Cells and methods for production of heme

Abstract

The present disclosure relates to yeasts, in particular Yarrowia cells, with enhanced production of heme. Also provided are methods for enhanced production of heme with Yarrowia cells.

Description

[0001] Cells and methods for production of heme

[0002] Technical field

[0003] The present disclosure relates to yeasts, in particular Yarrowia cells, with enhanced production of heme. Also provided are methods for enhanced production of heme with Yarrowia cells.

[0004] Background

[0005] Heme refers to a group of molecules found throughout all kingdoms of life. They are often bound as cofactors in proteins and are involved in a variety of functions such as oxygen transport, chemical catalysis, and electron transfer. Hemes consist of a porphyrin ring binding an iron ion. Common forms include heme A, heme B (Figure 1), heme C, and heme O, which differ in the functional groups attached to the core porphyrin ring structure.

[0006] Heme is also an important component of some foods such as red meat, where it is involved in flavor formation, appearance, and nutritional content.

[0007] In the yeast Saccharomyces cerevisiae, the heme B biosynthetic pathway consists of 8 enzymes, namely 5-aminolevulinic acid synthase (HEM1), 5-aminolevulinic acid dehydratase (HEM2), porphobilinogen deaminase (HEM3), uroporphyrinogen III synthase (HEM4), uroporphyrinogen decarboxylase (HEM12), coproporphyrinogen III oxidase (HEM13), protoporphyrinogen oxidase (HEM14), and ferrochelatase (HEM15). Together these 8 enzymes convert the precursors glycine and succinyl-CoA to Heme B.

[0008] Additionally, S. cerevisiae has a heme oxygenase gene (HMX1) involved in the degradation of heme. Heme oxygenases cleave the heme porphyrin structure and release the bound iron ion, for example converting heme B into biliverdin.

[0009] Heme regulatory motifs (HRM) have been described in several species in various proteins, such as the 5-Aminolevulinate synthase 1 (ALAS'!) of Quail (Munakata et al., 2004) and heme oxygenase (HMX) of humans (Fleischhacker et al., 2020). HRMs consist of a 5 amino acid motif where the core cysteine-proline (CP) motif is conserved (Munakata et al., 2004). Studies on the Quail ALAS1 demonstrated that the HRMs and their interaction with heme is involved in regulating ALAS1 mitochondrial import. The mutation of the ALAS1 HRMs resulted in increased ALAS1 mitochondrial import and insensitivity to heme feedback inhibition (Munakata et al., 2004).

[0010] Summary

[0011] The present disclosure relates to yeast cells of the genus Yarrowia capable of producing heme and / or hemoproteins, as well as methods for producing heme and / or hemoproteins in Yarrowia cells. Thus, provided herein are Yarrowia cells with increased heme content, enhanced heme synthesis, altered heme profile and / or up- regulated heme biosynthesis, and methods of producing such Yarrowia cells.

[0012] Indeed, an effect of increasing the content of heme and / or hemoprotein in Yarrowia cells according to the present disclosure, is that the appearance and flavour of said cells, as well as other chemical characteristics, change. Yarrowia cells with increased content of heme and / or one or more hemoproteins according to this disclosure may appear more red and / or brown themselves. Interestingly, this may also be reflected in biomass and / or compositions comprising said Yarrowia cells. Furthermore, “browning” is increased when Yarrowia cells according to this disclosure are cooked (heated). They may therefore be useful as meat alternatives, for example sustainable meat alternatives.

[0013] In a main aspect is provided a Yarrowia cell producing or capable of producing one or more hemes and / or one or more hemoproteins, said cell comprising one or more of the following modifications, such as two or three of the following modifications: i. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased activity is the result of a mutation, further optionally wherein said heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3; ii. increased activity of a 5-aminolevulinic acid synthase (ALAS) relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said increased activity is the result of a mutation, further optionally wherein said ALAS is HEM 1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 ; and / or iii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased sensitivity is the result of a mutation, such as a mutation of at least one heme-regulatory motif (HRM) of said ALAS, further optionally wherein said ALAS is HEM 1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 , preferably wherein the production of said one or more hemes and / or said one or more hemoproteins is improved relative to the production of said one or more hemes and / or said one or more hemoproteins by a corresponding Yarrowia cell without said modification but otherwise identical, when incubated and optionally propagated in similar conditions. In some embodiments, the Yarrowia cell is a Y. lipolytica cell or a Y. porcina cell. In some embodiments, the heme is heme B.

[0014] Thus, in some embodiments, the Yarrowia cell comprises all of the three modifications above; in other embodiments, the cell comprises modifications i. and ii. above; in other embodiments, the cell comprises modifications ii. and iii. above; in other embodiments, the cell comprises modifications i. and iii. above.

[0015] In some embodiments, the Yarrowia cell produces or is capable of producing one or more hemes. In some embodiments, the Yarrowia cell produces or is capable of producing one or more hemoproteins. In some embodiments, the Yarrowia cell produces or is capable of producing one or more hemes and one or more hemoproteins.

[0016] The Yarrowia cell according to the present disclosure preferably comprises, produces or is capable of producing one or more hemoproteins. Thus, provided herein are also one or more hemes and / or one or more hemoproteins obtainable by a method and / or produced by a Yarrowia cell disclosed herein. The one or more hemoproteins may be hemoproteins endogenous to the Yarrowia cell and / or hemoproteins heterologous to the Yarrowia cell, for example one or more globins and / or one or more cytochromes. In another main aspect, provided herein is a method of producing and / or increasing production of one or more hemes and / or one or more hemoproteins in a Yarrowia cell, comprising the steps of: i. providing a Yarrowia cell disclosed herein; and ii. incubating and optionally propagating said Yarrowia cell in a medium, whereby said one or more hemes and / or one or more hemoproteins is produced or whereby production of said one or more hemes and / or one or more hemoproteins is increased.

[0017] Provided herein is also a method of manufacturing biomass comprising Yarrowia cells with increased intracellular concentration and / or content of one or more hemes and / or one or more hemoproteins, said method comprising the steps of: i. providing a Yarrowia cell with increased intracellular concentration and / or content of a hemoprotein, compared to a corresponding cell without the one or more modifications, such as two or three modifications, disclosed herein, but otherwise identical, cell as disclosed herein; and ii. incubating and optionally propagating said Yarrowia cell in a medium; and iii. optionally recovering biomass comprising said Yarrowia cell, whereby said biomass comprising Yarrowia cells with increased intracellular concentration of heme and / or a hemoprotein is obtained.

[0018] In another aspect, provided is a method of manufacturing a meat alternative and / or ingredient therefor, comprising the steps of: i. providing a Yarrowia cell as disclosed herein; ii. incubating and optionally propagating said Yarrowia cell in a medium, iii. recovering biomass comprising said Yarrowia cell and / or fermentation liquid comprising biomass comprising said Yarrowia cell; and / or iv. converting said biomass and / or fermentation liquid comprising biomass comprising said Yarrowia cell into a meat alternative and / or ingredient therefor, whereby a meat alternative and / or ingredient therefor is manufactured.

[0019] It is also an aspect of the present disclosure to provide various compositions comprising a Yarrowia cell, one or more hemes and / or one or more hemoproteins according to the present disclosure. Thus, provided herein is a composition comprising a Yarrowia cell disclosed herein, and / or one or more hemes and / or one or more hemoproteins obtainable by a method disclosed herein.

[0020] Provided is also a biomass comprising Yarrowia cells disclosed herein, such as Yarrowia cells with increased intracellular concentration and / or content of one or more hemes and / or one or more hemoproteins compared to a corresponding cell without the one or more modifications, such as two or three modifications, disclosed herein, but otherwise identical, obtainable by a method disclosed herein.

[0021] Further provided is the use of a composition as disclosed herein as a food product, food material, food ingredient and / or feed ingredient.

[0022] In particular, provided in the present disclosure is the use of a composition as disclosed herein in a meat alternative and / or in a method for producing a meat alternative.

[0023] Provided is also a meat alternative and / or ingredient therefor obtainable by a method as disclosed herein.

[0024] The present disclosure also provides a food composition comprising one or more hemes and / or one or more hemoproteins from a Yarrowia cell as disclosed herein.

[0025] Provided herein is also the use of a Yarrowia cell and / or biomass as disclosed herein, for production of meat alternative and / or ingredient therefor, for production of one or more hemes and / or one or more hemoproteins, and / or for production of food and / or feed containing one or more hemes and / or one or more hemoproteins.

[0026] In another aspect, provided is also an expression system for expression in a Yarrowia cell, comprising a nucleic acid encoding an ALAS, optionally wherein the ALAS is HEM1 as set forth in SEQ ID NO: 1 and / or HEM1_mutHRM as set forth in SEQ ID NO: 2, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 and / or SEQ ID NO: 2, respectively.

[0027] Description of Drawings

[0028] Figure 1. Heme B molecular structure. Figure 2. Overexpression of HEM1 and knockout of HMX1 improve heme production. Relative quantification of coproporphyrin (intermediate), heme B, and biliverdin (degradation product) compared to reference strain (Ref.) without any heme-related modifications. Overexpression of HEM1 and knockout of HMX1 result in an increased heme B production. Additionally, the decrease in biliverdin in the HMX1 knockout (KO) strain experimentally validates YALI1_D33552 as the Y. lipolytica heme oxygenase, HMX1.

[0029] Figure 3. Identification of heme regulatory motifs in HEM1. N-terminal sequence of HEM1 (YIHEM1, SEQ ID NO: 1) with the three identified heme regulatory motifs (HRMs) annotated.

[0030] Figure 4. HRM removal in HEM1 increases heme production. Overexpression of a mutated HEM1 lacking the HRMs results in an increased heme accumulation.

[0031] Figure 5. Sequence alignment showing HRMs in ALAS. Clustal omega sequence alignment of C-terminal part of ALAS from four species of Yarrowia. The three conserved HRMs are annotated above the corresponding sequence.

[0032] Figure 6. Heme B quantification in Y. lipolytica strains expressing the hemoprotein leghemoglobin A from Glycine max (Gm_LBA, SEQ ID NO: 4 encoded by SEQ ID NO: 9) in varying copy number. Relative quantification of heme B compared to reference strain (Reference). Increasing the copy number of Gm_LBA results in increased heme B production.

[0033] Figure 7. Effect of HMX1 deletion on heme production in S. cerevisiae. Data plotted as average of biological triplicates. Error bars represent standard deviation, n.s. = not significant.

[0034] Figure 8. Heme content in heme overproducer strain. Data plotted as average of biological triplicates. Error bars represent standard deviation. WW = wet weight. Detailed description

[0035] Definitions

[0036] Heme as term herein refers to an enzyme capable of degrading heme.

[0037] Heme oxygenase is capable of catalysing the reaction (EC 1.14.14.18): protoheme + 3 [reduced NADPH-hemoprotein reductase] + 3 O2 = biliverdin + Fe2++ CO + 3 [oxidized NADPH-hemoprotein reductase] + 3 H2O

[0038] Heme oxygenase may also be referred to as haem oxygenase, and the terms may be used interchangeably herein.

[0039] 5-aminolevulinic acid as term herein refers to an enzyme capable of synthesizing 5-aminolevulinate. 5-aminolevulinic acid synthase may also be referred to as ALAS or ALAS1. ALAS is capable of catalysing the reaction (EC 2.3.1.37): glycine + H++ succinyl-CoA = 5-aminolevulinate + CO2 + CoA 5-aminolevulinate may also be referred to as 5-aminolevulinic acid, 5ALA, 5-ALA and / or ALA, and the terms may be used interchangeably herein.

[0040] Heme herein refers to a molecule or coordination complex comprising an iron cation

[0041] (ferrous iron, Fe"), which is coordinated to porphyrin, acting as a tetradentate ligand, and to one or two axial ligands. Heme is an essential prosthetic group for various heme-binding proteins, including hemoglobin. Different types of heme exist, for example heme A, heme B, heme C and heme D. Derivatives of heme B are for example heme / , heme m, heme D and Heme S. Heme may also be referred to as haem and the terms may be used interchangeably herein.

[0042] Hemoprotein herein refers to metalloproteins capable of binding heme covalently and / or noncovalently. Heme is a prosthetic groups of hemoproteins. Examples of hemoproteins are hemoglobin, myoglobin, cytochrome P450s, cytochrome c oxidases, catalases, ligninases and peroxidases. Hemoprotein may also be referred to as hemoprotein, hemeprotein, haemprotein, haemoprotein, or heme-binding protein, and the terms may be used interchangeably herein.

[0043] Heme motif herein refers to a five amino acid motif comprising a conserved cysteine-proline (CP) motif. Heme-regulatory motif (HRM) is known for being involved in the regulatory function of heme. The CP motif or CP dipeptide can ligate Fe3+-heme via the cysteine residue. Heme-regulatory motif may also be referred to as HRM or heme-binding motif, and the terms may be used interchangeably herein.

[0044] Meat alternative herein refers to an alternative food product to meat of animal origin, which may be used (consumed or eaten) as a replacement for meat. Non-limited alternative words for meat alternative are meat substitute, mock meat, replica meat, or fake meat.

[0045] Identity, homology or similarity with respect to a nucleic acid sequence (polynucleotide, DNA, RNA) or polypeptide (protein), are defined herein as the percentage of nucleotides or amino acids, respectively, in the candidate sequence that are identical, homologous or similar, respectively, to the residues of a corresponding native (may be codon-optimised) nucleotide or amino acid sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity I similarity, and considering any conservative substitutions according to the NCIIIB rules ([https: / / iubmb.qmul.ac.uk / misc / naseq.html; NC-llIB, Eur J Biochem (1985)]) as part of the sequence identity. In particular, the percentage of similarity refers to the percentage of residues conserved with similar physiochemical properties. Neither 5' or 3' extensions nor insertions (for nucleic acids) or N’ or C’ extensions nor insertions (for polypeptides) result in a reduction of identity, similarity or homology. Methods and computer programs for the alignments are well known in the art.

[0046] Generally, a given identity between two sequences implies that the similarity between these sequences is at least equal to the identity; for example, if two sequences are 70% identical to one another, they cannot be less than 70% similar to one another - but could be sharing 80% similarity. Thus, throughout the present disclosure, it will be understood that any variant, such as a functional variant, or homologue said to have at least 70% sequence identity, similarity, or homology to a specified sequence (nucleic acid sequence (polynucleotide) or polypeptide) refers to a sequence having at least 70%, such as at least 71 %, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81 %, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity, similarity, or homology thereto.

[0047] The terms nucleic acid, nucleic acid sequence, nucleic acid constructs, nucleic acid molecules, oligonucleotide, and polynucleotides may be used interchangeably herein.

[0048] Functional variant as term herein refers to a functional variant of an enzyme which retains at least some of the activity of the parent enzyme. It follows that a functional variant of an ALAS can catalyse the same conversion as an ALAS from which it is derived, although the efficiency of the conversion reaction may be different, e.g. the efficiency is decreased or increased compared to the parent enzyme or the substrate specificity may be modified. as term refers herein to a cell which does not comprise the modifications disclosed herein for production or overproduction of heme and / or hemoprotein, but is otherwise identical. The term may for example be used in relation to the Yarrowia cell disclosed herein, where the corresponding cell is a cell of the same genus and species, which has not been modified to produce or overproduce heme and / or one or more hemoproteins. A corresponding cell may also be referred to as reference cell or wild-type cell, and the terms may be used interchangeably herein.

[0049] Native as term when referring to a polypeptide such as an enzyme or to a polynucleotide, such as a gene, coding sequence of a gene or genetic element, shall herein be construed to refer to an amino acid sequence (polypeptide) or a nucleic acid sequence (polynucleotide), such as a gene, which is naturally present in a wild-type cell.

[0050] Heterologous as term when referring to a polypeptide such as an enzyme or to a polynucleotide, such as a gene, coding sequence of a gene or genetic element, shall herein be construed to refer to an amino acid sequence (polypeptide) or a nucleic acid sequence (polynucleotide), such as a gene, which is not naturally present in a wild-type cell.

[0051] Mutation as term when used herein in the context of nucleic acids herein refers to a change in nucleic acid compared to the parent nucleic acid. The term mutation covers single nucleotide mutations, but also insertions and deletions of multiple nucleotides, i.e. any change that leads to a different nucleic acid than the parent nucleic acid. The term mutation thus encompasses deletions, such as deletions of a whole gene or of a coding sequence of a gene, or a fragment / fraction of a gene or of a coding sequence of a gene. The term mutation when used herein in the context of polypeptides, such as enzymes, refers to a change in amino acid sequence compared to the parent amino acid sequence. The term mutation covers single amino acid mutations, but also insertions and deletions of multiple amino acids, i.e. any change that leads to a different amino acid sequence than the parent amino acid sequence. The term mutation thus encompasses amino acid substitutions, i.e. where an amino acid is exchanged for a different amino acid.

[0052] Decreased as term may herein refers to a total or partial loss of activity of a given polypeptide, such as an enzyme. In some cases, the polypeptide is encoded by a non-essential gene, and the activity may be decreased or it may be completely lost, e.g. as a consequence of a total or partial deletion of the gene encoding the polypeptide or as a consequence of the introduction of a nonsense mutation in the gene encoding the polypeptide. “Decreased activity” may also be referred to as “reduced activity” and the terms may be used interchangeably herein. as term may herein refer to an improvement of activity of a given polypeptide, such as an enzyme. Said improvement in activity may be assessed based on the activity of the unmodified, corresponding polypeptide serving as a reference. The improvement of activity may not be present at all times and / or in all conditions. Thus, the improvement of activity may be dependent on the condition wherein it is assessed such as a growth condition and therefore be considered conditiondependent. However, the improvement of activity may also be condition-independent and present in all conditions, optionally with varying improvement. Improvement of activity may be a consequence of a mutation of the gene encoding the polypeptide and / or a mutation in one or more genetic elements influencing the expression of the gene and / or the activity of the polypeptide. Improvement of activity may be achieved by modifying a transcription factor, promoter and / or terminator of the gene encoding the polypeptide. “Increased activity” may also be referred to as “improved activity”, “enhanced activity”, and the terms may be used interchangeably herein. Titer as term herein refers to the concentration of a compound that accumulates inside a cell (intracellular) and / or in the fermentation liquid (broth / supernatant) during incubation, and optionally propagation, of the cell.

[0053] Overexpression as term herein refers to a process by which a nucleic acid, such as a gene, encoding a polypeptide is artificially expressed in a modified cell to produce a level of expression of the encoded polypeptide that exceeds the level of expression of the same polypeptide in an unmodified cell, also termed reference cell or corresponding cell herein, which unmodified cell is otherwise identical to the modified cell. Thus, while the term is typically used in conjunction with a nucleic acid, the term "overexpression" may also be used in conjunction with a polypeptide, such as an enzyme, to refer to the increased level of a polypeptide resulting from the overexpression of its encoding nucleic acid. In some embodiments, overexpression of a nucleic acid encoding a polypeptide is achieved by increasing the number of copies of the nucleic acid that encodes the polypeptide. In other embodiments, overexpression of a nucleic acid encoding a polypeptide is achieved by increasing the binding strength of the promoter region and / or the ribosome binding site in such a way to increase the transcription and / or the translation of the nucleic acid that encodes the polypeptide. In some embodiments, the overexpression of a nucleic acid encoding a polypeptide is achieved by increasing the activity of a transcription factor controlling the level of transcription of said nucleic acid. In some embodiments, the overexpression of a nucleic acid encoding a polypeptide results from the expression of at least one copy of the corresponding encoding nucleic acid (polynucleotide) present on a multicopy plasmid that has been introduced into a Yarrowia cell. In other embodiments, the overexpression of a nucleic acid encoding a polypeptide results from the expression of two or more copies of the corresponding encoding nucleic acid (polynucleotide) that are integrated into the genome of the Yarrowia cell. For expression of polypeptides heterologous to the cell, where no native expression level exists for comparison, “overexpression” refers to expressing the polypeptide at a medium or strong level in the cell, or at least at a level such that the polypeptide can be detected and / or its activity (when the polypeptide is an enzyme) can be measured. For expression of a modified variant of a native polypeptide, where no native expression level of said modified variant exists for comparison, “overexpression” is determined relative to the expression level of the native polypeptide without said modification. Classification of promoter strength as “strong” refers to the degree to which the promoter alters the rate of transcription initiation from the associated nucleic acid, such as the associated gene, compared to the endogenous level or the level obtained using a normal promoter, such as the promoters of the RNA processing protein YALI0D05577g (CUS1) (prCUSI), the actin capping enzyme YALI0C20735g (CAP2) (prCAP2), or the RNA splicing enzyme YALI0D09955g (prPRP2). Thus, herein a given promoter is considered “strong”, if its rate of transcription initiation of an associated nucleic acid is greater than the rate of transcription initiation of said nucleic acid by at least one of prCAP2 and prPRP2, when the strength is determined under the same conditions and using a corresponding Yarrowia cell otherwise identical. Per definition a strong promoter results in an increase in the associated gene transcription compared to endogenous levels. The skilled person knows whether a given promoter is classified as a strong promoter and / or how to determine the promoter strength of a uncharacterised promoter. For instance, RNA levels can be measured using RNA sequencing, or protein levels can be quantified with a reporter protein, such as gene-fluorescence- protein (GFP), and used for determining the strength of the promoter. A strong promoter may either be a native promoter or a non-native promoter of the organism, such as the Yarrowia cell, that it is comprised within. A non-native promoter may be a promoter that is native to another organism, for example native to another yeast species, or it may be a synthetic promoter. Examples of strong promoters are provided in section “Strong promoter”.

[0054] Yarrowia cell

[0055] This disclosure relates to Yarrowia cells capable of producing heme and / or hemoprotein. In particular, the disclosure relates to modified Yarrowia cells with increased content of heme and / or a hemoprotein compared to corresponding cells without one or more of the modifications described herein. The disclosure, also relates to biomass of said Yarrowia cells and to use of the Yarrowia cells and / or biomass, for example use thereof in food compositions.

[0056] In a main aspect, the present disclosure discloses a Yarrowia cell capable of producing heme, said cell comprising one or more of the following modifications: i. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased activity is the result of a mutation, further optionally wherein said heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3; ii. increased activity of a 5-aminolevulinic acid synthase (ALAS) relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said increased activity is the result of a mutation, further optionally wherein said ALAS is HEM 1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 ; and / or iii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased sensitivity is the result of a mutation, such as a mutation of at least one heme-regulatory motif (HRM) of said ALAS, further optionally wherein said ALAS is HEM 1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1.

[0057] Thus, in some embodiments, the present disclosure relates to a Yarrowia cell producing or capable of producing heme and optionally one or more hemoproteins, said cell comprising two or three of the following modifications: i. increased activity of a 5-aminolevulinic acid synthase (ALAS) relative to the activity of said ALAS in a corresponding Yarrowia cell without said two or three modifications but otherwise identical, optionally wherein said increased activity is the result of a mutation, optionally wherein said ALAS is HEM1 as set forth in SEQ ID NO: 1, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1; ii. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said two or three modifications but otherwise identical, optionally wherein said decreased activity is the result of a mutation, optionally wherein said heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3; and / or iii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said two or three modifications but otherwise identical, optionally wherein said decreased sensitivity is the result of a mutation, such as a mutation of at least one heme- regulatory motif (HRM) of said ALAS, optionally wherein said ALAS is HEM1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1.

[0058] Preferably, the yeast cell comprises at least i. and ii.

[0059] In preferred embodiments, the production of said heme is improved relative to the production of heme by a corresponding Yarrowia cell without said modification but otherwise identical, when cultivated in similar conditions.

[0060] The term “cultivated” herein implies “incubated and optionally propagated”, and vice versa.

[0061] Said heme may be selected from heme A, heme B, heme C and heme O. In preferred embodiments of the present disclosure, the heme is heme B. In other embodiments, said heme may comprise a combination of one or more hemes selected from heme A, heme B, heme C and heme O.

[0062] In some embodiments, the ALAS is HEM1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1. In some embodiments, the heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3

[0063] The Yarrowia cells disclosed herein producing or capable of producing heme and / or hemoprotein, might also be referred to as production organisms, microbial factories, microbial production organisms, hosts, host cells, host organisms, production hosts, cell factories, and the like.

[0064] Various Yarrowia species may be useful according to the present disclosure. In preferred embodiments, the Yarrowia cell is a non-pathogenic Yarrowia cell. Preferably, the Yarrowia cell is generally-regarded-as-safe (GRAS) and / or has safe-to- consume status. The Yarrowia cell according to the present disclosure may be a genetically modified (GMO) cell or a non-GMO cell. In other words, the Yarrowia cell may be modified for example using mutagenesis, including random and targeted mutagenesis, and / or genetic engineering using genetic engineering tools, such as CRISPR / Cas. Thus, in some embodiments, the Yarrowia cell is a non-GMO cell. In other words, in some embodiments, the Yarrowia cell is a non-transgenic cell. In other embodiments, the Yarrowia cell is a GMO cell.

[0065] In some embodiments, the Yarrowia cell belongs to the species of Yarrowia lipolytica, Yarrowia porcina, Yarrowia bubula, Yarrowia deformans, Yarrowia yakushimensis, Yarrowia parophonii, Yarrowia galli, Yarrowia oslonensis, Yarrowia alimentaria, Yarrowia hollandica, or Yarrowia phangngaensis. In preferred embodiments, the Yarrowia cell is a Yarrowia lipolytica cell, or a Yarrowia porcina cell.

[0066] The specific nucleic acids, for example genes, identified in Y. lipolytica, as described herein, encode specific polypeptides, such as proteins and / or enzymes. In other Yarrowia species, the specific polypeptides or nucleic acids may be differently annotated, but however still share a similar function, encode a similar enzyme and / or protein or a functional variant sharing a similar function, respectively. Thus, the knowledge from Y. lipolytica can be transferred to other Yarrowia species, for example Y. porcina. The skilled person will know how to identify the corresponding polypeptides, such as enzymes, or nucleic acids, such as genes, to be modified, mutated, deleted, down-regulated, up-regulated, or overexpressed, based on the information provided herein for Y. lipolytica.

[0067] In preferred embodiments, the Yarrowia cell further produces or is capable of producing one or more hemoproteins. Thus, disclosed herein is also in another aspect, a Yarrowia cell capable of producing heme and hemoprotein, said cell comprising one or more of the following modifications: i. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased activity is the result of a mutation, further optionally wherein said heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3; ii. increased activity of a 5-aminolevulinic acid synthase (ALAS) relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said increased activity is the result of a mutation, further optionally wherein said ALAS is HEM 1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 ; and / or iii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased sensitivity is the result of a mutation, such as a mutation of at least one heme-regulatory motif (HRM) of said ALAS, further optionally wherein said ALAS is HEM 1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1.

[0068] Thus, in some embodiments the present disclosure relates to a Yarrowia cell producing or capable of producing heme and hemoprotein, said cell comprising two or three of the following modifications: i. increased activity of a 5-aminolevulinic acid synthase (ALAS) relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said increased activity is the result of a mutation, optionally wherein said ALAS is HEM1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ I D NO: 1 ; ii. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased activity is the result of a mutation, optionally wherein said heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3; and / or iii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased sensitivity is the result of a mutation, such as a mutation of at least one heme-regulatory motif (HRM) of said ALAS, optionally wherein said ALAS is HEM1 as set forth in SEQ ID NO: 1, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1.

[0069] In preferred embodiments, the production of said heme and said hemoprotein is improved relative to the production of said heme and said hemoprotein by a corresponding Yarrowia cell without said modification but otherwise identical, when incubated and optionally propagated in similar conditions.

[0070] Disclosed is also a Yarrowia cell capable of producing a hemoprotein, said cell comprising one or more of the following modifications: i. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased activity is the result of a mutation, further optionally wherein said heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3; ii. increased activity of a 5-aminolevulinic acid synthase (ALAS) relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said increased activity is the result of a mutation, further optionally wherein said ALAS is HEM 1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 ; and / or iii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased sensitivity is the result of a mutation, such as a mutation of at least one heme-regulatory motif (HRM) of said ALAS, further optionally wherein said ALAS is HEM 1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1. In some embodiments, the present disclosure relates to a Yarrowia cell producing or capable of producing a hemoprotein, said cell comprising two or three of the following modifications: i. increased activity of a 5-aminolevulinic acid synthase (ALAS) relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said increased activity is the result of a mutation, optionally wherein said ALAS is HEM1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 ; ii. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased activity is the result of a mutation, optionally wherein said heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3; and / or iii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased sensitivity is the result of a mutation, such as a mutation of at least one heme-regulatory motif (HRM) of said ALAS, optionally wherein said ALAS is HEM1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1.

[0071] In preferred embodiments, the production of said hemoprotein is improved relative to the production of said hemoprotein by a corresponding Yarrowia cell without said modification but otherwise identical, when cultivated in similar conditions.

[0072] Hemoproteins are further described in the section “Hemoprotein”. In some embodiments, the hemoprotein is an endogenous hemoprotein. In other embodiments, the hemoprotein is a polypeptide, such as a protein, heterologous to the Yarrowia cell. Said hemoprotein may be a globin, such as myoglobin, hemoglobin, or leghemoglobin. In other embodiments, said hemoprotein may be a cytochrome. In preferred embodiments, the Yarrowia cell is incubated in the presence of glycine, alpha-ketoglutarate, succinyl-CoA and / or iron, such as an iron salt.

[0073] In some embodiments, the Yarrowia cell is capable of synthesizing glycine, alpha- ketoglutarate and / or succinyl-CoA. In other embodiments, the Yarrowia cell is supplemented with glycine, alpha-ketoglutarate, and / or succinyl-CoA. For example, in some embodiments, the Yarrowia cell is capable of synthesizing succinyl-CoA and is supplemented with alpha-ketoglutarate.

[0074] In other embodiments, the Yarrowia cell is supplemented with iron, such as an iron salt, for example ferric chloride, iron sulfate, iron nitrate, and / or ferric citrate. For example, in some embodiments, the Yarrowia cell is capable of synthesizing succinyl-CoA and is supplemented with alpha-ketoglutarate and iron.

[0075] Suitable media and conditions for growth are detailed further elsewhere herein, for example in the section “Medium and growth conditions”.

[0076] Heme oxygenase

[0077] The Yarrowia cell capable of producing heme and / or hemoprotein may comprise one or more modifications resulting in decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical. Preferably, said Yarrowia cell capable of producing heme and / or hemoprotein does not comprise an active heme oxygenase. In preferred embodiments, the production of said heme is improved relative to the production of heme by a corresponding Yarrowia cell without said modification but otherwise identical, when incubated and optionally cultivated in similar conditions.

[0078] Heme oxygenases can catalyse degradation of heme. Thus, the Yarrowia cell according to the present disclosure is preferably not capable of degrading heme. In some embodiments, the Yarrowia cell thus has decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical. A heme oxygenase is capable of converting heme to biliverdin, iron (or ferrous ion), and carbon monoxide. The heme oxygenase of the present disclosure may be a heme oxygenase with EC 1.14.14.18. The heme oxygenase may be an endogenous heme oxygenase. In other words, the heme oxygenase may be a heme oxygenase natively expressed by the Yarrowia cell. Thus, in preferred embodiments, the heme oxygenase is native to the Yarrowia cell.

[0079] In some embodiments, the heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 3. In some embodiments, the gene encoding the heme oxygenase is HMX1 as set forth in SEQ ID NO: 8, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 8.

[0080] In some embodiments, said decreased activity is the result of a mutation.

[0081] The one or more modifications resulting in decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, may comprise or consist of: i. partial or total deletion of a gene encoding the heme oxygenase; ii. down-regulation of the expression of a gene encoding the heme oxygenase; and / or iii. down-regulation of the activity of the heme oxygenase, whereby the activity of the heme oxygenase is decreased.

[0082] In some embodiments, the one or more modifications resulting in decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, comprises or consists of: i. a mutation in a nucleic acid sequence upstream of the gene encoding the heme oxygenase, such as a mutation in a promoter of said gene, such as a partial or total deletion of said upstream nucleic acid sequence, such as of said promoter; ii. a mutation of a transcription factor controlling regulation of the transcription of the gene encoding the heme oxygenase, such as a partial or total deletion of said transcription factor, wherein the mutation results in partial or total loss of activity of said promoter and / or transcription factor, respectively, whereby the expression of the gene encoding the heme oxygenase is down-regulated.

[0083] In some embodiments, the one or more modifications resulting in decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, comprises or consists of: i. a partial or total loss-of-function mutation of the gene encoding the heme oxygenase; and / or ii. inhibition of the heme oxygenase, such as reversible or irreversible inhibition, whereby the activity of the heme oxygenase is down-regulated.

[0084] In some embodiments, the one or more modifications resulting in decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, comprise or consist of a mutation in a nucleic acid sequence upstream of the gene encoding the heme oxygenase, such as a mutation in a promoter of said gene, such as a partial or total deletion of said upstream nucleic acid sequence, such as of said promoter, wherein the mutation results in partial or total loss of activity of said promoter, whereby the expression of the gene encoding the heme oxygenase is down-regulated.

[0085] In other embodiments, the one or more modifications resulting in decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, comprise or consist of a mutation of a transcription factor controlling regulation of the transcription of the gene encoding the heme oxygenase, such as a partial or total deletion of said transcription factor, wherein the mutation results in partial or total loss of activity of said transcription factor, whereby the expression of the gene encoding the heme oxygenase is down- regulated.

[0086] In further embodiments, the one or more modifications may comprise or consist of a partial or total loss-of-function mutation of the gene encoding heme oxygenase, whereby the activity of the heme oxygenase is down-regulated. In some embodiments, one or more modifications may comprise or consist of inhibition of the heme oxygenase, such as reversible or irreversible inhibition, whereby the activity of the heme oxygenase is down-regulated. In other embodiments, the one or more modifications may comprise or consist of a partial or total loss-of-function mutation of the gene encoding heme oxygenase, and of inhibition of the heme oxygenase, such as reversible or irreversible inhibition, whereby the activity of the heme oxygenase is down-regulated.

[0087] In some embodiments, where the Yarrowia cell comprises one or more modifications, such as two or more modifications, wherein one of the modifications is decreased activity of a heme oxygenase, the cell does not comprise a modification in a hemedependent repressor of hypoxic genes, such as R0X1 (accession no. Q6CFZ3, database Uniprot). In other embodiments, where the Yarrowia cell comprises one or more modifications, such as two or more modifications, wherein one of the modifications is decreased activity of a heme oxygenase, the cell does not comprise a modification in a vacuolar proteinase, such as PEP4 (accession no. Q6C080, database Uniprot). In other embodiments, where the Yarrowia cell comprises one or more modifications, such as two or more modifications, wherein one of the modifications is decreased activity of a heme oxygenase, the cell does not also comprise a modification in a heme-dependent repressor of hypoxic genes, such as R0X1, and a modification in a vacuolar proteinase, such as PEP4.

[0088] 5-aminolevulinic acid synthase (ALAS)

[0089] The Yarrowia cell capable of producing heme and / or hemoprotein, and comprising one or more modifications resulting in decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, may further comprise or alternatively comprise increased activity of a 5-aminolevulinic acid synthase (ALAS) relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical.

[0090] Thus, the Yarrowia cell capable of producing heme and / or hemoprotein may comprise one or more modifications resulting in increased activity of an ALAS relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical. In preferred embodiments, said increased activity of an ALAS results in improved production of heme relative to the production of heme by a corresponding Yarrowia cell without said modification but otherwise identical, when cultivated in similar conditions.

[0091] ALAS is capable of catalysing synthesis of 5-aminolevulinate (ALA). Thus, the ALAS may be capable of converting succinyl-CoA and glycine to ALA. The ALAS may be an ALAS with EC 2.3.1.37.

[0092] In some embodiments, the ALAS is an ALAS native to the Yarrowia cells. In other words, the ALAS may be an endogenous ALAS. In other words, the ALAS may be an ALAS native to the Yarrowia cell.

[0093] In some embodiments, the endogenous ALAS is a Yarrowia ALAS, for example a Yarrowia lipolytica ALAS such as HEM1 as set forth in SEQ ID NO: 1, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 1. HEM1 as set forth in SEQ ID NO: 1 may also be referred to herein as YIHEM1. In some embodiments, the gene encoding the ALAS is HEM1 as set forth in SEQ ID NO: 6, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 6.

[0094] The Yarrowia cell may comprise at least 2 copies of the gene encoding the ALAS, such as at least 3 copies, such as at least 4 copies, such as at least 5 copies, such as at least 6 copies, such as at least 7 copies, such as at least 8 copies, such as at least 9 copies, such as at least 10 copies, or more.

[0095] In other embodiments, the ALAS is an ALAS heterologous to the Yarrowia cell. Said ALAS heterologous to the Yarrowia cell, may be an ALAS of Saccharomyces cerevisiae, such as ScALAS (Uniprot ID: P09950), of Schizosaccharomyces pombe, such as SpALAS (Uniprot ID: 014092), of Neurospora crassa, such as NcALAS (Uniprot ID: Q7RVY5), of Agaricus bisporus, such as AbALAS (Uniprot ID: Q92403), of Gallus gallus, such as GgALASI (Uniprot ID: P07997) or GgALAS2 (Uniprot ID: P18080), or an ALAS of Bos taurus, such as BtALAS (Uniprot ID: A6QLI6), or BtALAS2 (Uniprot ID: Q3ZC31).

[0096] In some embodiments, said increased activity is the result of a mutation.

[0097] In some embodiments, the Yarrowia cell comprises one or more modifications, wherein the one or more modifications comprises or consists of i. overexpression of a gene encoding the ALAS; ii. increased production of the ALAS; and / or iii. gain-of-function mutation of a gene encoding the ALAS, whereby the activity of ALAS is increased.

[0098] In one embodiment, the Yarrowia cell comprises one or more modifications, wherein the one or more modifications comprises or consists of overexpression of a gene encoding the ALAS, whereby the activity of ALAS is increased. In another embodiment, the Yarrowia cell comprises one or more modifications, wherein the one or more modifications comprises or consists of increased production of the ALAS, whereby the activity of ALAS is increased. In further one embodiment, the Yarrowia cell comprises one or more modifications, wherein the one or more modifications comprises or consists of gain-of-function mutation of a gene encoding the ALAS, whereby the activity of ALAS is increased. In some embodiments, the one or more modifications comprises or consists of increasing the copy number of ALAS. Thus, in some embodiments, the Yarrowia cell carries at least one additional copy of said ALAS, such as at least two additional copies, such as at least four additional, such as at least five additional copies, such as at least six additional copies, such as at least seven additional copies, such as at least eight additional copies, such as at least nine additional copies, such as at least ten additional copies, or more copies of said ALAS.

[0099] In other embodiments, the overexpression of a gene encoding said ALAS results from the expression of at least one copy of the corresponding encoding nucleic acid present on a multicopy plasmid that has been introduced into a Yarrowia cell. In other embodiments, the overexpression of a nucleic acid encoding a polypeptide results from the expression of two or more copies of the corresponding encoding nucleic acid that are integrated into the genome of the Yarrowia cell.

[0100] In other embodiments, the one or more modifications comprises or consists of a mutation resulting in increased transcription of the gene encoding the ALAS, whereby the gene encoding the ALAS is overexpressed.

[0101] In some embodiments, the one or more modifications comprises or consists of prolonging the half-life of mRNA transcribed from a gene encoding the ALAS, whereby the production of ALAS is increased.

[0102] In some embodiments, the expression of the gene encoding ALAS is modified, preferably increased, such as by introducing a mutation in the native promoter of ALAS, for example a partial or total substitution of a nucleic acid sequence upstream of ALAS, or by operably linking the gene encoding ALAS to a non-native promoter, whereby expression of the gene encoding ALAS is modified, preferably increased, compared to expression from a reference cell, wherein expression of the gene encoding ALAS is not modified in said reference cell, and / or wherein the one or more modifications comprises or consists of a mutation in a nucleic acid sequence upstream of the gene encoding ALAS, such as a mutation in a promoter of said gene, for example a partial or total substitution of said upstream nucleic acid sequence, such as of said promoter, preferably said promoter of ALAS is substituted, such as replaced, by a stronger promoter relative to said promoter of ALAS, where transcription from said strong promoter is higher compared to transcription from the native ALAS promoter under the same conditions.

[0103] In some embodiments, the expression of the gene encoding ALAS is increased, whereby expression of the gene encoding ALAS is increased, compared to expression from a reference cell, wherein expression of the gene encoding ALAS is not increased in said reference cell. Increased expression of said gene may be obtained by introducing a mutation in the native promoter of ALAS, for example a partial or total substitution of a nucleic acid sequence upstream of ALAS, or by operably linking the gene encoding ALAS to a non-native promoter.

[0104] In other embodiments, the Yarrowia cell comprises one or more modifications, said modifications comprising or consisting of a mutation in a nucleic acid sequence upstream of the gene encoding ALAS, such as a mutation in a promoter of said gene, for example a partial or total substitution of said upstream nucleic acid sequence, such as of said promoter, preferably said promoter of ALAS is substituted, such as replaced, by a stronger promoter relative to said promoter of ALAS, where transcription from said strong promoter is higher compared to transcription from the native ALAS promoter under the same conditions.

[0105] In some embodiments, said nucleic acid sequence upstream of ALAS, such as said promoter of ALAS or fragment thereof, for example said native ALAS promoter or fragment thereof, is replaced by prEXPI as set forth in SEQ ID NO: 11, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 11. Heme-regulatory motifs (HRMs)

[0106] The Yarrowia cell capable of producing heme and / or hemoprotein, and comprising increased activity of an ALAS relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, may further comprise or alternatively comprise one or more modifications, such as one or more mutations, resulting in decreased sensitivity of an ALAS to feedback inhibition by heme. Thus, the Yarrowia cell may comprise or even overexpress an ALAS with decreased sensitivity to feedback inhibition by heme. Said decreased sensitivity may be the result of one or more mutations in the ALAS, for example one or more mutations described in the present section. The Yarrowia cell may further comprise one or more modifications resulting in decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, as described herein above.

[0107] The ALAS may be an ALAS as described elsewhere herein, for example in the section “5-aminolevulinic acid synthase (ALAS)”. For example, the ALAS may be an endogenous ALAS and / or an ALAS heterologous to the Yarrowia cell.

[0108] In some embodiments, the ALAS is a Yarrowia ALAS, for example HEM1 (SEQ ID NO: 1), or a functional variant thereof having at least 70% sequence identity, similarity or homology to SEQ ID NO: 1. In other embodiments, the ALAS is an ALAS heterologous to the Yarrowia cell, such as an ALAS of Saccharomyces cerevisiae, such as ScALAS (Uniprot ID: P09950), of Schizosaccharomyces pombe, such as SpALAS (Uniprot ID: 014092), of Neurospora crassa, such as NcALAS (Uniprot ID: Q7RVY5), of Agaricus bisporus, such as AbALAS (Uniprot ID: Q92403), of Gallus gallus, such as GgALASI (Uniprot ID: P07997) or GgALAS2 (Uniprot ID: P18080), or an ALAS of Bos taurus, such as BtALAS (Uniprot ID: A6QLI6), or BtALAS2 (Uniprot ID: Q3ZC31).

[0109] In some embodiments, said one or more mutations resulting in decreased sensitivity of an ALAS to feedback inhibition by heme are one or more mutations resulting in total loss of sensitivity to feedback inhibition by heme. Accordingly, the one or more mutations may, in other embodiments, result in partial loss of sensitivity to feedback inhibition by heme. The mutation resulting in decreased sensitivity of an ALAS to feedback inhibition by heme may be a mutation in at least one HRM of the ALAS.

[0110] In some embodiments, the ALAS comprises at least one HRM, such as at least two HRMs, such as at least three HRMs, or more. In preferred embodiments, the at least one HRM of the ALAS comprises a cysteine-proline (CP) motif.

[0111] The HRM of an ALAS may be determined by performing a sequence alignment of said ALAS with another ALAS comprising at least one known HRM. Sequence alignments may be performed by software known to the skilled person, such as Clustal Omega. Figure 5 is such a sequence alignment, wherein the C-terminal of ALAS from four Yarrowia species have been aligned and the three conserved HRMs have been annotated. Thus, the CP motif of the three HRMs of HEM 1 (SEQ ID NO: 1) are residues 12 to 13 (denoted HRM1), 38 to 39 (denoted HRM2), and 76 to 77 (denoted HRM3). Figure 3 shows the position of said motifs in the N-terminal part of HEM1 as set forth in SEQ ID NO: 1.

[0112] The mutation in at least one HRM of the ALAS may comprise or consist of a single amino acid substitution, preferably a single amino acid substitution of the CP-motif of said at least one HRM.

[0113] In some embodiments, the mutation in at least one HRM of the ALAS is a single- nucleotide-polymorphism (SNP) and / or a substitution of one or more nucleotides in the gene encoding ALAS. Preferably, said SNP and / or substitution is a missense SNP and / or missense substitution. In other embodiments, the mutation in the gene encoding ALAS comprises or consists of at least one mutation, such as at least two mutations, such as at least three mutations, such as at least four mutations, such as at least five mutations, such as at least six mutations, or more. Said SNP and / or substitution preferably results in a single amino acid substitution of the CP-motif of said at least one HRM.

[0114] In preferred embodiments, the mutation in the at least one HRM of the ALAS comprises or consists of a mutation resulting in a single amino acid substitution. In some embodiments, the mutation in the at least one HRM of the ALAS comprises or consists of a substitution of cysteine (C) of the CP motif of the at least one HRM to a different amino acid and / or a substitution of proline (P) of the CP motif of the at least one HRM to a different amino acid. Thus, in one embodiment, the mutation in the at least one HRM of the ALAS comprises or consists of a substitution of cysteine (C) of the CP motif of the at least one HRM to a different amino acid. In another embodiment, the mutation in the at least one HRM of the ALAS comprises or consists of a substitution of proline (P) of the CP motif of the at least one HRM to a different amino acid.

[0115] With respect to said mutation resulting in a single amino acid substitution to a different amino acid, the different amino acid may be selected from a polar uncharged amino acid, a nonpolar aliphatic amino acid, an aromatic amino acid, a positively charged amino acid and / or a negatively charged amino acid. In one embodiment, said polar uncharged amino acid is selected from serine, threonine, asparagine, and glutamine. In another embodiment, said nonpolar aliphatic amino acid is selected from glycine, proline, alanine, isoleucine, leucine, methionine, and valine. In further another embodiment, said aromatic amino acid is selected from phenylalanine, tryptophan, and tyrosine. In yet another embodiment, said positively charged amino acid is selected from arginine, histidine, and lysine. In another embodiment, said negatively charged amino acids is glutamic acid (glutamate), and aspartic acid (aspartate).

[0116] In some embodiments, the single amino acid substitution may be a substitution from a cysteine (C) to a different amino acid, for example to arginine, histidine, lysine, serine, threonine, asparagine, glutamine, glycine, proline, alanine, isoleucine, leucine, methionine, glutamic acid (glutamate), aspartic acid (aspartate), phenylalanine, tryptophan, tyrosine, or valine. In other embodiments, the single amino acid substitution may be a substitution from a proline (P) to a different amino acid, for example to arginine, histidine, lysine, serine, threonine, asparagine, glutamine, glycine, cysteine, alanine, isoleucine, leucine, methionine, glutamic acid (glutamate), aspartic acid (aspartate), phenylalanine, tryptophan, tyrosine, or valine.

[0117] In some embodiments, the mutation in the at least one HRM of the ALAS is a substitution of cysteine of the CP motif of the at least one HRM to a serine. In some embodiments, the mutation in the at least one HRM of the ALAS is a substitution of cysteine of the CP motif of the at least one HRM to a serine, and the ALAS is an ALAS as described in the section “5-aminolevulinic acid synthase (ALAS)” herein. In other embodiments, the Yarrowia cell comprises an ALAS comprising at least one HRM, such as several HRMs, for example three HRMs, wherein each HRM comprises a mutation, further wherein said mutation results in a single amino acid substitution to a different amino acid, wherein said different amino acid is the same for each of the HRMs, such as each of the three HRMs. Preferably, said amino acid substitution is a substitution of either cysteine, proline or both cysteine and proline of the CP-motif of said at least one HRM

[0118] In further some embodiments, the Yarrowia cell comprises an ALAS comprising at least one HRM, such as several HRMs, for example three HRMs, wherein each HRM comprises a mutation, further wherein said mutation results in a single amino acid substitution to a different amino acid, wherein the different amino acid is different for each of the HRMs, such as each of the HRMs.

[0119] In other embodiments, the Yarrowia cell comprises an ALAS comprising several HRMs, such as three HRMs, wherein each HRM comprises a mutation, further wherein said mutation results in a single amino acid substitution to a different amino acid, wherein the different amino acid is the same for two of the HRMs.

[0120] In other embodiments, the Yarrowia cell comprises an ALAS comprising several HRMs, such as three HRMs, wherein each HRM comprises a mutation, further wherein said mutation results in a single amino acid substitution to a different amino acid, wherein each HRM comprises at least one substitution of cysteine of the CP motif of each HRM to a serine.

[0121] In one embodiment, the Yarrowia cell capable of producing heme and / or hemoprotein comprises an ALAS with decreased sensitivity to feedback inhibition by heme, wherein said ALAS is HEM1_mutHRM as set forth in SEQ ID NO: 2, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 2.

[0122] The gene encoding said HEM1_mutHRM may be HEM1_mutHRM as set forth in SEQ ID NO: 7, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91 %, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 7.

[0123] Specific combinations

[0124] In some embodiments, the Yarrowia cell is capable of producing one or more hemes and / or one or more hemoproteins, said cell comprises all of the following modifications: i. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, wherein said heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3, and wherein said decreased activity is the result of a mutation; ii. increased activity of an ALAS relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, wherein said ALAS is HEM1 (SEQ ID NO: 1) and / or HEM1_mutHRM (SEQ ID NO: 2), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and wherein said increased activity is the result of a mutation; and iii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, wherein said decreased sensitivity is the result of a mutation of at least one HRM of said ALAS, wherein said ALAS is HEM 1 (SEQ ID NO: 1), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 , for example said ALAS with decreased sensitivity to feedback inhibition is HEM1_mutHRM (SEQ ID NO: 2), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 2.

[0125] In some embodiments, the Yarrowia cell comprises modifications i. and ii. above; in other embodiments, the cell comprises modifications ii. and iii. above; in other embodiments, the cell comprises modifications i. and iii. above.

[0126] Thus, in some embodiments, the Yarrowia cell produces or is capable of producing one or more hemes and / or one or more hemoproteins, said cell comprises all of the following modifications: i. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, wherein said heme oxygenase is HMX1 (SEQ ID NO: 3), or a functional variant thereof having at least 70% sequence identity, similarity, or homology thereto, and wherein said decreased activity is the result of a mutation; and ii. increased activity of an ALAS relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, wherein said ALAS is HEM1 (SEQ ID NO: 1) and / or HEM1_mutHRM (SEQ ID NO: 2), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and wherein said increased activity is the result of a mutation.

[0127] In other embodiments, the Yarrowia cell produces or is capable of producing one or more hemes and / or one or more hemoproteins, said cell comprises all of the following modifications: i. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, wherein said heme oxygenase is HMX1 (SEQ ID NO: 3), or a functional variant thereof having at least 70% sequence identity, similarity, or homology thereto, and wherein said decreased activity is the result of a mutation; and ii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, wherein said decreased sensitivity is the result of a mutation of at least one HRM of said ALAS, wherein said ALAS is HEM 1 (SEQ ID NO: 1), or a functional variant thereof having at least 70% sequence identity, similarity, or homology thereto.

[0128] In other embodiments, the Yarrowia cell produces or is capable of producing one or more hemes and / or one or more hemoproteins, said cell comprises all of the following modifications: i. increased activity of an ALAS relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, wherein said ALAS is HEM1 (SEQ ID NO: 1) and / or HEM1_mutHRM (SEQ ID NO: 2), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and wherein said increased activity is the result of a mutation; and ii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without modification but otherwise identical, wherein said decreased sensitivity is the result of a mutation of at least one HRM of said ALAS, wherein said ALAS is HEM 1 (SEQ ID NO: 1), or a functional variant thereof having at least 70% sequence identity, similarity, or homology thereto.

[0129] In embodiments where the activity of an ALAS has been increased and wherein sensitivity of an ALAS to feedback inhibition by heme has been decreased, this may concern the same ALAS or two different ALAS.

[0130] In some embodiments, the ALAS having decreased sensitivity to feedback inhibition by heme is identical to the ALAS with increased activity. Said increased activity may be obtained by overexpression of said ALAS having decreased sensitivity to feedback inhibition by heme. Thus, in some embodiments, the Yarrowia cell is capable of producing heme and / or hemoprotein, said cell comprises a modification resulting in increased activity of an ALAS relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical and decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased sensitivity is the result of a mutation of at least one HRM of said ALAS.

[0131] Thus, in some embodiments, the Yarrowia cell is capable of producing one or more hemes and / or one or more hemoproteins, said cell comprises the following modifications: i. partial or total deletion of a gene encoding HMX1 (SEQ ID NO: 3), such as partial or total deletion of HMX1 (SEQ ID NO: 8), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3 and SEQ ID NO: 8, respectively; and ii. overexpression of HEM1 (SEQ ID NO: 1), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1.

[0132] In other embodiments, the Yarrowia cell is capable of producing one or more hemes and / or one or more hemoproteins, said cell comprises the following modifications: i. partial or total deletion of a gene encoding HMX1 (SEQ ID NO: 3), such as partial or total deletion of HMX1 (SEQ ID NO: 8), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3 and SEQ ID NO: 8, respectively; and ii. overexpression of HEM1 (SEQ ID NO: 1), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 ; and iii. overexpression of HEM1_mutHRM (SEQ ID NO: 2), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 2.

[0133] In other embodiments, the Yarrowia cell is capable of producing one or more hemes and / or one or more hemoproteins, said cell comprises the following modifications: i. partial or total deletion of a gene encoding HMX1 (SEQ ID NO: 3), such as partial or total deletion of HMX1 (SEQ ID NO: 8), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3 and SEQ ID NO: 8, respectively; and ii. overexpression of HEM1_mutHRM (SEQ ID NO: 2), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 2. Amount of heme and / or hemoprotein

[0134] The Yarrowia cells disclosed herein are capable of producing heme and / or a hemoprotein. Yarrowia cells harbouring the one or more modifications, such as two or three modifications, disclosed herein may have increased heme content, enhanced heme synthesis, altered heme profile and / or up-regulated heme biosynthesis compared to a corresponding Yarrowia cells not harbouring said one or more modifications, but otherwise corresponding to each other and being treated similarly, such as incubated and optionally propagated in similar conditions.

[0135] Thus, in some embodiments, the amount of heme in a Yarrowia cell is increased by at least 2-fold, such as at least 3-fold, such as at least 3.5-fold, such as at least 4-fold, such as at least 4.5-fold, such as at least 5-fold, such as at least 6-fold, such as at least 10-fold, or more, compared to the amount produced by a corresponding Yarrowia cell without the one or more modifications, such as two or three modifications, as disclosed herein elsewhere. Preferably, said heme comprises heme B. In other embodiments, the amount of a hemoprotein in a Yarrowia cell is increased by at least 2-fold, such as at least 3-fold, such as at least 3.5-fold, such as at least 4-fold, such as at least 4.5-fold, such as at least 5-fold, such as at least 6-fold, such as at least 10-fold, or more, compared to the amount produced by a corresponding Yarrowia cell without the one or more modifications, such as two or three modifications, as disclosed herein elsewhere. Said one or more hemoproteins may be a globin, such as leghemoglobin, for example leghemoglobin A (LBA), and / or a cytochrome.

[0136] In some embodiments, said amount of heme and / or said hemoprotein in a Yarrowia cell is the sum of intracellular heme and / or said hemoprotein and the extracellular heme and / or said hemoprotein. In some embodiments, the amount is the sum of intracellular heme and / or said hemoprotein and the extracellular heme and / or said hemoprotein. Thus, in some embodiments, said amount of heme and said hemoprotein in a Yarrowia cell is the sum of intracellular heme and said hemoprotein and the extracellular heme and said hemoprotein. In other embodiments, said amount of heme in a Yarrowia cell is the sum of intracellular heme and extracellular heme. In other embodiments, said amount of said hemoprotein in a Yarrowia cell is the sum of intracellular hemoprotein and extracellular hemoprotein. In preferred embodiments, said heme is heme B. In other embodiments, the heme and / or a hemoprotein is produced with a titer that is at least 25% increased, such as at least 30% increased, such as at least 35% increased, such as at least 40% increased, such as at least 45% increased, such as at least 50% increased, such as at least 55% increased, such as at least 60% increased, such as at least 75% increased, such as at least 80% increased, such as at least 90% increased, such as at least 100% increased, such as at least 150% increased, such as at least 200% increased, such as at least 250% increased, such as at least 300% increased, such as at least 350% increased, such as at least 400% increased, such as at least 450% increased, such as at least 500% increased, such as at least 550% increased, such as at least 600% increased, such as at least 650% increased, such as at least 700% increased, such as at least 800% increased, or more increased, compared to the titer produced by a corresponding Yarrowia cell without the one or more modifications, such as two or three modifications, as disclosed herein elsewhere, optionally when cultivated in similar conditions.

[0137] The term “similar conditions” referred to herein, is in some embodiments the “same conditions” or “identical conditions”. “Similar conditions” implies similar parameters and type of cultivation or incubation, such as similar cultivation medium, the cultivation / growth temperature, and type of cultivation, e.g. shake flask, batch, continuous fermentation, or fed-batch cultivation.

[0138] Heme may be quantified as described in the section “Example 1”. For example, liquidchromatography (LC) coupled with mass spectrometry (MS) / mass spectrometry (MS / MS), i.e. LC-MS / MS, can be used therefor, such as ultra-high performance LC- MS / MS (UHPLC- MS / MS). Depending on the nature of the hemoprotein, said protein may be determined by analytical methods available to the skilled person in the art. The skilled person is capable of identifying a suitable analytical method for quantifying the amount of hemoprotein.

[0139] The titer and / or amount of heme and / or hemoprotein may be the total titer and / or amount of said compounds, such as the sum of intracellular heme and / or hemoproteins and the extracellular heme and / or hemoproteins. Within food / nutritional analysis, the amount / content of an ingredient or nutrient is usually specified as g / 100g product, since this most closely represents what is actually eaten by the consumer. In some embodiments, the Yarrowia cell disclosed herein comprises at least 15 mg heme per 100 g biomass (wet weight (W / W)), or more. Preferably, the heme is heme B. Thus, disclosed herein is a Yarrowia cell comprising heme in an amount of at least 15 mg / 100 g biomass (W / W), such as at least 20 mg / 100 g biomass (W / W), such as at least 21 mg / 100 g biomass (W / W), such as at least 22 mg / 100 g biomass (W / W), such as at least 23 mg / 100 g biomass (W / W), such as at least 24 mg / 100 g biomass (W / W), such as at least 25 mg / 100 g biomass (W / W), such as at least 26 mg / 100 g biomass (W / W), such as at least 27 mg / 100 g biomass (W / W), such as at least 28 mg / 100 g biomass (W / W), such as at least 29 mg / 100 g biomass (W / W), such as at least 30 mg / 100 g biomass (W / W), such as at least 32 mg / 100 g biomass (W / W), such as at least 35 mg / 100 g biomass (W / W), such as at least 37 mg / 100 g biomass (W / W), such as at least 40 mg / 100 g biomass (W / W), such as at least 45 mg / 100 g biomass (W / W), such as at least 50 mg / 100 g biomass (W / W), such as at least 55 mg / 100 g biomass (W / W), such as at least 60 mg / 100 g biomass (W / W), such as at least 65 mg / 100 g biomass (W / W), such as at least 70 mg / 100 g biomass (W / W), such as at least 80 mg / 100 g biomass (W / W), such as at least 85 mg / 100 g biomass (W / W), such as at least 90 mg / 100 g biomass (W / W), such as at least 100 mg / 100 g biomass (W / W), or more.

[0140] In some embodiments, the Yarrowia cell comprises heme in an amount between 14 mg / 100 g biomass (W / W) to 85 mg / 100 g biomass (W / W), such as between 15 mg / 100 g biomass (W / W) to 85 mg / 100 g biomass (W / W), such as between 15 mg / 100 g biomass (W / W) to 50 mg / 100 g biomass (W / W), such as between 15 mg / 100 g biomass (W / W) to 45 mg / 100 g biomass (W / W), such as between 15 mg / 100 g biomass (W / W) to 40 mg / 100 g biomass (W / W), such as between 15 mg / 100 g biomass (W / W) to 35 mg / 100 g biomass (W / W), such as between 16 mg / 100 g biomass (W / W) to 30 mg / 100 g biomass (W / W), such as between 17 mg / 100 g biomass (W / W) to 27 mg / 100 g biomass (W / W), such as between 20 mg / 100 g biomass (W / W) to 25 mg / 100 g biomass (W / W).

[0141] Nucleic acids and expression systems

[0142] Provided herein are expression systems useful for obtaining a Yarrowia cell capable of producing heme and / or a hemoprotein as disclosed herein. Provided are also nucleic acids and / or Yarrowia cell comprising said nucleic acids useful for producing and / or increasing production of heme and / or hemoprotein. The present nucleic acids disclosed herein may be provided as one or more nucleic acid molecules, for example they may be comprised in one or more vectors and / or expression systems. Such nucleic acids and / or expression systems may be introduced in the Yarrowia cell by methods known in the art. The terms nucleic acid, nucleic acid sequence, nucleic acid constructs, nucleic acid molecules, oligonucleotide, and polynucleotides may be used interchangeably herein. Said nucleic acids and / or expression systems may be useful for expression in, engineering and / or modification of a Yarrowia cell.

[0143] In one aspect, the present disclosure provides an expression system for expression in a Yarrowia cell, comprising one or more nucleic acids encoding one or more ALAS, optionally wherein the ALAS is HEM1 (SEQ ID NO: 1) and / or HEM1_mutHRM (SEQ ID NO: 2), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 and / or SEQ ID NO: 2, respectively.

[0144] In some embodiments, the nucleic acid encoding ALAS comprises or consists of HEM1 as set forth in SEQ ID NO: 6, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 6.

[0145] In other embodiments, the nucleic acid encoding ALAS comprises or consists of HEM1_mutHRM as set forth in SEQ ID NO: 7, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 7.

[0146] The expression system may further comprise one or more nucleic acids encoding one or more hemoproteins, such as a heme B-containing protein, a cytochrome, and / or a globin, for example myoglobin and / or leghemoglobin. Said leghemoglobin may be leghemoglobin A (LBA).

[0147] In some embodiments, the globin is Bos taurus myoglobin as set forth in SEQ ID NO: 5 (Bt_myoglobin) or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 5, and the nucleic acid encoding myoglobin comprises or consists of SEQ ID NO: 10, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 10.

[0148] In other embodiments, the globin is Glycine max LBA as set forth in SEQ ID NO: 4 (Gm_LBA) or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 4 and the nucleic acid encoding myoglobin comprises or consists of SEQ ID NO: 9, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 9. In some embodiments, the nucleic acid encoding Gm_LBA further comprises a promoter, preferably a strong promoter. For example, the nucleic acid encoding Gm_LBA (SEQ ID NO: 4) or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 4, may further comprise prEXPI (SEQ ID NO: 11) or prGPDI (SEQ ID NO: 12), or functional variants of any of the aforementioned having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 11 or SEQ ID NO: 12, respectively.

[0149] The nucleic acid encoding the one or more ALAS and / or hemoproteins may be present in high copy number. For example, the nucleic acid encoding HEM1_mutHRM (SEQ ID NO: 2), such as HEM1_mutHRM (SEQ ID NO: 7), or functional variants to any of the aforementioned having at least 70% sequence identity, similarity, or homology thereto, may be present in high copy number. In some embodiments, the copy number of said gene and / or nucleic acid encoding ALAS is at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, or more. Preferably, the increase in copy number of ALAS results in an increase in the encoded activity. In other embodiments, the copy number of said gene and / or nucleic acid encoding LBA, preferably Gm_LBA (SEQ ID NO: 4) or a functional variant thereof having at least 70% sequence identity, similarity, or homology thereto, is at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, or more. In some embodiments, the copy number of said gene and / or nucleic acid encoding LBA, preferably Gm_LBA (SEQ ID NO: 4) or a functional variant thereof having at least 70% sequence identity, similarity, or homology thereto, is at least 2, such as at least 3, such as at least 4 copies, or more.

[0150] In some embodiments, the nucleic acid encoding ALAS further comprises a promoter, such as a strong promoter. Strong promoters and examples thereof are further disclosed in the section “Strong promoter” herein. For example, the nucleic acid encoding ALAS, such as HEM1 and / or HEM1_mutHRM, may further comprise prEXPI (SEQ ID NO: 11) or prGPDI (SEQ ID NO: 12), or functional variants of any of the aforementioned having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 11 or SEQ ID NO: 12, respectively.

[0151] In preferred embodiments, the Yarrowia cell is a Yarrowia cell disclosed herein, for example in the section “Yarrowia cell” herein above. Preferably, a Yarrowia cell comprising an expression system disclosed herein, is capable of producing heme. In some embodiments, a Yarrowia cell comprising an expression system disclosed herein, is capable of producing a hemoprotein.

[0152] The nucleic acids and / or expression systems disclosed herein may be designed to be integrated within the genome of the Yarrowia cell and / or they may be comprised in one or more plasmids comprised within the Yarrowia cell and / or a combination of both integrated within the genome and within one or more plasmids comprised in the Yarrowia cell.

[0153] In some embodiments, one or more of the nucleic acids and / or expression systems may be integrated in the genome of said Yarrowia cell. Methods for integrating a nucleic acid molecule are well known in the art. The skilled person will recognise that such methods include, but are not limited to: cloning and homologous recombinationbased methods. Cloning methods may involve the design and construction of a plasmid e.g. in an organism such as Escherichia coli. The plasmid may be an integrative or a non-integrative vector. Cloning-free methods comprise homologous recombinationbased methods such as adaptamer-mediated PCR or gap repair. Such methods often result in integration of the nucleic acid in the genome of the Yarrowia cell.

[0154] The nucleic acid encoding a polypeptide disclosed herein, may be codon-optimised, in particular codon-optimised for expression in a Yarrowia cell, such as a Yarrowia lipolytica cell.

[0155] The nucleic acids encoding any one of the polypeptides disclosed herein may be present in high copy number. The nucleic acids encoding any of the polypeptides disclosed herein of interest may be present in low copy number.

[0156] In some embodiments, the nucleic acid and / or expression system further comprises or consists of one or more plasmids, such as an integrative plasmid or a replicative plasmid. In some embodiments, the plasmid is a high copy replicative plasmid. In some embodiments, the plasmid is a low copy replicative plasmid. In some embodiments, the plasmid is an episomal plasmid.

[0157] Strong promoter

[0158] As detailed elsewhere herein, overexpression of a nucleic acid, such as a gene, may be achieved using a strong promoter for transcription initiation of said nucleic acid, such as said gene.

[0159] Several strong promoters are available in the field, for example prEXPI (SEQ ID NO: 11), prTEFI (promoter of TEF1 (YALI1_C 12642g)), prGPDI (SEQ ID NO: 12promoter of GPD1 (YALI1_B04433g)), and prRPL18 (promoter of RPL18 (YALI1_B11934g)). Said strong promoter may either be a native protomer of said Yarrowia cell or a nonnative promoter of said Yarrowia cell, for example a synthetic promoter. The YALI1 ID is a standard way of referring to Yarrowia genes in the field. YALI1 refers to well annotated genomes, in particular the genome of Yarrowia lipolytica W29 (Magnan et al., 2016). With respect to overexpression of ALAS, expression thereof is defined as “overexpression”, if the expression level of ALAS obtained after promoter replacement / substitution is significantly higher than the expression level obtained using the native promoter of ALAS, i.e. prALAS. Overexpression of ALAS may be achieved by introducing one or more copies of ALAS into the Yarrowia cell, optionally wherein said one or more copies of ALAS is under the control of a native or non-native strong promoter.

[0160] The skilled person knows have to determine and compared promoter strengths, either by comparing expression levels using for example RNA sequencing or a reporter gene, such as gene-fluorescence-protein (GFP).

[0161] Examples of Y. lipolytica genes (specified using the YALI1 gene IDs from Y. lipolytica

[0162] W29) that are herein considered to have a strong promoter are:

[0163] YALI1_E09438g, YALI1_C00464g, YALI1_D26051g, YALI1_E01865g, YALI1_C 12642g, YALI1_C26377g, YALI1_A22369g, YALI1_E41367g, YALI1_B26377g, YALI1_D09712g, YALI1_C32844g, YALI1_E31389g, YALI1_E23659g, YALI1_C08262g, YALI1_B30382g, YALI1_F08260g, YALI1_F13373g, YALI1_C16851g, YALI1_C 15964g, YALI1_E02007g, YALI1_B27615g, YALI1_C32850g, YALI1_D12513g, YALI1_A21775g, YALI1_F08431g, YALI1_C07638g, YALI1_F27175g, YALI1_E27880g, YALI1_F31531g, YALI1_C14450g, YALI1_D12135g, YALI1_D 18673g, YALI1_F33331g, YALI1_F00821g, YALI1_E29930g, YALI1_F32792g, YALI1_C31549g, YALI1_A20739g, YALI1_D10535g, YALI1_C33299g, YALI1_E17243g, YALI1_E31396g, YALI1_D05343g, YALI1_B19897g, YALI1_F02309g, YALI1_F22978g, YALI1_C22922g, YALI1_F32305g, YALI1_E36179g, YALI1_C16108g, YALI1_E29150g, YALI1_B03797g, YALI1_D32153g, YALI1_E27279g, YALI1_F03573g, YALI1_F19249g, YALI1_D06233g, YALI1_C09150g, YALI1_E39796g, YALI1_F33345g, YALI1_E30886g, YALI1_C05133g, YALI1_A22454g, YALI1_B16996g, YALI1_E14988g, YALI1_B18707g, YALI1_B27485g, YALI1_D 10600g, YALI1_C08106g, YALI1_A09798g, YALI1_B05646g, YALI1_E32722g, YALI1_C06511g, YALI1_A 17957g, YALI1JD11769g, YALI1_F14729g, YALI1_E22238g, YALI1_B17008g, YALI1_F09932g, YALI1_A10827g, YALI1_F32090g, YALI1_D17108g, YALI1_E27871g, YALI1_B28659g, YALI1_F11689g, YALI1_C 12788g, YALI1_A 18557g, YALI1_F20914g, YALI1_B05798g, YALI1_C06597g, YALI1_E32842g, YALI1_A21372g, YALI1_D 11984g, YALI1_E 13453g, YALI1_F34429g, YALI1_E17466g, YALI1_F31587g, YALI1_A03322g, YALI1_B21493g, YALI1_A20370g, YALI1_E24442g, YALI1_B26596g, YALI1_D20676g, YALI1_B05024g, YALI1_F38629g, YALI1_C07170g, YALI1_F22436g, YALI1_C 10555g, YALI1_D22124g, YALI1_F28333g, YALI1_F03215g, YALI1_A01864g, YALI1_C23948g, YALI1_D16029g, YALI1_F12874g, YALI1_A12437g, YALI1_E40361g, YALI1_E30914g, YALI1_F34397g, YALI1JD12930g, YALI1_E14967g, YALI1_B18930g, YALI1_B20462g, YALI1_E34344g, YALI1_F08635g, YALI1_C09230g, YALI1_B05364g, YALI1_D04128g, YALI1_C30130g, YALI1_B02696g, YALI1_E37295g, YALI1_F02758g, YALI1_E41061g, YALI1_F32476g, YALI1_C15832g, YALI1_D26146g, YALI1_E16731g, YALI1_E26336g, YALI1_F37234g, YALI1_F27556g, YALI1_F22935g, YALI1_F34281g, YALI1_F32043g, YALI1_E37886g, YALI1_B04433g, YALI1_D14639g, YALI1_D26263g, YALI1_A00406g, YALI1_F11930g, YALI1_B04032g, YALI1_B27929g, YALI1_E25317g, YALI1_E41315g, YALI1_A00102g, YALI1_F08284g, YALI1_B09994g, YALI1_E23610g, YALI1_F25001g, YALI1_E29959g, YALI1_E23912g, YALI1_B11934g, YALI1_F14495g, YALI1_D17030g, YALI1_B27320g, YALI1_B18499g, YALI1_E17214g, YALI1_F12842g, YALI1_B07937g, YALI1_E16255g, YALI1_B08885g, YALI1_D 16249g, YALI1_C24707g, YALI1_E37930g, YALI1_D13388g, YALI1_E35963g, YALI1_D07379g, YALI1_B 10635g, YALI1_D15734g, YALI1_D17206g, YALI1_E18441g, YALI1_A22330g, YALI1_D09566g, YALI1_C30320g, YALI1JD10424g, YALI1_E16649g, YALI1_B25868g, YALI1_B04138g, YALI1_B27399g, YALI1_F33301g, YALI1_F06236g, YALI1_F32597g, YALI1_D 15458g, YALI1_F10115g, YALI1_F18105g, YALI1_B06839g, YALI1_E40467g, YALI1_C30946g, YALI1_E28033g, YALI1_E03584g, YALI1_A14736g, YALI1_E17849g, YALI1_F07847g, YALI1_F14712g, YALI1_C11862g, YALI1_A01824g, YALI1_B09266g, YALI1_B15996g, YALI1_C29810g, YALI1_E03300g, YALI1_D30256g, YALI1_E16556g, YALI1_E27207g, YALI1_C16333g, YALI1_B04853g, YALI1_D28273g, YALI1_D06094g, YALI1_F03407g, YALI1_E32033g, YALI1_D18037g, YALI1_F24773g, YALI1_B19932g, YALI1_B14382g, YALI1_D24054g, YALI1_E00574g, YALI1_B13699g, YALI1_B27534g, YALI1_C09950g, YALI1_B05995g, YALI1_B19844g, YALI1_F11583g, YALI1_F02518g, YALI1_D09103g, YALI1_C22986g, YALI1_E31947g, YALI1_D09203g, YALI1_D26496g, YALI1_C24124g, YALI1_F11049g, YALI1_E18927g, YALI1_D06302g, YALI1_F04433g, YALI1_B22677g, YALI1_F07344g, YALI1_A10934g, YALI1_E07744g, YALI1_E11529g, YALI1_E38131g, YALI1_E38178g, YALI1_E09817g, YALI1_D05873g, YALI1_C09094g, YALI1_B02487g, YALI1_E30028g, YALI1_E28046g, YALI1_A06434g, YALI1_F23664g, YALI1_F04455g, YALI1_E41421g, YALI1_D05594g, YALI1_F26967g, YALI1_C29506g, YALI1_F37463g, YALI1_E07099g, YALI1_E17484g, YALI1_D36161g, YALI1_D34893g, YALI1_B26808g, YALI1_F14933g, YALI1_D32754g, YALI1_E19172g, YALI1_F04110g, YALI1_D07348g, YALI1_F00654g, YALI1_E28107g, YALI1_D07888g, YALI1_F02545g, YALI1_C05386g, YALI1_D32268g, YALI1_F19090g, YALI1_F39051g, YALI1_E19852g, YALI1_B28150g, YALI1_C07423g, YALI1_F37320g, YALI1_E27249g, YALI1_A06311g, YALI1_A10318g, YALI1_F05089g, YALI1_E27313g, YALI1_B19079g, YALI1_E23635g, YALI1_E39171g, YALI1_F09230g, YALI1_B15092g, YALI1_A07649g, YALI1_F33103g, YALI1_F32441g, YALI1_B 19044g, YALI1_C04067g, and / or YALI1_B20378g.

[0164] Methods

[0165] Disclosed herein are also useful methods for production or manufacturing of heme, hemoprotein, biomass of containing said heme and / or hemoprotein, and / or Yarrowia cells containing said heme and / or hemoprotein. Such biomass can be used in food or feed composistions, or as meat alternative(s) or as ingredient(s) to prepare meat alternative(s).

[0166] In one aspect, the present disclosure provides a method of producing heme and / or a hemoprotein in a Yarrowia cell, comprising the steps of: i. providing a Yarrowia cell disclosed herein; and ii. incubating and optionally propagating said Yarrowia cell in a medium, whereby said heme and / or hemoprotein is produced. In preferred embodiments, said heme is heme B. In other embodiments, said hemoprotein is LBA, such as Gm_LBA (SEQ ID NO: 4) or a functional variant thereof having at least 70% sequence identity, similarity, or homology thereto.

[0167] In one aspect, the present disclosure provides a method of increasing production of heme and / or a hemoprotein in a Yarrowia cell, comprising the steps of: i. providing a Yarrowia cell disclosed herein; and ii. incubating and optionally propagating said Yarrowia cell in a medium, whereby production of said heme and / or hemoprotein is increased. In preferred embodiments, said heme is heme B.

[0168] For example, in some embodiments, the method of increasing production of heme in a Yarrowia cell, comprises the steps of: i. providing a Yarrowia cell; and ii. incubating and optionally propagating said Yarrowia cell in a medium, whereby production of said heme is increased, wherein said Yarrowia cell carries at least two copies of a nucleic acid encoding said hemoprotein, for example a heterologous hemoprotein, such as at least three copies, such as at least four copies, such as at least five copies, such as at least six copies, such as at least seven copies, such as at least eight copies, such as at least nine copies, such as at least ten copies, or more copies a nucleic acid encoding said hemoprotein, preferably wherein said hemoprotein is a leghemoglobin, such as leghemoglobin A (LBA), for example Glycine max LBA as set forth in SEQ ID NO: 4 (Gm_LBA), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 4. The Yarrowia cell may be any Yarrowia cell described elsewhere herein, such as in the section “Yarrowia cell”. In some embodiments, the method further comprises a step of recovering the heme and / or the hemoprotein. Recovery is further detailed in the section “Recovery”.

[0169] In some embodiments, the method disclosed herein produces the heme and / or the hemoprotein in an increased amount. This is further detailed in the section “Amount of heme and / or hemoprotein” herein.

[0170] Yarrowia cells useful for producing heme and / or a hemoprotein are provided in the present disclosure, for example in the section “Yarrowia cell”.

[0171] Method for manufacturing of a biomass and / or meat-alternative

[0172] Another main aspect of the present disclosure is to provide Yarrowia cells and biomass comprising said Yarrowia cells for manufacturing of meat alternatives and / or ingredients for the manufacture of meat alternatives.

[0173] Thus, provided herein is a method of manufacturing biomass comprising Yarrowia cells disclosed herein, such as Yarrowia cells with increased intracellular concentration and / or content of heme and / or a hemoprotein, comprising the steps of: i. providing a Yarrowia cell disclosed herein, such as a Yarrowia cell with increased intracellular concentration and / or content of said heme and / or said hemoprotein compared to a corresponding cell without the one or more modifications, such as two or three modifications, disclosed herein, but otherwise identical, as disclosed herein; and ii. incubating and optionally propagating said Yarrowia cell in a medium; and iii. optionally recovering biomass comprising said Yarrowia cell, whereby said biomass comprising Yarrowia cells, such as Yarrowia cells with increased intracellular concentration and / or content of heme and / or a hemoprotein, is obtained.

[0174] Provided is also a method of manufacturing a meat alternative and / or ingredient therefor, comprising the steps of: i. providing a Yarrowia cell as disclosed herein; ii. incubating and optionally propagating said Yarrowia cell in a medium, iii. recovering biomass comprising said Yarrowia cell and / or fermentation liquid comprising biomass comprising said Yarrowia cell; and / or iv. converting said biomass and / or fermentation liquid comprising biomass comprising said Yarrowia cell into a meat alternative and / or ingredient therefor whereby a meat alternative and / or ingredient therefor is manufactured.

[0175] Herein biomass or cell biomass refers to the mass of cells obtained during and / or after propagation of a Yarrowia cell. Other terms for biomass are cell pellet, cake, or composition comprising Yarrowia cells. Biomass may be obtained from a fermentation liquid / broth after propagating cells in a suitable medium, by use of techniques such as centrifugation or filtration. Biomass may both comprise cells that are dead or alive. Said dead cells may for example be lysed cells or cell debris. Biomass comprising living cells or cells capable of propagating may be deactivated by methods known in the art. For example, it may be treated with heat, pressure or sterilized in another way.

[0176] Biomass may be wet or dried to various degrees. Methods for drying biomass are known to the skilled person.

[0177] The ingredient may be a food ingredient and / or a feed ingredient.

[0178] Medium and growth conditions

[0179] The incubation may be performed aerobically or anaerobically, at temperatures and at pH suitable for supporting growth of the Yarrowia cells. The medium should include the required nutrients, and may be supplemented with precursors, nutrients, carbon and energy sources as applicable. The time of incubation will vary depending on which Yarrowia cell is used, but can easily be adapted by the skilled person. The medium and growth conditions should thus be such that they allow propagation of the Yarrowia cells, as is known in the art. The term “medium” may also be referred to as “cultivation medium” herein.

[0180] In some embodiments, the medium comprises iron, such as an iron salt, for example ferric chloride, iron sulfate, iron nitrate, and / or ferric citrate. Thus, in some embodiments the medium comprises ferric chloride. In other embodiments, the medium comprises glycine. The medium may comprise succinyl-CoA. In other embodiments, the medium comprises alpha-ketoglutarate. For the manufacture of biomass for food compositions or products, for example for meat alternatives or ingredients therefor, food grade medium components may be used as appropriate.

[0181] Amount of heme and / or hemoprotein produced with methods

[0182] The methods disclosed herein yield one or more hemes and / or one or more hemoproteins, as well as biomass and / or meat-alternatives comprising heme and / or hemoprotein. Employment of the Yarrowia cells harbouring the one or more modifications, such as two or three modifications, disclosed herein in said methods, may result in production of increased amounts of heme and / or hemoprotein, compared to the amounts produced using corresponding Yarrowia cells without the one or more modifications disclosed herein.

[0183] Accordingly, in some embodiments, the amount of heme produced in or with a method disclosed herein, is increased by at least 2-fold, such as at least 3-fold, such as at least 3.5-fold, such as at least 4-fold, such as at least 4.5-fold, such as at least 5-fold, such as at least 6-fold, such as at least 10-fold, or more, compared to the amount produced by a corresponding Yarrowia cell without the one or more modifications, such as two or three modifications, as disclosed herein elsewhere. Preferably, said heme comprises heme B. In other embodiments, the amount of one or more hemoproteins produced in or with a method disclosed herein is increased by at least 2-fold, such as at least 3- fold, such as at least 3.5-fold, such as at least 4-fold, such as at least 4.5-fold, such as at least 5-fold, such as at least 6-fold, such as at least 10-fold, or more, compared to the amount produced by a corresponding Yarrowia cell without the one or more modifications, such as two or three modifications, as disclosed herein elsewhere. The hemoprotein may be a heme B-containing hemoprotein, globin, such as leghemoglobin, for example leghemoglobin A (LBA), and / or a cytochrome.

[0184] In other embodiments, the heme and / or one or more hemoproteins are produced with a titer that is at least 25% increased, such as at least 30% increased, such as at least 35% increased, such as at least 40% increased, such as at least 45% increased, such as at least 50% increased, such as at least 55% increased, such as at least 60% increased, such as at least 75% increased, such as at least 80% increased, such as at least 90% increased, such as at least 100% increased, such as at least 150% increased, such as at least 200% increased, such as at least 250% increased, such as at least 300% increased, such as at least 350% increased, such as at least 400% increased, such as at least 450% increased, such as at least 500% increased, such as at least 550% increased, such as at least 600% increased, such as at least 650% increased, such as at least 700% increased, such as at least 800% increased, or more increased, compared to the titer produced by a corresponding Yarrowia cell without the one or more modifications, such as two or three modifications, as disclosed herein elsewhere, when incubated and optionally propagated in similar conditions.

[0185] The term “similar conditions” referred to herein, is in some embodiments the “same conditions” or “identical conditions”. “Similar conditions” implies similar parameters and type of cultivation or incubation, such as similar cultivation medium, the cultivation / growth temperature, and type of cultivation, e.g. shake flask, batch, continuous fermentation, or fed-batch cultivation.

[0186] Recovery

[0187] Methods for recovering and / or purifying heme and / or hemoprotein obtained by Yarrowia cells and / or methods of the present disclosure are known in the art.

[0188] The step of recovering heme and / or hemoprotein, may also be referred to isolation, extraction, and / or purification.

[0189] Heme and / or hemoprotein may be extracted from the liquid phase and / or solid phase obtained after incubation of a Yarrowia cell according to the present disclosure.

[0190] For example, the step of recovering the heme and / or the hemoprotein may comprise separating the cell culture, obtained by incubation of a Yarrowia cell, in a solid phase to obtain a cell phase and in a liquid phase to obtain a supernatant. The the heme and / or the hemoprotein may be present in the supernatant and / or the cell phase. The cell culture is also known as fermentation broth or fermentation liquid. The cell phase is also known as pellet or cell pellet. Extracellular heme and / or hemoprotein is usually present in the supernatant. Intracellular heme and / or hemoprotein is usually present in the cell pellet.

[0191] Heme may be extracted from the cell pellet by extraction solvent, such as HCI in acetone or HCI in ethanol. Food compositions and products

[0192] In another aspect the present disclosure provides biomass and compositions comprising Yarrowia cells disclosed herein, which are particularly useful for food purposes.

[0193] Thus, provided is a composition comprising a Yarrowia cell disclosed herein, for example in the section “Yarrowia cell”, and / or one or more hemes obtainable by a method disclosed herein, for example in the section “Methods”, and / or one or more hemoproteins obtainable by a method disclosed herein, for example in the section “Methods”.

[0194] In some embodiments, provided is a composition comprising a Yarrowia cell disclosed herein, for example in the section “Yarrowia cell”. In other embodiments, provided herein is a composition comprising heme obtainable by a method disclosed herein, for example in the section “Methods”. Preferably, said heme is heme B or a derivative thereof. In further embodiments, provided is a composition comprising a hemoprotein disclosed herein, for example in the section “Hemoprotein”. Thus, provided herein is heme obtainable by a method disclosed herein, for example in the section “Methods”. Preferably, said heme is heme B or a derivative thereof. Provided is also a hemoprotein obtainable by a method disclosed herein, for example in the section “Methods”.

[0195] Provided herein is a biomass comprising Yarrowia cells with increased intracellular concentration and / or content of heme, compared to a corresponding cell without the one or more modifications, such as two or three modifications, disclosed herein, but otherwise identical, wherein said biomass is obtainable by a method as disclosed herein, for example in the section “Methods”. Herein provided is also a biomass comprising Yarrowia cells with increased intracellular concentration and / or content of a hemoprotein, compared to a corresponding cell without the one or more modifications, such as two or three modifications, disclosed herein, but otherwise identical, obtainable by a method disclosed herein, for example in the section “Methods”. That the concentration of heme and / or hemoprotein is increased may imply, that the amount of heme and / or hemoprotein is increased. Provided herein is also a meat alternative and / or ingredient therefor obtainable by a method disclosed herein, for example in the section “Method for manufacturing of a biomass and / or meat-alternative”.

[0196] Provided is also a food composition comprising heme and / or a hemoprotein from a Yarrowia cell disclosed herein, for example in the section “Yarrowia cell” herein above.

[0197] In some embodiments the food material, food product, food composition, and / or food ingredient is a meat substitute, such as a beef substitute, a chicken substitute, or a fish substitute.

[0198] Uses

[0199] Further disclosed herein are various uses of the Yarrowia cells, biomass comprising the Yarrowia cells, hemes, and / or hemoproteins, and / or compositions comprising any one or more of the Yarrowia cells, biomass comprising the Yarrowia cells, hemes, and / or hemoproteins.

[0200] Disclosed is also the use of a composition as a food product, food material, food ingredient and / or feed ingredient, wherein said composition comprises a Yarrowia cell disclosed herein, for example in the section “Yarrowia cell” herein above, and / or one or more hemes obtainable by a method disclosed herein, for example in the section “Methods”, and / or one or more hemoproteins obtainable by a method disclosed herein, for example in the section “Methods”. In preferred embodiments, disclosed is the use of a composition comprising a Yarrowia cell disclosed herein, for example in the section “Yarrowia cell” herein above, as a food product.

[0201] Also disclosed herein is the use of a composition in a meat alternative and / or in a method for producing a meat alternative, wherein said composition comprises a Yarrowia cell disclosed herein, for example in the section “Yarrowia cell” herein above, and / or heme obtainable by a method disclosed herein, for example in the section “Methods”, and / or a hemoprotein obtainable by a method disclosed herein, for example in the section “Methods”. In preferred embodiments, disclosed is the use of a composition comprising a Yarrowia cell disclosed herein, for example in the section “Yarrowia cell” herein above, in a meat alternative and / or in a method for producing a meat alternative In some embodiments, the use of a Yarrowia cell disclosed herein, for example in the section “Yarrowia cell” herein above, is for production of meat alternative and / or ingredient therefor.

[0202] Provided is also use of the Yarrowia cell and / or biomass disclosed herein, for example in the section “Yarrowia cell” herein above, for production of one or more hemoproteins or for production of food and / or feed containing one or more hemoproteins.

[0203] Also provided herein is the use of nucleic acids and / or expression systems disclosed anywhere herein, such as in “Nucleic acids and expression systems” herein above, for production of one or more hemes and / or one or more hemoproteins.

[0204] Hemoprotein

[0205] Disclosed are is Yarrowia cells, biomass, products and compositions comprising hemoprotein, and / or methods and uses producing and / or obtaining hemoprotein.

[0206] In some embodiments, the hemoprotein is an endogenous hemoprotein. In other embodiments, the hemoprotein is a hemoprotein heterologous to the Yarrowia cell. In preferred embodiments, the hemoprotein is heme B-containing protein.

[0207] In some embodiments, the endogenous hemoprotein is a catalase, a peroxidase, or a cytochrome. Said endogenous hemoprotein may be functionally inactivated, for example by mutating the active site so that the active site becomes disrupted.

[0208] In some embodiments, the hemoprotein heterologous to the Yarrowia cell is a globin, such as myoglobin, hemoglobin, or leghemoglobin. Said leghemoglobin may be leghemoglobin A (LBA).

[0209] According to the present disclosure, the inventors have found that increased production of heme may be obtained by overexpression of a hemoprotein, such as overexpression of an endogenous hemoprotein or a heterologous hemoprotein. The inventors have exemplified this by increasing the copy number of Gm_LBA (SEQ ID NO: 4) (Figure 6) thereby obtaining increased heme B production. Thus, in some embodiments, the Yarrowia cell carries at least two copies of a nucleic acid encoding said hemoprotein, such as at least three copies, such as at least four copies, such as at least five copies, such as at least six copies, such as at least seven copies, such as at least eight copies, such as at least nine copies, such as at least ten copies, or more copies a nucleic acid encoding said hemoprotein. Said hemoprotein may for example be LBA.

[0210] Leghemoglobin A (LBA) may for example be Glycine max leghemoglobin A as set forth in SEQ ID NO: 4 (Gm_LBA), or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 4. The nucleic acid encoding Gm_LBA (SEQ ID NO: 4), or a functional variant thereof having at least 70% identity, similarity, or homology to SEQ ID NO: 4, may comprise or consist of SEQ ID NO: 9, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 9.

[0211] Myoglobin may for example be Bos taurus myoglobin as set forth in SEQ ID NO: 5 (Bt_myoglobin), or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 5. The nucleic acid encoding Bt_myoglobin (SEQ ID NO: 5), or a functional variant thereof having at least 70% identity, similarity, or homology to SEQ ID NO: 5, may comprise or consist of SEQ ID NO: 10, or a functional variant thereof having at least 70%, such as at least 71%, such as at least 72%, such as at least 73%, such as at least 74%, such as at least 75%, such as at least 76%, such as at least 77%, such as at least 78%, such as at least 79%, such as at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% identity, similarity, or homology to SEQ ID NO: 10.

[0212] In other embodiments, the hemoprotein heterologous to the Yarrowia cell is a cytochrome.

[0213] Examples

[0214] Example 1

[0215] A BLASTp search was conducted to identify Y. lipolytica genes involved in heme biosynthesis. The following genes were selected:

[0216] • YALI1_C07816g - HEM1, 5-aminolevulinic acid synthase

[0217] • YALI1_F27686g - HEM2, 5-aminolevulinic acid dehydratase

[0218] • YALI1_F34051g - HEM3, porphobilinogen deaminase

[0219] • YALI1_F13039g - HEM4, uroporphyrinogen III synthase

[0220] • YALI1_C02391g - HEM12, Uroporphyrinogen decarboxylase

[0221] • YALI1_E35410g - HEM13, coproporphyrinogen III oxidase

[0222] • YALI1_D30196g - HEM14, Protoporphyrinogen oxidase

[0223] • YALI1_F25924g - HEM15, Ferrochelatase Additionally, the Y. lipolytica protein YALI1_D33552p shares a 38% identity with the S. cerevisiae heme oxygenase Hmxlp, an enzyme involved in degradation of heme.

[0224] DNA assembly and strain construction was carried out according to the EasyCloneYALI toolbox (Holkenbrink et al., 2018). The parent / reference strain was Yarrowia lipolytica Y-1095, carrying genome integrated transcriptional units for Cas9 and DsdA (D-serine deaminase) according to the EasyClone toolbox. The insertion of the Cas9 and DsdA transcriptional units disrupts the function of the DNA repair protein KU70. Specifically, overexpression of the selected heme biosynthetic genes were done by assembling the corresponding coding sequences under the control of the strong promoter prEXPI (SEQ ID NO: 11) and the terminator tPEX20. The resulting transcriptional units were genomically integrated at location I ntF_3. Heterologous expression of the hemoprotein leghemoglobin A from Glycine max (Gm_LBA, SEQ ID NO: 4) was done by assembling the corresponding coding sequences (SEQ ID NO: 9) under the control of a strong promoter using either prGPD (SEQ ID NO: 12) or prEXPI (SEQ ID NO: 11) and the terminators tPEX20 or tLIP2. The resulting transcriptional units were genomically integrated at locations lntE_3 and lntD_1. Knockout was carried out by whole coding sequence removal, using Cas9-based engineering (Holkenbrink et al., 2018). Overexpression of HEM4 resulted in a growth defect, and was not further analyzed.

[0225] To determine the heme production, strains were cultivated in liquid media. Heme was extracted and analyzed by LC-MS. Specifically the strains were cultivated for 87 hours in shaking incubators set to 30 °C and 250 RPM. The liquid media contained the following components: Glucose, 40 g / L; Ammonium sulfate, 5 g / L; Biotin, 2 pg / L; Calcium pantothenate, 400 pg / L; folic acid, 2 pg / L; inositol, 2 mg / L; nicotinic acid, 400 pg / L; paminobenzoic acid, 200 pg / L; pyridoxine HCI, 400 pg / L; riboflavin, 200 pg / L; thiamine HCI, 400 pg / L; boric acid, 500 pg / L; copper sulfate, 40 pg / L; potassium iodine, 100 pg / L; ferric chloride, 33 mg / L; manganese sulfate, 400 pg / L; sodium molybdate, 200 pg / L; zinc sulfate, 400 pg / L; potassium phosphate monobasic, 1 g / L; magnesium sulfate, 0.5 g / L; sodium chloride, 0.1 g / L; calcium chloride, 0.1 g / L; Phosphate buffer pH 6.0, 12 g / L. approximately 20 mg biomass was sampled and rapidly transferred into tubes containing methanol precooled to -79 °C. The tubes were centrifuged in a cooled centrifuge at -9 °C and 18000 g for 5 minutes. The supernatant was removed and the pellet was stored at -80 °C until further analysis. To extract the heme, pellets from the previous step were extracted by adding 1 mL extraction solvent (2 % HCI in acetone), and vortexing at 1500 RPM for 30 minutes. The extraction solvent was evaporated in a SpeedVac overnight. Pellets were resuspended in 250 pl 70 % ethanol, and pulse vortexed for 10 min followed by centrifugation at 18000 g for 10 min. The supernatant was transferred to a microtiter plate and analyzed by LC-MS. The LC-MS / MS analysis for detection of COP, heme and biliverdin was performed on a Vanquish Duo LIHPLC binary system (Thermo Fisher Scientific, USA) coupled to the IDXOrbitrap Mass Spectrometer (Thermo Fisher Scientific, USA). The analytes were separated using a Waters ACQUITY BEH C18 (10 cm x 2.1 mm, 1.7 pm) column equipped with an ACQUITY BEH C18 guard column kept at 40 C. The mobile phases consisted of MilliQ© water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B). The initial composition was 2%B held for 0.8 min, followed by a linear gradient till 5% in 3.3 min, and to 100%B in 10 min held for 1 min before going back to initial conditions. Reequilibration time was 2.7 min. The flow rate was set at 0.35 mL / min. The MS measurement was done in positive heated electrospray ionization (HESI) mode with a voltage of 3500 V acquiring in full MS / MS spectra (Data dependent Acquisition driven MS / MS) in the m / z of 400-1000. The peak corresponding to heme B was verified with a hemin analytical standard.

[0226] The cultivated strains expressed 7 of the biosynthetic enzymes (HEM1, HEM2, HEM3, HEM12, HEM13, HEM14, HEM15) or had the HMX1 missing (Figure 2). This revealed that HEM1 is the key bottleneck in the heme biosynthetic pathway, and its overexpression is crucial for high production. Additionally, knockout of the gene YALI1_D33552g (tentative HMX1) resulted in an increased accumulation of heme B, as well as a drastic decrease in biliverdin concentration, confirming its role as a Y. lipolytica heme oxygenase (Figure 2, Table 1).

[0227] Table 1 : Heme production as result of overexpression of HEM1 and knockout of HMX1 in Y. lipolytica. Relative quantification of coproporphyrin (intermediate), heme B, and biliverdin (degradation product) compared to Y. lipolytica reference strain (Ref.) without any heme-related modifications.

[0228] Heme regulatory motifs (HRM) were discovered in the N-terminal region of Hemi p (Figure 3). Overexpression of a mutated form of HEM1 (HEM1_mutHRM as set forth in SEQ ID NO: 2), lacking all three HRMs, resulted in an increased heme accumulation compared to overexpression of the native HEM1 (Figure 4). The disruption of the HRMs was accomplished by mutating the cysteine in the HRM cysteine-proline motif to serine.

[0229] The cultivated strains expressed one (1x), two (2x), or three (3x) copies of the hemoprotein leghemoglobin A from Glycine max (Gm_LBA, SEQ ID NO: 4 encoded by SEQ ID NO: 9) (Figure 6). Integration of the gene copies into the genome resulted in an increased abundance of heme B, with each additional gene copy integration increasing in further accumulation of heme B, when heme B was quantified relative to a Y. lipolytica reference strain (Reference) without any Gm_LBA expression. Thus, this revealed that increasing the number of gene copies of a hemoprotein integrated in the genome resulted in an increased production of the hemoprotein and accumulation of heme B.

[0230] Table 2: Heme production as result of expression of hemoprotein (Gm_LBA, SEQ ID NO: 4 encoded by SEQ ID NO: 9) at varying copy numbers (one (1x), two (2x), or three (3x) gene copies). Relative quantification of heme B compared to Y. lipolytica reference strain (Reference) without any Gm_LBA expression. Example 2

[0231] A Saccharomyces cerevisiae hmxlA strain was constructed using well-established Cas9-mediated engineering methods Jessop-Fabre et al., 2016. The knockout was constructed by complete CDS removal.

[0232] The reference strain (S. cerevisiae) and engineered strain (S. cerevisiae hmxlA) were grown in shake flasks for 48h in minimal media at 28 C in a shaking incubator. Heme was extracted from the biomass and analyzed by LC-MS.

[0233] In contrast to the finding for Y. lipolytica hmxlA, where an increased heme content was observed compared to the reference strain (Table 1), deletion of the heme oxygenase (Hmx1; accession no. P32339, database Uniprot) in the model yeast S. cerevisiae did not result in a significant increase in the heme (heme B) content (Figure 7).

[0234] Example 3

[0235] To put the heme (heme B) producing capability in context, the heme content of a heme overproducer Y. lipolytica strain was quantified and compared to a reference Y. lipolytica strain and beef.

[0236] We constructed a heme overproducer strain (ST10240 [YALI73_HMX1::6xLBA_HEM1mut]) combining three modifications: disruption of heme degradation (hmxlA), multicopy overexpression of heme binding proteins (6 copies of LBA), and expression of HEM1 lacking HRM motifs (HEMImut).

[0237] The heme (heme B) content was higher for the heme overproducing strain ST10240 than the Y. lipolytica reference strain, and the heme overproducing strain also contained more heme than beef (Figure 8). When provided as a meat alternative, the heme overproducing strain ST10240 (78 mg / 100g biomass) has significant potential to contribute toward the recommended daily intake of iron (Men: 8mg Fe / 88mg heme, Women: 18mg Fe / 198mg heme).

[0238] Within food / nutritional analysis, the amounts / content of ingredients and nutrients are typically specified as g / 100g product, as this most closely represents what is actually eaten. Hence, in this example the heme content is provided as mg / 100g wet weight (WW), which corresponds to the weight of the actual food product (as opposed to dry weight, for which the weight contribution of the water has been removed). The moisture content of the three tested samples (biomasses and beef) are within a similar range.

[0239] Sequence overview

[0240] SEQ ID NO: 13 corresponds to the sequence labelled “YIHEM1” of Figure 3. SEQ ID

[0241] NO: 14 corresponds to the sequence labelled “ALAS_Y.lipolytica” of Figure 5. SEQ ID

[0242] NO: 15 corresponds to the sequence labelled “ALAS_Y.galli” of Figure 5. SEQ ID NO: 16 corresponds to the sequence labelled “ALAS_Y.divulgata” of Figure 5. SEQ ID NO:

[0243] 17 corresponds to the sequence labelled “ALAS_Y.porcina” of Figure 5.

[0244] References

[0245] Munakata et al. Role of the Heme Regulatory Motif in the Heme-Mediated Inhibition of Mitochondrial Import of 5-Aminolevulinate Synthase. J. Biochem. (Tokyo) 136, 233- 238 (2004).

[0246] Fleischhacker et al. The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation. J. Biol. Chem. 295, 5177-5191 (2020). Holkenbrink et al. EasyCloneYALI: CRISPR / Cas9-Based Synthetic Toolbox for

[0247] Engineering of the Yeast Yarrowia lipolytica. Biotechnol. J. 13, 1700543 (2018).

[0248] Magnan et al., Sequence Assembly of Yarrowia lipolytica Strain W29 / CLIB89 Shows Transposable Element Diversity. PLoS ONE, 2016; 11(9): e0162363.

[0249] Jessop-Fabre et al., EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. J. Biotechnol. Volume 11 , Issue 8, Pages 1110-1117 (2016).

[0250] Items

[0251] 1 . A Yarrowia cell producing or capable of producing heme, said cell comprising one or more of the following modifications, such as two or three of the following modifications: i. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased activity is the result of a mutation, further optionally wherein said heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3; ii. increased activity of a 5-aminolevulinic acid synthase (ALAS) relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said increased activity is the result of a mutation, further optionally wherein said ALAS is HEM 1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 ; and / or iii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased sensitivity is the result of a mutation, such as a mutation of at least one heme-regulatory motif (HRM) of said ALAS, further optionally wherein said ALAS is HEM1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1, preferably wherein the production of heme is improved relative to the production of heme by a corresponding Yarrowia cell without said modification but otherwise identical, when cultivated in similar conditions. A Yarrowia cell producing or capable of producing heme and a hemoprotein, said cell comprising one or more of the following modifications, such as two or three of the following modifications,: i. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased activity is the result of a mutation, further optionally wherein said heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3; ii. increased activity of a 5-aminolevulinic acid synthase (ALAS) relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said increased activity is the result of a mutation, further optionally wherein said ALAS is HEM 1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1; and / or iii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased sensitivity is the result of a mutation, such as a mutation of at least one heme-regulatory motif (HRM) of said ALAS, further optionally wherein said ALAS is HEM1 as set forth in SEQ ID NO: 1, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1, preferably wherein the production of said heme and said hemoprotein is improved relative to the production of said heme and said hemoprotein by a corresponding Yarrowia cell without said modifications but otherwise identical, when cultivated in similar conditions. A Yarrowia cell producing or capable of producing a hemoprotein, said cell comprising one or more of the following modifications, such as two or three of the following modifications: i. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased activity is the result of a mutation, further optionally wherein said heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3; ii. increased activity of a 5-aminolevulinic acid synthase (ALAS) relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said increased activity is the result of a mutation, further optionally wherein said ALAS is HEM 1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 ; and / or iii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said modification but otherwise identical, optionally wherein said decreased sensitivity is the result of a mutation, such as a mutation of at least one heme-regulatory motif (HRM) of said ALAS, further optionally wherein said ALAS is HEM1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 , preferably wherein the production of said hemoprotein is improved relative to the production of said hemoprotein by a corresponding Yarrowia cell without said modifications but otherwise identical, when cultivated in similar conditions. The Yarrowia cell according to any one of the preceding items, wherein: i. the heme oxygenase is a heme oxygenase with EC 1.14.14.18; ii. the ALAS is an ALAS with EC 2.3.1.37. 5. The Yarrowia cell according to any one of the preceding items, wherein said heme is selected from heme A, heme B, heme C and heme O, preferably wherein the heme is heme B.

[0252] 6. The Yarrowia cell according to any one of the preceding items, wherein the ALAS having decreased sensitivity to feedback inhibition by heme is identical to the ALAS with increased activity, optionally wherein said increased activity is obtained by overexpression of said ALAS having decreased sensitivity to feedback inhibition by heme.

[0253] 7. The Yarrowia cell according to any one of the preceding items, wherein said heme oxygenase is capable of converting heme to biliverdin, carbon monoxide, and iron, preferably wherein the heme oxygenase is native to said cell.

[0254] 8. The Yarrowia cell according to any one of the preceding items, wherein the one or more modifications, such as two or three modifications, comprises or consists of: i. partial or total deletion of a gene encoding the heme oxygenase; ii. down-regulation of the expression of a gene encoding the heme oxygenase; and / or iii. down-regulation of the activity of the heme oxygenase, whereby the activity of the heme oxygenase is decreased.

[0255] 9. The Yarrowia cell according to item 7, wherein the one or more modifications, such as two or three modifications, comprises or consists of: i. a mutation in a nucleic acid sequence upstream of the gene encoding the heme oxygenase, such as a mutation in a promoter of said gene, such as a partial or total deletion of said upstream nucleic acid sequence, such as of said promoter; ii. a mutation of a transcription factor controlling regulation of the transcription of the gene encoding the heme oxygenase, such as a partial or total deletion of said transcription factor, wherein the mutation results in partial or total loss of activity of said promoter and / or transcription factor, respectively, whereby the expression of the gene encoding the heme oxygenase is down- regulated. The Yarrowia cell according to any one of items 7 to 9, wherein the one or more modifications, such as two or three modifications, comprises or consists of: i. a partial or total loss-of-function mutation of the gene encoding the heme oxygenase; and / or ii. inhibition of the heme oxygenase, such as reversible or irreversible inhibition, whereby the activity of the heme oxygenase is down-regulated. The Yarrowia cell according to any one of the preceding items, wherein the heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3. The Yarrowia cell according to any one of the preceding items, wherein the gene encoding the heme oxygenase is HMX1 as set forth in SEQ ID NO: 8, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 8. The Yarrowia cell according to any one of the preceding items, wherein said ALAS is capable of converting succinyl-CoA and glycine to 5-aminolevulinate (ALA). The Yarrowia cell according to any one of the preceding items, wherein the ALAS is an ALAS heterologous to the Yarrowia cell and / or an ALAS native to the Yarrowia cell. The Yarrowia cell according to any one of the preceding items, wherein said ALAS is an ALAS heterologous to the Yarrowia cell, such as an ALAS of Saccharomyces cerevisiae, such as ScALAS (Uniprot ID: P09950), of Schizosaccharomyces pombe, such as SpALAS (Uniprot ID: 014092), of Neurospora crassa, such as NcALAS (Uniprot ID: Q7RVY5), of Agaricus bisporus, such as AbALAS (Uniprot ID: Q92403), of Gallus gallus, such as GgALASI (Uniprot ID: P07997) or GgALAS2 (Uniprot ID: P18080), or an ALAS of Bos taurus, such as BtALAS (Uniprot ID: A6QLI6), or BtALAS2 (Uniprot ID: Q3ZC31).

[0256] 16. The Yarrowia cell according to any one of the preceding items, wherein the one or more modifications comprises or consists of: i. overexpression of a gene encoding the ALAS; ii. increased production of the ALAS; and / or iii. gain-of-function mutation of a gene encoding the ALAS, whereby the activity of ALAS is increased.

[0257] 17. The Yarrowia cell according to any one of the preceding items, wherein the one or more modifications is two modifications selected from: i. overexpression of a gene encoding the ALAS; ii. increased production of the ALAS; and / or iii. gain-of-function mutation of a gene encoding the ALAS, whereby the activity of ALAS is increased, preferably wherein the two modifications are i. and ii.

[0258] 18. The Yarrowia cell according to any one of the preceding items, wherein the cell comprises at least two copies of the gene encoding the ALAS, such as at least three copies, such as at least four copies, such as at least five copies, or more.

[0259] 19. The Yarrowia cell according to any one of items 16 to 18, wherein the one or more modifications, such as two or three modifications, comprises or consists of: i. increased transcription of the gene encoding the ALAS, whereby the gene encoding the ALAS is overexpressed.

[0260] 20. The Yarrowia cell according to any one of items 16 to 19, wherein the one or more modifications, such as two or three modifications, comprises or consists of: i. prolonged half-life of mRNA transcribed from the gene encoding the ALAS, whereby the production of ALAS is increased. 21. The Yarrowia cell according to any one of items 16 to 20, wherein expression of the gene encoding ALAS is modified, preferably increased, such as by introducing a mutation in the native promoter of ALAS, for example a partial or total substitution of a nucleic acid sequence upstream of ALAS, or by operably linking the gene encoding ALAS to a non-native promoter, whereby expression of the gene encoding ALAS is modified, preferably increased, compared to expression from a reference cell, wherein expression of the gene encoding ALAS is not modified in said reference cell, and / or wherein the one or more modifications, such as two or three modifications, comprises or consists of a mutation in a nucleic acid sequence upstream of the gene encoding ALAS, such as a mutation in a promoter of said gene, for example a partial or total substitution of said upstream nucleic acid sequence, such as of said promoter, preferably said promoter of ALAS is substituted, such as replaced, by a stronger promoter relative to said promoter of ALAS, where transcription from said strong promoter is higher compared to transcription from the native ALAS promoter under the same conditions.

[0261] 22. The Yarrowia cell according to item 21 , wherein said nucleic acid sequence upstream of ALAS, such as said promoter of ALAS or fragment thereof, for example said native ALAS promoter or fragment thereof, is replaced by prEXPI as set forth in SEQ ID NO: 11 , or a functional variant thereof having at least 70% sequence identity, similarity or homology to SEQ ID NO: 11.

[0262] 23. The Yarrowia cell according to any one of the preceding items, wherein the endogenous ALAS is a Yarrowia ALAS, optionally a Yarrowia lipolytica ALAS such as HEM1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity or homology to SEQ ID NO: 1.

[0263] 24. The Yarrowia cell according to any one of the preceding items, wherein the ALAS is HEM 1 as set forth in SEQ ID NO: 1, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1. 25. The Yarrowia cell according to any one of the preceding items, wherein the gene encoding the ALAS is HEM1 as set forth in SEQ ID NO: 6, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 6.

[0264] 26. The Yarrowia cell according to any one of the preceding items, wherein the modification resulting in decreased sensitivity of an ALAS to feedback inhibition by heme resulting in decreased sensitivity of an ALAS to feedback inhibition by heme is one or more modifications or a mutation resulting in total loss of sensitivity of an ALAS to feedback inhibition by heme.

[0265] 27. The Yarrowia cell according to any one of the preceding items, wherein the ALAS comprises at least one HRM, such as at least two HRMs, such as at least three HRMs, or more.

[0266] 28. The Yarrowia cell according to any one of the preceding items, wherein the mutation resulting in decreased sensitivity of an ALAS to feedback inhibition by heme is a mutation in at least one HRM of the ALAS.

[0267] 29. The Yarrowia cell according to any one of the preceding items, wherein the mutation in at least one HRM of the ALAS is a single amino acid substitution.

[0268] 30. The Yarrowia cell according to any one of the preceding items, wherein the mutation in at least one HRM of the ALAS is a single-nucleotide-polymorphism and / or a substitution of one or more nucleotides in the gene encoding ALAS.

[0269] 31. The Yarrowia cell according to item 30, wherein the mutation in the gene encoding ALAS comprises or consists of at least one mutation, such as at least two mutations, such as at least three mutations, such as at least four mutations, such as at least five mutations, such as at least six mutations, or more.

[0270] 32. The Yarrowia cell according to any one of the preceding items, wherein the at least one HRM of the ALAS comprises a cysteine-proline motif. 33. The Yarrowia cell according to any one of the preceding items, wherein the mutation in the at least one HRM of the ALAS comprises or consists of a mutation resulting in a single amino acid substitution, optionally a substitution of cysteine of the cysteine-proline motif of the at least one HRM to a different amino acid and / or a substitution of proline of the cysteine-proline motif of the at least one HRM to a different amino acid.

[0271] 34. The Yarrowia cell according to item 33, wherein the different amino acid is selected from a polar uncharged amino acid, a nonpolar aliphatic amino acid, an aromatic amino acid, a positively charged amino acid and / or a negatively charged amino acid.

[0272] 35. The Yarrowia cell according to item 34, wherein the polar uncharged amino acid is selected from serine, threonine, asparagine, and glutamine.

[0273] 36. The Yarrowia cell according to item 34, wherein the nonpolar aliphatic amino acid is selected from glycine, proline, alanine, isoleucine, leucine, methionine, and valine.

[0274] 37. The Yarrowia cell according to item 34, wherein the aromatic amino acid is selected from phenylalanine, tryptophan, and tyrosine.

[0275] 38. The Yarrowia cell according to item 34, wherein the positively charged amino acid is selected from arginine, histidine, and lysine.

[0276] 39. The Yarrowia cell according to item 34, wherein the negatively charged amino acids is glutamic acid (glutamate), and aspartic acid (aspartate).

[0277] 40. The Yarrowia cell according to item 29, wherein the single amino acid substitution is a substitution from a cysteine to a different amino acid, for example to arginine, histidine, lysine, serine, threonine, asparagine, glutamine, glycine, proline, alanine, isoleucine, leucine, methionine, glutamic acid, aspartic acid, phenylalanine, tryptophan, tyrosine, or valine. The Yarrowia cell according to any one of the preceding items, wherein the mutation in the at least one HRM of the ALAS is a substitution of cysteine of the cysteine-proline motif of the at least one HRM to a serine, optionally wherein the ALAS is an ALAS according to any one of items 1 to 4 and / or 13 to 24. The Yarrowia cell according to any one of the preceding items, comprising an ALAS comprising at least one HRM, such as three HRMs, wherein the different amino acid is the same for each of the HRMs. The Yarrowia cell according to any one of the preceding items, comprising an ALAS comprising at least one HRM, such as three HRMs, wherein the different amino acid is different for each of the HRMs. The Yarrowia cell according to any one of the preceding items, comprising an ALAS comprising at least one HRM, such as three HRMs, wherein the different amino acid is the same for two of the three HRMs. The Yarrowia cell according to any one of the preceding items, comprising an ALAS comprising at least one HRM, such as three HRMs, wherein each of the HRMs comprises at least one substitution of cysteine of the cysteine-proline motif of each of the HRMs to a serine. The Yarrowia cell according to any one of the preceding items, wherein the ALAS having decreased sensitivity to feedback inhibition by heme is HEM1_mutHRM as set forth in SEQ ID NO: 2, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 2. The Yarrowia cell according to item 46, wherein the gene encoding said HEM1_mutHRM is HEM1_mutHRM as set forth in SEQ ID NO: 7, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 7. The Yarrowia cell according to any one of the preceding items, wherein the Yarrowia cell is generally-regarded-as-safe (GRAS), has safe-to-consume status, and / or is a non-pathogenic Yarrowia cell. 49. The Yarrowia cell according to any one of the preceding items, wherein the Yarrowia cell is genetically modified.

[0278] 50. The Yarrowia cell according to any one of the preceding items, wherein the Yarrowia cell belongs to the species of Yarrowia lipolytica, Yarrowia porcina, Yarrowia bubula, Yarrowia deformans, Yarrowia yakushimensis, Yarrowia parophonii, Yarrowia galli, Yarrowia oslonensis, Yarrowia alimentaria, Yarrowia hollandica, or Yarrowia phangngaensis.

[0279] 51. The Yarrowia cell according to any one of the preceding items, further producing a hemoprotein.

[0280] 52. The Yarrowia cell according to any one of the preceding items, wherein the cell is capable of synthesizing glycine, alpha-ketoglutarate and / or succinyl-CoA.

[0281] 53. The Yarrowia cell according to any one of the preceding items, wherein the amount of heme and / or the hemoprotein is increased by at least 2-fold, such as at least 3-fold, such as at least 3.5-fold, such as at least 4-fold, such as at least 4.5-fold, such as at least 5-fold, such as at least 6-fold, such as at least 10-fold, or more, compared to the amount produced by a corresponding Yarrowia cell without the one or more modifications, such as the two or three modifications, according to any one of items 1 to 50, optionally wherein the amount is the sum of intracellular heme and / or hemoprotein and the extracellular heme and / or hemoprotein.

[0282] 54. The Yarrowia cell according to any one of the preceding items, wherein the similar conditions is the same conditions.

[0283] 55. The Yarrowia cell according to any one of the preceding items, wherein the cell does not comprise a modification in a heme-dependent repressor of hypoxic genes, such as R0X1.

[0284] 56. The Yarrowia cell according to any one of the preceding items, wherein the cell does not comprise a modification in a vacuolar proteinase, such as PEP4. 57. The Yarrowia cell according to any one of the preceding items, wherein the one or more modifications comprises decreased activity of a heme oxygenase, and wherein the cell does not comprise a modification in a heme-dependent repressor of hypoxic genes, such as R0X1, and / or a modification in a vacuolar proteinase, such as PEP4.

[0285] 58. The Yarrowia cell according to any one of the preceding items, wherein the one or more modifications is two or more modifications comprising decreased activity of a heme oxygenase, and wherein the cell does not comprise a modification in a heme-dependent repressor of hypoxic genes, such as R0X1, and / or a modification in a vacuolar proteinase, such as PEP4.

[0286] 59. An expression system for expression in a Yarrowia cell, comprising a nucleic acid encoding an ALAS, optionally wherein the ALAS is HEM1 as set forth in SEQ ID NO: 1 and / or HEM1_mutHRM as set forth in SEQ ID NO: 2, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1 and / or SEQ ID NO: 2, respectively.

[0287] 60. The expression system according to item 59, wherein the copy number of said nucleic acid encoding ALAS is at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, or more.

[0288] 61. The expression system according to any one of items 59 to 60, wherein the nucleic acid encoding ALAS comprises or consists of HEM1 as set forth in SEQ ID NO: 6, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 6.

[0289] 62. The expression system according to any one of items 59 to 61 , wherein the nucleic acid encoding ALAS comprises or consists of HEM1_mutHRM as set forth in SEQ ID NO: 7, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 7. 63. The expression system according to any one of items 59 to 62, wherein the nucleic acid further comprises a promoter, such as a strong promoter, for example prEXPI as set forth in SEQ ID NO: 11 or prGPDI as set forth in SEQ ID NO: 12, or functional variants of any of the aforementioned having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 11 or SEQ ID NO: 12, respectively.

[0290] 64. The expression system according to any one of items 59 to 63, further comprising a nucleic acid encoding a myoglobin, optionally wherein the myoglobin is Bos taurus myoglobin as set forth in SEQ ID NO: 5 (Bt_myoglobin), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 5.

[0291] 65. The expression system according to item 64, wherein the nucleic acid encoding Bt_myoglobin (SEQ ID NO: 5) comprises or consists of SEQ ID NO: 10, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 10.

[0292] 66. The expression system according to any one of items 59 to 65, further comprising a nucleic acid encoding a leghemoglobin, optionally wherein the leghemoglobin is Glycine max LBA as set forth in SEQ ID NO: 4 (Gm_LBA) or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 4.

[0293] 67. The expression system according to item 66, wherein the nucleic acid encoding Gm_LBA (SEQ ID NO: 4) comprises or consists of SEQ ID NO: 9, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 9.

[0294] 68. The expression system according to any one of items 64 to 67, wherein the copy number of said nucleic acid encoding a myoglobin and / or a hemoglobin, preferably Gm_LBA (SEQ ID NO: 4) or a or a functional variant thereof having at least 70% sequence identity, similarity, or homology thereto, is at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 8, such as at least 9, such as at least 10, or more. The expression system according to any one of items 59 to 67, wherein the Yarrowia cell is as defined in any one of items 1 to 54. The Yarrowia cell according to any one of items 1 to 58, said cell comprising the expression system according to any one of items 59 to 69, whereby said Yarrowia cell is capable of producing heme and / or hemoprotein. A method of producing and / or increasing production of heme and / or a hemoprotein in a Yarrowia cell, comprising the steps of: i. providing a Yarrowia cell according to any one of items 1 to 58, 70 or 89 to 91 ; and ii. incubating and optionally propagating said Yarrowia cell in a medium, whereby said heme and / or hemoprotein is produced or whereby production of said heme and / or hemoprotein is increased. The method according to item 71 , further comprising a step of recovering the heme and / or the hemoprotein. A method of manufacturing biomass comprising Yarrowia cells with increased intracellular concentration and / or content of heme and / or a hemoprotein: i. providing a Yarrowia cell according to any one of items 1 to 58, 70 or 89 to 91 , having increased intracellular concentration and / or content of a hemoprotein, compared to a corresponding cell without the one or more modifications, such as two or three modifications, disclosed herein, but otherwise identical; and ii. incubating and optionally propagating said Yarrowia cell in a medium; and iii. optionally recovering biomass comprising said Yarrowia cell, whereby said biomass comprising Yarrowia cells with increased intracellular concentration and / or content of heme and / or a hemoprotein is obtained. 74. The method according to item 73, further comprising a step of converting said biomass into a meat alternative and / or ingredient therefor, whereby a meat alternative and / or ingredient therefor is manufactured.

[0295] 75. A method of manufacturing a meat alternative and / or ingredient therefor, comprising the steps of: i. providing a Yarrowia cell according to any one of items 1 to 58, 70 or 89 to 91 ; ii. incubating and optionally propagating said Yarrowia cell in a medium, iii. recovering biomass comprising said Yarrowia cell and / or fermentation liquid comprising biomass comprising said Yarrowia cell; and / or iv. converting said biomass and / or fermentation liquid comprising biomass comprising said Yarrowia cell into a meat alternative and / or ingredient therefor, whereby a meat alternative and / or ingredient therefor is manufactured.

[0296] 76. The method according to item 75, wherein the ingredient is a food ingredient and / or a feed ingredient.

[0297] 77. The method according to any one of items 71 to 76, wherein the medium comprises: i. iron, such as an iron salt, for example ferric chloride, iron sulfate, iron nitrate, and / or ferric citrate; ii. glycine; iii. alpha-ketoglutarate; and / or iv. succinyl-CoA.

[0298] 78. The method according to any one of items 71 to 77, wherein the heme and / or the hemoprotein is produced with a titer that is at least 25% increased, such as at least 30% increased, such as at least 35% increased, such as at least 40% increased, such as at least 45% increased, such as at least 50% increased, such as at least 55% increased, such as at least 60% increased, such as at least 75% increased, such as at least 80% increased, such as at least 90% increased, such as at least 100% increased, such as at least 150% increased, such as at least 200% increased, such as at least 250% increased, such as at least 300% increased, such as at least 350% increased, such as at least 400% increased, such as at least 450% increased, such as at least 500% increased, such as at least 550% increased, such as at least 600% increased, such as at least 650% increased, such as at least 700% increased, such as at least 800% increased, or more increased, compared to the titer produced by a corresponding Yarrowia cell without the one or more modifications, such as two or three modifications, as defined in any one of items 1 to 54, 70 or 89 to 91.

[0299] 79. A composition comprising the Yarrowia cell according to any one of items 1 to 58, 70 or 89 to 91 , and / or the biomass, heme, and / or hemoprotein obtainable by a method according to any one of items 71 to 78 or 89 to 91.

[0300] 80. Heme and / or a hemoprotein obtained or obtainable by a method according to any one of items 71 to 78 or 89 to 91.

[0301] 81. A biomass obtained or obtainable by a method according to any one of items 73 to 74, and / or a biomass comprising Yarrowia cells as defined in any one of items 1 to 54, 70 or 89 to 91.

[0302] 82. A meat alternative and / or ingredient therefor obtained or obtainable by a method according to any one of items 75 to 78 or 89 to 91.

[0303] 83. A food composition comprising heme and / or hemoprotein from the Yarrowia cell according to any one of items 1 to 58or 70, 70 or 89 to 91.

[0304] 84. Use of a composition according to item 79 or 83, as a food product, food material, food ingredient, and / or feed ingredient.

[0305] 85. The food material, food product, food composition, ingredient, and / or food ingredient according to any one of the preceding items, wherein the food material, food product, food composition, ingredient, and / or food ingredient is a meat substitute, such as a beef substitute, a chicken substitute, or a fish substitute. 86. Use of a composition according to any one of items 79 and 83 in a meat alternative and / or in a method for producing a meat alternative.

[0306] 87. Use of a Yarrowia cell according to any one of items 1 to 58, 70 or 89 to 91 , a biomass obtainable by a method according to any one of items 73 to 74, and / or a biomass according to item 81 , for production of meat alternative and / or ingredient therefor.

[0307] 88. Use of a Yarrowia cell according to any one of items 1 to 58, 70 or 89 to 91 , for production of a hemoprotein, or production of a food and / or a feed containing hemoprotein.

[0308] 89. The Yarrowia cell, the method, the composition, the biomass, and / or the use according to any one of the preceding items, wherein the hemoprotein is an endogenous hemoprotein or a hemoprotein heterologous to the Yarrowia cell, preferably wherein the hemoprotein is a heme B-containing protein, and optionally wherein the Yarrowia cell carries at least two copies of a nucleic acid encoding said hemoprotein, such as at least three copies, such as at least four copies, such as at least five copies, such as at least six copies, such as at least seven copies, such as at least eight copies, such as at least nine copies, such as at least ten copies, or more copies a nucleic acid encoding said hemoprotein, preferably wherein said hemoprotein is a leghemoglobin, such as leghemoglobin A (LBA), for example Glycine max LBA as set forth in SEQ ID NO: 4 (Gm_LBA), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 4.

[0309] 90. The Yarrowia cell, the method, the composition, the biomass, and / or the use according to any one of the preceding items, wherein the hemoprotein is a hemoprotein heterologous to the Yarrowia cell, for example a globin, or a cytochrome.

[0310] 91. The Yarrowia cell, the method, the composition, the biomass, and / or the use according to item 90, wherein the globin is hemoglobin, myoglobin, such as Bos taurus myoglobin as set forth in SEQ ID NO: 5 (Bt_myoglobin), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 5, or leghemoglobin, such as leghemoglobin A (LBA), for example Glycine max LBA as set forth in SEQ ID NO: 4 (Gm_LBA), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 4. The Yarrowia cell, the method, the composition, the biomass, and / or the use according to any one of the preceding items, wherein the hemoprotein is a myoglobin, cytochrome P450, cytochrome c oxidase, catalase, ligninase or peroxidase. The use and / or the food composition according to any one of the preceding items, wherein the food material, food product, food composition, ingredient, and / or food ingredient is a meat substitute, such as a beef substitute, a chicken substitute, or a fish substitute.

Claims

Claims1. A Yarrowia cell producing or capable of producing heme and optionally one or more hemoproteins, said cell comprising two or three of the following modifications: i. increased activity of a 5-aminolevulinic acid synthase (ALAS) relative to the activity of said ALAS in a corresponding Yarrowia cell without said two or three modifications but otherwise identical, optionally wherein said increased activity is the result of a mutation, further optionally wherein said ALAS is HEM1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1; ii. decreased activity of a heme oxygenase relative to the activity of said heme oxygenase in a corresponding Yarrowia cell without said two or three modifications but otherwise identical, optionally wherein said decreased activity is the result of a mutation, further optionally wherein said heme oxygenase is HMX1 as set forth in SEQ ID NO: 3, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 3; and / or iii. decreased sensitivity of an ALAS to feedback inhibition by heme relative to the activity of said ALAS in a corresponding Yarrowia cell without said two or three modifications but otherwise identical, optionally wherein said decreased sensitivity is the result of a mutation, such as a mutation of at least one heme-regulatory motif (HRM) of said ALAS, further optionally wherein said ALAS is HEM1 as set forth in SEQ ID NO: 1 , or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 1, preferably wherein the production of said heme and optionally said one or more hemoproteins is improved relative to the production of said heme and optionally said one or more hemoproteins by a corresponding Yarrowia cell without said two or three modifications but otherwise identical, when cultivated in similar conditions, further optionally wherein said heme is heme B.

2. The Yarrowia cell according to claim 1 , wherein the two or three modifications comprise or consist of:i. partial or total deletion of a gene encoding the heme oxygenase; ii. down-regulation of the expression of a gene encoding the heme oxygenase; and / or iii. down-regulation of the activity of the heme oxygenase, whereby the activity of the heme oxygenase is decreased.

3. The Yarrowia cell according to any one of the preceding claims, wherein the ALAS having decreased sensitivity to feedback inhibition by heme is identical to the ALAS with increased activity, optionally wherein said increased activity is obtained by overexpression of said ALAS having decreased sensitivity to feedback inhibition by heme.

4. The Yarrowia cell according to any one of the preceding claims, wherein the ALAS having decreased sensitivity to feedback inhibition by heme is HEM1_mutHRM as set forth in SEQ ID NO: 2, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 2, optionally wherein the gene encoding said HEM1_mutHRM is HEM1_mutHRM as set forth in SEQ ID NO: 7, or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 7.

5. The Yarrowia cell according to any one of the preceding claims, wherein the mutation resulting in decreased sensitivity of an ALAS to feedback inhibition by heme is a mutation in at least one HRM of the ALAS, optionally wherein the mutation in at least one HRM of the ALAS is a single amino acid substitution, further optionally wherein the mutation in at least one HRM of the ALAS is a single-nucleotide-polymorphism and / or a substitution of one or more nucleotides in the gene encoding ALAS.

6. The Yarrowia cell according to any one of the preceding claims, wherein the Yarrowia cell belongs to the species of Yarrowia lipolytica, Yarrowia porcina, Yarrowia bubula, Yarrowia deformans, Yarrowia yakushimensis, Yarrowia parophonii, Yarrowia galli, Yarrowia oslonensis, Yarrowia alimentaria, Yarrowia hollandica, or Yarrowia phangngaensis, preferably wherein the Yarrowia cell is a Yarrowia lipolytica cell.

7. The Yarrowia cell according to any one of the preceding claims, wherein the amount of heme and / or the hemoprotein is increased by at least 2-fold, such as at least 3-fold, such as at least 3.5-fold, such as at least 4-fold, such as at least 4.5-fold, such as at least 5-fold, such as at least 6-fold, such as at least 10-fold, or more, compared to the amount produced by a corresponding Yarrowia cell without the two or three modifications according to any one of claims 1 to 6, optionally wherein the amount is the sum of intracellular heme and / or hemoprotein and the extracellular heme and / or hemoprotein.

8. The Yarrowia cell according to any one of the preceding claims, wherein the hemoprotein is an endogenous hemoprotein or a hemoprotein heterologous to the Yarrowia cell, for example a globin, or a cytochrome, and / or wherein the hemoprotein is a heme B-containing protein, optionally wherein the globin is hemoglobin, myoglobin, such as Bos taurus myoglobin as set forth in SEQ ID NO: 5 (Bt_LBA), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 5, or leghemoglobin, such as leghemoglobin A (LBA), for example Glycine max LBA as set forth in SEQ ID NO: 4 (Gm_LBA), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 4.

9. The Yarrowia cell according to any one of the preceding claims, wherein the Yarrowia cell carries at least two copies of a nucleic acid encoding said hemoprotein, such as at least three copies, such as at least four copies, such as at least five copies, such as at least six copies, such as at least seven copies, such as at least eight copies, such as at least nine copies, such as at least ten copies, or more copies a nucleic acid encoding said hemoprotein, optionally wherein said hemoprotein is Glycine max LBA as set forth in SEQ ID NO: 4 (Gm_LBA), or a functional variant thereof having at least 70% sequence identity, similarity, or homology to SEQ ID NO: 4.

10. A method of producing and / or increasing production of heme and / or a hemoprotein in a Yarrowia cell, comprising the steps of: i. providing a Yarrowia cell according to any one of claims 1 to 9; and ii. incubating and optionally propagating said Yarrowia cell in a medium,whereby said heme and / or hemoprotein is produced or whereby production of said heme and / or hemoprotein is increased.

11. A method of manufacturing biomass comprising Yarrowia cells with increased intracellular concentration and / or content of heme and / or a hemoprotein: i. providing a Yarrowia cell according to any one of claims 1 to 9, having increased intracellular concentration and / or content of a hemoprotein, compared to a corresponding cell without the two or three modifications disclosed herein, but otherwise identical; and ii. incubating and optionally propagating said Yarrowia cell in a medium; and iii. optionally recovering biomass comprising said Yarrowia cell; and iv. further optionally, comprising a step of converting said biomass into a meat alternative and / or ingredient therefor, whereby a meat alternative and / or ingredient therefor is manufactured.

12. The method according to any one of claims 10 to 11 , wherein the medium comprises: i. iron, such as an iron salt, for example ferric chloride, iron sulfate, iron nitrate, and / or ferric citrate; ii. glycine; iii. alpha-ketoglutarate; and / or iv. succinyl-CoA.

13. A biomass obtainable by the method according to claim 11, and / or a biomass comprising Yarrowia cells as set forth in any one of claims 1 to 9.

14. A food composition comprising heme and / or hemoprotein from the Yarrowia cell as defined in any one of claims 1 to 9, optionally wherein the food composition is a meat substitute, such as a beef substitute, a chicken substitute, or a fish substitute.

15. Use of a Yarrowia cell as defined in any one of claims 1 to 9, a biomass as defined in claim 13, and / or a biomass obtainable by the method according to claim 11,for production of meat alternative and / or ingredient therefor, for production of one or more hemoproteins, and / or for production of food and / or feed containing one or more hemoproteins.

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