CARRP enzyme variants and their use in producing carotenoids and apocarotenoids

Abstract

The present invention relates to the increased accumulation of carotenoids and their derivatives through the manipulation and heterologous expression of CarRP from Mucor circinelloides.

Description

Detailed Description of the Invention

[0001] The present invention relates to the increased accumulation of carotenoids and their derivatives through the manipulation and heterologous expression of CarRP from Mucor circinelloides.

[0002] Carotenoids, including C-40 isoprenoid compounds such as carotenes and xanthophylls, as well as cleavage products such as apocarotenoids, are responsible for the orange color of carrots, the pink color of flamingos and salmon, the red color of lobsters or shrimp, and further important applications in the food, feed, cosmetic, or pharmaceutical industries. Furthermore, beta-carotene is an important precursor or intermediate in the synthesis of vitamin A.

[0003] Retinoids, which belong to the apocarotenoid class, are one of the most important and essential nutrients for both humans and animals, which need to be supplied through diet.Retinoids promote the health of humans / animals, especially in terms of vision, immune system, and growth.

[0004] Since the chemical synthesis of carotenoids or retinoids has several major drawbacks, namely the consumption of energy and / or water, organic and / or inorganic solvents, the synthesis of undesirable by-products, and the growing global demand for natural products for use, for example, as colorants or dietary supplements, there is a strong need for the biotechnological production of such compounds.

[0005] Carotenoids, including carotenes and xanthophylls, and apocarotenoids, including retinoids and ionones, are naturally produced by certain organisms, including photosynthetic organisms (e.g., plants, algae, cyanobacteria (particularly for the production of carotenoids) and some fungi, such as Mucor circinelloides, Yarrowia, and Saccharomyces (for both carotenoid and retinoid production), as well as in bacteria, such as E. coli or Paracoccus. However, these systems are industrially cumbersome and / or produce the compounds at such low levels that industrial-scale isolation is not feasible.

[0006] A key enzyme in both carotenoid and apocarotenoid biosynthesis is the bifunctional enzyme CarRP, which catalyzes the conversion of geranylgeranyl pyrophosphate (GGPP) to phytoene (i.e., acts as phytoene synthase) and the conversion of lycopene to beta-carotene (i.e., acts as lycopene cyclase). A widely used enzyme with good performance is derived from Mucor circinelloides (McCarRP), but lycopene accumulation can lead to feedback inhibition of CarRP and, consequently, reduced production of beta-carotene and xanthophylls.

[0007] Therefore, there is a strong need for more efficient biological production of carotenoids and apocarotenoids, including but not limited to retinal or retinol, where the respective genes are (over)expressed in generally recognized as safe (GRAS) host cells, such as oleaginous yeast, to reduce or eliminate known bottlenecks in such production processes.

[0008] Surprisingly, the present inventors have identified amino acid residues in CarRP from Mucor circinelloides (McCarRP) that are essential for the formation of phytoene and / or beta-carotene, and thus for the production of carotenoids or apocarotenoids, thereby reducing known feedback inhibition. Introduction of one or more amino acid substitutions located in both the lycopene cyclase (R)-domain and the phytoene synthase (P)-domain of the enzyme results in an increase in the formation of the carotenoid compounds listed above, in the range of at least about 5%, e.g., in the range of 20-75%, and even more, compared to the wild-type (unmodified) McCarRP of SEQ ID NO: 1 or shown in FIG. 1 .

[0009] Specifically, the present invention relates to modified bifunctional enzymes involved in the synthesis of phytoene and acting as lycopene cyclases, as well as to such modified enzymes, i.e., enzymes that catalyze the conversion of geranylgeranyl pyrophosphate (GGPP) to phytoene and / or the conversion of lycopene to beta-carotene, specifically, enzymes that have one or more modifications, e.g., at least about 20%, e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 6 Regarding a method for producing a CarRP comprising an amino acid substitution introduced into a sequence having 2, 95, 97, 98, 99%, or up to 100% identity, or a sequence shown in Figure 1, the one or more amino acid substitutions are introduced at positions corresponding to amino acid residues selected from the group consisting of positions 7, 33, 153, 159, 167, 194, 305, 330, 430, 431, 432, 476, 547, 579, and combinations thereof, of SEQ ID NO: 1 or in the polypeptide shown in Figure 1.

[0010] More specifically, the present invention relates to a modified CarRP as defined herein comprising one or more amino acid substitutions at positions defined herein, wherein the amino acid residue at the position corresponding to position 7 of SEQ ID NO:1 or as shown in Figure 1 is different from glutamic acid or glutamine, the amino acid residue at the position corresponding to position 33 of SEQ ID NO:1 or as shown in Figure 1 is different from alanine or tryptophan, the amino acid residue at the position corresponding to position 153 of SEQ ID NO:1 or as shown in Figure 1 is different from alanine, the amino acid residue at the position corresponding to position 159 of SEQ ID NO:1 or as shown in Figure 1 is different from leucine, and the amino acid residue at the position corresponding to position 167 of SEQ ID NO:1 or as shown in Figure 1 is tyrosine. wherein the amino acid residue at a position corresponding to position 194 or 476 of SEQ ID NO: 1 or as shown in Figure 1 is different from isoleucine; the amino acid residue at a position corresponding to position 305 of SEQ ID NO: 1 or as shown in Figure 1 is different from threonine; the amino acid residue at a position corresponding to position 330 of SEQ ID NO: 1 or as shown in Figure 1 is different from aspartic acid; the amino acid residue at a position corresponding to position 430 or 431 of SEQ ID NO: 1 or as shown in Figure 1 is different from serine; the amino acid residue at a position corresponding to position 432 or 547 of SEQ ID NO: 1 or as shown in Figure 1 is different from valine; and / or the amino acid residue at a position corresponding to position 579 of SEQ ID NO: 1 or as shown in Figure 1 is different from arginine.

[0011] In some embodiments, the use of such modified enzymes in processes for the production of carotenoids and apocarotenoids, including carotenes, xanthophylls, retinoids, and ionones, wherein said modified enzymes are expressed, particularly heterologously expressed, in suitable host cells, particularly fungal carotenoid and / or retinoid producing host cells, more particularly beta-carotene producing host cells, results in an increase in titer of such products, based on the total carotenoid / apocarotenoid / retinoid present in / produced by said modified host cells, by at least about 5%, such as in the range of 5-20%, or even 5-75%, compared to a process using the same conditions but with the unmodified CarRP enzyme of SEQ ID NO: 1 or shown in FIG. 1 .

[0012] The terms "CarRP," "phytoene synthase," "lycopene cyclase," and "CrtYB" are used interchangeably herein and refer to a bifunctional enzyme involved in the biosynthetic pathway from GGPP to beta-carotene, which can catalyze the conversion of GGPP to phytoene, i.e., function as a phytoene synthase [EC 2.5.1.32], and / or can catalyze the conversion of lycopene to beta-carotene, i.e., function as a lycopene beta-cyclase [EC 5.5.1.19]. An exemplary and suitable enzyme that can be used to generate the modified enzymes of the invention is McCarRP as shown in SEQ ID NO: 1 or in FIG. 1, or an enzyme having at least about 20% identity to SEQ ID NO: 1 or the enzyme shown in FIG. 1, e.g., the enzyme encoded by the polynucleotide of SEQ ID NO: 2.

[0013] A "modified" CarRP as defined herein, based on an unmodified CarRP, particularly an enzyme having at least about 20% identity to McCarRP of SEQ ID NO: 1 or shown in FIG. 1, exhibits increased enzymatic activity, i.e., increased activity toward the formation of phytoene and / or beta-carotene, and thus increased formation of apocarotenoids, including retinoids, and / or carotenoids, including xanthophylls, as defined herein, particularly an increase of at least about 5%, for example 5-75%, particularly in the range of 20-75% and greater (particularly with respect to retinoid production), compared to the formation of the above products using the enzyme of SEQ ID NO: 1 or shown in FIG. 1.

[0014] The term "carotenoid" as used herein is well known in the art. It encompasses long 40-carbon conjugated isoprenoid polyenes (C-40 isoprenoids) naturally formed by the ligation of two 20-carbon GGPP molecules. These include, but are not limited to, phytoene, lycopene, beta-carotene, alpha-carotene, gamma-carotene, rhodoxanthin, canthaxanthin, zeaxanthin, astaxanthin, beta-cryptoxanthin, or lutein. The biosynthesis of carotenoids is described, for example, in International Publication No. 2006 / 102342. The term carotenoid also includes the group of "xanthophylls," i.e., oxidized carotenoid derivatives, such as lutein, zeaxanthin, or beta-cryptoxanthin.

[0015] As used herein, "apocarotenoids" are carotenoid cleavage products and are therefore defined as carotenoids below C40, and include, but are not limited to, retinoids or ionones, such as retinal, retinol, retinyl acetate, beta-ionone, or alpha-ionone.

[0016] As used herein, retinoids include, but are not limited to, retinal, retinoic acid, retinol, retinoic acid methoxide, retinyl acetate, retinyl esters, 4-keto-retinoids, 3-hydroxy-retinoids, or combinations thereof. As used herein, long-chain retinyl esters are defined as hydrocarbon esters of retinol and fatty acids, wherein the fatty acids contain at least about 8 carbon atoms, for example, 9, 10, 12, 13, 15, or 20 carbon atoms, and up to about 26 carbon atoms, for example, 25, 22, 21, or less, and preferably up to about 6 unsaturated bonds, for example, 0, 1, 2, 4, 5, or 6 unsaturated bonds. The fatty acid in the long-chain retinyl esters includes, but is not limited to, linoleic acid, oleic acid, or palmitic acid. The biosynthesis of retinoids is described, for example, in WO 2008042338 or WO 2019058000, which disclose the enzymatic conversion of beta-carotene to retinal, then to retinol, then to retinyl acetate in strains of Yarrowia lipolytica expressing the corresponding heterologous genes.

[0017] The terms "conversion" and "enzymatic conversion" in relation to enzymatic catalysis of GGPP and / or lycopene are used interchangeably herein and refer to the action of modified or unmodified CarRP used as a biocatalyst in the conversion of GGPP to phytoene or lycopene, as defined herein, and thence to beta-carotene, and thus including the synthase or cyclase activity of CarRP as described herein.

[0018] Suitable host cells according to the present invention include fungal host cells. As used herein, the term "fungal host cell" specifically includes GRAS host cells, particularly yeast cells, which are carotenoid and / or apocarotenoid producing host cells, particularly beta-carotene and / or retinol producing fungal host cells, including, but not limited to, Yarrowia or Saccharomyces, such as Yarrowia lipolytica or Saccharomyces cerevisiae.

[0019] The modified enzymes may be used in isolated form (e.g., in a cell-free system) or may be expressed in a suitable host cell, such as, for example, a carotenoid- and / or apocarotenoid-producing host cell, particularly a fungal host cell as defined herein. The enzymes may be expressed as endogenous enzymes or as heterologous enzymes. Preferably, the modified enzymes described herein are introduced and expressed as heterologous enzymes in a suitable host cell, such as, for example, a carotenoid- and / or apocarotenoid-producing host cell, preferably a carotene- and / or retinol-producing host cell, particularly a fungal host cell as defined herein.

[0020] In one embodiment, the modified CarRP enzyme defined herein comprises an amino acid substitution at a position corresponding to residue 7 in SEQ ID NO:1 or in the polypeptide shown in Figure 1, specifically the introduction of an aspartic acid (E7D), e.g., via substitution of glutamic acid, wherein the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO:1 or shown in Figure 1. Using such a modified enzyme comprising the above mutations in a fermentation process, wherein the modified enzyme is introduced and expressed under suitable conditions using a carotenoid or apocarotenoid producing host cell as identified herein, and wherein the formation of carotenoids may be increased by at least about 5-30%, e.g., 10, 15, 20, 25, 30, or more, and the formation of apocarotenoids, particularly retinoids, may be increased by at least about 5-67%, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or more, compared to a corresponding process using an unmodified McCarRP of SEQ ID NO: 1 or as shown in FIG. 1, which comprises McCarRP of SEQ ID NO: 1 or as shown in FIG. 1 but which contains a glutamine at position 7 in the polypeptide of SEQ ID NO: 1 or as shown in FIG. 1.

[0021] In one embodiment, the modified CarRP enzyme defined herein comprises an amino acid substitution at a position corresponding to residue 33 in SEQ ID NO:1 or in the polypeptide shown in Figure 1, specifically the introduction of an asparagine (A33N), e.g., via alanine substitution, wherein the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO:1 or shown in Figure 1. Using such a modified enzyme comprising the above mutations in a fermentation process, wherein the modified enzyme is introduced and expressed under suitable conditions using a carotenoid or apocarotenoid producing host cell as identified herein, and wherein the formation of carotenoids may be increased by at least about 5-30%, e.g., 10, 15, 20, 25, 30, or more, and the formation of apocarotenoids, particularly retinoids, may be increased by at least about 20-46%, e.g., 25, 30, 35, 40, 45, 50, or more, compared to a corresponding process using an unmodified McCarRP of SEQ ID NO: 1 or as shown in FIG. 1, which comprises McCarRP of SEQ ID NO: 1 or as shown in FIG. 1 but which comprises tryptophan at position 33 in the polypeptide of SEQ ID NO: 1 or as shown in FIG. 1.

[0022] In some embodiments, a modified CarRP enzyme defined herein comprises an amino acid substitution at a position corresponding to residue 153 in the polypeptide of SEQ ID NO: 1 or shown in FIG. 1, specifically the introduction of a serine (A153S), e.g., via an alanine substitution, where the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO: 1 or shown in FIG. 1. Using such a modified enzyme comprising such a mutation in a fermentation process, where the modified enzyme is introduced and expressed under suitable conditions using a carotenoid- or apocarotenoid-producing host cell identified herein, the formation of carotenoids may be increased by at least about 5-30%, e.g., 10, 15, 20, 25, 30, or more, and the formation of apocarotenoids, specifically retinoids, may be increased by at least about 20-43%, e.g., 25, 30, 35, 40, 45, or more, compared to a corresponding process using unmodified CarRP of SEQ ID NO: 1 or shown in FIG. 1.

[0023] In some embodiments, a modified CarRP enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 159 in the polypeptide of SEQ ID NO: 1 or shown in FIG. 1, specifically the introduction of a valine (L159V), e.g., via a substitution of a leucine, wherein the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO: 1 or shown in FIG. 1. Using such a modified enzyme comprising such a mutation in a fermentation process, wherein the modified enzyme is introduced and expressed under suitable conditions using a carotenoid- or apocarotenoid-producing host cell identified herein, the formation of carotenoids may be increased by at least about 5-30%, e.g., 10, 15, 20, 25, 30, or more, and the formation of apocarotenoids, specifically retinoids, may be increased by at least about 20-41%, e.g., 25, 30, 35, 40, 45, or more, compared to a corresponding process using unmodified CarRP of SEQ ID NO: 1 or shown in FIG. 1.

[0024] In one embodiment, the modified CarRP enzyme defined herein comprises an amino acid substitution at a position corresponding to residue 167 in the polypeptide of SEQ ID NO: 1 or shown in Figure 1, specifically the introduction of a phenylalanine (Y167F), e.g., via substitution of a tyrosine, where the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO: 1 or shown in Figure 1. Using such a modified enzyme comprising such a mutation in a fermentation process, where the modified enzyme is introduced and expressed under suitable conditions using a carotenoid or apocarotenoid-producing host cell identified herein, the formation of carotenoids may be increased by at least about 5-30%, e.g., 10, 15, 20, 25, 30, or more, and the formation of apocarotenoids, specifically retinoids, may be increased by at least about 20-24%, compared to a corresponding process using unmodified CarRP of SEQ ID NO: 1 or shown in Figure 1.

[0025] In one embodiment, the modified CarRP enzyme defined herein comprises an amino acid substitution at a position corresponding to residue 194 in the polypeptide of SEQ ID NO: 1 or shown in Figure 1, specifically the introduction of a leucine (I194L), e.g., via a substitution of an isoleucine, wherein the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO: 1 or shown in Figure 1. Using such a modified enzyme comprising such a mutation in a fermentation process, wherein the modified enzyme is introduced and expressed under suitable conditions using a carotenoid or apocarotenoid-producing host cell identified herein, the formation of carotenoids may be increased by at least about 5-30%, e.g., 10, 15, 20, 25, 30, or more, and the formation of apocarotenoids, specifically retinoids, may be increased by at least about 20-35%, e.g., 25, 30, 35, 40, or more, compared to a corresponding process using unmodified CarRP of SEQ ID NO: 1 or shown in Figure 1.

[0026] In one embodiment, the modified CarRP enzyme defined herein comprises an amino acid substitution at a position corresponding to residue 305 in the polypeptide of SEQ ID NO: 1 or shown in Figure 1, specifically the introduction of an alanine (T305A), e.g., via a threonine substitution, wherein the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO: 1 or shown in Figure 1. Using such a modified enzyme comprising the above mutation in a fermentation process, wherein the modified enzyme is introduced and expressed under suitable conditions using a carotenoid or apocarotenoid-producing host cell identified herein, the formation of carotenoids may be increased by at least about 5-30%, and the formation of apocarotenoids, specifically retinoids, may be increased by at least about 20-23%, compared to a corresponding process using unmodified CarRP of SEQ ID NO: 1 or shown in Figure 1.

[0027] In one embodiment, the modified CarRP enzyme defined herein comprises an amino acid substitution at a position corresponding to residue 330 in the polypeptide of SEQ ID NO: 1 or shown in FIG. 1, specifically the introduction of glutamic acid or asparagine, e.g., via substitution of aspartic acid (D330E or D330N), wherein the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO: 1 or shown in FIG. 1. Using such a modified enzyme comprising such a mutation in a fermentation process, wherein the modified enzyme is introduced and expressed under suitable conditions using a carotenoid- or apocarotenoid-producing host cell identified herein, the formation of carotenoids may be increased by at least about 5-30%, and the formation of apocarotenoids, specifically retinoids, may be increased by at least about 5-27%, e.g., 10, 15, 20, 25, 30, or more, compared to a corresponding process using unmodified CarRP of SEQ ID NO: 1 or shown in FIG. 1.

[0028] In one embodiment, a modified CarRP enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 430 in the polypeptide of SEQ ID NO: 1 or shown in FIG. 1, specifically the introduction of an alanine (S430A), e.g., via a serine substitution, wherein the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO: 1 or shown in FIG. 1. Using such a modified enzyme comprising the above mutation in a fermentation process, wherein the modified enzyme is introduced and expressed under suitable conditions using a carotenoid or apocarotenoid-producing host cell identified herein, the formation of carotenoids may be increased by at least about 5-30%, and the formation of apocarotenoids, specifically retinoids, may be increased by at least about 16-26%, e.g., 20, 25, 30, or more, compared to a corresponding process using unmodified CarRP of SEQ ID NO: 1 or shown in FIG. 1.

[0029] In one embodiment, a modified CarRP enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 431 in the polypeptide of SEQ ID NO: 1 or shown in Figure 1, specifically the introduction of a threonine (S431T), e.g., via a serine substitution, wherein the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO: 1 or shown in Figure 1. Using such a modified enzyme comprising the above mutation in a fermentation process, wherein the modified enzyme is introduced and expressed under suitable conditions using a carotenoid or apocarotenoid-producing host cell identified herein, the formation of carotenoids may be increased by at least about 5-30%, and the formation of apocarotenoids, specifically retinoids, may be increased by at least about 20-42%, e.g., 25, 30, 35, 40, 45%, or more, compared to a corresponding process using unmodified CarRP of SEQ ID NO: 1 or shown in Figure 1.

[0030] In one embodiment, a modified CarRP enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 432 in the polypeptide of SEQ ID NO: 1 or depicted in FIG. 1, specifically the introduction of an isoleucine (V432I), e.g., via a substitution of valine, where the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO: 1 or depicted in FIG. 1. Using such a modified enzyme comprising such a mutation in a fermentation process, where the modified enzyme is introduced and expressed under suitable conditions using a carotenoid or apocarotenoid-producing host cell identified herein, the formation of carotenoids may be increased by at least about 5-30%, and the formation of apocarotenoids, specifically retinoids, may be increased by at least about 20-65%, e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70%, or more, compared to a corresponding process using unmodified CarRP of SEQ ID NO: 1 or depicted in FIG. 1.

[0031] In one embodiment, a modified CarRP enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 476 in the polypeptide of SEQ ID NO: 1 or depicted in FIG. 1, specifically the introduction of an alanine (I476A), e.g., via a substitution of an isoleucine, wherein the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO: 1 or depicted in FIG. 1. Using such a modified enzyme comprising the above mutation in a fermentation process, wherein the modified enzyme is introduced and expressed under suitable conditions using a carotenoid- or apocarotenoid-producing host cell identified herein, the formation of carotenoids may be increased by at least about 5-30%, and the formation of apocarotenoids, specifically retinoids, may be increased by at least about 20-25%, compared to a corresponding process using unmodified CarRP of SEQ ID NO: 1 or depicted in FIG. 1.

[0032] In one embodiment, a modified CarRP enzyme as defined herein comprises an amino acid substitution at a position corresponding to residue 547 in the polypeptide of SEQ ID NO: 1 or shown in FIG. 1, specifically the introduction of an isoleucine (V547I), e.g., via a substitution of valine, where the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO: 1 or shown in FIG. 1. Using such a modified enzyme comprising such a mutation in a fermentation process, where the modified enzyme is introduced and expressed under suitable conditions using a carotenoid or apocarotenoid-producing host cell identified herein, the formation of carotenoids may be increased by at least about 5-30%, and the formation of apocarotenoids, specifically retinoids, may be increased by at least about 20-35%, e.g., 25, 30, 35, 40%, or more, compared to a corresponding process using unmodified CarRP of SEQ ID NO: 1 or shown in FIG. 1.

[0033] In one embodiment, the modified CarRP enzyme defined herein comprises an amino acid substitution at a position corresponding to residue 579 in the polypeptide of SEQ ID NO: 1 or shown in Figure 1, specifically the introduction of a lysine (R579K), e.g., via substitution of an arginine, wherein the modified enzyme is derived from an enzyme with at least about 20% identity to McCarRP of SEQ ID NO: 1 or shown in Figure 1. Using such a modified enzyme comprising the above mutation in a fermentation process, wherein the modified enzyme is introduced and expressed under suitable conditions using a carotenoid or apocarotenoid-producing host cell identified herein, the formation of carotenoids may be increased by at least about 5-30%, and the formation of apocarotenoids, specifically retinoids, may be increased by at least about 20-38%, e.g., 25, 30, 35, 40%, or more, compared to a corresponding process using unmodified CarRP of SEQ ID NO: 1 or shown in Figure 1.

[0034] Thus, in some embodiments, the modified enzyme comprises two or more mutations, i.e., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, etc., amino acid substitutions, selected from at least 2 to 14 mutations at positions corresponding to positions 7, 33, 153, 159, 167, 194, 305, 330, 430, 431, 432, 476, 547, or 579 in SEQ ID NO: 1 or in the polypeptide depicted in FIG. 1, wherein the at least 2 to 14 amino acids introduced at positions corresponding to the loci in SEQ ID NO: 1 or in the polypeptide depicted in FIG. 1 are different from E7, Q7, A33, W33, A153, L159, Y167, 1194, T305, D330, S430, S431, V432, 1476, V547, R579, in any combination.

[0035] In a preferred embodiment, the present invention relates to a modified CarRP derived from the McCarRP set forth in SEQ ID NO: 1 or shown in Figure 1, and to methods for producing such modified CarRPs described herein, wherein the modified CarRP comprises 1 to 14 amino acid substitutions selected from the group consisting of E7D, A33N, A135S, L159V, Y167F, I194L, T305A, D330E or D330N, S430A, S431T, V432I, I476A, V547I, R579K, and combinations thereof. Upon expression of such a modifying enzyme in a suitable retinoid-producing host cell, the percentage of retinoid can be increased by 5-75%, such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80% or more, compared to a process in which the retinoid-producing host cell expresses CarRP of SEQ ID NO: 1 or shown in Figure 1. As used herein, the term "at least two mutations" means that the modifying enzyme described herein contains two or more mutations, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 mutations, at locations corresponding to the specified positions in SEQ ID NO: 1 or in the polypeptide shown in Figure 1. Specifically, the modified CarRP enzymes described above are expressed in apocarotenoid-, e.g., retinoid-, and / or carotenoid-producing host cells, preferably those selected from Yarrowia, Saccharomyces, or Escherichia, and most preferably in Yarrowia lipolytica, which produces carotenoids and / or apocarotenoids, e.g., retinoids, known in the art. Most preferably, the modified CarRPs defined herein are codon-optimized for expression in the respective host cells.

[0036] Specifically, the present invention relates to modified CarRPs derived from McCarRP as set forth in SEQ ID NO: 1 or as set forth in FIG. 1, including proteins having at least about 20% identity, and methods for producing such modified CarRPs described herein, wherein the modified CarRPs include E7D, A33N, A135S, L159V, Y167F, I194L, T305A, D330E or D330N, S430A, S431T, V432I, I476A, V547I, and and R579K, and any combination thereof, and more specifically, a combination of E7D with A33N, Y167F, V547I, and R579K, or a combination of E7D with A33N, Y167F, D330N, and V432I, or a combination of E7D with D330N, S430A, V547I, and R579K, or a combination of E7D with A33N, Y167F, V547I, and R579K, or a combination of E7D with A33N, Y167F, V547I, and R579K. , Y167F, T305A, and R576K, or a combination of E7D and A33N, Y167F, S431T, and R579K, or a combination of E7D, A33N, Y167F, S430A, and R579K, or a combination of E7D and A33N, Y167F, S430A, and V547I, or a combination of E7D and A33N, Y167F, T305A, and S430A, or a combination of E7D and A33N, S430A, V547I, and R5 and R579K, or a combination of E7D with A33N, Y167F, T305A, and D33N, or a combination of A33N with Y167F, S430A, V547I, and R579K, or a combination of E7D with Y167F, S430A, V547I, and R579K, or a combination of E7D with Y167F, S431T, V547I, and R579K, or a combination of E7D with A33N, Y167F, V431I, and V547I.

[0037] In some preferred embodiments, the present invention relates to modified CarRPs derived from McCarRP as set forth in SEQ ID NO: 1 or as set forth in FIG. 1, including proteins having at least about 20% identity thereto, and methods for producing such modified CarRPs as described herein, wherein the modified CarRPs comprise at least one mutation, e.g., V432I, or a combination of at least five mutations, including a combination of E7D with A33N or Y167F, S430I, V547I, and R579K; and through the introduction of such amino acid substitutions and expression of a suitable apocarotenoid, e.g., retinoid, and / or the expression of such modifying enzymes in a carotenoid-producing host cell, the percentage of retinoids, for example, can be increased by about 60-70% and greater compared to a host cell expressing an unmodified CarRP as defined herein, e.g., McCarRP as set forth in SEQ ID NO: 1 or as set forth in FIG. 1.

[0038] According to all embodiments of the present invention, modifications of amino acids corresponding to the specified positions in SEQ ID NO: 1 or in the polypeptide shown in Figure 1, such as amino acid substitutions at positions corresponding to residues 7, 33, 153, 159, 167, 194, 305, 330, 430, 431, 432, 476, 547 or 579 in SEQ ID NO: 1 or in the polypeptide shown in Figure 1 (wherein one or more of the above amino acids are modified, for example in particular where at least 1, 2, 3, 4, 5 amino acids are modified), when used in the respective host cell, i.e., carotenoid- and / or apocarotenoid-producing host cell as defined herein, results in increased formation of carotenoids and / or apocarotenoids, preferably retinoids, wherein the carotenoid or retinoid titer may be increased by at least about 5% compared to the respective host cell in which the particular amino acid is not substituted in a manner defined herein, i.e., compared to a host cell expressing the polypeptide of SEQ ID NO: 1 or shown in Figure 1. Specifically, apocarotenoid, and particularly retinoid, titers may be increased by about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75%, or more, with an increase of at least about 40-70% in retinoids relative to total retinoids being achieved by single mutations at positions corresponding to V432I, A33N, L159V, A153S in SEQ ID NO: 1 or the polypeptide shown in Figure 1, and an increase of at least about 25-75%, e.g., 40-75%, and more, may be achieved using a modified CarRP comprising a quintuple mutation as defined herein, e.g., including one or more of the amino acid substitutions corresponding to E7D, A33N, S430A, V547I, R579K, Y167F in SEQ ID NO: 1 or the polypeptide shown in Figure 1, compared to a corresponding host cell expressing the polypeptide of SEQ ID NO: 1 or the polypeptide shown in Figure 1.Specifically, the titer of certain carotenoids, such as zeaxanthin, canthaxanthin, astaxanthin, rhodoxanthin, beta-cryptoxanthin, etc., may be increased by about 5, 10, 15, 20, 25, 30, 35, 40%, or more, with an increase of at least about 5-35% in total carotenoids being achieved by single mutations at positions corresponding to V432I, V547I, S431T, D330E, and T305 in SEQ ID NO: 1 or the polypeptide shown in Figure 1, and an increase of at least about 5-40% in canthaxanthin, based on total carotenoids, being achieved by single mutations at positions corresponding to V432I, V547I, S431T, D330E, and T305 in SEQ ID NO: 1 or the polypeptide shown in Figure 1, compared to a corresponding host cell expressing the polypeptide of SEQ ID NO: 1 or the polypeptide shown in Figure 1.

[0039] The modified host cells defined herein comprise one or more copies of the modified enzymes defined herein, and preferably the modified enzymes are heterologously expressed in the modified host cells. Modifications to cause the host cells defined herein to produce more copies of a gene and / or protein, e.g., more copies of modified CarRP, may include the use of a strong promoter, suitable transcriptional and / or translational enhancers, or the introduction of one or more gene copies into the carotenoid and / or apocarotenoid-producing host cells, particularly fungal host cells, resulting in increased accumulation of the respective enzymes within a given time period. Those skilled in the art will know which techniques to use depending on the host cell. Increased and decreased gene expression can be measured by various methods, such as, for example, Northern, Southern, or Western blot techniques known in the art.

[0040] The generation of mutations in nucleic acids or amino acids, i.e., mutagenesis, can be carried out in a variety of ways, for example, by random or site-directed mutagenesis, physical damage caused by factors such as radiation, chemical treatment, or insertion of genetic elements. Methods for introducing mutations are known to those skilled in the art.

[0041] Thus, the present invention relates to the carotenoid- and / or apocarotenoid-producing host cells described herein, as well as processes for producing such host cells, particularly fungal host cells, which contain an expression vector or polynucleotide encoding a modified CarRP described herein integrated into the chromosomal DNA of the host cell (see also WO2009126890, particularly Example 1A). Such modified host cells, particularly fungal host cells, containing either a heterologous polynucleotide on an expression vector or a heterologous polynucleotide integrated into chromosomal DNA encoding a modified CarRP described herein are referred to as recombinant or modified host cells. Carotenoid- and / or apocarotenoid-producing host cells, particularly fungal host cells, can contain one or more copies of a gene encoding a modified CarRP defined herein, comprising a mutation as defined herein that results in overexpression of such a gene encoding the modified CarRP defined herein. Increased gene expression can be measured by various methods known in the art, such as, for example, Northern, Southern, or Western blot techniques, transcriptomics, genome sequencing, or proteomics.

[0042] The present invention particularly relates to the use of these novel modified CarRPs in processes for the production of carotenoids and / or apocarotenoids, particularly retinoids, including processes in which the retinoids comprise a mixture of retinol, retinal, and retinyl acetate, particularly a percentage of at least about 40% by weight based on the total retinoids being retinyl acetate, particularly reducing the formation of long-chain retinyl esters. Those skilled in the art know how to create such conditions (see, for example, WO2021136689 or WO2022090548).

[0043] The terms "sequence identity" and "% identity" are used interchangeably herein. For purposes of the present invention, to determine the percentage of sequence identity between two amino acid sequences or two nucleic acid sequences, it is herein defined that the sequences are aligned for optimal comparison. Gaps may be introduced into either of the two sequences being compared to optimize the alignment between the two sequences. Such alignments can be performed over the entire length of the sequences being compared. Alternatively, alignments can be performed over shorter lengths, for example, over about 20, about 50, about 100, or more nucleic acids / bases or amino acids. Sequence identity is the percentage of perfect matches between the two sequences over the reported alignment region. The percent sequence identity between two amino acid sequences or two nucleotide sequences can be determined using the Needleman-Wunsch algorithm for aligning the two sequences (Needleman, S.B. and Wunsch, C.D. (1970) J. Mol. Biol. 48, 443-453). This algorithm can align both amino acid and nucleotide sequences. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE. For the purposes of the present invention, the NEEDLE program from the EMBOSS package was used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, Longden and Bleasby, Trends in Genetics 16, (6) pp276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 is used for the substitution matrix. For nucleotide sequences, EDNAFULL is used. Optional parameters used are a gap opening penalty of 10 and a gap extension penalty of 0.5.Those skilled in the art will understand that all of these different parameters will produce slightly different results, but the overall percentage identity of the two sequences will not change significantly when using different algorithms.

[0044] After alignment by the program NEEDLE described above, the percentage of sequence identity between a query sequence and a sequence of the invention is calculated as follows: [the number of corresponding positions in the alignment that show identical amino acids or identical nucleotides in both sequences] divided by [the total length of the alignment after subtracting the total number of gaps in the alignment]. Identity, as defined herein, can be obtained from NEEDLE by using the NOBRIEF option and is labeled "longest identity" in the program's output. If both amino acid sequences being compared do not differ in any of their amino acids, they are identical, or have 100% identity.

[0045] The modified enzymes defined herein may contain further amino acid substitutions that do not alter the enzymatic activity, i.e., they exhibit the same properties as phytoene synthase and / or lycopene beta-cyclase for the enzymes defined herein and catalyze the conversion of GGPP to phytoene and / or lycopene to beta-carotene to the same extent as modified enzymes having only one or more of the amino acid substitutions described herein. Such mutations are also called "silent mutations" that do not alter the (enzymatic) activity of the enzymes according to the invention.

[0046] Expression of an enzyme / polynucleotide encoding one of the modified CarRPs defined herein can be carried out in any host system, including a (micro)organism, that is suitable for the production of carotenoids and / or apocarotenoids and that allows the expression of a nucleic acid encoding one of the modified enzymes disclosed herein, including functional equivalents or derivatives as described herein. Examples of suitable carotenoid / apocarotenoid-producing host (micro)organisms are bacteria, algae, fungi such as yeast, plant or animal cells. Preferred bacteria are those of the genus Escherichia, e.g., Escherichia coli. coli), Streptomyces, Pantoea (Erwinia), Bacillus, Flavobacterium, Synechococcus, Lactobacillus, Corynebacterium, Micrococcus, Myxococcus coccus, Brevibacterium, Bradyrhizobium, Gordonia, Dietzia, Muricauda, ​​Sphingomonas, Synochocystis, Paracoccus, e.g., Paracoccus zeaxanthinifaciens.Preferred eukaryotic microorganisms, particularly fungi such as yeasts, include Saccharomyces such as Saccharomyces cerevisiae, Aspergillus such as Aspergillus niger, Pichia such as Pichia pastoris, Hansenula such as Hansenula polymorpha, Kluyveromyces such as Kluyveromyces lactis, and Phycomyces brackenhausenus. The host cell is selected from Phycomyces, such as Blakeslea blakesleanus, Mucor, Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea, such as Blakeslea trispora, or Yarrowia, such as Yarrowia lipolytica. Expression in fungal host cells, such as Yarrowia or Saccharomyces, or in Escherichia, more preferably Yarrowia lipolytica or Saccharomyces cerevisiae, is particularly preferred.

[0047] In the context of the present invention, organisms such as microorganisms, fungi, algae, or plants are also understood to include synonyms or basonyms of such species having the same physiological properties as defined by the International Code of Nomenclature of Prokaryotes or the International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). Thus, for example, the Lachancea mirantina strain is a synonym of the Zygosaccharomyces sp. strain IFO 11066 originating from Japan.

[0048] In some embodiments, the present invention relates to the production of retinoids, including but not limited to, the production of retinal, retinol, and retinyl acetate, as described in, for example, WO2019058001, wherein the percentage of retinyl acetate is at least about 40-80% by weight based on total retinoids, using host cells expressing modified CarRP enzymes described herein, whereby retinoid production is increased by at least about 5%, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75%, and more, compared to a process using the corresponding unmodified CarRP enzyme of SEQ ID NO: 1 or shown in FIG. 1. The produced retinyl acetate can be isolated and, optionally, further purified from the medium and / or host cells. The above acetylated retinoids defined herein can be used as building blocks to provide vitamin A.

[0049] The modified host cells defined herein can be cultured under aerobic or anaerobic conditions in aqueous media supplemented with appropriate nutrients, as known to those skilled in the art for the respective carotenoid and / or apocarotenoid-producing host cells. Optionally, such cultures are in the presence of proteins and / or cofactors involved in electron transfer known in the art. Carbon sources suitable for the purposes of the present invention can be selected from glucose, fructose, raffinose, lactose, galactose, glycerol, xylose, arabinose, sucrose, maltose, vegetable oils, including but not limited to, oleic acid or linoleic acid, with or without ethanol, and specifically from glucose, galactose, or xylose. Specific culture conditions for producing apocarotenoids, specifically retinoids, can include batch and feed run, with the batch phase having 5% (w / v) glucose and 1% ethanol (w / v) and the feed phase having 100% (w / v). The host cells can be cultured / grown in batch, fed-batch, semi-continuous, or continuous mode, specifically fed-batch, for 80, 90, 100, 110, 120, or 130 hours under appropriate culture conditions. As known to those skilled in the art, conditions can be adapted depending on the host cell and the carotenoids and / or apocarotenoids to be produced. For example, the culture and isolation of carotenoid- and / or apocarotenoid-producing host cells, such as those selected from Yarrowia and Saccharomyces, are described, for example, in WO 2008042338. For the production of beta-carotene and retinoids in host cells selected from E. coli, methods are described, for example, in U.S. Patent Application Publication No. 20070166782.

[0050] In some embodiments, apocarotenoid-producing host cells expressing modified CarRPs as defined herein are cultured in a two-phase system, and apocarotenoids, particularly retinoids including, but not limited to, retinol and / or retinyl acetate (preferably at least about 40-80% retinyl acetate based on total retinoids), are harvested in a suitable lipophilic phase and subsequently isolated therefrom. Specific conditions and lipophilic solvents are disclosed in WO2022090548 or WO2022090549.

[0051] In some embodiments, the present invention relates to two-phase fermentation with retinoid-producing strains expressing modified CarRPs as defined herein, using lipophilic solvents such as isopar or corn oil as the second phase in addition to known solvents including Drakeol®, silicone, or n-dodecane (see Jang et al., Microbial Cell Factories 10:59, 2011).

[0052] As used herein, the term "specific activity" or "activity" in reference to an enzyme refers to its catalytic activity, i.e., its ability to catalyze the formation of a product from a given substrate. Specific activity defines the amount of substrate consumed and / or product produced per defined amount of protein at a defined temperature in a given time. Specific activity is usually expressed as μmol of substrate consumed or product formed per mg of protein per minute. μmol / min is usually abbreviated as U (=unit). Thus, the unit definitions of specific activity μmol / min / (mg of protein) or U / (mg of protein) are used interchangeably throughout this document. An enzyme is active if it performs its catalytic activity in vivo, i.e., in a host cell as defined herein, or in an appropriate (cell-free) system in the presence of a suitable substrate. Those skilled in the art know how to measure enzyme activity. Analytical methods for assessing the potency of suitable bifunctional CarRPs as defined herein are known in the art, for example, as described in Ma et al. (Nature Communications, 2022, 13:572, https: / / doi.org / 10.1038 / s41467-022-28277-w). For example, the titer of apocarotenoids or carotenoid-containing products, such as retinyl acetate, retinol, trans-retinal, cis-retinal, beta-carotene, canthaxanthin, zeaxanthin, astaxanthin, rhodoxanthin, lycopene, phytoene, beta-ionone, etc., can be measured by HPLC.

[0053] The general construction of carotenoid producing host cells, particularly beta-carotene producing host cells, is known in the art, for example as described in WO2006102342.

[0054] As used herein, a "retinoid-producing host cell" refers to a particular apocarotenoid-producing host cell in which the respective polypeptides are expressed and active in vivo, resulting in the production of retinoids as defined herein, including, for example, retinal, retinol, and / or retinyl acetate, via the enzymatic conversion of beta-carotene to retinol via retinal, and optionally further to retinyl acetate. These polypeptides include enzymes that catalyze the conversion of beta-carotene to retinal (see, e.g., WO2019057999), enzymes that catalyze the conversion of retinal to retinol (see, e.g., WO2019057998), and optionally enzymes that catalyze the conversion of retinol to retinyl acetate (see, e.g., WO2019058001).

[0055] "Vitamin A," as used herein, can refer to any chemical form of vitamin A found in aqueous solutions, solids, and formulations, including retinol, retinyl acetate, and retinyl esters. Also included is retinoic acid, either undissociated in the free acid form or dissociated as an anion.

[0056] As used herein, "retinal" is known by its IUPAC name (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)-nona-2,4,6,8-tetraenal and includes both cis- and trans-isoforms, e.g., 11-cis retinal, 13-cis retinal, trans-retinal, and all-trans retinal. [Brief explanation of the drawings]

[0057] [Figure 1]1 is the amino acid sequence of M. circinelloides CarRP (McCarRP; SEQ ID NO: 1), where amino acid residues selected for amino acid substitutions described in this application are marked in bold / underlined, and amino acids substituting the original amino acids shown in SEQ ID NO: 1 are marked in italics and are shown above the respective original amino acids.

[0058] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way. The contents of all references, patent applications, patents, and published patent applications cited throughout this application are hereby incorporated by reference (specifically, International Publication Nos. WO 2014096992, WO 2019058001, WO 2021136689, WO 2022090548, WO 2008042338, U.S. Patent Application Publication No. 20070166782, WO 2022090549, WO 2006102342, WO 2016172282, WO 2019058000, WO 2019057999, WO 2019057998, and U.S. Patent Application Publication No. 20180148697).

[0059] [Example] Example 1: General methods, strains, and plasmids All basic molecular biology and DNA manipulation procedures described herein are generally performed according to Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press: New York (1989), or Ausubel et al. (eds). Current Protocols in Molecular Biology. Wiley: New York (1998).

[0060] Strains, Plasmids, and Sequences: The strains used as host cells and the respective plasmids used to express CarRP, as well as other enzymes or constructs identified below, are listed in Tables 1, 2, and 4 and in the Sequence Listing. Figure 1 shows the wild-type CarRP (McCarRP) amino acid sequence (SEQ ID NO: 1), highlighting the residues selected for modification as defined herein.

[0061] [Table 1]

[0062] [Table 2]

[0063] [UPLC Reversed-Phase Method] For rapid screening, this method does not separate cis isomers, but only the major functional groups. Samples were injected using a Waters Acquity UPLC with PDA detection (or similar) with an autosampler. Retinoids and / or carotenoids were separated using an Acquity UPLC HSS T3 1.8µm column, P / N 186003539. The mobile phase consisted of either 1000mL of hexane, 30mL of isopropanol, or 0.1mL of acetic acid in the case of retinoid-related compounds, including carotenoids. The flow rate for each was 0.6mL / min. The column temperature was 20°C. The injection volume was 5µL. The detector was a photodiode array detector collecting light from 210 to 600nm. Analytes were detected as shown in Table 3. Rhodoxanthin, astaxanthin, zeaxanthin, and ionones can be quantified as described in the following references: Royer et al., (Sci. Adv. 2020; Vol. 6, Issue 17), WO 2014096992, and U.S. Patent Application Publication No. 20180148697, respectively.

[0064] [Table 3]

[0065] [Table 4]

[0066] Calibration Method: The method is calibrated for the carotenoid retinyl acetate, and retinol and retinal are quantified relative to retinyl acetate using the specified response factors. Using a volumetric flask, dissolve retinyl acetate in THF at approximately 200 μg / ml for the stock solution. Using volumetric flasks, make 20x, 50x, and 100x dilutions of the stock solution with 50 / 50 methanol / MTBE. The UV absorbance of retinyl acetate becomes nonlinear fairly quickly, so care must be taken to stay within the linear range. Consequently, lower concentrations may be better. Retinyl palmitate can also be used as a retinyl ester calibration. Retinyl acetate peaks at approximately 3 minutes and retinyl esters (long-chain retinyl esters) peak at approximately 3.5 minutes. For measurements of other apocarotenoids or carotenoids, this can be adapted accordingly.

[0067] Sample preparation: Samples were prepared in various ways depending on the conditions. For whole broth or washed broth samples, the broth was placed in a Precellys® tube, weighed, and mobile phase was added. Briefly, a 2 ml Precellys® tube was charged with 25 μl of thoroughly mixed broth and 975 μl of THF. The sample was then processed in a Precellys® homogenizer (Bertin Corp, Rockville, MD, USA) according to the manufacturer's instructions at the highest setting three times, typically 3 × 15 × 7500 tpms. For washed pellets, the sample was spun in a 1.7 ml tube in a microcentrifuge at 10,000 rpm for 1 minute, the broth decanted, 1 ml of water added, mixed, pelleted, decanted, and returned to the original volume. The mixture was pelleted again, placed back in the appropriate amount of mobile phase, and processed by Precellys® bead beating. For analysis of silicone oil fractions, samples were spun at 4000 RPM for 10 minutes, the oil was decanted and the top portion removed with a positive displacement pipette (Eppendorf, Hauppauge, NY, USA), diluted in mobile phase mixed by vortexing, and the concentration of retinoid (or other compound of interest) determined by UPLC analysis.

[0068] Example 2: Retinoid production in Yarrowia lipolytica expressing mutant carRP To evaluate the carRP allele, strain ML18743 was transformed with plasmid MB10866 (SEQ ID NO: 4), which encodes a synthetic guide RNA (sgRNA) and SpCas9 protein to direct mutagenesis in the carRP sequence present in the genome. Strain ML19637, a carRP mutant, was identified by its white color. Strain ML19637 was passaged on nonselective medium, and hygromycin-sensitive isolates were identified. One of these isolates was further mutagenized with plasmid MB9282 (SEQ ID NO: 5), which encodes a sgRNA and SpCas9 protein to direct mutagenesis at the Ku70 locus, to generate ML19836. The uracil auxotroph, ML19836-ura, was isolated from ML19836 by selection on 5-fluoroorotic acid (5-FOA)-containing medium. Strain ML19836-ura was transformed with SfiI-linearized DNA from plasmid MB10157 (SEQ ID NO: 3) expressing wild-type McCarRP and mutant derivatives MB10157-1 through MB10157-29, whose polypeptide sequences are shown in Tables 4A and 4B and highlighted in bold / underline in Figure 1, and selected for uracil prototrophy.

[0069] Transformants were grown in shaker plates as described elsewhere (see, e.g., WO2022090549). Typically, 200 μl of 0.075% yeast extract, 0.25% peptone (0.25X YP) was inoculated with 10 μl of freshly grown Yarrowia and overlaid with 200 μl of Drakeol 5 (Penreco, Karns City, PA, USA) mineral oil, silicone oil, or corn oil (containing 2% oleic acid or 2% glucose) as the carbon source. Clonal isolates of transformants were grown in 24-well plates (Multitron, 30°C, 800 RPM) in YPD medium containing one of the above overlays for 4 days. Overlay fractions were removed from the shaker plate wells and analyzed by HPLC on a normal-phase column using a photodiode array detector. Retinoid production was measured (Table 4) and the percentage of retinoids using SEQ ID NO: 1 or the polynucleotide expressing reference McCarRP in Figure 1 expressed on plasmid MB10157 (total retinoid titer from the shake plate assay described above) was set to 100%.

[0070] [Table 5]

[0071] [Table 6]

[0072] Example 3: Production of various carotenoids or apocarotenoids in Yarrowia lipolytica expressing mutant carRP. Strains expressing mutant CarRP and specific genes for producing canthaxanthin, zeaxanthin, beta-cryptoxanthin, rhodoxanthin, or beta-ionone are constructed as follows: The beta-carotene producing strain ML15710 is transformed with the CRE-recombinase-containing plasmid MB6128 (SEQ ID NO: 6) and selected on geneticin-containing medium. Hygromycin-sensitive isolates are identified among the transformants by replica plating onto selective and non-selective media. One hygromycin-sensitive isolate is further grown on non-selective media, and then geneticin-sensitive isolates are identified by replica plating onto selective and non-selective media. One such isolate is transformed with one of the following PvuII-linearized plasmids: MB7076 (for canthaxanthin production; SEQ ID NO: 7), MB7190 (for zeaxanthin production; SEQ ID NO: 8), MB9931 (for beta-cryptoxanthin production; SEQ ID NO: 9), MB7918 (for rhodoxanthin production; SEQ ID NO: 10), or MB6806 (for beta-ionone production; SEQ ID NO: 11) and selected on hygromycin-containing medium to generate the canthaxanthin-producing strain ML15710+crtW, the zeaxanthin-producing strain ML15710+crtZ, the beta-cryptoxanthin-producing strain ML15710+Lfreq-CrtZ, the rhodoxanthin-producing strain ML15710+bhy-21, or the beta-ionone-producing strain ML15710+CCD1. These strains are transformed with the CRE-recombinase-containing plasmid MB6128 and selected on geneticin-containing medium. Hygromycin-sensitive isolates are identified among the transformants by replica plating onto selective and non-selective media. One hygromycin-sensitive isolate (for all carotenoids / apocarotenoids specified above) is further grown on non-selective media, and then geneticin-sensitive isolates are identified by replica plating onto selective and non-selective media.These strains were then transformed with MB10866 as described in Example 1 to generate the white carRP mutant strains ML15710+crtW+carRP- (for canthaxanthin production), ML15710+crtZ+carRP- (for zeaxanthin production), ML15710+Lfreq-CrtZ+carRP- (for beta-cryptoxanthin production), ML15710+bhy-21+carRP- (for rhodoxanthin production), or ML15710+CCD1+carRP- (for beta-ionone production). Hygromycin-sensitive isolates were identified by passage on nonselective medium and replica plating onto selective and nonselective media. These strains were then transformed with plasmid MB9282 as described in Example 1 to generate ku70. - Mutant strain ML15710+crtW+carRP - +ku70 - (for canthaxanthin production), ML15710+crtZ+carRP - +ku70 - (for zeaxanthin production), ML15710+Lfreq-CrtZ+carRP - +ku70 - (for beta-cryptoxanthin production), ML15710+bhy-21+carRP - +ku70 - (for rhodoxanthin production), or ML15710+CCD1+carRP - +ku70 - Strains auxotrophic for uracil were isolated from these strains by selection on 5-FOA, and these were designated ML15710+xx+carRP (for beta-ionone production). - +ku70 - +ura3 -where "xx" represents the respective carotenoid / apocarotenoid-specific gene. This white uracil auxotrophic strain is then transformed with plasmid MB10157 expressing wild-type McCarRP as well as with the respective plasmids expressing mutant forms of McCarRP listed in Table 4 (see Example 2) to generate canthaxanthin-, zeaxanthin-, beta-cryptoxanthin-, rhodoxanthin-, or beta-ionone-producing strains that also express heterologous CarRP (wt or mutant). Strains are grown in microtiter plates or fermentation as described in more detail below for canthaxanthin-producing strains, but are applicable mutatis mutandis for the production of other carotenoids, such as zeaxanthin, beta-cryptoxanthin, rhodoxanthin, or beta-ionone.

[0073] Strain ML15710+crtW+CarRP containing plasmid MB101570 or the plasmids shown in Table 5 - +ku70 - +ura3 - The resulting transformants were grown in microtiter plates as described in WO2022090549 or above in Example 2, except for the use of Phase 2 and glucose as the sole carbon source. Fermentation and carotenoid analysis were performed according to previously described methods (see, e.g., Examples 2-4 of U.S. Pat. No. 7,851,199). Introduction of plasmids MB101570-7, MB10157-11, and MB10157-14 resulted in an increase in both the percentage of total carotenoids (total carotenoid titer) measured by the shake plate assay described above, and the percentage of total carotenoid yield and canthaxanthin yield, i.e., yield relative to carbon (g total carotenoids / g carbon source and g canthaxanthin / g carbon source, respectively), measured by fermentation, where the percentages using SEQ ID NO: 1 expressed on plasmid MB10157 or a polynucleotide expressing reference McCarRP in Figure 1 are set to 100%.

[0074] [Table 7]

[0075] Example 4: Astaxanthin production in Yarrowia lipolytica expressing mutant carRP The beta-carotene-producing strain ML15710 is transformed with the CRE-recombinase-containing plasmid MB6128 and selected on geneticin-containing medium. Hygromycin-sensitive isolates are identified among the transformants by replica plating on selective and non-selective media. One hygromycin-sensitive isolate is further grown on non-selective media, and then geneticin-sensitive isolates are identified by replica plating on selective and non-selective media. One such isolate is transformed with PvuII-linearized plasmid MB7082 (SEQ ID NO: 12) and selected on nourseothricin-containing media to generate the astaxanthin-producing strain ML15710+crtW. This strain is then transformed with PvuII-linearized MB9930 (SEQ ID NO: 13) and selected on hygromycin medium to generate ML15710+crtW+crtZ. This strain is transformed with the CRE-recombinase-containing plasmid MB6128 and selected on geneticin-containing medium. Hygromycin- and nourseothricin-sensitive isolates are identified among the transformants by replica plating onto selective and nonselective media. One hygromycin- and nourseothricin-sensitive isolate is further grown on nonselective media, and then a geneticin-sensitive isolate is identified by replica plating onto selective and nonselective media. This strain is then transformed with MB10866 as described in Example 1 to produce the white carRP mutant ML15710+crtW+crtZ+carRP. - Hygromycin-sensitive isolates are identified by passage on non-selective media and replica plating onto selective and non-selective media. This strain is then transformed with MB9282 as in Example 1 to produce ku70.- Mutant strain ML15710+crtW+crtZ+carRP - +ku70 - A uracil auxotrophic strain was isolated from this strain by selection on 5-FOA, which is ML15710+crtW+crtZ+carRP. - +ku70 - +ura3 - This white uracil auxotrophic strain is then transformed with a plasmid expressing a heterologous wt CarRP or mutant listed in Table 4 (Example 2) to generate an astaxanthin-producing strain that also expresses a mutant CarRP. Compared to an astaxanthin-producing strain expressing the wt McCarRP of SEQ ID NO: 1 or FIG. 1, the astaxanthin titer can be increased by at least about 5% in the strain expressing the CarRP mutant (not shown).

[0076] Example 5: Lycopene production in Yarrowia lipolytica expressing mutant carRP CarRP mutants expressing the DNA listed in Table 2 were combined with the mutation E78G using a DNA synthesis provider (Genscript). This E78G mutation inactivates the lycopene cyclase domain of CarRP (see, e.g., WO2014151748). The beta-carotene-producing strain ML15710 was transformed with the CRE-recombinase-containing plasmid MB6128 and selected on geneticin-containing medium. Hygromycin-sensitive isolates were identified among the transformants by replica plating on selective and non-selective media. One hygromycin-sensitive isolate was further grown on non-selective media, and then geneticin-sensitive isolates were identified by replica plating on selective and non-selective media. One such isolate was transformed with MB10866 as described in Example 1 to generate the white carRP mutant strain ML15710+carRP. -Hygromycin-sensitive isolates are identified by passage on non-selective media and replica plating onto selective and non-selective media. This strain is then transformed with MB9282 as in Example 1 to produce ku70. - Mutant strain ML15710+carRP - +ku70 - A uracil auxotrophic strain was isolated from this strain by selection on 5-FOA, which was then transformed into ML15710+carRP. - +ku70 - +ura3 - This strain is called McCarRP. This strain is transformed with SfiI-linearized DNA containing the mutations in Table 2 in combination with the E78G mutation to generate a lycopene-producing strain that also expresses a mutant CarRP. This strain is then transformed with a plasmid expressing a heterologous wt CarRP or mutant listed in Table 4 (Example 2) in combination with the E78G mutation to generate a lycopene-producing strain that also expresses a mutant CarRP. Compared to a lycopene-producing strain expressing the wt McCarRP of SEQ ID NO: 1 or FIG. 1, lycopene titers can be increased by at least about 5% in strains expressing the CarRP mutants (not shown).

[0077] Example 7: Phytoene production in Yarrowia lipolytica expressing mutant carRP The beta-carotene-producing strain ML15710 is transformed with the CRE-recombinase-containing plasmid MB6128 and selected on geneticin-containing medium. Hygromycin-sensitive isolates are identified among the transformants by replica plating on selective and non-selective media. One hygromycin-sensitive isolate is further grown on non-selective media, and then a geneticin-sensitive isolate is identified by replica plating on selective and non-selective media. One such isolate is then transformed with MB10866 as described in Example 1 to produce the white carRP mutant strain ML15710+carRP. -Hygromycin-sensitive isolates are identified by passage on non-selective medium and replica plating onto selective and non-selective media. One such isolate is then transformed with MB7522 (SEQ ID NO: 14) to yield the carB mutant strain ML15710+carRP. - +carB - Hygromycin-sensitive isolates are identified by passage on non-selective media and replica plating onto selective and non-selective media. This strain is then transformed with MB9282 as in Example 1 to produce ku70. - Mutant strain ML15710+carRP - +carB - +ku70 - A uracil auxotrophic strain was isolated from this strain by selection on 5-FOA, which was then transformed into ML15710+carRP. - +carB - +ku70 - +ura3 - This white uracil auxotrophic strain is then transformed with a plasmid expressing a heterologous wt CarRP or mutant listed in Table 4 (Example 2) to generate a phytoene-producing strain that also expresses a mutant CarRP. Compared to a phytoene-producing strain expressing the wt McCarRP of SEQ ID NO: 1 or FIG. 1, phytoene titers can be increased by at least about 8% in strains expressing the CarRP mutants (not shown).

[0078] Example 8: Beta-carotene production in Yarrowia lipolytica expressing mutant carRP The beta-carotene-producing strain ML15710 is transformed with the CRE-recombinase-containing plasmid MB6128 and selected on geneticin-containing medium. Hygromycin-sensitive isolates are identified among the transformants by replica plating on selective and non-selective media. One hygromycin-sensitive isolate is further grown on non-selective media, and then a geneticin-sensitive isolate is identified by replica plating on selective and non-selective media. One such isolate is then transformed with MB10866 as described in Example 1 to produce the white carRP mutant strain ML15710+carRP. - Hygromycin-sensitive isolates are identified by passage on non-selective medium and replica plating onto selective and non-selective media. One such isolate is then transformed into MB9282 as in Example 1 to produce ku70. - Mutant strain ML15710+carRP - +ku70 - A uracil auxotrophic strain was isolated from this strain by selection on 5-FOA, which was then transformed into ML15710+carRP. - +ku70 - +ura3 - This white uracil auxotrophic strain is then transformed with a plasmid expressing a heterologous wt CarRP or mutant listed in Table 4 (Example 2) to generate a beta-carotene producing strain that also expresses a mutant CarRP. Compared to a beta-carotene producing strain expressing the wt McCarRP of SEQ ID NO: 1 or FIG. 1, the beta-carotene titer can be increased by at least about 5% in the strain expressing the CarRP mutant (not shown).

Claims

1. 1. A modified bifunctional enzyme that catalyzes the conversion of geranylgeranyl pyrophosphate (GGPP) to phytoene and / or the conversion of lycopene to beta-carotene, the enzyme comprising one or more amino acid substitutions, e.g., amino acid substitutions introduced into a sequence having at least about 20%, e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99%, or up to 100% identity to SEQ ID NO: 1, or the sequence shown in Figure 1, wherein the one or more amino acid substitutions are introduced at positions corresponding to amino acid residues selected from the group consisting of positions 7, 33, 153, 159, 167, 194, 305, 330, 430, 431, 432, 476, 547, 579, and combinations thereof, of SEQ ID NO: 1 or in the polypeptide shown in Figure 1.

2. the amino acid residue at the position corresponding to position 7 of SEQ ID NO: 1 or shown in Figure 1 is different from glutamic acid or glutamine, the amino acid residue at the position corresponding to position 33 of SEQ ID NO: 1 or shown in Figure 1 is different from alanine or tryptophan, the amino acid residue at the position corresponding to position 153 of SEQ ID NO: 1 or shown in Figure 1 is different from alanine, the amino acid residue at the position corresponding to position 159 of SEQ ID NO: 1 or shown in Figure 1 is different from leucine, the amino acid residue at the position corresponding to position 167 of SEQ ID NO: 1 or shown in Figure 1 is different from tyrosine, the amino acid residue at the position corresponding to position 194 or 476 of SEQ ID NO: 1 or shown in Figure 1 is different from alanine 2. The modified enzyme of claim 1, wherein the amino acid residue is different from isoleucine, the amino acid residue at the position corresponding to position 305 of SEQ ID NO: 1 or shown in Figure 1 is different from threonine, the amino acid residue at the position corresponding to position 330 of SEQ ID NO: 1 or shown in Figure 1 is different from aspartic acid, the amino acid residue at the position corresponding to position 430 or 431 of SEQ ID NO: 1 or shown in Figure 1 is different from serine, the amino acid residue at the position corresponding to position 432 or 547 of SEQ ID NO: 1 or shown in Figure 1 is different from valine, and / or the amino acid residue at the position corresponding to position 579 of SEQ ID NO: 1 or shown in Figure 1 is different from arginine.

3. The modified enzyme of claim 1 or 2, wherein the amino acid residues corresponding to positions 7, 33, 153, 159, 167, 194, 305, 330, 430, 431, 432, 476, 547, 579, and / or combinations thereof in SEQ ID NO: 1 or in the polypeptide shown in Figure 1 are selected from D7, N33, S153, V159, F167, L194, A305, E330, N330, A430, T431, I432, A476, I547, and / or K579.

4. 4. The modified enzyme of claim 3, comprising one or more amino acid substitutions selected from the group consisting of E7D, A33N, A153S, L159V, Y167F, I194L, T305A, D330E, D330N, S430A, S431T, V432I, I476A, V547I, R579K, and combinations thereof, introduced into a sequence having at least about 20%, e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99%, or up to 100% identity to SEQ ID NO: 1, or the sequence shown in Figure 1.

5. The modified enzyme according to any one of claims 1 to 4, which is derived from Mucor circinelloides CarRP.

6. The modified enzyme according to any one of claims 1 to 5, which is introduced and expressed in a suitable carotenoid and / or apocarotenoid producing host cell, in particular a retinoid producing host cell.

7. 7. The modified enzyme of any one of claims 1 to 6, wherein the catalytic activity for the production of apocarotenoids, in particular retinoids, when expressed in a suitable apocarotenoid / retinoid producing host cell is increased by at least about 5%, such as in the range of 20-65%, or more, compared to a corresponding host cell expressing CarRP of SEQ ID NO: 1, or as shown in Figure 1.

8. 7. The modified enzyme of any one of claims 1 to 6, wherein the catalytic activity for the production of carotenoids, when expressed in a suitable carotenoid-producing host cell, is increased by at least about 5%, such as in the range of 5 to 30%, compared to a corresponding host cell expressing CarRP of SEQ ID NO: 1, or as shown in Figure 1.

9. A carotenoid and / or apocarotenoid producing host cell heterologously expressing the modifying enzyme according to any one of claims 1 to 8.

10. 10. The host cell of claim 9, which is a fungal host cell or is selected from E. coli, preferably Yarrowia or Saccharomyces.

11. 11. The host cell of claim 9 or 10, which heterologously expresses a gene involved in the biosynthesis of a carotenoid and / or an apocarotenoid selected from the group consisting of beta-carotene, lycopene, phytoene, beta-ionone, beta-cryptoxanthin, canthaxanthin, astaxanthin, zeaxanthin, rhodoxanthin, retinal, retinol, retinyl acetate, and mixtures thereof.

12. 1. A process for producing carotenoids or apocarotenoids in a suitable host cell, comprising: (a) culturing a host cell according to any one of claims 9 to 11 under suitable culture conditions for expressing the modifying enzyme according to any one of claims 1 to 8; (b) isolating and optionally purifying the carotenoid or apocarotenoid from the culture medium; A process wherein the percentage of carotenoids or apocarotenoids is increased by at least about 5% compared to a process using a host cell expressing CarRP of SEQ ID NO: 1, or as shown in Figure 1, instead of the modifying enzyme.

13. 13. The process of claim 12, wherein the carotenoid or aporacotenoid is selected from the group consisting of beta-carotene, lycopene, phytoene, beta-ionone, beta-cryptoxanthin, canthaxanthin, astaxanthin, zeaxanthin, rhodoxanthin, retinal, retinol, retinyl acetate, and mixtures thereof.

14. 1. A method for increasing the productivity of an apocarotenoid-producing host cell, particularly a retinoid-producing host cell, comprising: (a) providing a host cell that expresses genes involved in the biosynthesis of apocarotenoids, particularly retinoids, including but not limited to the biosynthesis of retinal, retinol, and / or retinyl acetate; (b) transforming the host cell with a polynucleotide that expresses the modifying enzyme according to any one of claims 1 to 8; (c) isolating and optionally purifying apocarotenoids, particularly retinoids, including but not limited to retinal, retinol, and / or retinyl acetate, from said host cells; The method, wherein said productivity is increased by at least about 20-75% compared to the host cell of step (b) transformed with a polynucleotide according to SEQ ID NO: 2 instead of a polynucleotide expressing said modifying enzyme.

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