Universal metalloporphyrin compound production platform as well as establishment method and application thereof

Abstract

The invention provides a universal metalloporphyrin compound production platform and an establishment method and application thereof. The invention establishes a production platform for in-vitro production of metalloporphyrin compounds. The production platform comprises a fermentation module and a catalysis module. The invention also provides a strategy for producing the metalloporphyrin compounds in vitro, and various metalloporphyrin compounds can be obtained. According to the strategy disclosed by the invention, various metalloporphyrin compounds which are not synthesized in microorganisms can be efficiently, conveniently and flexibly produced, the purpose of producing the metalloporphyrin compounds by adding the metals is achieved, and the production platform disclosed by the invention provides a new production strategy for efficient production of the metalloporphyrin compounds.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering and biosynthesis, and more specifically, the present invention relates to a universal metal porphyrin compound production platform, a method for establishing the platform and an application thereof. Background Art

[0002] Porphyrin compounds are a class of aromatic macrocyclic molecules, which are composed of four pyrrole rings connected by methyl groups to form a large π-conjugated structure ( Figure 1 ). The 8 β positions on the pyrrole ring and the 4 hydrogen atoms inside the porphyrin ring can be replaced by other groups to form porphyrin derivatives, and different metal ions can be chelated in the center of the porphyrin ring to form different metal porphyrin compounds. All kinds of metal elements in the periodic table, including rare earth metal elements, can chelate with the porphyrin ring. Porphyrin compounds are widely present in nature, and because they can show different colors by chelating different metal ions, they are called "pigments of life." Previously, people have long known heme and chlorophyll that participate in the transport of oxygen by hemoglobin and photosynthesis in chloroplasts. They represent porphyrins and porphyrin derivatives in nature, respectively ( Figure 1 ).

[0003] In the last century, metal porphyrin compounds have been widely used in the fields of medicine, functional materials, catalysis, analytical chemistry, etc. due to their unique π-electron conjugated structure and the diversity of modifiable sites. In the medical field, metal porphyrin compounds are mainly used for cancer cell localization and treatment; in functional materials, metal porphyrin compounds can change the emission wavelength and be designed into photosensitivity organic photoelectric materials; in biomimetic catalysis, the molecules of metal porphyrin structure are very similar to the structure of metal organic framework materials (COFs), so they can be used in catalytic reactions such as hydrogen evolution reaction, oxygen evolution reaction, and carbon dioxide reduction; in analytical chemistry, since the porphyrin ring can display different colors when chelated with metal ions, it can be used to monitor metal ions in sewage.

[0004] Existing metal porphyrin compound synthesis is mainly based on chemical synthesis and animal and plant extraction, but chemical synthesis will produce irregular assembly, resulting in very low yield, and the target product and by-products are not easy to separate, the synthesis process is complicated, the reaction conditions are harsh and other problems, and the animal and plant extraction method uses organic reagents to extract, which will cause reagent waste and environmental pollution, and its yield is very low, which cannot meet market demand. With the development of molecular biology and synthetic biology, it has become a trend to produce metal porphyrin compounds with microorganisms as cell factories. Microbial synthesis can produce both naturally occurring porphyrins and non-natural porphyrin compounds. The most representative is the microbial synthesis of heme of iron porphyrin, and other metal chelated porphyrin compounds are gradually involved by people. This makes it possible to produce metal porphyrin compounds by microbial metabolic engineering.

[0005] However, the existing microbial synthesis can only produce one metal porphyrin compound at a time, and the strains must be complexly modified. The early chassis modification is time-consuming and labor-intensive, and the later tank is not ideal due to the heavy metabolic burden of the strains. If different metal porphyrin compounds are to be synthesized in microorganisms, there must be enough metal ions in the microorganisms to chelate the porphyrin macrocyclic structure. However, the metal ions are too similar, causing non-specific chelation of metal chelating enzymes. In particular, divalent metal ions lack shape, they have similar valence states and highly overlap with the geometric sites of enzyme binding. Therefore, each metal is interconnected and interdependent, that is, the change of one metal ion will affect the effects of several other metal ions and the effects of their response enzymes. Therefore, it is impossible to produce multiple metal porphyrin compounds in microorganisms at the same time. Summary of the invention

[0006] The purpose of the present invention is to provide a universal metal porphyrin compound production platform, its establishment method and application.

[0007] In a first aspect of the present invention, a method for producing a metal porphyrin compound is provided, comprising: (1) providing a metal porphyrin compound production system (platform), which comprises a fermentation module and a catalytic module; the fermentation module is used to cultivate porphyrin compound production bacteria and produce porphyrin compounds as substrates for the catalytic module; preferably, the substrate is coproporphyrin III; the catalytic module is used to chelate the porphyrin compound with metal ions using metal chelate enzymes as catalytic enzymes to obtain metal porphyrin compounds; (2) using the metal porphyrin compound production system of (1) to produce metal porphyrin compounds.

[0008] In the first aspect of the present invention, a metal porphyrin compound production system (platform) is provided, which includes a fermentation module and a catalytic module; the fermentation module is used to cultivate porphyrin compound production bacteria and produce porphyrin compounds as substrates for the catalytic module; preferably, the substrate is coproporphyrin III; the catalytic module is used to use metal chelate enzymes as catalytic enzymes to chelate porphyrin compounds with metal ions to obtain metal porphyrin compounds.

[0009] In one or more preferred embodiments, the metallochelatase is a ferrochelatase HemH; preferably, the ferrochelatase HemH is HemH of Staphylococcus aureus (S. aureus); preferably, the HemH includes a variant thereof, and the amino acid sequence of the variant is shown in SEQ ID NO: 1;

[0010] Preferably, the ferrochelatase HemH is an enzyme produced by recombinant expression, including: a crude enzyme or a purified enzyme; preferably, the prokaryotic expression includes recombinant expression in prokaryotic cells; preferably, the prokaryotic cells include Escherichia coli cells.

[0011] In one or more preferred embodiments, the metal ions include metal ions selected from the group consisting of Fe 2+ , Fe 3+ 、Co 2+ 、Ni 2+ , Cu 2+ 、Zn 2+ Mg 2+ or Mn 2+ .

[0012] In one or more preferred embodiments, the metal ions include metal ions selected from the group consisting of Fe 2+ , Fe 3+ 、Co 2+ 、Ni 2+ Mg 2+ or Mn 2+ .

[0013] In one or more preferred embodiments, the porphyrin compound producing bacteria are modified producing bacteria, and their hemN gene is down-regulated; preferably, the hemN gene is down-regulated by methods including gene knockout / silencing and gene interference; preferably, the hemN gene is knocked out / silenced by homologous recombination or gene editing.

[0014] In one or more preferred embodiments, the porphyrin compound-producing bacteria include bacteria selected from the following group: photosynthetic bacteria, denitrifying Paracoccus, Bacillus subtilis, Agrobacterium tumefaciens, Sinorhizobium, Methanobacterium barkleyi, Escherichia coli or Corynebacterium glutamicum; preferably, the photosynthetic bacteria include bacteria selected from the following group: spherical red bacteria, capsule red bacteria, nitrogen-fixing red bacteria or gelatinous red longevity bacteria.

[0015] In one or more preferred embodiments, the fermentation module includes a reactor body one, which includes a fermentation broth (a fermentation broth for producing a substrate, preferably containing glucose), and the porphyrin compound is produced in the reactor body one by adding porphyrin compound producing bacteria to the fermentation broth; the porphyrin compound is produced in the reactor body one as a substrate.

[0016] In one or more preferred embodiments, the catalytic module includes a reactor body 2, which includes a reaction solution, and the reaction solution includes a substrate (from the fermentation module), metal ions, and metal chelate enzymes; the metal porphyrin compound is obtained by reaction in the reactor body 2.

[0017] In one or more preferred embodiments, the method has improved production efficiency; preferably, the ferrochelatase HemH is used to improve production efficiency and shorten catalysis time; preferably, the HemH includes a variant thereof, and the amino acid sequence of the variant is shown in SEQ ID NO:1.

[0018] In one or more preferred embodiments, in the method or production system, the time for producing the metal porphyrin compound using the catalytic module is within 2 hours, preferably within 1.5 hours, more preferably within 1.2 hours, such as 1 hour (for example, within 0.01-2 hours, specifically 0.05, 0.1, 0.2, 0.3, 0.5, 1, 1.2, 1.4, 1.6, 1.8 hours).

[0019] In another aspect of the present invention, a HemH variant is provided, wherein the amino acid sequence of the variant is shown in SEQ ID NO:1.

[0020] In another aspect of the present invention, a polynucleotide or an expression vector or a host containing the polynucleotide is provided, wherein the polynucleotide encodes the HemH variant.

[0021] In another aspect of the present invention, any of the above methods or production systems, or the use of the HemH variant, the polynucleotide encoding the same, or the expression vector or host containing the polynucleotide, is provided for producing a metal porphyrin compound; preferably, the metal ion comprises a metal ion or a combination thereof selected from the group consisting of: Fe 2+ , Fe 3+ 、Co 2+ 、Ni 2+ Mg 2+ , Mn 2 + .

[0022] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 , molecular structure and application of metal porphyrin compounds.

[0024] Figure 2 , schematic diagram of the method for establishing a metal porphyrin compound production platform.

[0025] Figure 3A , SDS-PSGE analysis of proteins in the crude enzyme solution of SaH induced by IPTG. The left side is the supernatant and the right side is the precipitate.

[0026] Figure 3B , comparison of enzyme activity between the recombinantly expressed HemH mutant and the wild type.

[0027] Figure 4 、Metal ion Fe 2+ , Fe 3+ and Mg 2+ Reaction with the substrate coproporphyrin III. Fe 2+ , Fe3+ and Mg 2+ HPLC images (A, C, E) and full spectrum scans (B, D, F) of porphyrin compounds after reaction with coproporphyrin III; chromatograms from bottom to top: coproporphyrin III, buffer, pET28a(+), SaH (reaction with enzyme for 60 min, reaction without enzyme for 5 h), chromatogram at 390 nm.

[0028] Figure 5 、Metal ion Zn 2+ HPLC chart (A) and full spectrum scan chart (B) after reaction with substrate coproporphyrin III. Figure 4 Same as in.

[0029] Figure 6 、Metal ion Ni 2+ , Mn 2+ and Co 2+ HPLC images (A, C, E) and full spectrum scans (B, D, F) after reaction with substrate coproporphyrin III; reaction conditions and Figure 4 Same as in.

[0030] Figure 7 、Metal ion Cu 2+ HPLC chart (A) and full spectrum scan chart (B) after reaction with substrate coproporphyrin III; reaction conditions and Figure 4 Same as in.

[0031] Figure 8 , hemN knockout plasmid and verification diagram.

[0032] Fig. 9 , fermentation tank status diagram for hemN production after 74 hours of fermentation. DETAILED DESCRIPTION

[0033] After in-depth research, the inventors have established a production platform for in vitro production of metal porphyrin compounds, which includes a fermentation module and a catalytic module. The present invention also provides a strategy for in vitro production of metal porphyrin compounds, which can obtain a variety of metal porphyrin compounds. The strategy described in the present invention can efficiently, conveniently and flexibly produce various metal porphyrin compounds that are not synthesized in microorganisms, and achieve the purpose of producing any metal porphyrin compound by adding any metal. The production platform described in the present invention provides a new production strategy for the efficient production of metal porphyrin compounds.

[0034] As used herein, the term "in vitro" refers to the production of a target compound in an "ex vivo" state, where part or all of the production process is performed outside of a cell or animal body. For example, the production is performed in a solution / buffer.

[0035] As used in the present invention, the "platform" or "system" are used interchangeably and refer to a device for the efficient production of metal porphyrin compounds, preferably also including corresponding fermentation systems, solution / buffer systems, metal chelates, metal ions, culture media and other components required to implement the technical solution of the present invention.

[0036] As used herein, the "porphyrin compound" includes ferrocoprophyrin III, coproporphyrin III, heme, etc. The heme is a downstream product of ferrocoprophyrin III or coproporphyrin III. Further, the "porphyrin compound" may also include protoporphyrin IX, hematin, vitamin B12, chlorophyll, etc. The "porphyrin compound" may also be a variation or derivative form based on the compound disclosed in the present invention, for example, the parent core structure of the compound remains unchanged, but a group (such as an aliphatic hydrocarbon group containing 1-4 carbon atoms (preferably 1-2 carbon atoms)) is substituted at individual (such as 1-3, 1-2) positions.

[0037] As used herein, the "gene" or "coding gene" may be a polynucleotide that encodes the protein, or may be a polynucleotide that also includes additional coding and / or non-coding sequences.

[0038] In order to solve the technical problem of limited production of various metal porphyrin compounds in microorganisms, the present invention establishes a production platform for producing various metal porphyrin compounds in vitro, which includes a fermentation module and a catalytic module; the fermentation module is used to cultivate porphyrin compound-producing bacteria and produce porphyrin compounds as substrates of the catalytic module; the catalytic module is used to use metal chelate enzymes as catalytic enzymes to chelate porphyrin compounds with metal ions to obtain metal porphyrin compounds.

[0039] The fermentation module comprises a reactor body (such as a shell) which can contain a fermentation liquid, and the fermentation liquid can be used to produce a substrate. Preferably, the porphyrin compound is produced in the reactor body as a substrate by adding porphyrin compound producing bacteria to the fermentation liquid.

[0040] The reactor is not particularly limited in scale, and can be small, medium or relatively large, for example, 0.5-1000 L, more specifically 1, 2, 5, 10, 15, 20, 50, 100, 200, 500, 800 L, etc.

[0041] The catalytic module includes a reactor body (such as a shell) in which a reaction solution can be contained to facilitate the chelation reaction of metal ions and substrates to obtain metal porphyrin compounds. Preferably, the catalytic module may include substrates (from the fermentation module), metal ions, and metal chelating enzymes.

[0042] It is understood by those skilled in the art that the reactor may include devices required for adjusting reaction conditions, such as temperature control, feeding, discharging, adding, stirring, dissolved oxygen control, heat preservation, etc., to facilitate the fermentation or reaction. The reactor may be mechanical, automatic or semi-automatic.

[0043] In a particularly preferred embodiment of the present invention, in the catalytic module, the metal chelate enzyme is a ferrochelate enzyme HemH. Although it is a ferrochelate enzyme, the inventors have found that it plays an excellent role in the chelation of various metal ions with porphyrin compounds. When HemH is present, the metal ion Fe 2+ or Fe 3+ The reaction time with the substrate coproporphyrin III was greatly shortened to 1 hour, and the conversion rate could reach 100%. 2+ The reaction time with the substrate coproporphyrin III is significantly shortened, showing a chelating effect. HemH can also accelerate the chelation of metal ions Ni 2+ The reaction with the substrate coproporphyrin III increases the substrate conversion rate. 2+ The reaction with coproporphyrin III is also faster under the enzyme catalysis of HemH. 2+ The reaction with coproporphyrin III is also accelerated under the catalysis of SaH enzyme, and the substrate coproporphyrin III is 100% converted within 1 hour after adding HemH enzyme solution. More preferably, the HemH is HemH enzyme (SaH) derived from Staphylococcus aureus.

[0044] In a particularly preferred embodiment of the present invention, by down-regulating (including deleting) the hemN gene of the porphyrin compound producing bacteria, the strain that originally does not produce porphyrin compounds can efficiently produce porphyrin compounds, including ferroporphyrin III and coproporphyrin III. The down-regulation can be knocking out the hemN gene or silencing (such as using a gene interference method).

[0045] After knowing the role of the hemN gene as a target regulatory gene, various methods well known to those skilled in the art can be used to reduce the expression of the hemN gene or make it lack expression, such as using homologous recombination technology or gene editing technology to knock out the hemN gene, or delivering an expression unit (such as an expression vector or virus, etc.) carrying an antisense hemN gene to the target site, so that the host bacteria do not express or reduce the expression of HemN protein. Or a down-regulator that down-regulates hemN gene transcription, protein expression or protein activity is transferred into the host bacteria.

[0046] As a preferred embodiment of the present invention, the hemN gene is knocked out by using a homologous recombination method. After the gene knockout target is determined in the present invention, a recombinant vector with the same fragment (homologous arm) is designed for the specific fragments at both ends of the target, and after the recombinant vector is introduced into the host cell, the exogenous recombinant vector and the same fragment in the host cell will undergo homologous recombination, thereby introducing an exogenous gene fragment or deleting part of the gene at the target to achieve gene knockout.

[0047] As another embodiment of the present invention, CRISPR / Cas (such as CRISPR / Cas9) system is used for gene editing to knock out the hemN gene. Suitable sgRNA target sites will bring higher gene editing efficiency. Before embarking on gene editing, it is critical to design and find suitable target sites. After designing specific target sites, in vitro cell activity screening is also required to obtain effective target sites for subsequent experiments. For example, the method of knocking out the hemN gene may include: integrating the coding gene of the Cas enzyme into the host genome, transferring sgRNA or a nucleic acid that can form the sgRNA into the host bacteria, or transferring sgRNA or a nucleic acid that can form the sgRNA, Cas mRNA or a nucleic acid that can form the CasmRNA into the host bacteria; obtaining a transformant strain. After determining the target site, known methods can be used to introduce sgRNA and Cas into the cell.

[0048] The present invention also relates to down-regulators of HemN protein or hemN gene (such as gene knockout reagents, and also such as antisense hemN gene, miRNA, shRNA) and uses thereof.

[0049] Any substance that can downregulate the activity of HemN protein, downregulate the stability of HemN protein, inhibit the expression of HemN protein, reduce the effective action time of HemN protein, or reduce the transcription and translation of hemN gene can be used in the present invention as an effective substance that can be used to transform host bacteria.

[0050] The modes of small molecule interference include, but are not limited to, gene silencing regulated by miRNA, cosuppression caused by sense RNA, antisense RNA inhibition, virus-mediated gene silencing (VIGS), dsRNA, small interfering RNA, hairpin RNA (hpRNA)-mediated gene silencing, etc., which can also be applied to the present invention.

[0051] The starting strain on which the transformation of the present invention is based can be a wild-type strain, a genetically engineered strain and / or a chemically mutated strain.

[0052] In a specific embodiment of the present invention, there is provided Figure 2 The platform shown mainly involves a fermentation module and a catalytic module. First, the substrate coprophyrin III required for metalloporphyrin is produced by the fermentation module, and then metal chelates and different metal ions are added to the catalytic module to generate a variety of metalloporphyrin compounds. The metalloporphyrin compound production platform of the present invention has three advantages: first, the chassis advantage of the engineered strain is used. Preferably, the spherical red bacteria are used, which is a photosynthetic bacterium. The cells naturally produce a large amount of chlorophyll and photosynthetic pigments, and the porphyrin metabolism in the body is vigorous, which is an advantageous chassis for producing metalloporphyrin compounds. The second is the accumulation advantage of a large amount of substrates. By deleting the hemN gene in the industrial spherical red bacteria, 1.2g / L of coprophyrin can be accumulated at the shake flask level, and these large amounts of coprophyrin III are secreted to the extracellular, which is conducive to the application of the catalytic module. By a 5L tank small test fermentation, the output of coprophyrin III can be increased to 19.6g / L, providing a large amount of substrates for the production of metalloporphyrin compounds. The third is to produce a variety of metalloporphyrin compounds by enzyme catalysis in vitro. Due to the broad recognition of metal substrates by metal chelatase, it is possible to determine in vitro what metal is added and what metal porphyrin compound is produced, making the production platform more flexible, convenient and efficient.

[0053] The present invention also provides a variant (mutant) of the HemH enzyme, which has a better catalytic effect than the wild type, and is conducive to further improving production efficiency and shortening catalysis time.

[0054] The present invention also provides a polynucleotide sequence encoding a variant of the present invention or a conservative variant protein thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The polynucleotide encoding the mature protein of the variant includes: a coding sequence encoding only a mature protein; a coding sequence of a mature protein and various additional coding sequences; a coding sequence of a mature protein (and optional additional coding sequences) and a non-coding sequence.

[0055] The present invention also relates to vectors comprising the polynucleotides of the present invention, host cells produced by genetic engineering using the vectors or variant coding sequences of the present invention, and methods for producing the mutant enzymes of the present invention by recombinant technology. The polynucleotide sequences of the present invention can be used to express or produce recombinant variants by conventional recombinant DNA technology. Generally, the following steps are included: (1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the variant of the present invention, or with a recombinant expression vector containing the polynucleotide; (2). Cultivate the host cell in a suitable culture medium; (3). Isolate and purify the protein from the culture medium or cells.

[0056] The vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform appropriate host cells to enable them to express proteins.

[0057] Those skilled in the art will appreciate how to select appropriate vectors, promoters, enhancers and host cells.

[0058] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0059] Example 1: Establishment of production platform

[0060] Since the production of various metal porphyrin compounds in microorganisms is limited, the present invention establishes a production platform for producing various metal porphyrin compounds in vitro.

[0061] like Figure 2 As shown, the platform set up by the inventors mainly includes a fermentation module and a catalytic module. First, the fermentation module produces the substrate required for metalloporphyrin (such as coproporphyrin III (CPIII)), and then metal chelate enzymes and different metal ions are added to the catalytic module to generate a variety of metalloporphyrin compounds.

[0062] Specifically, the fermentation module is mainly used for producing substrates by fermentation. The module mainly includes: a basic bioreactor body 1, which includes a fermentation liquid (a fermentation liquid for producing substrates, such as containing glucose). The substrate is produced by adding an engineered strain to the fermentation liquid and reacting under conditions suitable for the reaction.

[0063] Specifically, the catalytic module is mainly used to catalyze the substrate to obtain the product. The module mainly includes: a basic bioreactor body 2, which includes a reaction liquid, to which fermentation liquid containing the substrate (from the fermentation module), metal ions, and enzymes (crude enzymes) are added to obtain metal porphyrin compounds.

[0064] This production platform was used for the production of metalloporphyrin compounds in subsequent examples.

[0065] Example 2: Inducible expression of metallochelatase

[0066] Due to the differences in the size and binding sites of transition metals, metallochelatase often cannot effectively identify different metal ions. After analysis, the inventors chose to use ferrochelatase HemH as metallochelatase.

[0067] HemH is used for in vitro enzyme catalysis reaction, using crude enzyme solution, and it is necessary to explore its protein expression level and catalytic activity of crude enzyme solution. After screening and experimental analysis, the inventors used HemH from Staphylococcus aureus (S. aureus) with high enzyme activity (wild-type sequence see Uniprit ID: Q2FXA4) for crude enzyme reaction.

[0068] Based on the wild-type HemH, the inventors carried out sequence modification. The amino acid sequence of the modified HemH variant (SEQ ID NO: 1; the underlined part is the mutation site):

[0069] MTKKMGLLVMA V GTPYKE A DIEPYYTDIRHGKRPSEEELQDLKD AF EFIGGL A PLAGTTDDQADALVSALNKAYADVEFKLYLGLKHISPFIEDAVEQMHNDGITEAITVVLAPHYSSFSVGS R DKRADEEAAKYGIQLTHVKHYYEQPK A IEYWTNKVNETLAQIPE V EHKDTVLVVSA R SLPKGLIEKNNDPYPQELEHTALLIKEQSNIEHIAIGWQSEGNTGTPWLGPDVQDLTRDLYEKHQYKNFIYTPVGFVCEHLAVLYDNDYECKVVCDDIGANYYRPKMPNTHPLFIGAIIDEIKSIF

[0070] The protein of the above sequence is called HemH-mut, and is subsequently also referred to as SaH protein.

[0071] Construction of recombinant plasmid: The coding nucleic acid of SaH protein was directly synthesized into pET28a vector, and the His tag coding sequence was added to the coding nucleic acid corresponding to its C-terminus to obtain a recombinant plasmid. At the same time, a plasmid containing wild-type HemH (HemH-mut) was constructed.

[0072] Transformation of recombinant plasmid: First, the plasmid was transformed into the protein expression host BL21 (DE3), and IPTG was used to induce the expression of the target protein. After the bacteria were collected, they were broken by a high-pressure homogenizer to release the protein, which was directly used for catalysis.

[0073] Use SDS-PAGE to verify whether the protein is successfully expressed. Figure 3AAs shown, SaH in the crude enzyme solution was soluble and expressed, and a thick band appeared at its theoretical molecular weight of 35KDa, indicating that the induced expression of SaH protein was successful and its expression level was high, which could be well used for subsequent crude enzyme catalysis reactions. The same method was used to express HemH-mut.

[0074] Take the same amount of crude enzyme solution of mutant HemH and wild-type HemH to ensure that the concentration of the target protein is 0.05 μM, and add 50 μM Fe 2+ , 50 μM substrate coproporphyrin III, react at 37°C for 1 min, then use acidic acetone to stop the enzymatic reaction and extract the porphyrin compound after the reaction, and use HPLC to detect the amount of product generated. Figure 3B As shown, wild-type HemH has enzymatic activity; HemH-mut (SaH) has a very significant improvement in enzymatic activity compared to wild-type HemH. Both can be applied to the platform of the present invention, and SaH has a more ideal effect.

[0075] Example 3: Production of coproporphyrin III using a fermentation module

[0076] The fermentation module of the production platform in Example 1 was used to ferment and produce coproporphyrin III.

[0077] 1. Strain transformation and verification

[0078] Using wild-type Rhodobacter sphaeroides (ATH 2.4.1) as the starting strain, a hemN gene knockout strain was established by homologous recombination ( Figure 8 ). The left and right homologous arms of hemN (hemN-left, hemN-right) are fragments extending 500 bp outward from the hemN gene. Using the genome of Rhodobacter sphaeroides as a template, GXL polymerase was used to amplify the left and right homologous arms of hemN, and the amplification primers were introduced into 20 bp overlapping with the adjacent fragments at both ends of the left and right homologous arms; the resistance screening marker was AmpR, and AmpR and lox sites were amplified by GXL polymerase, and the amplification primers were introduced into 20 bp overlapping with the adjacent fragments. The fragment was prepared, and the sequence of the fragment was as follows:

[0079] hemN-left (SEQ ID NO: 2):

[0080] ATCGGAGCTGCGAACGATCTGCGCGAGATGATCGCGGCCTTCGACTGCGGCGCGCGGAGCTACCTGCCCTCGTCGGTGGGGATCGACGATATCGTCGAGGCGAGCCGCCTTGCGACCTTCGACGGGATCTTCCTGCCGACCGCCAGTCTCACGGCGCTGCGCGGCGCCCTGCCCGATCGGGCAGAGCCGCAGATCGCCGTGGACGAGCATCTGACCGAGCGCCAGCGGGCCGTCTGCGACGCGCTCCGCCGGGGCAAGTCCAACAAGATCATCGCCGGAGAGCTCAATCTCTGCGAAAGCACGGTGAAGGTCCATATCCGCAACATCATGAAGAAGCTCGGGGCGAAGAACCGCACCGAGGCTGCCTTCAAGCTGAATTCCAGCATGTTCGCGGGAGAAGGTCGCCTCTCCTGAGCCGGAGCGGCGGATGCCCCGGCCCCCTCGGGGGCCGGGGCATTGCCTTTCACGAGGCCTGCGAGTAGCGGGCTTCCGGCGTCTCGTAGGC

[0081] hemN-Right(SEQ ID NO:3):

[0082] AGGAGGGCGATGTTTGTCATGCTGAACCCAATAACGGTTTCACGAGACGTTAGACTTTGCGCAGGATCAAACGCCCCGGACAGGGTGTCGCAGGCTAGAGCGGCAGCGGCGAGACGCCCAGCAGGATCGGATGCAGCGTCAGCGCCGCGGCCCAGAGCGCCACGACCGCCAGAAGCCGCGGGAAGAGATGGTCCTCGTTCTGCTTCAGCCAGTGCGCATCGACCAGCGGGCGCAG CGAGAAGAGCGCGGTGGCCCCGAAGGCCGCGAGCGCCTCGACCGGCGTCAGGGCGGCGGCGGGCCCGGGCGTCGAACATCGGGATGGCCGCCAGCGAGAGGCCGAAGAAGGTGCCGAACAGGATGACATGCGCCAGATCGCCGTTCGGAAAAAGATGGGCCGCCGACCAGAGCATCAAGGCGATCAGCAGCGGGTGGCGCGTGATGGCCGCAAAGCCCGGCCGCTCGGGATCGAAA

[0083] The commercial vector pk18mobsacB was linearized using restriction enzymes BamHI and EcoRI. The prepared fragments were assembled by nova Rec plus one-step recombinase to obtain pk18mobsacB-hemN (abbreviated as pk18-hemN).

[0084] The obtained pk18mobsacB-hemN was transferred into DH10b competent cells, and transformants were picked after overnight culture to select positive transformants. The knockout plasmid pk18mobsacB-hemN that was verified and sequenced correctly was introduced into Rhodobacter sphaeroides by conjugative transfer, and multiple rounds of screening were performed on sucrose culture plates. The strain with successful hemN knockout (ΔhemN strain) was obtained by colony PCR verification.

[0085] The ΔhemN strain was subcultured for 2 generations to stabilize its properties. After knocking out the hemN gene, the colony color changed from the original dark green to a lighter dark green, and there was a dark red secretion in the culture medium. The colonies were picked for shake flask fermentation. Shake flask fermentation process: 4% glucose, 0.4% corn syrup powder, 0.3% sodium glutamate, 0.3% (NH4)2SO4, 0.28% NaCl, 0.3% KH2PO4 and 0.63% MgSO4, after adjusting the pH to 6.5-6.9, 0.2% CaCO3 was added. 1mg / L thiamine hydrochloride, 1mg / L niacin and 15μg / L biotin were added during fermentation, and cultured at 32°C, 220rpm for 48h.

[0086] The results are shown in Table 1. After 48 hours of shake flask fermentation, the color of the fermentation broth of the knockout strain ΔhemN changed significantly. The color of the fermentation broth of the control strain WT was dark green, while the color of the fermentation broth of the ΔhemN strain turned dark red. After centrifugation of the fermentation broth, the WT supernatant was light yellow, and the dark green was the color of the bacteria itself; while the supernatant of the ΔhemN strain was still dark red, indicating that after the hemN gene was knocked out, new compounds were produced and secreted outside the cell.

[0087] Table 1

[0088]

[0089] The supernatant of the fermentation broth of Rhodobacter sphaeroides WT and ΔhemN strains was filtered with a 0.22 μm microporous filter membrane and then analyzed by LC-MS. The knockout strain ΔhemN had two more absorption peaks than the control strain, and the ultraviolet absorption peaks of the two peaks were consistent, indicating that the two substances were of the same class. The positive and negative source ion peaks of the two substances were extracted. The positive and negative source ion peaks of substance 1 were 706.6 and 708.1, respectively, and the positive and negative source ion peaks of substance 2 were 653.1 and 655.3, respectively. Therefore, the molecular weights of substances 1 and 2 were 708 and 654, respectively, which were consistent with the molecular weights of ferroporphyrin III and coproporphyrin III.

[0090] In order to further confirm the above two compounds, LC-HRMS was performed on the supernatant of the fermentation broth of Rhodobacter sphaeroides ΔhemN. The results showed that there were two obvious absorption peaks at 370nm, and the ion chromatograms with molecular weights between 654.5-655.5 and 707.5-708.5 were extracted. The extracted ion chromatograms were compared using QE software. The absorption intensity of peak 1 reached 2.99E8, the molecular weight was 708.71133, and the chemical structure was compared to C 36 H 36 O8N4Fe, which is consistent with the molecular formula of ferrocoprophyrin III. The absorption intensity of peak 2 is 2.37E9, the molecular weight is 655.26074, and the chemical structure comparison is C 36 H39 O8N4, which is consistent with the molecular formula of coproporphyrin III.

[0091] After the above LC-HRMS analysis, after knocking out the hemN gene, a large amount of dark red substances secreted in the supernatant of the fermentation broth of the strain were coproporphyrin III and ferroporphyrin III.

[0092] Therefore, the WT strain does not produce visible coproporphyrin III and ferrocoproporphyrin III; after the hemN gene was knocked out, the accumulation of coproporphyrin III was higher than 1000 mg / L (shake flask level).

[0093] 2. Production of coproporphyrin III using fermentation module

[0094] Fermentation is carried out in a bioreactor, the schematic diagram of which is shown in FIG. Figure 2 Left. Fermentation process:

[0095] Culture medium: glucose, enzymatic corn syrup, monosodium glutamate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, and calcium carbonate.

[0096] Fermentation conditions: Under the conditions of pH 5-8.5 and temperature 28-35°C, the glucose content (residual sugar) and dissolved oxygen in the fermentation broth are controlled in stages; wherein the stepwise control of the glucose content in the fermentation broth includes: within the range of glucose concentration of 4-20 g / L, reducing dissolved oxygen and increasing glucose concentration in the early stage of fermentation, and increasing dissolved oxygen and reducing glucose concentration in the middle and late stages of fermentation.

[0097] Produced by fermentation ( Fig. 9 ), collect the fermentation liquid, centrifuge to remove the bacteria, and the obtained fermentation liquid supernatant contains a large amount of coproporphyrin III. The test results show that the accumulated amount of coproporphyrin III can reach 19.6g / L.

[0098] Example 4: Production of metal porphyrin compounds using a catalytic module

[0099] The inventors analyzed the metal ions in the third period, such as Fe 2+ , Fe 3+ 、Co 2+ 、Ni 2+ , Cu 2+ 、Zn 2+ Mg 2+ and Mn 2+ Since the sizes and binding sites of transition state metals are similar, metallochelatase cannot effectively recognize different metal ions, and the ferrochelatase HemH was used for the analysis of metallochelatase.

[0100] In order to verify the reaction of coproporphyrin III with other metal ions under the catalysis of HemH, this experiment used BL21 (DE3) carrying the empty plasmid pET28a (+) without enzyme as a strict control to explore the reaction effect of metal ions with the substrate coproporphyrin III when no enzyme was added and SaH was added.

[0101] The inventors have verified that the first type of reaction involves the metal ion Fe 2+ , Fe 3+ Mg 2+ and Zn 2+ .

[0102] Reaction groups and reaction systems:

[0103] Group 1: 50 μM (final concentration) of coproporphyrin III produced in Example 3, buffer (50 mM Tris-HCl, added in an amount of 250 μL);

[0104] Group 2: Coproporphyrin III 50 μM produced in Example 3, selected from Fe 2+ , Fe 3+ Mg 2+ or Zn 2+ Metal ions 50 μM (final concentration), buffer;

[0105] Group 3: Coproporphyrin III 50 μM produced in Example 3, selected from Fe 2+ , Fe 3+ Mg 2+ or Zn 2+ 50 μM metal ions, pET28a empty plasmid fermentation broth (the wet weight of the crude enzyme solution was consistent with SaH), buffer;

[0106] Group 4: Coproporphyrin III 50 μM produced in Example 3, selected from Fe 2+ , Fe 3+ Mg 2+ or Zn 2+ Metal ions 50 μM, SaH crude enzyme solution 0.6 μM (SaH final concentration), buffer.

[0107] Reaction conditions: temperature was 37°C, reaction time was 60 min with enzyme, and reaction time was 5 h without enzyme.

[0108] When the reaction system contains only metal ions Fe 2+ or Fe 3+When the SaH crude enzyme solution was added to the reaction system, even if the reaction time was shortened to 1 hour, the chromatographic peak of iron coproporphyrin III appeared in the HPLC chart, and the strongest characteristic absorption peak of coproporphyrin III at 402 nm in the full spectrum scan disappeared, and the strongest characteristic absorption peak of iron coproporphyrin III at 390 nm appeared. Therefore, the metal ion Fe 2+ or Fe 3+ React with the substrate, and the conversion rate can reach 100%, such as Figure 4 AD. At the same time, the reaction rate accelerated significantly under the catalysis of SaH enzyme.

[0109] Other reactions belonging to the first category include Mg 2+ When only Mg is present in the reaction system 2+ When Mg and coproporphyrin III were added, the HPLC graph showed only the chromatographic peak of substrate coproporphyrin III and the full spectrum graph did not change. 2+ There is no spontaneous reaction with coproporphyrin III. When the crude enzyme reaction of SaH is added to the reaction system, the chromatographic peak of the metal chelation of the substrate coproporphyrin III appears in HPLC, but the chromatographic peak of the substrate coproporphyrin III does not disappear, but the peak height is slightly reduced, indicating that the metal ion Mg 2+ Chelation with coproporphyrin III occurs, but the reaction is not complete, e.g. Figure 4 EF. Therefore, considering the completeness of the reaction, Mg 2+ Not as compatible with SaH as Fe 2+ or Fe 3+ At the same time, the reaction rate was accelerated under the catalysis of SaH enzyme.

[0110] Other reactions belonging to the first category include Zn 2+ ,like Figure 5 As shown, regardless of Zn 2+ Whether the reaction was spontaneous with the substrate coproporphyrin III for 5 h or with the addition of SaH crude enzyme solution, the chromatographic peak of coproporphyrin III in the HPLC chart did not decrease or disappear, and the full spectrum scanning peak of the reaction product did not show the characteristic absorption peak of zinc coproporphyrin III (405, 535, 570 nm), and the characteristic absorption peak of the substrate coproporphyrin III (402 nm) did not decrease. This indicates that Zn 2+ It does not react spontaneously with coproporphyrin III, nor does it react under the catalysis of SaH enzyme. Ferrochelatase cannot recognize Zn 2+ It is chelated into the porphyrin ring of coproporphyrin III. Therefore, considering the completeness of the reaction, Zn 2+The compatibility with SaH is not ideal.

[0111] Example 5, Second type of reaction: HemH accelerates the production rate of metal porphyrin compounds

[0112] The inventors have verified the second type of reaction, the metal ion involved is Ni 2+ , Mn 2+ and Co 2+ The inventors found that when HemH is added, the reaction can be accelerated. 2+ , Mn 2+ and Co 2+ .

[0113] The reaction system is the same as in Example 4, but the metal ion is replaced by Ni 2+ , Mn 2+ or Co 2+ .

[0114] like Figure 6 As shown in A, when the metal ion Ni 2+ When the reaction time with substrate coproporphyrin III was extended to 5 h, the peak of nickel coproporphyrin III appeared. However, when SaH crude enzyme solution was added for 1 h, Ni 2+ It can react completely with the substrate coproporphyrin III. Figure 6 B also proved that nickel coproporphyrin III was produced. Therefore, the catalysis of SaH can accelerate the Ni 2+ Reacts with substrate coproporphyrin III to increase substrate conversion rate.

[0115] Mn 2+ The reaction with coproporphyrin III is also faster under the enzyme catalysis of SaH. Figure 6 C, Mn 2+ The substrate conversion rate of spontaneous reaction with coproporphyrin III for 5 h was the same as that of reaction with crude SaH enzyme solution for 1 h. Figure 6 The substrate coproporphyrin III and Mn 2+ It can react directly or with the substrate coproporphyrin III after adding enzyme, but SaH can accelerate the reaction rate, which reflects the advantage of enzyme as a catalyst.

[0116] Co 2+ The reaction with coproporphyrin III was also accelerated under the catalysis of SaH enzyme. After adding SaH enzyme solution, the substrate coproporphyrin III was completely converted within 1 hour, while the spontaneous reaction lasted for 5 hours and some substrates were still not converted. Figure 6 The formation of cobalt coproporphyrin III can be demonstrated in EF.

[0117] Example 6, third type of reaction

[0118] The inventors have verified the third type of reaction, in which the metal ion involved is Cu 2+ .

[0119] The reaction system is the same as in Example 4, but the metal ion is replaced by Cu 2+ .

[0120] like Figure 7 As shown in A, regardless of whether the crude enzyme solution SaH is added or not, the substrate coproporphyrin III can be converted into copper coproporphyrin III, and the crude enzyme solution control of BL21 (DE3) carrying the empty plasmid pET28a (+), the substrate coproporphyrin III is also converted into copper coproporphyrin III. In addition, in the full spectrum scan, the characteristic peak of the substrate coproporphyrin III is reduced, as shown in FIG. Figure 7 B. Therefore, for Cu 2+ , the catalytic effect of SaH enzyme is not obvious. However, even though SaH enzyme may not be used, the platform system of the present invention can still be used for the preparation of copper coproporphyrin III.

[0121] In the catalytic reaction between the above metal ions and the substrate coproporphyrin III, metal ions such as Fe 2+ , Fe 3+ and Mg 2+ It is a metal chelation reaction that SaH enzyme depends on, Zn 2+ The reaction with coproporphyrin III may be a zinc chelatase-dependent reaction, Ni 2+ 、Co 2 + and Mn 2+ It reacts spontaneously with coproporphyrin III, but its metal chelation reaction is accelerated in the presence of SaH. 2+ The spontaneous reaction with coproporphyrin III is extremely fast, and the catalysis of SaH has little effect on the reaction rate. Different metal ions have different reaction rates with the substrate coproporphyrin III. The present invention can quickly obtain a variety of metalloporphyrin compounds at one time by adding metal chelate enzymes and different metal ions, showing the superiority of the metalloporphyrin compound production platform established by the present invention.

[0122] Summarize

[0123] The present invention establishes an efficient metal porphyrin compound production platform, takes advantage of the chassis of the vigorous metabolism of porphyrin compounds of Rhodobacter sphaeroides, and ferments the engineered strain with inactivated hemN gene in a tank, and the accumulation of the substrate coproporphyrin III can reach 19.6g / L. Then, enzyme catalysis is carried out in vitro to produce a variety of metal porphyrin compounds. For example, the metal ions in the third cycle, Fe 2+ , Fe 3+ 、Co 2+ 、Ni 2+ , Cu 2+ 、Zn 2+Mg 2+ and Mn 2+ All of them can react with the substrate coproporphyrin III with the participation of metal chelate enzymes or spontaneously to produce corresponding metal porphyrin compounds. Various metal porphyrin compounds can be obtained efficiently and conveniently through in vitro enzyme catalysis, which broadens the application of metal porphyrin compounds.

[0124] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A method for producing a metal porphyrin compound, comprising: (1) Providing a metal porphyrin compound production system, which includes a fermentation module and a catalytic module; The fermentation module is used to cultivate porphyrin compound production bacteria and produce porphyrin compounds as substrates for the catalytic module; preferably, the substrate is coproporphyrin III; The catalytic module is used to chelate the porphyrin compound with the metal ion using the metal chelate enzyme as the catalytic enzyme to obtain the metal porphyrin compound; (2) Producing a metalloporphyrin compound using the metalloporphyrin compound production system of (1).

2. A metalloporphyrin compound production system, comprising a fermentation module and a catalytic module; The fermentation module is used to cultivate porphyrin compound production bacteria and produce porphyrin compounds as substrates for the catalytic module; preferably, the substrate is coproporphyrin III; The catalytic module is used to chelate the porphyrin compound with the metal ion using the metal chelate enzyme as the catalytic enzyme to obtain the metal porphyrin compound.

3. The method according to claim 1 or the production system according to claim 2, characterized in that: The metallochelatase is a ferrochelatase HemH; preferably, the ferrochelatase HemH is HemH of Staphylococcus aureus (S. aureus); preferably, the HemH includes a variant thereof, and the amino acid sequence of the variant is shown in SEQ ID NO: 1; Preferably, the ferrochelatase HemH is an enzyme produced by recombinant expression, including: a crude enzyme or a purified enzyme; preferably, the prokaryotic expression includes recombinant expression in prokaryotic cells; preferably, the prokaryotic cells include Escherichia coli cells.

4. The method according to claim 1 or the production system according to claim 2, characterized in that: The metal ions include metal ions or combinations thereof selected from the group consisting of Fe 2+ , Fe 3+ 、Co 2+ 、Ni 2+ , Cu 2+ 、Zn 2+ Mg 2+ or Mn 2+ ; Preferably, the metal ions include metal ions or combinations thereof selected from the group consisting of Fe 2+ , Fe 3+ 、Co 2+ 、Ni 2+ Mg 2+ or Mn 2+ .

5. The method according to claim 1 or the production system according to claim 2, characterized in that: The porphyrin compound producing bacteria are modified producing bacteria, and the hemN gene thereof is down-regulated; preferably, the hemN gene is down-regulated by methods including gene knockout / silencing and gene interference; preferably, the hemN gene is knocked out / silenced by homologous recombination or gene editing; Preferably, the porphyrin compound-producing bacteria include bacteria selected from the following group: photosynthetic bacteria, denitrifying Paracoccus, Bacillus subtilis, Agrobacterium tumefaciens, Sinorhizobium, Methanobacterium barkleyi, Escherichia coli or Corynebacterium glutamicum; preferably, the photosynthetic bacteria include bacteria selected from the following group: Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodobacter nitrogen-fixing or Rhodobacter colloidalus.

6. The method according to claim 1 or the production system according to claim 2, characterized in that: The fermentation module comprises a reactor body 1, which comprises a fermentation broth, and the porphyrin compound is produced in the reactor body 1 as a substrate by adding porphyrin compound producing bacteria to the fermentation broth; The catalytic module comprises a second reactor body, which comprises a reaction solution, wherein the reaction solution comprises a substrate, metal ions and metal chelate enzyme; a metal porphyrin compound is obtained by reaction in the second reactor body.

7. The method according to claim 1 or the production system according to claim 2, characterized in that: The method has improved production efficiency; preferably, the ferrochelatase HemH is used to improve production efficiency and shorten catalysis time; preferably, the HemH includes a variant thereof, and the amino acid sequence of the variant is shown in SEQ ID NO: 1; Preferably, in the method or production system, the time for producing the metal porphyrin compound using the catalytic module is within 2 hours, preferably within 1.5 hours, more preferably within 1.2 hours, such as 1 hour.

8. A HemH variant, the amino acid sequence of which is shown in SEQ ID NO:

1.

9. A polynucleotide or an expression vector or a host containing the polynucleotide, wherein the polynucleotide encodes the HemH variant according to claim 8.

10. The method or production system according to any one of claims 1 to 7, or the HemH variant according to claim 8 or 9, the polynucleotide encoding the same, or the expression vector or host containing the polynucleotide, for producing a metal porphyrin compound; preferably, the metal ion comprises a metal ion or a combination thereof selected from the group consisting of: Fe 2+ , Fe 3+ 、Co 2+ 、Ni 2+ Mg 2+ , Mn 2+ .

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