WO2021151891A1

WO2021151891A1 - Novel phytoene desaturase variants to produce neurosporene and/or zeta-carotene

Info

Publication number: WO2021151891A1
Application number: PCT/EP2021/051759
Authority: WO
Inventors: Sandra CASTANG; Emmanuelle CAMBON; Georges Gaudriault; Marie GRAINDORGE; Guillaume Letellier
Original assignee: Deinove
Priority date: 2020-01-27
Filing date: 2021-01-26
Publication date: 2021-08-05
Also published as: WO2021151891A9

Abstract

The present invention relates to phytoene desaturase variants exhibiting modified product specificity towards neurosporene and/or zeta-carotene. The present invention also relates to a method of producing neurosporene and/or zeta-carotene using said variants.

Description

Novel phytoene desaturase variants to produce neurosporene and/or zeta-carotene

FIELD OF THE INVENTION

The present invention relates to the field of microbiology and in particular to the field of biosynthetic pathway engineering. More specifically, the present invention relates to the field of production of neurosporene and/or zeta-carotene using phytoene desaturase variants and genetically modified bacteria.

BACKGROUND OF THE INVENTION

Carotenoids are a class of natural pigments that are synthesized by all photosynthetic organisms and in some heterotrophic growing bacteria and fungi. Because animals are unable to synthetize de novo these molecules, carotenoids have been widely used commercially as food supplements, animal feed additives or nutraceuticals. They have also found various applications as colorants or for cosmetic and pharmaceutical purposes.

One of these molecules, namely neurosporene, was found to exhibit various biological activities such as antioxidant. Neurosporene is an intermediate in the synthesis of lycopene from phytoene. Indeed, phytoene is isomerized to all-trans-phytoene and then undergoes a series of desaturation reactions. The number of these reactions depends on the species and the enzyme. Some enzymes stop after two steps resulting in the formation of zeta-carotene, some catalyze three steps resulting in the formation of neurosporene, and some continue with a fourth step, resulting in the formation of lycopene.

Unlike lycopene or beta-carotene, microbial production of neurosporene or zeta-carotene has been rarely described, probably because these compounds are often bound intermediates of phytoene desaturase Crtl. Indeed, in most bacteria, Crtl phytoene desaturase catalyzes a four- step desaturation converting phytoene into lycopene without any detectable amounts of intermediate products, i.e. phytofluene, zeta-carotene and neurosporene. Other phytoene desaturases such as Crtl from Rubrivivax gelatinosus, may catalyze simultaneously a three- and four- step de saturation producing both neurosporene and lycopene.

Neurosporene was shown to be accumulated as a major carotenoid by a mutant strain of Rhodobacter capsulatus (Scolnik et al. 1980, J Biol Chem 255:2427-2432) or was genetically engineered using a phytoene desaturase gene (crtl) cluster of Rubrivivax gelatinosus and expressed in Escherichia coli (Harada et al., 2001, Plant Cell Physiol. 42: 1112-1118). However, both of these two processes yielded low amounts of neurosporene due to unstable strains. Natural and stable neurosporene accumulating, phototrophic purple non-sulfur bacterium Rhodobacter viridis has also been described (Ramaprasad et al. 2013, Biotechnol Lett. 35:1093-1097) but such strain is not adapted to industrial production. Some studies disclosed the production of phytoene desaturase variants with modi(ie< product specificity (Stickforth and Sandmann, Arch Biochem Biophys. 2011 Jan 1 ;505(1): 118 22; Schmidt-Dannert et al. Nat Biotechnol. 2000, 18, 750-753) but the need for enzymes t< efficiently produce neurosporene or zeta-carotene remains largely unsatisfied. SUMMARY OF THE INVENTION

The invention aims to provide variants of Deinococcus geothermalis phytoene desaturasi Crtl which are able to produce detectable amounts of neurosporene and/or zeta-carotene iron phytoene. In particular, these variants exhibit a modified product specificity toward: neurosporene and/or zeta-carotene as compared to the wild-type phytoene desaturase. With thi wild-type enzyme, neurosporene and zeta-carotene are bound intermediates of the desaturatioi pathway to lycopene and are thus non-detectable products. Using the variants of the invention significant amounts of neurosporene and/or zeta-carotene are produced. On the other side, thi production of lycopene is dramatically reduced.

Accordingly, the present invention relates to a phytoene desaturase variant comprising ; sequence (i) having at least 65% identity to the full length amino acid sequence set forth in SE( ID NO: 1 and (ii) comprising at least one substitution at position corresponding to residue G521 R171, A253 or G172 of SEQ ID NO: 1, preferably at position corresponding to residue G521 R171 or G172 of SEQ ID NO: 1, wherein said variant exhibits a modified product specificity towards neurosporene and/or zeta-carotene, preferably neurosporene, as compared to phytocm desaturase of SEQ ID NO: 1. In particular, the phytoene desaturase variant of the presen invention produces neurosporene and/or zeta-carotene, preferably neurosporene.

The variant may comprise a substitution at position corresponding to residue G521 o SEQ ID NO: 1. In particular, the variant may comprise a substitution at position correspond to residue G521 of SEQ ID NO: 1 and the residue at position corresponding to residue G521 o SEQ ID NO: 1 may be substituted by serine, threonine, alanine, valine, methionine, leucine an isoleucine, preferably serine, threonine or alanine. Preferably, the variant comprises : substitution at position corresponding to residue G521 of SEQ ID NO: 1 and the residue a position corresponding to residue G521 of SEQ ID NO: 1 is substituted by serine, threonine alanine, valine, methionine, leucine, isoleucine, asparagine, cysteine or glutamine, preferably serine, threonine or alanine, leucine, isoleucine, asparagine, cysteine or glutamine, mori corresponding to residue R171 of SEQ ID NO: 1 is substituted by phenylalanine, tyrosine tryptophan, glycine, alanine, serine, threonine, leucine, isoleucine, methionine or valine, in on preferably by phenylalanine, tyrosine or tryptophan.

Alternatively, or in addition, the variant may comprise a substitution at positioi corresponding to residue A253 of SEQ ID NO: 1 and preferably the residue at positioi corresponding to residue A253 of SEQ ID NO: 1 is substituted by threonine, glycine or serine.

Alternatively, or in addition, the variant may comprise a substitution at positioi corresponding to residue G172 of SEQ ID NO: 1 and preferably the residue at positioi corresponding to residue G172 of SEQ ID NO: 1 is substituted by valine, methionine, leucine o isoleucine, more preferably by valine.

In some preferred embodiments, the variant of the invention produces neurosporene a: main product and comprises a substitution at position corresponding to residue G521 of SEQ IE NO: 1, wherein the residue at position corresponding to residue G521 of SEQ ID NO: 1 i preferably substituted by serine, threonine, alanine, methionine, leucine, isoleucine, asparagine cysteine or glutamine, more preferably by serine, asparagine or isoleucine. In some othe preferred embodiments, the variant of the invention produces neurosporene as main product am comprises a substitution at position corresponding to residue R171 of SEQ ID NO: 1, whereii the residue at position corresponding to residue R171 of SEQ ID NO: 1 is preferably substitute! by phenylalanine, tyrosine or tryptophan. Preferably, in the variant of the invention, the residue at position corresponding to residui

E383 of SEQ ID NO: 1 is glutamic acid or glutamine and/or the residue at position correspond! ni to residue L308 of SEQ ID NO: 1 is leucine.

In particular, the variant may comprise a sequence having at least 69% identity to thi amino acid sequence from position 11 to position 543 of SEQ ID NO: 1 and/or having at leas 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity to the full length amino acid sequence of a wild-type phytoene desaturase of a Deinococcu, bacterium, preferably selected from the group consisting of SEQ ID No. 1 to 31. In particular the variant may comprise a sequence that differs from a sequence of a wild-type phytoem desaturase of a Deinococcus bacterium, preferably selected from the group consisting of SEQ IE No. 1 to 31, by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 substitutions, insertions and/o deletions. The present invention also relates to a method of producing a phytoene desaturase varian of the invention comprising:

(a) culturing the host cell according to the invention in a suitable culture medium unde suitable conditions to produce phytoene desaturase variant; and (b) recovering said variant from the cell culture.

The present invention also relates to a method of producing neurosporene and/or zeta carotene, preferably neurosporene, comprising contacting phytoene with a phytoene desaturasi variant of the invention or a host cell of the invention, and optionally recovering neurosporem and/or zeta-carotene, preferably neurosporene. It also relates to a method of produci neurosporene and/or zeta-carotene, preferably neurosporene, comprising culturing a host cell o the invention under conditions suitable to produce neurosporene and/or zeta-carotene, preferabh neurosporene, and optionally recovering neurosporene and/or zeta-carotene, preferabh neurosporene, from the culture.

The present invention further relates to the use of a phytoene desaturase variant of th invention or a host cell of the invention to produce neurosporene and/or zeta-carotene, preferabh neurosporene.

DETAILED DESCRIPTION OF THE INVENTION

Deinococcus bacteria are non-pathogen bacteria that were firstly isolated in 1956 b; Anderson and collaborators. These extremophile organisms have been proposed for use ii various industrial processes. However, bacteria of this genus remain poorly studied b; comparison with the others and this often results in a lack of suitable tools, such as enzymati· tools, to perform efficient production of compounds of interest with Deinococcus bacteria.

Based on their solid knowledge of Deinococcus metabolism and genetic, the inventor: used a recombinant phytoene-producer D. geothermalis strain wherein the endogenous phytoem desaturase gene ( crtl) was knockout, to screen a library of D. geothermalis Crtl variants obtaine< by saturation mutagenesis approach. The wild-type D. geothermalis Crtl enzyme is a four-ste_j phytoene desaturase and does not produce any detectable amount of neurosporene or zeta carotene. The inventors thus selected Crtl variants with modified product specificity, in particula variants which are able to produce significant amounts of neurosporene and/or zeta-carotene. h particular, in experimental conditions used for the screening, neurosporene produced by selecte< of lycopene as compared with the wild-type D. geothermalis Crtl. These variants may product phytofluene, zeta-carotene, neurosporene and lycopene in different proportions.

Definitions

Herein, the terms “peptide”, “oligopeptide”, “polypeptide” and “protein” are employee interchangeably and refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain.

The term “wild-type protein” as used herein, refers to the non-mutated version of ; polypeptide as it appears naturally in a species. As used herein, the term “wild-type D geothermalis Crtl” refers to the phytoene desaturase of SEQ ID NO: 1. The amino acids are herein represented by their one-letter or three-letter code according t< the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleuchn (lie); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: prolim (Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Val) W: tryptophan (Trp ) and Y: tyrosine (Tyr).

The term "substitution", as used herein in relation to a position or amino acid, means tha the amino acid in the particular position has been replaced by another amino acid or that an amine acid different from the one of the wild-type protein is present. Preferably, the term “substitution' refers to the replacement of an amino acid residue by another selected from the naturally occurring standard 20 amino acid residues, rare naturally occurring amino acid residues (e.g hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylysine, N-ethylglycine, N methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylv aline pyroglutamine, aminobutyric acid, ornithine), and non-naturally occurring amino acid, oftei made synthetically, (e.g. norleucine, norvaline and cyclohexyl-alanine). Preferably, the tern “substitution” refers to the replacement of an amino acid residue by another selected from th naturally-occurring standard 20 amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R Q, N, E, D, S and T). The sign “+” indicates a combination of substitutions. In the presen document, the following terminology is used to designate a substitution: G521S denotes tha amino acid residue at position 521 of SEQ ID No. 1 (glycine, G) is changed to a serine (S). Th substitution can be a conservative or non-conservative substitution. Examples of conservativt (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine an threonine).

As used herein, the term “Crtl” or “phytoene desaturase” or “phytoene dehydrogenase' refers to an enzyme catalyzing desaturation steps to convert phytoene to zeta-carotene neurosporene and lycopene. Wild-type D. geothermalis Crtl (SEQ ID NO: 1) catalyzes a four step desaturation converting phytoene into lycopene (EC 1.3.99.31, lycopene forming phytoem desaturase). On the other side, Crtl variants of the invention exhibit neurosporene-formin_| phytoene desaturase activity (EC 1.3.99.28) and optionally zeta-carotene-forming phytocm desaturase (EC 1.3.99.29) and/or lycopene forming phytoene desaturase activity (EC 1.3.99.31) These activities may be easily detected or measured by the skilled person, for example as detailet in the experimental section or by contacting the enzyme to be tested with phytoene and detecting or measuring the products, i.e. neurosporene, zeta-carotene and/or lycopene, for example usin_| HPLC analysis. More specifically, neurosporene-forming phytoene desaturase activity can b detected or measured by contacting the enzyme to be tested with phytoene and detecting o measuring the production of neurosporene. Zeta-carotene-forming phytoene desaturase activit; can be detected or measured by contacting the enzyme to be tested with phytoene and detectim or measuring the production of zeta-carotene. Lycopene forming phytoene desaturase activit; can be detected or measured by contacting the enzyme to be tested with phytoene and detectim or measuring the production of lycopene. The enzyme may be contacted with phytoene in vivo e.g. by culturing a phytoene producer bacterium expressing said enzyme, or in vitro, e.g. b; contacting the purified or isolated enzyme or a cellular extract of a bacterium expressing sak enzyme, with phytoene. In particular, the phytoene desaturase activity may be assessed b; incubating phytoene desaturase with phytoene in the presence of catalase and glucose oxidase adding a mixture of methanol and KOH and heating at 60°C for 15 min to terminate the reaction extracting the products from the incubation mixture with diethyl ether/light petroleum evaporating the solvent phase and redissolving the residue in cool acetone/methanol, an< identifying products by HPLC (see e.g. Xu et al., Microbiology, 153, 1642-1652, 2007). All thcs techniques are well-known by the skilled person.

The term "Crtl variant" or “phytoene desaturase variant”, as used herein, refers to ai enzyme which is derived from a phytoene desaturase, preferably from an enzyme catalyzing : four-sten desaturation converting nhvtoene into Ivconene (EC 1.3.99.31. Ivconene formi "deletion", used in relation to a position or an amino acid, means that the amino acid in th« particular position has been deleted or is absent. The term "insertion", used in relation to ; position or amino acid, means that one or more amino acids have been inserted or are presen adjacent to and immediately following the amino acid occupying the particular position. Th variant may be obtained by various techniques well known in the art. In particular, examples o techniques for altering the DNA sequence encoding the wild-type protein, include, but are no limited to, site-directed mutagenesis, random mutagenesis and synthetic oligonucleotidi construction.

As used herein, the term “sequence identity” or “identity” refers to the number (%) o matches (identical amino acid residues) in positions from an alignment of two polypcptid sequences. The sequence identity is determined by comparing the sequences when aligned so a to maximize overlap and identity while minimizing sequence gaps. In particular, sequenci identity may be determined using any of a number of mathematical global or local alignmen algorithms, depending on the length of the two sequences. Sequences of similar lengths an preferably aligned using a global alignment algorithm (e.g. Needleman and Wunsch algorithm Needleman and Wunsch, 1970) which aligns the sequences optimally over the entire length while sequences of substantially different lengths are preferably aligned using a local alignmen algorithm (e.g. Smith and Waterman algorithm (Smith and Waterman, 1981) or Altschu algorithm (Altschul et al., 1997; Altschul et al., 2005)). Alignment for purposes of determinm_] percent amino acid sequence identity can be achieved in various ways that are within the skill ii the art, for instance, using publicly available computer software available on internet web site: such as http://blast.ncbi.nlm.nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Those skilled h the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximal alignment over the full length of the sequences being compared Preferably, for purposes herein, % amino acid sequence identity values refers to values generatet using the pair wise sequence alignment program EMBOSS Needle that creates an optimal globa alignment of two sequences using the Needleman-Wunsch algorithm, wherein all searcl parameters are set to default values, i.e. Scoring matrix = BLOSUM62, Gap open = 10, Ga_j extend = 0.5, End gap penalty = false, End gap open = 10 and End gap extend = 0.5. A "recombinant nucleic acid" designates a nucleic acid which has been engineered and i: not found as such in wild tvne bacteria. Tn some narticular embodiments this term mav refer t< by e.g., recombinant, enzymatic and/or chemical techniques, and subsequently replicated in ; host cell or an in vitro system. The gene typically comprises an open reading frame encoding ; desired protein. The gene may contain additional sequences such as a transcription terminator o a signal peptide. The term "expression", as used herein, refers to any step involved in the production of ; polypeptide including, but being not limited to, transcription, post-transcriptional modification translation, post-translational modification, and secretion.

The term “expression cassette” denotes a nucleic acid construct comprising at least om coding region, i.e. at least one gene, and a regulatory region, i.e. comprising one or more contro sequences, operably linked. Preferably, the control sequences are suitable for Deinococcus hos cells.

As used herein, the term "expression vector" means a DNA or RNA molecule tha comprises an expression cassette of the invention. Preferably, the expression vector is a linear o circular double stranded DNA molecule. The term "operably linked" means a configuration in which a control sequence is placet at an appropriate position relative to a coding sequence, e.g. a nucleic acid of the invention, ii such a way that the control sequence directs expression of the coding sequence.

The term "control sequences" means nucleic acid sequences necessary for expression o a gene. Control sequences may be native or heterologous. Well-known control sequences ant currently used by the person skilled in the art will be preferred. Such control sequences include but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter ribosome binding site, signal peptide sequence, and transcription terminator. Preferably, thi control sequences include a promoter and a transcription terminator.

As used herein, the term “native” or “endogenous”, with respect to a bacterium, refers t< a genetic element or a protein naturally present in said bacterium. The term “heterologous”, wit! respect to a bacterium, refers to a genetic element or a protein that is not naturally present in sak bacterium.

As used herein, the term “purified” or “isolated”, in relation to a polypeptide or nuclei* acid, refers to a polypeptide or nucleic acid which is not in its natural medium or form. The tern "isolated" thus includes a polypeptide or nucleic acid removed from its original environment e.e.. the natural environment if it is naturallv occurring. For instance an isolated nolvnentide i partially purified form, the recombinant polypeptide, the polypeptide which is expressed o secreted by a cell, as well as the polypeptide in a heterologous host cell or culture. In relation t< a nucleic acid, the term isolated or purified indicates e.g., that the nucleic acid is not in its natura genomic context (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced in a heterologous host cell).

In the context of the invention, the term “ Deinococcus ” includes wild type or varian strains of Deinococcus , e.g., strains obtained through accelerated evolution, by DNA-shufflm_] technologies, mutagenesis or recombinant strains obtained by insertion of eukaryotic prokaryotic and/or synthetic nucleic acid(s), strains genetically and/or chemically modified by any process known per se in the art or any genetic engineering technology. Deinococcus bacteri; can designate any bacterium of the genus Deinococcus , such as without limitation. D actinosclerus, D. aerius, D. aerolatus, D. aerophilus, D. aetherius, D. alpinitundrae, D altitudinis, D. antarcticus, D. apachensis, D. aquaticus, D. aquaticus, D. aquatilis, D aquiradiocola, D. caeni, D. carri, D. cellulosilyticus, D. citri, D. claudionis, D. daejeonensis D. depolymerans, D. deserti, D. enclensis, D. ficus, D.frigens, D. geothermalis, D. gobiensis, D grandis, D. guangriensis, D. guilhemensis, D. hohokamensis, D. hopiensis, D. humi , D. indicus D. maricopensis, D. marmoris, D. metalli, D. metallilatus, D. misasensis, D. murrayi, D navajonensis, D. papagonensis, D. peraridilitoris, D. phoenicis, D. pimensis, D. piscis, D proteolyticus, D. puniceus, D. radiodurans, D. radiomollis, D. radiophilus, D. radiopugnans, D radioresistens, D. radiotolerans, D. reticulitermitis, D. roseus, D. sahariens, D. saxicola, D. soli D. sonorensis, D. swuensis, D. wulumuqiensis, D. xinjiangensis, D. xibeiensis and D yavapaiensis bacterium, or any combinations thereof. Preferably, the term “ Deinococcus ” refer: to D. geothermalis, D. murrayi, D. grandis, D. aquaticus, D. indicus, D. cellulosilyticus, D depolymerans, D. radiodurans, D. gobiensis, D. humi, D. yunweiensis or D. wulumuqiensis More preferably, the term “ Deinococcus ” refers to D. geothermalis. In some preferret embodiments, the term “ Deinococcus ” refers to a thermophilic Deinococcus, i.e. a Deinococcu, which is able to grow at a temperature of more than 40°C, preferably between 40°C and 50°C more preferably between 42°C and 48°C, and even more preferably at about 45°C. In particular the thermophilic Deinococcus may be selected from the group consisting of D. murrayi, D maricopensis and D. geothermalis. Preferably, the thermophilic Deinococcus is D. geothermalis As used herein the term “related bacterium” or “ Deinococcus related bacterium” refer: bacterium” may refer to a Deinobacterium, Truepera, Thermus, Meiothermus, Marinithermus Oceanithermus, Vulcanithermus, Bacillus, Microbacterium, Cellulosimicrobium Methylobacterium, Sphingobacterium, Pseudomonas, Caldimonas, Paenibacillus, Gordonia Rhodococcus, Stenotrophomonas, Novosphingobium, Sphingomonas, Flavobacterium Sphingobium, Sphingopyxis, Tepidimonas, Exiguobacterium, Nocardia, Arthrobacter Kineococcus, Williamsia, Porphyrobacter, Geodermatophylus, Hymenobacter, Kineococcus Kocuria, Methylobacterium, Halobacterium salinarum, Chroococcidiopsis, Pyrococcus abissi or Lactobacillus plantarum bacterium. Preferably, this term refers to a bacterium belonging t< the phylum of Deinococcus-Thermus such as Deinobacterium, Truepera, Thermus Meiothermus, Marinithermus, Oceanithermus or Vulcanithermus bacteria. More preferably, thi term refers to a bacterium belonging to the genus Meiothermus.

As used herein, the term “neurosporene” refers to a 11 - trans-n c uro s pore n c and to cis-tran, isomers thereof, i.e. isomers of all -trans- neurosporene wherein one or several double bonds an in cis configuration. The term “zeta-carotene” refers to all-fra/M-zcta-caiOtcnc and to cis-tran, isomers thereof, i.e. isomers of a 11 - fra/rs- zcta-c aro tc n c wherein one or several double bonds an in cis configuration.

In a first aspect, the present invention relates to a phytoene desaturase (Crtl) varian comprising a sequence (i) having at least 65% identity to the full length amino acid sequence o SEQ ID NO: 1 and (ii) comprising at least one substitution at position corresponding to residui G521, R171, A253 or G172 of SEQ ID NO: 1. The positions are numbered by reference to thi amino acid sequence set forth in SEQ ID NO: 1.

As compared to phytoene desaturase of SEQ ID NO: 1, the Crtl variant of the inventioi exhibits a modified product specificity towards neurosporene and/or zeta-carotene, preferabl; towards neurosporene and optionally zeta-carotene. Indeed, in contrast to phytoene desaturase o SEQ ID NO: 1 which only produces lycopene from phytoene, the variant of the inventioi produces neurosporene and/or zeta-carotene from phytoene, i.e. detectable amounts o neurosporene and/or zeta-carotene. Thus, as used herein, the term “modified product sped rich; towards neurosporene” means that the Crtl variant produces and releases neurosporene, i.e produces neurosporene as an unbound/final product, and the term “modified product specificit; towards zeta-carotene” means that the Crtl variant nroduces and releases zeta-carotene. i.e (optionally along with phytofluene and/or lycopene). Thus, contrary to wild-type enzyme, thi Crtl variant produces detectable amounts (preferably amounts detectable by HPLC) o neurosporene and/or zeta-carotene. In other embodiments, the Crtl variant exhibits an increasi of the production of neurosporene and/or zeta-carotene of at least 10%, preferably at least 25% at least 50%, at least 75% or at least 100%, by comparison to the wild-type enzyme.

The activity of the Crtl variant of the invention may be easily assessed. For example, ; Crtl variant may be expressed in a recombinant Deinococcus bacterium which is able to product phytoene and wherein the wild-type Crtl has been deleted. After 24 hours of culture, carotenoid: produced may be detected using any routine method such as HPLC. Preferably, neurosporene produced by the Crtl variant of the invention represents at leas

10% (w/w) of the Crtl variant products (phytofluene, zeta-carotene, neurosporene and lycopene) More preferably, neurosporene produced by the Crtl variant of the invention represents at leas 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% (w/w) o the Crtl variant products (phytofluene, zeta-carotene, neurosporene and lycopene). In preferret embodiments, neurosporene produced by the Crtl variant of the invention represents at least 40°/ (w/w) of the Crtl variant products, more preferably represents at least 50% (w/w) of the Crt variant products.

Alternatively, or in addition, the ratio for the formation of neurosporene vs lycopene (N/L w/w) for the Crtl variant of the invention may be of at least 0.1, preferably at least 0.5, at least 1 at least 2, at least 3, at least 10, at least 20, at least 30, at least 50, at least 70, at least 100 or a least 200. In preferred embodiments, the ratio for the formation of neurosporene vs lycopcm (N/L, w/w) for the Crtl variant of the invention is of at least 1. In a particular embodiment, thi: ratio is of at least 20.

Zeta-carotene produced by the Crtl variant of the invention may represent at least 10°/ (w/w) of the Crtl variant products (phytofluene, zeta-carotene, neurosporene and lycopene) More preferably, zeta-carotene produced by the Crtl variant of the invention represents at leas 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at leas 60%, at least 70% or at least 80% (w/w) of the Crtl variant products (phytofluene, zeta-carotene neurosporene and lycopene). In some particular embodiments, zeta-carotene produced by th Crtl variant of the invention represents at least 30% (w/w) of the Crtl variant products in on nreferablv renresents at least 50% ( w/w 1 of the Crtl variant nroducts. embodiments, the ratio for the formation of zeta-carotene vs lycopene (Z/L, w/w) for the Crt variant of the invention is of at least 20. In a particular embodiment, this ratio is of at least 50.

Percentages of Crtl variant products as well as N/L and Z/L ratios can be easily measuret by the skilled person, for example as detailed in the examples, i.e. by culturing a phytocm producing Acrtl Deinococcus strain (i.e. a phytoene producing Deinococcus strain wherein thi wild-type crtl has been deleted) expressing a Crtl variant of the invention in a well of 96-wel plate for 24 h and quantifying Crtl variant products by HPLC.

The Crtl variant of the invention comprises a sequence having at least 65% identity to thi full length amino acid sequence of SEQ ID NO: 1. Preferably, the Crtl variant of the inventioi comprises a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90% at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the full lengtl amino acid sequence of SEQ ID NO: 1. More preferably, the Crtl variant of the inventioi comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96% at least 97%, at least 98%, at least 99% identity to the full length amino acid sequence of SEC ID NO: 1. In particular, the Crtl variant may comprise a sequence having at least 69% identity h the amino acid sequence from position 11 to position 543 of SEQ ID NO: 1, preferably at leas 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at leas 97%, at least 98%, at least 99% identity to the amino acid sequence from position 11 to positioi 543 of SEQ ID NO: 1. The variant may comprise, or consist of, a sequence that differs from the sequence se forth in SEQ ID No. 1 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 substitutions, insertion and/or deletions, preferably by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and/o deletions, more preferably by 1, 2, 3, 4 or 5 substitutions, insertions and/or deletions, and evei more preferably by 1, 2 or 3 substitutions, insertions and/or deletions. More particularly, thi variant may comprise, or consist of, a sequence that differs from the sequence set forth in SEC ID No. 1 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 substitutions, preferably by 1, 2, 3, 4 5, 6, 7, 8, 9 or 10 substitutions, more preferably by 1, 2, 3, 4 or 5 substitutions, and even mon preferably by 1, 2 or 3 substitutions.

In some other embodiments, the Crtl variant of the invention comprises, or consists of, i sequence having at least 65% identity to the full length amino acid sequence of SEQ ID NO: and at least 80%. nreferablv at least 90%. at least 95% or at least 99%. identitv to the full lenetl C1D2Z4; SEQ ID NO: 3), D. gobiensis (Uniprot accession number: H8GYF5; SEQ ID NO: 4) D. maricopensis (Uniprot accession number: E8UAM7; SEQ ID NO: 5), D. peraridilitori, (Uniprot accession number: L0A6E5; SEQ ID NO: 6), D. proteolyticus (Uniprot accessioi number: F0RJ97; SEQ ID NO: 7), D. puniceus (Uniprot accession number: A0A172TAT8; SEC ID NO: 8), D. radiodurans (Uniprot accession number: Q9RW08; SEQ ID NO: 9), D. sol (Uniprot accession number: A0A0F7JTT9; SEQ ID NO: 10), D. marmoris (Uniprot accessioi number: A0A1U7NYC6; SEQ ID NO: 11), D. swuensis (Uniprot accession number A0A0A7KJM4 ; SEQ ID NO: 12), D. reticulitermitis (Uniprot accession number: A0A1H6WPE ; SEQ ID NO: 13), D. koreensis (Uniprot accession number: A0A2K3UU91 ; SEQ ID NO: 14 , D. aerius (Uniprot accession number: A0A2I9DDX8 ; SEQ ID NO: 15) , D. hopiensis (Unipro accession number: A0A1W1VDD1 ; SEQ ID NO: 16), D. phoenicis (Uniprot accession number A0A016QSL2 ; SEQ ID NO: 17), D. metalUlatus (Uniprot accession number: A0A4Q0W521 SEQ ID NO: 18), D. irradiatisoli (Uniprot accession number:A0A2Z3JP54 ; SEQ ID NO: 19) D. wulumuqiensis R12 (Uniprot accession number: A0A0D5Z8R8 ; SEQ ID NO: 20), D radiophdus (Uniprot accession number: A0A3S0I4K1; SEQ ID NO: 21), D. ficus (Unipro accession number: A0A221SZ37 ; SEQ ID NO: 22), D. yavapaiensis KR-236 (Uniprot accessioi number: A0A318S6H4 ; SEQ ID NO: 23), D. grandis (Uniprot accession number A0A117DNB2 ; SEQ ID NO:24), D. indicus (Uniprot accession number: A0A246BPB 1 ; SE( ID NO: 25) and D. radiopugnans ATCC 19172 (Uniprot accession number: A0A5C4Y7X2 SEQ ID NO: 26), D. planocerae (NCBI accession number : WP_102127525.1; SEQ ID NO: 27) D. apachensis (NCBI accession number : WP_019584972.1; SEQ ID NO: 28), D. murray (NCBI accession number : WP_051363338.1; SEQ ID NO: 29), D. aquatilis (NCBI accessioi number : WP_019009610.1; SEQ ID NO: 30) and D. frigens (NCBI accession number WP_029476927.1 ; SEQ ID NO: 31). Preferably, in these embodiments, the Crtl variant comprises a sequence having at leas

69% identity to the amino acid sequence from position 11 to position 543 of SEQ ID NO: 1 preferably at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, a least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence iron position 11 to position 543 of SEQ ID NO: 1, and at least 70%, preferably at least 80%, at leas 90%, at least 95% or at least 99%, identity to the full length amino acid sequence of a wild-typi nhvtoene desaturase of a Dcinococcus bacterium nreferablv selected from the eroun consisti sequence of a wild-type phytoene desaturase of a Deinococcus bacterium, preferably selecte< from the group consisting of SEQ ID No. 1 to 31, by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 o 15 substitutions, insertions and/or deletions, preferably by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 1( substitutions, insertions and/or deletions, more preferably by 1, 2, 3, 4 or 5 substitutions insertions and/or deletions, and even more preferably by 1, 2 or 3 substitutions, insertions and/o deletions. More particularly, in these embodiments, the variant may comprise, or consist of, ; sequence having at least 65% identity to the full length amino acid sequence of SEQ ID NO: and that differs from a sequence of a wild-type phytoene desaturase of a Deinococcus bacterium preferably selected from the group consisting of SEQ ID No. 1 to 31, by 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12, 13, 14 or 15 substitutions, preferably by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions more preferably by 1, 2, 3, 4 or 5 substitutions, and even more preferably by 1, 2 or ' substitutions.

The variant of the invention comprises at least one substitution at position correspondim to residue G521, R171, A253 or G172 of SEQ ID NO: 1.

The position in a polypeptide corresponding to a specific residue of SEQ ID NO: 1 ma; be easily determined by the skilled person, for example using a global alignment algorithm sucl as Needleman and Wunsch algorithm.

In a preferred embodiment, the variant of the invention comprises at least one substitutioi at position corresponding to residue G521 of SEQ ID NO: 1. Preferably, the residue at positioi corresponding to residue G521 of SEQ ID NO: 1 is substituted by serine, threonine, alanine valine, methionine, leucine, isoleucine, asparagine, cysteine or glutamine, in particular by serine threonine, alanine, valine, methionine, leucine or isoleucine. In a more preferred embodiment the residue at position corresponding to residue G521 of SEQ ID NO: 1 is substituted by serine threonine, alanine, leucine, isoleucine, asparagine, cysteine or glutamine, in particular serine threonine or alanine. In a particularly preferred embodiment, the residue at positioi corresponding to residue G521 of SEQ ID NO: 1 is substituted by serine, isoleucine o asparagine, preferably by serine. In another embodiment, the residue at position correspondim to residue G521 of SEQ ID NO: 1 is substituted by valine, methionine, leucine or isoleucine preferably by valine.

In another embodiment the variant of the invention comnrises at least one substitution a preferably by phenylalanine, tyrosine, tryptophan, glycine or leucine, more preferably b; phenylalanine, tyrosine or tryptophan.

In another embodiment, the variant of the invention comprises at least one substitution a position corresponding to residue A253 of SEQ ID NO: 1. Preferably, the residue at positioi corresponding to residue A253 of SEQ ID NO: 1 is substituted by threonine, glycine or serine preferably by threonine.

In a particular embodiment, the variant of the invention comprises at least tw< substitutions at positions corresponding to residues R171 and A253 of SEQ ID NO: 1. Preferably the residue at position corresponding to residue R171 of SEQ ID NO: 1 is substituted b; phenylalanine, tyrosine, tryptophan, glycine, alanine, serine, threonine, leucine, isoleucim methionine or valine, preferably by phenylalanine, tyrosine or tryptophan, glycine or leucine more preferably by phenylalanine, tyrosine or tryptophan, and the residue at positioi corresponding to residue A253 of SEQ ID NO: 1 is substituted by threonine, glycine or serine preferably by threonine. More particularly, the residue at position corresponding to residue R17 of SEQ ID NO: 1 may be substituted by phenylalanine and the residue at position correspond to residue A253 of SEQ ID NO: 1 may be substituted by threonine.

In another embodiment, the variant of the invention comprises at least one substitution a position corresponding to residue G172 of SEQ ID NO: 1. Preferably, the residue at positioi corresponding to residue G172 of SEQ ID NO: 1 is substituted by valine, methionine, leucine o isoleucine, preferably by valine.

In a particular embodiment, the Crtl variant of the invention produces neurosporene am zeta-carotene and comprises at least one substitution at position corresponding to residue G521 R171, A253 or G172 of SEQ ID NO: 1

- wherein the residue at position corresponding to residue R171 of SEQ ID NO: 1 i: substituted by phenylalanine, tyrosine, tryptophan, leucine, valine or isoleucine, preferably phenylalanine, tyrosine, tryptophan or leucine, more preferably by phenylalanine, tyrosine o tryptophan,

- wherein preferably the residue at position corresponding to residue G521 of SEQ IE NO: 1 is substituted by serine, threonine, alanine, valine, methionine, leucine, asparagine cysteine or glutamine, more preferably by serine, threonine, valine, alanine, methionine o leucine even more nreferablv bv serine or valine. - wherein preferably, the residue at position corresponding to residue G172 of SEQ IE NO: 1 is substituted by valine, methionine, leucine or isoleucine, more preferably by valine.

Preferably, in this embodiment, zeta-carotene produced by the Crtl variant represents a least 10% (w/w) of the Crtl variant products and/or neurosporene produced by the Crtl varian represents at least 20% (w/w) of the Crtl variant products. Alternatively or additionally, the rath for the formation of zeta-carotene vs lycopene (Z/L, w/w) for the Crtl variant may be of at leas 0.1 and/or the ratio for the formation of neurosporene vs lycopene (N/L, w/w) for the Crtl varian may be of at least 0.1.

In another particular embodiment, the Crtl variant of the invention produces neurosporem and/or zeta-carotene as main products and comprises at least one substitution at positioi corresponding to residue G521, R171, A253 or G172 of SEQ ID NO: 1

- wherein preferably the residue at position corresponding to residue R171 of SEQ IE NO: 1 is substituted by phenylalanine, tyrosine or tryptophan,

- wherein preferably the residue at position corresponding to residue G521 of SEQ IE NO: 1 is substituted by serine, threonine, alanine, valine, methionine, leucine, cysteine o glutamine, more preferably by serine, threonine, valine, alanine, methionine, leucine o isoleucine, more preferably by serine or valine,

- wherein preferably the residue at position corresponding to residue A253 of SEQ IE NO: 1 is substituted by threonine, glycine or serine, more preferably by threonine, and - wherein preferably, the residue at position corresponding to residue G172 of SEQ IE

NO: 1 is substituted by valine, methionine, leucine or isoleucine, more preferably by valine.

Preferably, in this embodiment, zeta-carotene produced by the Crtl variant represents a least 50% (w/w) of the Crtl variant products and/or neurosporene produced by the Crtl varian represents at least 40% (w/w) of the Crtl variant products. Alternatively or additionally, the rath for the formation of zeta-carotene vs lycopene (Z/L, w/w) for the Crtl variant may be of at leas 20 and/or the ratio for the formation of neurosporene vs lycopene (N/L, w/w) for the Crtl varian may be of at least 1.

In a preferred embodiment, the Crtl variant of the invention produces zeta-carotene a: main product and comprises at least one substitution at position corresponding to residue G52 or G172 of SEQ ID NO: 1. Preferably, the residue at position corresponding to residue G172 o SEO ID NO: 1 is substituted bv valine methionine leucine or isoleucine more nreferablv h_' zeta-carotene produced by the Crtl variant represents at least 50% (w/w) of the Crtl varian products and/or the ratio for the formation of zeta-carotene vs lycopene (Z/L, w/w) for the Crt variant is of at least 20.

In another preferred embodiment, the Crtl variant of the invention comprises at least om substitution at position corresponding to residue G521 or G172 of SEQ ID NO: 1, wherein thi residue at position corresponding to residue G172 of SEQ ID NO: 1 is substituted by valine methionine, leucine or isoleucine, more preferably by valine, and the residue at positioi corresponding to residue G521 of SEQ ID NO: 1 is substituted by valine, methionine, leucine cysteine or glutamine, preferably valine or methionine, more preferably by valine. Preferably, ii this embodiment, zeta-carotene produced by the Crtl variant represents at least 20%, preferably at least 30%, at least 40%, or at least 50%, (w/w) of the Crtl variant products and/or the ratio fo the formation of zeta-carotene vs lycopene (Z/L, w/w) for the Crtl variant is of at least 20 preferably at least 30, at least 50, at least 90, at least 100, at least 200, at least 500.

In a further preferred embodiment, the Crtl variant of the invention produce: neurosporene as main product and comprises at least one substitution at position corrcspondim to residue R171 of SEQ ID NO: 1. Preferably, the residue at position corresponding to residui R171 of SEQ ID NO: 1 is substituted by phenylalanine, tyrosine or tryptophan. Optionally th Crtl variant may further comprise a substitution at position corresponding to residue A253 o SEQ ID NO: 1. Preferably, the residue at position corresponding to residue A253 of SEQ ID NO 1 is substituted by threonine, glycine or serine, more preferably by threonine. Preferably, in thi embodiment, neurosporene produced by the Crtl variant represents at least 40% (w/w) of the Crt variant products and/or the ratio for the formation of neurosporene vs lycopene (N/L, w/w) fo the Crtl variant is of at least 1.

In a further preferred embodiment, the Crtl variant of the invention produce: neurosporene as main product and comprises at least one substitution at position corrcspondim to residue G521 of SEQ ID NO: 1. Preferably, the residue at position corresponding to residui G521 of SEQ ID NO: 1 is substituted by serine, threonine, alanine, methionine, leucine isoleucine, asparagine, cysteine or glutamine, in particular by serine, threonine, alanine, leucine isoleucine, asparagine, cysteine or glutamine. Preferably, the residue at position correspond^ to residue G521 of SEQ ID NO: 1 is substituted by serine, asparagine or isoleucine, mon nreferablv bv serine. Preferablv. in this embodiment neurosnorene nroduced bv the Crtl varian Preferably, in all embodiments disclosed above, the residue at position corresponding t< residue E383 of SEQ ID NO: 1 is glutamic acid or glutamine and/or the residue at positioi corresponding to residue L308 of SEQ ID NO: 1 is leucine. In some embodiments, the variant of the invention may be fused at its N-terminus and/o

C-terminus to another polypeptide to create a hybrid polypeptide or fusion polypeptide Techniques for producing fusion polypeptides are known in the art, and include ligating thi coding sequences encoding the variant and the addition region of another polypeptide so tha they are in frame and that expression of the fusion polypeptide is under control of the sarm promoter(s) and terminator. The addition region of the fusion polypeptide can be selected in orde to enhance the stability of the enzyme, to promote the secretion (such as a N-termina hydrophobic signal peptide) of the fusion protein from a cell (such as a bacterial cell or a yeas cell), or to assist in the purification of the fusion protein. More particularly, the additional regioi can be a tag useful for purification or immobilization of the enzyme. Such a tag is well-knowi by the person skilled in the art, for instance a His tag (His₆), a FLAG tag, a HA tag (epitopi derived from the Influenza protein haemagglutinin), a maltose-binding protein (MPB), a MY( tag (epitope derived from the human proto-oncoprotein MYC) or a GST tag (small glutathione S-transferase). A fusion polypeptide can further comprise a cleavage site for proteases o chemical agents, between the enzyme and the addition region. Upon secretion of the fusioi protein, the site is cleaved releasing the two separate polypeptides.

The variant of the invention may also be fused at their N-terminus and/or C-terminus t< one or several polypeptides exhibiting distinct enzymatic activity.

The variant of the invention may be produced by recombinant techniques. In particular it may be expressed, secreted, isolated, or purified from a host cell, preferably a Deinococcu, bacterium. Optionally, it may be also modified (e.g., chemically, enzymatically, physically, etc. to improve one of its features such as stability or activity. In some embodiments, the variant o the invention may be in isolated or purified form. In particular, it may be isolated or purified b; techniques known per se in the art, and stored under conventional techniques.

The variant of the invention may be in soluble form or on solid phase. In particular, i may be bound to cell membranes or lipid vesicles, or to synthetic supports such as glass, plastic nolvmers. filter membranes e.e.. in the form of beads columns nlates and the like. a mixture of the two. It can be in single stranded form or in duplex form or a mixture of the two It can comprise modified nucleotides, comprising for example a modified bond, a modifie< purine or pyrimidine base, or a modified sugar. It can be prepared by any method known to om skilled in the art, including chemical synthesis, recombination, and mutagenesis. The nucleic ack according to the invention may be deduced from the sequence of the peptide according to thi invention and codon usage may be adapted according to the host cell in which the nucleic aci< shall be transcribed. These steps may be carried out according to methods well known to one o skill in the art and some of which are described in the reference manual Sambrook et al (Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, Third Edition Col< Spring Harbor).

The present invention further relates to an expression cassette comprising a nucleic ack according to the invention, i.e. an expression cassette comprising a nucleic acid according to thi invention operably linked to one or more control sequences that direct the expression of sak nucleic acid in a suitable host cell under conditions compatible with the control sequences.

The control sequence may include a promoter that is recognized by a host cell or an ii vitro expression system for expression of a nucleic acid of the invention. The promoter contain: transcriptional control sequences that mediate the expression of the variant. The promoter ma; be any polynucleotide that shows transcriptional activity in the host cell including mutant truncated, and hybrid promoters, and may be obtained from genes encoding extracellular o intracellular polypeptides either endogenous or heterologous to the host cell. The promoter ma; be an endogenous or heterologous promoter. The promoter may be a strong, weak, constitutive or inducible promoter. In a particular embodiment, the promoter is heterologous to the nuclei· acid of the invention, i.e. is not operably linked in nature to said nucleic acid or is operably linke< at a different location in nature. Preferably, the promoter is a polynucleotide that show: transcriptional activity in Deinococcus bacteria. In this regard, various promoters have beei studied and used for gene expression in Deinococcus bacteria. Examples of suitable promoter: include P tufA and PtufB promoters from the translation elongation factors Tu genes tup ( DR0309 ) and tufB ( DR2050 ), the promoter of the res U gene located in pI3, the promoter regioi PgroESL of the groESL operon (Lecointe, et al. 2004. Mol Microbiol 53: 1721-1730 ; Meima e al. 2001. T Bacteriol 183: 3169-31751. or derivatives thereof. used in the present invention. Preferably, the terminator is functional in Deinococcus bacteria Examples of such terminator are disclosed in Lecointe et al, 2004, supra. Usually, the terminate is chosen in correlation with the promoter.

The control sequence may also be a signal peptide coding sequence that encodes a signa peptide linked to the N-terminus of an encoded polypeptide and directs the polypeptide into thi cell's secretory pathway, i.e. for secretion into the extracellular (or periplasmic) space. Any signa peptide coding sequence that directs the expressed polypeptide into the secretory pathway of ; host cell may be used.

It may also be desirable to add regulatory sequences that regulate expression of the varian relative to the growth of the host cell. Examples of regulatory systems are those that causi expression of the gene to be turned on or off in response to a chemical or physical stimulus including the presence of a regulatory compound. Regulatory systems in prokaryotic system: include the lac, tac, and trp operator systems.

Typically, the expression cassette comprises, or consists of, a nucleic acid according te the invention operably linked to a transcriptional promoter and a transcription terminator.

The expression cassette of the invention may be used directly to transform a host cell preferably a Deinococcus host cell, and enable the expression of the nucleic acid of the inventioi in said cell. Preferably, the expression cassette, or a part thereof comprising a nucleic acid of thi invention, is inserted into the genome of the host cell. In a particular embodiment, the expressioi cassette is integrated in the genome of the host cell, preferably a Deinococcus host cell. Thi expression cassette may be integrated, for example, in the gene encodi phosphoacetyltransferase (pta ) or into an IS sequence present in the genome of the host cell (sei e.g. WO 2015/092013). The present invention also relates to an expression vector comprising a nucleic acid or ai expression cassette according to the invention.

The expression vector of the invention may be used to transform a host cell, preferably ; Deinococcus host cell, and enable the expression of the nucleic acid of the invention in said cell The choice of the vector will typically depend on the compatibility of the vector with the hos cell into which the vector is to be introduced. The vector may be an autonomously replicatm_] vector i.e.. a vector that exists as an extra-chromosomal entitv. the renlication of which i integrated into the genome and replicated together with the chromosome(s) into which it has beei integrated. Preferably, the vector, or a part thereof comprising a nucleic acid of the invention e.g. the expression cassette of the invention, is inserted in the genome of the host cell. In ; particular embodiment, the vector or expression cassette is integrated in the genome of the hos cell, preferably a Deinococcus host cell, in the gene encoding phosphoacetyltransferase (pta) o into an IS sequence present in the genome of the host cell (see e.g. WO 2015/092013).

The vector preferably comprises one or more selectable markers that permit easy selectioi of host cells comprising the vector. A selectable marker is a gene the product of which provide for antibiotic resistance, resistance to heavy metals, prototrophy to auxotrophy, and the like. The vector preferably comprises an element that permits integration of the vector into tin host cell's genome or autonomous replication of the vector in the cell independent of the genome When integration into the host cell genome occurs, integration of the sequences into the genomt may rely on homologous or non-homologous recombination. In one hand, the vector may contaii additional polynucleotides for directing integration by homologous recombination at a precist location into the genome of the host cell. These additional polynucleotides may be any sequenct that is homologous with the target sequence in the genome of the host cell. On the other hand the vector may be integrated into the genome of the host cell by non-homologous recombination

For autonomous replication, the vector may further comprise an origin of replicatioi enabling the vector to replicate autonomously in the host cell in question. The origin o replication may be any plasmid replicator mediating autonomous replication that functions in ; cell.

The methods for selecting these elements according to the host cell in which expressioi is desired, are well known to one of skill in the art. The vectors may be constructed by th« classical techniques of molecular biology, well known to one of skill in the art.

The present invention further relates to the use of a nucleic acid, an expression casseth or an expression vector according to the invention to transform, transfect or transduce a cell. Tin present invention also relates to a host cell comprising a nucleic acid, a cassette or an expressioi vector according to the invention. The host cell may be transformed, transfected or transduced in a transient or stabh manner. The resulting host cell is a recombinant host cell. As used herein the term "recombinan heterologous nucleic acid, expression cassette or vector of the invention, i.e. a nucleic acid tha does not naturally occur in said cell.

An expression cassette or vector of the invention may be introduced into a host cell s< that the cassette or vector is maintained as a chromosomal integrant or as a self-replicating extra chromosomal vector as described earlier. The term "host cell" also encompasses any progeny o a parent host cell that is not identical to the parent host cell due to mutations that occur durin_] replication.

The nucleic acid, expression cassette or expression vector according to the invention ma; be introduced into the host cell by any method known by the skilled person, such a electroporation, conjugation, transduction, competent cell transformation, protoplas transformation, protoplast fusion, biolistic "gene gun" transformation, PEG-mediate< transformation, lipid-assisted transformation or transfection, chemically mediated transfection lithium acetate-mediated transformation or liposome-mediated transformation.

Optionally, more than one copy of a nucleic acid, cassette or vector of the presen invention may be inserted into the host cell to increase production of the variant.

The host cell may be a prokaryote or eukaryote cell.

The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium Examples of suitable bacterial expression hosts include, but are not limited to, Deinococcus an< related bacteria, Escherichia (e.g. E. coli ), Pseudomonas (e.g. P. fluorescens or P. stutzerei ) Proteus (e.g. P. mirabilis), Ralstonia (e.g. R. eutropha), Streptomyces, Staphylococcus (e.g. S carnosus ), Lactococcus (e.g. L. lactis ), ox Agrobacterium (e.g. A. aurantiacum ), Halobacteriun (e.g. H. salinarium), Corynebacterium (e.g. C. glutamicum), Paracoccus (e.g. P carotinifaciens), Gordonia (e.g. G. jacobea ), Bacillus ( B . subtilis, B. megaterium, B licheniformis , etc.), Pantoea (e.g. P. ananatis), Rubrivivax (e.g. R. gelatinosus ), Microbacteriun (e.g. M. paraoxydans ), Rhodobacter (e.g. R. sphaeroides, R. viridis ), Rhodovulum and Kocurit bacteria.

The host cell may also be a non-human eukaryotic cell, such as a yeast, fungal mammalian, insect, plant or algal cell. Examples of suitable yeast expression hosts include, bu are not limited to, Saccharomyces (e.g. S. cerevisiae, S. carlsbergensis, S. diastaticus, S douglasii, S. kluyveri, S. norbensis, S. oviformis), Xanthophyllomyces (e.g. X. dendrorhous) Rhodotorula (e.e. R. plutinis V Schizosaccharomvces fc.p. S. nombe). Yarrowia (e.e. V Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium Fusarium (e.g. F. sporotrichioides), Humicola, Magnaporthe, Mucor, Myceliophthora Neocallimastix, Neurospora (e.g. N. crassa ), PaecUomyces, Penicillium, Phanerochaete Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia Tolypocladium or Trametes cells. Examples of suitable plant or algal expression hosts include but are not limited to, Chlorella, Scenedesmus, Botryococcus (e.g. B. braunii), Dunaliella (e.g D. salind), Haematococcus (e.g. H. pluvialis), Phaeodactylum (e.g. P. tricornutum ) o Desmodesmus cells.

Preferably, the host cell is a bacterium, more preferably a Deinococcus bacterium o related bacterium. Even more preferably, the host cell is a Deinococcus bacterium.

In preferred embodiments, the host cell of the invention is able to produce phytoene preferably to naturally produce phytoene, i.e. expresses a phytoene synthase.

As used herein, the term “CrtB” or “phytoene synthase” refers to a phytoene synthasi enzyme (EC 2.5.1.32) encoded by a crtB gene which catalyzes the condensation of two molecule: of geranylgeranyl diphosphate (GGPP) to give phytoene.

The crtB gene expressed or overexpressed in the host cell of the invention may encode ai endogenous or heterologous phytoene synthase.

In a particular embodiment, the polypeptide exhibiting phytoene synthase activity ma; be selected from CrtB from Pantoea agglomerans (GenBank accession number: AFZ89043.1,) CrtB from Paracoccus sp_N81106 (GenBank accession number: BAE47469.1) or phytoem synthases from Deinococcus bacteria. Examples of Deinococcus phytoene synthases (CrtB include, but are not limited to, phytoene synthases from D. geothermalis (Uniprot accessioi number: Q1J109), D. actinosclerus (Uniprot accession number: A0A0U3KC93), D. desert (Uniprot accession number: C1D2Z3), D. gobiensis (Uniprot accession number: H8GYF6), D maricopensis (Uniprot accession number: E8UAM8), D. peraridilitoris (Uniprot accessioi number: L0A567), D. puniceus (Uniprot accession number: A0A172TDE8), D. radioduran, (Uniprot accession number: Q9RW07), D. soli (Uniprot accession number: A0A0F7JV05) an D. swuensis (Uniprot accession number: A0A0A7KGT0). The polypeptide exhibiting phytocm synthase activity may also be any polypeptide exhibiting phytoene synthase activity and havin_] at least 60 %. nreferablv 65. 70. 75. 80. 85. 90. 95. 96. 97. 98 or 99%. identitv to anv nhvtoem The host cell of the invention may also be genetically modified in order to increase th production of geranylgeranyl diphosphate (GGPP).

In particular, the host cell of the invention may be genetically modified to increase th carbon flux to isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP and/or to increase the conversion of IPP and DMAPP to geranylgeranyl diphosphate (GGPP).

The carbon flux to IPP and DMAPP may be increased by enhancing the 2-C-methyl-D erythritol 4-phosphate/l-deoxy-D-xylulose 5-phosphate (MEP/DXP) pathway. As used herein the term “MEP pathway” or “MEP/DXP pathway” refers to the biosynthetic pathway leading t< the formation of IPP and DMAPP from the condensation of pyruvate and D-glyceraldehyde 3 phosphate to 1-deoxy-D-xylulose 5- phosphate (DXP). This pathway involves the following enzymes: 1-deoxy-D-xylulose 5- phosphate synthase (EC 2.2.1.7), 1-deoxy-D-xylulose 5 phosphate reductoisomerase (EC 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphat cytidylyltransferase (EC 2.7.7.60), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (EC 2.7.1.148), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (EC 4.6.1.12), 4-hydroxy-3 methylbut-2-en-l-yl diphosphate synthase (EC 1.17.7.1), 4-hydroxy-3-methylbut-2-eny diphosphate reductase (EC 1.17.1.2), and isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2)

This pathway may be enhanced by any method known by the skilled person, for exampL by a method described in the patent application WO 2015/189428. In particular, this pathway may be enhanced by increasing at least one enzymatic activity selected from the group consisti of DXP synthase (DXS), DXP reductoisomerase (DXR), IspD, IspE, IspF, IspG, IspH and IPI isomerase activities (IDI), preferably by increasing at least DXP synthase and IPP isomeras activities.

An enzymatic activity (e.g. DXS, DXR, IspD, IspE, IspF, IspG, IspH, IDI or FPPi activity) may be increased, for example, by overexpression of an endogenous gene or expressioi of a heterologous gene.

The term “DXS” or “DXP synthase” refers to the enzyme 1-deoxy-D-xylulose 5 phosphate synthase (EC 2.2.1.7) encoded by the dxs gene which catalyzes the condensation o pyruvate and D-glyceraldehyde 3-phosphate to 1-deoxy-D-xylulose 5- phosphate (DXP). Th names of gene product, “DXP synthase”, “DXS” or “DXPS”, are used interchangeably in thi annlication. The term "DXP reductoisomerase" or “DXR” refers to the enz.vme 1 -deoxv-D D-erythritol kinase, (EC 2.7.1.148) encoded by the ispE gene. The term "IspF" refers to thi enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (EC 4.6.1.12) encoded by th ispF gene. The term "IspG" refers to the enzyme 4-hydroxy-3-methylbut-2-en-l-yl diphosphah synthase (EC 1.17.7.1) encoded by the ispG gene. The term "IspH" refers to the enzyme 4 hydroxy-3-methylbut-2-enyl diphosphate reductase, also named hydroxymethylbuteny pyrophosphate reductase, (EC 1.17.1.2) encoded by the ispH gene. The term "IDI", ‘ΊR1 isomerase” or “ isopentenyl pyrophosphate isomerase” refers to the enzyme isopentenyl diphosphate delta-isomerase (EC 5.3.3.2) encoded by the idi gene that catalyzes the l,3-allyli< rearrangement of the homoallylic substrate isopentenyl (IPP) to its allylic isomer, dimethylally diphosphate (DMAPP). According to the organism, the nomenclature of the above idcnti fic enzymes and encoding genes may vary. However, for the sake of clarity, in the presen specification, these terms are used independently from the origin of the enzymes or genes.

Preferably, at least one gene selected from the group consisting of dxs, dxr, ispD, ispE ispF, ispG, ispH and idi genes, is overexpressed, more preferably at least dxs and/or idi genes and even more preferably at least dxs and idi genes are overexpressed. These genes may h endogenous or heterologous, preferably endogenous dxs, dxr, ispD, ispE, ispF, ispG, ispH am idi genes of the host cell of the invention.

In a particular embodiment, the host cell of the invention, preferably a Deinococcu, bacterium, is genetically modified to overexpress an endogenous dxs gene or to express ; heterologous dxs gene, and to overexpress an endogenous idi gene or to express a heterologou: idi gene.

In particular, the overexpressed endogenous or expressed heterologous dxs gene may h from a Deinococcus bacterium. Examples of dxs genes from Deinococcus bacteria include, bu are not limited to, the dxs genes from D. geothermalis (UniProt accession number: Q1IZP0), D yunweiensis (SEQ ID NO: 32), D. deserti (NCBI Accession number: WP_012692944.1 GenBank: AC045821.1; UniProt accession number: C1D1U7), D. radiodurans (UniPro accession number: Q9RUB5; NCBI Accession number: WP_010888114.1) and D. radiopugnan, (SEQ ID NO: 33). Any polypeptide, preferably from a Deinococcus bacterium, having at leas 70%, preferably 80%, more preferably 90%, sequence identity to any of the polypeptide: encoded by those genes and having a DXS activity may also be used in the present invention.

The overexnressed endogenous or exnressed heterologous idi gene mav also be from : accession number: Q9RVE2.3) or D. radiopugnans (SEQ ID NO: 36). Any polypeptide preferably from a Deinococcus bacterium, having at least 70%, preferably 80%, more preferably 90%, sequence identity to any of the polypeptides encoded by those genes and having an ID activity may also be used in the present invention. Recombinant Deinococcus bacteria genetically modified in order to increase tin production of geranylgeranyl diphosphate (GGPP) are disclosed in the patent application WC 2015/189428, which is herein incorporated by reference.

In addition, or alternatively, the host cell of the invention may also be genetically modified in order to increase the conversion of IPP and DMAPP to GGPP, preferably by increasing the FPP synthase activity by comparison to the wild-type host cell.

As used herein, the term "IspA", FDPS’ “ FPPS ” or “FPP synthase ” refers to an cnzynn encoded by the fdps (or crtE) gene and exhibiting farnesyl diphosphate synthase activity (EC 2.5.1.10), dimethylallyltranstransferase activity (EC 2.5.1.1) and geranylgeranyl diphosphati synthase activity (EC 2.5.1.29).

Preferably, the FPP synthase activity is increased in the host cell by overexpression of ai endogenous gene or expression of a heterologous fdps gene. In particular, the host cell of tin invention may comprise a heterologous nucleic acid encoding a polypeptide exhibiting FP1 synthase activity and/or may overexpress an endogenous nucleic acid encoding a polypeptidi exhibiting FPP synthase activity.

The polypeptide exhibiting FPP synthase activity may be any known FPP synthase h some preferred embodiments, in particular when the host cell is a Deinococcus bacterium, tin FPP synthase expresses in the host cell is from a Deinococcus bacterium. Examples o Deinococcus FPP synthases include, but are not limited to, FPP synthases from D. geothermali (NCBI Accession number: ABF45913), D. radiodurans (NCBI Accession number: NP_295118 and D. deserti (NCBI Accession number: AC046371). The polypeptide exhibiting FPP synthas activity may also be any polypeptide exhibiting FPP synthase activity and having at least 60 % preferably 65, 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99%, identity to any FPP synthase listed above

Recombinant Deinococcus bacteria genetically modified in order to increase tin conversion of IPP and DMAPP to geranylgeranyl diphosphate (GGPP) are disclosed in the paten annlication WO 2015/189428. which is herein incomorated bv reference. (DMAPP) and to increase the conversion of IPP and DMAPP to geranylgeranyl diphosphat (GGPP).

In another aspect, the present invention relates to a method of producing a phytocm desaturase variant of the invention comprising expressing a nucleic acid encoding the variant an< recovering the variant.

In particular, the present invention relates to in vitro methods of producing the variant o the present invention comprising (a) contacting a nucleic acid, cassette or vector of the inventioi with an in vitro expression system; and (b) recovering the variant. In vitro expression system: are well-known by the person skilled in the art and are commercially available.

Preferably, the present invention relates to a method of producing a phytoene desaturasi variant of the invention comprising

(a) culturing a host cell of the invention in a suitable culture medium under suitabli conditions to produce said variant; and (b) recovering said variant from the cell culture.

Optionally, the method further comprises purifying said variant.

The host cell may be cultivated in a nutrient medium suitable for production of the varian using methods known in the art. For example, it may be cultivated by shake flask cultivation, o small-scale or large-scale fermentation (including continuous, batch, fed- batch, or solid stati fermentations) in laboratory or industrial fermenters, performed in a suitable medium and unde conditions allowing the variant to be expressed and/or isolated. The cultivation takes place in ; suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, usin_| procedures known in the art. Suitable media are available from commercial suppliers or may b prepared according to published compositions (e.g., in catalogues of the American Type Culturt Collection).

The variant may be recovered using any method known in the art. If the variant of th invention is secreted into the nutrient medium, it can be recovered directly from the cultun supernatant. If the variant is not secreted, it can be recovered from cell lysates or afte permeabilisation. For example, the variant may be recovered from the nutrient medium b; conventional procedures including, but not limited to, collection, centrifugation, filtration extraction snrav-drvine. evanoration. or nrecinitation. preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation) SDS-PAGE, or extraction to obtain substantially pure polypeptides.

In another aspect, the present invention also relates to a method for preparing a variant o the invention immobilized on a solid support comprising producing the variant as detailed abovi and immobilizing the variant on a solid support. The present invention also relates to a soli< support, a variant of the invention being immobilized on the solid support. Immobilization mean are well-known to the person skilled in the art (see e.g. 'Enzyme Technology' by Martin Chaplii and Christopher Bucke, Cambridge University Press, 1990). The variant according to the presen disclosure can be immobilized on the solid support by any convenient mean, in particula adsorption, covalent binding, entrapment or membrane confinement. A wide variety of insolubli materials may be used to immobilize the variant. These are usually inert polymeric or inorganh matrices. The solid support can be for instance membranous, particulate or fibrous. Mon particularly, the solid support is preferably a bead, e.g., micro- or nanobeads. The variant can hi immobilized on a polyurethane matrix, on activated sepharose, alginate, amberlite resin Sephadex resin or Duolite resin. Other solid supports useful for the invention include resins wit! an acrylic type structure, polystyrene resins, macroreticular resins and resins with basi< functional groups. The immobilized variant may then be used in a reactor. Examples of reacto include, but are not limited to, an enzyme reactor, a membrane reactor, a continuous flow reacto such as a stirred tank reactor, a continuously operated packed bed reactor, a continuously operated fluidized bed reactor, and a packed bed reactor.

The present invention also relates to compositions comprising a variant or a host cell o the invention. In an embodiment, the composition comprises a variant of the invention Preferably, the composition further comprises components suitable for enzyme preservation. Thi variant may be free or immobilized on a solid support, preferably beads. The composition can hi liquid or dry. It may comprise the variant according to the invention in a purified or cnrichc form. In particular, the composition may further comprise stabilizers like glycerol, sorbitol o monopropylene glycol, additives like salts, sugar, preservatives or buffering agents, a redox agen such as DTT (Dithiothreitol), or a sequester such as EDTA (Ethylenediaminetetraacetic acid) h a narticular embodiment the comnosition is liouid and comnrises at least 10. 20. 30. 40 or 50 °/ In a particular embodiment, the composition is the supernatant of the culture mediun used to produce the variant of the invention from a host cell of the invention. In this embodiment the variant is secreted into the extracellular space or is released after permeabilisation or lysis o host cells. In another embodiment, the composition comprises a host cell of the invention. Th composition can be liquid (e.g. suspension) or dry (e.g. freeze-dried composition). Preferably the composition comprising the host cell is kept frozen (e.g at about -20°C) until use. Preferably the composition further comprises components suitable for cell preservation, in particular if cell: are frozen. The composition of the invention may comprise one or several host cells of thi invention, and optionally one or several additional cells.

The present invention further relates to a cell extract of a host cell according to th invention. As used herein, the term “cell extract” refers to any fraction obtained from a host cel using any method known by the skilled person such as centrifugation, ultracentrifugation ultrafiltration, homogenization, micro fluidization, crushing, lyophilization, and/or atomization In particular, the cell extract may be a cell lysate (obtained after lysis of the cells) or cel supernatant (e.g. obtained after lysis of the cells and elimination of the cell debris b; centrifugation), a cell debris, cell walls, DNA extract, enzymes or enzyme preparation or am preparation derived from host cells by chemical, physical and/or enzymatic treatment, which i: essentially free of living cells. Preferably, the cell extract comprises at least one variant of th invention.

The present invention also relates to a cosmetic composition comprising a host cell of thi invention and/or a cell extract thereof. It further relates to the use of a host cell of the inventioi and/or a cell extract thereof, to prepare a cosmetic composition. In the context of the invention cosmetic compositions, or beauty products, relate to compositions suitable for application on a least a part of the body, for cosmetic effects. The cosmetic composition of the invention ma; further contain one or more cosmetically acceptable carriers or diluents and/or one or mon additional active ingredients. In a particular embodiment, the cosmetic composition is intended to be topically used nreferablv as a cream lotion or make-un. comprising nutritional oral ingredients and which is formulated and marketed specifically fo beauty purposes.

The present invention also relates to a feed or food composition or a nutraceutica composition, comprising, or consisting of, a host cell of the invention and/or a cell extract thereol It further relates to the use of a host cell of the invention and/or a cell extract thereof, to prepan a feed or food composition, or a nutraceutical composition. Methods to produce sue! compositions are well-known by the skilled person, see e.g. WO 2013/092645. As used herein the term “nutraceutical composition” refers to a composition comprising nutrients isolated o purified and having a beneficial effect on the health of the consumer.

The present invention also relates to a method of producing a cosmetic, feed, food o nutraceutical composition comprising neurosporene and/or zeta-carotene, preferably neurosporene, said method comprising culturing a host cell of the invention under condition: suitable to produce neurosporene and/or zeta-carotene, preferably neurosporene, rccovcri neurosporene and/or zeta-carotene, preferably neurosporene, from the culture, and incorporate said neurosporene and/or zeta-carotene, preferably neurosporene, in a cosmetic, feed, food o nutraceutical composition. The composition may further comprise a host cell, a cell extract, a ert variant or a nucleic acid of the invention.

In another aspect, the present invention further relates to the use of a variant or a host cel of the invention to produce neurosporene, and optionally zeta-carotene. It also relates to a metho< of producing neurosporene and optionally zeta-carotene, comprising

- contacting phytoene with a phytoene desaturase variant or a host cell of the invention or

- culturing a host cell of the invention under conditions suitable to produce neurosporene and optionally recovering neurosporene.

The method may further comprise isolating or purifying said neurosporene and optionally zeta-carotene. The variant or the host cell of the invention produces neurosporene but it may als< nroduce nhvtofluene. zeta-carotene and/or Ivconene. nreferablv zeta-carotene. at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% (w/w) of thi Crtl variant products (phytofluene, zeta-carotene, neurosporene and lycopene). In preferret embodiments, neurosporene produced by the method of the invention represents at least 40°/ (w/w) of the Crtl variant products. In a preferred embodiment, the Crtl variant of the invention used in the method o producing neurosporene is a Crtl variant as defined above and comprising at least om substitution at position corresponding to residue G521, R171, A253 or G172 of SEQ ID NO: 1

- wherein preferably the residue at position corresponding to residue R171 of SEQ IE NO: 1 is substituted by phenylalanine, tyrosine or tryptophan, - wherein preferably the residue at position corresponding to residue G521 of SEQ IE

NO: 1 is substituted by serine, threonine, alanine, valine, methionine, leucine, isoleucine asparagine, cysteine or glutamine, in particular by serine, threonine, valine, alanine, methionine leucine and isoleucine, more preferably by serine or valine,

- wherein preferably the residue at position corresponding to residue A253 of SEQ IE NO: 1 is substituted by threonine, glycine or serine, more preferably by threonine, and

- wherein preferably, the residue at position corresponding to residue G172 of SEQ IE NO: 1 is substituted by valine, methionine, leucine or isoleucine, more preferably by valine.

In a more preferred embodiment, the Crtl variant of the invention used in the method o producing neurosporene is a Crtl variant as defined above and comprising at least om substitution at position corresponding to residue G521, wherein preferably the residue at positioi corresponding to residue G521 of SEQ ID NO: 1 is substituted by serine, threonine, alanine valine, methionine, leucine, isoleucine, asparagine, cysteine or glutamine, in particular by serine threonine, valine, methionine, leucine or isoleucine, more preferably by serine. The present invention further relates to the use of a variant or a host cell of the inventioi to produce zeta-carotene. It also relates to a method of producing zeta-carotene, comprising

- culturing a host cell of the invention under conditions suitable to produce zeta-carotene and optionally recovering zeta-carotene.

The method mav further comnrise isolating or nurifving said zeta-carotene. Preferably, zeta-carotene produced by the method of the invention represents at least 10°/ (w/w) of the Crtl variant products, i.e. phytofluene, zeta-carotene, neurosporene and lycopene More preferably, zeta-carotene produced by the method of the invention represents at least 20% at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% (w/w) of thi Crtl variant products (phytofluene, zeta-carotene, neurosporene and lycopene). In preferret embodiments, zeta-carotene produced by the method of the invention represents at least 50°/ (w/w) of the Crtl variant products.

In a preferred embodiment, the Crtl variant of the invention used in this method produce: neurosporene and zeta-carotene and comprises at least one substitution at position correspond ni to residue G521, R171, A253 or G172 of SEQ ID NO: 1 wherein preferably the residue a position corresponding to residue R171 of SEQ ID NO: 1 is substituted phenylalanine, tyrosine tryptophan, leucine, isoleucine, methionine or valine by preferably by phenylalanine, tyrosine tryptophan or leucine, more preferably by phenylalanine, tyrosine or tryptophan, whereii preferably the residue at position corresponding to residue G521 of SEQ ID NO: 1 is substitute< by serine, threonine, valine methionine, leucine, asparagine, cysteine or glutamine, in on preferably by serine or valine, wherein preferably the residue at position corresponding to residui A253 of SEQ ID NO: 1 is substituted by threonine, glycine or serine, more preferably b; threonine, and wherein preferably, the residue at position corresponding to residue G172 of SEC ID NO: 1 is substituted by valine, methionine, leucine or isoleucine, more preferably by valine. Conditions suitable to produce neurosporene and/or zeta-carotene may be easib determined by the skilled person according to the host cell used. Preferably, the host cell is ; bacterium, more preferably a Deinococcus bacterium.

In particular, the carbon source may be selected from the group consisting of C5 sugar: such as xylose and arabinose, C6 sugars such as glucose, cellobiose, saccharose and starch. In ; preferred embodiment, the carbon source is glucose. In more particular embodiment, the host cel is a bacterium, preferably a Deinococcus bacterium, and is cultured in aerobiosis and in thi presence of glucose as carbon source.

Alternatively, neurosporene and/or zeta-carotene may be produced from renewable biologically derived carbon sources such as cellulosic biomass. As used herein, the tern “cellulosic biomass” refers to any biomass material, preferably vegetal biomass, comprism_] cellulose hemicellulose and/or lienocellulose. nreferablv comnrisine cellulose an< bunch from oil palm and date palm, agave bagasse, from tequila industry), perennial grasse: (switchgrass, miscanthus, canary grass, erianthus, napier grass, giant reed, and alfalfa); municipa solid waste (MSW), aquatic products such as algae and seaweed, wastepaper, leather, cotton hemp, natural rubber products, and food processing by-products. Preferably, if the cellulosi· biomass comprises lignocellulose, this biomass is pre-treated before hydrolysis. Thi pretreatment is intended to open the bundles of lignocelluloses in order to access the polyme chains of cellulose and hemicellulose. Pretreatment methods are well known by the skilled persoi and may include physical pretreatments (e.g. high pressure steaming, extrusion, pyrolysis o irradiation), physicochemical and chemical pretreatments (e.g. ammonia fiber explosion treatments with alkaline, acidic, solvent or oxidizing agents) and/or biological pretreatments.

Temperature conditions can also be adapted depending on the host cell, in particula depending on the use of mesophilic or thermophilic bacteria.

In an embodiment, the host cell is a thermophilic Deinococcus bacterium such as fo example D. geothermalis or D. murrayi, and the culture of the host cell under conditions suitabli to produce neurosporene and/or zeta-carotene is performed at a temperature comprised betweei 30°C and 55°C, preferably between 35 and 50°C, more preferably between 40°C and 50°C, am even more preferably between 45 and 48°C.

In another embodiment, the host cell is a mesophilic Deinococcus , such as for exampL D. grandis, D. aquaticus, D. indicus, D. cellulosilyticus or D. depolymerans, and the culture o the host cell under conditions suitable to produce neurosporene and/or zeta-carotene is performe< at a temperature comprised between 20°C and 40°C, preferably between 28 and 35°C, mort preferably at about 30°C.

In a preferred embodiment, at least 1 mg/g DCW of neurosporene, preferably at least 1( mg/g DCW of neurosporene, and more preferably at least 15 mg/g DCW of neurosporene, an produced and/or recovered with the method of the invention.

In another preferred embodiment, at least 0.1 mg/g DCW of zeta-carotene, preferably a least 1 mg/g DCW of zeta-carotene, more preferably at least 5 mg/g DCW of zeta-carotene an produced and/or recovered with the method of the invention. The methods of the invention ma; be performed in a reactor. By “reactor” is meant a conventional fermentation tank or an; apparatus or system for biomass conversion, typically selected from bioreactors, biofilters, rotar; biological contactors and other gaseous and/or liouid nhase bioreactors. The annaratus whicl Further aspects and advantages of the present invention will be described in the folio wins examples, which should be regarded as illustrative and not limiting.

EXAMPLES

MATERIALS AND METHODS

A Deinococcus geothermalis strain was genetically engineered to produce phytoene. Th recombinant D. geothermalis producing phytoene was obtained by disrupting a part of thi carotenoid pathway, i.e. the phytoene desaturase (E.C. 1.3.99.26, 1.3.99.2g, 1.3.99.29, 1.3.99.31 ( crtl ) gene was knockout. This modification in the resulting mutant strain was checked IT sequencing.

Saturation mutagenesis technique was used to produce all possible amino acids at selectet positions of the D. geothermalis Crtl enzyme. The resulted mutated crtl genes encoding mutatet D. geothermalis phytoene desaturases were then introduced into the chromosome of the phytoem producing strain at an ectopic chromosome site and under the control of a constitutive promoter

To make seed cultures, 20pL of glyceroled cultures were inoculated into 600 pL o defined medium (NH₄)₂S0₄ < 100 mM ; NaH₂P0₄.H₂0 < 10 mM ; KC1 < 10 mM ; Na₂S0_{4 <} 10 mM ; Acide citrique < 30 mM ; MgCl₂.6H₂0 < 10 mM ; CaCl₂.2H₂0 < 10 mM ; ZnCl₂ < 5( mg/L ; FeS0₄.7H20 <50 mg/L ; MnCl₂.4H₂0 <50 mg/L; CuS0₄ < 50 mg/L; COC1₂.6H₂0 < 5( mg/L ; H3BO3 < 5 mg/L ; MES < 200 mM ; (NH₄)6Mq₇q₂₄.4H₂0 < 0,5 mM ; Glucose or dextrosi < 30 g/L (166 mM); Yeast Extract 5 g/L and cultivated at 48°C and 700 rpm. After 24H o growth, cultures were then diluted 30 times in a 96-well plate to inoculate the same fresh medium The cultures for carotenoids production were performed at 48 °C and shaked at 700 rpm for 24 h

After 24h of culture, 200pL of cultures were centrifuged and carotenoids were extractet by adding dichloromethane / ethanol (60:40, v/v) and heating 30 minutes at 60°C. Afte centrifugation, the organic phase was analyzed by HPLC (Column C18 poroshell agilent 2.1mn xl50 mm *2.7pm, mobile phase : methanol/acetonitrile/TBME). Quantification of lycopene neurosporene, zeta-carotene (all-trans-zeta carotene), phytofluene and phytoene were performet using the standard curves established with the corresponding commercial standards. Standan solution concentrations were measured using their specific absorption coefficient A^1% (15-cis phytoene (A^1% = 760 at 286 nm in MeOH); neurosporene (A^1% = 2920 at 440 nm hexane); zeta A second zeta-carotene isomer was identified. Assuming that the molar extinctioi coefficients were equivalent for both isomers of zeta-carotene, the standard curve of all -trans zeta-carotene was used to quantify the second isomer. Quantities of a 11 - trans - ze ta- c a ro tone an< said second isomer were added to obtained the total amount of zeta-carotene. Strains producing neurosporene were selected and sequenced to identify thi corresponding mutation.

RESULTS

Crtl products obtained with wild-type Crtl of SEQ ID NO: 1 and variants thereo comprising the following substitutions: G521S, R171F+A253T, R171F, R171Y, R171W R171G, R171F, G172V, G521V, G521A, G521T, G521F, G521I, G521M, G521N, G521C o G521Q are presented in the tables below.

Variant G521S: Crtl of SEQ ID NO: 1 wherein G521 is substituted by serine.

Variant R171F+A253T: Crtl of SEQ ID NO: 1 wherein R171 is substituted b; phenylalanine and A253 is substituted by threonine. Variant R171F: Crtl of SEQ ID NO: 1 wherein R171 is substituted by phenylalanine.

Variant R171Y: Crtl of SEQ ID NO: 1 wherein R171 is substituted by tyrosine.

Variant R171W: Crtl of SEQ ID NO: 1 wherein R171 is substituted by tryptophan.

Variant R171G: Crtl of SEQ ID NO: 1 wherein R171 is substituted by glycine.

Variant R171L: Crtl of SEQ ID NO: 1 wherein R171 is substituted by leucine. Variant G172V: Crtl of SEQ ID NO: 1 wherein G172 is substituted by valine.

Variant G521V: Crtl of SEQ ID NO: 1 wherein G521 is substituted by valine.

Variant G521A: Crtl of SEQ ID NO: 1 wherein G521 is substituted by alanine.

Variant G521T: Crtl of SEQ ID NO: 1 wherein G521 is substituted by threonine.

Variant G521L: Crtl of SEQ ID NO: 1 wherein G521 is substituted by leucine. Variant G521I: Crtl of SEQ ID NO: 1 wherein G521 is substituted by isoleucine.

Variant G521M: Crtl of SEQ ID NO: 1 wherein G521 is substituted by methionine.

Variant G521N: Crtl of SEQ ID NO: 1 wherein G521 is substituted by asparagine.

Variant G521C: Crtl of SEQ ID NO: 1 wherein G521 is substituted by cysteine.

Variant G521Q: Crtl of SEQ ID NO: 1 wherein G521 is substituted by glutamine. Table 1 : quantity of each carotenoid represented as a percentage (w/w) of the sum o

Crtl products, i.e. lycopene , neurosporene , zeta-carotene and phytofluene.

Table 2 : Ratios for the formation of neurosporene and zeta-carotene vs lycopene (N/l and Z/L, w/w) for the Crtl variants

Claims

1. A phytoene desaturase variant comprising a sequence (i) having at least 65% identity to the full length amino acid sequence set forth in SEQ ID NO: 1 and (ii) comprising at least om substitution at position corresponding to residue G521, R171, A253 or G172 of SEQ ID NO: 1 wherein said variant produces neurosporene.

2. The variant according to claim 1, wherein said variant comprises at least om substitution at position corresponding to residue G521, R171 or G172 of SEQ ID NO: 1.

3. The variant according to claim 1 or 2, wherein said variant comprises a substitution a position corresponding to residue G521 of SEQ ID NO: 1.

4. The variant according to claim 1 or 2, wherein said variant comprises a substitution a position corresponding to residue G521 of SEQ ID NO: 1 and wherein the residue at positioi corresponding to residue G521 of SEQ ID NO: 1 is substituted by serine, threonine, alanine valine, methionine, leucine, isoleucine, asparagine, cysteine or glutamine, preferably serine threonine or alanine, leucine, isoleucine, asparagine, cysteine or glutamine, more preferably b; serine.

5. The variant according to any of claims 1 to 4, wherein said variant comprises : substitution at position corresponding to residue R171 of SEQ ID NO: 1 and preferably whereii the residue at position corresponding to residue R171 of SEQ ID NO: 1 is substituted b; phenylalanine, tyrosine, tryptophan, glycine, alanine, serine, threonine, leucine, isoleucine methionine or valine, preferably by phenylalanine, tyrosine or tryptophan.

6. The variant according to any of claims 1 to 5, wherein said variant comprises : substitution at position corresponding to residue A253 of SEQ ID NO: 1 and preferably whereii the residue at position corresponding to residue A253 of SEQ ID NO: 1 is substituted b; threonine, glycine or serine. position corresponding to residue G172 of SEQ ID NO: 1 is substituted by valine, methionine leucine or isoleucine, preferably by valine.

8. The variant according to claim 3, wherein said variant produces neurosporene as mail product and wherein the residue at position corresponding to residue G521 of SEQ ID NO: 1 i: substituted by serine, threonine, alanine, methionine, leucine, isoleucine, asparagine, cysteine o glutamine, preferably by serine, asparagine or isoleucine.

9. The variant according to claim 5, wherein said variant produces neurosporene as mail product and wherein the residue at position corresponding to residue R171 of SEQ ID NO: 1 i: substituted by phenylalanine, tyrosine or tryptophan.

10. The variant according to any of claims 1 to 9, wherein the residue at positioi corresponding to residue E383 of SEQ ID NO: 1 is glutamic acid or glutamine.

11. The variant according to any of claims 1 to 10, wherein the residue at positioi corresponding to residue L308 of SEQ ID NO: 1 is leucine.

12. The variant according to any of claims 1 to 11, wherein said variant comprises : sequence having at least 69% identity to the amino acid sequence from position 11 to positioi

543 of SEQ ID NO: 1.

13. The variant according to any of claims 1 to 12, wherein said variant comprises : sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95°/ or at least 99% identity to the full length amino acid sequence of a wild-type phytoene desaturasi of a Deinococcus bacterium, preferably selected from the group consisting of SEQ ID No. 1 t< 31.

14. The variant according to any of claims 1 to 13, wherein said variant comprises ; sequence that differs from a sequence of a wild-type phytoene desaturase of a Deinococcu, bacterium nreferablv selected from the eroun consisting of SEO ID No. 1 to 31. bv 1. 2. 3. 4. 5

16. An expression cassette or vector comprising the nucleic acid of claim 15.

17. A host cell comprising the nucleic acid of claim 15 or the expression cassette or vecto of claim 16.

18. The host cell of claim 17, wherein said host cell is able to produce phytoene.

19. A method of producing a phytoene desaturase variant as defined in any of claims 1 1< 14, comprising:

(a) culturing the host cell according to claim 17 or 18 in a suitable culture medium unde suitable conditions to produce said phytoene desaturase variant; and

(b) recovering said phytoene desaturase variant from the cell culture. 20. A method of producing neurosporene comprising contacting phytoene with : phytoene desaturase variant of any of claims 1 to 14 or a host cell according to claim 17 or 18 and optionally recovering neurosporene.

21. A method of producing neurosporene comprising culturing a host cell of claim 17 o 18 under conditions suitable to produce neurosporene, and optionally recovering neurosporem from the culture.

22. Use of a phytoene desaturase variant of any of claims 1 to 14 or a host cell according to claim 17 or 18 to produce neurosporene.