阅读说明：本技术 菌株及其发酵生产麦角生物碱的应用 (Bacterial strain and application thereof in fermentation production of ergot alkaloid ) 是由 高书山 姚永鹏 王伟 安春艳 张军 颜芮 于 2021-08-05 设计创作，主要内容包括：本发明涉及微生物工程技术领域,特别涉及菌株及其发酵生产麦角生物碱的应用。本发明以构巢曲霉为底盘细胞,异源重构的麦角生物碱的生物合成途径,获得了9种不同结构的麦角生物碱化合物和2种途径中间体。通过途径优化、前体供应、P450电子传递链优化获得多株麦角生物碱高产菌株。基于上述,本发明实现了在构巢曲霉中异源高产麦角生物碱类化合物。(The invention relates to the technical field of microbial engineering, in particular to a strain and application thereof in producing ergot alkaloid by fermentation. The invention uses aspergillus nidulans as a chassis cell, and obtains 9 ergot alkaloid compounds with different structures and 2 pathway intermediates by a biosynthetic pathway of heterologous reconstructed ergot alkaloids. Multiple ergot alkaloid high-producing strains are obtained through path optimization, precursor supply and P450 electron transfer chain optimization. Based on the above, the present invention achieves heterologous high-yield ergot alkaloid compounds in aspergillus nidulans.)

1. Use of a gene or overexpression of a gene in the fermentative production of ergot alkaloids or in the construction of a strain for the fermentative production of ergot alkaloids;

the genes include:

(I) the method comprises the following steps One or more of easF, dmaW, easE (A.fum) thmgR, samS, or trpS; and/or

(II): one or more of dmaW, easF, easE (a.fum), or easC (a.fum); and/or

(III): one or more of dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur) or easC (ajap); and/or

(IV): one or more of dmaW, easF, easD, easE (a.fum), easc (ajap), or easA (a.fum), and knock-out or knock-down of easG (a.fum); and/or

(V): one or more of dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), cloA (c.afr), CPR (a.ary), or CYB 5; and/or

(VI): one or more of dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), cloA (c.pur), CPR (a.ter), or CYB 5; and/or

(VII): one or more of dmaW, easF, easD, easE (a.fum), easC (a.fum), easA (a.fum), cloA (c.afr), CPR (c.pur), or CYB 5.

2. The use of claim 1, wherein the genes comprise:

(A) the method comprises the following steps easF, dmaW, easE (A.fum) thmgR, samS, and trpS; and/or

(B) The method comprises the following steps dmaW, easF, easE (a.fum) and easC (a.fum); and/or

(C) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur) and easC (ajap); and/or

(D) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easc (ajap), and easA (a.fum), and knock-out or knock-down of easG (a.fum); and/or

(E) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), cloA (c.afr), CPR (a.ory), and CYB 5; and/or

(F) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), cloA (c.pur), CPR (a.ter) and CYB 5; and/or

(G) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easA (a.fum), cloA (c.afr), CPR (c.pur), and CYB 5.

3. The use according to claim 1 or 2, wherein the ergot alkaloids comprise one or more of N-Me-DMAT, Prechanoclavine (PCC), chanoclavine (CC), Agroclavine (AC), Festucaine (FC), Pyroclavine (PC), 6-nor-agroclavine (6-nor-AC), Elymocaline (EC), Lysogic Acid (LA), Dihydroelysergol (DHLG), dihydrolysergic acid (DHLA);

the starting strain of the strain comprises aspergillus nidulans.

4. A genetic element for the fermentative production of ergot alkaloids, comprising any element or combination of:

(a) the method comprises the following steps easF, dmaW, easE (A.fum) thmgR, samS, and trpS; and/or

(b) The method comprises the following steps dmaW, easF, easE (a.fum) and easC (a.fum); and/or

(c) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur) and easC (ajap); and/or

(d) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easc (ajap), and easA (a.fum), and knock-out or knock-down of easG (a.fum); and/or

(e) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), cloA (c.afr), CPR (a.ory), and CYB 5; and/or

(f) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), cloA (c.pur), CPR (a.ter) and CYB 5; and/or

(g) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easA (a.fum), cloA (c.afr), CPR (c.pur), and CYB 5.

5. An expression vector constructed from the genetic element of claim 4.

6. The expression vector of claim 5, comprising any plasmid or combination of the following;

the pEA01 plasmid, pYTU + easF; and/or

The pEA02 plasmid, pYTR + dmaW; and/or

The pEA03 plasmid, pYTP + easE (a.fum); and/or

The pEA10 plasmid, pYTU + dmaW + easF; and/or

The pEA12 plasmid, pYTU + dmaW + easF + easE (A.fum) + easC (A.fum); and/or

The pEA13 plasmid, pYTP + easF + easE (A.fum) + easC (A.fum); and/or

The pEA14 plasmid, pYTR + dmaW + easE (a.fum) + easC (a.fum); and/or

The pEA15 plasmid, pYTR + easD + easE (A.fum) + easC (A.fum); and/or

The pEA16 plasmid, pYTP + easG (A.fum) + easA (C.pur); and/or

The pEA31 plasmid, pYTP + easG (A.fum) + easA (A.fum); and/or

The pEA38 plasmid, pYTP + easG (A.fum) + easA (C.pur) + cloA (C.afr); and/or

The pEA39 plasmid, pYTP + easG (A.fum) + easA (C.pur) + cloA (C.pur); and/or

The pEA41 plasmid, pYTP + easA (A.fum) + cloA (C.afr).

7. A host transformed with the expression vector of claim 5 or 6.

8. The host of claim 7, comprising the following strains or combinations thereof:

strain An06, transformed with the pEA01 plasmid, the pEA02 plasmid and the pEA03 plasmid, introduces additional copies of the easF, dmaW and easE (a.fum) genes, and overexpresses the aspergillus nidulans endogenous genes thmgR, samS and trpS; and/or

Strain An10 transformed with plasmid pEA12, plasmid pEA13 and plasmid pEA 14; and/or

Strain An27 transformed with plasmid pEA10, plasmid pEA15 and plasmid pEA 16; replacing easC (a.fum) with easC (ajap); the strain An27 comprises two copies of easE (a.fum) and easc (ajap); and/or

Strain An32 transformed with the plasmid pEA10, pEA15 and pEA 31; and knocking out or knocking down the easG gene, and replacing easC (A.fum) with easC (Ajap); the strain An32 comprising 3 copies of easE (A.fum) and two copies of easC (A.jap); and/or

Strain An54 transformed with plasmid pEA10, plasmid pEA15 and plasmid pEA 38; cloA (c.afr) in the pEA38 plasmid was fitted to CPR (a.ory); and overexpresses the CYB5 gene endogenous to Aspergillus nidulans; and/or

Strain An55 transformed with plasmid pEA10, plasmid pEA15 and plasmid pEA 39; cloA (C.pur) in the pEA39 plasmid was adapted to CPR (C.pur); and overexpresses the CYB5 gene endogenous to Aspergillus nidulans; and/or

Strain An56 transformed with plasmid pEA10, plasmid pEA15 and plasmid pEA 39; cloA (c.pur) in the pEA39 plasmid was adapted to CPR (a.ter); and overexpresses the CYB5 gene endogenous to Aspergillus nidulans;

the starting strain of the strain comprises aspergillus nidulans.

9. Use of the genetic element of claim 4, the expression vector of claim 5 or 6, the host of claim 7 or 8 for the fermentative production of ergot alkaloids;

the ergot alkaloids include one or more of N-Me-DMAT, Prechanoclavine (PCC), chanoclavine (CC), Agroclavine (AC), Festucavine (FC), Pyroclavine (PC), 6-nor-agroclavine (6-nor-AC), Elymoclavine (EC), Lysergic Acid (LA), Dihydroelysergol (DHLG), and dihydrolysergic acid (DHLA).

10. A process for the fermentative production of ergot alkaloids, characterized in that ergot alkaloids are obtained by fermentation with a host according to claim 7 or 8, collection of the fermentation broth and purification.

Technical Field

The invention relates to the technical field of microbial engineering, in particular to a strain and application thereof in producing ergot alkaloid by fermentation.

Background

Parkinson's Disease (PD) is a progressive, neurodegenerative dyskinesia-like disorder caused by the loss of brain cells that can produce dopamine, which severely affects the daily life and work of the patient. According to statistics, nearly 300 million PD patients account for half of the PD patients in China, and are the most patients in the world. And the disease rate of newly increased patients in China is about 10 ten thousand per year, the disease rate is 1.7 percent above 65 years old, the disease rate reaches 3 to 5 percent above 70 years old, and the medicine is a third killer of middle-aged and elderly people who are excreted after tumors and cardiovascular and cerebrovascular diseases. Currently, levodopa is mainly used for treating PD, but long-term administration of the PD can cause motor and non-motor symptoms (switching phenomenon) of patients. However, new drug development of PD is difficult, for example, in 1 month 2018, Pfizer (Pfizer) published a statement by the largest pharmaceutical company worldwide, which announces that drug development for treating PD will be suspended due to insufficient technical ability. This results in PD patients still needing to use levodopa and dopamine agonist drugs to control their condition. Therefore, the improved medicine for treating the Parkinson disease is efficiently obtained and developed, so that the pain of a patient can be relieved, and the social contribution and the economic value are important.

Drugs derived from microorganisms play an important role in fighting against the hazards of diseases in human beings, such as the hypolipidemic lovastatin and derivatives thereof, broad-spectrum antibacterial drugs penicillin and cephalosporin antibiotics, immunosuppressive drugs cyclosporin and gliotoxin, and the like. Ergot alkaloids are secondary metabolites produced by the invasion of gramineous plants by ergot genera (Claviceps), and are known as one of the most important natural drug molecules. Studies have shown that, due to the structural similarity of the ergoline tetra-fused ring in the core structure of ergot alkaloids (fig. 1) to neurotransmitters (dopamine, tryptamine, 5-hydroxytryptamine, etc.), it can specifically bind to various neurotransmitter receptors in the human brain, activating or inhibiting the relevant nerve signal transduction. Thus, ergot alkaloid drugs, which are widely used in the medical market for treating parkinson's disease as dopamine receptor agonists, are mainly bromoergot cyclic peptide (bromocriptine), pergolide (pergolide), cabergoline (cabergoline), and the like (fig. 1). As shown in figure 1, ergot alkaloid drugs are mainly obtained by chemical modification and semi-synthesis with ergot acid as a core skeleton [3 ]. Compared with the traditional levodopa medicine, the lysergic acid has a more complex ring system structure, and provides more targets for the improvement and improvement of the activity of the medicine. Therefore, the acquisition mode of the lysergic acid determines the development and production cost of the Parkinson's disease medicine taking the lysergic acid as a lead compound and the yield of the medicine.

At present, there are two main ways of obtaining lysergic acid: direct chemical synthesis and natural product extraction plus chemical treatment. The direct chemical synthesis method has complicated synthesis steps, thirty or more steps are needed for synthesizing the core skeleton, and the highest yield of the final product is only 12 percent. Extraction of natural products plus chemical treatment is a common production method in industry at present. The method comprises infecting rye in large scale with C.purpurea or performing shallow water static culture with special C.purpurea strain, extracting natural ergot alkaloid from infected rye or production strain, and performing chemical conversion to obtain ergot acid. The method has long period, low yield and easy degeneration of the strain; a great amount of ergot alkaloid structural analogues exist in fermentation products, the separation difficulty is high, and the fermentation products are seriously dependent on chemical means. More importantly, the domestic fermentation strain yield is low, the international ergot raw material medicine market is monopolized by international enterprises, and the research and development and optimization of ergot alkaloid medicines are severely restricted by the strain patent protection barriers set by developed countries; in the example of nicergoline, the pharmaceutical companies of Longdeny and the company of Pesper occupy the majority of the market share. In conclusion, the development of the new method for producing the lysergic acid is a core for getting rid of the key of foreign technology dependence, improving the production efficiency and reducing the production and research and development costs, and has important scientific significance and application prospect.

The Microbial cell factory (Microbial cell factory) is to use Microbial cells as a production chassis and realize the high-efficiency production of target compounds in the Microbial cells by means of genetic modification, heterologous reconstruction of biosynthetic pathways and the like. For example, artemisinin sells up to 15 billion dollars per yearThe traditional production mode mainly depends on direct extraction from artemisia annua. In 2003, the institute of professor Jay D.Keasling, university of California, USA reported that the biosynthesis pathway of amorpha-4, 11-diene, which is an artemisinin precursor, is constructed in Escherichia coli, and the yield of amorpha-diene reaches 112 mg/L; in 2013, they further realized the production of artemisinin precursor arteannuic acid by microbial fermentation using yeast cell factory, and then successfully converted arteannuic acid into artemisinin using singlet oxygen. In the range of less than 100m³The fermentation plant of (2) can produce artemisinin up to 35t annually, corresponding to a planting yield of nearly 5 ten thousand acres of cultivated land, so the work is considered as a milestone work of synthetic biology. For another example, morphine and other opioid drugs are clinically important analgesics, and their production mainly comes from the extraction of poppy plants, with low productivity. The Christina Smolke professor of Stanford university in America introduces functional genes derived from bacteria, plants, animals and yeasts into yeast cells through functional element excavation and optimization to reconstruct a biosynthesis pathway, realizes the production of opioid drugs such as morphine by a microbial fermentation method, and the related work is published on Science and is evaluated as one of the ten great progresses of global life Science in 2015. In addition, in recent years, domestic researchers have attracted attention in the field, and the construction and optimization of cell factories of products such as a tanshinone key precursor miltirane and a ginsenoside precursor protopanaxadiol are realized successively. The above cases all show that the construction of a natural drug microbial cell factory is an important frontier field of current synthetic biology, can effectively reduce the drug production cost, generate great economic benefit, and promote the research and development of new drugs for human diseases including Parkinson's disease.

Filamentous fungi, as a cell factory chassis for natural drug production from eukaryotic sources, have unique advantages: the method has an intron splicing system, can directly express genome DNA (gDNA), and does not need to obtain cDNA firstly like Escherichia coli and saccharomyces cerevisiae; ② the product has phosphopantetheinyl transferase (PPTase), can directly express biological synthesis gene of polyketone and non-ribosome peptide; thirdly, the complete biosynthesis gene cluster (dozens to dozens of kb) of the natural product of the fungus can be expressed, and one-step heterologous synthesis of the product is realized; fourthly, the primary metabolic system is strong, and cheap carbon sources such as starch, cane sugar and the like can be utilized to realize the reutilization of industrial and agricultural wastes; the requirements for growth environment are wide, and the product has strong tolerance to temperature, pH and micromolecule products in a large range. Various filamentous fungi have been successfully used as underplate cells in the enzyme preparation, food and pharmaceutical industries, including Aspergillus nidulans, Penicillium chrysogenum, Aspergillus oryzae, Aspergillus niger and the like.

Aspergillus nidulans (a. nidulans) has extremely strong secondary metabolite production capacity and contains 56 secondary metabolite biosynthesis gene clusters; and the aspergillus nidulans protoplast is a monocyte, so that a high-efficiency genetic operation system is established. The aspergillus nidulans mutant strain LO8030 obtained by combined modification of professor Nancy Keller, professor Clay Wang and professor Berl Oakley deletes 8 main secondary metabolite biosynthesis gene clusters including variegated aspergillin (sterimatostatin) and orcellic acid (orsellic acid), simplifies the genome 244061bp, greatly reduces the complexity of metabolite spectrum, and makes it become a very potential cell factory underpan cell.

Disclosure of Invention

In view of this, the present invention achieves heterologous high yield of ergot alkaloid compounds in aspergillus nidulans.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an application of genes or overexpression of the genes in fermentation production of ergot alkaloids or construction of bacterial strains for fermentation production of the ergot alkaloids;

the genes include:

(I) the method comprises the following steps One or more of easF, dmaW, easE (A.fum) thmgR, samS, or trpS; and/or

(II): one or more of dmaW, easF, easE (a.fum), or easC (a.fum); and/or

(III): one or more of dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), or easC (a.jap); and/or

(IV): one or more of dmaW, easF, easD, easE (a.fum), easC (a.jap) or easA (a.fum), and knock-out or knock-down of easG (a.fum); and/or

(V): one or more of dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), cloA (c.afr), CPR (a.ary), or CYB 5; and/or

(VI): one or more of dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), cloA (c.pur), CPR (a.ter), or CYB 5; and/or

(VII): one or more of dmaW, easF, easD, easE (a.fum), easC (a.fum), easA (a.fum), cloA (c.afr), CPR (c.pur), or CYB 5.

In some embodiments of the invention, the genes comprise:

(A) the method comprises the following steps easF, dmaW, easE (A.fum) thmgR, samS, and trpS; and/or

(B) The method comprises the following steps dmaW, easF, easE (a.fum) and easC (a.fum); and/or

(C) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur) and easC (a.jap); and/or

(D) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.jap) and easA (a.fum), and knock-out or knock-down of easG (a.fum); and/or

(E) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), cloA (c.afr), CPR (a.ory), and CYB 5; and/or

(F) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), cloA (c.pur), CPR (a.ter) and CYB 5; and/or

(G) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easA (a.fum), cloA (c.afr), CPR (c.pur), and CYB 5.

In some embodiments of the invention, the ergot alkaloids comprise one or more of N-Me-DMAT, Prechanoclavine (PCC), chanoclavine (cc), Agroclavine (AC), festucavine (fc), pyroclavine (pc), 6-nor-agroclavine (6-nor-AC), elymoclavine (ec), Lysergic Acid (LA), dihydroelysergol (dhlg), dihydrolysergic acid (DHLA).

In some embodiments of the invention, the starting strain of said strain comprises aspergillus nidulans.

The invention also provides genetic elements for the fermentative production of ergot alkaloids, comprising any or a combination of the following:

(a) the method comprises the following steps easF, dmaW, easE (A.fum) thmgR, samS, and trpS; and/or

(b) The method comprises the following steps dmaW, easF, easE (a.fum) and easC (a.fum); and/or

(c) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur) and easC (a.jap); and/or

(d) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.jap) and easA (a.fum), and knock-out or knock-down of easG (a.fum); and/or

(e) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), cloA (c.afr), CPR (a.ory), and CYB 5; and/or

(f) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easG (a.fum), easA (c.pur), cloA (c.pur), CPR (a.ter) and CYB 5; and/or

(g) The method comprises the following steps dmaW, easF, easD, easE (a.fum), easC (a.fum), easA (a.fum), cloA (c.afr), CPR (c.pur), and CYB 5.

Based on the research, the invention also provides an expression vector constructed by the gene element.

In some embodiments of the invention, the expression vector comprises any plasmid or combination of the following;

the pEA01 plasmid, pYTU + easF; and/or

The pEA02 plasmid, pYTR + dmaW; and/or

The pEA03 plasmid, pYTP + easE (a.fum); and/or

The pEA10 plasmid, pYTU + dmaW + easF; and/or

The pEA12 plasmid, pYTU + dmaW + easF + easE (A.fum) + easC (A.fum); and/or

The pEA13 plasmid, pYTP + easF + easE (A.fum) + easC (A.fum); and/or

The pEA14 plasmid, pYTR + dmaW + easE (a.fum) + easC (a.fum); and/or

The pEA15 plasmid, pYTR + easD + easE (A.fum) + easC (A.fum); and/or

The pEA16 plasmid, pYTP + easG (A.fum) + easA (C.pur); and/or

The pEA31 plasmid, pYTP + easG (A.fum) + easA (A.fum); and/or

The pEA38 plasmid, pYTP + easG (A.fum) + easA (C.pur) + cloA (C.afr); and/or

The pEA39 plasmid, pYTP + easG (A.fum) + easA (C.pur) + cloA (C.pur); and/or

The pEA41 plasmid, pYTP + easA (A.fum) + cloA (C.afr).

More importantly, the invention also provides a host transformed with the expression vector.

In some embodiments of the invention, the host comprises the following strains or combinations thereof:

strain An06, transformed with the pEA01 plasmid, the pEA02 plasmid and the pEA03 plasmid, introduces additional copies of the easF, dmaW and easE (a.fum) genes, and overexpresses the aspergillus nidulans endogenous genes thmgR, samS and trpS; and/or

Strain An10 transformed with plasmid pEA12, plasmid pEA13 and plasmid pEA 14; and/or

Strain An27 transformed with plasmid pEA10, plasmid pEA15 and plasmid pEA 16; replacing easC (a.fum) with easC (a.jap); the strain An27 comprises two copies of easE (a.fum) and easC (a.jap); and/or

Strain An32 transformed with the plasmid pEA10, pEA15 and pEA 31; and knocking out or knocking down the easG gene, and replacing the easC (A.fum) with the easC (A.jap); the strain An32 comprising 3 copies of easE (A.fum) and two copies of easC (A.jap); and/or

Strain An54 transformed with plasmid pEA10, plasmid pEA15 and plasmid pEA 38; cloA (c.afr) in the pEA38 plasmid was fitted to CPR (a.ory); and overexpresses the CYB5 gene endogenous to Aspergillus nidulans; and/or

Strain An55 transformed with plasmid pEA10, plasmid pEA15 and plasmid pEA 39; (ii) cloA (C.pur) in the pEA39 plasmid is reassorted with CPR (C.pur); and overexpresses the CYB5 gene endogenous to Aspergillus nidulans; and/or

Strain An56 transformed with plasmid pEA10, plasmid pEA15 and plasmid pEA 39; cloA (c.pur) in the pEA39 plasmid was adapted to CPR (a.ter); and overexpresses the CYB5 gene endogenous to Aspergillus nidulans;

the starting strain of the strain comprises aspergillus nidulans.

Based on the research, the invention also provides the application of the gene element, the expression vector and the host in the fermentation production of the ergot alkaloid; the ergot alkaloids include one or more of N-Me-DMAT, Prechanoclavine (PCC), chanoclavine (CC), Agroclavine (AC), Festucavine (FC), Pyroclavine (PC), 6-nor-agroclavine (6-nor-AC), Elymoclavine (EC), Lysergic Acid (LA), Dihydroelysergol (DHLG), and dihydrolysergic acid (DHLA).

In addition, the invention also provides a method for producing the ergot alkaloid by fermentation, which uses the host to ferment, collects fermentation liquor and purifies to obtain the ergot alkaloid.

The invention uses aspergillus nidulans as a chassis cell, and obtains 10 ergot alkaloid compounds with different structures and 2 pathway intermediates by a biosynthetic pathway of heterologous reconstructed ergot alkaloids. Multiple ergot alkaloid high-producing strains are obtained through path optimization, precursor supply and P450 electron transfer chain optimization. Based on the above, the present invention achieves heterologous high-yield ergot alkaloid compounds in aspergillus nidulans.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the ergot alkaloid active skeleton and ergot alkaloid class of drugs;

FIG. 2 shows AC and FC A.nidulans producing strains; wherein FIG. 2(A) shows the production of N-Me-DMAT, PCC, CC and AC in a continuously engineered AC-producing strain; FIG. 2(B) shows the production of PCC in strains containing different easE genes; FIG. 2(C) shows CC production in strains containing different easC genes; FIG. 2(D) shows the yield of N-Me-DMAT, PCC, CC and AC after 6 days of shake flask culture of strain An 27; FIG. 2(E) shows the production of N-Me-DMAT, PCC, CC and FC in a continuously engineered FC-producing strain; FIG. 2(F) shows the yield of N-Me-DMAT, PCC, CC and FC after 6 days of shake flask culture of strain An 32;

FIG. 3 shows the LA and DHLA Aspergillus nidulans producing strains; wherein FIG. 3(A) shows the continuous oxidation of AC and FC by P450 CloA to produce LA and DHLA; FIG. 3(B) shows EC production in strains containing different cloA genes; FIG. 3(C) shows the production of DHLG in strains containing different cloA genes; figure 3(D) shows EC production in strains containing CloA (c.afr) and CPR genes from different sources; figure 3(E) shows EC production in strains containing CloA (c.pur) and CPR genes from different sources; figure 3(F) shows the production of DHLG in strains containing CloA (c.afr) and CPR genes from different sources;

FIG. 4 shows the heterologous reconstitution of the ergot alkaloid biosynthesis pathway in Aspergillus nidulans;

FIG. 5 shows LC-MS detection of EC and LA in strains containing different 450CloA genes; wherein FIG. 5(A) shows [ M + H ] in different strains]⁺Test result of 255 (EC); FIG. 5(B) shows [ M + H ] in different strains]⁺269 (LA); FIG. 5(C) shows MS profiles of EC and LA;

FIG. 6 shows LC-MS detection of DHLG and DHLA in strains containing different 450CloA genes; FIG. 6(A) shows [ M + H ] in different strains]⁺Test result of 257 (DHLG); FIG. 6(B) shows [ M + H ] in different strains]⁺The result of detection of 271 (DHLA); FIG. 6(C) shows MS profiles of DHLG and DHLA;

FIG. 7 shows the hydrogen spectrum (500MHz) of compound AC in deuterated methanol;

FIG. 8 shows the hydrogen spectrum (500MHz) of compound 6-nor-AC in deuterated DMSO;

FIG. 9 shows the hydrogen spectrum (500MHz) of compound FC in deuterated methanol;

FIG. 10 shows the hydrogen spectrum (500MHz) of compound PC in deuterated methanol;

figure 11 shows the hydrogen spectrum (500MHz) of compound EC in deuterated methanol;

figure 12 shows the hydrogen spectrum (500MHz) of compound DHLG in deuterated DMSO.

Detailed Description

The invention discloses a bacterial strain and application thereof in fermentation production of ergot alkaloid, and a person skilled in the art can use the content for reference and appropriately improve process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The gene source strains Aspergillus fumigatus 3.772, A.oryzae 3.334, A.niger 3.1454, A.terreus 3.15736 and Claviceps purpurea 3.1003 used in the present invention are all microorganisms studied in the national academy of sciences of China to which the present applicant belongs. The gene expression promoters used in the present invention are glaA, gpdA, amyB, tef 1. All gene expression of the present invention is carried out on a plasmid vector. The genes involved in the present invention are listed in Table 1. The plasmids constructed in the present invention are listed in Table 2. The strains constructed in the present invention are listed in Table 3. All primer sequences of the present invention are listed in Table 4.

TABLE 1 genes related to the present invention

TABLE 2 plasmids constructed according to the invention

TABLE 3 strains constructed according to the invention

TABLE 4 primers related to the present invention

In the application of the strain and the fermentation production of the ergot alkaloid, raw materials and reagents used by the strain can be purchased from the market.

The invention is further illustrated by the following examples:

example 1

(1) Extraction of fungal genomic DNA

Culturing fungus strain on PDA plate for 3 days, scraping surface hypha, washing with distilled water twice, freeze drying, and storing at-20 deg.C. The freeze-dried mycelia were ground to a fine powder using a liquid nitrogen pre-cooled mortar, resuspended in 500. mu.l of lysis buffer (40. mu. mol/L Tris-acetate, 20. mu. mol/L sodium acetate, 1. mu. mol/L EDTA, 1% w/v SDS, pH 7.8) and pipetted until the viscosity of the suspension decreased significantly and foam formed. RNase A was added and incubated at 37 ℃ for 5 minutes, followed by addition of 165. mu.l of 5. mu. mol/L NaCl solution and mixing. 13000 rpm for 20min, the supernatant was immediately transferred to a new tube and 400. mu.l chloroform and 400. mu.l phenol were added. The tube was gently inverted until the solution became milky. After centrifugation for 20 minutes, the aqueous phase was removed and extracted with an equal volume of chloroform. The DNA in the supernatant was precipitated with two volumes of 95% ethanol. The precipitated DNA was washed 3 times with 70% ice-cold ethanol, dried and dissolved in 50. mu.L of TE buffer (10. mu. mmol/L Tris-HCl, 0.1. mu. mmol/L EDTA pH 7.8) and stored at-20 ℃.

(2) Amplification of fragments of interest

Designing primers according to the sequence of each gene on an Aspergillus fumigatus ergot alkaloid biosynthesis gene cluster, and carrying out PCR amplification to obtain a gene fragment for construction of a subsequent plasmid.

(3) Extraction of pYTU, pYTR and pYTP vectors

Extracting pYTU, pYTR and pYTP vectors by using a full-scale gold Plasmid MiniPrep Kit (Trans easy pure Plasmid MiniPrep Kit), and specifically comprising the following steps of:

overnight culturing escherichia coli containing pYTU, pYTR and pYTP vectors respectively, taking 2-4ml of bacterial liquid, centrifuging at 10000rpm for 1min, and pouring out supernatant as far as possible.

② adding 250 microliter RB solution (containing RNase A) into the collected thalli, and whirlpool shaking until the thalli are completely resuspended.

③ adding 250 mu L of LB solution into the centrifuge tube, turning the centrifuge tube upside down for 5-6 times, and standing for 3-5min at room temperature.

And fourthly, adding 350 mu L of NB solution into the centrifuge tube, turning the centrifuge tube upside down for 4-6 times, and standing for 2min at room temperature.

Fifthly, centrifuging for 10min at the maximum rotating speed, carefully sucking the supernatant, adding the supernatant into a centrifugal column, centrifuging for 1min at 10000 Xg, and pouring the waste liquid in the collecting pipe.

Sixthly, 650 mu L of WB solution (80 mL of absolute ethyl alcohol is added before use) is added into the centrifugal column, and the centrifugal column is centrifuged for 1min at 12000 Xg, and waste liquid in the collecting pipe is poured off. And repeating the steps once.

Seventhly, putting the centrifugal column into a clean 1.5ml centrifugal tube, adding 40 mu L of EB solution or sterile water (preheated at 60-70 ℃) into the centrifugal column, and standing for 2min at room temperature.

Then centrifuge at 12000 Xg for 1min, and store the tube at-20 deg.C.

(4) Digestion of the vector and recovery of the vector and the target fragment

The pYTP/pYTR vector was digested with both SwaI and BamHI endonucleases in NEBuffer 3.1. The pYTU vector was double digested with SwaI and NotI in NEBuffer 3.1. Wherein the SwaI enzyme cutting temperature is 25 ℃, the BamHI enzyme cutting temperature and the NotI enzyme cutting temperature are 37 ℃, and the enzyme cutting time is 4 h. The enzyme digestion system is as follows:

both the cleaved vector and the target fragment were recovered using an OMEGA Kit (Gel Extraction Kit). The method comprises the following specific steps:

put the cut gel block into a 2ml centrifuge tube, add equal volume of Binding buffer (XP2), and heat at 60 ℃ until the gel is completely dissolved.

② transferring the solution to a DNA binding column, centrifuging at 12000 Xg for 1min, and pouring the waste liquid of the collecting tube.

③ adding 700. mu.L of SPW Wash Buffer (100 mL of ethanol is added before the use of the SPW Wash Buffer) into the DNA binding column, centrifuging at 12000 Xg for 1min, and pouring off the waste liquid in the collecting tube. And repeating the steps once.

Fourthly, placing the empty DNA binding column into a collecting pipe, and centrifuging for 2min at 12000 Xg.

Fifthly, the DNA binding column is put into a clean 1.5mL centrifuge tube, 15-30mL EB buffer solution or distilled water (preheated at 65 ℃) is added, and the mixture is kept stand for 2min at room temperature.

Sixthly, centrifuging for 1min at 12000 Xg, and storing the centrifuge tube filled with DNA at-20 ℃.

(5) The enzyme-digested vector is connected with a target fragment

Mixing the cut pYTU, pYTR and pYTP vectors, promoter fragments and gene fragments, and assembling the mixture by yeast to obtain the plasmid.

(6) Yeast competent preparation

Making competence for yeast strain BJ5464, preparing a reference kit: zymo research-Yeast Transformation II Kit Catalog No: t2001. The detailed steps are as follows:

firstly, 15ul of yeast is inoculated to 10ml of YPD to culture yeast at 30 ℃ and 220rpm till logarithmic phase, and the yeast is cultured for 18-22h till OD600 is 0.8-1.0;

② centrifuging at 500g for 4min at room temperature and collecting yeast;

③ at room temperature, 5ml EZ 1solution is used for cleaning the yeast sediment, 500g is used for centrifugation for 4min to collect the yeast;

fourthly, cleaning the yeast sediment again by 5ml EZ 1solution at room temperature, centrifuging for 4min by 500g and collecting the yeast;

fifthly, at room temperature, 1ml EZ 2solution is used for resuspending yeast precipitation, 25ul of each yeast is subpackaged, and the yeast precipitation is packaged into a plastic packaging bag, the name and the time are noted, and the yeast precipitation is directly frozen at-70 ℃ or below for storage and standby.

(7) Yeast homologous recombination

Melting at room temperature;

adding 0.2-1ug (5 ug at most) of DNA with conversion into 25ul of competence, adding 300ul of EZ 3solution, shaking and mixing well;

③ incubating for 1 hour at 30 ℃, and oscillating and mixing evenly once every 15 minutes; the incubation time can be extended to 2 hours.

Fourthly, the transformation system is completely coated and cultured for 2 to 4 days at the temperature of 30 ℃.

(8) Identification of transformants

4-5 transformants were selected for identification using colony PCR. Each clone was transferred to a 1X1 cm2 plate and one day later, the expanded yeast monoclonal transformants were subjected to PCR colony validation. The pretreatment method of the yeast colony PCR thallus comprises the following steps:

preparing a reagent: 0.2mM lithium acetate in 1% SDS solution, 100% ethanol, 70% ethanol

② scraping yeast monoclonals from the plate into 1.5ml EP tube, suspending the cells in 100ul solution in 0.2mM lithium acetate solution containing 1% SDS, incubating for 5min at 70 ℃

③ adding 300ul of 96-100 percent ethanol into 1.5ml of EP, and uniformly mixing by a vortex apparatus

Fourthly, 1.5ml of EP is put into a centrifuge, the rotating speed is 15000g, and the centrifugation is carried out for 3min

Fifthly, pouring the supernatant, adding 200ul 70% ethanol into the precipitate of 1.5ml EP to wash the residue, centrifuging the residue for 2-3min by a centrifuge at the rotating speed of 15000G, discarding the supernatant, putting the precipitate in a 65-degree oven for 5min to volatilize ethanol

Sixthly, the residue is resuspended in 15ul H2O, mixed thoroughly on a vortex apparatus, centrifuged at 15000g for 15s

Seventhly, taking 1ul of supernatant as a PCR template for plasmid assembly verification

The pEA01 plasmid was verified using the pYTU-SF/SR primer; the pEA01 plasmid was verified using the pYTR-SF/SR primer, and the pEA03 plasmid was verified using the pYTP-SF/SR primer, which had the following primer sequences:

TABLE 5

And (4) carrying out yeast plasmid extraction on the yeast transformants which are verified to be correct.

(9) Extraction of Yeast plasmid

Correct plasmids will be identified, yeast scraped from a 1x1 cm2 plate, extracted with reference to kit: zymo research Zymoprep II Yeast Plasmid Miniprep II Kit Catalog No: d2004

(10) Transformation of Yeast plasmids into E.coli

The yeast plasmid was introduced into E.coli Top10 for plasmid amplification as follows:

first, commercial chemical competence was removed from the-80 ℃ freezer and thawed on ice.

② adding 6 μ L yeast plasmid, gently shaking and mixing, and placing on ice for 30 min.

③ putting into 42 ℃ water bath for 60-90s, taking out and putting on ice for 2 min.

Adding 200 mu L of LB culture medium (10g/L peptone, 5g/L yeast powder, 10g/L NaCl), and putting the mixture into a shaker at 37 ℃ for incubation for 1 h.

Fifthly, the culture medium is spread on an LB solid plate containing 100 mug/mL ampicillin resistance and cultured overnight.

Sixthly, the grown clone is cultured for 12 hours by LB liquid culture medium containing 100 mug/mL ampicillin resistance, and plasmid is extracted.

Seventhly, sequencing the extracted plasmid, and verifying that the primer is consistent with the yeast.

Comparing the sequence obtained by sequencing with the target sequence, and indicating that the constructed plasmid is correct if the sequences are consistent.

(11) Transformation of plasmid into Aspergillus nidulans

The method for preparing the aspergillus nidulans protoplast comprises the following steps:

A.A.nidulans. DELTA.ST.DELTA.EM strain is spread or streaked on Solid CD Medium containing 0.5ug/ml pyridoxine HCl, 0.125ug/ml riboflavin, 10mM uridine, at 37 ℃ for about 4 days, and after the plate is grown full, 2ml of 0.1% Tween-80 is used.

b. Scraping spores on the left and right half plates by using a cotton swab, and filtering by using a spore filter; and (6) performing microscopic examination.

c.37 deg.C, 250rpm, 100ml, spore concentration about 10⁷Culturing Liquid CD Medium (containing 0.5ug/mL pyridoxine HCl, 0.125ug/mL riboflavin, and 10mM uridine nutrients) with shaking for 6.5h, and germinating spore, wherein the best state of spore germination is that mycelia grow to 3 times of the size of the dilated spore, and spores germinate after being gathered.

d. The mycelia in the Medium were centrifuged at 8000rpm using a centrifuge, 15mL of Osmatic Medium was added, and washing was repeated 3 times.

e. Adding 10ml mixed enzymolysis liquid (30mg dissolving Enzyme, 20mg Yatalase dissolved in 10ml Osmotic Medium buffer), culturing in shaking table at 30 deg.C and 80rpm, observing hypha enzymolysis condition with microscope, the protoplast after enzymolysis is about twice of spore volume, has uniform shape, and is in thin wall state. The enzymolysis time is generally about 2.5 h.

f. Adding an equal volume of tipping buffer, centrifuging at 2000rpm for 10min, and collecting the protoplast in the middle layer

g. Adding 3 times volume of STC buffer, centrifuging at 5000rpm for 10min, and removing liquid

h. Resuspend with addition of a small amount of STC buffer to give a protoplast concentration of about 10⁸-10⁹Per mL, 100. mu.L of each tube was dispensed into 1.5mL centrifuge tubes. All above operations were on ice or at 4 ℃.

Secondly, the plasmids are transformed into protoplasts together, and the steps are as follows:

a. take 5. mu.g of each plasmid, add to 100. mu.L of protoplast, mix gently, and place on ice for 60 min.

b. 1.25mL of 60% PEG Solution was added, gently mixed with a pipette, and left at room temperature for 20 min.

c. Spreading on Solid CD-Sorbitol Medium (added with corresponding nutrients) plate gently, and mixing and spreading gently. Culturing at 37 deg.C for 1d, and culturing for 1-2 days to obtain clone.

(12) Aspergillus nidulans product identification

Taking a monoclonal transformant to Solid CD Medium, and culturing a transformant strain at 37 ℃ for seed conservation;

inoculating the thallus into Liquid culture Medium of Liquid CD-ST Medium, culturing at 37 deg.c and 250rpm for three days;

extracting and detecting the product. The mycelium and the supernatant were extracted with ethyl acetate 3 times, and the organic phases were combined. Rotary evaporation at 30 ℃ until completely dry, dissolution in 300. mu.L of methanol and filtration through 0.22 μm filter. The detection method is HPLC or LC-MS.

And fourthly, HPLC detection conditions: the analysis was performed on a Shimadzu LC-2030C 3D Plus system (Shimadzu, Japan) with mobile phases of acetonitrile (v/v, 0.1% formic acid) and water (v/v, 0.1% formic acid) at a flow rate of 1 mL/min. 0-25min, 10% -100% acetonitrile; 25-30min, 100% acetonitrile; equilibrate with 10% acetonitrile for 8 min. The sample size was 5. mu.L.

LC-MS detection conditions: LC-MS analysis was performed on an Agilent-1200HPLC/6520QTOFMS (USA) system with mobile phases of acetonitrile (v/v, 0.1% formic acid) and water (v/v, 0.1% formic acid) at a flow rate of 0.3 mL/min. 0-15min, 10% -100% acetonitrile; 15-20min, 100% acetonitrile. Equilibrate with 10% acetonitrile for 5 min. The sample size was 5. mu.L. The Q-TOF uses dual ESI as the ion source interface, and the ESI source operates in positive ionization mode. The scan range is m/z 100-.

EXAMPLE 2 heterologous production of N-Me-DMAT and Prechanoclavine (PCC) in A.nidulans

Primers were designed based on the sequence of each gene in the Aspergillus fumigatus ergot alkaloid biosynthesis gene cluster, and dmaW, easF, and easE (a.fum) gene fragments were obtained by PCR amplification and used for the construction of the plasmids pEA01, pEA02, and pEA03, respectively.

The digested pYTU, pYTR and pYTP vectors are mixed with the gene fragments easF, dmaW and easE (A.fum), and then assembled by yeast to obtain pEA01, pEA02 and pEA03 plasmids. Then the plasmid mixture with correct sequencing verification is transferred into aspergillus nidulans to obtain a transformed strain An01, and the product detection and quantification are carried out by fermentation.

Introducing additional copies of the easF, dmaW, easE (A.fum) genes on the basis of strain An01 to give strain An 02; respectively overexpressing endogenous genes thmgR, samS and trpS of aspergillus nidulans on the basis of the strain An02 to obtain strains An03, An04 and An 05; endogenous genes thmgR, samS and trpS of the aspergillus nidulans are commonly overexpressed in the strain An02 to obtain a new strain An 06.

The above strains were fermented under conditions of 10mL of liquid CD-ST medium in 50mL of Falcon tube, incubated at 37 ℃ and 250rpm for three days, and product detection and quantification were performed. The highest yields of N-Me-DMAT and prechanoclavine were found in strain An 06. Then, the strain An06 was subjected to shake flask fermentation, 100mL of liquid CD-ST medium was placed in a 250mL Erlenmeyer flask, and cultured at 37 ℃ and 250rpm for six days, to obtain N-Me-DMAT and Prechanoclavine (PCC) yields of 260.1mg/L and 333.8mg/L, respectively.

Example 3 heterologous production of chanoclavine (CC) in A.nidulans

Primers are designed according to the sequence of each gene on an Aspergillus fumigatus ergot alkaloid biosynthesis gene cluster, and gene segments of dmaW, easF, easE (A.fum) and easC (A.fum) are obtained by PCR amplification and are used for constructing plasmids required by channoclavine expression. dmaW and easF were co-constructed into pYTU to give plasmid pEA 10; construction of easC (A.fum) into pYTR yielded plasmid pEA 11; the dmaW, easF, easE (a.fum) and easC (a.fum) were co-constructed to pYTU to give plasmid pEA 12; jointly constructing easF, easE (A.fum) and easC (A.fum) into pYTP to obtain a plasmid pEA 13; dmaW, easE (a.fum) and easC (a.fum) were co-constructed to pYTR to give plasmid pEA 14.

And (3) mixing plasmids with correct sequencing verification and transferring into aspergillus nidulans, wherein: pEA10, pEA03 and pEA11 are co-transformed to obtain a strain An 07; pEA12, pEA13 co-transformation obtains a strain An 08; pEA12, pEA13 and pEA09 are co-transformed to obtain a strain An 09; pEA12, pEA13, pEA14 were co-transformed to obtain the strain An 10.

The above strains were fermented under conditions of 10mL of liquid CD-ST medium in 50mL of Falcon tube, incubated at 37 ℃ and 250rpm for three days, and product detection and quantification were performed. The highest channoclavine (CC) production was found in strain An 10. Then, the strain An10 was subjected to shake flask fermentation, 100mL of liquid CD-ST medium was placed in a 250mL Erlenmeyer flask, and cultured at 37 ℃ and 250rpm for six days, giving a CC yield of 240.1 mg/L.

Example 4 heterologous production of Agroclavine (AC) in Aspergillus nidulans

Primers were designed based on the sequence of each gene in the Aspergillus fumigatus ergot alkaloid biosynthesis gene cluster, and the dmaW, easF, easE (A.fum), easC (A.fum) easD, and easG (A.fum) gene fragments were obtained by PCR amplification. Designing primers according to sequences of genes on Claviceps purpurea ergot alkaloid biosynthesis gene cluster, and amplifying to obtain an easA (C.pur) gene fragment for constructing a plasmid required for expressing the compound AC. Jointly constructing easD, easE (A.fum) and easC (A.fum) into pYTR to obtain a plasmid pEA 15; easG (A.fum) and easA (C.pur) were co-constructed into pYTP to obtain plasmid pEA 16.

And (3) mixing plasmids with correct sequencing verification and transferring into aspergillus nidulans, wherein: pEA10, pEA15, pEA16 were co-transformed to obtain the strain An 11.

And (3) fermenting the strain, wherein the fermentation condition is that 100mL of liquid CD-ST culture medium is filled in a 250mL triangular flask, culturing is carried out for three days at 37 ℃ and 250rpm, and product detection and quantification are carried out. The AC production in strain An11 was found to be 20.2mg/L, while more N-Me-DMAT (18.6mg/L) and PCC (18.9mg/L) were accumulated (FIG. 2A), indicating that EasE and EasC are the rate-limiting steps in AC production. To address this rate limiting step, we cloned the homologous genes for easE and easC from different EA producing strains. First, the easE (A.fum) gene in PCC producing strain An01 was replaced with the easE homologous gene, and the most active easE (A.fum) gene in A.nidulans was obtained by measuring the production amount of the final product PCC (FIG. 2B). Then, the easC (A.fum) gene in CC producing strain An07 was replaced with the easC homologous gene, and the yield of the final product CC was measured to obtain the most active easC (A.jap) gene in A.nidulans (FIG. 2C). Next, easC (a.jam) was used to replace easC (a.fum) in strain An11 and tested for the effect of different copy numbers of easE (a.fum) and easC (a.jap) on final product AC yield. The AC high producing strain An27, containing two copies of easE (a.fum) and easC (a.jap), was finally obtained. The strain An27 was then subjected to shake flask fermentation, 100mL of liquid CD-ST medium was filled in a 250mL Erlenmeyer flask and cultured at 37 ℃ and 250rpm for six days to give An AC yield of 78.7mg/L (FIG. 2D). In addition, we isolated the compound 6-nor-agroclavine (6-nor-AC) in the strain An 27.

The hydrogen spectrum (500MHz) of compound AC in deuterated methanol is shown in FIG. 7; the hydrogen spectrum (500MHz) of compound 6-nor-AC in deuterated DMSO is shown in FIG. 8.

Example 5 heterologous production of Festucvine (FC) in Aspergillus nidulans

Primers are designed according to the sequence of each gene on the Aspergillus fumigatus ergot alkaloid biosynthesis gene cluster, and dmaW, easF, easE (a.fum), easC (a.fum) easD, easG (a.fum) and easA (a.fum) gene fragments are obtained by PCR amplification and are used for constructing plasmids required for expressing the compound FC. easG (A.fum) and easA (A.fum) were co-constructed to pYTP to obtain plasmid pEA 31.

And (3) mixing plasmids with correct sequencing verification and transferring into aspergillus nidulans, wherein: pEA10, pEA15, pEA31 were co-transformed to obtain the strain An 28.

And (3) fermenting the strain, wherein the fermentation condition is that 100mL of liquid CD-ST culture medium is filled in a 250mL triangular flask, culturing is carried out for three days at 37 ℃ and 250rpm, and product detection and quantification are carried out. The strain An28 is found to have less FC yield, and the main product is chiral isomer of FC, namely pyrroclavine (24.5 mg/L). We tried to replace easG from a different source and found that the main product was still PC. Finally, we deleted the easG gene in strain An28 to give strain An31, which was found to be FC as the main product. Next, we replaced easC (a.fum) in strain An31 with easC (a.jap), and performed multiple copy tests on easE (a.fum) and easC (a.jap) to finally obtain strain An32, which has the greatest FC yield (fig. 2E), and which contains 3 copies of easE (a.fum) and two copies of easC (a.jap). Strain An32 was subjected to shake flask fermentation, 100mL of liquid CD-ST medium was filled in a 250mL Erlenmeyer flask and cultured at 37 ℃ and 250rpm for six days to give An FC yield of 99.2mg/L (FIG. 2F).

The hydrogen spectrum (500MHz) of compound FC in deuterated methanol is shown in FIG. 9. The hydrogen spectrum (500MHz) of compound PC in deuterated methanol is shown in FIG. 10.

Example 6 heterologous production of Elymoclavine (EC) and Lysergic Acid (LA) in Aspergillus nidulans

Primers were designed based on the sequence of each gene in the Aspergillus fumigatus ergot alkaloid biosynthesis gene cluster, and the dmaW, easF, easE (A.fum), easC (A.fum) easD, and easG (A.fum) gene fragments were obtained by PCR amplification. Primers are designed according to the sequence of each gene on the Claviceps purpurea ergot alkaloid biosynthesis gene cluster, and an easA (C.pur) gene fragment is obtained through amplification. According to the literature reports, cDNA sequences of functional cytochrome P450 genes cloA (E.typ), cloA (C.afr) and cloA (C.pur) are synthesized and used for constructing plasmids required for expressing EC and LA of compounds. Jointly constructing easG (A.fum), easA (C.pur) and cloA (E.typ) into pYTP to obtain a plasmid pEA 37; jointly constructing easG (A.fum), easA (C.pur) and cloA (C.afr) into pYTP to obtain a plasmid pEA 38; easG (A.fum), easA (C.pur) and cloA (C.pur) were co-constructed into pYTP to obtain plasmid pEA 39.

And (3) mixing plasmids with correct sequencing verification and transferring into aspergillus nidulans, wherein: pEA10, pEA15 and pEA37 are co-transformed to obtain a strain An 33; pEA10, pEA15, pEA38 were co-transformed to obtain the strain An 34. pEA10, pEA15, pEA39 were co-transformed to obtain the strain An 35.

And (3) fermenting the strain, wherein the fermentation condition is that 100mL of liquid CD-ST culture medium is filled in a 250mL triangular flask, culturing is carried out for three days at 37 ℃ and 250rpm, and product detection and quantification are carried out. It was found that EC was produced by all three strains (fig. 3B), and a small amount of LA was detected by strains An34 and An 35. This result indicates that the synthetic cloA genes can function in A.nidulans.

The fungal cytochrome P450 proteins often require the chaperonin CPR to transfer electrons from NADPH to the catalytic substrate when they function as oxidation catalysts, and may also contain a third protein CYB5 which also transfers electrons (FIG. 3A). Although aspergillus nidulans contains endogenous CPR, it may not be well-adapted to heterologous CloA, resulting in low EC and LA production. Therefore, to improve the yield of EC, we screened CloA (c.afr) and CloA (c.pur) for adapted CPR. Different sources of CPR were first PCR amplified and then cloned into strains An34 and An35, respectively. As a result, the EC yields were significantly improved in three strains An43(cloA (C.afr) -CPR (A.ory)), An44(cloA (C.pur) -CPR (C.pur)), and An46(cloA (C.pur) -CPR (A.ter)), reaching 1.7mg/L, 2.0 mg/L, and 1.9mg/L, respectively (FIGS. 3D and 3E), which were 1.9-fold, 2.8-fold, and 2.9-fold, respectively, of the starting strain. Next, the endogenous CYB5 gene of Aspergillus nidulans was overexpressed in the strains An43, An44 and An46 to obtain new strains An54, An55 and An56, and the yield of compound EC was further increased to 2.1mg/L (An54), 8.2mg/L (An55) and 2.8 mg/L (An56) (FIGS. 3D and 3E).

The results of EC and LA detection are shown in FIG. 5, in which FIG. 5(A) shows [ M + H ] in different strains]⁺Test result of 255 (EC); FIG. 5(B) shows [ M + H ] in different strains]⁺269 (LA); FIG. 5(C) shows MS spectra of EC and LA.

The hydrogen spectrum (500MHz) of compound EC in deuterated methanol is shown in FIG. 11.

Example 7 heterologous production of Dihydrolysergol (DHLG) and Dihydroserinic acid (DHLA) in Aspergillus nidulans

Primers were designed based on the sequence of each gene in the Aspergillus fumigatus ergot alkaloid biosynthesis gene cluster, and dmaW, eaxF, easE (A.fum), easC (A.fum) easD, and easA (A.fum) gene fragments were obtained by PCR amplification. According to literature reports, cDNA sequences of functional cytochrome P450 genes cloA (E.typ), cloA (C.afr) and cloA (C.pur) are synthesized and used for constructing plasmids required for expressing compounds DHLG and DHLA. Jointly constructing easA (A.fum) and cloA (E.typ) into pYTP to obtain a plasmid pEA 40; the easA (A.fum) and cloA (C.afr) are jointly constructed to pYTP to obtain a plasmid pEA 41; the easA (A.fum) and cloA (C.pur) were co-constructed into pYTP to obtain plasmid pEA 42.

And (3) mixing plasmids with correct sequencing verification and transferring into aspergillus nidulans, wherein: pEA10, pEA15 and pEA40 are co-transformed to obtain a strain An 36; pEA10, pEA15, pEA41 were co-transformed to obtain the strain An 37. pEA10, pEA15, pEA42 were co-transformed to obtain the strain An 38.

And (3) fermenting the strain, wherein the fermentation condition is that 100mL of liquid CD-ST culture medium is filled in a 250mL triangular flask, culturing is carried out for three days at 37 ℃ and 250rpm, and product detection and quantification are carried out. It was found that strains An37 and An38 could produce DHLG (fig. 3C), and that strain An37 could detect a small amount of DHLA.

To increase DHLG production, we screened CloA (c.afr) for adapted CPR. Different sources of CPR were first PCR amplified and then cloned into strain An37, respectively. As a result, it was found that the amount of DHLG produced by the strain An49(cloA (C. afr) -CPR (C.pur)) was significantly increased to 1.36mg/L (FIG. 3F), which was 2.5 times that of the starting strain. Subsequently, the endogenous CYB5 gene of Aspergillus nidulans is over-expressed in the strain An49 to obtain a new strain An57, and the yield of the compound DHLG is further improved to 4.0mg/L which is 7.3 times that of the strain An37 (FIG. 3F).

The results of DHLG and DHLA measurements are shown in FIG. 6. Wherein FIG. 6(A) shows [ M + H ] in different strains]⁺Test result of 257 (DHLG); FIG. 6(B) shows [ M + H ] in different strains]⁺The result of detection of 271 (DHLA); FIG. 6(C) shows MS profiles of DHLG and DHLA.

The hydrogen spectrum (500MHz) of compound DHLG in deuterated DMSO is shown in figure 12.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.