Camptothecin 9-hydroxylases NtCPT9H and NnCPT9H, genes and uses thereof

Abstract

The application discloses camptothecin 9-hydroxylase NtCPT9H and NnCPT9H from Nothaphoebe pubescens and N. odorata, genes and applications thereof. The amino acid sequence of NtCPT9H is shown as SEQ ID NO. 4, and the nucleotide sequence is shown as SEQ ID NO. 3. The amino acid sequence of NnCPT9H is shown as SEQ ID NO. 8, and the nucleotide sequence is shown as SEQ ID NO. 7. After heterologous expression in Nicotiana benthamiana and activity screening, the results show that NtCPT9H and NnCPT9H can catalyze camptothecin to generate 9-hydroxyl camptothecin. The application can be used for the production of camptothecin derivatives in synthetic biology, the production of 9-hydroxyl camptothecin and derivatives thereof in a biosynthesis pathway, or for guiding the molecular breeding of related medicinal plants.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically involving camptothecin 9-hydroxylases NtCPT9H and NnCPT9H, their genes, and applications. Background Technology

[0002] Camptothecin is a class of pentacyclic monoterpenoid indole alkaloids with a six-membered α-hydroxy lactone ring structure. It is mainly distributed in plants of the genera *Camptotheca* (family Davidia), *Pseudorascarida* (family Cornaceae), and *Hedyotis diffusa* (family Rubiaceae), such as *Camptotheca acuminata*, *Pseudorascarida odorata*, *Pseudorascarida pubescens*, *Hedyotis diffusa*, and *Hedyotis diffusa var. spp.* It possesses good cytotoxic activity and a significant inhibitory effect on malignant tumors. Its mechanism of action involves specifically binding to and inhibiting the activity of highly active topoisomerase I in tumor cells, thereby causing DNA double-strand breaks and exerting cytotoxic effects. However, camptothecin's poor water solubility, unstable lactone ring leading to inactivation, and significant side effects limit its direct clinical application. To date, three camptothecin-based drugs—irinotecan, topotecan, and beloteccan—have been successfully developed and marketed, and are widely used clinically for the treatment of various malignant tumors, including colorectal cancer, ovarian cancer, and small cell lung cancer.

[0003] Based on previous studies on the structural modification and antitumor activity of camptothecin A ring, it was found that compounds with substitutions at the 9 and 10 positions of camptothecin A ring exhibit the best antitumor effects. These compounds also improve the water solubility of camptothecin, reduce toxicity, and increase the stability of the lactone ring. However, the content of 9-hydroxycamptothecin in camptothecin-producing plants is extremely low, making it difficult to meet market demand. Currently, 9-hydroxycamptothecin is mainly obtained through chemical semi-synthesis. For example, it can be synthesized by hydrolyzing 9-methoxycamptothecin with hydrobromic acid, or by nitrifying camptothecin with concentrated sulfuric acid / nitric acid, hydrogenating with H2-Pd / C, and reducing it with SnHCl to obtain 9-aminocamptothecin, which is then converted to 9-hydroxycamptothecin under the action of NaNO2 / H2SO4. However, industrial applications require large amounts of strong acid and toxic reagents, which can cause environmental harm if not handled properly. Analyzing the biosynthetic pathways of active ingredients in medicinal plants, discovering key biosynthetic components, and achieving targeted and efficient heterologous production of active ingredients through metabolic engineering are new ideas and strategies for the sustainable development and utilization of medicinal plant resources. Therefore, the discovery of camptothecin 9-hydroxylase also provides a new solution for the structural diversification synthesis of camptothecin derivatives.

[0004] To date, there are no reports in the existing technology regarding the identification of camptothecin 9-hydroxylases NtCPT9H and NnCPT9H and their encoding genes from *Pseudolarix amabilis* and *Pseudolarix odoratum* of the Cornaceae family, nor on the application of camptothecin 9-hydroxylase. Summary of the Invention

[0005] In view of the above-mentioned shortcomings in the prior art,

[0006] One of the objectives of this invention is to provide camptothecin 9-hydroxylase;

[0007] The second objective of this invention is to provide a camptothecin 9-hydroxylase gene;

[0008] A third objective of this invention is to provide a recombinant expression vector containing the camptothecin 9-hydroxylase gene;

[0009] The fourth objective of this invention is to provide a transgenic recombinant bacterium containing camptothecin 9-hydroxylase;

[0010] The fifth objective of this invention is to provide the application of camptothecin 9-hydroxylase in vivo or in vitro to catalyze the production of 9-hydroxycamptothecin;

[0011] The sixth objective of this invention is to provide applications of the camptothecin 9-hydroxylase gene, recombinant expression vector, and transgenic recombinant bacteria in constructing a camptothecin biosynthetic pathway in prokaryotes or eukaryotes that do not possess a camptothecin biosynthetic pathway, or in increasing camptothecin production in prokaryotes or eukaryotes that possess a camptothecin biosynthetic pathway.

[0012] The seventh objective of this invention is to provide applications of the camptothecin 9-hydroxylase gene, recombinant expression vector, and transgenic recombinant bacteria in constructing a biosynthetic pathway for 9-hydroxycamptothecin and its derivatives in prokaryotes or eukaryotes that do not have the ability to catalyze the hydroxylation of camptothecin and its derivatives, or in increasing the yield of 9-hydroxycamptothecin and its derivatives in the biosynthetic pathway.

[0013] To achieve the above objectives, the present invention provides the following technical solution:

[0014] Provide camptothecin 9-hydroxylase, which is a protein having one of the following amino acid residue sequences:

[0015] a) Proteins having the amino acid residue sequence shown in SEQ ID NO.4 or SEQ ID NO.8;

[0016] b) An amino acid sequence derived from a) having the enzyme function of a) by substitution and / or deletion and / or addition of one or more amino acid residues in SEQ ID NO.4 or SEQ ID NO.8.

[0017] c) A protein derived from a) that has more than 80% homology to the amino acid sequence defined in a) or b) and has the enzyme function of a).

[0018] d) Derived proteins whose sequence contains the amino acid sequence of a) or b).

[0019] Provide the camptothecin 9-hydroxylase gene, which is a polynucleotide having one of the following nucleotide sequences:

[0020] a) Polynucleotides with nucleotide sequences as shown in SEQ ID NO.3 or SEQ ID NO.7;

[0021] b) Polynucleotides that encode the above proteins.

[0022] c) Polynucleotides encoding amino acid sequences as shown in SEQ ID NO.4 or SEQ ID NO.8.

[0023] d) Having more than 80% homology with the nucleotide sequence defined in a), b) or c) and encoding a polynucleotide that has the function of camptothecin 9-hydroxylase catalyzing the hydroxylation of camptothecin and its derivatives;

[0024] e) Polynucleotides complementary to sequences a), b), or c).

[0025] Provide a recombinant expression vector containing the camptothecin 9-hydroxylase gene.

[0026] Furthermore, the recombinant expression vector is obtained by inserting the camptothecin 9-hydroxylase gene into a prokaryotic or eukaryotic expression vector to obtain a recombinant expression vector expressing camptothecin 9-hydroxylase.

[0027] Furthermore, the recombinant expression vector is obtained by ligating the camptothecin 9-hydroxylases NtCPT9H and NnCPT9H, as shown in SEQ ID NO.3 and SEQ ID NO.7, into the pEAQ-HT vector via AgeI and XhoI restriction sites.

[0028] Provide transgenic recombinant bacteria containing camptothecin 9-hydroxylase.

[0029] Furthermore, the transgenic recombinant bacteria is Agrobacterium LBA4404, or other bacteria and fungi, including but not limited to Escherichia coli, Agrobacterium tumefaciens, Bacillus subtilis, Pichia pastoris, and Saccharomyces cerevisiae.

[0030] This invention provides the application of camptothecin 9-hydroxylase in the in vivo or in vitro catalytic hydroxylation of camptothecin and its derivatives, as well as its application in the in vivo or in vitro catalytic production of camptothecin.

[0031] This study aims to provide applications of the camptothecin 9-hydroxylase gene or recombinant vector in prokaryotes or eukaryotes that lack the ability to synthesize camptothecin, as well as applications to increase camptothecin production in prokaryotes or eukaryotes that possess the camptothecin biosynthetic pathway.

[0032] This invention provides applications of camptothecin 9-hydroxylase genes or recombinant vectors in prokaryotes or eukaryotes that lack the ability to catalyze the hydroxylation of camptothecin and its derivatives, as well as applications to increase the yield of 9-hydroxycamptothecin and its derivatives in prokaryotes or eukaryotes that possess a synthetic pathway for camptothecin and its derivatives.

[0033] This invention provides the application of transgenic recombinant bacteria or transgenic cell lines containing camptothecin 9-hydroxylase in the preparation of 9-hydroxycamptothecin and its derivatives or in the production of camptothecin.

[0034] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention discloses camptothecin 9-hydroxylase. Research has revealed that the amino acid sequences of camptothecin 9-hydroxylase are NtCPT9H as shown in SEQ ID NO.4 and NnCPT9H as shown in SEQ ID NO.8, encoding the nucleotides NtCPT9H as shown in SEQ ID NO.3 and NnCPT9H as shown in SEQ ID NO.7. Eukaryotic expression of camptothecin 9-hydroxylase can catalyze the production of 9-hydroxycamptothecin from camptothecin. Previously, there were no identifications of the enzyme responsible for the synthesis of 9-hydroxym-camptothecin and its encoding gene, nor were there any reports on the applications of camptothecin 9-hydroxylase. The discovery of camptothecin 9-hydroxylase provides more fundamental components for the synthetic biology of natural products and provides guidance and basis for the rational design of such enzymes, showing great promise for industrialization. This invention improves the understanding of camptothecin biosynthesis in plants of the genus *Pseudodra*. Attached Figure Description

[0035] Figure 1 The chemical reaction equations for enzyme reactions catalyzed by NtCPT9H and NnCPT9H are given.

[0036] Figure 2 Plasmid maps of NtCPT9H-pEAQ-HT and NnCPT9H-pEAQ-HT;

[0037] Figure 3 LC-MS analysis of NtCPT9H and NnCPT9H catalyzing the production of 9-hydroxycamptothecin from camptothecin. Detailed Implementation

[0038] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0039] Example 1

[0040] Cloning of the camptothecin 9-hydroxylase (CPT9H) gene.

[0041] (1) Extraction of total RNA from tender roots of *Pterocarya stenoptera* (or *Pterocarya stenoptera*) and synthesis of the first strand of cDNA.

[0042] A suitable amount of root tissue from *Dendrobium nobile* (or *Dendrobium odoratum*) was collected, ground in liquid nitrogen, and total RNA was extracted using the Vazyme Polysaccharide and Polyphenol Plant Total RNA Rapid Extraction Kit according to the manufacturer's instructions. RNA concentration and quality were detected using a Thermo Scientific NanoDrop spectrophotometer, and RNA quality was assessed by agarose gel electrophoresis.

[0043] cDNA was synthesized using total RNA as a template, following the instructions in the Vazyme HiScript III 1st Strand cDNA Synthesis Kit.

[0044] (2) Cloning of NtCPT9H and NnCPT9H genes.

[0045] Design specific primers, with the specific primer sequences as follows:

[0046] NtCPT9H-F:5'-atggatctttttcttcaac-3'(SEQ ID NO.1)

[0047] NtCPT9H-R:5'-tcacagataaagacttgga-3'(SEQ ID NO.2)

[0048] NnCPT9H-F:5'-atggatcattttcttcaac-3'(SEQ ID NO.5)

[0049] NnCPT9H-R:5'-tcacagataaagacttgga-3'(SEQ ID NO.6)

[0050] The genes NtCPT9H and NnCPT9H were amplified by PCR using cDNA from root tissue as a template and sequenced. The nucleotide sequences of the NtCPT9H and NnCPT9H genes are as follows: NtCPT9H is shown in SEQ ID NO.3, with the start codon ATG and the stop codon TGA; the translated protein-coding sequence is shown in SEQ ID NO.4; NnCPT9H is shown in SEQ ID NO.7, with the start codon ATG and the stop codon TGA; the translated protein-coding sequence is shown in SEQ ID NO.8.

[0051] Example 2

[0052] The functions of the NtCPT9H and NnCPT9H genes were verified by eukaryotic expression.

[0053] The NtCPT9H and NnCPT9H genes were introduced into the restriction enzyme sites. Primers were designed for the NtCPT9H and NnCPT9H genes. The forward primer for NtCPT9H contained a homologous arm sequence as shown in SEQ ID NO. 9, and the reverse primer contained a homologous arm sequence as shown in SEQ ID NO. 10. The forward primer for NnCPT9H contained a homologous arm sequence as shown in SEQ ID NO. 11, and the reverse primer contained a homologous arm sequence as shown in SEQ ID NO. 12. After obtaining the NtCPT9H and NnCPT9H genes with homologous arm sequences at both ends by PCR amplification, the complete coding sequences of NtCPT9H and NnCPT9H were ligated into the plasmid pEAQ-HT using homologous recombinase (ClonExpress Ultra One Step Cloning Kit) to obtain the recombinant expression vectors NtCPT9H-pEAQ-HT and NnCPT9H-pEAQ-HT, respectively.

[0054] The primer sequences are as follows:

[0055] NtCPT9H-AF:5'-tgcccaaattcgcgaccggtatggatctttttcttcaac-3'(SEQ ID NO.9)

[0056] NtCPT9H-AR:5'-ccagagttaaaggcctcgagtcacagataaagacttgga-3'(SEQ IDNO.10)

[0057] NnCPT9H-AF:5'-tgcccaaattcgcgaccggtatggatcattttcttcaac-3'(SEQ IDNO.11)

[0058] NnCPT9H-AR:5'-ccagagttaaaggcctcgagtcacagataaagacttgga-3'(SEQ IDNO.12)

[0059] The constructed plasmids NtCPT9H-pEAQ-HT and NnCPT9H-pEAQ-HT were transformed into the eukaryotic expression strain Agrobacterium LBA4404. The plasmids were plated on LB agar plates containing kanamycin, rifampin, and streptomycin. Single colonies were picked and cultured, and PCR was performed using universal primers to verify the results. Positive clones were screened to obtain the eukaryotic engineered strains NtCPT9H-pEAQ-HT-LBA4404 and NnCPT9H-pEAQ-HT-LBA4404. 10 μL of the bacterial culture was inoculated into 5 mL of LB liquid medium containing 50 μg / L kanamycin, 50 μg / L streptomycin, and 50 μg / L rifampin, and cultured at 30°C and 200 rpm. When OD... 600 When the OD value reaches approximately 0.5, centrifuge at 4000 rpm for 10 min and collect the bacterial cells. Resuspend the bacterial cells in sterile double-distilled water, centrifuge again, and collect the bacterial cells. Resuspend the bacterial cells in MMA buffer (20 mM MES pH 5.6, 20 mM MgCl2), and adjust the OD value of the bacterial cells accordingly. 600 The pH was adjusted to 0.3, and then acetylsyleugenone was added to a final concentration of 0.2 μM. The mixture was shaken at room temperature for 2 hours. Four- to six-week-old *Nicotiana benthamiana* plants were selected. A small hole was punctured on the underside of the tobacco leaves using a sterile syringe needle. Using a syringe without the needle, *Agrobacterium* LBA4404 resuspended in MMA buffer carrying the target plasmid DNA was slowly injected into the tobacco leaves through the punctured hole. After culturing at 25°C for 16 hours under light for 4 days, 0.2 mM camptothecin was injected into the leaves. One day later, tobacco leaves were collected, ground into powder with an appropriate amount of liquid nitrogen, and extracted with 25 ml of 95% ethanol by sonication for 2 hours. After centrifugation at 4000 rpm for 30 minutes, the supernatant was collected and extracted three times with an equal volume of petroleum ether. The ethanol phase was concentrated under reduced pressure and evaporated to dryness. Then, 1 ml of methanol was added to dissolve the extract, and the mixture was centrifuged at 12000 rpm for 10 minutes. 20 μl of the extract was analyzed by LC-MS using an Agilent 1290 / 6530 system and a YMC-Triart C column. 18 (ID 4.6×250mm), column temperature 25℃, flow rate 1mL / min, mobile phase elution program: 95%-0% phase A (water containing 0.1% formic acid), 5%-100% phase B (acetonitrile), gradient elution for 20min, mass spectrometer is electrospray ionization source (ESI), positive ion mode scanning, scan range (m / z): 100-500.

[0060] The test results showed that NtCPT9H and NnCPT9H catalyze the production of 9-hydroxycamptothecin from camptothecin (m / z: 365 [M+H)). + Under the analytical conditions, the retention time was 11.26 min. Figure 3 As shown.

[0061] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0062] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Camptothecin 9-hydroxylase, characterized in that, Its amino acid residue sequence is shown in SEQ ID NO.4 or SEQ ID NO.

8.

2. Camptothecin 9-hydroxylase gene, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.3 or SEQ ID NO.

7.

3. A recombinant expression vector containing the camptothecin 9-hydroxylase gene as described in claim 2, characterized in that, The recombinant expression vector is obtained by inserting the camptothecin 9-hydroxylase gene into a prokaryotic or eukaryotic expression vector to obtain a recombinant expression vector expressing camptothecin 9-hydroxylase.

4. The application of the camptothecin 9-hydroxylase as described in claim 1 in the catalytic production of 9-hydroxycamptothecin in *Dendrobium nobile*, *Dendrobium odoratum* and tobacco.

5. The application of the camptothecin 9-hydroxylase gene of claim 2 and the recombinant expression vector of claim 3 in constructing the 9-hydroxycamptothecin synthetic pathway in tobacco that does not have the ability to catalyze the hydroxylation of camptothecin.

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