CARRP enzyme variants and their use in production of carotenoids and apoprosthetic carotenoids

Abstract

The present invention relates to increasing the accumulation of carotenoids and derivatives thereof via manipulation and heterologous expression of CarRP derived from Mucor circinelloids.

Description

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

[0002] Carotenoids include C-40 isoprenoid compounds (such as carotenes and xanthophylls) and cleavage products (such as apocarotenoids), which are responsible for the orange color of carrots, the pink color of flamingos and salmon, and the red color of lobsters or shrimp, and are used for further important applications in the food, feed, cosmetics or pharmaceutical industries. In addition, beta-carotene is a key precursor or intermediate in the synthesis of vitamin A.

[0003] Retinoids (a class of apocarotenoids) are very important and essential nutritional factors for humans and animals that must be provided through the diet. Retinoids promote human and animal health, especially in the areas of vision, immune system, and growth.

[0004] Since the chemical synthesis of carotenoids or retinoids has some major disadvantages, namely the consumption of energy and / or water, organic and / or inorganic solvents, the synthesis of unwanted by-products, and the worldwide increasing demand for natural products useful as, for example, colorants or nutritional supplements, there is a strong demand for biotechnological production of such compounds.

[0005] Carotenoids (including carotenes and xanthophylls) and apocarotenoids (including retinoids and ionones) are produced naturally by certain organisms, including photosynthetic organisms (e.g., plants, algae, cyanobacteria, particularly with respect to the production of carotenoids) and some fungi (e.g., Mucor circinelloides, Yarrowia, Saccharomyces (both for the production of carotenoids and retinoids) and bacteria (e.g., Escherichia coli or Paraccocus). However, these systems are industrially difficult to handle and / or produce the compounds at such low levels that commercial-scale isolation is not feasible.

[0006] A key enzyme in the biosynthesis of both carotenoids and apocarotenoids is the bifunctional enzyme CarRP, which catalyzes the conversion of geranylgeranyl pyrophosphate (GGPP) to phytoene (i.e., acts as phytoene synthase) and catalyzes the conversion of lycopene to β-carotene (i.e., acts as lycopene cyclase). A widely used enzyme with good performance is derived from Mucor circinelloides (McCarRP), however, accumulation of lycopene leads to feedback inhibition of CarRP, thereby reducing the production of β-carotene and xanthophylls.

[0007] Therefore, there is a strong need for more efficient bioproduction of carotenoids and apocarotenoids (including but not limited to retinal or retinol), wherein the corresponding genes are (over)expressed in generally recognized as safe (GRAS) host cells (e.g. oleagenous yeast), while reducing or eliminating known bottlenecks in such production processes.

[0008] Surprisingly, we have now identified amino acid residues in CarRP (McCarRP) from Mucor circinelloides that are essential for the formation of phytoene and / or β-carotene and therefore for the production of carotenoids or apocarotenes, whereby the known feedback inhibition is reduced. Figure 1 The introduction of one or more amino acid substitutions located in both the lycopene cyclase (R)- and phytoene synthase (P)-domains of the enzyme results in an increase in the formation of the carotenoid compound by at least about 5%, such as in the range of 20-75% and more, compared to wild-type (unmodified) McCarRP as shown or according to SEQ ID NO: 1.

[0009] In particular, the present invention relates to a modified bifunctional enzyme involved in the synthesis of phytoene and acting as a lycopene cyclase, and to a method for producing such a modified enzyme, wherein the modified bifunctional enzyme is an enzyme that catalyzes the conversion of geranylgeranyl pyrophosphate (GGPP) to phytoene and / or catalyzes the conversion of lycopene to β-carotene, in particular a CarRP comprising one or more modifications, such as those introduced into Figure 1 or SEQ ID NO:1 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, said one or more amino acid substitutions being introduced into the sequence corresponding to that in Figure 1at a position selected from the group consisting of amino acid residues 7, 33, 153, 159, 167, 194, 305, 330, 430, 431, 432, 476, 547, 579, and combinations thereof, of the polypeptide as shown in or according to SEQ ID NO: 1.

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

[0011] In some embodiments, the modified enzyme is used in a method for producing carotenoids and apocarotenoids (including carotenes, xanthophylls, retinoids and ionones), wherein the modified enzyme is expressed, in particular heterologously expressed, in a suitable host cell (in particular a host cell producing fungal carotenoids and / or retinoids, more particularly a host cell producing β-carotene), and the use results in the same conditions as using Figure 1 The titer of the product is increased by at least about 5%, such as 5 to 20% or even 5 to 75%, based on the total carotenoids / apocarotenoids / retinoids present in / produced by the modified host cell, compared to the method of producing an unmodified CarRP enzyme as shown in SEQ ID NO: 1 or according to SEQ ID NO: 1.

[0012] The terms "CarRP", "phytoene synthase", "lycopene cyclase", "CrtYB" are used interchangeably herein and refer to a bifunctional enzyme involved in the biosynthetic pathway from GGPP to β-carotene, which is capable of catalyzing the conversion of GGPP to phytoene (i.e., functioning as a phytoene synthase [EC 2.5.1.32]) and / or catalyzing the conversion of lycopene to β-carotene (i.e., functioning as a lycopene β-cyclase [EC 5.5.1.19]). Exemplary and suitable enzymes that can be used to produce modified enzymes according to the present invention are as follows: Figure 1 or McCarRP shown in SEQ ID NO: 1 or Figure 1 or SEQ ID NO: 1, including enzymes encoded by the polynucleotide according to SEQ ID NO: 2.

[0013] Based on unmodified CarRP, especially based on Figure 1 A "modified" CarRP as defined herein that has at least about 20% identity to the McCarRP shown in SEQ ID NO: 1 or according to SEQ ID NO: 2 shows increased enzymatic activity, i.e., is more effective than using Figure 1 1 or according to SEQ ID NO: 1, thereby increasing the formation of apocarotenoids (including retinoids) and / or carotenoids (including xanthophylls) as defined herein, in particular by at least about 5%, such as in the range of 5-75%, in particular by 20-75% and more (in particular with respect to retinoid production).

[0014] As used herein, the term "carotenoid" is well known in the art. It includes long 40-carbon conjugated isoprenoid polyenes (C-40 isoprenoids), which are formed in nature by the linkage of two 20-carbon GGPP molecules. These include, but are not limited to, phytoene, lycopene, β-carotene, α-carotene, γ-carotene, lutein, zeaxanthin, astaxanthin, β-cryptoxanthin or lutein. The biosynthesis of carotenoids is described, for example, in WO2006102342. The term "carotenoid" also includes the group of "xanthophylls", i.e., oxidized carotenoid derivatives such as lutein, zeaxanthin or β-cryptoxanthin.

[0015] As used herein, "apocarotenoid" is a cleavage product of a carotenoid and is thus defined as <C40-carotenoid, including but not limited to retinoids or ionones such as retinal, retinol, retinol acetate, β-ionone or α-ionone.

[0016] Retinoids used herein include, but are not limited to, retinal, retinoic acid, retinol, retinoic methoxide, retinol acetate, retinyl esters, 4-oxo-retinoids, 3-hydroxy-retinoids or combinations thereof. Long-chain retinyl esters used herein are defined as hydrocarbon esters of retinol and fatty acids, where the fatty acid consists of at least about 8, such as 9, 10, 12, 13, 15 or 20 carbon atoms and at most about 26, such as 25, 22, 21 or fewer carbon atoms, preferably at most about 6 unsaturated bonds, such as 0, 1, 2, 4, 5, 6 unsaturated bonds. Fatty acids in long-chain retinyl esters include, but are not limited to, linoleic acid, oleic acid or palmitic acid. The biosynthesis of retinoids is described, for example, in WO2008042338 or WO2019058000, which disclose the enzymatic conversion of β-carotene to retinal, to retinol, to retinol acetate in Yarrowia lipolytica strains expressing the corresponding heterologous genes.

[0017] The terms "conversion", "enzymatic conversion", which are associated with the enzymatic catalysis of GGPP and / or lycopene, are used interchangeably herein and refer to the action of modified or unmodified CarRP as a biocatalyst in the conversion of GGPP to phytoene or lycopene to β-carotene as defined herein, and thus include 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" particularly includes GRAS host cells (e.g., yeast cells), wherein the cell is a host cell that produces carotenoids and / or apocarotenoids, particularly fungal host cells that produce β-carotene and / or retinol, including but not limited to host cells of the genus Yarrowia or Saccharomyces, such as host cells of Yarrowia lipolytica or Saccharomyces cerevisiae.

[0019] The modified enzyme can be used in isolated form (e.g., in a cell-free system) or can be expressed in a suitable host cell, for example, in a host cell that produces carotenoids and / or apocarotenoids, in particular in a fungal host cell as defined herein. The enzyme can be expressed as an endogenous enzyme or a heterologous enzyme. Preferably, the modified enzyme as described herein is introduced as a heterologous enzyme and expressed in a suitable host cell, for example, in a host cell that produces carotenoids and / or apocarotenoids, preferably a host cell that produces carotene and / or retinol, in particular in a fungal host cell as defined herein.

[0020] In one embodiment, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1 or according to SEQ ID NO: 1, in particular the introduction of aspartic acid, for example via substitution of glutamic acid for aspartic acid (E7D), wherein the modified enzyme is derived from Figure 1 or according to SEQ ID NO: 1. Such modified enzymes comprising said mutations are used in fermentation processes wherein said modified enzymes are introduced and expressed under suitable conditions using a host cell producing carotenoids or apocarotenoids as specified herein, as compared to using a host cell according to Figure 1 or according to SEQ ID NO: 1 (including Figure 1 or according to SEQ ID NO: 1 but in the McCarRP Figure 1 7 or a glutamine at position 7 in the polypeptide according to SEQ ID NO: 1), the formation of carotenoids can be increased by at least about 5-30%, for example 10%, 15%, 20%, 25%, 30% or more, and the formation of apocarotenoids, in particular retinoids, can be increased by at least about 5-67%, for example 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more.

[0021] In one embodiment, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1 or according to SEQ ID NO: 1, in particular the introduction of asparagine, for example via the substitution of alanine for asparagine (A33N), wherein the modified enzyme is derived from Figure 1 or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell for producing carotenoids or apocarotenoids as specified herein, Figure 1 or according to SEQ ID NO: 1 (including Figure 1 or according to SEQ ID NO: 1 but in the McCarRP Figure 1 33 in the polypeptide as shown in SEQ ID NO: 1 or having a tryptophan at position 33 in the polypeptide, the formation of carotenoids can be increased by at least about 5-30%, such as 10%, 15%, 20%, 25%, 30% or more, and the formation of apocarotenoids, in particular retinoids, can be increased by at least about 20-46%, such as 25%, 30%, 35%, 40%, 45%, 50% or more.

[0022] In some embodiments, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1 or according to SEQ ID NO: 1, in particular the introduction of serine, for example the introduction of serine via substitution of alanine (A153S), wherein the modified enzyme is derived from Figure 1 or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell for producing carotenoids or apocarotenoids as specified herein, Figure 1 The formation of carotenoids can be increased by at least about 5-30%, for example 10%, 15%, 20%, 25%, 30% or more, and the formation of apocarotenoids, in particular retinoids, can be increased by at least about 20-43%, for example 25%, 30%, 35%, 40%, 45% or more, compared to the corresponding method of the unmodified CarRP shown in SEQ ID NO: 1 or according to SEQ ID NO: 1.

[0023] In some embodiments, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1or according to SEQ ID NO: 1, in particular the introduction of valine, for example via substitution of leucine for valine (L159V), wherein the modified enzyme is derived from Figure 1 or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell for producing carotenoids or apocarotenoids as specified herein, Figure 1 The formation of carotenoids can be increased by at least about 5-30%, for example 10%, 15%, 20%, 25%, 30% or more, and the formation of apocarotenoids, in particular retinoids, can be increased by at least about 20-41%, for example 25%, 30%, 35%, 40%, 45% or more, compared to the corresponding method of the unmodified CarRP shown in SEQ ID NO: 1.

[0024] In one embodiment, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1 or according to SEQ ID NO: 1, in particular the introduction of phenylalanine, for example via substitution of tyrosine for phenylalanine (Y167F), wherein the modified enzyme is derived from Figure 1 or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell producing carotenoids or apocarotenoids as specified herein, Figure 1 The formation of carotenoids can be increased by at least about 5-30%, such as 10%, 15%, 20%, 25%, 30% or more, and the formation of apocarotenoids, in particular retinoids, can be increased by at least about 20-24%, compared to the corresponding method of the unmodified CarRP shown in SEQ ID NO: 1 or according to SEQ ID NO: 1.

[0025] In one embodiment, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1 or according to SEQ ID NO: 1, in particular the introduction of a leucine, for example by substitution of an isoleucine for a leucine (I194L), wherein the modified enzyme is derived from Figure 1or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell for producing carotenoids or apocarotenoids as specified herein, Figure 1 The formation of carotenoids can be increased by at least about 5-30%, for example 10%, 15%, 20%, 25%, 30% or more, and the formation of apocarotenoids, in particular retinoids, can be increased by at least about 20-35%, for example 25%, 30%, 35%, 40% or more, compared to the corresponding method of the unmodified CarRP shown in SEQ ID NO: 1.

[0026] In one embodiment, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1 or according to SEQ ID NO: 1, in particular the introduction of alanine, for example via substitution of tyrosine for alanine (T305A), wherein the modified enzyme is derived from Figure 1 or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell for producing carotenoids or apocarotenoids as specified herein, Figure 1 The formation of carotenoids can be increased by at least about 5-30%, and the formation of apocarotenoids, in particular retinoids, can be increased by at least about 20-23%, compared to the corresponding method using the unmodified CarRP shown in SEQ ID NO: 1 or according to SEQ ID NO: 1.

[0027] In one embodiment, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1 or according to SEQ ID NO: 1, in particular the introduction of glutamic acid or asparagine, for example by substitution of aspartic acid for glutamic acid or asparagine (D330E or D330N), wherein the modified enzyme is derived from Figure 1 or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell for producing carotenoids or apocarotenoids as specified herein, Figure 1Compared to the corresponding method of the unmodified CarRP shown in SEQ ID NO: 1 or according to SEQ ID NO: 1, the formation of carotenoids can be increased by at least about 5-30%, and the formation of apocarotenoids, especially retinoids, can be increased by at least about 5-27%, for example 10%, 15%, 20%, 25%, 30% or more.

[0028] In one embodiment, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1 or according to SEQ ID NO: 1, in particular the introduction of alanine, for example via substitution of serine for alanine (S430A), wherein the modified enzyme is derived from Figure 1 or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell for producing carotenoids or apocarotenoids as specified herein, Figure 1 Compared to the corresponding method of the unmodified CarRP shown in SEQ ID NO: 1 or according to SEQ ID NO: 1, the formation of carotenoids can be increased by at least about 5-30%, and the formation of apocarotenoids, especially retinoids, can be increased by at least about 16-26%, such as 20%, 25%, 30% or more.

[0029] In one embodiment, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1 or according to SEQ ID NO: 1, in particular the introduction of a threonine, for example the introduction of a threonine via substitution of a serine (S431T), wherein the modified enzyme is derived from Figure 1 or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell for producing carotenoids or apocarotenoids as specified herein, Figure 1 Compared to the corresponding method of the unmodified CarRP shown in SEQ ID NO: 1 or according to SEQ ID NO: 1, the formation of carotenoids can be increased by at least about 5-30%, and the formation of apocarotenoids, especially retinoids, can be increased by at least about 20-42%, for example 25%, 30%, 35%, 40%, 45% or more.

[0030] In one embodiment, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1or according to SEQ ID NO: 1, in particular the introduction of isoleucine, for example the introduction of isoleucine via substitution of valine (V432I), wherein the modified enzyme is derived from Figure 1 or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell producing carotenoids or apocarotenoids as specified herein, Figure 1 The formation of carotenoids can be increased by at least about 5-30%, and the formation of apocarotenoids, in particular retinoids, can be increased by at least about 20-65%, for example 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more, compared to the corresponding method of the unmodified CarRP shown in SEQ ID NO: 1.

[0031] In one embodiment, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1 or according to SEQ ID NO: 1, in particular the introduction of alanine, for example via substitution of isoleucine for alanine (I476A), wherein the modified enzyme is derived from Figure 1 or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell for producing carotenoids or apocarotenoids as specified herein, Figure 1 The formation of carotenoids can be increased by at least about 5-30%, and the formation of apocarotenoids, in particular retinoids, can be increased by at least about 20-25%, compared to the corresponding method using the unmodified CarRP shown in or according to SEQ ID NO: 1.

[0032] In one embodiment, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1 or according to SEQ ID NO: 1, in particular the introduction of isoleucine, for example the introduction of isoleucine via substitution of valine (V547I), wherein the modified enzyme is derived from Figure 1or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell producing carotenoids or apocarotenoids as specified herein, Figure 1 Compared to the corresponding method of the unmodified CarRP shown in SEQ ID NO: 1 or according to SEQ ID NO: 1, the formation of carotenoids can be increased by at least about 5-30%, and the formation of apocarotenoids, especially retinoids, can be increased by at least about 20-35%, for example 25%, 30%, 35%, 40% or more.

[0033] In one embodiment, the modified CarRP enzyme as defined herein is comprised in a region corresponding to Figure 1 or according to SEQ ID NO: 1, in particular the introduction of lysine, for example via substitution of arginine for lysine (R579K), wherein the modified enzyme is derived from Figure 1 or an enzyme having at least about 20% identity to McCarRP as shown in SEQ ID NO: 1. Such a modified enzyme comprising said mutation is used in a fermentation process, wherein said modified enzyme is introduced and expressed under suitable conditions using a host cell for producing carotenoids or apocarotenoids as specified herein, Figure 1 Compared to the corresponding method of the unmodified CarRP shown in SEQ ID NO: 1 or according to SEQ ID NO: 1, the formation of carotenoids can be increased by at least about 5-30%, and the formation of apocarotenoids, especially retinoids, can be increased by at least about 20-38%, for example 25%, 30%, 35%, 40% or more.

[0034] Thus, in some embodiments, the modified enzyme comprises two or more mutations, i.e., amino acid substitutions, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 mutations selected from the group consisting of Figure 1 at least 2 to 14 mutations at positions 7, 33, 153, 159, 167, 194, 305, 330, 430, 431, 432, 476, 547 or 579 of the polypeptide shown in or according to SEQ ID NO: 1, and wherein at positions corresponding to Figure 1The at least 2 to 14 amino acids introduced at the positions of the loci shown in or according to SEQ ID NO: 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 Figure 1 or SEQ ID NO: 1, 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, and methods for preparing the modified CarRPs described herein. Such modified enzymes are expressed in a suitable retinoid-producing host cell, wherein the host cell expresses the retinoid-producing enzyme. Figure 1 The percentage of retinoids can be increased by 5 to 75% and more, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or more, compared to the method shown in SEQ ID NO: 1 or according to the CarRP of SEQ ID NO: 1. As used herein, the term "at least two mutations" means that the modified enzyme described herein has at least two mutations in the corresponding Figure 1 1 or according to SEQ ID NO: 1 comprises 2 or more mutations, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 mutations at the positions indicated in the polypeptide. In particular, the modified CarRP enzyme is expressed in a host cell producing apocarotenoids (e.g., retinoids) and / or carotenoids, such as preferably selected from the genus Yarrowia, Saccharomyces or Escherichia, most preferably the modified CarRP enzyme is expressed in Yarrowia lipolytica, a carotenoid and / or apocarotenoid (e.g., retinoid) producing yeast known in the art. Most preferably, the modified CarRP as defined herein is codon-optimized for expression in the corresponding host cell.

[0036] In particular, the present invention relates to Figure 1or a modified CarRP of the McCarRP shown in SEQ ID NO: 1, including proteins having at least about 20% identity, and methods for producing the modified CarRP as described herein, comprising at least 1, 2, 3, 4, 5 amino acid substitutions, preferably 5 amino acid substitutions, selected from E7D, A33N, A135S, L159V, Y167F, I194L, T 305A, D330E or D330N, S430A, S431T, V432I, I476A, V547I and R579K, more particularly comprising 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, D330N and V432I. 67F, T305A and R579K, or the combination of E7D with A33N, Y167F, S431T and R579K, or the combination of E7D with A33N, Y167F, S430A and R579K, or the combination of E7D with A33N, Y167F, S430A and V547I, or the combination of E7D with A33N, Y167F, T305A and S430A, or the combination of E7D with A33N, S430A, V547I and R or a combination of E7D with A33N, Y167F, T305A, and D330N, 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 Figure 1 or McCarRP as shown in SEQ ID NO: 1, including proteins having at least about 20% identity, and methods for producing said modified CarRP as described herein, said modified CarRP comprising at least one mutation (e.g., V432I) or a combination of at least 5 mutations (including a combination of E7D and A33N or Y167F, S430I, V547I and R579K), wherein said amino acid substitutions are introduced into a suitable apocarotenoid (e.g., retinoid)-producing and / or carotenoid-producing host cell and said modified enzyme is expressed, as compared to expressing an unmodified CarRP as defined herein (e.g., Figure 1For example, the percentage of retinoid can be increased by about 60-70% and more compared to a host cell expressing the McCarRP shown in SEQ ID NO: 1 or according to SEQ ID NO: 1.

[0038] According to all embodiments of the present invention, in the case corresponding to Figure 1 or according to SEQ ID NO: 1) in the amino acid positions indicated in the polypeptide (e.g., at positions corresponding to Figure 1 1 or according to SEQ ID NO: 1) results in an increased formation of carotenoids and / or apocarotenoids, preferably retinoids, when used in a corresponding host cell (i.e. a host cell producing carotenoids and / or apocarotenoids as defined herein), wherein the titer of the carotenoid or retinoid may be increased by at least about 5% compared to a corresponding host cell (in which the specified amino acid is not substituted in the manner defined herein). In particular, the titer of apocarotenoids, particularly retinoids, can be increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more, based on total retinoids, by increasing the titer of apocarotenoids in the presence of a retinoid ... Figure 1 Single mutations at positions V432I, A33N, L159V, A153S in the polypeptide shown in or according to SEQ ID NO: 1 achieve an increase in retinoids of at least about 40 to 70%, wherein the expression Figure 1 or according to SEQ ID NO: 1, using a modified CarRP comprising a quintuple mutation as defined herein (e.g. comprising a polypeptide corresponding to Figure 1 33N, S430A, V547I, R579K, Y167F in the polypeptide shown in SEQ ID NO: 1 or according to SEQ ID NO: 2), an increase of at least about 25% to 75%, such as about 40% to 75% or even more can be achieved. In particular, the titer of a specific carotenoid (e.g., zeaxanthin, canthaxanthin, astaxanthin, anthoxanthin, β-cryptoxanthin) can be increased by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or more, wherein in the case of a polypeptide corresponding to Figure 1Single mutations at positions V432I, V547I, S431T, D330E, T305 in the polypeptide shown in or according to SEQ ID NO: 1 achieve an increase in total carotenoids by at least about 5% to 35%, wherein the expression Figure 1 Compared to the corresponding host cells of the polypeptide shown in or according to SEQ ID NO: 1, using Figure 1 Single mutations at positions V432I, V547I, S431T, D330E, T305 in the polypeptide shown in or according to SEQ ID NO: 1 achieve an increase in canthaxanthin by at least about 5% to 40%.

[0039] The modified host cell as defined herein comprises one or more copies of the modified enzyme as defined herein, preferably wherein the modified enzyme is heterologously expressed in the modified host cell. In order to produce more copies of the gene and / or protein in the host cell as defined herein, for example, the modification of more copies of the modified CarRP can include the use of a strong promoter, a suitable transcription enhancer and / or translation enhancer, or one or more gene copies are introduced into a host cell, particularly a fungal host cell, that produces carotenoids and / or apocarotenoids, resulting in an increase in the accumulation of the corresponding enzyme within a given time. The skilled person knows which technology to use depending on the host cell. The increase or decrease in gene expression can be measured by various methods, such as Northern, Southern or Western blotting techniques known in the art.

[0040] The generation of mutations in nucleic acids or amino acids (i.e., mutagenesis) can be performed in different ways, for example by random or lateral mutagenesis, physical damage caused by an agent such as radiation, chemical treatment or insertion of a genetic element. The skilled person knows how to introduce mutations.

[0041] Thus, the present invention relates to host cells and methods for producing carotenoids and / or apocarotenoids as described herein, host cells, in particular fungal host cells, comprising an expression vector or polynucleotide encoding a modified CarRP as described herein (see also WO2009126890, in particular Ex. 1A), which has been integrated into the chromosomal DNA of the host cell. Such modified host cells, in particular fungal host cells, comprising a heterologous polynucleotide on an expression vector or integrated into the chromosomal DNA encoding a modified CarRP as described herein are referred to as recombinant or modified host cells. Host cells, in particular fungal host cells, that produce carotenoids and / or apocarotenoids may contain one or more copies of a gene encoding a modified CarRP as defined herein, comprising a mutation as defined herein, resulting in overexpression of such a gene encoding the modified CarRP as defined herein. The increase in gene expression can be measured by various methods, such as Northern, Southern or Western blotting techniques, transcriptomics, genomic sequencing or proteomics known in the art.

[0042] The present invention particularly relates to the use of this novel modified CarRP in a method for producing carotenoids and / or apocarotenoids, in particular retinoids, including methods wherein the retinoid comprises a mixture of retinol, retinal and retinyl acetate, in particular the percentage of retinyl acetate based on the total retinoids is at least about 40% by weight, and wherein the formation of long-chain retinyl esters is particularly reduced. The skilled person knows how to generate such conditions (see, for example, WO2021136689 or WO2022090548).

[0043] The terms "sequence identity" and "% identity" are used interchangeably herein. For the purposes of the present invention, it is defined herein that in order to determine the sequence identity percentage of two amino acid sequences or two nucleic acid sequences, sequences are aligned for optimal comparison purposes. In order to optimize the comparison between the two sequences, a gap can be introduced in any of the two sequences being compared. This comparison can be carried out over the full length of the compared sequence. Alternatively, the comparison can be carried out over a shorter length, for example, over about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Sequence identity is the percentage of identical matches between the two sequences on the reported comparison region. The sequence identity percentage between two amino acid sequences or between two nucleotide sequences can be determined using the Needleman and Wunsch algorithm for comparing the two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol Biol. 48, 443-453). Both amino acid sequences and nucleotide sequences can be compared by an algorithm. The Needleman-Wunsch algorithm has been implemented in the computer program NEEDLE. For the purposes of the present invention, the NEEDLE program from the EMBOSS software package (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 / ) is used. 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. The skilled person will understand that all of these different parameters will produce slightly different results, but the overall percent identity of the two sequences will not change significantly when different algorithms are used.

[0044] After alignment by the program NEEDLE as described above, the percentage of sequence identity between the query sequence and the sequence of the invention is calculated as follows: the number of corresponding positions in the alignment showing the same amino acid or the same nucleotide in the two 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 using the NOBRIEF option and is labeled "longest identity" in the program's output. Two amino acid sequences are identical or have 100% identity if they do not differ in any of their amino acids.

[0045] The modified enzyme as defined herein may comprise further amino acid substitutions that do not alter the activity of the enzyme, i.e., it exhibits the same properties as the phytoene synthase and / or lycopene beta cyclase relative to the enzyme as defined herein and catalyzes the conversion of GGPP to phytoene and / or lycopene to beta-carotene to the same extent as a modified enzyme that only carries one or more amino acid substitutions as described herein. Such mutations are also referred to as "silent mutations" which do not alter the (enzymatic) activity of the enzyme according to the invention.

[0046] The expression of an enzyme / polynucleotide encoding one of the modified CarRPs as defined herein can be accomplished in any host system, including (microbial) organisms that are suitable for carotenoid and / or apocarotenoid production and that allow expression of a nucleic acid encoding one of the enzymes as described herein, including functional equivalents or derivatives as described herein. Examples of suitable host (microbial) organisms that produce carotenoids and / or apocarotenoids are bacteria, algae, fungi (including yeast), plant or animal cells. Preferred bacteria are those of the genus Escherichia, for example Escherichia coli, Streptomyces, Pantoea (Erwinia), Bacillus, Flavobacterium, Synechococcus, Lactobacillus, Corynebacterium, Micrococcus, Mixococcus, Brevibacterium, Bradyrhizobium, Gordonia, Dioscorea, Murine Tau, Sphingomonas, Synechocystis, Paracoccus, for example Paracoccus zeaxanthinifaciens. Preferred eukaryotic microorganisms, in particular fungi including yeast, are selected from the group consisting of: 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; Phycomyces, such as Phycomyces blakesleanus; Mucor; Rhodotorula; Sporoblosarcoma; Phaffia; Blakeslea trispora; or Yarrowia, such as Yarrowia lipolytica. Particularly preferred is expression in a fungal host cell (e.g., Yarrowia or Saccharomyces), or expression in Escherichia, more preferably in Yarrowia lipolytica or Saccharomyces cerevisiae.

[0047] In connection with the present invention, it is understood that organisms (e.g., microorganisms, fungi, algae, or plants) also include synonyms or substantial synonyms 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, strain Lachancea mirantina is a synonym for strain Zygosaccharomyces sp. IFO 11066 originating from Japan.

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

[0049] As known to those skilled in the art for corresponding host cells producing carotenoids and / or apocarotenoids, the modified host cells as defined herein can be cultured under aerobic or anaerobic conditions in an aqueous medium supplemented with appropriate nutrients. Optionally, such culture is carried out in the presence of proteins and / or cofactors involved in electron transfer, as known in the art. Suitable carbon sources 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) (in the presence or absence of ethanol), in particular from glucose, galactose or xylose. Specific culture conditions for the production of apocarotenoids, in particular retinoids, can comprise batch and feed operations, with a glucose concentration of 5% (w / v) and an ethanol concentration of 1% (w / v) in the batch phase and a concentration of 100% (w / v) in the feed phase. The host cells can be cultured / grown in batch, fed-batch, semi-continuous, or continuous mode under suitable culture conditions, particularly in fed-batch mode for 80, 90, 100, 110, 120, or 130 hours. Depending on the host cell and the carotenoid and / or apocarotenoid to be produced, the conditions can be adjusted as known to those skilled in the art. The culture and isolation of host cells (e.g., selected from the genera Yarrowia and Saccharomyces) that produce carotenoids and / or apocarotenoids are described, for example, in WO2008042338. Methods for producing β-carotene and retinoids in host cells selected from Escherichia coli are described, for example, in US20070166782.

[0050] In some embodiments, a host cell expressing a modified CarRP as defined herein that produces apocarotenoids is cultured in a two-phase system, wherein apocarotenoids, particularly retinoids (including but not limited to retinol and / or retinyl acetate), are collected in a suitable lipophilic phase and then separated therefrom, preferably at a retinyl acetate content of at least about 40-80% by weight based on total retinoids. Specific conditions and lipophilic solvents are disclosed in WO2022090548 or WO2022090549.

[0051] In some embodiments, the present invention relates to a two-phase fermentation using a lipophilic solvent as the second phase and using a retinoid-producing strain expressing a modified CarRP as defined herein, wherein the lipophilic solvent comprises In addition to known solvents such as silicone or n-dodecane, such as isopars or corn oil (see Jang et al., Microbial Cell Factories 10:59, 2011).

[0052] As used herein, the term "specific activity" or "activity" with respect to an enzyme refers to its catalytic activity, i.e., the ability of its catalysis to form a product from a given substrate. Specific activity defines the amount of substrate consumed and / or product produced within a given time period and the protein per defined amount at a defined temperature. Typically, specific activity is expressed as μmol substrate per minute per mg protein formed or consumed. Typically, μmol / min is abbreviated as U (=unit). Therefore, the unit definition of specific activity of μmol / min / (mg protein) or U / (mg protein) is used interchangeably in this document. If the enzyme is in vivo, i.e., in a host cell as defined herein or in a suitable (cell-free) system, and performs its catalytic activity in the presence of a suitable substrate, the enzyme is active. Technicians know how to measure enzyme activity. Assays for evaluating the ability 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). The titer of a product comprising apocarotenoids or carotenoids (e.g., retinyl acetate, retinol, trans-retinal, cis-retinal, β-carotene, canthaxanthin, zeaxanthin, astaxanthin, anthoxanthin, lycopene, phytoene, β-ionone, etc.) can be measured by HPLC.

[0053] The general construction of host cells for producing carotenoids, in particular host cells for producing β-carotene, is known in the art and is described, for example, in WO2006102342.

[0054] As used herein, a "retinol-producing host cell" is a specific host cell that produces apocarotenoids, wherein the corresponding polypeptide is expressed and active in vivo, resulting in the production of retinoids as defined herein (e.g., including retinal, retinol, and / or retinol acetate) by enzymatic conversion of β-carotene to retinaldehyde and optionally further conversion to retinol acetate. These polypeptides include enzymes that catalyze the conversion of β-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 retinol acetate (see, e.g., WO2019058001).

[0055] As used herein, "vitamin A" can be any chemical form of vitamin A found in aqueous solutions, solids, and formulations, and includes retinol, retinyl acetate, and retinyl esters. It also includes retinoic acid, e.g., undissociated, in its free acid form, or dissociated into anions.

[0056] As used herein, "retinal" is known by the IUPAC name (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohexen-1-yl)-nona-2,4,6,8-tetraenal and includes cis and trans isomers, such as 11-cis retinal, 13-cis retinal, trans retinal, and all-trans retinal.

[0057] Attached photos

[0058] Figure 1 : The amino acid sequence of M. circinelloides CarRP (McCarRP; SEQ ID NO: 1), wherein the amino acid residues selected for amino acid substitution as described in the present application are marked in bold / underlined, and wherein the amino acids replacing the original amino acids as shown in SEQ ID NO: 1 are marked in italics and shown on top of the corresponding original amino acids.

[0059] The following examples are merely illustrative and are not intended to limit the scope of the present invention in any way. The contents of all references, patent applications, patents and disclosed patent applications cited throughout this application are incorporated herein by reference, particularly WO2014096992, WO2019058001, WO2021136689, WO2022090548, WO2008042338, US20070166782, WO2022090549, WO2006102342, WO2016172282, WO2019058000, WO2019057999, WO2019057998 and US20180148697. Example

[0060] Example 1: General methods, strains and plasmids

[0061] All basic molecular biology and DNA manipulation procedures described herein are generally 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).

[0062] Strains, plasmids, and sequences. The strains used as host cells and the corresponding plasmids used for expression of CarRP and other enzymes or constructs specified below are listed in Tables 1, 2 and 4 and in the Sequence Listing. Figure 1 The wild-type CarRP (McCarRP) amino acid sequence (SEQ ID NO: 1) is shown with specific indication of the residues selected for modification as defined herein.

[0063] Table 1: List of Yarrowia strains (denoted by "ML") used for the production of carotenoids and / or retinoids carrying heterologous (unmodified or modified) CarRP genes. See the text for more details.

[0064]

[0065]

[0066] Table 2: List of plasmids used to construct strains carrying heterologous genes expressing the designated inserts ("Inserts") comprising a polynucleotide sequence expressing McCarRP according to SEQ ID NO: 1 (SEQ ID NO: 2) and other genes for producing retinoids and / or carotenoids. All modified CarRPs are based on Figure 1 or according to McCarRP of SEQ ID NO: 1. See text for more details.

[0067] plasmids Insert markers MB10157 MccarRP URA3+ARG4 MB10866 Cas9+sgCarRP HygR MB9282 Cas9+sgKu70 HygR MB6128 CRE-recombinase G418 / Geneticin MB7076 wxya HygR MB7190 ZlUT NatR MB7082 wxya NatR MB9930 ZlUT HygR MB7522 Cas9+sgcarB HygR MB9931 Lfreq-crtZ HygR MB7918 bhy-21 HygR MB6806 cCD1 HygR

[0068] UPLC reverse phase method. For rapid screening, this method does not separate cis isomers, only the major functional groups. A Waters Acquity UPLC with PDA detection (or similar) with an autosampler was used to inject the sample. Retinoids and / or carotenoids were separated using an Acquity UPLC HSS T31.8um P / N 186003539. The mobile phase consisted of 1000mL hexane, 30mL isopropanol, and 0.1mL acetic acid for 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 from 210 to 600nm. Analytes were detected according to Table 3. Anthoxanthin, astaxanthin, zeaxanthin and ionone can be quantified as in the following references: Royer et al. (Sci. Adv. 2020; Vol. 6, No. 17), WO2014096992 and US20180148697, respectively.

[0069] Table 3A: List of analytes used in the reversed-phase method. Depending on the measurement, the sum of all added intermediates gives the total amount of retinoids or carotenoids. β-Carotene* can be detected at 325 nm and interferes with retinyl ester quantification, so care must be taken to observe the carotene peak and not include it in the retinoid quantification. "N / A" indicates "not available." See the text for more details.

[0070] intermediates Retention time [min] λ maximum value [nm] Response Factor Retinyl Acetate 2.93 325 1.00 Retinyl esters 3.2-3.8 325 1.68 Retinaldehyde 2.77 325 0.87 Retinol 2.73 325 0.87 beta-carotene* 3.56 450 N / A Canthaxanthin 3.12 470 N / A

[0071] Table 3B: UPLC method gradient, solvent A: water; solvent B: acetonitrile; solvent C: methanol; solvent D: tert-butyl methyl ether.

[0072]

[0073]

[0074] Method calibration. The method is calibrated on carotenoids, retinyl acetate, retinol, and retinal, and quantified using the specified response factors relative to retinyl acetate. Retinyl acetate is dissolved in THF at ~200 μg / ml as a stock solution using a volumetric flask. Using a volumetric flask, x20, x50, and x100 dilutions of the stock solution in 50 / 50 methanol / MTBE are prepared. The UV absorbance of retinyl acetate becomes nonlinear quite quickly, so care must be taken to stay within the linear range. Therefore, lower concentrations may be better. Retinyl palmitate can also be used as a retinyl ester calibrant. The peak for retinyl acetate is at approximately 3 minutes, and the peak for retinyl esters (long-chain retinyl esters) is at approximately 3.5 minutes. This can be adjusted accordingly for measurements of other carotenoids or carotenoids.

[0075] Sample preparation. Samples were prepared by various methods depending on the conditions. For whole broth or washed broth samples, the broth was placed in tube, weigh it, and add mobile phase. In a tube, 25 μl of well-mixed fermentation broth and 975 μl of THF were added. The homogenizer (Bertin Corp, Rockville, MD, USA) was processed at the highest setting 3X according to the manufacturer's instructions, typically 3 × 15 × 7500 TPMS. For the washed pellet, the sample was spun at 10,000 rpm for 1 minute in a microcentrifuge in a 1.7 ml tube, the fermentation broth was decanted, 1 ml of water was added, mixed, precipitated and decanted, and adjusted to the original volume. The mixture was pelleted again and placed in an appropriate amount of mobile phase and passed through Bead beating. To analyze the silicone oil fraction, the sample was spun at 4000 RPM for 10 minutes, and the oil was decanted from the top by a positive displacement pipette (Eppendorf, Hauppauge, NY, USA) and diluted into the mobile phase mixed by vortexing, and the retinoid (or other test compound) concentration was measured by UPLC analysis.

[0076] Example 2: Production of retinoids in Yarrowia lipolytica expressing mutant carRP

[0077] To evaluate carRP alleles, strain ML18743 was transformed with plasmid MB10866 (SEQ ID NO: 4), which encodes a synthetic guide RNA (sgRNA) and SpCas9 protein to guide mutagenesis of the carRP sequence present in the genome. Strain ML19637 is a mutant of carRP, confirmed by its white color. Strain ML19637 was passaged on non-selective medium and hygromycin-sensitive isolates were confirmed. One such isolate was further mutagenized with plasmid MB9282 (SEQ ID NO: 5), which encodes an sgRNA and SpCas9 protein to guide mutagenesis at the Ku70 locus, to produce ML19836. The uracil auxotrophic strain ML19836-ura was isolated from ML19836 by selection on medium containing 5-fluoroorotic acid (5-FOA). Strain ML19836-ura was transformed with Sfil linearized DNA from plasmid MB10157 (SEQ ID NO: 3) expressing the proteins shown in Tables 4A and 4B and in Figure 1 Wild-type McCarRP and mutant derivatives MB10157-1 to MB10157-29 are highlighted in bold / underline in the polypeptide sequences shown in and were selected for uracil prototrophy.

[0078] As described elsewhere, transformants were grown in shaking plates, see for example WO2022090549. Typically, 200 μl of 0.075% yeast extract, 0.25% peptone (0.25x YP) were inoculated with 10 μL of freshly grown Yarrowia yeast and covered with 200 μL of Drakeol 5 (Penreco, Karns City, PA, USA) mineral oil, silicone oil or corn oil with 2% oleic acid or 2% glucose as a carbon source. The clonal isolates of the transformants were grown for 4 days in YPD medium in 24-well plates (Multitron, 30 ° C, 800 RPM) with one of the previously indicated overlays. The overlay fractions were removed from the shaking plate wells and analyzed by HPLC on a normal phase column with a photodiode array detector. Retinoid production (Table 4) was measured using expression vectors expressed on plasmid MB10157 according to SEQ ID NO: 1 or Figure 1 The percentage of retinoid (total retinoid titer from the shake plate assay described above) was set to 100% based on the reference McCarRP polynucleotide.

[0079] Table 4A: Effect of carRP alleles on retinoid output compared to single mutations of CarRP as indicated by "Insert" (e.g., E7D, A33N, etc.; plasmids MB10157-1 to MB10157-15 carrying mutated carRP genes) using wild-type carRP according to SEQ ID NO: 2 (expressing retinoids according to SEQ ID NO: 1 or Figure 1 The fraction of output or titer obtained with the wtCarRP plasmid MB10157) was used.

[0080] See the main text for more details.

[0081] plasmids Insert Retinoids [%] MB10157 MccarRP 100 MB10157-1 MccarRP_E7D 105 MB10157-2 MccarRP_A33N 146 MB10157-3 MccarRP_A153S 143 MB10157-4 MccarRP_L159V 141 MB10157-5 MccarRP_Y167F 124 MB10157-6 MccarRP_I194L 135 MB10157-7 MccarRP_T305A 123 MB10157-8 MccarRP_D330E 136 MB10157-9 MccarRP_D330N 105 MB10157-10 MccarRP_S430A 116 MB10157-11 MccarRP_S431T 130 MB10157-12 MccarRP_V432I 165 MB10157-13 MccarRP_I476A 125 MB10157-14 MccarRP_V547I 135 MB10157-15 MccarRP_R579K 138

[0082] Table 4B: Effect of carRP alleles on retinoid output using wild-type carRP according to SEQ ID NO: 2 (expressing retinoids according to SEQ ID NO: 1 or 2) compared to quintuple mutations of carRP as indicated by "Insert" (plasmids MB10157-16 to MB10157-29 carrying quintuple-mutated carRP genes). Figure 1 The titers obtained with the wt CarRP plasmid MB10157 are fractions of the output. See the text for more details.

[0083] plasmids Insert Retinoids [%] MB10157 MccarRP 100 MB10157-16 MccarRP_E7D_A33N_Y167F_V547I_R579K 159 MB10157-17 MccarRP_E7D_A33N_Y167F_D330N_V432I 149 MB10157-18 MccarRP_E7D_D330N_S430A_V547I_R579K 147 MB10157-19 MccarRP_E7D_A33N_Y167F_T305A_R579K 125 MB10157-20 MccarRP_E7D_A33N_Y167F_S431T_R579K 148 MB10157-21 MccarRP_E7D_A33N_Y167F_S430A_R579K 146 MB10157-22 MccarRP_E7D_A33N_Y167F_S430A_V547I 138 MB10157-23 MccarRP_E7D_A33N_Y167F_T305A_S430A 135 MB10157-24 MccarRP_E7D_A33N_S430A_V547I_R579K 175 MB10157-25 MccarRP_E7D_A33N_Y167F_T305A_D330N 128 MB10157-26 MccarRP_A33N_Y167F_S430A_V547I_R579K 155 MB10157-27 MccarRP_E7D_Y167F_S430A_V547I_R579K 172 MB10157-28 MccarRP_E7D_Y167F_S431T_V547I_R579K 151 MB10157-29 MccarRP_E7D_A33N_Y167F_V432I_V547I 136

[0084] Example 3: Production of various carotenoids or apocarotenoids in Yarrowia lipolytica expressing mutant carRP

[0085] Strains expressing mutant CarRP and specific genes for producing canthaxanthin, zeaxanthin, β-cryptoxanthin, anthoxanthin, or β-ionone were constructed as follows:

[0086] The β-carotene-producing strain ML15710 was transformed with plasmid MB6128 (SEQ ID NO: 6) containing the CRE recombinase and selected on medium containing geneticin. Hygromycin-sensitive isolates were confirmed among the transformants by replica plating onto selective and non-selective medium. One hygromycin-sensitive isolate was further propagated on non-selective medium and confirmed as geneticin-sensitive by replica plating onto selective and non-selective medium. One such isolate was 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 β-cryptoxanthin production; SEQ ID NO: 9), MB7918 (for anthaxanthin production; SEQ ID NO: 10), or MB6806 (for β-ionone production; SEQ ID NO: 11), and selected on medium containing hygromycin to produce canthaxanthin-producing strain ML15710+crtW, zeaxanthin-producing strain ML15710+crtZ, β-cryptoxanthin-producing strain ML15710+Lfreq-CrtZ, anthaxanthin-producing strain ML15710+bhy-21, or β-ionone-producing strain ML15710+CCD1. These strains were transformed with plasmid MB6128 containing CRE recombinase and selected on a culture medium containing geneticin. Hygromycin-sensitive isolates were confirmed in the transformants by copy-plating onto selective and non-selective culture media. A hygromycin-sensitive isolate (as noted above, for each apocarotenoid / carotenoid) was further propagated on non-selective culture media and confirmed by copy-plating onto selective and non-selective culture media. These strains were then transformed with MB10866 as described in Example 1 to produce white carRP mutant strains ML15710+crtW+carRP- (for canthaxanthin production), ML15710+crtZ+carRP- (for zeaxanthin production), ML15710+Lfreq-CrtZ+carRP- (for β-cryptoxanthin production), ML15710+bhy-21+carRP- (for anthocyanin production) or ML15710+CCD1+carRP- (for β-ionone production). Hygromycin-susceptible isolates were confirmed by passage on nonselective medium and replica plating onto selective and nonselective media.These strains were then transformed with plasmid MB9282 as in Example 1 to generate ku70- mutant strains ML15710+crtW+carRP-+ku70- (for canthaxanthin production), ML15710+crtZ+carRP-+ku70- (for zeaxanthin production), ML15710+Lfreq-CrtZ+carRP-+ku70- (for β-cryptoxanthin production), ML15710+bhy-21+carRP-+ku70- (for anthoxanthin production), or ML15710+CCD1+carRP-+ku70- (for β-ionone production). Uracil auxotrophic strains were isolated from these strains by selection on 5-FOA and designated ML15710+xx+carRP-+ku70-+ura3-, where "xx" represents the corresponding carotenoid / apocarotenoid specific gene. This white uracil auxotrophic strain was then transformed with plasmid MB10157 expressing wild-type McCarRP and with the corresponding plasmids expressing the mutant forms of McCarRP listed in Table 4 (see Example 2) to generate strains that further express heterologous CarRP (wild-type or mutant) canthaxanthin, zeaxanthin, β-cryptoxanthin, anthoxanthin or β-ionone. The strains were cultured in microtiter plates or fermentations as described in more detail below for canthaxanthin production, but mutatis mutandis for production of other carotenoids, such as zeaxanthin, β-cryptoxanthin, anthoxanthin or β-ionone:

[0087] As described in WO2022090549 or Example 2 above, transformants from strain ML15710+crtW+CarRP-+ku70-+ura3- were grown in microtiter plates with plasmid MB101570 or the plasmids shown in Table 5, except that a second phase was used and glucose was used as the sole carbon source. Fermentation and carotenoid analysis were performed according to previously described methods (see, for example, US7851199 Examples 2-4). Introduction of plasmids MB101570-7, MB10157-11, and MB10157-14 resulted in an increase in the percentage of total carotenoids (titer of total carotenoids), as measured by the shake plate assay described above, and an increase in the total carotenoid yield and the yield of canthaxanthin, i.e., the yield on carbon (g total carotenoids / g carbon source and g canthaxanthin / g carbon source, respectively), using expression vectors expressed on plasmid MB10157 according to SEQ ID NO: 1 or Figure 1 The percentage of the reference McCarRP polynucleotide was set to 100%.

[0088] Table 5: Effect of CarRP alleles on carotenoid export using wild-type carRP according to SEQ ID NO: 2 (expressing carotenoids according to SEQ ID NO: 1 or 2) compared to single mutations of carRP as indicated by "Insert" Figure 1 "CXN" refers to canthaxanthin. "Carotenoids [%]" reflects the carotenoid titer (total carotenoids) measured from the microtiter plate. "Carotenoid yield [%]" and "CXN yield [%]" mean the yield on carbon (g product / g carbon source) measured in the fermentation. For more details, see the text.

[0089]

[0090] Example 4: Production of astaxanthin in Yarrowia lipolytica expressing mutant carRP

[0091] The beta-carotene-producing strain ML15710 was transformed with plasmid MB6128 containing CRE recombinase and selected on a culture medium containing geneticin. Hygromycin-sensitive isolates were confirmed in the transformants by replica plating onto selective and non-selective culture media. A hygromycin-sensitive isolate was further propagated on non-selective culture media and confirmed by replica plating onto selective and non-selective culture media. A strain was transformed with PvuII linearized plasmid MB7082 (SEQ ID NO: 12) and selected on a culture medium containing nostril to generate the astaxanthin-producing strain ML15710+crtW. This strain was then transformed with PvuII linearized MB9930 (SEQ ID NO: 13) and selected on a culture medium containing geneticin to generate ML15710+crtW+crtZ. This strain was transformed with plasmid MB6128 containing CRE recombinase and selected on a culture medium containing geneticin. Hygromycin and nodularin sensitive isolates were confirmed in the transformants by replica plating onto selective and non-selective media. One isolate sensitive to hygromycin and nodularin was further propagated on non-selective media and confirmed as geneticin sensitive by replica plating onto selective and non-selective media. This strain was then transformed with MB10866 as described in Example 1 to generate the white carRP mutant strain ML15710+crtW+crtZ+carRP-. Hygromycin sensitive isolates were confirmed by passage on non-selective media and replica plating onto selective and non-selective media. This strain was then transformed with MB9282 as in Example 1 to generate the ku70- mutant strain ML15710+crtW+crtZ+carRP-+ku70-. A uracil auxotrophic strain was isolated from this strain by selection on 5-FOA and designated ML15710+crtW+crtZ+carRP-+ku70-+ura3-. The white uracil auxotrophic strain was then transformed with the plasmids expressing heterologous wt CarRP or mutants listed in Table 4 (Example 2) to generate an astaxanthin-producing strain that further expresses the mutant CarRP. Figure 1 Compared with the astaxanthin-producing strain expressing wt McCarRP, the astaxanthin titer can be increased by at least about 5% in the strain expressing the CarRP mutant (not shown).

[0092] Example 5: Lycopene production in Yarrowia lipolytica expressing mutant carRP

[0093] The DNA expressing the CarRP mutants described in Table 2 was combined with the mutation E78G by a DNA-synthesis provider (Genscript). The E78G mutation inactivates the lycopene cyclase domain of CarRP (see, for example, WO2014151748). The β-carotene-producing strain ML15710 was transformed with the plasmid MB6128 containing the CRE recombinase and selected on a culture medium containing geneticin. Hygromycin-sensitive isolates were confirmed in the transformants by replica plating onto selective and non-selective culture media. A hygromycin-sensitive isolate was further propagated on non-selective culture media and confirmed by replica plating onto selective and non-selective culture media. Then, one such isolate was transformed with MB10866 as described in Example 1 to generate a white carRP mutant strain ML15710+carRP-. Hygromycin-sensitive isolates were confirmed by passage on non-selective culture media and replica plating onto selective and non-selective culture media. This strain was then transformed with MB9282 as in Example 1 to generate the ku70- mutant strain ML15710+carRP-+ku70-. A uracil auxotrophic strain was isolated from this strain by selection on 5-FOA and designated ML15710+carRP-+ku70-+ura3-. This strain was transformed with SfiI linearized DNA containing the mutations in Table 2 in combination with the E78G mutation to generate a lycopene-producing strain that further expresses the mutant CarRP. This strain was then transformed with plasmids expressing heterologous wt CarRP or mutants in combination with the E78G mutation listed in Table 4 (Example 2) to generate a lycopene-producing strain that further expresses the mutant CarRP. Figure 1 Compared with the lycopene-producing strain expressing the wtMcCarRP, the lycopene titer can be increased by at least about 5% in the strain expressing the CarRP mutant (not shown).

[0094] Example 7: Production of phytoene in Yarrowia lipolytica expressing mutant carRP

[0095] The beta-carotene-producing strain ML15710 was transformed with plasmid MB6128 containing CRE recombinase and selected on a medium containing geneticin. Hygromycin-sensitive isolates were confirmed in the transformants by replica plating on selective and non-selective media. A hygromycin-sensitive isolate was further propagated on non-selective media and confirmed by replica plating on selective and non-selective media. Then, one such isolate was transformed with MB10866 as described in Example 1 to generate a white carRP mutant strain ML15710+carRP-. Hygromycin-sensitive isolates were confirmed by passage on non-selective media and replica plating on selective and non-selective media. Then, one such isolate was transformed with MB7522 (SEQ ID NO: 14) to generate a carB mutant strain ML15710+carRP-+carB-. Hygromycin-sensitive isolates were confirmed by passage on non-selective media and replica plating on selective and non-selective media. This strain was then transformed with MB9282 as in Example 1 to generate the ku70- mutant strain ML15710+carRP-+carB-+ku70-. A uracil auxotrophic strain was isolated from this strain by selection on 5-FOA and designated ML15710+carRP-+carB-+ku70-+ura3-. This white uracil auxotrophic strain was then transformed with the plasmids expressing heterologous wt CarRP or mutants listed in Table 4 (Example 2) to generate a phytoene-producing strain that further expresses a mutant CarRP. Figure 1 Compared with the phytoene-producing strain expressing the wtMcCarRP, the titer of phytoene can be increased by at least about 8% in the strain expressing the CarRP mutant (not shown).

[0096] Example 8: Production of β-carotene in Yarrowia lipolytica expressing mutant carRP

[0097] The beta-carotene-producing strain ML15710 was transformed with plasmid MB6128 containing CRE recombinase and selected on a culture medium containing geneticin. Hygromycin-sensitive isolates were confirmed in the transformants by replica plating onto selective and non-selective culture media. A hygromycin-sensitive isolate was further propagated on non-selective culture media and confirmed as a geneticin-sensitive isolate by replica plating onto selective and non-selective culture media. Then, such an isolate was transformed with MB10866 as described in Example 1 to generate a white carRP mutant strain ML15710+carRP-. Hygromycin-sensitive isolates were confirmed by passage on non-selective culture media and replica plating onto selective and non-selective culture media. Then, such an isolate was transformed with MB9282 as in Example 1 to generate a ku70- mutant strain ML15710+carRP-+ku70-. A uracil auxotrophic strain was isolated from this strain by selection on 5-FOA and designated ML15710+carRP-+ku70-+ura3-. This white uracil auxotrophic strain was then transformed with the plasmids expressing heterologous wt CarRP or mutants listed in Table 4 (Example 2) to generate a β-carotene-producing strain that further expresses a mutant CarRP. Figure 1 Compared with the β-carotene-producing strain expressing wt McCarRP, the titer of β-carotene can be increased by at least about 5% in the strain expressing the CarRP mutant (not shown).

Claims

1. A modified bifunctional enzyme that catalyzes the conversion of geranylgeranyl pyrophosphate (GGPP) to phytoene and / or catalyzes the conversion of lycopene to β-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 the sequence shown in Figure 1 or SEQ ID NO: 1, wherein the one or more amino acid substitutions are introduced into a sequence corresponding to the sequence shown in Figure 1 or according to SEQ ID NO:

1. At a position of the amino acid residue in the polypeptide of NO: 1 selected from positions 7, 33, 153, 159, 167, 194, 305, 330, 430, 431, 432, 476, 547, 579 and combinations thereof.

2. The modified enzyme according to claim 1 , wherein the amino acid residue at the position corresponding to position 7 as shown in FIG. 1 or in SEQ ID NO: 1 is other than glutamic acid or glutamine, wherein the amino acid residue at the position corresponding to position 33 as shown in FIG. 1 or in SEQ ID NO: 1 is other than alanine or tryptophan, wherein the amino acid residue at the position corresponding to position 153 as shown in FIG. 1 or in SEQ ID NO: 1 is other than alanine, wherein the amino acid residue at the position corresponding to position 159 as shown in FIG. 1 or in SEQ ID NO: 1 is other than leucine, wherein the amino acid residue at the position corresponding to position 167 as shown in FIG. 1 or in SEQ ID NO: 1 is other than tyrosine, wherein the amino acid residue at the position corresponding to position 194 or 476 as shown in FIG. 1 or in SEQ ID NO: 1 is other than isoleucine, wherein the amino acid residue at the position corresponding to position 305 as shown in FIG. 1 or in SEQ ID NO: 1 is other than threonine, wherein the amino acid residue at the position corresponding to position 330 as shown in FIG. 1 or in SEQ ID NO: 1 is other than aspartic acid, wherein the amino acid residue at the position corresponding to position 157 as shown in FIG. 1 or in SEQ ID NO: 1 is other than aspartic acid, The amino acid residue at position 430 or 431 in NO: 1 is different from serine, wherein the amino acid residue at the position corresponding to position 432 or 547 shown in Figure 1 or in SEQ ID NO: 1 is different from valine, and / or wherein the amino acid residue at the position corresponding to position 579 shown in Figure 1 or in SEQ ID NO: 1 is different from arginine.

3. The modified enzyme according to 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 a combination thereof in the polypeptide depicted in Figure 1 or according to SEQ ID NO: 1 are selected from D7, N33, S153, V159, F167, L194, A305, E330, N330, A430, T431, 1432, A476, 1547 and / or K579.

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, 1194L, T305A, D330E, D330N, S430A, S431T, V432I, 1476A, 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 the sequence shown in Figure 1 or to SEQ ID NO:

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 claim 1 , which is introduced into and expressed in a suitable host cell producing carotenoids and / or apocarotenoids, in particular a host cell producing retinoids.

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

1.

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

1.

9. A host cell for producing carotenoids and / or apocarotenoids heterologously expressing the modified enzyme according to any one of claims 1 to 8.

10. The host cell according to claim 9, which is a fungal host cell or a fungal host cell selected from Escherichia coli, preferably selected from Yarrowia or Saccharomyces.

11. The host cell according to claim 9 or 10, heterologously expressing a gene involved in the biosynthesis of carotenoids and / or apocarotenoids selected from the group consisting of β-carotene, lycopene, phytoene, β-ionone, β-cryptoxanthin, canthaxanthin, astaxanthin, zeaxanthin, anthoxanthin, retinal, retinol, retinyl acetate, and mixtures thereof.

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

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

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

Images