / IDR.S249864). As such, the CDC now rates it as an “urgent threat / ’ and a cause for the highest level of concern (Lyman et al, 2023 Ann Intern Med. doi: 10.7326 / M22-3469.). The rise of C. auris has also made clear the need for a wider antifungal armamentarium given the resistance observed to all three major classes of systemic antifungals (Forsberg et al. 2018. Med. My col, 57: 1-12; doi.org / 10.1093 / mmy / myy054).

[0007] The emergence of virulent pathogens such as Candida auris, and the increasing prevalence of multi-drug resistant strains now makes it an urgent priority to develop new antifungal agents. Underscoring this need, both Candida auris and Candida albicans feature in the WHO’s recently published “Critical group” of top 5 most fungal pathogens of highest concern (WHO, 2022, WHO fungal priority pathogens list to guide research, development, and public health action, www.who.int / publications / i / item / 9789240060241). Natural products have long been recognized as a valuable source of drugs and crop protection chemicals (Newman, D.J. and Cragg. G.M., 2020. J. Nat. Prod. 83: 770-803; Sparks, T. C., et. al., 2019. Pesticide Biochem. Physiol, doi: 10.1016 / j. pestbp.2019.09.002; Katz, L. and Baltz, R. H., 2016. J. Ind. Microbiol. Biotechnol. 43: 155-176). Pyridoxatin is a natural product that was originally identified from fungi and which has been described as having activity against Candida albicans (Chang et al. (2015) Biochimica et Biophysica Acta 1850 (2015) 1762-1771. However, the molecule has not previously been demonstrated as effective against auris. Furthermore, it is widely considered that auris is generally a much tougher pathogen to control than albicans and thus one cannot expect that a compound that has efficacy against albicans will also have efficacy against auris.

[0008] Pyridoxatin A

[0009] The chemical structure of pyridoxatins shown after Ohashi et al., 2020, Nature 586, 64). A range of different analogs of the pyridoxatin molecule class can be produced by introducing substituent chemical groups at the numbered atoms. Such substituent groups can be introduced by traditional organic chemical synthesis or by so called synthetic biology based “gene- shuffling” approaches whereby component genes from within the pyridoxatin gene clusters from different fungal strains are reassorted to generate novel clusters that produce a new functional analog.

[0010] Many secondary metabolites are produced in insufficient quantities by their native source organisms, or they are too complex for complete or semi-synthesis and these barriers have hindered the development of many promising molecule classes. Traditionally, attempts to overcome such challenges typically involved either classic genetic selection of a strain that produces elevated levels of a target molecule, optimizing the media / fermentation of the native producer organism, co-cultivation with antagonistic or mutualistic organisms to stimulate production, and / or semisynthesis from naturally -produced precursors followed by subsequent modification. Recent advances in genomics and synthetic biology now offer the possibility of either genetically engineering source organisms to produce desired metabolites at tractable levels or transferring the requisite biosynthesis genes into heterologous fermentation microbes (Milne, N., et al. 2020. Metabol. Eng. doi.org / 10.1016 / j.ymben.2019.12.007; Choi, K. R. et al. 2019. Trends Biotechnol. doi: 10.1016 / j.tibtech.2019.01.003; Cravens, A. et al. 2019. Nature Comm. 10: doi: 10.1038 / s41467-019-09848-w; Luo. X. et al. 2019. Nature. doi: 10.1038 / s41586- 019-0978-9; Galame, S, et al. 2015. Science 349: 1095-1 100. doi: 10.1126 / science.aac9373; Schafer, H., and Wink, M., 2009. Prog. Alkaloid Biosynth. Biotechnol. J. 4: 1684-1703. doi: 10. 1002 / biot.200900229.).

[0011] Many natural products produce general cellular toxicity, which can hamper their use as drugs. In such instances a chemical synthesis (or semi-synthesis approach) can be used to generate analogs of the original natural product which retain the desired activity but which have reduced general toxicity. Pyridoxatin is such a molecule; it is challenging and costly to produce by chemical synthesis and has been reported to show toxicity against mammalian cells. Shang et al.. 2015 noted that pyridoxatin exhibited potent but non-selective cytotoxicity (IC50 0.3-0.4 pM) against three human cancer cell lines (Shang et al. Org. Biomol. Chem., 2015, D01: 10.1039 / C50B01058F). Additionally, it was reported that pyridoxatin inhibited activated gelatinase A with an IC50 of 15.2uM. It also had a strong cytotoxicity against human cancer cell lines in vitro and inhibited DNA synthesis with an IC50 of 2.92 pM in PC-3 prostate cancer cells (Lee et al., 1996, Journal of Microbiology and Biotechnology 6, 445 -450). Other researchers, however, note that pyridoxatin shows relatively low toxicity in human cell lines; the compound has been reported as non-toxic when tested up to 121.6 pM in non-neoplastic, immortalized human prostatic epithelial (RWPE-1) cells and normal human bronchial epithelium (HBE) cells (Winter et al., Phytochemistry Letters 55 (2023) 88-96, and references therein).

[0012] Pyridoxatins are produced naturally in small quantities by certain genera of fungi, including Tolypocladium sp. (Winter et al., Phytochemistry Letters 55 (2023) 88-96). Albophoma sp. and Aspergillus sp. (Ohashi et al., 2020, supra). Herein we provide methods of use of pyridoxatin to control Candida auris and methods of producing novel analogs of pyridoxatin which have activity against Candida auris but a reduced level of toxicity in mammalian cells.

[0013] The genomes from the pyridoxatin producing strains of the above genera can be compared to closely related non-pyridoxatin producing strains from the same genera in bioinformatic analyses to identify the biosynthetic gene clusters (BGCs) that encode for the biosynthesis of these molecules. The component genes from these gene clusters are then “mixed and matched’" via a “gene shuffling” approach and are then transformed into heterologous organisms to produce novel analogs through fermentation at scale.

[0014] SUMMARY

[0015] Advances in low-cost DNA sequencing / synthesis and powerful bioinformatic tools now enable a predictive, genomics-driven approach to drug discovery (Foulston, L., 2019. Curr. Opin. Microbiol. 51 : 1—8; Baltz, R. H., 2019. J. Indust. Microbiol. Biotechnol. 46:281-299; Medema M.H. and Fischbach M.A. 2015. Nat. Chem. Biol. 11:639-648). Because many microbially-derived complex natural products are encoded by chromosomally linked biosynthetic gene clusters (BGCs), it is possible to identify and annotate the genes responsible for production of these valuable natural products from fungi (Blin, K., et al., 2019. Nucl. Acids Res. 47(W1), W81-W87; Tran, P. N„ et al., 2019Appl. Microbiol. Biotechnol. 103:3277-3287. doi: 10. 1007 / s00253-019-09708-z; Van der Lee, T. A. J., and Medema. M. H„ 2016. Fungal Gen. Biol. 89: 29-36. doi: 10.1016 / j.fgb.2016.01.006).

[0016] Many natural products exhibit “biological activity” which is a term used in pharmacology to describe the beneficial or adverse effects of a molecule or substance of interest on living cells. Among the various properties of a chemical compound, biological activity is of particular interest since it suggests the potential utility of the compound in medical applications. However, a chemical compound may show adverse and toxic effects which may prevent its use in medical practice. Activity is generally dependent on the applied dose of a compound. Furthermore, it is common to have effects ranging from beneficial to adverse for a compound when going from low to high doses. Such activity ranges must be determined in experimental tests. Activity depends critically on fulfillment of the ADME criteria. To be an effective drug, a compound not only must be active against a target disease, but also possess the appropriate ADME (Absorption, Distribution, Metabolism, and Excretion) properties necessary to make it suitable for use as a drug. Additionally, a compound often requires extensive formulation and / or optimization via semisynthetic analoging (i.e., applying chemical synthesis in the lab to change the side groups on a molecular backbone produced through fermentation) to fine tune its ADME properties. Thus, development of a compound of interest into a drug requires access to substantial quantities of the compound for optimization work and ultimately for inclusion in extensive experimental trials. The methods detailed herein provide a new means of producing a natural product molecule at sufficient scale so as to be able to test its biological activity and potential utility and then develop it into drug in an efficient and cost-effective manner. Despite the promise of pyridoxatin-as an antifungal drug, its use has been hampered by the fact that native fungi that produce it do so at relatively low levels, and by the fact that it is challenging to make de novo via traditional chemical synthesis. To further evaluate and develop pyridoxatin for therapeutic drug use, a larger supply of the compound is needed for both in vivo studies and analog development via semisynthesis. The disclosure herein addresses that challenge and details a platform for production of pyridoxatins in engineered fermentation microbes.

[0017] Methods and compositions for production of pyridoxatin and other natural products in fermentation microbes are described and the utility of genes isolated from fungi in biosynthesizing novel pyridoxatin analogs is presented. A putative pyridoxatin gene cluster has recently been reported (Ohashi et al., 2020, Nature 586. 64). However, to date its use in heterologous fermentation to produce pyridoxatin and or its analogs has not been demonstrated.

[0018] DETAILED DESCRIPTION

[0019] Pyridoxatin was discovered by Teshima et al., in 1991. The molecule has also shown antimicrobial activity against methicillin-resistant Staphylococcus aureus and Gram-negative Escherichia and an in vivo model with Caenorhabditis elegans showed effectiveness of pyridoxatin as an antifungal agent, promoting survival at doses comparable to fluconazole. The compound has been reported as non-toxic when tested up to 121.6 pM in non-neoplastic, immortalized human prostatic epithelial (RWPE-1) cells and normal human bronchial epithelium (HBE) cells (Winter et al., Phytochemistry Letters 55 (2023) 88-96, and references therein).

[0020] Pyridoxatins occur in nature as a suite of variants built on a common backbone. In this application, we show for the first time, that a pyridoxatin class molecule inhibits the growth of Candida auris. This activity, combined with the potential low toxicity of pyridoxatin and / or its analogs implies that molecules of the class can be included as active ingredients in drug formulations used to treat Candida auris infections.

[0021] Research and possible wide-spread use of this high potential molecule class has been hindered by of the high cost of samples of the compounds for testing, which in turn stems challenges in fermentation of the native producer strains and the complexity of the chemical structure for traditional synthesis. Typically, “analoging” involves the chemical synthesis of a set of molecules (which are often called structural “analogs” or “analogues”) with common core structural features, such as the backbone shown in structure of pyridoxatin (supra) and then testing to select specific molecules from the set which have a high level of a desired biological activity (e.g., an in vivo effect such as inhibiting growth in a Candida assay, while minimizing non-specific toxic effects. Such an approach is difficult to pursue in cases when the chemical synthesis is technically challenging and / or costly.

[0022] Here we present heterologous expression for producing a refactored fungal pyridoxatin biosynthetic gene cluster in commercially viable fermentation microbes (e.g., in yeast, Aspergillus, E. coli or Streptomyces) . This enables a plug-and-play approach to create new pyridoxatin analogs derived from related gene clusters discovered from across multiple fungal strains and even those of other kingdoms to create a diversity of molecules for screening and development and further semisynthetic lead optimization studies.

[0023] Yeast and several model fungal species (e.g., A. nidulans and A. oryzae) have been successfully deployed to express fungal gene clusters (Harvey, C. J.et al. 2018. Sci. Advances 4: eaar5459; Alberti, F. et al., 2017. Appl. Microbiol. Biotechnol. 101:493-500; Skellam E. 2018. Trends Biotechnol. 37:416-427. doi: 10. 1016 / j.tibtech.2018.09.003). However, the yeast chassis requires extensive engineering for the proper expression of fungal gene clusters and may suffer from low yield. On the other hand, the model fungal species are generally slow growing and demand long experimental timelines to achieve any desirable genetic optimization. In contrast, Streptomyces species are prokaryotes that grow relatively quickly and produce an enormous array of structurally and functionally diverse natural products, including a large number of compounds with the dioxopiperazine core (e.g., thaxtomins). However, Streptomyces have not been reported as natural producers of pyridoxatins and heterologous expression of pyridoxatins in either Streptomyces or in any other fermentation microbe has not previously been described.

[0024] DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0025] As used herein and in the appended claims, the singular forms "a." "an." and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a plant” includes a plurality of such plants, and a reference to “a stress” is a reference to one or more stresses and equivalents thereof known to those skilled in the art, and so forth.

[0026] Laboratory analytical procedures related to chemical analysis of metabolites and selection of microbial strains for metabolgenomic studies have been published by Amrine, C.S.M. et al., 2018. J. Ind. Microbiol. Biotechnol. 45: 1053-1065 (and references therein)

[0027] Mycology7: Fungal cultures referenced herein were originally obtained from Mycosynthetix, Inc., via an ongoing project to identify diverse natural product study material as a source of anticancer drug leads (Kinghom A.D. et al.. 2016. Anticancer Res. 36:5623-5637). The following Mycosynthetix strains (designated MSX strains) were identified as potential pyridoxatin producers from metabolomics studies (see Table 1, below). Table 1. Mycosynthetix strains identified as potential pyridoxatin producers from metabolomics studies

[0028] The pyridoxatin BGCs from the above strains provide a tool-box for creating artificially constructed pyridoxatin BGCs for expression in heterologous fermentation microbes to produces a variety of analogs.

[0029] For genomic and synthetic biology based approaches we reference the following; PCR protocols: A guide to methods and applications. Academic Press, San Diego). Methods for BLAST search in NCBI GenBank and Maximum Likelihood phylogenetic analysis, as utilized to identify fungal strains, have been detailed recently (Raja. H.A. et al. 2017. supra

[0030] Media and fermentations: For the analysis of microbial cultures in Petri dishes, seven types of agar media were used, and ingredients to make these were purchased from Difco™, Acros Organics™, and Research Products International (RPI): malt extract agar, potato dextrose agar, yeast extract soy peptone dextrose agar, Spezieller Nahrstoffarmer agar, Sabouraud dextrose agar, potato dextrose mushroom and oatmeal agar (See Table IS of Amrine et al., 2018, supra, for compositions). For example, a small piece (~ 0.5 cm2) of fresh culture grown on potato dextrose agar was cut from the leading edge of 2-week-old colony from two strains and was used to inoculate various types of agar media in duplicate, respectively. The plates were then incubated at room temperature until the cultures showed good growth as noted by the expansion of the colony over the surface of the medium (~ 3 to 4 weeks).

[0031] Rice fermentations for culture of fungal strains [(1: 1 commercial white rice (variety: Calrose Botan; 5 g) and (variety: Sona Masoori; 5 g)] were prepared by adding 10 g of rice to a Petri dish or a 250 mL flask with 25 mL of deionized H2O, followed by autoclaving at 221 °C for 30 min. Oatmeal media (Old fashioned Quaker oats) was prepared using 10 g of rolled oats and ~ 17 mL of deionized FLO, followed by autoclaving at 221 °C for 30 min. The flasks containing the rice or oatmeal media were inoculated with a seed culture grown in 10 mL of YESD broth media (2% soy peptone, 2% dextrose and 1% yeast extract) for about 5 days with agitation (100 rpm) at room temperature. The inoculated cultures were grown at room temperature for ~ 4-5 weeks, except for the time progression studies, where each duplicate of the oatmeal culture was extracted every few days, from days 2 to 35. Strains were scaled-up in a similar way, using 2.8 L Fembach flasks (Coming, Inc., Coming, NY, USA). The medium was prepared using 150 g of either rice or oatmeal, with 300 rnL or 250 rnL of deionized H2O added for rice medium and oatmeal medium, respectively, and both sterilized at 221 °C for 30 min. In addition, and as noted in more detail in the supplemental information of Amrine et al. 2018 (See Fig. S7 of Amrine et al., 2018, supra), augmenting the oatmeal media with amino acids was also examined. However, this did not result in significant differences in the biosynthesis of pyridoxatins, and thus, this line of inquiry was not pursued further.

[0032] Extraction of microbial cultures for chemical analysis: Each culture was chopped with a spatula to ensure thorough extraction. For the cultures grown on Petri dishes, the chopped pieces were transferred into 150 rnL flasks. Solutions of 60 rnL of 1:1 chloroform:methanol (CHClwMeOH) were poured in each flask, followed by overnight shaking (100 rpm) at room temperature. The cultures were filtered by vacuum, and then the filtrates were mixed with 90 mL of CHCh and 150 mL of H2O and stirred for 30 min. The mixtures were transferred into a separatory funnel, where the bottom organic layer was evaporated in vacuo and reconstituted with 100 mL of 1 : 1 acetonitrile:methanol (CHsCN:MeOH) and 100 rnL hexanes. Again, the defatted organic layers w ere collected and evaporated in vacuo.

[0033] In situ analysis of metabolite profiles can be undertaken via the droplet probe coupled with UPLC-PDA-HRMSMS / MS: The use of the droplet probe with microbial cultures, including fungal cultures, has been detailed previously (Sica V.P. et al., 2015. J. Nat. Prod .78: 1926-1936) and several examples have been published (Paguigan, N.D. et al., 2016. Phytochem. 126:59-65; Sica, V.P. et al., 2016a. Anal. Methods 8:6143-6149; Sica, V.P. et al., 2016b. J. Ind. Microbiol. Biotechnol. 43: 1 149-1157; Sica V.P. et al., 2017. Phytochem. 143:45- 53; Sica V.P. et al., 2016c. Front. Microbiol .7:544. doi.org / 10.3389 / fmicb.2016.00544). A CTC / LEAP HTC Pal auto-sampler converted to a droplet probe with assistance from colleagues at Oak Ridge National Laboratories (Kertesz, V., and Berkel, G.J.V., 2014. Rapid Commun. Mass Spectrom. 28: 1553-1560; Kertesz, V., and Van Berkel. G.J., 2010. Anal. Chem. 82:5917- 5921). Microextractions (3-5 pL) are performed on precise spots directly on fungal cultures using a 1: 1 solution of Me0H:H20. The extractions are injected onto a UPLC, and the chromatographic method followed previous protocols (ELElimat. et al. 2013. J. Nat. Prod. 76: 1709-1716) used in the dereplication of fungal extracts. A Waters Acquity UPLC system is used with a BEH C 18 column (Waters; 50 mm x 2. 1 mm x 1.8 pm) heated to 40 °C and a mobile phase using a gradient from 15 to 100% CH3CN; the other solvent was 0.1% formic acid-I I2O. The run utilized a flow rate of 0.3 mL / min for 10 min. Spectrometer data are collected from 190 to 500 nm and the eluent split into a Thermo Fisher Scientific Q Exactive™ Plus mass spectrometer via electrospray ionization (ESI). MS data are collected from 150 to 2000 m / z at a resolution of 70,000 while alternating between positive and negative modes. MS / MS was performed with an HCD value of 35, and Xcalibur™ software was used for the primary analysis.

[0034] Analysis of microbial metabolite profiles via UPLC-PDA-HRMSMS / MS: Extracts of the plates and flasks are dissolved separately in 1 :1 MeOH:dioxane to elaborate concentrations of 2.5 x 10-2, 0.5 or 2.0 mg per mL; 3 pL are injected directly onto the UPLC-PDA- HRMSMS / MS system. In addition to HRMS and MSMS data, retention times and ultraviolet data are used as mutually supportive data.

[0035] Bioinformatic analysis: The present invention may include an integrated system, computer or computer readable medium that comprises an instruction set for determining the identity of one or more sequences in a database. In addition, the instruction set can be used to generate or identify sequences that meet any specified criteria. Furthermore, the instruction set may be used to associate or link certain functional benefits, such improved characteristics, with one or more identified sequence.

[0036] For example, the instruction set can include, e.g., a sequence comparison or other alignment program, e.g., an available program such as, for example, the Wisconsin Package Version 10.0, such as BLAST. FASTA, PILEUP, FINDPATTERNS or the like (GCG. Madison, WI). Public sequence databases such as GenBank, EMBL, Swiss-Prot and PIR or private sequence databases can be searched.

[0037] Alignment of sequences for comparison can be conducted by the local homology algorithm of Smith, T.F., and Waterman, M.S., 1981. Adv. Appl. Math. 2: 482-489, by the homology alignment algorithm of Needleman, S.B. and Wunsch, C.D., 1970. J. Mol. Biol. 48: 443-453, by the search for similarity method of Pearson, W.R. and Lipman, D.J., 1988. Proc. Natl. Acad. Sci. 85: 2444-2448, by computerized implementations of these algorithms. After alignment, sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a comparison window to identify' and compare local regions of sequence similarity. The comparison window can be a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 contiguous positions. A description of the method is provided in Current Protocols in Molecular Biology. Ausubel et al. eds.. Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2000). A variety of methods for determining sequence relationships can be used, including manual alignment and computer assisted sequence alignment and analysis. This later approach is a preferred approach in the present disclosure, due to the increased throughput afforded by computer assisted methods. As noted above, a variety of computer programs for performing sequence alignment are available, or can be produced by one of skill.

[0038] One example algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul, S.F., et al., 1990. J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available, e.g., through the National Library' of Medicine's National Center for Biotechnology7Information (www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 1 1, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff, S., and Henikoff, J.G., 1992. Proc. Natl. Acad. Sci. 89: 10915-10919). Unless otherwise indicated, "‘sequence identity” here refers to the % sequence identity generated from a tblastx using the NCBI version of the algorithm at the default settings using gapped alignments with the filter “off’ (see, for example, NIH NLM NCBI website at www.ncbi.nlm.nih.gov / ).

[0039] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin, S. and Altschul, S.F., 1993. Proc. Natl. Acad. Sci. 90: 5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence (and, therefore, in this context, homologous) if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, or less than about 0.01, and or even less than about 0.001. An additional example of a useful sequence alignment algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. The program can align, e g., up to 300 sequences of a maximum length of 5,000 letters.

[0040] The integrated system, or computer typically includes a user input interface allowing a user to selectively view one or more sequence records corresponding to the one or more character strings, as well as an instruction set which aligns the one or more character strings with each other or with an additional character string to identify one or more region of sequence similarity. The system may include a link of one or more character strings with a particular phenotype or gene function. Typically, the system includes a user readable output element that displays an alignment produced by the alignment instruction set.

[0041] The methods of this disclosure can be implemented in a localized or distributed computing environment. In a distributed environment, the methods may be implemented on a single computer comprising multiple processors or on a multiplicity of computers. The computers can be linked, but more preferably the computer(s) are nodes on a network. The network can be a generalized or a dedicated local or wide-area network and, in certain preferred embodiments, the computers may be components of an intra-net or an internet, or ‘'cloud” computing platforms like that offered by Amazon Web Services.

[0042] Thus, the disclosure provides methods for identifying a sequence similar or homologous to one or more polynucleotides as noted herein, or one or more target polypeptides encoded by the polynucleotides, or otherwise noted herein and may include linking or associating a given phenofype such as the capacity for cellular biosynthesis of a target molecule with a sequence. In the methods, a sequence database is provided (locally or across an inter or intranet) and a query is made against the sequence database using the relevant sequences herein and associated phenotypes or functions in the cellular biosynthesis of target molecules.

[0043] Any sequence herein can be entered into the database, before or after query ing the database. This provides for both expansion of the database and, if done before the querying step, for insertion of control sequences into the database. The control sequences can be detected by the query to ensure the general integrity of both the database and the query. As noted, the query can be performed using a web browser-based interface. For example, the database can be a centralized public database such as GenBank, or a private database, and the querying can be done from a remote terminal or computer across an internet or intranet.

[0044] Any sequence herein can be used to identify a similar, homologous sequence in a fungal genome by the methods identified above. A homologous polynucleotide sequence that has a conserv ed or equivalent functionality in the cellular biosynthesis of a target molecule such as a pyndoxatin typically has at least 70%. 71%. 72%. 73%. 74%. 75%. 76%. 77%. 78%. 79%. 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% to a polynucleotide sequence from a BGC of a producer strain that is known to produce the target molecule. A homologous polypeptide that has a conserved or equivalent functionality in the cellular biosynthesis of a target molecule, such as a pyridoxatin, which is homologous to a polypeptide encoded by a polynucleotide selected from a BGC of a producer strain that is known to produce the target molecule, typically possesses at least 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,

[0045] 72%, 73%, 74%, 75%, 76%. 77%, 78%. 79%. 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%.

[0046] 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or about 100% amino acid identity' to the polypeptide encoded by the polynucleotide selected from a BGC of a producer strain.

[0047] Genome mining to identify biosynthetic gene clusters (BGCs): An example of using a comparative analysis approach to identify BGCs needed to produce a target metabolite, by comparing the genomes of producer strains with non-producing strains was published by Villani, A., et al., 2019. BMC Genomics, 20. doi: 10.1186 / sl2864-019-5567-7, who were able to identify' critical functional elements within BGCs that control the expression levels and structure of secondary metabolite mycotoxins. Such an approach can be applied to identify BGCs for pyridoxatins in a target genome.

[0048] Typically, up to 5 pg of DNA is purified from each selected strain, with DNA samples being prepared at a concentration in the range of 2-200 ng / pL for sequencing. Standard methods are used for DNA extraction that utilize commercially available kits (with instructions provided therein) such as the ZYMO RESEARCH CORP. Quick-DNA™ Fungal / Bacterial Miniprep Kit Catalog No. D6005 (www. zymoresearch. com).

[0049] Various service providers offer commercial DNA sequencing services, including SeqMatic (Fremont, CA: www. seqmatic.com / ) which provides Illumina® -based short-read sequencing that can be used to generate fungal genome sequence datasets. In some instances, nanopore sequencing is also be used and a combined analysis of short read and long-read sequences is then performed to identify intact BGCs. BGC identification takes advantage of the processing capability- of an Amazon Web Sendees (AWS) cloud infrastructure (or comparable system) to perform analyses both iteratively (to improve the outcomes based on feedback) and in parallel (to examine a large number of samples). Specifically, paired-read FASTQ files are screened using FastQC to gather statistics on per-base sequence quality, read length, and GC percentage (Andrews, S., 2010. www.bioinformatics.babraham.ac.uk / projects / fastqc ). These metrics are used to select samples for further investigation and Trimmomatic (Bolger A.M., et al., 2014. Bioinformatics. 30:2114-2120) is used to remove remaining Illumina ® adapter sequences. The reads are then be assembled into contigs and scaffolds using Velvet and the Velvet Optimiser, which runs various assemblies to choose the best candidate (Zerbino D.R. and Birney E„ 2008. Genome Res. 185:821-829. doi: 10. 1101 / gr.074492.107; Gladman S.L. et al. 2009. www.vicbioinformatics.com / software.shtml). AWS Parallelcluster, AWS Elastic Container Service, and AWS Batch, collectively enable rapid analyses at reasonable cost. BLAST searches are then conducted against known genomic databases for quality evaluation and gene discovery (Altschul, et al., 1990. supra). Contigs and draft assemblies are then analyzed with Fungal antiSMASH (Blin, K., et al., 2019. supra) to identify putative BGCs. Using known BGCs from publicly-available sequences as “ground truth” cases, one skilled in the art can calibrate the search parameters and use those settings for BGC discovery process. Additional tools are also deployed to identify and classify BGCs based on the presence of a candidate gene encoding the potential target of the lead molecule within the biosynthetic cluster, e.g., ARTS (Alanjary, M, et al., 2017. Nucleic Acids Res. 2017. Jul 3;45(W1):W42-W48).

[0050] Generation of genetically modified microorganisms and genetically modified cells: Transgenic cells, microorganisms, yeasts, fungi, (or plant cells, or plant explants, or plant tissues) incorporating the polynucleotides of the disclosure and / or expressing the polypeptides of the disclosure can be produced by a variety of well-established techniques. Following construction of a vector, most typically an expression cassette, including one or more polynucleotides from an identified BGC, e.g., encoding an enzyme or a transcription factor or a homolog thereof, of the disclosure, standard techniques can be used to introduce the polynucleotide into a cell to create a genetically modified cell or cell hne. Optionally, in the case that the organism is capable of multicellular grow th, such as in the case of a plant cell, the plant cell, explant, or tissue can be regenerated to produce a transgenic plant.

[0051] Transformation and multiplication and / or regeneration of cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the organism to be transformed; those skilled in the art will recognize the suitability of particular methods for given organism types. Suitable methods may include, but are not limited to: electroporation of protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; Li-mediated transformation, transformation using viruses; micro-injection of cells; micro-projectile bombardment of cells; vacuum infiltration; and Agrobacterium tumefaciens mediated transformation. In this disclosure, transformation involves introducing a recombinant nucleotide sequence into a host cell in a manner to cause stable or transient expression of the sequence so as to result in expression of the encoded polypeptide which in turn results in the production of a desired target molecule.

[0052] Successful methodologies for the generation of genetically modified microorganisms, including genetically modified fungi are provided in Kawai, et al., 2010, Bioeng. Bugs. 1: 395- 403. Exemplary methods of transformation of Streptomyces are provided by Pigac and Schrempf, 1995, Appl Environ Microbiol. 61 : 352-356.

[0053] Following transformation, genetically modified cells may be preferably selected using a dominant selectable marker incorporated into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed cells, and selection of transformants can be accomplished by exposing the cells to appropriate concentrations of the antibiotic or herbicide. Alternatively, color-based markers such as GFP or GUS may be used to select transformed cells, or transformed cells may be selected based on detection of expression of the polynucleotide in the introduced expression cassette by RT-PCR or detection of a target molecule produced by the genetically modified cell.

[0054] Generation of genetically modified organisms through targeted gene editing: a variety of methods are available to a skilled practitioner to enhance the activity of genes within BGCs, including BGCs for pyridoxatin biosynthesis, through targeted modification of the native loci in the native producer microorganism. Such methods include deletion, insertion and / or substitution of one or more nucleotides, site-specific mutagenesis, chemical mutagenesis, targeting induced local lesions in genomes (TILLING), gene editing techniques using a transcription activator-like effector nuclease (TALEN) or zinc finger nuclease (ZFN) based method, or gene editing using a CRISPR-Cas endonuclease technique that utilizes a nuclease selected from Cas nuclease, Cas9 nuclease, Casl2 nuclease, CasX nuclease, CasY nuclease, a Cpfl nuclease, a C2cl nuclease, a C2c2 nuclease (Casl3a nuclease), or a C2c3 nuclease, NgAgo nuclease, or gene editing using a technique that utilizes base editing deaminases, engineered site-specific meganucleases, or Argonaute. A guide to the application of CRISPR is provided at .addgene.org / guides / crispr / . Nodvig et al. (PLoS One. 2015; 10: e0133085) have described a CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi, which can be deployed to introduce changes to BGCs of the disclosure to increase production of target molecules such as pyridoxatins in the native producer strains.

[0055] To confirm that the changes in expression levels or activity of the polypeptide or polynucleotide of the disclosure are associated with the production of a target molecule by the genetically modified microorganism, analysis of mRNA expression can be performed using Northern blots. RT-PCR, or microarrays, or RNASeq or protein expression using immunoblots or Western blots or gel shift assays.

[0056] EXAMPLES

[0057] The invention, now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention. It will be recognized by one of skill in the art that a genetic modification that is associated with a particular first trait may also be associated with at least one other, unrelated and inherent second trait which was not predicted by the first trait.

[0058] EXAMPLE 1A. Identification of Novel Pyridoxatin Class Molecule Producing Biosynthetic Gene Clusters

[0059] DNA is extracted from a fermentation of a strain of fungus selected from a genus of Tolypocladium, Albophoma, Aspergillus, Acremonium, Diaporthe, or Verticilhum and obtained at a concentration above 10 ng / pL. The DNA is subjected to short read Illumina sequencing and / or Nanopore sequencing. Contigs are assembled and mined to identify BGCs. A novel pyridoxatin BGC is identified, which contains new features as compared to the putative pyridoxatin BGC previously postulated by Ohashi et al. 2020, supra.

[0060] EXAMPLE IB. Specific Identification of a PYR Candidate Gene Cluster From a Tolypocladium Strain.

[0061] The full genome of strain 50556. a Tolypocladium species (SEQ ID NO 12), was examined bioinformatically and a BGC encoding orthologs for the pyridoxatin gene cluster in Albophoma (adx) as proposed by Ohashi et al. 2020 supplemental) was discovered using BLAST. These protein sequences are listed in Table 2. Table 2. ADX orthologs in strain 50556

[0062] EXAMPLE 2. Demonstration of Anti-Candida Auris Activity of PYR and PYR-Producing Strains Using a Bioassay.

[0063] Isolates of antibiotic-resistant Candia auris strains were obtained from the CDC and used in a bioassay adapted from Vinuesa and Fernandez, 2021. One C. auris strain, (#1101) has relatively low levels of antibiotic resistance (e.g., 0.047 ug / ml minimum inhibitory' concentration (MIC) for Amphotericin B). Candida auris strains 381 and 390 have increasing levels of AmpB resistance (0.38 or 4.0 ug / ml / MIC, respectively). This assay can be used to test pure antifungal compounds for their efficacy against resistant C. auris or other related pathogens, as well as to identify antifungal activity in complex natural product broths, such as fungal fermentation extracts.

[0064] In one assay, all wells of a 96-well microplate contained 200 pL of 1x106cells / mL C. auris strain #1101, and 5 pL of test solution (control, or experimental fungal extract from Clue Library ). Normal Candida growth was observable as a layer of clumped cells as seen in DMSO negative control wells. Inhibition of C. auris growth was observed as a reduction or absence of clumped cells compared to the DMSO control, as seen in the Amphotericin positive control wells. Treatments were arranged as below:

[0065] Column 1 A-H: Amphotericin B: 8, 4, 2, 1 , 0.5, 0.25, 0.125, 0.0625 pg / mL

[0066] Column 2A-H: Amphotericin B: 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625 pg / mL

[0067] Column 3A-H: Pyridoxatin: 12.5, 6.25, 3.125, 1.56, 0.78, 0.39, 0.20, 0.10 pg / mL

[0068] Column 4A-H: DMSO: 0.025%. 0.0125%, 0.006%, 0.003%, 0.002%. 0.001%, 0.0004%. 0%, (in water) Positive AmpB controls showed expected levels of control, aligned with the reported MICs for each strain. Pyridoxatin showed good efficacy above a concentration of 3.125 pg / mL. and was similarly effective against C. auris strains of varying levels of AmpB resistance.

[0069] In another use of the bioassay, 283 different fungal extracts were screened. These extracts were initially selected based on previously-observed activity of each of these 283 extracts against a related pathogen, Candida albicans, in a similarly performed bioassay. Here, the assay was used to determine if anti-Candida albicans activity is a reliable predictor of antiCandida auris activity. Of the 283 extracts, 42 showed strong inhibitory effects on the three different Candida auris strains described above; that is, only -15% of the antifungal extracts showed activity against the more difficult to control Candida auris pathogen.

[0070] Among the extracts tested in the above experiment were two from fungal strains known to be pyridoxatin producing isolates. Strain 50556 (SEQ ID NO 12) and strain 41370 (SEQ ID NO 11) both showed strong or moderate activity, respectively, against the three different Candida auris strains tested.

[0071] EXAMPLE S. EXAMPLE S. Expression of Novel Pyridoxatin Class Molecules by Heterologous Expression in Aspergillus or another fermentation microbe

[0072] Expression of a refactored pyridoxatin BGC is demonstrated in Aspergillus, or other fermentation microbe. Portions of. and the entire pyridoxatin gene cluster, are expressed in the Aspergillus or other microbial system to produce a biosynthetic intermediate of pyridoxatin. This enables the practitioner to take advantage of BGCs from a diverse set of fungal strains to produce a spectrum of pyridoxatins. Full genome sequences from a selection of the strains are used to identity' the pyridoxatin BGC from each strain using the bioinformatic and genome mining techniques detailed herein. Novel pyridoxatins with novel activity’ can then be produced by a mix and match approach. The variations in analogous gene cluster member sequences from the strains enables the design of optimized expression cassettes for a variety of pyridoxatin analogs that form a library of lead molecules for anti-Candida screening and further semisynthetic lead optimization studies.

[0073] Heterologous expression of a pyridoxatin gene cluster is demonstrated by re-engineering and codon-optimization of the pyridoxatin gene cluster (originally postulated by Ohashi et al. 2020, supra) for expression in an Aspergillus or other microbial host. The BGC member genes can be introduced into the target strain in the same expression cassette, or they can be added in a stepwise manner by subsequent transformations via additional expression cassettes.

[0074] Following expression of one or more of the pyridoxatin BGC member genes in the genetically modified microorganism, fermentation extracts are analyzed via LC-MS alongside a positive control extract prepared from a native pyridoxatin producer strain (see Table 1, supra), and / or a wild-type strain of the genetically modified microorganism, and / or a pure pyridoxatin standard, to demonstrate and quantify the production of either the intermediates or the final pyridoxatin class molecule itself.

[0075] EXAMPLE 4. Approaches to Increase the Level of Pyridoxatin Produced by a Fungal or other Microbial Fermentation

[0076] The approaches detailed herein demonstrate a substantial improvement on the prior art and enable the production of a broad diversify pyridoxatin class molecules at levels greater than 0.001 mg per g, 0.01 mg per g, 0. 1 mg per g, 1 mg per g, or greater than 2 mg per g, or greater than 3 mg per g, or greater than 4 mg per g, or greater than 5 mg per g, or greater than 6 mg per g, or greater than 7 mg per g, or greater than 8 mg per g, or greater than 9 mg per g, or greater than 10 mg per g, or greater than 20 mg per g, or greater than 30 mg per g, or greater than 40 mg per g, or greater than 50 mg per g, or greater than 60 mg per g, or greater than 80 mg per g, or greater than 100 mg per g, or greater than 200 mg per g of microbial fermentation extract. Or alternatively, from liquid fermentation pyridoxatin class molecules can be produced at levels of greater than 0.001 mg per rnL, 0.01 mg per mL, 0. 1 mg per mL, 1 mg per mL, or greater than 2 mg per mL, or greater than 3 mg per mL, or greater than 4 mg per mL, or greater than 5 mg per mL, or greater than 6 mg per mL, or greater than 7 mg per mL, or greater than 8 mg per mL, or greater than 9 mg per mL, or greater than 10 mg per mL, or greater than 20 mg per mL, or greater than 30 mg per rnL, or greater than 40 mg per mL, or greater than 50 mg per rnL, or greater than 60 mg per rnL, or greater than 80 mg per mL, or greater than 100 mg per mL, or greater than 200 mg per mL of microbial fermentation extract.

[0077] EXAMPLE 5. Generation of Analogs of Pyridoxatin by Chemical Synthesis or Semisynthesis Using A Molecule with the Same Backbone as Pyridoxatin as a Feedstock

[0078] Pyridoxatin was the first pyridoxatin class molecule to be described. Many additional analogs can be created artificially via the methods detailed herein through combinatorial expression of specific pyridoxatin BGC gene members, and / or semi-synthesis approaches or traditional synthetic approaches to substitute groups at the atomic positions numbered on the pyridoxatin class molecule shown, supra.

[0079] EXAMPLE 6. Identification of Specific Fungal Isolates That Are Previously Unrecognized Producers of Pyridoxatin Class Molecules from Collection of Candidate Fungal Isolates. Fungal genomic DNA is isolated from candidate isolates (strains) using the Quick-DNA Fungal / Bacterial Miniprep Kit from Zymo Research. PCR is then used to amplify the ITS 1 region according to methods by Raja et al., 2017 (Raja et al., 2017: J. Nat. Prod. 2017, 80, 3, 756-770, doi.org / 10.1021 / acs.jnatprod.6b01085) =. Resulting PCR products (“barcodes”) are DNA-sequenced (Eurofins), and barcodes are aligned to the barcodes from one or more of the barcodes from the strains shown in Table 1 using BLASTN (Zheng Zhang, Scott Schwartz, Lukas Wagner, and Webb Miller (2000), J. Comput. Biol. 2000; 7(l-2):203-14.) and / or the rRNA_typestrains / ITS_RefSeq_Fungi database at NCBI (BioProject PRJNA177353) (Table 3). Generally, the hit with the lowest E-value is selected as the best taxonomic match for each barcode. Fungal strains that are identified with 98% or higher level of identity' in the barcode to a strain from Table 1 are then regrown in “scintillation” vials containing growth medium for several days and the presence of pyridoxatins is confirmed by HPLC and / or mass spectrometry.

[0080] Table 3. PYR-producing Strain Barcodes Used to Assign Genus and Species

[0081] EXAMPLE 7. Methods for Cloning of and Heterologous Expression of Pyridoxatin Molecule Class Producing Biosynthetic Gene Clusters

[0082] Intact pyridoxatin BGCs are cloned (using the putative BGC postulated by Ohashi et al. 2020 supra, from source microbial strains in Table 1 and expressed in a heterologous expression microbe such as Aspergillus using the following general method:

[0083] Materials:

[0084] Source microbial strain with the desired pyridoxatin biosynthetic gene cluster:

[0085] • CRISPR / Cas9 system plasmids (e.g., pCas9 and pUC19-sgRNA)

[0086] • Restriction enzymes

[0087] • E. coli DH5a cells

[0088] • Agar plates with appropriate antibiotics

[0089] • Aspergillus nidulans or other heterologous host

[0090] • Transformation reagents specific to the host

[0091] Protocol: 1. Design of guide RNAs: Design guide RNAs (sgRNAs) that target the desired pyridoxatin biosynthetic gene cluster using commercially available software, such as Benchling or CRISPOR. The sgRNAs should target regions flanking the gene cluster and include restriction enzyme recognition sites.

[0092] 2. Cloning of the sgRNAs: Clone the sgRNAs into the plasmid pUC 19-sgRNA using restriction enzymes. Transform the ligation mixture into E. coli DH5a cells, and plate onto agar plates containing appropriate antibiotics. Select colonies that contain the recombinant plasmid with the sgRNAs.

[0093] 3. CRISPR / Cas9 cleavage: Transform the Pyridoxatin producer strain with the pCas9 plasmid expressing the Cas9 protein using transformation reagents specific to the host. Transfect the sgRNA plasmid to express the sgRNA that guides the Cas9 protein to cleave the target genomic region, including the desired pyridoxatin biosynthetic gene cluster.

[0094] 4. Cloning of the gene cluster: Digest the genomic DNA from the CRISPR / Cas9 treated Pyridoxatin producer strain using the restriction enzymes specific to the sgRNA sites. Purify the digested genomic DNA and the vector using commercially available DNA purification kits. Ligate the digested genomic DNA into the vector using DNA ligase. Transform the ligation mixture into E. coli DH5a cells, and plate onto agar plates containing appropriate antibiotics. Select colonies that contain the recombinant vector with the intact pyridoxatin biosynthetic gene cluster.

[0095] 5. Heterologous expression: Transform the recombinant vector containing the intact pyridoxatin biosynthetic gene cluster into the heterologous host microbe (e.g., Aspergillus nidulans) using transformation reagents appropriate to the host. Plate onto agar plates containing appropriate antibiotics to select for transformants.

[0096] Screening: Screen the transformants for expression of the desired pyridoxatin products by analyzing the culture supernatants or extracts for the presence of the compounds using methods such as high-performance liquid chromatography (HPLC) or mass spectrometry (MS). Confirm the identify of the compounds by comparing their spectral data with that of the known pyridoxatin compounds

[0097] Although the specification has been described with reference to specific embodiments and examples, it should be understood that one of ordinary skill can make various modifications without departing from the spirit of this specification.

Claims

CLAIMSWhat is claimed is:

1. A method of inhibiting the grow th of Candida auris cells, wherein C. auris cells are contacted with a chemical formulation that contains a molecule of the pyridoxatin class.

2. The method of claim 1, wherein the molecule of the pyridoxatin class is present at a concentration of at least 3. 125 pg / mL.

3. The method according to claim 1 or claim 2, wherein the pyridoxatin class molecule is pyridoxatin.

4. A genetically modified microorganism comprising either:(i) a recombinant polynucleotide that includes a pyridoxatin biosynthetic gene cluster (BGC) nucleotide, or(ii) a non-naturally occurring polynucleotide that has been produced by gene editing, or selection of a spontaneous or induced mutation; and w herein the genetically modified microorganism produces an increased level of a target pyridoxatin class molecule compared to a control microorganism that does not include the recombinant polynucleotide.

5. The genetically modified microorganism of claim 4, wherein the microorganism is a strain of a genus of fungus selected from the set: Tolypocladium, Albophoma, Aspergillus, Acremonium, Diaporthe, and Verticillium.

6. The genetically modified microorganism of claim 4, wherein the recombinant or non- naturally occurring polynucleotide encodes a polypeptide with at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to a polypeptide selected from the group SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

7. The genetically modified microorganism of claim 4, wherein one or more open reading frames within the recombinant polynucleotide is obtained from a fungal strain of the genus Tolypocladium, Albophoma, Aspergillus, Acremonium, Diaporthe, or Verticillium, or a fungal strain which has been selected based on its genome sequence harboring an ITS sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NO: 1 1.

12. 13, 14, 15, and 16.

8. The genetically modified microorganism of claim 4, wherein the genetically modified microorganism is selected from the group consisting of a yeast, a fungus, a bacterium, a cell line, and an alga.

9. The genetically modified microorganism of claim 4, wherein the genetically modified microorganism is selected from the genus Streptomyces, the genus Saccharomyces, the genus Aspergillus.

10. The genetically modified microorganism of claim 4, wherein the pyridoxatin class molecule obtained from fermentation of the microorganism is used to inhibit the growth of a fungus of the genus Candida.

11. The genetically modified microorganism of claim 4, wherein growth of the fungus is inhibited is Candida auris.

12. The genetically modified microorganism of claim 4, wherein the increased level of the target pyridoxatin class molecule is at least 5%, at least 10% higher, at least 25% higher, at least 50% higher, at least 75% higher, at least 100% higher, at least 200% higher, at least 500% higher or at least 1000% higher than in the control microorganism.

13. The genetically modified microorganism of claim 4, wherein the target pyridoxatin class molecule is produced at a level of greater than 0.001 mg per g, 0.01 mg per g, 0. 1 mg per g, 1 mg per g, or greater than 2 mg per g, or greater than 3 mg per g, or greater than 4 mg per g, or greater than 5 mg per g, or greater than 6 mg per g, or greater than 7 mg per g, or greater than 8 mg per g, or greater than 9 mg per g, or greater than 10 mg per g, or greater than 20 mg per g, or greater than 30 mg per g, or greater than 40 mg per g, or greater than 50 mg per g, or greater than 60 mg per g, or greater than 80 mg per g. or greater than 100 mg per g, or greater than 200 mg per g of microbial fermentation extract.

14. The genetically modified microorganism of claim 4, wherein the target pyridoxatin class molecule is produced at a level of greater than 0.001 mg per mL, 0.01 mg per mL, 0.1 mg per mL, 1 mg per mL, or greater than 2 mg per mL, or greater than 3 mg per mL, or greater than 4 mg per mL, or greater than 5 mg per mL, or greater than 6 mg per mL, or greater than 7 mg per mL, or greater than 8 mg per mL, or greater than 9 mg per mL, or greater than 10 mg per mL, or greater than 20 mg per mL, or greater than 30 mg per mL, or greater than 40 mg per mL. or greater than 50 mg per mL, or greater than 60 mg per mL, or greater than 80 mg per mL. or greater than 100 mg per mL, or greater than 200 mg per mL of microbial fermentation extract.

15. The genetically modified microorganism of claim 4, wherein the genetic modification is introduced by a process of transformation, gene editing, gene insertion, gene re-arrangement, gene shuffling, homologous recombination, or mutagenesis.

16. The recombinant polynucleotide of claim 4, wherein one or more open reading frames within the recombinant polynucleotide is obtained from a fungal strain that was identified based on it harboring within its genome a nucleotide sequence with at least 98%, at least 99% or 100% identity to a sequence selected from the group SEQ ID NO: 1 1, 12, 13, 14, 15, and 16.

17. A microbial biomass produced from the genetically modified microorganism of claim 4.

18. A biologically active pyridoxatin class molecule produced by purification of the microbial biomass of claim 17.

19. A method for producing a target pyridoxatin class molecule, the method comprising: selecting a fungal strain that produces the target molecule; preparing DNA from a fermentation of the fungal strain at a concentration of at least 10 ng / pL, at least 20 ng / pL. at least 30 ng / pL, at least 40 ng / pL; at least 50 ng / pL, at least 75 ng / pL, at least 100 ng / pL, at least 500 ng / pL or at least 1000 ng / pL; sequencing the DNA preparation and conducting bioinformatic assembly of the resulting sequence reads to obtain contiguous sequences of the genome of the fungal strain; computationally identity ing a biosynthetic gene cluster that contains at least one gene that encodes a polypeptide that functions in the biosynthesis of the target pyridoxatin class molecule: introducing into a host microorganism a recombinant polynucleotide that includes the biosynthetic gene cluster or introducing into the fungal strain that produces the target molecule a gene edit that modifies the activity of biosynthetic gene cluster; and selecting a cell of the microorganism that contains an increased level of the target molecule compared to a control cell of the host microorganism that lacks the recombinant polynucleotide, and then using the selected microorganism to produce the target molecule in a fermentation broth from which it can be purified from.

20. A method of producing a pyridoxatin class molecule, comprising the step of fermentation of a microorganism of the genus selected from the group consisting of Tolypocladium, Albophoma, Aspergillus, Acremonium, Diaporthe, and Verticillium.

21. A method of producing a pyridoxatin class molecule comprising:(i) growing a collection of candidate isolates of fungi;(ii) extracting DNA from one or more isolates;(iii) sequencing the DNA from regions comprising ITS sequences;(iv) selecting an isolate where the ITS sequence obtained is at least 98% identical to a reference ITS sequence from a species of one of the list of genera: Tolypocladium, Albophoma. Aspergillus, Acremonium, Diaporthe, and Verticillium;(v) growing the selected isolate in a fermentation process; and(vi) confirming the presence of the pyridoxatin class molecule in the fermentation product by a process that comprises chromatography, mass spectrometry and / or nuclear magnetic resonance.

22. The method of claim 21, wherein the ITS sequence has at least 98%, at least 99% or 100% identity to a sequence selected from the group consisting of SEQ ID NO: 11, 12, 13, 14, 15, and 16.

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