FIELD OF THE INVENTION

The invention relates to a recombinant fungal cell. The invention further relates to a culturing method. The invention further relates to a production method. The invention further relates to a growth medium. The invention further relates to a use of the recombinant fungal cell.

BACKGROUND OF THE INVENTION

Anaerobic cultivation of fungal cells is known in the art. Snoek and Steensma, 2007, describes that for S. cerevisiae the biosynthesis of sterols requires oxygen, and that for the synthesis of one molecule of ergosterol, 12 molecules of molecular oxygen are needed. It further describes that under anaerobic conditions the S. cerevisiae cells no longer synthesize sterols, but instead import them, and that this sterol uptake is essential under anaerobic conditions.

Labate Valde Da Costa, et al., “Forever panting and forever growing: physiology of Saccharomyces cerevisiae at extremely low oxygen availability in the absence of ergosterol and unsaturated fatty acids”, FEMS Yeast Research, Volume 19, Issue 6, September 2019, describes an investigation regarding the dependence of the growth of Saccharomyces cerevisiae under low-oxygen conditions on the anaerobic growth factors ergosterol and oleic acid.

Dekker et al., “Anaerobic growth of Saccharomyces cerevisiae CEN.PK113-7D does not depend on synthesis or supplementation of unsaturated fatty acids”, FEMS Yeast Research, Volume 19, Issue 6, September 2019, describes an evaluation of the dependence of a S. cerevisiae strain on unsaturated fatty acid and sterol supplementation during anaerobic conditions.

Macy and Miller, “Anaerobic growth of Saccharomyces cerevisiae in the absence of oleic acid and ergosterol?”, Archives of Microbiology, 134, 64-67, 1983, describes anaerobic cultivation of S. cerevisiae on a Yeast Nitrogen Base medium under a CO2- atmosphere.

Campbell and Msongo, “GROWTH OF AEROBIC WILD YEASTS”, Journal of the Institute of Brewing, 1991, describes anaerobic cultivation of wild yeasts of the genera Debaryomyces, Hansenula and Pichia , as well as of S. cerevisiae.

Dumitru et al., “Defined Anaerobic Growth Medium for Studying Candida albicans Basic Biology and Resistance to Eight Antifungal Drugs”, Antimicrobial Agents and Chemotherapy, 2004, describes a chemically defined anaerobic growth medium for four strains of Candida albicans.

Huseyin et al., “The Fungal Frontier: A Comparative Analysis of Methods Used in the Study of the Human Gut Mycobiome”, frontiers in Microbiology, 2017, describes cultivation of fungi from the human gut on four different media.

US20030207317A1 describes nucleic acids isolated from Tetrahymena which code for a ciliate-specific triterpenoid cyclase, and describes the use of nucleic acids for the regulation of triterpenoid cyclase expression in a host organism, as well as the targeted knockout or repriming of the triterpenoid cyclase gene.EP3042960Al describes a method for producing ambrein, comprising reacting a tetraprenyl-P-curcumene cyclase with 3- deoxyachilleol A to obtain ambrein. W02018157021A1 describes a squalene hopene cyclase isolated from Gluconobacter morbifer as well as variants thereof and a method for using the G. morbifer SHC to biocatalytically convert homofamesol to ambroxan.

SUMMARY OF THE INVENTION

Saccharomyces yeasts have a long history in anaerobic biotechnology. Already, S. cerevisiae may be responsible for what may be the single largest product of modern industrial biotechnology: (‘bio’-)ethanol as an automotive biofuel. The world-wide production of this yeast fermentation product, which may predominantly be made from com starch and cane sugar, may currently be ca. 65 million tonnes/year. Concerns about competition with food and feed production may provide strong incentives for development of production processes for desired products, such as ethanol, from non-food agricultural residues and energy crops.

Most fungi can ferment sugars to ethanol and may not depend on respiration for their energy metabolism. However, with few exceptions, fungi may not grow under anaerobic conditions due to non-respiratory oxygen requirements. Saccharomyces species may be rare among yeasts and, indeed, among fungi, for their ability to grow (well) in the absence of molecular oxygen. However, even Saccharomyces species may need supplemented growth media to support anaerobic growth, or may otherwise rely on intracellular sterol accumulated during an aerobic precultivation phase and/or natural sterol contents of plant-biomass-based industrial growth media. Supplemented growth media for anaerobic growth may be more complex than media used for aerobic growth, which may be disadvantageous with regards to medium preparation. Supplementation, as well as reliance on cellular sterol reserves or sterol contents of industrial plant-biomass-based media can also negatively affect predictability and reproducibility of growth outcomes. Further, the supplements may add additional costs to the media preparation, which may be particularly disadvantageous for the production of bulk chemicals, such as ethanol.

Further, many fungi, including many yeasts, such as the facultatively fermentative yeast species Hansenula (Ogataea) polymorpha and Kluyveromyces marxianus, may not grow (well) anaerobically in these supplemented growth media. These fungi may include species with industrially highly attractive properties, such as thermotolerance, broad substrate specificity and robustness to inhibitors present in plant biomass hydrolysates, but prior art strains may be unsuitable for simple, low-cost anaerobic processes.

The (non-respiratory) requirements for molecular oxygen may also be adverse in industrial production processes under (micro-)oxic conditions. Specifically, aeration of bioreactors to provide a sufficient level of dissolved oxygen may be energy-demanding and expensive, and the required level of dissolved oxygen may at least partially be due to the (non- respiratory) need for molecular oxygen.

Hence, it is an aspect of the invention to provide a recombinant fungal cell, which preferably further at least partly obviates one or more of above-described drawbacks. The present invention may have as object to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

Hence, in a first aspect the invention may provide a recombinant fungal cell comprising at least one genome modification relative to a parental fungal cell. The genome modification may especially comprise (an insertion of) an exogenous gene. In embodiments, the exogenous gene may provide the recombinant fungal cell with the ability to synthesize a (suitable) sterol surrogate under anaerobic conditions. In embodiments, the exogenous gene may encode a protein selected from the group comprising a protein having squalene- tetrahymanol cyclase activity and a protein having squalene-hopene cyclase activity.

Surprisingly, the inventors discovered that a recombinant Saccharomyces cerevisiae cell expressing a gene encoding a squalene-tetrahymanol cyclase may grow anaerobically without sterol supplements. Similarly, the inventors discovered that a recombinant Saccharomyces cerevisiae cell expressing a gene encoding a squalene-hopene cyclase may grow anaerobically without sterol supplements. Further, the inventors surprisingly discovered that a recombinant Kluyveromyces marxianus cell expressing a gene encoding a squalene-tetrahymanol cyclase may grow anaerobically, even though Kluyveromyces marxianus may not grow anaerobically even in a growth medium supplemented with sterols.

Sterols are a class of hydrophobic triterpenoid compounds, representatives of which may be important constituents in almost all eukaryotic membranes. Sterols may affect membrane fluidity and permeability as well as localization of specific membrane proteins. In particular, ergosterol may be the major sterol in most fungi. In aerobic conditions, S. cerevisiae may synthesize ergosterol from squalene in a multi -reaction process, which may consume 12 molecules of molecular oxygen for each molecule of ergosterol produced. Given the need for molecular oxygen, S. cerevisiae may not produce ergosterol in anoxic conditions. The term “anoxic conditions” may herein especially refer to conditions wherein the oxygen concentration is below a detection threshold. In particular, the term “anoxic conditions” may refer to conditions wherein the available oxygen is insufficient for a parental S. cerevisiae cell to grow in the absence of sterol supplementation. Hence, in anoxic conditions, the growth media for S. cerevisiae may be supplemented with ergosterol - or with one or more other sterols - to facilitate anaerobic growth. Specifically, S. cerevisiae may incorporate one or more sterols from the supplemented growth medium in order to grow anaerobically.

S. cerevisiae has been intensively used as a model organism for studying in vivo eukaryotic sterol function and biosynthesis. Sterol synthesis mutants of S. cerevisiae have revealed a wide range of cellular processes impacted by sterol content and composition, which may include endocytosis, intracellular trafficking and excretion of proteins and nutrient uptake. In addition, sterols may affect resistance to external stresses. Consistent with the importance of sterols for fungal growth, many fungicides may target ergosterol biosynthesis. Further, several studies have reported that the completion of the yeast cell cycle requires trace (‘sparking’) amounts of specific sterols. These studies may include, for example: Rodriguez et al. 1982; Pinto and Nes 1983; Lorenz et al. 1989; and Nes et al. 1993.

Hence, the invention may provide a recombinant fungal cell comprising at least one genome modification relative to a parental fungal cell (also: “parent fungal cell”). In particular, the recombinant fungal cell may be derived from the parental fungal cell. The term “parental fungal cell” may herein also refer to a plurality of parental fungal cells (of a parental fungal strain), especially to the parental fungal strain.

The term “recombinant fungal cell” may refer herein to a fungal cell comprising at least one genome modification, especially relative to a (corresponding) parental fungal cell. In embodiments, the recombinant fungal cell may especially be a yeast cell. In further embodiments, the recombinant fungal cell may especially be a filamentous fungus cell, i.e. a cell of a filamentous fungus. The term “recombinant fungal cell” may herein also refer to a plurality of recombinant fungal cells (of a recombinant fungal strain), especially to the recombinant fungal strain. The term “genome modification” may be used herein to refer to a difference between two genomes, especially wherein the difference was deliberately introduced. In embodiments, the genome modification may comprise one or more of an insertion (also “addition”), a deletion and a substitution (of nucleotides). In embodiments, the genome modification may especially comprise an exogenous gene, i.e., especially an insertion of an exogenous gene. The term “genome modification” may also refer to a plurality of genome modifications.

In embodiments, the parental fungal cell may be incapable of anaerobic growth.

In further embodiments, the parental fungal cell may be auxotrophic for sterols in anoxic conditions, especially for one or more specific sterols (see above), or especially wherein any one sterol from a group of specific sterols is sufficient to complement the auxotrophy of the parental fungal cell.

The term “auxotrophic” may herein refer to the inability of an organism to synthesize a particular compound required for its growth. The parental fungal cell may especially be selected from the group comprising

Ascomycetous and Basidiomycetous fungi.

The parental fungal cell may especially be a yeast cell. In further embodiments, the recombinant fungal cell may especially be a filamentous fungus cell. It will be clear to the person skilled in the art that the parental fungal cell and the recombinant fungal cell will essentially be the same species. In embodiments, the parental fungal cell may be selected from the group comprising Saccharomycetaceae, such as Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces beticus, Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces uvarum and Saccharomyces bayanus; Torulaspora, such as Torulaspora delbrueckii; Kluyveromyces, such as Kluyveromyces marxianus and Kluyveromyces lactis; Pichia, such as Pichia stipitis (also known as Scheffersomyces stipitis), Pichia pastoris; Ogataea, such as Ogataea parapolymorpha; Zygosaccharomyces, such as Zygosaccharomyces bailii; Brettanomyces, such as Brettanomyces intermedius, Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custersianus, Brettanomyces naardenensis, Brettanomyces nanus, Dekkera bruxellis and Dekkera anomala; Metschnikowia; Issatchenkia, such as Issatchenkia orientalis; and Kloeckera, such as Kloeckera apiculata. In further embodiments, the parental fungal cell may be selected from the group comprising Schizosaccharomycetaceae, such as Schizosaccharomyces, such as Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus and Schizosaccharomyces cryophilus, especially selected from the group comprising Schizosaccharomyces pombe, Schizosaccharomyces octosporus and Schizosaccharomyces cryophilus; or from the group of Dothideomycetes, such as Aureobasidium pullulans ; or from the group of Dipodascaceae, such as Yarrowia lipolytica.

In further embodiments, the parental fungal cell may comprise a Saccharomycetaceae species. In further embodiments, the parental fungal cell may comprise a Schizosaccharomyces species. In further embodiments, the parental fungal cell may comprise a Torulaspora species. In further embodiments, the parental fungal cell may comprise a Kluyveromyces species. In further embodiments, the parental fungal cell may comprise a Pichia species. In further embodiments, the parental fungal cell may comprise an Ogataea species. In further embodiments, the parental fungal cell may comprise a Zygosaccharomyces species. In further embodiments, the parental fungal cell may comprise a Brettanomyces species. In further embodiments, the parental fungal cell may comprise a Metschnikowia species. In further embodiments, the parental fungal cell may comprise an Issatchenkia species. In further embodiments, the parental fungal cell may comprise a Kloeckera species. In further embodiments, the parental fungal cell may comprise an Aureobasidium species. In further embodiments, the parental fungal cell may comprise a Yarrowia species.

In particular, good results may have been achieved with recombinant fungal cells derived from either a parental Saccharomyces cerevisiae cell or from a parental Kluyveromyces marxianus cell (see below). Hence, in embodiments, the recombinant fungal cell may be a yeast cell. In further embodiments, the recombinant fungal cell may be selected from the group comprising Saccharomycetaceae, such as Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces eubayanus, Saccharomyces jurei, Saccharomyces beticus, Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces uvarum and Saccharomyces bayanus; Torulaspora, such as Torulaspora delbrueckii; Kluyveromyces, such as Kluyveromyces marxianus and Kluyveromyces lactis; Pichia, such as Pichia stipitis (also known as Scheffersomyces stipitis), Pichia pastoris; Ogataea, such as Ogataea parapolymorpha; Zygosaccharomyces, such as Zygosaccharomyces bailii; Brettanomyces, such as Brettanomyces intermedius, Brettanomyces bruxellensis, Brettanomyces anomalus, Brettanomyces custer sianus, Brettanomyces naardenensis, Brettanomyces nanus;, Dekkera bruxellis and Dekkera anomala; Metschnikowia; Issatchenkia, such as Issatchenkia orientalis; and Kloeckera, such as Kloeckera apiculata. In further embodiments, the parental fungal cell may be selected from the group comprising Schizosaccharomycetaceae, such as Schizosaccharomyces, such as Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus and Schizosaccharomyces cryophilus, especially selected from the group comprising Schizosaccharomyces pombe, Schizosaccharomyces octosporus and Schizosaccharomyces cryophilus; or from the group of Dothideomycetes, such as Aureobasidium pullulans ; or from the group of Dipodascaceae, such as Yarrowia lipolytica. In particular, good results may have been achieved with a recombinant Saccharomyces cerevisiae cell and with a recombinant Kluyveromyces marxianus cell (see below).

In further embodiments, the recombinant fungal cell may be a (recombinant) filamentous fungus cell. In further embodiments, the fungal cell may be selected from the group comprising Aspergillus spp., Penicillium spp. and Trichoderma spp. In further embodiments the parental fungal cell may be a filamentous fungus cell. In further embodiments, the parental fungal cell may be selected from the group comprising Aspergillus spp., Rhizopus spp., Myceliophthera spp., Thielavia spp., Penicillium spp. and Trichoderma spp. In general, the recombinant fungal cell and the parental fungal cell are the same species.

In embodiments, the recombinant fungal cell may be selected from the group comprising Saccharomyces and Kluyveromyces, especially from the group comprising Saccharomyces, such as the group comprising Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces eubayanus, Saccharomyces jurei, Saccharomyces beticus, Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces uvarum and Saccharomyces bayanus, or especially from the group comprising Kluyveromyces, such as the group comprising Kluyveromyces marxianus and Kluyveromyces lactis.

In embodiments, the parental fungal cell may be selected from the group comprising Saccharomyces and Kluyveromyces, especially from the group comprising Saccharomyces, such as the group comprising Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces eubayanus, Saccharomyces jurei, Saccharomyces beticus, Saccharomyces fermentati, Saccharomyces paradoxus, Saccharomyces uvarum and Saccharomyces bayanus, or especially from the group comprising Kluyveromyces, such as the group comprising Kluyveromyces marxianus and Kluyveromyces lactis

The invention is herein, for explanatory purposes, primarily described with respect to S. cerevisiae and K. marxianus. However, it will be clear to the person skilled in the art that the invention is not limited to S. cerevisiae and K. marxianus.

In embodiments, the genome modification may comprise an exogenous gene. The term “exogenous gene” may herein especially refer to a gene exogenous to the parental fungal cell, especially to the parental fungal species. Hence, the genome of the recombinant fungal species and the genome of the parental fungal cell may differ in at least that the genome of the recombinant fungal species comprises (an insertion of) the exogenous gene, wherein the exogenous gene is absent from the genome of the parental fungal cell, especially from the parental fungal species. In particular, the exogenous gene may encode a protein having an activity, wherein the activity is absent from the proteins encoded by expressible native genes of the parental fungal cell. For example, in embodiments wherein the exogenous gene encodes a protein having squalene-tetrahymanol cyclase activity, the squalene-tetrahymanol cyclase activity may be absent from the proteins encoded by (expressible) native genes of the parental fungal cell, i.e., the parental fungal cell may (be selected to) lack a gene encoding a protein having the squalene-tetrahymanol cyclase activity.

In further embodiments, the recombinant fungal cell may be configured to express the exogenous gene. In particular, the recombinant fungal cell may be configured to transcribe the exogenous gene into (exogenous) mRNA, and especially to translate the (exogenous) mRNA into an (exogenous) protein. It will be clear to the person skilled in the art how to configure the recombinant fungal cell such that the exogenous gene is expressed. The exogenous gene may, for example, be provided with or be arranged at a site providing a suitable promoter.

In further embodiments, the exogenous gene may encode a protein, especially an exogenous protein, selected from the group comprising a protein having squalene- tetrahymanol cyclase activity and a protein having squalene-hopene cyclase activity. In further embodiments, the exogenous gene may encode a protein, especially an exogenous protein, having squalene-tetrahymanol cyclase activity. In further embodiments, the exogenous gene may encode a protein, especially an exogenous protein, having squalene-hopene cyclase activity.

The phrase “a protein having activity”, such as in the phrase “a protein having squalene-tetrahymanol cyclase activity” may herein especially refer to the protein catalyzing the corresponding reaction, irrespective of whether the protein is annotated as a protein catalyzing the indicated reaction. The activity may especially be inferred from the presence of the (direct) product of the reaction. In particular, the activity may be inferred from the presence of the (direct) product of the reaction in a cell, or in a culture broth comprising the cell, expressing a gene encoding the protein, especially by comparing the quantity of the (direct) product with the quantity thereof in a second cell, or in a culture broth comprising the second cell, devoid of the gene (or not expressing it).

For example, the activity of an exogenous protein having squalene-tetrahymanol cyclase activity may be inferred from the presence of tetrahymanol in a recombinant S. cerevisiae cell, wherein the recombinant S. cerevisiae cell expresses an exogenous gene encoding the exogenous protein. Similarly, the activity of an exogenous protein having squalene-hopene cyclase activity may be inferred from the presence of one or more hopanoids in a recombinant S. cerevisiae cell expressing an exogenous gene encoding the exogenous protein, especially from the presence of one or more of hop-22(29)-ene, hopan-22-ol, hop- 17(21)-ene, hop-21-ene. Further, the activity of an exogenous protein having squalene-hopene cyclase activity may, for example, also be inferred from the presence of tetrahymanol in a fungus expressing a native or (second) exogenous gene encoding a protein having a tetrahymanol synthase (hopene) activity.

The phrase “a protein having squalene-tetrahymanol cyclase activity” may herein especially refer to a protein capable of catalyzing the reaction squalene + H ₂ 0 tetrahymanol

, such as especially a protein with enzyme classification EC 4.2.1.123.

The phrase “a protein having squalene-hopene cyclase activity” may herein especially refer to a protein capable of catalyzing the reaction squalene hopene

, such as especially a protein with enzyme classification EC 5.4.99.17; and/or to a protein capable of catalyzing the reaction squalene + H ₂ 0 hopanol

, such as especially a protein with enzyme classification EC 4.2.1.129. In embodiments, the protein having squalene-hopene cyclase activity may especially be capable of catalyzing the reaction squalene hopene. In further embodiments, the protein having squalene-hopene cyclase activity may especially be capable of catalyzing the reaction squalene + H2O hopanol.

The term “hopanoid” may herein especially refer to a pentacyclic compound based on the chemical structure of hopane, especially wherein an outermost (E-)ring consists of 5 carbon atoms, and especially belonging to the class of cyclic triterpenoids and synthesized by cyclization of the branched terpenoid hydrocarbon squalene.

The phrase “a protein having tetrahymanol synthase activity” may herein especially refer to a protein capable of catalyzing the reaction hopene + H ₂ 0 tetrahymanol

, such as especially a protein with enzyme classification EC 4.2.1.B28. In embodiments, the recombinant fungal cell may be capable of producing a desired product. The term “desired product” may herein refer to a (bulk) chemical as defined below. However, in embodiments, the desired product may also be biomass.

Hence, in specific embodiments, the invention may provide a recombinant fungal cell comprising at least one genome modification relative to a parental fungal cell, wherein the genome modification comprises an exogenous gene encoding a protein selected from the group comprising a protein having squalene-tetrahymanol cyclase activity and a protein having squalene-hopene cyclase activity.

In embodiments, the exogenous gene may encode the (exogenous) protein with squalene-tetrahymanol cyclase activity.

In general, if two proteins comprise (highly) similar amino acid sequences, these two proteins may be likely to perform a similar, especially the same, biological function. This relation between amino acid sequence and protein function may, for example, be used to predict the function of a protein based on its sequence identity with proteins of known function (annotation by sequence homology-based inference). The term “sequence identity” herein refers to the percentage of the characters (such as amino acids for protein sequences) of two sequences matching in a sequence alignment (also see below) of the two sequences. The higher the sequence identity between two proteins, the higher the chance may be that these two proteins have the same or a similar function. Although there may not be a hard rule for inferring functional identity or similarity based on a threshold value for sequence identity, especially as the threshold value may need to be adjusted based on (relative) sequence length and/or protein function, proteins may have been successfully annotated based on a common rule-of-thumb threshold of at least 30-40% sequence identity. Hence, proteins comprising an amino acid sequence similar to a protein with known squalene-tetrahymanol cyclase activity may be likely to also have squalene-tetrahymanol cyclase activity.

Hence, in further embodiments, the protein with squalene-tetrahymanol cyclase activity may have a first amino acid sequence, especially wherein the first amino acid sequence has a sequence identity of at least 30% with respect to a sequence alignment with a (first) reference amino acid sequence of a reference squalene-tetrahymanol cyclase, such as an amino acid identity of at least 40%, especially at least 50%, such as at least 60%, especially at least 70%, such as at least 80%, especially at least 90%, such as at least 95%, including 100%. In further embodiments, the sequence alignment may have a length of at least 30% of the (first) reference amino acid sequence, such as at least 40%, especially at least 50%, such as at least 60%, especially at least 70%, such as at least 80%, especially at least 90%, such as at least 95%, including 100%. In further embodiments, the reference squalene-tetrahymanol cyclase may be derived from an organism selected from the group comprising Neocallimastigomycota , Tetrahymena spp., and Oleandra spp..

A sequence alignment of two (or more) sequences may be obtained using, for example, BLASTp. BLASTp is a Basic Local Alignment Search Tool for proteins and will be familiar to the person skilled in the art. BLASTp may be used to align a query amino acid sequence against another amino acid sequence, especially with default settings, and may provide an alignment indicating the “query coverage” of the query amino acid sequence(s) (the percentage of the query amino acid sequence successfully aligned with the other amino acid sequence) and a sequence identity. Hence, the phrase “a sequence alignment having a sequence length > 50% of the sequence length of a reference amino acid sequence” may especially refer to a “query coverage” of > 50% for a BLASTp alignment with the reference amino acid sequence as the query amino acid sequence. For example, two sequences may be aligned via BLASTp, especially using default algorithm parameters, such as using a BLOSUM62 matrix with a gap cost of 11 : 1 (existence:extension).

In embodiments, the exogenous gene may encode a protein with squalene- hopene cyclase activity. In further embodiments, the protein with squalene-hopene cyclase activity has a second amino acid sequence, wherein the second amino acid sequence has a sequence identity of at least 30% with respect to a sequence alignment with a (second) reference amino acid sequence of a reference squalene-hopene cyclase, such as an amino acid identity of at least 40%, especially at least 50%, such as at least 60%, especially at least 70%, such as at least 80%, especially at least 90%, such as at least 95%, including 100%. In further embodiments, the sequence alignment may have a length of at least > 30% of the (second) reference amino acid sequence, such as at least 40%, especially at least 50%, such as at least 60%, especially at least 70%, such as at least 80%, especially at least 90%, such as at least 95%, including 100%. In further embodiments, the reference squalene-hopene cyclase may be derived from an organism selected from the group comprising Alicyclobacillus acidocaldarius, Zymomonas mobilis, Bradyrhizobium japonicum, Sinorhizobium fredii, Rhodopseudomonas palustris, Streptomyces peucetius, Methylococcus capsulatus, Schizosaccharomyces japonicus, Adiantum capillus, and Dryopteris crassirhizoma.

In embodiments, the exogenous gene may encode a protein with tetrahymanol synthase activity. In further embodiments, the protein with tetrahymanol synthase activity has a third amino acid sequence, wherein the third amino acid sequence has a sequence identity of at least 30% with respect to a sequence alignment with a (third) reference amino acid sequence of a reference tetrahymanol synthase, such as an amino acid identity of at least 40%, especially at least 50%, such as at least 60%, especially at least 70%, such as at least 80%, especially at least 90%, such as at least 95%, including 100%. In further embodiments, the sequence alignment may have a length of at least > 30% of the (third) reference amino acid sequence, such as at least 40%, especially at least 50%, such as at least 60%, especially at least 70%, such as at least 80%, especially at least 90%, such as at least 95%, including 100%. In further embodiments, the reference tetrahymanol synthase may be derived from an organism selected from the group comprising Methylomicrobium alcaliphilum, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Nitrobacter hamburgensis, Afipia broomeae, Agromonas oligotrophica, Rhodovulum sp. PH 10, Methylobacterium nodulans, and Desulfovibrio africanus.

In further embodiments, the genome modification, especially the exogenous gene, may comprise a plurality of exogenous genes. Especially, the exogenous gene may comprise one or more of a first exogenous gene, a second exogenous gene, and a third exogenous gene. The first exogenous gene may especially encode a protein having squalene- tetrahymanol cyclase activity. The second exogenous gene may especially encode a protein having squalene-hopene cyclase activity. The third exogenous gene may especially encode a protein having tetrahymanol synthase activity.

In further embodiments, the exogenous gene may comprise the second exogenous gene and the third exogenous gene. Thereby, the recombinant fungal cell may convert squalene to tetrahymanol via hopene.

In further embodiments, the exogenous gene may encode a protein catalyzing a chemical reaction, wherein the protein enables the recombinant fungal cell to produce a sterol- surrogate in an oxygen-independent manner, especially a sterol-surrogate selected from the group comprising tetrahymanol and a hopanoid, wherein the parental fungal cell is incapable of producing the sterol-surrogate.

The term “sterol-surrogate” may herein especially refer to a compound suitable to compensate for a deficiency in one or more sterols in a fungus, especially in the recombinant fungal cell, especially suitable to compensate for a deficiency in all sterols in the fungus.

In embodiments, the recombinant fungal cell may comprise one or more compounds selected from the group comprising tetrahymanol and a hopanoid.

The expression of a gene encoding a squalene-tetrahymanol cyclase or a squalene-hopene cyclase in S. cerevisiae relieved the sterol requirement of S. cerevisiae in anoxic conditions (see further below). Hence, it appears that tetrahymanol and hopanoids can at least partially replace sterols in S. cerevisiae cells. Generally, ergosterol, a main sterol in fungi, may be found in lipid structures, such as cell membranes and/or lipid globules, of fungi.

Hence, in embodiments, the recombinant fungal cell may comprise a lipid structure, wherein the lipid structure comprises one or more compounds selected from the group comprising tetrahymanol and a hopanoid, especially tetrahymanol, or especially a hopanoid. In further embodiments, the lipid structure may comprise a membrane, especially an (outer) cell membrane. In further embodiments, the lipid structure may comprise a lipid globule.

In embodiments, the genome modification may further comprise a deletion of a squalene epoxidase gene, especially a deletion of all copies of a squalene epoxidase gene, more especially the deletion of all copies of all squalene epoxidase genes in the genome of the parental fungal cell. Squalene epoxidase may generally catalyze the first oxygen-consuming reaction in sterol synthesis, thereby consuming squalene and providing oxidosqualene. Thus, by deleting the squalene epoxidase gene, the recombinant fungal cell may be made incapable of sterol synthesis. This may provide the benefit that there may be reduced competition from the squalene epoxidase for the synthesis of tetrahymanol and/or a hopanoid from squalene. Further, in the absence of sterol synthesis, the recombinant fungal cell may have a more consistent performance, as access to small amounts of oxygen would not result in sterol synthesis, which could otherwise affect the cell membrane. Further, by preventing flux through the sterol synthesis pathway, the otherwise consumed molecular oxygen may remain available for other metabolic processes, thereby effectively reducing the need for molecular oxygen under (micro-)aerobic conditions.

The genome modification may result in changes in characteristics of the fungus. For example, it has surprisingly been observed that the expression of a gene encoding a protein having squalene-tetrahymanol cyclase activity may result in an increased tolerance to high temperatures, which may be beneficial, especially in industrial settings. For example, S. cerevisiae may have a temperature optimum for growth and fermentation of about 30-35°C. However, fungal enzymes typically used for hydrolysis of cellulose and other plant polymers may generally function optimally at temperatures above 50 °C. Therefore, especially for production of biofuels such as ethanol, n-butanol and isobutanol from agricultural residues, it may be attractive to perform fermentation processes at a higher temperature. A high temperature during fermentation may reduce the need to cool hydrolysates prior to fermentation and may support simultaneous saccharification and fermentation strategies. Moreover, higher fermentation temperatures may decrease costs for product recovery by distillation and may reduce microbial contamination risks. Similarly, the invention may be particularly beneficial with regards to fungi naturally endowed with a (high) tolerance to high temperatures. Thereby, the recombinant fungal cell may both be tolerant to high temperatures and be capable of synthesizing a sterol- surrogate in an oxygen-independent manner. Hence, in embodiments, the parental fungal cell may, for example, be a K. marxianus cell.

In embodiments, the recombinant fungal cell may comprise an exogenous gene encoding a protein having tetrahymanol synthase activity. Such embodiments may be particularly beneficial with regards to recombinant fungal cells further comprising an exogenous gene encoding a protein having squalene-hopene cyclase activity, and/or with regards to a recombinant fungal cell derived from a parental fungal cell, wherein the parental fungal cell comprises an (expressible) native gene encoding a protein having squalene-hopene cyclase activity.

Hence, in specific embodiments, the recombinant fungal cell comprises at least one genome modification relative to a parental fungal cell, wherein the genome modification comprises an exogenous gene encoding a protein having tetrahymanol synthase activity, wherein: - the genome modification further comprises an exogenous gene encoding a protein having squalene-hopene cyclase activity; or wherein the parental fungal cell comprises an (expressible) native gene encoding a protein having squalene-hopene cyclase activity.

Hence, in embodiments, the parental fungal cell may be selected from the group comprising Aspergilli and Schizosaccharomyces, especially Schizosaccharomyces japonicus. These fungi may comprise an (expressible) native gene encoding a protein having squalene- hopene cyclase activity. In a second aspect, the invention may provide a culturing method for culturing a recombinant fungal cell according to any one of the preceding claims. The culturing method may comprise inoculating a growth medium with the recombinant fungal cell.

In particular, the recombinant fungal cell may be capable of producing a sterol- surrogate, such as tetrahymanol or a hopanoid, especially wherein the sterol-surrogate may at least partially compensate for a deficiency in one or more sterols, especially all sterols. Especially, the recombinant fungal cell may be capable of producing the sterol-surrogate in microoxic and anoxic conditions, especially in anoxic conditions.

Hence, in embodiments, the growth medium may especially comprise < 5 mg/L of sterols, such as < 1 mg/L, especially < 0.1 mg/L, such as < 0.05 mg/L, especially < 0.01 mg/L, such as < 0.001 mg/L, including (essentially) no sterols.

In further embodiments, the growth medium may, especially during at least part of the culturing method, comprise < 0.5 mg/L dissolved oxygen, such as < 0.3 mg/L, especially < 0.1 mg/L, such as < 0.05 mg/L dissolved oxygen, especially < 0.01 mg/L dissolved oxygen, such as < 0.001 mg/L dissolved oxygen, including (essentially) no dissolved oxygen. Hence, in embodiments the growth medium may be anoxic, especially during at least part of the culturing method.

In a further aspect, the invention may provide a production method for producing a desired product using the recombinant fungal cell. The production method may especially comprise the culturing method according to the invention.

In embodiments, the desired product may be biomass, i.e., the biomass generated by the growth of the recombinant fungal cell.

In embodiments, the desired product may be a chemical compound. In embodiments, the desired product may be ethanol. In further embodiments, the desired product may be a non-ethanolic fermentation product, especially a bulk or fine chemical, such as a bulk or fine chemical that is producible by a eukaryotic microorganism, especially by the recombinant fungal cell, such as by a recombinant yeast cell or a recombinant filamentous fungus cell. In further embodiments, the desired product may comprise a chemical compound selected from the group comprising ethanol, isobutanol, ethene, n-butanol, lactic acid, succinic acid, malic acid, and tetrahymanol, especially selected from the group comprising ethanol, isobutanol, ethene, n-butanol, lactic acid, succinic acid, malic acid, or especially tetrahymanol.

In further embodiments, the production method may comprise separating a fraction containing the desired product, especially a fraction containing the chemical compound.

The parental fungal cell may be auxotrophic for one or more sterols in anoxic conditions. However, the recombinant fungal cell may grow anaerobically without sterol supplement, thereby enabling the recombinant fungal cell to anaerobically grow in growth media devoid of sterols. These growth media may be cheaper and/or easier to provide than the growth media used for anaerobic growth of the parental fungal cell.

Hence, in a further aspect, the invention may provide a growth medium for anaerobic growth, especially exponential anaerobic growth, of the recombinant fungal cell. In embodiments, the growth medium may comprise nicotinic acid, biotin, pantothenic acid and thiamine. In further embodiments, the growth medium may comprise < 0.5 mg/L of sterols, such as < 1 mg/L, especially < 0.1 mg/L, such as < 0.01 mg/L, including (essentially) no sterols.

In a further aspect, the invention may provide the use of the recombinant fungal cell to produce a desired product. In embodiments, the use may comprise, especially during at least part of the production, producing the desired product in a growth medium comprising < 0.1 mg/L dissolved oxygen, , such as < 0.05 mg/L dissolved oxygen, especially < 0.01 mg/L dissolved oxygen, such as < 0.001 mg/L dissolved oxygen, including (essentially) no dissolved oxygen. Hence, in embodiments the growth medium may be anoxic, especially during at least part of the use.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which: Fig. 1A-D schematically depict experimental data obtained using a parental fungal cell and an embodiment of the recombinant fungal cell. Fig. 2A-F schematically depict experimental data obtained using a parental fungal cell and embodiments of the recombinant fungal cell. Fig. 3A-B schematically depict experimental data obtained using a parental fungal cell and an embodiment of the recombinant fungal cell. Fig. 4 schematically depicts experimental data obtained using a parental fungal cell and an embodiment of the recombinant fungal cell. Fig. 5A-C schematically depict experimental data obtained using a parental fungal cell and an embodiment of the recombinant fungal cell.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Experimental procedures

Maintenance and media - For the examples below, stock cultures were propagated in different media as indicated below. The synthetic medium SMD herein refers to the synthetic medium as described in Verduyn et al. 1992, which is hereby herein incorporated by reference, with 20 g L ¹ glucose as carbon source. The synthetic urea medium (SMD-urea) refers to SMD in which ammonium sulfate is replaced by 2.3 g L ¹ urea and 6.6 g L ¹ K2SO4. The yeast peptone dextrose medium (YPD) refers to a medium comprising 10 g L ¹ Bacto yeast extract, 20 g L ¹ Bacto peptone and 20 g L ¹ glucose. Where indicated, media were supplemented with ergosterol (> 95%, Sigma-Aldrich, St. Louis, MO) as a sterol source and/or Tween 80 (polyethylene glycol sorbate monooleate, Merck, Darmstadt, Germany) as a source of unsaturated fatty acids, to final concentrations of 10 mg L ¹ and 420 mg L ¹ , respectively. Concentrated stock solutions (800 x) of these supplements were prepared by adding 8.4 g of Tween 80 and/or 0.2 g of ergosterol to 17 mL of absolute ethanol. These suspensions were heated at 80°C for 20 min prior to addition to growth media to ensure proper solubilization of sterols. Stock cultures of E. coli DH5a and derived strains were propagated in Lysogeny Broth (LB, 10 g L ¹ Bacto tryptone, 5 g L ¹ Bacto yeast extract and 5 g L ¹ NaCl), where relevant supplemented with 100 mg L ¹ ampicillin. After adding sterile glycerol (30 % v/v), samples were frozen and stored at -80 °C.

The synthetic medium SMD-phosphate herein refers to the synthetic medium as described in Verduyn et al. 1992, but with 14.4 g L ¹ of potassium dihydrogen phosphate, and with 20 g L ¹ glucose.

Anaerobic pre-cultures - Anaerobic pre-cultures were grown to stationary phase in SMD-urea with Tween 80 and ergosterol and were washed twice with sterile demineralized water and were used to inoculate an anaerobic pre-culture in a shake flask containing SMD- urea with 50 g L ¹ glucose and with Tween 80 at an initial OD660 of 0.20. Optical density at 660 nm was measured with a 7200 visible spectrophotometer (Jenway, Staffordshire, UK). When the OD660 of the anaerobic pre-culture no longer increased, it was used to inoculate either an anaerobic growth experiment on SMD-urea with Tween 80 and with 20 g L ¹ glucose, or an aerobic growth experiment with SMD-urea and 20 g L ¹ glucose at an initial OD660 of 0 2

Anaerobic bioreactor cultivation - Anaerobic sequential batch reactor (SBR) experiments were performed in 2-L bioreactors with a working volume of 1.2 L according to the procedure described in Dekker et al. 2019, which is hereby herein incorporated by reference. Cultures were grown on SMD-urea at 30°C and stirred at 800 rpm with an initial pH of 6.0. The outlet gas of the bioreactors was cooled to 4°C in a condenser and dried with a PermaPure PD- 50T-12MPP dryer (Permapure, Lakewood, NJ) prior to analysis with a NGA 2000 Rosemount gas analyzer (Emerson, St. Lois, MO). Experiments were initiated with a batch cultivation cycle on medium without ergosterol and Tween 80 to deplete endogenous reserves of sterols and unsaturated fatty acids. On-line monitoring of CO2 concentrations in the outlet gas of reactors was used to monitor growth. When growth ceased, three consecutive batch cycles were performed on SMD-urea, either containing both supplements or only Tween 80 as indicated below. New cultivation cycles were initiated upon reaching stationary phase by emptying the vessel at the end of a previous cycle and refilling it with fresh medium. Shortly before CO2 concentrations in the outlet gas returned to zero, residual air-saturated medium was flushed back from the medium inlet tubing by relieving pressure from the vessel, and subsequently a next SBR cycle was initiated. The 5-L glass medium reservoir from which the cultures were refilled was kept anoxic by continuous sparging with N5.5 grade N2. Precultures for the SBR experiments - the precultures were prepared by inoculating aerobic shake-flask cultures (100 mL) on SMD containing 20 g L ¹ glucose with frozen glycerol stock cultures. After overnight cultivation at 30 °C, a sample from these cultures was used to inoculate a second 100-mL aerobic shake-flask pre-culture on the same medium. For SBR experiments with strain IMK870 (see below), an anaerobic shake-flask culture on SMD with Tween 80 and ergosterol, containing 20 g L ¹ glucose, was inoculated from a frozen glycerol stock. After overnight incubation at 30 °C, this culture was used to inoculate a second anaerobic shake flask culture. When this second pre-culture was growing exponentially, biomass was harvested by centrifugation at 3000 x g, washed with sterile demineralised water and used to inoculate the bioreactors at an initial OD660 of 0.2.

Analytical methods - metabolites: metabolite concentrations in culture supernatants were analyzed by high-performance liquid chromatography as described by Verhoeven et al. (2017), which is hereby herein incorporated by reference.

Analytical methods - biomass: Biomass dry weight measurements were performed using pre-weighed nitrocellulose filters (0.45 pm, Gelman Laboratory, Ann Arbor, MI). Filters were rinsed with demineralized water, used for filtration of 10- or 20-mL culture samples, and washed with demineralized water prior to drying in a microwave oven (20 min at 360 W).

Analytical methods - triterpenoid fraction: Isolation of the triterpenoid fraction of biomass was based on the procedure published described by Miiller et al., 2017, which is hereby herein incorporated by reference, with the following modifications. Biomass for membrane analysis was harvested at the end of a cultivation cycle and/or during mid exponential phase by centrifuging 45 mL of culture broth (5 min at 3000 x g) and washed once with demineralized water. After lyophilizing the biomass pellets overnight (Alpha 1-4 LD Plus freeze dryer, Christ, Osterode am Harz, Germany), 10-30 mg of lyophilized material was weighed into glass methylation tubes (Article no. 10044604, PYREX™ Borosilicate glass, Thermo Fisher Scientific). Then 1 mL of 2 M NaOH (Article No. 72068, Sigma-Aldrich) was added and suspensions were heated for at 1 h at 70 °C, without sonication but with vortexing. After cooling to room temperature, the content of the tube was transferred to a 2-mL plastic Eppendorf tube (Greiner Bio-One, Alphen aan den Rijn, The Netherlands), and afterwards extraction with /er/-butyl-methyl ether (/BME) was performed. After extraction, the dried sterol fraction was dissolved in 0.1 mL of tBME and directly used for analysis without trimethyl- silylation. Sterols were analysed by gas-chromatography with flame-ionization detection (GC- FID) on an Agilent Technologies 7890A GC-FID system equipped with a FID-1000-220 Gas Station (Parker Balston, Haverhill, MA, USA) and an Agilent Technologies 7693 Autosampler. A VF-5ms column (30 m, 0.25 mm internal diameter, 0.25 pm film thickness, Agilent part no. CP9013) was used for separation, and N2 was used as a carrier gas at a constant flow of 1 mL min ¹ . The initial oven temperature was 80 °C and was kept constant for 1 min, then increased to 280 °C at 50 °C min ¹ , and finally increased to 320 °C at 6 °C min ¹ and kept constant for another 15 min. The inlet temperature was set at 150 °C, and the FID temperature at 280 °C. The GC-FID system was calibrated for squalene (> 98%, Sigma-Aldrich), ergosterol (> 98%, Boom B.V), 5a-cholestane (internal standard; > 97%, Sigma-Aldrich), lanosterol (> 99%, Avanti Polar Lipids, Alabaster AL, United States of America), and tetrahymanol (> 99%, ALB Technologies), using a 10-point calibration curve for all compounds.

For analysis of triterpenoids in yeast biomass by gas chromatography with flame ionization detection (GC-FID), synthetic reference material for squalene (>98%), ergosterol (> 95.0%), 5a-cholestane (> 97.0%) and hop-22(29)-ene (0.1 mg/mL in isooctane, analytical standard) was used (obtained from Sigma-Aldrich).

Unless specified otherwise, the examples outlined below are performed according to the experimental procedures defined hereabove.

Examples 1 and 2 relate to experiments wherein the parent fungal cell is a parent S. cerevisiae cell, hereinafter referred to as IMX585, as described (as IMX585) in Mans et al., 2015, which is hereby herein incorporated by reference.

Example 1 : expression of squalene-tetrahymanol cyclase in S. cerevisiae.

Example 1 relates to an embodiment of the recombinant fungal cell, wherein the recombinant fungal cell is a recombinant S. cerevisiae cell, hereinafter referred to as IMX1438, derived from IMX585. IMX1438 comprises a genome modification relative to IMX585, wherein the genome modification comprises (an insertion of) an exogenous gene encoding a protein having squalene-tetrahymanol cyclase activity. Specifically, the exogenous gene, hereinafter also referred to as TtTHCl , was derived from the squalene-tetrahymanol cyclase gene THC1 of Tetrahymena thermophila (GenBank accession no. XM OO 1026696.2) by codon-optimization for expression in S. cerevisiae using the Jcat algorithm, and corresponds to SEQ ID NO:l. IMX1438 was constructed by inserting an expression cassette comprising TtTHCl into the ri'Crii-locus of IMX585 using Cas9-mediated genome editing with 500 ng of CRISPR plasmid and 400 ng of linear insert. Specifically, the expression cassette corresponding to SEQ ID NO:3 was integrated between nucleotide positions 172264 and 174002 of chromosome IX of the S. cerevisiae genome (CEN.PK113-7D, NCBI Accession number PRJNA393501, as described in Salazar et al., 2017, which is hereby herein incorporated by reference). In particular, the Cas9-mediated genome editing was performed as described in Mans et al., 2015, which is hereby herein incorporated by reference.

Example 1 further relates to a further embodiment of the recombinant fungal cell, wherein the recombinant fungal cell is a recombinant S. cerevisiae cell, hereinafter referred to as IMK870, derived from IMX585. IMK870 comprises a genome modification relative to IMX585, wherein the genome modification comprises (an insertion of) an exogenous gene encoding a protein having squalene-tetrahymanol cyclase activity, and wherein the genome modification further comprises a deletion of a squalene epoxidase gene. IMK870 was constructed using the same procedure as outlined hereabove for IMX1438, followed by a deletion of the ERG1 gene (encoding squalene epoxidase). In particular, the ERG1 gene was deleted by inserting an expression cassette corresponding to SEQ ID NO: 5 between nucleotide positions 872978 and 874956 of chromosome VII of the S. cerevisiae genome (CEN.PK113- 7D, NCBI Accession number PRJNA393501, as described in Salazar et al., 2017).

The inserted expression cassette comprised a KanMX gene under control of the TEFL -promotor and TEFL -terminator (AgTEFlp-KanMX-AgTEFlt) as a marker to select the recombinant cells.

Hence, the S. cerevisiae strains used in example 1 are:

Fig. 1A-D schematically depict experimental data obtained using IMX585 and IMX1438 in growth media supplemented with ergosterol. Specifically, Fig. 1A-D depict CO2 percentage P (%C0 ₂ in off-gas; Fig. 1A, Fig. 1C), and measurement values of biomass B (in g/L) and compounds C (in g/L; Fig. IB, Fig. ID) over time (in hours) for IMX585 (Fig. 1A, Fig. IB) and IMX1438 (Fig. 1C, Fig. ID). Fig. 1A-D each represent data from a single representative experiment of three anaerobic sequential batch reactor (SBR) experiments (see above) at 30°C on SMD-urea.

Fig. 1A, Fig. 1C indicate the CO ₂ percentage P during a carry-over phase on medium without anaerobic growth factors (Bo) and the first (Bi), second (B ₂ ) and third (B ₃ ) batch cycles on growth medium supplemented with Tween 80 and ergosterol. The phrase “without anaerobic growth factors” herein refers to the absence of unsaturated fatty acids, including Tween 80, as well as to the absence of sterols, including ergosterol. Fig. IB, Fig. ID indicate measurement values of biomass B and compounds C during the second batch cycle, wherein line Li represents biomass, line L2 represents glucose, line L3 represents ethanol, and line L4 represents glycerol.

As can be seen, both the recombinant fungal cell and the parental fungal cell were observed to exhibit exponential growth in anoxic conditions in a growth medium supplemented with ergosterol.

Fig. 2A-F schematically depict experimental data obtained using IMX585, IMX1438 and IMK870 in growth media without ergosterol. Specifically, Fig. 2A-F depict CO2 percentage P (%C0 ₂ in off-gas; Fig. 2A, Fig. 2C; Fig. 2E), and measurement values of biomass B (in g/L) and compounds C (in g/L; Fig. 2B, Fig. 2D; Fig. 2F) over time (in hours) for IMX585 (Fig. 2 A, Fig. 2B), IMX1438 (Fig. 2C, Fig. 2D), and IMK870 (Fig. 2E, Fig. 2F). Fig. 2A-F each represent data from a single representative experiment of three anaerobic sequential batch reactor (SBR) experiments (see above) at 30°C on SMD-urea.

Fig. 2A, Fig. 2C, Fig. 2E indicate the CO2 percentage P during a carry-over phase on medium without anaerobic growth factors (Bo) and the first (Bi), second (B2) and third (B3) batch cycles on growth medium supplemented with Tween 80.

Fig. 2B, Fig. 2D, Fig. 2F indicate measurement values of biomass B and compounds C during the second batch cycle, wherein line Li represents biomass, line L2 represents glucose, line L3 represents ethanol, and line L4 represents glycerol.

As can be seen from the off-gas profiles in Fig. 2A, IMX585 was not observed to grow exponentially in the absence of sterol supplementation. Although IMX585 was observed to eventually consume all glucose, this took approximately 100 hours (see Fig. 2B). In contrast, IMX1438 and IMK870 were observed to grow exponentially and to consume the glucose in approximately 35 and 50 hours respectively.

Further, IMX585, IMX1438, and IMK870 were grown anaerobically on glucose synthetic medium with Tween 80, in presence or absence of ergosterol. The observed specific growth rates and biomass yields are provided in the table below, represented as average values and standard deviations based on at least two replicate experiments: Hence, the expression of a gene encoding a protein having squalene- tetrahymanol cyclase activity is observed to enable a recombinant fungal cell to anaerobically grow (exponentially) in the absence of sterol supplementation.

In Fig 2B, some increase in biomass may be observed for IMX585 in an anaerobic sequential batch reactor experiment despite the absence of sterol supplementation. Hence, the triterpenoid fraction of IMX585, IMX1438 and IMK870 were analyzed using GC- FID (also see above), and the observed triterpenoids are summarized in the table below:

Lanosterol may be the first cyclic compound after epoxidation of squalene in the natural ergosterol synthesis pathway in S. cerevisiae and the synthesis thereof may require molecular oxygen. Hence, the presence of lanosterol may imply the presence of some residual molecular oxygen, which may account for the observed biomass increase depicted in Fig. 2B.

For IMK870 no lanosterol was observed, consistent with the deletion of the squalene epoxidase gene ( ERG1 ) in IMK870. Hence, whereas IMX585 may exhibit some (non exponential) growth in the anaerobic sequential batch reactor experiment due to residual molecular oxygen, IMX1438 and IMX870 may grow in the anaerobic sequential batch reactor experiment in an oxygen-independent manner.

Example 2: expression of squalene-hopene cyclase in S. cerevisiae.

Example 2 relates to an embodiment of the recombinant fungal cell, wherein the recombinant fungal cell is a recombinant S. cerevisiae cell, hereinafter referred to as IMX2081, derived from IMX585. IMX2081 comprises a genome modification relative to IMX585, wherein the genome modification comprises (an insertion of) an exogenous gene encoding a protein having squalene-hopene cyclase activity. Specifically, the exogenous gene, hereinafter also referred to as SjSHC , was derived from the squalene-hopene cyclase gene SHC of Schizosaccharomyces japonicus yFS275 (Genbank Accession number: NW_011627861.1) by codon-optimization for use in S. cerevisiae using the online GeneOptimizer tool (GeneArt, Regensburg, Germany), and corresponds to SEQ ID NO:2. IMX2081 was constructed by inserting the exogenous gene under control of the TEFL -promoter and with the CYC1- terminator into the SGA /-locus of IMX585 using Cas9-mediated genome editing. Specifically, the expression cassette corresponding to SEQ ID NO:4 was integrated between nucleotide positions 172264 and 173934 of chromosome IX of the S. cerevisiae genome (CEN.PK113- 7D, NCBI Accession number PRJNA393501, as described in Salazar et al., 2017). The use of the TEFL -promoter (also “ZE ”) and the CTC7 -terminator for expression in yeast is described in Ronicke et al, 1997, which is hereby herein incorporated by reference. In particular, the Cas9-mediated genome editing was performed as described in Mans et al., 2015.

Hence, the S. cerevisiae strains used in example 2 are:

IMX585 and IMX2081 were grown aerobically overnight on SMD-urea. From this pre-culture, strains were transferred to fresh SMD-urea in the anaerobic chamber (carry over), for depletion of the internal sterol storage. The carry-over medium contained 50 g.L ¹ glucose. When stationary phase was reached, the cultures were transferred to SMD-urea containing Tween 80 and ergosterol (T/E), Tween 80 only (T/-), or neither Tween 80 nor ergosterol (-/-).

Fig. 3A-B schematically depicts the Oϋboo (O) of IMX585 (Fig. 3A) and IMX2081 (Fig. 3B) over time (in hours) in the carry-over medium, T/E, T /-, and -/-, wherein line Lii represents the observations from the carry-over medium, line L12 from T/E, line L13 from T /-, and line Lu from -/-.

As can be seen in Fig. 3 A-B, both IMX585 and IMX2081 were observed to grow anaerobically in SMD-urea supplemented with both Tween 80 and ergosterol. When supplied with Tween 80 only, minimal background growth was observed for the parental fungal cell IMX585, while the recombinant fungal cell IMX2081 harboring the SjSHC gene showed clear anaerobic growth. Also, on SMD-urea lacking both Tween 80 and ergosterol (-/-), IMX2081 was observed to grow anaerobically, whereas the parental strain IMX585 was not. These results demonstrate that the expression of an exogenous gene encoding a protein with squalene-hopene cyclase activity in S. cerevisiae enables anaerobic growth in the absence of ergosterol.

Example 3 : expression of squalene-tetrahymanol cyclase in K. marxianus.

Example 3 relates to an embodiment of the recombinant fungal cell, wherein the recombinant fungal cell is a recombinant K. marxianus cell, hereinafter referred to as IMS1111, derived from K. marxianus NBRC 1777, hereinafter referred to as NBRC 1777. NBRC 1777 was ordered from the Biological Resource Center, NITE (NBRC) (Chiba, Japan). IMS1111 comprises a genome modification relative to NBRC1777, wherein the genome modification comprises (an insertion of) an exogenous gene encoding a protein having squalene-tetrahymanol cyclase activity. Specifically, the exogenous gene, hereinafter also referred to as TtTHCl , was derived from the squalene-tetrahymanol cyclase gene THC1 of Tetrahymena thermophila (GenBank accession no. XM_001026696.2) by codon-optimization for expression in S. cerevisiae using the Jcat algorithm, and corresponds to SEQ ID NO:l.

First, recombinant fungal cell IMX2323 was constructed by transforming NBRC1777, using the LiAc procedure described by Gietz and Woods (2002), which is hereby herein incorporated by reference, with a Notl digested linear DNA fragment of an expression cassette comprising TtSHCl and transformants were selected on YPD-hygB plates (YPD plates with added hygromycin B as selective marker) for integration of SHCl. Specifically, an expression cassette with the exogenous gene TtSHCl under control of the KmPDCl -promoter and ScADHl -terminator, corresponding to SEQ ID NO:6, was integrated into the genome of NBRC1777. The use of the KmPDCl -promoter and the ScADHl terminator is described in Rajkumar et al, 2019 and Hassing el al, 2019, respectively, which are hereby herein incorporated by reference.

Then, IMX2323 was incubated in SMD-urea supplemented with Tween-80 in micro-oxic conditions. When, after 20 days of incubation, growth was observed, two transfers to fresh SMD-urea medium with Tween-80 were performed. Then, a single cell line was isolated by plating on three consecutive YPD agar plates containing hygromycin B. After the final restreak, this procedure yielded the single-colony isolate recombinant fungal cell IMS 1111.

Hence, the K. marxianus strains used in example 3 are:

Fig. 4 schematically depicts observed OD600 over time (in hours) of 5 serial transfers on SMD-urea of the K. marxianus parental strain and the recombinant fungal cell strains. Specifically, lines L21 represents IMS1111, lines L22 represents NBRC1777, and lines L23 represents IMX2323.

As depicted in Fig. 4, some growth is observed for all strains during the first of the serial transfers, which may at least partially be due to residual sterols from an aerobic pre cultivation. Further, it is observed that experimental evolution in micro-oxic conditions, as described above, may enhance the performance of the recombinant fungal strain. As depicted in Fig. 4, IMS1111 showed anaerobic growth during 5 serial transfers on SMD-urea supplemented with Tween 80. At the end of the growth phase of the 1st, 2nd and 3rd transfer biomass was harvested for triterpenoid analysis as described above. Tetrahymanol was detected in biomass of anaerobically cultivated IMS1111 in each of the sequential transfers, indicating the successful expression of the inserted exogenous gene. Hence, although K. marxianm , including strain NBRC1777, is incapable of anaerobic growth, even in a medium supplemented with sterols, the insertion of the exogenous gene encoding a protein having squalene-tetrahymanol cyclase activity enabled the recombinant K. marxianm cell to acquire the ability to grow (exponentially) in anoxic conditions.

Recombinant strain IMS1111 has been deposited at the Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT, the Netherlands, on January 29 2020 under accession number CBS 146403.

Example 4: expression of squalene-hopene cyclase and tetrahymanol synthase in S. cerevisiae.

Example 4 relates to an embodiment of the recombinant fungal cell, wherein the recombinant fungal cell is a recombinant S. cerevisiae cell, hereinafter referred to as IMX2616, derived from IMX2600.

Example 4 further relates to an embodiment of the recombinant fungal cell, wherein the recombinant fungal cell is a recombinant S. cerevisiae cell, hereinafter referred to as IMX2629, derived from IMX2616.

Strain IMX2600 was constructed by integration of cas9 and natNT2 expression cassettes in the CAN1 locus of CEN.PK113-7D. The integration/expression cassettes for cas9 and natNT2 were respectively obtained by PCR from p414-TEFlp-cas9-CYCl as described in Dicarlo et al., 2013 and pUG-natNT2 as described in Stefan de Kok et ak, 2012.

Strain IMX2616 ( MATa canl A::cas9-natNT2 sgal AwSjSHC) was constructed by genomic integration of an expression cassette for a codon-optimized squalene-hopene cyclase gene from Schizosaccharomyces japonicus CBS5679 in strain IMX2600.

Strain IMX2629 ( MATa canl A:\cas9-natNT2 sgal A:\SjSHC x2 A: \MaTHS) was constructed by subsequent genomic integration of an expression cassette for a codon-optimized tetrahymanol synthase gene MaTHS from Methylomicrobium alkaliphilum (Genbank Accession number: F0082060.1, locus tag MEALZ_1626) in strain IMX2629.

Hence, IMX2616 comprises a genome modification relative to IMX2600, wherein the genome modification comprises (an insertion of) an exogenous gene encoding a protein having squalene-hopene cyclase activity. Specifically, the exogenous gene, hereinafter also referred to as SjSHC 2, was derived from the squalene-hopene cyclase gene SHC of Schizosaccharomyces japonicus CBS5679 by codon-optimization for expression in S. cerevisiae using the GeneOptimizer tool, and corresponds to SEQ ID NO:7. IMX2616 was constructed by inserting an expression cassette comprising SjSHC 2 under control of the TEF1 promotor and the CYC1 terminator into the ri'Grif-locus of IMX2600. In particular, the expression cassette was inserted by co-transformation with ri'GVI /-targeting plasmid pUDRl 19 as described in Van Rossum et al, 2016. Specifically, the expression cassette corresponding to SEQ ID NO:9 was integrated between nucleotide positions 172264 and 173934 of chromosome IX of the S. cerevisiae genome (CEN.PK113-7D, also see above). Example 4 further relates to a further embodiment of the recombinant fungal cell, wherein the recombinant fungal cell is a recombinant S. cerevisiae cell, hereinafter referred to as IMX2629, derived from IMX2616. IMX2629 comprises a genome modification relative to IMX2616, wherein the genome modification comprises (an insertion of) an exogenous gene encoding a protein having tetrahymanol synthase activity. Hence, IMX2629 comprises a genome modification relative to IMX2600, wherein the genome modification comprises (the insertion of) a plurality of exogenous genes, wherein a (second) exogeneous gene encodes a protein having squalene- hopene cyclase activity, and wherein a (third) exogeneous gene a protein having tetrahymanol synthase activity. Specifically, the exogenous gene, hereinafter also referred to as MaTHS, was derived from the tetrahymanol synthase gene THS of Methylomicrobium alcaliphilum 20Z (GenBank accession no. F0082060.1, locus tag MEALZ 1626) by codon-optimization for expression in S. cerevisiae using the GeneOptimizer tool, and corresponds to SEQ ID NO:8. Specifically, IMX2629 was constructed by inserting an expression cassette comprising MaTHS under control of the TDH3 promotor and the ADH1 terminator into the X2 -locus of IMX2616 by co-transformation with X2-targeting plasmid pUDR538. Specifically, the expression cassette corresponding to SEQ ID NO: 10. was integrated between nucleotide positions 194944 and 195980 of chromosome X of the S. cerevisiae genome (CEN.PK113-7D, as represented on SGD (http s : //www. y eastgenome . or g/) ; also corresponding to the X2-locus as described by Mikkelsen et al.,).

Hence, the S. cerevisiae strains used in example 4 are:

To evaluate the impact of squalene-hopene cyclase expression in S. cerevisiae on anaerobic physiology, and to test if combined expression of the tetrahymanol synthase of Methylomicrobium alcaliphilum ( MaTHS) and the squalene hopene cyclase would enable tetrahymanol production, recombinant fungal cells IMX2616 and IMX2629, and parental fungal cell CEN.PK113-7D were grown anaerobically in shake flasks. Aerobically grown cultures of these strains were used to inoculate an anaerobic pre-culture on SMD-phosphate with a higher glucose concentration (50 g L ¹ ). The pre-cultures were grown until end- exponential phase to stimulate depletion of sterols originating from the preceding aerobic culture. These pre-cultures were used to inoculate fresh SMD-phosphate medium (with 20 g L ¹ glucose) in the absence of an exogenous source of unsaturated fatty acids or sterols, and SMD-phosphate medium to which only Tween 80 was added. Fig. 5A-C schematically depict the corresponding Oϋboo measurements over time T (in hours) with two biological duplicates, where Fig. 5A corresponds to parental fungal cell CEN.PK113-7D, Fig. 5B corresponds to IMX2616, and Fig. 5C corresponds to IMX2929. Specifically, the open circles (lines L52, L55, and L58) correspond to cultivation in the absence of fatty acids and sterols, whereas the filled circles correspond to cultivation media supplemented with Tween 80.

Hence, on media with Tween 80, CEN.PK113-7D reached an optical density of 1.1 in 58 h (see L51), while strains IMX2616 and IMX2629 reached OD ₆ oo’s of 2.1 and 2.4 in 33 hours, respectively (see L54 and L56). When transferred to fresh SMD-phosphate media with Tween 80, both recombinant strains sustained growth for another 2.8 doublings (see lines L54 and L57).

Whereas CEN.PK113-7D grown on medium with Tween 80 was not able to consume its glucose, residual glucose was low for IMX2629. Specifically, residual glucose was 1.08 ± 1.02 mM and 0.09 ± 0.09 mM vs. 35.54 ± 1.68 mM after 42 for IMX2616, IMX2629 and after 58 hours for CEN.PK113-7D, respectively.

The results of these experiments show that the expression of SjSHC 2, alone or combined with the expression of MaTHS stimulates growth of S. cerevisiae in the absence of added sterols.

Specifically, these experiments further demonstrate that different exogeneous genes encoding for different proteins with squalene-hopene cyclase activity may each be beneficial for anaerobic growth in fungi, especially in yeast species, such as in S. cerevisiae. To investigate whether co-expression of the squalene-hopene cyclase and tetrahymanol synthase enabled tetrahymanol production in S. cerevisiae , biomass of the anaerobically grown cultures of CEN.PK113-7D, IMX2616 and IMX2629 was harvested at the end of each shake-flask experiment, and the triterpenoid fraction was analysed by GC-FID. The biomass of IMX2616 and IMX2629 contained multiple additional triterpenoid compounds with reference to strain CEN.PK113-7D, of which one could be identified as hop-22(29)-ene (diploptene) based on synthetic reference material. The biomass of strain IMX2629 additionally contained tetrahymanol. Hence, these experiments demonstrate that the recombinant fungal cells indeed produced hopanoids, and that combined expression of genes encoding for a protein having squalene-hopene cyclase activity and for a protein having tetrahymanol synthase activity may enable the recombinant fungal cell to produce tetrahymanol.

The term “plurality” refers to two or more. Furthermore, the terms “a plurality of’ and “a number of’ may be used interchangeably.

The terms “substantially” or “essentially” herein, and similar terms, will be understood by the person skilled in the art. The terms “substantially” or “essentially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially or essentially may also be removed. Where applicable, the term “substantially” or the term “essentially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. Moreover, the terms ’’about” and “approximately” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%. For numerical values it is to be understood that the terms “substantially”, “essentially”, “about”, and “approximately” may also relate to the range of 90% - 110%, such as 95%-105%, especially 99%-101% of the values(s) it refers to.

The term “comprise” includes also embodiments wherein the term “comprises” means “consists of’.

The term “and/or” especially relates to one or more of the items mentioned before and after “and/or”. For instance, a phrase “item 1 and/or item 2” and similar phrases may relate to one or more of item 1 and item 2. The term "comprising" may in an embodiment refer to "consisting of but may in another embodiment also refer to "containing at least the defined species and optionally one or more other species".

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The devices, apparatus, or systems may herein amongst others be described during operation. As will be clear to the person skilled in the art, the invention is not limited to methods of operation, or devices, apparatus, or systems in operation.

The term “further embodiment” and similar terms may refer to an embodiment comprising the features of the previously discussed embodiment, but may also refer to an alternative embodiment.

It should be noted that the above-mentioned embodiments illustrate rather than limit the invention, and that those skilled in the art will be able to design many alternative embodiments without departing from the scope of the appended claims.

In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim.

Use of the verb "to comprise" and its conjugations does not exclude the presence of elements or steps other than those stated in a claim. Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, “include”, “including”, “contain”, “containing” and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

The article "a" or "an" preceding an element does not exclude the presence of a plurality of such elements.

The invention may be implemented by means of hardware comprising several distinct elements, and by means of a suitably programmed computer. In a device claim, or an apparatus claim, or a system claim, enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage.

The invention also provides a control system that may control the device, apparatus, or system, or that may execute the herein described method or process. Yet further, the invention also provides a computer program product, when running on a computer which is functionally coupled to or comprised by the device, apparatus, or system, controls one or more controllable elements of such device, apparatus, or system.

The invention further applies to a device, apparatus, or system comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. The invention further pertains to a method or process comprising one or more of the characterizing features described in the description and/or shown in the attached drawings. Moreover, if a method or an embodiment of the method is described being executed in a device, apparatus, or system, it will be understood that the device, apparatus, or system is suitable for or configured for (executing) the method or the embodiment of the method respectively.

The various aspects discussed in this patent can be combined in order to provide additional advantages. Further, the person skilled in the art will understand that embodiments can be combined, and that also more than two embodiments can be combined. Furthermore, some of the features can form the basis for one or more divisional applications.

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