JP2022101424A - Methods for producing biochemicals using enzyme genes derived from brevundimonas strain and compositions made thereby - Google Patents

Abstract

To provide crtW gene encoding a novel ketolase derived from a Brevundimonas strain for carotenoid synthesis and synthetic nucleic acid constructs and host organisms transformed with the synthetic nucleic acid constructs.SOLUTION: A vector comprising a nucleic acid of a specific sequence and a heterologous nucleic acid sequence is used.SELECTED DRAWING: None

Description

The present disclosure generally relates to the field of molecular biology and, more specifically, genetically manipulates the metabolic pathways of microorganisms to include a variety of gaseous feedstocks for the biological production of biochemicals. Regarding the use of feedstock.

In certain embodiments, nucleic acid sequences are provided to express a carotenoid product comprising any one or more of SEQ ID NOs: 1, 4, 5, 6 or 7. In certain frequent embodiments, a vector comprising the nucleic acid of SEQ ID NO: 1 and a heterologous nucleic acid sequence is provided.

In certain frequent embodiments, a nucleic acid sequence encoding an enzyme comprising an amino acid sequence that is at least 96% identical or homologous to SEQ ID NO: 2 or 3 is provided and the expressed enzyme converts β-carotene to canthaxanthin. can do. In certain related embodiments, the amino acid sequence is at least 97% identical or homologous to SEQ ID NO: 2 or 3, and the expressed enzyme is capable of converting β-carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 98% identical or homologous to SEQ ID NO: 2 or 3, and the expressed enzyme is capable of converting β-carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 99% identical or homologous to SEQ ID NO: 2 or 3, and the expressed enzyme is capable of converting β-carotene to canthaxanthin.

In a frequently included embodiment, a vector comprising one or more nucleic acid sequences encoding an enzyme comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 2 or 3 is provided and when expressed, the enzyme is β. -Can convert carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 97% identical or homologous to SEQ ID NO: 2 or 3, and the expressed enzyme is capable of converting β-carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 98% identical or homologous to SEQ ID NO: 2 or 3, and the expressed enzyme is capable of converting β-carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 99% identical or homologous to SEQ ID NO: 2 or 3, and the expressed enzyme is capable of converting β-carotene to canthaxanthin.

In a frequently included embodiment, a synthetic nucleic acid construct comprising a promoter, a ribosome binding site, and one of a plurality of nucleic acid sequences encoding an enzyme comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 2 or 3. Once provided and expressed, the enzyme can convert β-carotene to cantaxanthin. In certain related embodiments, the amino acid sequence is at least 97% identical or homologous to SEQ ID NO: 2 or 3, and the expressed enzyme is capable of converting β-carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 98% identical or homologous to SEQ ID NO: 2 or 3, and the expressed enzyme is capable of converting β-carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 99% identical or homologous to SEQ ID NO: 2 or 3, and the expressed enzyme is capable of converting β-carotene to canthaxanthin. Often, the synthetic nucleic acid construct is a vector containing a plasmid.

In frequent embodiments, transformed host organisms comprising the synthetic nucleic acid constructs described above and herein are provided, and the transformed host organisms are capable of heterologous expression of the synthetic nucleic acid constructs. Often, the expression host organism is a transformed bacterium adapted to grow in a chemically synthesized autotrophic mode. In certain embodiments, the expression host organism is Cupriavidus necator.

In certain embodiments, a nucleic acid sequence corresponding to the crtW carotenoid ketrase gene from Brebandimonas strain OB307, which encodes the amino acid sequence of SEQ ID NO: 2, is provided, which nucleic acid sequence is such to produce carotenoids in biological host cells. Included in the expression constructs adapted to. In certain frequent embodiments, the biological host cell can use CO ₂ and H ₂ to meet at least some of the carbon and energy requirements of the host cell.

In certain embodiments, the nucleic acid sequence corresponding to the crtZ-crtW carotenoid hydroxylase-ketrase gene fusion is provided, the crtW portion of the fusion being the ketrase gene from the Brebandimonas strain OB307 encoding amino acid SEQ ID NO: 2. be.

In certain embodiments, the nucleic acid sequence is provided encoding the crtZ-crtW carotenoid hydroxylase-ketrase fusion protein of SEQ ID NO: 3, and (a) the crtW portion of the fusion encodes the amino acid sequence of SEQ ID NO: 2. It is a ketorase gene derived from Brebandimonas strain OB307, and (b) the nucleic acid sequence is part of an expression construct adapted to produce carotenoids when functionally integrated into a biological host cell.

In certain embodiments, a nucleic acid sequence encoding the crtZ-crtW carotenoid hydroxylase-ketrase fusion protein of SEQ ID NO: 3 is provided, where (a) the crtW portion of the fusion encodes the amino acid sequence of SEQ ID NO: 2. A ketolase gene from Bandimonas strain OB307, (b) the nucleic acid sequence is part of an expression construct adapted to produce carotenoids when functionally integrated into a biological host cell, (c). Biological host cells can use CO ₂ and H ₂ to meet at least some of their carbon and energy requirements.

In certain embodiments, a transposon is used to provide a suicide vector construct adapted to insert a DNA sequence into the bacterial genome, wherein the suicide vector construct comprises (a) a DNA sequence, (b) a transposon. It contains an insertion adjacent DNA containing the nucleic acid sequence of SEQ ID NO: 8, and (c) a suicide plasmid skeleton.

In certain embodiments, a transformed host cell comprising a nucleic acid sequence encoding amino acid SEQ ID NO: 2 is provided, the nucleic acid sequence being part of an expression construct adapted to produce carotenoids in the host cell. be.

In certain embodiments, a method of forming a transformed host cell as contemplated herein is provided, the method comprising inserting an expression construct into the genome of the host cell using a transposon. .. In many cases, such insertions using transposons are random insertions.

In certain embodiments, a nucleic acid sequence corresponding to the crtW carotenoid ketrase gene from Brebandimonas strain OB307, which encodes the amino acid sequence of SEQ ID NO: 2, is provided and the nucleic acid sequence produces carotenoids in a cell-free expression system. It is part of an expression construct adapted as such.

In certain embodiments, a method of producing ketocarotenoids in a biological host cell is provided by heterologous expression of OB307-crtW in the host cell. In many cases, biological host cells contain hydrogen bacteria. Also, hydrogen bacteria are often selected from Cupriavidus, Rhodobacter, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Paracoccus or Hydrogenophaga. Includes strains that are to be treated. In a particular embodiment, the strain of hydrogen bacteria is Cupriavidus necator. In certain embodiments often included, biological host cells are cultured as part of a consortium of host cells of different species.

In certain embodiments, a method of producing ketocarotenoid in a biological host cell is provided, the step of transforming the biological host cell with a vector containing a crtZ-OB307-crtW fusion, and crtZ in the biological host cell. It comprises the step of heterologously expressing the -OB307-crtW fusion to synthesize the ketocarotenoid, or the step of heterologously expressing the crtZ-OB307-crtW fusion in a biological host cell to synthesize the ketocarotenoid. In many cases, biological host cells contain hydrogen bacteria. Also, hydrogen bacteria are often selected from Cupriavidus, Rhodobacter, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Paracoccus or Hydrogenophaga. Includes strains that are to be treated. In a particular embodiment, the strain of hydrogen bacteria is Cupriavidus necator. In certain embodiments often included, biological host cells are cultured as part of a consortium of host cells of different species.

In certain embodiments, a method of producing canthaxanthin from β-carotene in vitro is provided, wherein the method is in a solution containing β-carotene and is either SEQ ID NO: 1, 4, 5, 6, 7 or 8. The protein expression product catalyzes the conversion of at least a portion of the β-carotene to canthaxanthin, comprising contacting the nucleic acid sequence with a protein expression product that is at least 96% identical to the nucleic acid sequence. In most cases, the nucleic acid sequence is at least 90% identical to the nucleic acid sequence of any of SEQ ID NOs: 1, 4, 5, 6, 7 or 8. In most cases, the nucleic acid sequence is at least 91% identical to the nucleic acid sequence of any of SEQ ID NOs: 1, 4, 5, 6, 7 or 8. In most cases, the nucleic acid sequence is at least 92% identical to the nucleic acid sequence of any of SEQ ID NOs: 1, 4, 5, 6, 7 or 8. In most cases, the nucleic acid sequence is at least 93% identical to the nucleic acid sequence of any of SEQ ID NOs: 1, 4, 5, 6, 7 or 8. In most cases, the nucleic acid sequence is at least 94% identical to the nucleic acid sequence of any of SEQ ID NOs: 1, 4, 5, 6, 7 or 8. In most cases, the nucleic acid sequence is at least 95% identical to the nucleic acid sequence of any of SEQ ID NOs: 1, 4, 5, 6, 7 or 8. In most cases, the nucleic acid sequence is at least 97% identical to the nucleic acid sequence of any of SEQ ID NOs: 1, 4, 5, 6, 7 or 8. In most cases, the nucleic acid sequence is at least 98% identical to the nucleic acid sequence of any of SEQ ID NOs: 1, 4, 5, 6, 7 or 8. In most cases, the nucleic acid sequence is at least 99% identical to the nucleic acid sequence of any of SEQ ID NOs: 1, 4, 5, 6, 7 or 8. In certain frequent embodiments, the host organism is one that naturally produces β-carotene.

It is a schematic diagram of individual enzymes and products in the biosynthetic pathway between farnesyl diphosphate (FPP) and astaxanthin. It is a figure which shows the component of the system 1 astaxanthin operon. It is a figure which shows the component of the system 2 canthaxanthin operon. It is a figure which shows the component of the system 3 astaxanthin operon and the crtZW fusion gene. FIG. 3 is a detailed map of a synthetic carotenoid operon that uses tetracycline as an antibiotic resistance marker and produces canthaxanthin (including the OB307-crtW gene) along with the Tn5 transposase gene inserted into the suicide vector. Transposons are added and operons are randomly inserted into the host cell genome. It is a figure which shows the process of transformation and chromosome insertion of an operon into a host cell using transposon mutagenesis. Here, system 1 is used as an example. (ME = Mosaic Ends (reverse repeats); Pro = promoter; ori = origin of replication or transfer; term = transcription terminator). FIG. 5 shows an HPLC chromatogram (black trace) showing canthaxanthin produced by C. necator cells that heterologously express the canthaxanthin biosynthetic pathway. It is a figure which shows the corresponding UV-Vis spectrum of the canthaxanthin peak shown in FIG. 7. FIG. 3 shows an HPLC chromatogram (black trace) showing a carotenoid product derived from C. necator cells that heterologously express the astaxanthin biosynthetic pathway. The chromatogram of the purified astaxanthin standard is shown in red, which overlaps with the small peaks of the black trace. It is a figure which shows the corresponding UV-Vis spectrum of the astaxanthin peak shown in FIG.

Carotenoids are long-chain isoprenoid molecules with nutritional advantages as colorants and additives in fish feeds, animal feeds and dietary supplements that provide protection against oxidative damage to cells, especially free radicals and active oxygen species. .. Carotenoids can be expressed naturally and through the expression of one or more carotenoid genes encoding biosynthetic enzymes in plants, algae, paleontology, fungi and bacteria. Conventional production of 40 carbon (C40) tetraterpene carotenoids, including carotene and xanthophylls, has involved the extraction of natural molecules from various microorganisms and plants. However, some naturally occurring astaxanthin-producing strains, such as Xanthophyllomyces yeast, produce lower-value mirror isomers of astaxanthin and are more productive, such as Haematococcus pluvialis. The process of growing naturally occurring microalgae is difficult, time consuming, resource intensive and expensive.

Non-biological production of molecules such as astaxanthin and canthaxanthin has been achieved by chemical synthesis from petroleum sources (2002). However, these latter methods produce mixtures of astaxanthin and enantiomers, which are also of low value because they are inefficient radical quenchers and therapeutic agents, and these synthetic products are human in the EU. And face serious regulatory issues regarding animal consumption. Recently, genetically engineered organisms have been used to produce high-value canthaxanthin, astaxanthin and other C40 carotenoids as well as xanthophylls. FIG. 1 shows the carotenoid biosynthetic pathway from farnesyl diphosphate (FPP) to astaxanthin.

In addition to astaxanthin, canthaxanthin is a valuable carotenoid product that can be synthesized by transketolase enzymes, such as the bacterial crtW ketolase gene that acts on β-carotene as its substrate. Carotenoids such as canthaxanthin and astaxanthin can be produced by the crtW gene-encoded ketolase from various Brebandimonas species, which is considered to be the most active and effective carotenoid ketolase.

Since the yield and production rate of carotenoids per gram of dry weight of biomass are not high in natural or genetically modified organisms, an expression system capable of inexpensively and efficiently producing carotenoids using this CrtW enzyme is also required. Is.

Hydrogen bacteria are attractive hosts for carotenoid expression, because some species naturally produce larger intima than many other bacteria, and these membranes are highly lipophilic C40. Necessary for accumulating carotenoids.

Wider membrane capacity is also advantageous, as both CrtZ hydroxylase and CrtW ketolase enzymes are likely to be endogenous membrane proteins containing transmembrane (TM) helices that can span cell membranes.

In addition, certain hydrogen bacteria, such as Cupriavidus necator, do not naturally produce carotenoids and thus contain regulatory interference (eg, feedback inhibition) or (eg, the crtG gene, thus dihydroxyting the astaxanthin product. -There is less potential for unwanted enzymatic modification of products (such as Cupriavidus vesicularis strain DC263) that spontaneously hydroxylate to astaxanthin.

The carotenoids thus produced are either provided as part of the bacterial biomass or extracted from it to produce a substantially pure carotenoid product, or supercritical CO ₂ or solvent-based extraction, etc. Concentrates are formed by other extraction methods. In addition, carotenoids such as canthaxanthin are mixed with other ingredients such as sugars, cornstarch, lignosulfonates, binders and oils to produce products (eg, DSM Carophyll Red 10%). can do.

Bacterial CrtW enzymes are determined by the presence of His-rich motif HX (3 or 4) H, HX (2 or 3) HH and HX (2 or 3) HH: the following amino acid residues, ie His69, 6-8 of His73, His107, His110, His111, His225, His228 and His229 are used to bind a ferrous cofactor that catalyzes the oxygenation reaction. Based on mutagenesis studies, Asp118 may also be required. Thus, although not intended to be bound by any particular operating theory, natural or engineered versions of this enzyme should or must contain these ligands in order to have catalytic activity. It is thought that it will not be. Similarly, such enzymes may require a functional transmembrane sequence because there are putative TM helices that appear to organize iron binding sites inside the membrane.

Expressing such codon-optimized gene pathways in bacteria with high GC + C content, for example, because GC content makes it difficult to newly synthesize genes and operons for synthetic biology. , Has previously been proven to be a challenge.

The present disclosure describes a newly discovered crtW gene from a new strain of Brebandimonas, referred to herein as OB307, which encodes a novel transketolase for carotenoid synthesis. The present disclosure also provides an exemplary synthetic operon containing additional related carotenoid gene sequences, the expression of which is used to produce ketocarotenoids. Suitable DNA expression constructs derived from these sequences are inserted into the expression host for expression. The expression product is a transketolase enzyme that can be engineered to convert β-carotene to canthaxanthin and astaxanthin. The carotenoid products of this synthetic operon are Escherichia coli, B. B-14200, B-356, Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Cupriavidus necator. It has been expressed. Rhodopseudomonas palustris and Rhodobacter sphaeroides are commonly known as purple non-sulfur (PNS) bacteria. Rhodobacter capsulatus is another PNS bacterium that can be used as a host for these DNA expression constructs.

As disclosed herein, the CrtW ketolase enzymes currently disclosed include astaxanthin and cantaxanthin via cloning of the disclosed DNA sequences, including similar sequences with the attributes described herein. Often utilized in the production of ketocarotenoids, DNA is placed in constructs containing ribosome binding sites, promoters, and terminators, as well as other structural gene elements. Other enzyme genes according to this embodiment, such as crtZ, crtY, crtI, crtB, crtE, as well as additional structural and regulatory elements are also optionally incorporated into the construct to form an operon for carotenoid production. The construct is then introduced into a host organism, such as a host cell, as one or more small circular DNA vectors, such as a plasmid, or via integration into the genome of the organism, using methods known in the art. .. For organisms that are already producing beta-carotene, a gene encoding this single enzyme is introduced to produce this CrtW transketolase enzyme, converting some of the β-carotene to canthaxanthin. When the crtZ gene is also introduced, a gene product (ie, hydroxylase) can also be expressed, thereby converting at least a portion of canthaxanthin to astaxanthin.

The product of this crtW gene is used, for example, in a cell-free expression system in which β-carotene is enzymatically converted to canthaxanthin. When the crtZ gene and the crtW gene are expressed simultaneously or in combination, at least a part of the β-carotene substrate is converted to canthaxanthin and a part is converted to astaxanthin by the action of the enzymatic products of the two genes. The novel crtW and crtZ genes may be provided on two different segments of DNA or as a single DNA constituting the gene for the fusion protein encoding the function of both CrtW ketolase and CrtZ hydroxylase. May be.

Many different organisms can be considered as heterologous expression hosts for this novel crtW gene. Hosts that can utilize H ₂ and CO ₂ as energy and carbon sources, and hosts that cannot utilize H ₂ and CO ₂ as energy and carbon sources, are considered suitable heterologous expression hosts. For example, these include bacteria, plants, algae, archaea, and fungi. Bacteria such as Escherichia coli and Bacillus subtilis, fungi such as sprouting yeast and Koji mold, plants such as African rice, algae such as Chlorella vulgaris, and archaea such as Sulfolobus solfataricus. Classes, or other species, can be heterologous hosts for this novel crtW gene to produce the enzyme it encodes and the action of this enzyme to produce carotenoid products.

Heterologous expression of this enzyme and synthetic operon disclosed herein initially uses a broad host range of expression plasmids, initially E. coli, Cupriavidus nectifolia B-14200, Cupriavidus nectus B-356, Rhodopseudomonas palustris. It has been shown in Palstris), Rhodobacter sphaeroides and Cupriavidus necator. In each case, heterologous expression of the novel OB307-crtW gene was observed via the production of canthaxanthin in transformed bacteria (no canthaxanthin production in wild-type organisms). This transformation was performed using the same plasmid used in C. necator. The promoters disclosed herein are active in all of these strains. As mentioned above, E. coli cells were transformed using plasmid electroporation. Other strains were transformed using conjugation with E. coli strain S17-1 according to standard methods (see, eg, Phornphisutthimas et al, 2007; Gruber et al, 2015). The joined cells are first plated on LB agar medium and then resuspended on a serially diluted sterile liquid medium and then resuspended on the following agar plates, ie (1) LB + 50 μg / ml canamycin or 10 μg / ml for E. coli. Tetracycline; (2) MR2 medium + 2% fructose and 50 μg / ml canamycin for bacillus; (3) play on MR2 medium + 2% fructose and 500 μg / ml canamycin for C. necator (C. necatol) and PNS bacteria. Diluted. The surviving transconjugated colonies were then harvested and re-streaked on new plates until a pure single colony was obtained. Propagation in liquid culture was performed by inoculating LB + antibiotics (all strains) or MR2 + antibiotics (H2 oxidized PNS bacteria and C. necator (C. necatol)) with cells of a given mutant.

A fusion gene consisting of crtZ and crtW was created by constructing a fragment of synthetic DNA in which crtZ and crtW were linked by a linker sequence and incorporating this fusion sequence into a synthetic operon instead of the original crtW gene of the expression plasmid. This heterologous expression vector was then transformed into E. coli and Cupriavidus necator. In each case, the production of astaxanthin and canthaxanthin was confirmed. In addition, this synthetic operon was converted to C. necator using an allelic exchange system (NaCl-free agar medium containing 6% sucrose (w / v) was used for the counterselection of sacB levansucrase) and a suicide vector. Upon insertion into the genome, carotenoid production was reconfirmed.

C. necator (C. necatol) strain H16 has been used as an expression host as well as other C. necator (C. necatol) strains and strains of other hydrogen bacteria. Thus, carotenoid products can be produced by gas fermentation of transformed bacteria using inexpensive raw materials (eg, waste CO ₂ , H ₂ , O ₂ and inorganic salts) and of the process. Economic efficiency can be improved.

Further genera and species of hydrogen bacteria that can be transformed with the vectors and DNA constructs described herein for heterologous expression in the carotenoid pathway while growing on H2 _{- CO2} - _O2 . For example, Rhodobacter capsulatus and other Rhodobacter species, Paracocccus, Rhodococcus, Hydrogenophaga, Rhodospirillum, Rhodospirillum, Rhodopseu. And so on.

A novel strain of Brebandimonas OB307 was isolated from the agar plate as a red-orange contaminant colony in the laboratory. The 16S rRNA gene was sequenced (forward and reverse) and ClustalW was used to compare it to the 16S sequences of other Brebandimonas species. This analysis revealed that OB307 has 99.7-99.8% identity with the 16S sequences of B. vesicularis and B. nasdae. Genomic DNA is extracted from approximately 100 mg of wet cell paste, sequenced whole genome using 60x Illumina paired end sequencing (150 base pair reads), and sequenced by SNPsaurus Inc. (Eugene, Oregon). Assembled and annotated the Contig. From this sequence, BLAST searches identified multiple genes with high similarity to other published carotenoid biosynthetic genes.

One of the complete open reading frame sequences was first identified as "fatty acid desaturase" by annotation software. The fatty acid desaturase is known to have a structure similar to that of carotenoid ketrase, and further analysis reveals that this sequence has a high similarity to CrtW-type carotenoid ketrase, and subsequent expression cloning reveals its activity. Was confirmed. Therefore, the gene sequence is referred to herein as OB307-crtW (SEQ ID NO: 1). As can be seen from the translated sequence of OB307-CrtW, there are eight histidine motifs (highlighted in yellow) and Asp-118 that define the ferrous binding site of this type of ketrase (SEQ ID NO: 2). (Highlighted in blue) is included. SEQ ID NO: 3 shows the CrystalW2.1 amino acid sequence alignment between OB307-CrtW and CrtW of Brebandimonas strain DC263 (GenBank Accession No. ABC5016.1). Both proteins contain 241 amino acids, with 11 amino acid differences between them (approximately 95.5% identity). More recently, the putative crtW gene from the Brebandimonas strain SgAir0440 has been published as part of the genomic sequence of air-polluting bacteria (GenBank Accession No. QCR00114). This gene has 99.6% similarity to the amino acid sequence of OB307-crtW, but has not been reported to be cloned and expressed, and the function of the enzyme was analyzed to actually β-carotene. It has not been confirmed to be a trase.

The native OB307-crtW sequence was converted to a new codon-optimized sequence for expression on C. necator. This new sequence was included as part of a codon-optimized synthetic operon containing canthaxanthin, including crtE, crtY, crtI, crtB and crtW (FIG. 3). The construct for making astaxanthin also contained the complete crtZ sequence (Fig. 2). Other gene sequences within the pathway are sourced from various other bacteria and the GenBank accession numbers are as follows: the genes crtE, crtY, crtI and crtB are Pantoea agglomerans / Erwinia herbicola pAC-BETA plasmid, M8720. / Synthesized from the sequence of M99707, crtZ was synthesized from the sequence of Pantoea ananatis strains AJ13355, NC_017533, and crtW was synthesized from the sequence of OB307-crtW described herein.

Operon synthesis benefits from special procedures (eg, available from Aster Bioscience, Inc. (Livermore, Calif.)) Due to its very high G + C content (approximately 61% -70%). A highly active constitutive promoter in C. necator was placed upstream of the carotenoid gene to direct intracellular mRNA synthesis. Other suitable promoters are well known in the art and are also contemplated herein. Inducibility that can be used to control the timing of initiation of gene transcription by applying an external inducer molecule (eg, IPTG of the lac or tac promoter) or environmental stimuli (eg, nitrogen deficiency of the phaC1 promoter). Promoters can also be used if they are compatible with the host's metabolic and transport system. A ribosome binding site (RBS) optimized for C. necator was placed upstream of each gene sequence. To optimize overall expression, spacer sequences were added between the promoter and RBS of the crtE gene and between the RBS and start codon of individual genes. An end sequence (E. coli rrnB) was placed at the end of the operon to prevent unnecessary translation of downstream elements.

Synthetic operons (SEQ ID NOs: 5, 6 and 7) are first obtained by cloning them into a broad host range plasmid pBBR1MCS-2 (eg, kanamycin as a choice) using NdeI and AseI as adjacency restriction sites. The activity was tested. The ligated DNA product was electropo with Bio-Rad GenePulser II equipped with a Capacitance Extender Plus Pulse Controller II unit (Bio-Rad Inc., Hercules, Calif.). It was transformed into Escherichia coli by ration. Wash E. coli cells three times with 10% cold glycerol according to the method described in Belcher and Knight's online protocol (https://openwetware.org/wiki/Belcher/Knight:_Electrocompetent_Cells) to make E. coli cells electrocompetent. did. 50 μl of electrocompetent cells were added to a 1 mm gap of chilled sterile cuvette and mixed with 1 μl of DNA (about 1-50 ng). The electroporator settings are as follows: 1.2kV, 25μF, 200Ω. The time constant was usually 3-5 milliseconds. After the pulse, cells were transferred to preheated SOB medium in a small sterile tube and restored at 37 ° C. for 1 hour with shaking. Then, aliquots were plated on LB agar medium containing 50 μg / ml kanamycin to select antibiotics. After culturing at 30 ° C., colonies were collected and individually grown in LB broth. Plasmid DNA was isolated from various clones by standard methods. This DNA was cleaved with an appropriate restriction enzyme and analyzed by agarose gel electrophoresis to identify positive clones. Plasmid DNA from one correct clone was transformed into E. coli conjugation strain S17-1. After that, the above process was repeated to find the correct S17-1 clone. The S17-1 clone containing the synthetic canthaxanthin or astaxanthin operon on the plasmid pBBR1MCS-2 was then conjugated to the C. necator (C. necatol) host strain or other host strain by the standard method described above. When plated on solid MR2-fructose medium (Table 1) containing 500 μg / ml kanamycin, C. necator colonies appeared. Colonies showing a deep orange or red color were harvested and streaked back into kanamycin plates to confirm their color phenotype and antibiotic resistance. Selected clones were harvested and grown in liquid medium containing antibiotics.

As mentioned above, the processing capacity of the enzyme at the end of the astaxanthin production pathway can be improved by genetically fusing the crtZ and crtW genes to encode the chimeric protein. As shown in the map of FIG. 4, between the 3'end of the complete crtZ gene from Pantoea ananatis and the 5'end of the OB307-crtW gene (without N-terminal methionine) (amino acid sequence). The fusion protein was sequenced by inserting the DNA sequence of the short linker peptide (encoding GGGGSGGPGS), as well as SEQ ID NO: 3 and SEQ ID NO: 4. The crtZ-crtW fusion sequence was codon-optimized, synthesized and used to replace the crtW gene in the original operon construct to create an insert known as System 3 (SEQ ID NO: 7). When the expression plasmid encoding this sequence was transformed into a suitable host as described above, the cells expressed astaxanthin (FIGS. 9 and 10).

In certain embodiments, the pathways contemplated herein are by genetic modification, in particular by directed evolutionary methods such as random mutagenesis or library screening to identify improved variants. Will be improved. Strain engineering of the host genome can also be used to enhance the expression of recombinant pathway genes.

In certain embodiments, operons are semi-randomly inserted into the genome and screened for production levels. For carotenoid production, this screening can be done by looking for strong coloration in the colonies from the plated library of transformants. Therefore, restricted cloning with NdeI and NsiI allows the operon to be inserted between the mosaic ends of the phage Tn5 transposon (inverting the inner and outer end sequences of 19 bp) (Hmelo et al. Non-replicating alliogen exchange plasmid). Constructed a custom suicide vector (based on). The Tn5 transposase sequence was also inserted into the plasmid (using gateway cloning) along with a tetracycline resistant cassette acting as an antibiotic marker (see, eg, FIG. 5, FIG. 6 and SEQ ID NO: 8). A transposon suicide vector was assembled, transformed into E. coli strain S17-1, and then conjugated to C. necator strain H16 as described above. As mentioned above, transconjugate is plated on MR2 agar with 2% fructose and 10 μg / ml tetracycline, followed by LB agar with 50 μg / ml kanamycin or MR2 agar with 2%. E. coli donors were removed by plating with agar plus 50 μg / ml kanamycin. Colonies colored orange and red were collected and evaluated for further properties of carotenoids as described above. Various light and dark colonies are observed, indicating that the operon is inserted at a different genomic position in each carotenoid-expressing clone.

To rapidly confirm the initial expression of the pathway and the production of carotenoid products, shake a C. necator clone containing the pBBRMCS-2 expression plasmid with 20 g / L fructose as a carbon source. In a flask, 50 ml of sterile liquid minimum medium (MR2, pH 6.8) was inoculated at 30 ° C. After about 48 hours of growth, the culture had an A620 (optical density measured at 620 nm) of about 1.4 and exhibited a deep orange or red color due to the production of carotenoids. Other expression hosts transformed with expression plasmids such as Bacterial strain NRRLB-14200, Bacterial strain NRRL B-354, Rhodopseudomonas palustris strain NRRL B4276 and Rhodobacter sphaeroides strain NRRL B1727. Was also cultured in this way. The NRRL strain was obtained from the USDA-ARS Culture Collection (Peoria, Illinois).

To assess the production of carotenoids on gas, cells containing genomically integrated operons are placed at pH 6.8 in a capped stir flask (magnetic stir bar) with underwater gas inlet and outlet ports. Sterile MR2 minimal medium 200-500 ml (without carbon source) was inoculated. Sterilized external gas inlets, outlets, and rubber tubes were covered with a sterile disc filter (pore size 0.2 μm, cellulose acetate syringe filter, VWR) to prevent contamination from the outside air. A mixture of H ₂ : CO ₂ : O ₂ in a ratio of approximately 80:10:10 is supplied by a commercially available gas cylinder (Praxair, Inc.) or electrolytic hydrogen from a generator (Parker Dominick Hunter Model 40H; Charlotte, NC). Was supplied. In some embodiments, CO ₂ (often including other gases such as H ₂ , CO, SO _x , NO _x ) comes from cement production, fossil fuel combustion, petrochemical hydrocracking operations, and the like. It was collected as discharged CO ₂ waste and supplied to the pressurized cylinder. Gas mixing and gas flow were controlled by a small network of gas flow meters and mass flow controllers (Alicat Scientific, Inc. Tucson, Arizona). The stirring plate and flask were housed in an incubator kept at 30 ° C. The outlet gas was recovered and discharged to the outside air. The culture was grown for 72 hours until cells reached A620 of about 0.4 and the color turned red or orange. On a commercial scale, this type of culture takes place in a loop bioreactor specifically designed for mass cultures that grow entirely in gas. An example of a loop bioreactor for gas fermentation of methanotrofu (using methane and oxygen as raw materials) is provided by Petersen et al (2017, 2020). In another embodiment, fermentation and culture of host cells expressing the carotenoid gene is a consortium (ie, of different species) to improve the growth rate of the carotenoid-containing biomass or to improve the overall properties of the biomass. Mixture) is adopted.

Generation using a cell-free system. The enzymes and constructs provided in the present disclosure are intended to be used to express pathway enzymes and to produce carotenoid products using cell-free expression systems (Schneider et al., 2010; Gregorio). et al., 2019; Khambati et al., 2019). Such expression systems can be fed, for example, with simple precursors of carotenoid pathways such as IPP, DMAP, FPP and the like to convert these compounds into more valuable ketocarotenoid products. Cell-free expression refers to agents that, when combined with a polynucleotide, allow in vitro translation of the polypeptide or protein encoded by the polynucleotide. These expression systems are known in the art and exist for both eukaryotic and prokaryotic applications. Exemplary cell-free expression systems that can be used in connection with the present disclosure include, for example, extracts available from Invitrogen Ambion, Qiagen and Roche Molecular Diagnostics, Escherichia coli and other strains, as well as Cupriavidus. ), Rhodobacter, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Paracocccus or Hydrogenophaga (hydrogenophaga) cells extracted from hydrogen bacteria. Includes commercial kits for various species such as things.

Centrifugation was performed at 6,000 × g for 10 minutes, and cells were collected. After resuspension in phosphate buffered saline, cells were centrifuged again. Aliquots of washed cell pellet were extracted with n-hexane / methanol (1: 1 v / v) in a 1.5 ml microcentrifuge tube. Centrifugation was performed at 14,000 × g for 5 minutes to separate the solvent extract from the cell debris. Carotenoids can also be efficiently separated and purified from biomass using supercritical CO ₂ extraction (Valderrama et al, 2003; Di Sanzo et al, 2018).

Carotenoid analysis. To identify and assay carotenoid production, a 50 μl solvent-extracted sample was loaded via a syringe into a Symmetric C185 5 μm (4.6 x 250 mm) HPLC column, which was loaded with methanol / water 90:10 (v /). Pre-equalized with a solution containing v). The running solution consisted of water, ethyl acetate, and a gradation of water. The HPLC apparatus used was a Beckman System Gold equipped with a 168NM diode array detector. The execution conditions are as follows: Flow rate 1 mL / min; Temperature 30 ° C. Peaks were identified by comparing their retention times with a known carotenoid standard solution dissolved in n-hexane. Canthaxanthin standards were obtained from Honeywell Research, Inc. and astaxanthin was obtained from Abcam, Cambridge, Miami. Eluted components can also be identified, if possible, by their characteristic absorbance spectra. The sample chromatograms of canthaxanthin (FIG. 7) and astaxanthin (FIG. 9) produced using the expression system of the present disclosure are shown. From these experiments, it was confirmed that the OB307-crtW gene encodes β-carotene ketolase, and that constructs expressing the new OB307-crtW gene actually produce canthaxanthin and astaxanthin.

This crtW sequence requires codon optimization to produce a sufficient amount of active enzyme to catalyze the conversion of β-carotene to canthaxanthin when the gene is heterologously expressed in various expression hosts. I have something to do. This also applies to synthetic operons and constructs in which gene sequences are arranged to produce fusion proteins such as the crtZ-crtW fusion protein. In some embodiments of the present disclosure, the expression host is a plant. In some embodiments, the expression host is a fungus such as Saccharomyces cerevisiae. In some embodiments, the expression host is an algae such as Chlorella vulgaris. In some embodiments, the expression host is Methylobacterium extorquens (eg, Methylobacterium extorquens), Methylococcus capsulatus (eg, Methylococcus capsulatus), acetogen (eg, Clostridium autoethanogenum). Bacteria such as Genam), hydrogen bacteria (eg, Cupriavidus necator), or Rhodobacterillum rubrum, Rhodobacter sphearoides, Rhodobacter capsulatus or Rhodobacter capsulatus. Rhodobacter monas pulsetris) and other purple non-sulfur bacteria. Other potentially suitable bacterial hosts include Paracoccus species such as Rhodococcus opacus, Paracocccus zeaxanthinifaciens, or E. coli.

[Additional literature]
Phornphisutthimas, S et al. (2007) Mating in E. coli-laboratory training Biochem Mol Biol Educ 35: 440-5
Gruber, S et al. (2015) A plasmid-based general-purpose expression system for Gram-negative bacteria-the points illustrated by the bacterium Ralstonia eutropha H16. New Biotechnol 32: 552-8.

The present disclosure generally relates to the field of molecular biology and, more specifically, genetically manipulates the metabolic pathways of microorganisms to include a variety of gaseous feedstocks for the biological production of biochemicals. Regarding the use of feedstock.

In certain embodiments, nucleic acid sequences are provided to express a carotenoid product comprising any one or more of SEQ ID NOs: 1 , 5 , 6 , 7 or 8 . In certain frequent embodiments, a vector comprising the nucleic acid of SEQ ID NO: 1 and a heterologous nucleic acid sequence is provided.

In certain frequent embodiments, a nucleic acid sequence encoding an enzyme comprising an amino acid sequence that is at least 96% identical or homologous to SEQ ID NO: 2 is provided and the expressed enzyme converts β-carotene to canthaxanthin. Can be done. In certain related embodiments, the amino acid sequence is at least 97% identical or homologous to SEQ ID NO: 2 , and the expressed enzyme is capable of converting β-carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 98% identical or homologous to SEQ ID NO: 2 , and the expressed enzyme is capable of converting β-carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 99% identical or homologous to SEQ ID NO: 2 , and the expressed enzyme is capable of converting β-carotene to canthaxanthin.

In a frequently included embodiment, a vector comprising one or more nucleic acid sequences encoding an enzyme comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 2 is provided and when expressed, the enzyme is β-carotene. Can be converted to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 97% identical or homologous to SEQ ID NO: 2 , and the expressed enzyme is capable of converting β-carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 98% identical or homologous to SEQ ID NO: 2 , and the expressed enzyme is capable of converting β-carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 99% identical or homologous to SEQ ID NO: 2 , and the expressed enzyme is capable of converting β-carotene to canthaxanthin.

In a frequently included embodiment, a synthetic nucleic acid construct comprising one of a plurality of nucleic acid sequences encoding an enzyme comprising a promoter, a ribosome binding site, and an amino acid sequence that is at least 96% identical to SEQ ID NO: 2 is provided. Once expressed, the enzyme can convert β-carotene to cantaxanthin. In certain related embodiments, the amino acid sequence is at least 97% identical or homologous to SEQ ID NO: 2 , and the expressed enzyme is capable of converting β-carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 98% identical or homologous to SEQ ID NO: 2 , and the expressed enzyme is capable of converting β-carotene to canthaxanthin. In certain related embodiments, the amino acid sequence is at least 99% identical or homologous to SEQ ID NO: 2 , and the expressed enzyme is capable of converting β-carotene to canthaxanthin. Often, the synthetic nucleic acid construct is a vector containing a plasmid.

In frequent embodiments, transformed host organisms comprising the synthetic nucleic acid constructs described above and herein are provided, and the transformed host organisms are capable of heterologous expression of the synthetic nucleic acid constructs. Often, the expression host organism is a transformed bacterium adapted to grow in a chemically synthesized autotrophic mode. In certain embodiments, the expression host organism is Cupriavidus necator.

In certain embodiments, a nucleic acid sequence corresponding to the crtW carotenoid ketrase gene from Brebandimonas strain OB307, which encodes the amino acid sequence of SEQ ID NO: 2, is provided, which nucleic acid sequence is such to produce carotenoids in biological host cells. Included in the expression constructs adapted to. In certain frequent embodiments, the biological host cell can use CO ₂ and H ₂ to meet at least some of the carbon and energy requirements of the host cell.

In certain embodiments, the nucleic acid sequence corresponding to the crtZ-crtW carotenoid hydroxylase-ketrase gene fusion is provided, the crtW portion of the fusion being the ketrase gene from the Brebandimonas strain OB307 encoding amino acid SEQ ID NO: 2. be.

In certain embodiments, the nucleic acid sequence is provided encoding the crtZ-crtW carotenoid hydroxylase-ketrase fusion protein of SEQ ID NO: 4 , and (a) the crtW portion of the fusion encodes the amino acid sequence of SEQ ID NO: 2. It is a ketorase gene derived from Brebandimonas strain OB307, and (b) the nucleic acid sequence is part of an expression construct adapted to produce carotenoids when functionally integrated into a biological host cell.

In certain embodiments, a nucleic acid sequence encoding the crtZ-crtW carotenoid hydroxylase-ketrase fusion protein of SEQ ID NO: 4 is provided, where (a) the crtW portion of the fusion encodes the amino acid sequence of SEQ ID NO: 2. A ketolase gene from Bandimonas strain OB307, (b) the nucleic acid sequence is part of an expression construct adapted to produce carotenoids when functionally integrated into a biological host cell, (c). Biological host cells can use CO ₂ and H ₂ to meet at least some of their carbon and energy requirements.

In certain embodiments, a transposon is used to provide a suicide vector construct adapted to insert a DNA sequence into the bacterial genome, wherein the suicide vector construct comprises (a) a DNA sequence, (b) a transposon. It contains an insertion adjacent DNA containing the nucleic acid sequence of SEQ ID NO: 9 , and (c) a suicide plasmid skeleton.

In certain embodiments, a transformed host cell comprising a nucleic acid sequence encoding amino acid SEQ ID NO: 2 is provided, the nucleic acid sequence being part of an expression construct adapted to produce carotenoids in the host cell. be.

In certain embodiments, a method of forming a transformed host cell as contemplated herein is provided, the method comprising inserting an expression construct into the genome of the host cell using a transposon. .. In many cases, such insertions using transposons are random insertions.

In certain embodiments, a nucleic acid sequence corresponding to the crtW carotenoid ketrase gene from Brebandimonas strain OB307, which encodes the amino acid sequence of SEQ ID NO: 2, is provided and the nucleic acid sequence produces carotenoids in a cell-free expression system. It is part of an expression construct adapted as such.

In certain embodiments, a method of producing ketocarotenoids in a biological host cell is provided by heterologous expression of OB307-crtW in the host cell. In many cases, biological host cells contain hydrogen bacteria. Also, hydrogen bacteria are often selected from Cupriavidus, Rhodobacter, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Paracoccus or Hydrogenophaga. Includes strains that are to be treated. In a particular embodiment, the strain of hydrogen bacteria is Cupriavidus necator. In certain embodiments often included, biological host cells are cultured as part of a consortium of host cells of different species.

In certain embodiments, a method of producing ketocarotenoid in a biological host cell is provided, the step of transforming the biological host cell with a vector containing a crtZ-OB307-crtW fusion, and crtZ in the biological host cell. It comprises the step of heterologously expressing the -OB307-crtW fusion to synthesize the ketocarotenoid, or the step of heterologously expressing the crtZ-OB307-crtW fusion in a biological host cell to synthesize the ketocarotenoid. In many cases, biological host cells contain hydrogen bacteria. Also, hydrogen bacteria are often selected from Cupriavidus, Rhodobacter, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Paracoccus or Hydrogenophaga. Includes strains that are to be treated. In a particular embodiment, the strain of hydrogen bacteria is Cupriavidus necator. In certain embodiments often included, biological host cells are cultured as part of a consortium of host cells of different species.

In certain embodiments, a method of producing canthaxanthin from β-carotene in vitro is provided, wherein the method is in a solution containing β-carotene and is any of SEQ ID NOs: 1 , 5 , 6, 7 or 8. The protein expression product catalyzes the conversion of at least a portion of the β-carotene to canthaxanthin, comprising contacting the nucleic acid sequence with a protein expression product that is at least 96% identical to the nucleic acid sequence. In most cases, the nucleic acid sequence is at least 90% identical to the nucleic acid sequence of any of SEQ ID NOs: 1 , 5 , 6, 7 or 8. In most cases, the nucleic acid sequence is at least 91% identical to the nucleic acid sequence of any of SEQ ID NOs: 1 , 5 , 6, 7 or 8. In most cases, the nucleic acid sequence is at least 92% identical to the nucleic acid sequence of any of SEQ ID NOs: 1 , 5 , 6, 7 or 8. In most cases, the nucleic acid sequence is at least 93% identical to the nucleic acid sequence of any of SEQ ID NOs: 1 , 5 , 6, 7 or 8. In most cases, the nucleic acid sequence is at least 94% identical to the nucleic acid sequence of any of SEQ ID NOs: 1 , 5 , 6, 7 or 8. In most cases, the nucleic acid sequence is at least 95% identical to the nucleic acid sequence of any of SEQ ID NOs: 1 , 5 , 6, 7 or 8. In most cases, the nucleic acid sequence is at least 97% identical to the nucleic acid sequence of any of SEQ ID NOs: 1 , 5 , 6, 7 or 8. In most cases, the nucleic acid sequence is at least 98% identical to the nucleic acid sequence of any of SEQ ID NOs: 1 , 5 , 6, 7 or 8. In most cases, the nucleic acid sequence is at least 99% identical to the nucleic acid sequence of any of SEQ ID NOs: 1 , 5 , 6, 7 or 8. In certain frequent embodiments, the host organism is one that naturally produces β-carotene.

It is a schematic diagram of individual enzymes and products in the biosynthetic pathway between farnesyl diphosphate (FPP) and astaxanthin. It is a figure which shows the component of the system 1 astaxanthin operon. It is a figure which shows the component of the system 2 canthaxanthin operon. It is a figure which shows the component of the system 3 astaxanthin operon and the crtZW fusion gene. It is a detailed map of a synthetic carotenoid operon that produces astaxanthin (including the OB307-crtW gene) together with the Tn5 transposase gene inserted into the suicide vector using tetracycline as an antibiotic resistance marker. Transposons are added and operons are randomly inserted into the host cell genome. It is a figure which shows the process of transformation and chromosome insertion of an operon into a host cell using transposon mutagenesis. Here, system 1 is used as an example. (ME = Mosaic Ends (reverse repeats); Pro = promoter; ori = origin of replication or transfer; term = transcription terminator). FIG. 5 shows an HPLC chromatogram (black trace) showing canthaxanthin produced by C. necator cells that heterologously express the canthaxanthin biosynthetic pathway. It is a figure which shows the corresponding UV-Vis spectrum of the canthaxanthin peak shown in FIG. 7. FIG. 3 shows an HPLC chromatogram (black trace) showing a carotenoid product derived from C. necator cells that heterologously express the astaxanthin biosynthetic pathway. The chromatogram of the purified astaxanthin standard is shown in red, which overlaps with the small peaks of the black trace. It is a figure which shows the corresponding UV-Vis spectrum of the astaxanthin peak shown in FIG.

Carotenoids are long-chain isoprenoid molecules with nutritional advantages as colorants and additives in fish feeds, animal feeds and dietary supplements that provide protection against oxidative damage to cells, especially free radicals and active oxygen species. .. Carotenoids can be expressed naturally and through the expression of one or more carotenoid genes encoding biosynthetic enzymes in plants, algae, paleontology, fungi and bacteria. Conventional production of 40 carbon (C40) tetraterpene carotenoids, including carotene and xanthophylls, has involved the extraction of natural molecules from various microorganisms and plants. However, some naturally occurring astaxanthin-producing strains, such as Xanthophyllomyces yeast, produce lower-value mirror isomers of astaxanthin and are more productive, such as Haematococcus pluvialis. The process of growing naturally occurring microalgae is difficult, time consuming, resource intensive and expensive.

Non-biological production of molecules such as astaxanthin and canthaxanthin has been achieved by chemical synthesis from petroleum sources (2002). However, these latter methods produce mixtures of astaxanthin and enantiomers, which are also of low value because they are inefficient radical quenchers and therapeutic agents, and these synthetic products are human in the EU. And face serious regulatory issues regarding animal consumption. Recently, genetically engineered organisms have been used to produce high-value canthaxanthin, astaxanthin and other C40 carotenoids as well as xanthophylls. FIG. 1 shows the carotenoid biosynthetic pathway from farnesyl diphosphate (FPP) to astaxanthin.

In addition to astaxanthin, canthaxanthin is a valuable carotenoid product that can be synthesized by transketolase enzymes, such as the bacterial crtW ketolase gene that acts on β-carotene as its substrate. Carotenoids such as canthaxanthin and astaxanthin can be produced by the crtW gene-encoded ketolase from various Brebandimonas species, which is considered to be the most active and effective carotenoid ketolase.

Since the yield and production rate of carotenoids per gram of dry weight of biomass are not high in natural or genetically modified organisms, an expression system capable of inexpensively and efficiently producing carotenoids using this CrtW enzyme is also required. Is.

Hydrogen bacteria are attractive hosts for carotenoid expression, because some species naturally produce larger intima than many other bacteria, and these membranes are highly lipophilic C40. Necessary for accumulating carotenoids.

Wider membrane capacity is also advantageous, as both CrtZ hydroxylase and CrtW ketolase enzymes are likely to be endogenous membrane proteins containing transmembrane (TM) helices that can span cell membranes.

In addition, certain hydrogen bacteria, such as Cupriavidus necator, do not naturally produce carotenoids and thus contain regulatory interference (eg, feedback inhibition) or (eg, the crtG gene, thus dihydroxyting the astaxanthin product. -There is less potential for unwanted enzymatic modification of products (such as Cupriavidus vesicularis strain DC263) that spontaneously hydroxylate to astaxanthin.

The carotenoids thus produced are either provided as part of the bacterial biomass or extracted from it to produce a substantially pure carotenoid product, or supercritical CO ₂ or solvent-based extraction, etc. Concentrates are formed by other extraction methods. In addition, carotenoids such as canthaxanthin are mixed with other ingredients such as sugars, cornstarch, lignosulfonates, binders and oils to produce products (eg, DSM Carophyll Red 10%). can do.

Bacterial CrtW enzymes are determined by the presence of His-rich motif HX (3 or 4) H, HX (2 or 3) HH and HX (2 or 3) HH: the following amino acid residues, ie His69, 6-8 of His73, His107, His110, His111, His225, His228 and His229 are used to bind a ferrous cofactor that catalyzes the oxygenation reaction. Based on mutagenesis studies, Asp118 may also be required. Thus, although not intended to be bound by any particular operating theory, natural or engineered versions of this enzyme should or must contain these ligands in order to have catalytic activity. It is thought that it will not be. Similarly, such enzymes may require a functional transmembrane sequence because there are putative TM helices that appear to organize iron binding sites inside the membrane.

Expressing such codon-optimized gene pathways in bacteria with high GC + C content, for example, because GC content makes it difficult to newly synthesize genes and operons for synthetic biology. , Has previously been proven to be a challenge.

The present disclosure describes a newly discovered crtW gene from a new strain of Brebandimonas, referred to herein as OB307, which encodes a novel transketolase for carotenoid synthesis. The present disclosure also provides an exemplary synthetic operon containing additional related carotenoid gene sequences, the expression of which is used to produce ketocarotenoids. Suitable DNA expression constructs derived from these sequences are inserted into the expression host for expression. The expression product is a transketolase enzyme that can be engineered to convert β-carotene to canthaxanthin and astaxanthin. The carotenoid products of this synthetic operon are Escherichia coli, B. B-14200, B-356, Rhodopseudomonas palustris, Rhodobacter sphaeroides, and Cupriavidus necator. It has been expressed. Rhodopseudomonas palustris and Rhodobacter sphaeroides are commonly known as purple non-sulfur (PNS) bacteria. Rhodobacter capsulatus is another PNS bacterium that can be used as a host for these DNA expression constructs.

As disclosed herein, the CrtW ketolase enzymes currently disclosed include astaxanthin and cantaxanthin via cloning of the disclosed DNA sequences, including similar sequences with the attributes described herein. Often utilized in the production of ketocarotenoids, DNA is placed in constructs containing ribosome binding sites, promoters, and terminators, as well as other structural gene elements. Other enzyme genes according to this embodiment, such as crtZ, crtY, crtI, crtB, crtE, as well as additional structural and regulatory elements are also optionally incorporated into the construct to form an operon for carotenoid production. The construct is then introduced into a host organism, such as a host cell, as one or more small circular DNA vectors, such as a plasmid, or via integration into the genome of the organism, using methods known in the art. .. For organisms that are already producing beta-carotene, a gene encoding this single enzyme is introduced to produce this CrtW transketolase enzyme, converting some of the β-carotene to canthaxanthin. When the crtZ gene is also introduced, a gene product (ie, hydroxylase) can also be expressed, thereby converting at least a portion of canthaxanthin to astaxanthin.

The product of this crtW gene is used, for example, in a cell-free expression system in which β-carotene is enzymatically converted to canthaxanthin. When the crtZ gene and the crtW gene are expressed simultaneously or in combination, at least a part of the β-carotene substrate is converted to canthaxanthin and a part is converted to astaxanthin by the action of the enzymatic products of the two genes. The novel crtW and crtZ genes may be provided on two different segments of DNA or as a single DNA constituting the gene for the fusion protein encoding the function of both CrtW ketolase and CrtZ hydroxylase. May be.

Many different organisms can be considered as heterologous expression hosts for this novel crtW gene. Hosts that can utilize H ₂ and CO ₂ as energy and carbon sources, and hosts that cannot utilize H ₂ and CO ₂ as energy and carbon sources, are considered suitable heterologous expression hosts. For example, these include bacteria, plants, algae, archaea, and fungi. Bacteria such as Escherichia coli and Bacillus subtilis, fungi such as sprouting yeast and Koji mold, plants such as African rice, algae such as Chlorella vulgaris, and archaea such as Sulfolobus solfataricus. Classes, or other species, can be heterologous hosts for this novel crtW gene to produce the enzyme it encodes and the action of this enzyme to produce carotenoid products.

Heterologous expression of this enzyme and synthetic operon disclosed herein initially uses a broad host range of expression plasmids, initially E. coli, Cupriavidus nectifolia B-14200, Cupriavidus nectus B-356, Rhodopseudomonas palustris. It has been shown in Palstris), Rhodobacter sphaeroides and Cupriavidus necator. In each case, heterologous expression of the novel OB307-crtW gene was observed via the production of canthaxanthin in transformed bacteria (no canthaxanthin production in wild-type organisms). This transformation was performed using the same plasmid used in C. necator. The promoters disclosed herein are active in all of these strains. As mentioned above, E. coli cells were transformed using plasmid electroporation. Other strains were transformed using conjugation with E. coli strain S17-1 according to standard methods (see, eg, Phornphisutthimas et al, 2007; Gruber et al, 2015). The joined cells are first plated on LB agar medium and then resuspended on a serially diluted sterile liquid medium and then resuspended on the following agar plates, ie (1) LB + 50 μg / ml canamycin or 10 μg / ml for E. coli. Tetracycline; (2) MR2 medium + 2% fructose and 50 μg / ml canamycin for bacillus; (3) play on MR2 medium + 2% fructose and 500 μg / ml canamycin for C. necator (C. necatol) and PNS bacteria. Diluted. Surviving transconjugated colonies were then harvested and re-streaked on new plates until a pure single colony was obtained. Propagation in liquid culture was performed by inoculating LB + antibiotics (all strains) or MR2 + antibiotics (H2 oxidized PNS bacteria and C. necator (C. necatol)) with cells of a given mutant.

A fusion gene consisting of crtZ and crtW was created by constructing a fragment of synthetic DNA in which crtZ and crtW were linked by a linker sequence and incorporating this fusion sequence into a synthetic operon instead of the original crtW gene of the expression plasmid. This heterologous expression vector was then transformed into E. coli and Cupriavidus necator. In each case, the production of astaxanthin and canthaxanthin was confirmed. In addition, this synthetic operon was converted to C. necator using an allelic exchange system (NaCl-free agar medium containing 6% sucrose (w / v) was used for the counterselection of sacB levansucrase) and a suicide vector. Upon insertion into the genome, carotenoid production was reconfirmed.

C. necator (C. necatol) strain H16 has been used as an expression host as well as other C. necator (C. necatol) strains and strains of other hydrogen bacteria. Thus, carotenoid products can be produced by gas fermentation of transformed bacteria using inexpensive raw materials (eg, waste CO ₂ , H ₂ , O ₂ and inorganic salts) and of the process. Economic efficiency can be improved.

Further genera and species of hydrogen bacteria that can be transformed with the vectors and DNA constructs described herein for heterologous expression in the carotenoid pathway while growing on H2 _{- CO2} - _O2 . For example, Rhodobacter capsulatus and other Rhodobacter species, Paracocccus, Rhodococcus, Hydrogenophaga, Rhodospirillum, Rhodospirillum, Rhodopseu. And so on.

A novel strain of Brebandimonas OB307 was isolated from the agar plate as a red-orange contaminant colony in the laboratory. The 16S rRNA gene was sequenced (forward and reverse) and ClustalW was used to compare it to the 16S sequences of other Brebandimonas species. This analysis revealed that OB307 has 99.7-99.8% identity with the 16S sequences of B. vesicularis and B. nasdae. Genomic DNA is extracted from approximately 100 mg of wet cell paste, sequenced whole genome using 60x Illumina paired end sequencing (150 base pair reads), and sequenced by SNPsaurus Inc. (Eugene, Oregon). Assembled and annotated the Contig. From this sequence, BLAST searches identified multiple genes with high similarity to other published carotenoid biosynthetic genes.

One of the complete open reading frame sequences was first identified as "fatty acid desaturase" by annotation software. The fatty acid desaturase is known to have a structure similar to that of carotenoid ketrase, and further analysis reveals that this sequence has a high similarity to CrtW-type carotenoid ketrase, and subsequent expression cloning reveals its activity. Was confirmed. Therefore, the gene sequence is referred to herein as OB307-crtW (SEQ ID NO: 1). As can be seen from the translated sequence of OB307-CrtW, there are eight histidine motifs (highlighted in yellow) and Asp-118 that define the ferrous binding site of this type of ketrase (SEQ ID NO: 2). (Highlighted in blue) is included. SEQ ID NO: 3 shows the CrystalW2.1 amino acid sequence alignment between OB307-CrtW and CrtW of Brebandimonas strain DC263 (GenBank Accession No. ABC5016.1). Both proteins contain 241 amino acids, with 11 amino acid differences between them (approximately 95.5% identity). More recently, the putative crtW gene from the Brebandimonas strain SgAir0440 has been published as part of the genomic sequence of air-polluting bacteria (GenBank Accession No. QCR00114). This gene has 99.6% similarity to the amino acid sequence of OB307-crtW, but has not been reported to be cloned and expressed, and the function of the enzyme was analyzed to actually β-carotene. It has not been confirmed to be a trase.

The native OB307-crtW sequence was converted to a new codon-optimized sequence for expression on C. necator. This new sequence was included as part of a codon-optimized synthetic operon containing canthaxanthin, including crtE, crtY, crtI, crtB and crtW (FIG. 3). The construct for making astaxanthin also contained the complete crtZ sequence (Fig. 2). Other gene sequences within the pathway are sourced from various other bacteria and the GenBank accession numbers are as follows: the genes crtE, crtY, crtI and crtB are Pantoea agglomerans / Erwinia herbicola pAC-BETA plasmid, M8720. / Synthesized from the sequence of M99707, crtZ was synthesized from the sequence of Pantoea ananatis strains AJ13355, NC_017533, and crtW was synthesized from the sequence of OB307-crtW described herein.

Operon synthesis benefits from special procedures (eg, available from Aster Bioscience, Inc. (Livermore, Calif.)) Due to its very high G + C content (approximately 61% -70%). A highly active constitutive promoter in C. necator was placed upstream of the carotenoid gene to direct intracellular mRNA synthesis. Other suitable promoters are well known in the art and are also contemplated herein. Inducibility that can be used to control the timing of initiation of gene transcription by applying an external inducer molecule (eg, IPTG of the lac or tac promoter) or environmental stimuli (eg, nitrogen deficiency of the phaC1 promoter). Promoters can also be used if they are compatible with the host's metabolic and transport system. A ribosome binding site (RBS) optimized for C. necator was placed upstream of each gene sequence. To optimize overall expression, spacer sequences were added between the promoter and RBS of the crtE gene and between the RBS and start codon of individual genes. An end sequence (E. coli rrnB) was placed at the end of the operon to prevent unnecessary translation of downstream elements.

Synthetic operons (SEQ ID NOs: 6 , 7 and 8 ) are first obtained by cloning them into a broad host range plasmid pBBR1MCS-2 (eg, kanamycin as a choice) using NdeI and AseI as adjacency restriction sites. The activity was tested. The ligated DNA product was electropo with Bio-Rad GenePulser II equipped with a Capacitance Extender Plus Pulse Controller II unit (Bio-Rad Inc., Hercules, Calif.). It was transformed into Escherichia coli by ration. Wash E. coli cells three times with 10% cold glycerol according to the method described in Belcher and Knight's online protocol ( _{https://openwetware.org/wiki/Belcher/Knight:_Electrocompetent_Cells} ) to make E. coli cells electrocompetent. did. 50 μl of electrocompetent cells were added to a 1 mm gap of chilled sterile cuvette and mixed with 1 μl of DNA (about 1-50 ng). The electroporator settings are as follows: 1.2kV, 25μF, 200Ω. The time constant was usually 3-5 milliseconds. After the pulse, cells were transferred to preheated SOB medium in a small sterile tube and restored at 37 ° C. for 1 hour with shaking. Then, aliquots were plated on LB agar medium containing 50 μg / ml kanamycin to select antibiotics. After culturing at 30 ° C., colonies were collected and individually grown in LB broth. Plasmid DNA was isolated from various clones by standard methods. This DNA was cleaved with an appropriate restriction enzyme and analyzed by agarose gel electrophoresis to identify positive clones. Plasmid DNA from one correct clone was transformed into E. coli conjugation strain S17-1. After that, the above process was repeated to find the correct S17-1 clone. The S17-1 clone containing the synthetic canthaxanthin or astaxanthin operon on the plasmid pBBR1MCS-2 was then conjugated to the C. necator (C. necatol) host strain or other host strain by the standard method described above. When plated on solid MR2-fructose medium (Table 1) containing 500 μg / ml kanamycin, C. necator colonies appeared. Colonies showing a deep orange or red color were harvested and streaked back into kanamycin plates to confirm their color phenotype and antibiotic resistance. Selected clones were harvested and grown in liquid medium containing antibiotics.

As mentioned above, the processing capacity of the enzyme at the end of the astaxanthin production pathway can be improved by genetically fusing the crtZ and crtW genes to encode the chimeric protein. As shown in the map of FIG. 4, between the 3'end of the complete crtZ gene from Pantoea ananatis and the 5'end of the OB307-crtW gene (without N-terminal methionine) (amino acid sequence). The fusion protein was sequenced by inserting the DNA sequence of the short linker peptide (encoding GGGGSGGPGS) as well as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 8 . The crtZ-crtW fusion sequence was codon-optimized, synthesized and used to replace the crtW gene in the original operon construct to create an insert known as System 3 (SEQ ID NO: 7). When the expression plasmid encoding this sequence was transformed into a suitable host as described above, the cells expressed astaxanthin (FIGS. 9 and 10).

In certain embodiments, the pathways contemplated herein are by genetic modification, in particular by directed evolutionary methods such as random mutagenesis or library screening to identify improved variants. Will be improved. Strain engineering of the host genome can also be used to enhance the expression of recombinant pathway genes.

In certain embodiments, operons are semi-randomly inserted into the genome and screened for production levels. For carotenoid production, this screening can be done by looking for strong coloration in the colonies from the plated library of transformants. Therefore, restricted cloning with NdeI and NsiI allows the operon to be inserted between the mosaic ends of the phage Tn5 transposon (inverting the inner and outer end sequences of 19 bp) (Hmelo et al. Non-replicating alliogen exchange plasmid). Constructed a custom suicide vector (based on). The Tn5 transposase sequence was also inserted into the plasmid (using gateway cloning) along with a tetracycline resistant cassette acting as an antibiotic marker (see, eg, FIG. 5, FIG. 6 and SEQ ID NO: 9 ). A transposon suicide vector was assembled, transformed into E. coli strain S17-1, and then conjugated to C. necator strain H16 as described above. As mentioned above, transconjugate is plated on MR2 agar with 2% fructose and 10 μg / ml tetracycline, followed by LB agar with 50 μg / ml kanamycin or MR2 agar with 2%. E. coli donors were removed by plating with agar plus 50 μg / ml kanamycin. Colonies colored orange and red were collected and evaluated for further properties of carotenoids as described above. Various light and dark colonies are observed, indicating that the operon is inserted at a different genomic position in each carotenoid-expressing clone.

To rapidly confirm the initial expression of the pathway and the production of carotenoid products, shake a C. necator clone containing the pBBRMCS-2 expression plasmid with 20 g / L fructose as a carbon source. In a flask, 50 ml of sterile liquid minimum medium (MR2, pH 6.8) was inoculated at 30 ° C. After about 48 hours of growth, the culture had an A620 (optical density measured at 620 nm) of about 1.4 and exhibited a deep orange or red color due to the production of carotenoids. Other expression hosts transformed with expression plasmids such as Bacterial strain NRRLB-14200, Bacterial strain NRRL B-354, Rhodopseudomonas palustris strain NRRL B4276 and Rhodobacter sphaeroides strain NRRL B1727. Was also cultured in this way. The NRRL strain was obtained from the USDA-ARS Culture Collection (Peoria, Illinois).

To assess the production of carotenoids on gas, cells containing genomically integrated operons are placed at pH 6.8 in a capped stir flask (magnetic stir bar) with underwater gas inlet and outlet ports. Sterile MR2 minimal medium 200-500 ml (without carbon source) was inoculated. Sterilized external gas inlets, outlets, and rubber tubes were covered with a sterile disc filter (pore size 0.2 μm, cellulose acetate syringe filter, VWR) to prevent contamination from the outside air. A mixture of H ₂ : CO ₂ : O ₂ in a ratio of approximately 80:10:10 is supplied by a commercially available gas cylinder (Praxair, Inc.) or electrolytic hydrogen from a generator (Parker Dominick Hunter Model 40H; Charlotte, NC). Was supplied. In some embodiments, CO ₂ (often including other gases such as H ₂ , CO, SO _x , NO _x ) comes from cement production, fossil fuel combustion, petrochemical hydrocracking operations, and the like. It was collected as discharged CO ₂ waste and supplied to the pressurized cylinder. Gas mixing and gas flow were controlled by a small network of gas flow meters and mass flow controllers (Alicat Scientific, Inc. Tucson, Arizona). The stirring plate and flask were housed in an incubator kept at 30 ° C. The outlet gas was recovered and discharged to the outside air. The culture was grown for 72 hours until cells reached A620 of about 0.4 and the color turned red or orange. On a commercial scale, this type of culture takes place in a loop bioreactor specifically designed for mass cultures that grow entirely in gas. An example of a loop bioreactor for gas fermentation of methanotrofu (using methane and oxygen as raw materials) is provided by Petersen et al (2017, 2020). In another embodiment, fermentation and culture of host cells expressing the carotenoid gene is a consortium (ie, of a different species) to improve the growth rate of the carotenoid-containing biomass or to improve the overall properties of the biomass. Mixture) is adopted.

Generation using a cell-free system. The enzymes and constructs provided in the present disclosure are intended to be used to express pathway enzymes and to produce carotenoid products using cell-free expression systems (Schneider et al., 2010; Gregorio). et al., 2019; Khambati et al., 2019). Such expression systems can be fed, for example, with simple precursors of carotenoid pathways such as IPP, DMAP, FPP and the like to convert these compounds into more valuable ketocarotenoid products. Cell-free expression refers to agents that, when combined with a polynucleotide, allow in vitro translation of the polypeptide or protein encoded by the polynucleotide. These expression systems are known in the art and exist for both eukaryotic and prokaryotic applications. Exemplary cell-free expression systems that can be used in connection with the present disclosure include, for example, extracts available from Invitrogen Ambion, Qiagen and Roche Molecular Diagnostics, Escherichia coli and other strains, as well as Cupriavidus. ), Rhodobacter, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Paracocccus or Hydrogenophaga (hydrogenophaga) cells extracted from hydrogen bacteria. Includes commercial kits for various species such as things.

Centrifugation was performed at 6,000 × g for 10 minutes, and cells were collected. After resuspension in phosphate buffered saline, cells were centrifuged again. Aliquots of washed cell pellet were extracted with n-hexane / methanol (1: 1 v / v) in a 1.5 ml microcentrifuge tube. Centrifugation was performed at 14,000 × g for 5 minutes to separate the solvent extract from the cell debris. Carotenoids can also be efficiently separated and purified from biomass using supercritical CO ₂ extraction (Valderrama et al, 2003; Di Sanzo et al, 2018).

Carotenoid analysis. To identify and assay carotenoid production, a 50 μl solvent-extracted sample was loaded via a syringe into a Symmetric C185 5 μm (4.6 x 250 mm) HPLC column, which was loaded with methanol / water 90:10 (v /). Pre-equalized with a solution containing v). The running solution consisted of water, ethyl acetate, and a gradation of water. The HPLC apparatus used was a Beckman System Gold equipped with a 168NM diode array detector. The execution conditions are as follows: Flow rate 1 mL / min; Temperature 30 ° C. Peaks were identified by comparing their retention times with a known carotenoid standard solution dissolved in n-hexane. Canthaxanthin standards were obtained from Honeywell Research, Inc. and astaxanthin was obtained from Abcam, Cambridge, Miami. Eluted components can also be identified, if possible, by their characteristic absorbance spectra. The sample chromatograms of canthaxanthin (FIG. 7) and astaxanthin (FIG. 9) generated using the expression system of the present disclosure, and the corresponding UV-Vis spectra (FIG. 8 and FIG. 10) are shown. From these experiments, it was confirmed that the OB307-crtW gene encodes β-carotene ketolase, and that constructs expressing the new OB307-crtW gene actually produce canthaxanthin and astaxanthin.

This crtW sequence requires codon optimization to produce a sufficient amount of active enzyme to catalyze the conversion of β-carotene to canthaxanthin when the gene is heterologously expressed in various expression hosts. I have something to do. This also applies to synthetic operons and constructs in which gene sequences are arranged to produce fusion proteins such as the crtZ-crtW fusion protein. In some embodiments of the present disclosure, the expression host is a plant. In some embodiments, the expression host is a fungus such as Saccharomyces cerevisiae. In some embodiments, the expression host is an algae such as Chlorella vulgaris. In some embodiments, the expression host is Methylobacterium extorquens (eg, Methylobacterium extorquens), Methylococcus capsulatus (eg, Methylococcus capsulatus), acetogen (eg, Clostridium autoethanogenum). Bacteria such as Genam), hydrogen bacteria (eg, Cupriavidus necator), or Rhodobacterillum rubrum, Rhodobacter sphearoides, Rhodobacter capsulatus or Rhodobacter capsulatus. Rhodobacter monas pulsetris) and other purple non-sulfur bacteria. Other potentially suitable bacterial hosts include Paracoccus species such as Rhodococcus opacus, Paracocccus zeaxanthinifaciens, or E. coli.

[Additional literature]
Phornphisutthimas, S et al. (2007) Mating in E. coli-laboratory training Biochem Mol Biol Educ 35: 440-5
Gruber, S et al. (2015) A plasmid-based general-purpose expression system for Gram-negative bacteria-the points illustrated by the bacterium Ralstonia eutropha H16. New Biotechnol 32: 552-8.

In certain embodiments, the nucleic acid sequence is provided encoding the crtZ-crtW carotenoid hydroxylase-ketrase fusion protein of SEQ ID NO : 4, and (a) the crtW portion of the fusion comprises the amino acid sequence of SEQ ID NO: 2. It is a ketorase gene from the Brebandimonas strain OB307 that encodes, and (b) the nucleic acid sequence is part of an expression construct adapted to produce carotenoids when functionally integrated into a biological host cell. ..

As mentioned above, the processing capacity of the enzyme at the end of the astaxanthin production pathway can be improved by genetically fusing the crtZ and crtW genes to encode the chimeric protein. As shown in the map of FIG. 4, between the 3'end of the complete crtZ gene from Pantoea ananatis and the 5'end of the OB307-crtW gene (without N-terminal methionine) (amino acid sequence). The fusion protein was sequenced by inserting the DNA sequence of the short linker peptide (encoding GGGGSGGPGS) as well as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 8. The crtZ-crtW fusion sequence was codon-optimized, synthesized and used to replace the crtW gene in the original operon construct to create an insert known as System 3 (SEQ ID NO: 8 ). When the expression plasmid encoding this sequence was transformed into a suitable host as described above, the cells expressed astaxanthin (FIGS. 9 and 10).

Claims (31)

A vector containing the nucleic acid of SEQ ID NO: 1 and a heterologous nucleic acid sequence. Nucleic acid sequence for expressing a carotenoid product containing SEQ ID NO: 1. Nucleic acid sequence for expressing a carotenoid product, comprising SEQ ID NO: 5, 6, 7 or 8. A nucleic acid sequence encoding an enzyme comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 2 or 3, said enzyme capable of converting β-carotene to canthaxanthin or astaxanthin when expressed. arrangement. A vector comprising one or more nucleic acid sequences encoding an enzyme comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 2 or 3, said enzyme canthaxanthin or astaxanthin β-carotene when expressed. A vector that can be converted to. A synthetic nucleic acid construct comprising a promoter, a ribosome binding site, and one or more nucleic acid sequences encoding an enzyme comprising an amino acid sequence that is at least 96% identical to SEQ ID NO: 2 or 3, said enzyme. , A construct capable of converting β-carotene to cantaxanthin or astaxanthin. The synthetic nucleic acid construct according to claim 6, wherein the synthetic nucleic acid construct is a plasmid. A transformed expression host organism comprising the synthetic nucleic acid construct according to claim 6, wherein the transformed host organism is an expression host organism capable of heterologous expression of the synthetic nucleic acid construct. The transformed expression host organism according to claim 8, wherein the expression host organism is a transformed bacterium adapted to grow in a chemosynthetic autotrophic metabolism mode. The transformed expression host organism according to claim 8, wherein the expression host organism is Cupriavidus necator. A nucleic acid sequence corresponding to the crtW carotenoid ketolase gene from Brebandimonas strain OB307, which encodes the amino acid sequence of SEQ ID NO: 2, and the nucleic acid sequence is an expression adapted to produce carotenoids in a biological host cell. Nucleic acid sequence contained in the construct. 13. The nucleic acid sequence of claim 11, wherein the biological host cell can use CO ₂ and H ₂ to meet at least some of its carbon and energy requirements. A nucleic acid sequence corresponding to a crtZ-crtW carotenoid hydroxylase-ketrase gene fusion, wherein the crtW portion of the fusion is a ketrase gene derived from Brebandimonas strain OB307 encoding amino acid SEQ ID NO: 2. A nucleic acid sequence encoding the crtZ-crtW carotenoid hydroxylase-transketolase fusion protein of SEQ ID NO: 4.
(A) The crtW portion of the fusion is a transketolase gene derived from Brebandimonas strain OB307, which encodes the amino acid sequence of SEQ ID NO: 2.
(B) The nucleic acid sequence is part of an expression construct adapted to produce carotenoids when functionally integrated into a biological host cell. A nucleic acid sequence encoding the crtZ-crtW carotenoid hydroxylase-transketolase fusion protein of SEQ ID NO: 4.
(A) The crtW portion of the fusion is a transketolase gene derived from Brebandimonas strain OB307, which encodes the amino acid sequence of SEQ ID NO: 2.
(B) The nucleic acid sequence is part of an expression construct adapted to produce carotenoids when functionally integrated into a biological host cell.
(C) A nucleic acid sequence in which the biological host cell can use CO ₂ and H ₂ to meet at least some of its carbon and energy requirements. A suicide vector construct adapted to insert a DNA sequence into the bacterial genome using a transposon.
(A) DNA sequence,
(B) Insertion adjacent DNA containing the nucleic acid sequence of SEQ ID NO: 8 containing a transposon, and (c) a suicide vector construct containing a suicide plasmid skeleton. A transformed host cell comprising a nucleic acid sequence encoding amino acid SEQ ID NO: 2, wherein the nucleic acid sequence is part of an expression construct adapted to produce carotenoids in the host cell. 17. The method of forming a transformed host cell according to claim 17, comprising using a transposon to randomly insert the expression construct into the genome of the host cell. A nucleic acid sequence corresponding to the crtW carotenoid ketrase gene from Brebandimonas strain OB307, which encodes the amino acid sequence of SEQ ID NO: 2, wherein the nucleic acid sequence is adapted to produce carotenoids in a cell-free expression system. Nucleic acid sequence that is part of the construct. A method for producing a ketocarotenoid in a biological host cell, wherein the ketocarotenoid is synthesized by heterologous expression of OB307-crtW. The method of claim 20, wherein the biological host cell comprises hydrogen bacteria. The hydrogen bacteria are selected from Cupriavidus, Rhodobacter, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Paracocccus, or Hydrogenophaga. , The method according to claim 21. 22. The method of claim 22, wherein the strain of the hydrogen bacterium is Cupriavidus necator. 20. The method of claim 20, wherein the biological host cell is cultured as part of a consortium of different species. A method of producing ketocarotenoids in biological host cells.
(A) A step of transforming the biological host cell with a vector containing the crtZ-OB307-crtW fusion and heterologously expressing the crtZ-OB307-crtW fusion in the biological host cell to synthesize ketocarotenoid, or (b). ) A method comprising the step of heterologously expressing a crtZ-OB307-crtW fusion in the biological host cell to synthesize ketocarotenoid. 25. The method of claim 25, wherein the biological host cell comprises hydrogen bacteria. The hydrogen bacteria are selected from Cupriavidus, Rhodobacter, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Paracocccus, or Hydrogenophaga. , The method of claim 26. The method according to claim 27, wherein the strain of the hydrogen bacterium is Cupriavidus necator. 28. The method of claim 28, wherein the biological host cell is cultured as part of a consortium of different species. A method for producing canthaxanthin or astaxanthin from β-carotene in vitro, a protein expression product of a nucleic acid sequence that is at least 96% identical to the nucleic acid sequence of any of SEQ ID NOs: 1, 4, 5, 6, 7 or 8. The method comprising contacting the nucleic acid in a solution containing β-carotene, wherein the protein expression product catalyzes the conversion of at least a portion of β-carotene to canthaxanthin or astaxanthin. 30. The method of claim 30, wherein the host organism spontaneously produces β-carotene.

Images