WO2019016382A1

WO2019016382A1 - Novel ferredoxin reductase

Info

Publication number: WO2019016382A1
Application number: PCT/EP2018/069800
Authority: WO
Inventors: Bastien Chevreux; John Royer
Original assignee: Dsm Ip Assets B.V.
Priority date: 2017-07-21
Filing date: 2018-07-20
Publication date: 2019-01-24

Abstract

The present invention is related to novel protein involved in transfer of electrons, in particular in an enzymatic conversion of carotenoids into particular retro-carotenoids, more particularly in a conversion of beta-carotene into rhodoxanthin via the catalytic action of rhodoxanthin-producing hydroxylases (hereinafter also referred to as BHYRs). The invention also features polynucleotides and the corresponding polypeptides comprising the full-length sequences of the novel gene/polypeptide and fragments thereof, in particular functional equivalents of said gene/polypeptide. The invention further relates to genetically engineered carotene producing fungal host cells and their use for biotechnological production of rhodoxanthin.

Description

NOVEL FERREDOXIN REDUCTASE

The present invention is related to novel protein involved in transfer of electrons, in particular in an enzymatic conversion of carotenoids into particular retro- carotenoids, more particularly in a conversion of beta-carotene into rhodoxanthin via the catalytic action of rhodoxanthin-producing hydroxylases (hereinafter also referred to as BHYRs). The invention also features polynucleotides and the corresponding polypeptides comprising the full-length sequences of the novel gene/polypeptide and fragments thereof, in particular functional equivalents of said gene/polypeptide. The invention further relates to genetically engineered carotenoid-producing fungal host cells and their use for biotechnological production of rhodoxanthin.

Many cellular processes require the action of specific electron transfer systems. Examples of such compounds acting as donor and/or acceptor of electrons, include but are not limited to co-factors and/or proteins selected from the group consisting of NAD(H), NADP(H), ferredoxin, ferredoxin oxidoreductase, flavodoxin, flavodoxin oxidoreductase, putaredoxin, putaredoxin reductase,

monodehydroascorbate reductase, glutathione reductase, AOX (mitochondrial alternative oxidase), PTOX (plastid terminal oxidase) and adrenodoxin.

The selection of suitable proteins and/or co-factors acting as donors/acceptors of electrons is complex and likely dependent on several factors, such as e.g. the host cell, the enzyme, the substrate. There is probably no one-fits-for-all solution available.

Retro-carotenoids are carotenoids with a shift of one position of the single and double bonds of the respective conjugated polyene systems. There are indications that some of them have stronger antioxidative activity for lipid peroxidation induced by free radical and singlet oxygen than that of β-carotene, i.e. the non-retro type carotenoids.

Rhodoxanthin, an example of a retro-carotenoid, and which is found in nature in e.g. arils, berries, leaves or flowers of the poisonous yew (Taxus), Aloe or honeysuckle (Lonicera sp.), is widely used as a coloring material for foodstuffs and beverages as well as pharmaceutical and cosmetic preparations, imparting to them a yellow to red coloration. As a food additive, it is used under the E number E161f as a food coloring. Nowadays, there is a strong need for a biotechnological production of

rhodoxanthin and other retro-carotenoids, which can be used as e.g. coloring material in the food & beverage, pharmaceutical and cosmetic industry in order to replace the chemical produced (synthetic) rhodoxanthin.

A rhodoxanthin-producing beta-carotene hydroxylase (herein referred as BHYR) has been identified from the red berries of Lonicera (SEQ ID NO:4) with the aim to biotechnologically generate rhodoxanthin in the respective host cell, such as e.g. fungi, in particular strains of Yarrowia, more preferably Y. Iipolytica, Unexpectedly, it turned out that in-vivo activity of the BHYR, in particular in a process using carotenoid-producing fungi, including yeast, for production of retro-carotenoids, is highly dependent on specific/special electron transfer systems.

Thus, it is an ongoing task to optimize enzymatic conversion of carotenoids into rhodoxanthin in a suitable host cell, such as e.g. a carotenoid-producing fungal cell including yeast, in order to satisfy the need for biotechnologically produced

"natural" coloring material to be used in food, beverage, pharmaceutical and cosmetic industry.

Surprisingly, we have now isolated a novel ferredoxin NADP reductase homolog originating from Aloe which is in particular useful in a biotechnological production of rhodoxanthin in a suitable host cell, in particular in a carotenoid-producing fungal host, including yeast, expressing a beta-carotene hydroxylase herein also referred to as BHYR, with enzymatic activity towards biosynthesis of rhodoxanthin. Using a carotenoid-producing fungal host cell comprising the novel ferredoxin reductase homolog as described herein rhodoxanthin-peaks could be detected in HPLC.

In particular, the present invention is directed to a polynucleotide having electron transfer activity, i.e. ferredoxin reductase activity, which is selected from a polypeptide with at least 82%, such as e.g. 85, 90, 92, 95, 97, 99 or even up to 100% identity to SEQ ID NO:2, which might be encoded by a polynucleotide including, but not limited to SEQ ID NO:1 or SEQ ID NO:1 1 , in particular a recombinant nucleic acid molecule.

Furthermore, the present invention is directed to the use of the novel protein having ferredoxin reductase activity in a biotechnological production of

rhodoxanthin in a suitable host cell, in particular a carotenoid-producing fungal host including yeast, wherein said host cell furthermore is expressing a gene encoding a polypeptide having beta-carotene hydroxylase activity and which is selected from a polypeptide with at least 63 %, such as e.g. at least 65, 70, 75,80, 85, 90, 95, 97, 98, 99 or even 100% identity to SEQ ID NO:4, which might be encoded by a polynucleotide including, but not limited to SEQ ID NO:3, in particular a recombinant nucleic acid molecule.

In another embodiment, the present invention is directed to the use of the novel protein having ferredoxin reductase activity in a biotechnological production of rhodoxanthin in a suitable host cell, in particular a carotenoid-producing fungal host including yeast, wherein said host cell furthermore is expressing a gene encoding a polypeptide having beta-carotene hydroxylase activity and which is selected from a polypeptide with at least 78%, such as e.g. 80, 85, 90, 95, 97, 98, 99 or even 100% identity to SEQ ID NO:6, which might be encoded by a polynucleotide including, but not limited to SEQ ID NO:5, in particular a

recombinant nucleic acid molecule. Preferably, the protein having ferredoxin reductase activity is obtainable from Aloe as shown in SEQ ID NO:2, in particular encoded by a polynucleotide according to SEQ ID NO:1 . Based on the novel function, said protein with at least 82% identity to SEQ ID NO:2 is hereinafter defined as "Aloe ferredoxin reductase" or "Aloe FNR". The enzyme according to SEQ ID NO:2 shows an amino acid identity of only 81 .4% to the known ferredoxin- NADP reductase from Nicotiana tabacum Accession No. O04977.

According to the present invention, the terms "oxygenase", "hydroxylase", "beta- carotene hydroxylase" or "BHYR" are used interchangeably herein. In one embodiment the present invention is related to a process for conversion of beta- carotene into rhodoxanthin, said conversion being catalyzed by a protein having at least 63 %, such as e.g. 65, 70, 75, 80, 85, 90, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:4, which might be encoded by a polynucleotide including, but not limited to SEQ ID NO:3, in particular a recombinant nucleic acid molecule, said conversion being performed in the presence of the Aloe FNR as described herein.

The host cell used for production of rhodoxanthin as defined herein, i.e.

expressing a polypeptide having beta-carotene hydroxylase activity with at least 63 %, such as e.g. 65, 70, 75,80, 85, 90, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:4 together with a polypeptide having ferredoxin reductase activity with at least 82% identity to SEQ ID NO:2 can be selected from any fungal host suitable for carotenoid production, including carotenoid-producing yeast cell such as e.g. Yarrowia.

As used herein, a carotenoid-producing host cell is a host cell wherein the respective polypeptides are expressed and active in vivo leading to production of carotenoids. The genes and methods to generate carotenoid-producing host cells are known in the art. Depending on the carotenoid to be produced, different genes might be involved. As defined herein, a rhodoxanthin-producing host cell is capable of expressing a polypeptide having beta-carotene hydroxylase activity with at least 63%, such as e.g. 65, 70, 75, 80, 85, 90, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:4.

Particularly useful hosts are fungi, including hosts of the known culture collections as published by the World Federation for Culture Collections (WFCC). In particular, a fungal cell, including yeast, is selected from the group consisting of Saccharomyces, Aspergillus, Pichia, Hansenula, Phycomyces, Mucor,

Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea, and Yarrowia, preferably selected from the group consisting of Saccharomyces cerevisiae, Aspergillus niger, Pichia pastoris, Hansenula polymorpha, Phycomyces blakesleanus, Blakeslea trispora and Yarrowia lipolytica, In particularly preferred is expression in Yarrowia, most preferably expression in Yarrowia lipolytica.

It is understood that the above-mentioned microorganisms also 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).

As used herein, the term "% identity" and "identity" refers to the comparison of two amino acid sequences using a sequence analysis program as for instance Blast or Clustal Omega. Secondary structure prediction can be done by at least use of the Prime software from Schrodinger or by on-line software tools such as JPred. The term % identical refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical amino acids in the compared amino acid sequence. If both amino acid sequences which are compared do not differ in any of their amino acids, they are identical or have 100% identity. It is understood, that for such a comparison based on % identity in an area of at least 100 consecutive nucleotides/amino acids, such as at least 300, preferably at least 500 consecutive nucleotides/amino acids, is selected. As used herein, the present invention is directed to a novel ferredoxin reductase as defined herein involved in electron transfer in the enzymatic conversion of carotenoids into rhodoxanthin, said bioconversion being catalyzed by a beta- carotene hydroxylating enzyme such as BHYR as defined herein. The BHYr gene might be truncated and/or codon-optimized for expression in the fungal host cell, such as e.g. Yarrowia (see SEQ ID NO:5).

In one embodiment, the rhodoxanthin-producing host cell, in particular a

carotenoid-producing fungal host cell including yeast, preferably Yarrowia, furthermore comprises a protein having ferredoxin activity, in particular wherein the gene encoding said polypeptide being originating from Aloe, preferably with at least 59 %, such as e.g. at least 60, 65, 70, 75, 80, 90, 92, 95, 98, 99% or up to 100% identity to SEQ ID NO:8, which might be encoded by a polynucleotide including, but not limited to SEQ ID NO:7 or SEQ ID NO:12. Thus, a preferred fungal carotenoid producing host strain, including yeast, preferably Yarrowia, useful for conversion of beta-carotene into rhodoxanthin comprises (a) a

polynucleotide expressing a polypeptide having ferredoxin reductase activity with at least 82%, such as e.g. at least 85, 90, 92, 95, 97, 99% or up to 100% identity to SEQ ID NO:2, (b) a polynucleotide expressing a polypeptide having beta- carotene hydroxylase activity with at least 63 %, such as e.g. 65, 70, 75, 80, 85, 90, 95, 97, 98, 99 or even 100% identity to SEQ ID NO:4, and (c) a polynucleotide expressing a polypeptide having ferredoxin activity with at least 59%, such as e.g. 60, 65, 70, 75, 80, 90, 92, 95, 98, 99% or event up to 100% identity to SEQ ID NO:8.

In even a further embodiment, the rhodoxanthin-producing host cell, in particular a carotenoid-producing fungal host cell including yeast, preferably Yarrowia, furthermore comprises a protein having chloroplast processing enzyme (CPE) activity, such as, e.g. a gene encoding stromal processing peptidase (UniProtKB Q40983) from Pisum sativum, preferably with at least 80%, such as 85, 90, 92, 95, 97, 99 or even 100% identity to SEQ ID NO:10, which might be encoded by a polynucleotide including, but not limited to SEQ ID NO:9. Thus, a preferred carotenoid fungal host strain, including yeast, preferably Yarrowia, useful for conversion of carotenoid into rhodoxanthin comprises (a) a polynucleotide expressing a polypeptide having ferredoxin reductase activity with at least 82%, such as e.g. 85, 90, 92, 95, 97, 99% or up to 100% identity to SEQ ID NO:2, (b) a polynucleotide expressing a polypeptide having beta-carotene hydroxylase activity with at least 78%, such as e.g. 80, 85, 90, 95, 97, 98, 99 or even 100% identity to SEQ ID NO:4, (c) a polynucleotide expressing a polypeptide having ferredoxin activity with at least 59%, such as e.g. 60, 65, 70, 75, 80, 90, 92, 95, 98, 99% or event up to 100% identity to SEQ ID NO:8, and (d) a polynucleotide expressing a polypeptide having CPE activity with at least 80%, such as 85, 90, 92, 95, 97, 99 or even 100% identity SEQ ID NO:10.

As used herein, the term "ferredoxin activity" encompasses activity of all three ferredoxin isoforms, i.e. as ferredoxin 1 , 2 and/or 3, and both leaf and

heterotrophic versions of the proteins

Depending on the host cell, the polynucleotides expressing BHYR, AloeFNR, Aloe FD or CPE as defined herein, might be optimized for expression in the respective host cell. The skilled person knows how to generate such modified

polynucleotides. It is understood that the polynucleotides as defined herein also encompass such host-optimized nucleic acid molecules as long as they still express the polypeptide with the respective activities as defined herein.

The novel enzyme according to the present invention also encompasses enzymes carrying amino acid substitution(s) which do not alter enzyme activity, i.e. which show the same properties with respect to the wild-type enzyme and catalyze at least one of the abovementioned oxidation reduction reactions reactions. Such mutations are also called "silent mutations", which do not alter the (enzymatic) activity of the enzyme as described herein.

A nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence provided by the present invention, such as for instance the sequence shown in SEQ ID NO:1 1 , for example a fragment which may be used as a probe or primer or a fragment encoding a portion of a protein according to the invention. The nucleotide sequence determined from the cloning of the FDR gene allows for the generation of probes and primers designed for use in identifying and/or cloning other FDR family members, as well as FDR homologues from other species. The probe/primer typically comprises

substantially purified oligonucleotides which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least about 12 or 15, preferably about 18 or 20, more preferably about 22 or 25, even more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 or more consecutive nucleotides of a nucleotide sequence shown in SEQ ID NO:1 1 or a fragment or derivative thereof.

A preferred, non-limiting example of such hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 1 x SSC, 0.1 % SDS at 50°C, preferably at 55°C, more preferably at 60°C and even more preferably at 65°C.

Highly stringent conditions include, for example, 2 h to 4 days incubation at 42°C using a digoxigenin (DIG)-labeled DNA probe (prepared by using a DIG labeling system; Roche Diagnostics GmbH, 68298 Mannheim, Germany) in a solution such as DigEasyHyb solution (Roche Diagnostics GmbH) with or without 100 g/ml salmon sperm DNA, or a solution comprising 50% formamide, 5x SSC (150 mM NaCI, 15 mM trisodium citrate), 0.02% sodium dodecyl sulfate, 0.1 % N- lauroylsarcosine, and 2% blocking reagent (Roche Diagnostics GmbH), followed by washing the filters twice for 5 to 15 minutes in 2x SSC and 0.1 % SDS at room temperature and then washing twice for 15-30 minutes in 0.5x SSC and 0.1 % SDS or 0.1 x SSC and 0.1 % SDS at 65-68°C.

The host cell, such as e.g. a carotenoid-producing fungal cell, which is able to express the BHYR, FNR, FD with or without CPE according to the present invention may be cultured in an aqueous medium supplemented with appropriate nutrients under aerobic or anaerobic conditions and as known by the skilled person for the different host cells. Optionally, such cultivation is in the presence of proteins and/or co-factors involved in transfer of electrons, as defined herein. The cultivation/growth of the host cell may be conducted in batch, fed-batch, semi- continuous or continuous mode. Depending on the host cell, preferably, production of retro-carotenoids such as e.g. rhodoxanthin is performed in a fed-batch process using corn oil as carbon source.

In one embodiment, a process according to the present invention is directed to production of rhodoxanthin from beta-carotene, comprising the steps of:

(a) expression of ferredoxin reductase as defined herein from Aloe in a suitable carotenoid-producing (fungal) host cell, said protein being preferably a polypeptide according to SEQ ID NO:2 or a polypeptide with at least 82%, such as e.g. 85, 90, 92, 95, 97, 99% or up to 100% identity to SEQ ID NO:2;

(b) contacting said FDR with a polypeptide having activity towards conversion of beta-carotene into rhodoxanthin, such as BHYR as defined herein, preferably a BHYR with at least 63%, such as e.g. 65, 70, 75, 80, 85, 90, 95, 97, 98, 99 or even 100% identity to SEQ ID NO:4,

(c) optionally, expression of ferredoxin as defined herein from Aloe, said protein being preferably a polypeptide according to SEQ ID NO:8 or a polypeptide with at least 59%, preferably at least 60, 65, 70, 75, 80, 90, 92, 95, 98, 99 or event up to 100% identity to SEQ ID NO:8,

(d) optionally, expression of CPE as defined herein from Pisum sativum, said protein being preferably a polypeptide according to SEQ ID NO:10 or a

polypeptide with at least 80%, preferably at least 85, 90, 92, 95, 97, 99 or even 100% identity to SEQ ID NO:1 1 ,

(e) production of rhodoxanthin from beta-carotene under suitable culture conditions; and optionally;

(f) isolating the produced rhodoxanthin from the culture medium with or without further purification step(s).

As used herein, the term "specific activity" or "activity" with regards to enzymes means its catalytic activity, i.e. its ability to catalyze formation of a product from a given substrate. The specific activity defines the amount of substrate consumed and/or product produced in a given time period and per defined amount of protein at a defined temperature. Typically, specific activity is expressed in μιτιοΙ substrate consumed or product formed per min per mg of protein. Typically, mol/min is abbreviated by U (= unit). Therefore, the unit definitions for specific activity of mol/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. within the host cell as defined herein or within a system in the presence of a suitable substrate. The skilled person knows how to measure enzyme activity, in particular activity of an oxygenase as defined herein. With regards to the present invention, "FNR activity" is defined as the capability to facilitate the electron transfer in the oxidation of carotenoid beta-carotene into rhodoxanthin. FNR activity is also defined as the capability to facilitate the electron transfer in the production of other plant metabolites, carotenoid, retrocarotenoid such as eschscholtzanthin. Production of rhodoxanthin in the presence of the FNR homolog as defined herein can be measured by HPLC as known by the skilled person (see Figure 1 ). Rhodoxanthin as used herein includes any chemical form of rhodoxanthin found in aqueous solutions, including all isoforms. In particular, it includes a mixture of cis EZ-, EE and ZZ-rhodoxanthin.

The following examples are illustrative 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.

Figure 1. HPLC chromatogram (left side) and UV spectra for the individual peaks (right side) of Yarrowia lipolytica extracts. Y. lipolytica transformed with CarB and CarRP from Mucor circinellioides, and BHYr from Lonicera sp. (A) is compared to Y. lipolytica transformed with CarB and CarRP from Mucor circinellioides, BHYr from Lonicera sp, CPE from Pisum sativum, ferredoxin (Fd2) and ferredoxin reductase (FNR) from Aloe sp. (B). Peak 1 : beta-cryptoxanthin; peak 2:

rhodoxanthin cis zz isomer; peak 3: rhodoxanthin cis ez isomer; peak 4:

rhodoxanthin cis ee isomer; peak 5: zeaxanthin. Absorbance at 494 nm is shown on the y-axis, time in minutes is shown on the x-axis.

Figure 2. Sequences 1 to 12 as used herein (for more explanation see text).

Examples Example 1 : Cloning of ferredoxin reductase from Aloe

A transcriptomics approach was used to identify genes that were highly expressed in rhodoxanthin-producing flowers of Pink Blush Aloe. A gene (FNR SEQ ID NO:1 1 ) with homology to ferredoxin NADPH reductase was found to be highly expressed. A Yarrowia codon optimized version of the gene (SEQ ID No:1 )with restriction sites for cloning into Yarrowia expression vectors was synthesized (Genscript) and cloned into a hygromycin expression vector to generate plasmid pMB8056

Example 2: Screening for Aloe FNR homologs

Ferredoxin homologues can be identified from the publicly available protein and nucleic acid databases. Alternatively, the The Aloe FNR DNA sequence according to SEQ ID NO:1 1 can be used as probe in a Southern blot to identify homologous FNR clones from other organisms, in particular from organisms which are able to synthesize carotenoid, in particular rhodoxanthin or other retro carotenoids from beta-carotene. A standard protocol for Southern blot hybridization is described "Molecular cloning: A lab manual. Sambrook, J, E. F. Fritsch, T. Maniatis 1989"

Identified clones are further tested together for their activity on electron transfer in the presence of a rhodoxanthin forming protein (BHYR) as defined in SEQ ID NO:4.

Example 3: Expression of FNR and production of rhodoxanthin in a yeast system

Rhodoxanthin production in Yarrowia lipolytica was enabled by introducing the full- length Aloe FNR gene into a beta-carotene producing Yarrowia strain comprising DNA expressing BHYR, full-length Aloe ferredoxin along with DNA expressing the chloroplast processing enzyme from Pisum sativum (UniProtKB Q40983; SEQ ID NO:10) designed to cleave the chloroplast targeting signal from the ferredoxin and ferredoxin reductase.

The BHYr gene of Lonicera (SEQ ID NO:3) was truncated to remove the presumed chloroplast targeting signal, and modified by codon optimization for expression in Yarrowia to result in SEQ ID NO:5. An Nhel site was incorporated into the 5'-region, and an Mlul site was incorporated into the 3'-region to allow subcloning into the Yarrowia expression vector MB6157 to generate plasmid MB7918, where expression of BHYr is under the control of the Yarrowia Tef promoter. MB7918 was transformed into the beta-carotene producing strain ML2461 which contains Mucor circinellioides CarB (phytoene dehydrogenase; CAB40843.1 ) and M. circinellioides CarRP (lycopene cyclase/phytoene synthase; CAB60272.1 ) to generate strain ML17461 . ML17461 was grown on YPoil (yeast extract (10 g/l), peptone (20 g/l), tryptophan (0.15 g/l) and corn oil 2%) medium and samples were extracted and analyzed by normal phase HPLC. Small amounts of beta-cryptoxanthin, zeaxanthin, and tentative rhodoxanthin EZ were produced (Fig.l A).

DNA sequences for Aloe ferredoxin reductase, Aloe ferredoxin and P. sativum CPE genes were Yarrowia codon-optimized (see SEQ ID NOs:1 , 7, 9) and synthesized with an Nhel site at the 5'-ends and an Mlul site at the 3'-ends to allow cloning into Yarrowia expression vectors to generate pMB 8056, pMB8059, and pMB 7896. Expression of FD and FNR was with the TEF promoter, while expression of CPE was with the Alk1 promoter of Yarrowia . Strain ML 17769, which contains the Mucor CarB and CarRP genes for beta carotene production, the P. sativum chloroplast targeting enzyme (CPE), the Aloe ferredoxin and ferredoxin reductase, and the truncated BHYR gene was grown on YPoil (yeast extract (10 g/l), peptone (20 g/l), tryptophan (0.15 g/l) and corn oil 2%) medium and samples were extracted and analyzed by normal phase HPLC.

ML17769 produced significantly higher levels of rhodoxanthin than strain ML17461 containing CarB, CarRP and BHYR, alone (Fig.l B).

Claims

1 . A nucleic acid molecule encoding a polypeptide having ferredoxin reductase activity involved in conversion of beta-carotene into rhodoxanthin, said

polynucleotide being selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide with at least 82% identity to SEQ ID NO:2, and

(b) a polynucleotide the complementary strand of which hybridizes under stringent conditions to a polynucleotide defined in (a).

2. A nucleic acid molecule according to claim 1 which is operatively linked to an expression control element.

3. A polypeptide encoded by a polynucleotide according to claim 1 or 2.

4. Use of a polypeptide according to claim 3 in a process for conversion of beta- carotene into rhodoxanthin, wherein said polypeptide is acting as electron transfer protein.

5. A carotenoid-producing host cell, preferably a microorganism, more preferably a fungal strain, even more preferably a yeast, most preferably Yarrowia, comprising a nucleotide acid molecule according to claim 1 or 2.

6. A carotenoid-producing host cell according to claim 5, further comprising a nucleotide acid molecule encoding a polypeptide having beta-carotene

hydroxylase activity, said polypeptide having at least 63 % identity to a polypeptide according to SEQ ID NO:4.

7. A carotenoid-producing host cell according to claim 6, further comprising a polypeptide having ferredoxin 2 activity, said polypeptide having at least 59 % identity to a polypeptide according to SEQ ID NO:8.

8. A carotenoid-producing host cell according to claim 6 or 7, further comprising a polypeptide having CPE activity, said polypeptide having at least 80% identity to a polypeptide according to SEQ ID NO:10.

9. A process for conversion of a carotenoid, preferably a carotenoid selected from the group consisting of lutein, beta-carotene, cantaxanthin, cryptoxanthin, phytoene, lycopene, eschscholtzxanthin and zeaxanthin into rhodoxanthin, using a host cell according to any of claims 6 to 8.

10. A process for biosynthesis of rhodoxanthin comprising the steps:

(a) introduction of a nucleic acid molecule according to claim 1 or 2 into a carotenoid-producing host cell,

(b) introduction of a rhodoxanthin-synthesizing polynucleotide capable of conversion of beta-carotene into rhodoxanthin in the presence of a polypeptide according to claim 3,

(c) conversion of beta-carotene into rhodoxanthin under suitable conditions, and optionally

(d) isolation and/or purification of the rhodoxanthin from the host cell.

1 1 . A process according to claim 10, further comprising introduction of a nucleic acid molecule expressing a polypeptide having ferredoxin 2 activity, preferably a polypeptide with at least 59 % identity to SEQ ID NO:8.

12. A process according to claim 10 or 1 1 , further comprising introduction of a nucleic acid molecule expressing a polypeptide having CPE activity, preferably a polypeptide with at least 80% identity to SEQ ID NO:10.