CN106701857B - Construction and application of genetically engineered bacteria producing lovastatin and monacolin J - Google Patents

Abstract

The invention relates to the construction and application of genetically engineered bacteria for producing lovastatin and monacolin J. A series of exogenous genes are introduced into yeast engineering bacteria by gene recombination technology to obtain yeast engineering bacteria that can produce lovastatin or its intermediate products. The yeast engineering bacteria have the characteristics of low metabolic background, strong heterologous expression ability, whole-cell synthesis of end products, easy separation of end products and few by-products, etc., and provide a new idea for industrial production of statins.

Description

Construction and application of genetically engineered bacteria producing lovastatin and monacolin J

technical field

The invention belongs to the field of biotechnology, and more particularly, the invention relates to the construction and application of genetically engineered bacteria for producing lovastatin and monacolin J.

Background technique

Cardiovascular and cerebrovascular disease is a serious threat to human health, and its morbidity and mortality ranks first in many countries and regions. An effective way to prevent cardiovascular and cerebrovascular diseases is to inhibit excessive synthesis of cholesterol in the liver. In the cholesterol biosynthesis pathway, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key enzyme controlling the rate of cholesterol synthesis, catalyzing the conversion of HMG-CoA to mevalonate. Therefore, inhibiting the activity of HMG-CoA reductase can achieve the purpose of reducing or blocking the synthesis of cholesterol in vivo, thereby preventing and treating hyperlipidemia.

Statins work by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the limiting step in cells that catalyzes cholesterol biosynthesis. Lovastatin and simvastatin are the most common cholesterol-lowering drugs, which can effectively reduce TC and LDL-C in patients with primary hyperlipidemia, prevent the recurrence of heart disease and the development of atherosclerosis , reducing the risk of myocardial revascularization and non-fatal myocardial infarction surgery.

Lovastatin is mainly prepared by submerged fermentation of Aspergillus terreus or Monascus rubber, while simvastatin is a non-natural product. Its synthesis mainly includes chemical synthesis and biosynthesis, and chemical synthesis includes indirect methylation. Synthesis (side-chain synthesis) and direct methylation; biosynthesis is the synthesis of Monacolin J (MonacolinJ) and side-chain 2,2-dimethylbutylphthalide chloride. Both indirect methylation synthesis and biosynthesis require the drug intermediate monacolin J as a synthetic substrate.

The synthesis of monacolin J includes biological fermentation and chemical hydrolysis. Biological fermentation method is to interrupt the biosynthetic pathway of lovastatin in Aspergillus terreus by molecular means, and the mutant Aspergillus terreus strain accumulates the intermediate metabolite monacolin J during the fermentation process. Monacolin J was obtained after purification. The chemical hydrolysis method is to hydrolyze the natural product lovastatin to obtain monacolin J through four chemical reaction steps of deesterification, hydroxyl protection, re-esterification and deprotection.

In the above method, the secondary metabolites of Aspergillus terreus are produced in many backgrounds, the yield of target metabolites is low, the purification is complicated, and the production cost is high; while in the chemical synthesis method, there are many route steps, long production time, and substrates. The conversion rate is low, and there are many by-products in the deesterification reaction, and the overall yield is also low.

Therefore, further research and development of other production methods for lovastatin or its intermediate products are required in order to simplify the production process and reduce the production cost.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the construction and application of genetically engineered bacteria for producing lovastatin and monacolin J.

In a first aspect of the present invention, there is provided a method for producing lovastatin, the method comprising:

(1) providing a yeast engineering bacteria transformed with the expression cassettes of the exogenous lower group genes: LovB, LovC, LovG, LovA, NpgA, LovD, LovF; and

(2) culturing the yeast engineering bacteria of (1), thereby producing a lovastatin product.

In another aspect of the present invention, there is provided a method for producing lovastatin intermediate monacolin J, the method comprising:

(a) providing a yeast engineering bacteria transformed with the expression cassettes of the exogenous following genes: LovB, LovC, LovG, LovA, NpgA; and

(b) culturing the engineered yeast strain of (a) to produce the monacolin J product.

In a preferred example, the LovB, LovC, LovG, LovA, NpgA, LovD or LovF genes are derived from Aspergillus terreus.

In another preferred example, the LovB gene has the nucleotide sequence shown in SEQ ID NO:1, or a degenerate sequence thereof, or more than 70% (preferably 80%) of the sequence of SEQ ID NO:1 more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%) identical nucleotide sequences encoding isofunctional proteins.

In another preferred example, the LovC gene has the nucleotide sequence shown in SEQ ID NO:2, or a degenerate sequence thereof, or more than 70% (preferably 80%) of the sequence of SEQ ID NO:2 more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%) identical nucleotide sequences encoding isofunctional proteins.

In another preferred example, the LovG gene has the nucleotide sequence shown in SEQ ID NO:3, or a degenerate sequence thereof, or more than 70% (preferably 80%) of the sequence of SEQ ID NO:3 more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%) identical nucleotide sequences encoding isofunctional proteins.

In another preferred example, the NpgA gene has the nucleotide sequence shown in SEQ ID NO:4, or a degenerate sequence thereof, or more than 70% (preferably 80%) of the sequence of SEQ ID NO:4 more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%) identical nucleotide sequences encoding isofunctional proteins.

In another preferred example, the LovD gene has the nucleotide sequence shown in SEQ ID NO:5, or its degenerate sequence, or more than 70% (preferably 80%) of the sequence of SEQ ID NO:5 more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%) identical nucleotide sequences encoding isofunctional proteins.

In another preferred example, the LovF gene has the nucleotide sequence shown in SEQ ID NO:6, or a degenerate sequence thereof, or more than 70% (preferably 80%) of the sequence of SEQ ID NO:6 more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%) identical nucleotide sequences encoding isofunctional proteins.

In another preferred example, the LovA gene has the nucleotide sequence shown in SEQ ID NO:7, or its degenerate sequence, or more than 70% (preferably 80%) of the sequence of SEQ ID NO:7 more preferably more than 90%; more preferably more than 93%; more preferably more than 95%; more preferably more than 97%) identical nucleotide sequences encoding isofunctional proteins.

In another aspect of the present invention, there is provided a yeast engineering bacterium for producing lovastatin, the yeast engineering bacterium comprising an exogenous expression cassette of the following group of genes: LovB, LovC, LovG, LovA, NpgA, LovD , LovF.

In another aspect of the present invention, a yeast engineering bacterium for producing lovastatin intermediate monacolin J is provided, wherein the yeast engineering bacterium contains the expression cassettes of the exogenous genes of the following group: LovB, LovC , LovG, LovA, NpgA.

In a preferred example, the yeast engineering bacterium is Pichia pastoris.

In another preferred embodiment, the Pichia pastoris is Pichia pastoris GS115.

In another aspect of the present invention, there is provided a recombinant expression vector or expression construct for producing lovastatin, the recombinant expression vector comprising the expression cassettes of the following genes: LovB, LovC, LovG, LovA, NpgA, LovD, LovF.

In another aspect of the present invention, there is provided a recombinant expression vector or expression construct for the production of lovastatin intermediate monacolin J, the recombinant expression vector comprising the expression cassettes of the following genes: LovB, LovC , LovG, LovA, NpgA.

In another aspect of the present invention, there is provided the use of a gene combination, the gene combination comprising the following group of genes: LovB, LovC, LovG, LovA, NpgA, LovD, LovF, the gene combination is used for preparing lovastatin intermediate or its intermediate monacolin J.

In another aspect of the present invention, there is provided a kit for producing lovastatin, the kit comprising the yeast engineering bacteria.

In another aspect of the present invention, there is provided a kit for producing lovastatin intermediate monacolin J, the kit comprising the yeast engineering bacteria.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. High-performance liquid chromatography analysis of the fermentation product of recombinant monacolin J bacteria.

A is the high-performance liquid chromatogram of the fermentation broth sample of the negative control strain (without transferring the lovastatin polyketide synthase gene);

B is the high performance liquid chromatogram of the recombinant strain fermentation broth sample;

C is the high performance liquid chromatogram of monacolin J standard.

Figure 2. High-performance liquid chromatography analysis chart of fermentation products of recombinant lovastatin-producing bacteria.

A is the high-performance liquid chromatogram of the fermentation broth sample of the negative control strain (without transferring the lovastatin polyketide synthase gene);

B is the high performance liquid chromatogram of the recombinant strain fermentation broth sample;

C is the high performance liquid chromatogram of lovastatin standard.

Figure 3. High density fermentation profile of recombinant strains producing monacolin J and lovastatin.

A is the high-density fermentation process of the recombinant strain producing monacolin J, and records the cell biomass and monacolin J production as a function of fermentation time.

B is the high-density fermentation profile of the recombinant strain producing lovastatin, which records the cell biomass and monacolin J production as a function of fermentation time.

Figure 4. Genetic stability map of recombinant strains producing monacolin J and lovastatin.

A is the passage number of the recombinant strain producing monacolin J and the monacolin J yield of the strains of different passages;

B is the passage number of the recombinant strain producing lovastatin and the lovastatin yield of the strains of different passages.

Detailed ways

After in-depth research, the inventors introduced a series of exogenous genes into yeast engineering strains through gene recombination technology to obtain yeast engineering bacteria that can produce lovastatin or its intermediate products. The yeast engineering bacteria have the characteristics of low metabolic background, strong heterologous expression ability, whole-cell synthesis of end products, easy separation of end products and few by-products, etc., and provide a new idea for industrial production of statins.

the term

As used herein, the "expression cassette" or "gene expression cassette" refers to a gene expression system that contains all necessary elements for expressing a polypeptide of interest, typically it includes the following elements: a promoter, a gene sequence encoding a polypeptide, terminators; and optionally, signal peptide coding sequences, etc.; these elements are operably linked.

As used herein, "operably linked (linked)" or "operably linked (linked)" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, the promoter region is placed at a specific location relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region, and thus, the promoter region is "operably linked" to the nucleic acid sequence.

As used herein, an "expression construct" refers to a recombinant DNA molecule comprising the desired nucleic acid coding sequence, which may comprise one or more gene expression cassettes. The "construct" is usually contained in an expression vector.

As used herein, "foreign" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between proteins (or nucleic acids) from different sources and a host cell relationship between. For example, a nucleic acid is foreign to a host cell if the combination of the nucleic acid and the host cell does not normally occur in nature. A particular sequence is "foreign" to the cell or organism into which it is inserted.

Genes and their expression systems

In the present invention, the efficient production of lovastatin or its intermediate in the yeast engineering bacteria is achieved by transforming the five-gene combination or the seven-gene combination into the yeast engineering bacteria. The five-gene combination includes the following genes: LovB, LovC, LovG, LovA, NpgA; the seven-gene combination includes the following genes: LovB, LovC, LovG, LovA, NpgA, LovD and LovF. The above-mentioned genes are all genes known in the art.

In the present invention, the above-mentioned genes may be naturally occurring, for example, they may be isolated or purified from animals, plants or microorganisms. In addition, the gene can also be artificially prepared, for example, the gene can be obtained according to conventional genetic engineering recombination techniques, or the gene can be obtained by artificial synthesis.

The nucleotide sequences of the above-mentioned genes may be the same as those shown in SEQ ID NOs: 1 to 7, or may be degenerate variants thereof. As used herein, a "degenerate variant" in the present invention refers to a nucleic acid sequence that encodes a protein having the same function, but differs from the sequence selected from SEQ ID NOs: 1-7.

The gene may include: a coding sequence encoding only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

The present invention also relates to the variant of the gene, the encoded polypeptide is different from its corresponding wild-type polypeptide in amino acid sequence, and is a fragment, analog or derivative of the wild-type polypeptide. Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides that does not substantially alter the function of the encoded polypeptide .

The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. In particular, the present invention relates to polynucleotides that are hybridizable under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, more Hybridization occurs when it is above 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the corresponding wild-type polypeptide.

It should be understood that although each gene of the present invention is preferably obtained from Aspergillus terreus, those obtained from other microorganisms are highly homologous to the corresponding genes in Aspergillus terreus (eg have more than 70%, such as 80%, 90%, 95%, or even 98%) % sequence identity) other genes are also contemplated by the present invention. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.

The full-length sequences or fragments thereof of each gene of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and the relevant sequences can be obtained by amplification. When the sequence is longer, two or more PCR amplifications can be performed, and then the fragments amplified by each time can be spliced together in the correct order.

The present invention also relates to vectors comprising said polynucleotides, and to host cells genetically engineered with said vectors.

In the present invention, the sequence of each gene can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as it is replicable and stable in the host. An important feature of expression vectors is that they typically contain an origin of replication, a promoter, marker genes and translational control elements.

The sequences of each gene can be inserted into the recombinant expression vector separately, and multiple recombinant expression vectors can be co-transfected into the host cell; the expression cassettes of multiple genes can also be inserted into the same recombinant expression vector in a tandem manner, and then transformed into the host cell. The recombinant expression vector may also contain an expression control sequence operably linked to the sequence of the gene to facilitate protein expression. It should be understood that after those skilled in the art understand the technical content of the present invention, the recombinant expression vector can be easily constructed. The obtained recombinant expression vector is also included in the present invention.

In the expression control sequences or expression cassettes, according to different needs, inducible or constitutive promoters can be used. Inducible promoters can achieve more controllable protein expression and compound production, which is beneficial to industrial application.

As a preferred mode of the present invention, there is provided an expression vector (expression construct,) which includes the expression cassettes of the following genes: LovB, LovC, LovG, LovA, NpgA; and also provides a kind of expression vector (expression construct,) It includes expression cassettes for the following genes: LovD and LovF genes.

The establishment of expression vectors (expression constructs) is now a technique familiar to those skilled in the art. Therefore, the establishment of expression constructs can be easily performed by those skilled in the art after knowing the desired gene for selection. Gene sequences can be inserted into different expression constructs (such as expression vectors), or into the same expression construct, as long as the encoded polypeptide can be efficiently expressed and active after being transferred into cells . As a preferred mode of the present invention, the expression vectors are pPIC Z, pPIC3.5K and pAG32.

Vectors comprising the appropriate gene sequences described above, together with appropriate promoter or control sequences, can be used to transform appropriate host cells so that they can express the protein. In the present invention, the host cell is preferably a yeast engineering bacterium, more preferably Pichia pastoris, such as Pichia pastoris GS115.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformants can be cultured by conventional methods, and the medium used in the culture can be a yeast medium well known in the art. The cultivation is carried out under conditions suitable for the growth of yeast cells.

The recombinant Pichia strain obtained by the gene recombination technology has the characteristics of low metabolic background, strong heterologous expression ability, whole-cell synthesis of end products, easy separation of end products and few by-products, etc. The problems existing in the synthesis of biological and chemical methods provide new ideas for the industrial production of statins.

Method for synthesizing lovastatin or its intermediate

The structural formula of lovastatin is shown in the following formula (I), and the structural formula of its intermediate monacolin J is shown in the following formula (II).

The invention discloses a method for producing lovastatin or its intermediate by heterologous synthesis of microorganisms. The method includes: transforming five genes (LovB, LovC, LovG, LovA, NpgA) into yeast engineering bacteria to produce Lovastatin intermediate monacolin J; or, transforming seven genes (LovB, LovC, LovG) , LovA, NpgA, LovD, LovF) transformed yeast engineering bacteria to produce lovastatin.

The fermentation culture of recombinant yeast engineering bacteria can adopt yeast fermentation methods known in the art. A preferred method is: in liquid YPD culture at 30 ° C and a rotation speed of 200 r/min, the recombinant bacteria are cultured to logarithmic phase and collected. The cells were washed twice with sterile water and then transferred to MM medium, and cultured at 30°C at a speed of 200r/min for 72-96h. During the fermentation process, 0.5% methanol was added to the liquid medium every 24 h.

After the fermentation product is obtained, techniques known in the present invention can be used to extract lovastatin or its intermediate from the fermentation product. The product can be analytically identified using high performance liquid chromatography to confirm that the desired compound has been obtained.

This patent uses recombinant Pichia pastoris to heterologously produce lovastatin and monacolin J, which not only solves the problems of large secondary metabolism background of Aspergillus terreus, a natural producing bacterium in the biological fermentation method, and difficult product separation, but also avoids the Unfavorable factors such as many by-products, difficult purification, and large environmental pollution in the chemical synthesis method have opened up a new way for the industrial production of hypolipidemic drugs lovastatin and simvastatin to synthesize intermediate monacolin J. Therefore, the recombinant Pichia strain of the present invention has the potential to industrially produce lovastatin and monacolin J, a statin precursor.

The invention realizes the technological breakthrough of using recombinant yeast for whole-cell biosynthesis of monacolin J and lovastatin, and provides a new way for the production of blood lipid-lowering drugs lovastatin, simvastatin and other blood pressure-lowering drug derivatives.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, 2002, or according to the conditions described by the manufacturer. the proposed conditions.

culture medium

YPD liquid medium: glucose 20.0g/L, peptone 20.0g/L, yeast extract 10.0g/L;

YPD solid medium: glucose 20.0g/L, peptone 20.0g/L, yeast extract 10.0g/L, agar 20g/L;

MM liquid medium: methanol 5mL/L, YNB 13.4g/L.

Embodiment 1, the construction of expression plasmid

1. Construction of polyketide synthase related genes integrated into AOX1 promoter expression plasmid

Design specific primers, take the Aspergillus terreus genome as a template, and use the corresponding primers in Table 1 to obtain lovastatin polyketide synthase-related genes (LovB, LovC, LovG, NpgA, LovD, LovF) by PCR amplification, using EcoRI and The plasmid pPIC Z and each gene fragment were digested with XhoI, and each gene was integrated into the AOX1 promoter downstream of the plasmid pPIC ZB (Invitrogen) using ligase.

Wherein, the gene sequence of LovB is shown in SEQ ID NO:1;

The gene sequence of LovC is shown in SEQ ID NO:2;

The gene sequence of LovG is shown in SEQ ID NO:3;

The gene sequence of NpgA is shown in SEQ ID NO:4;

The gene sequence of LovD is shown in SEQ ID NO:5;

The gene sequence of LovF is shown in SEQ ID NO:6.

The primers used for PCR amplification are shown in Table 1.

Table 1

2. Construction of plasmid pPIC Z_BCGN

The plasmid constructed in "1" was double digested with BamH I and Bgl II, respectively, and the complete expression cassettes of LovB, LovC, LovG and NpgA genes including promoter and terminator were recovered respectively.

Similarly, the plasmid pPIC Z was digested with BamH I and Bgl II, and the complete expression cassettes of each gene were integrated into the plasmid head-to-tail by using ligase, and the plasmid pPIC Z_BCGN containing LovB, LovC, LovG and NpgA genes was obtained.

3. Construction of plasmid pPIC 3.5K_A

Taking the Aspergillus terreus genome as a template, with the corresponding primers in Table 1, the gene LovA fragment was obtained by PCR amplification, using Not I and Bln I double enzyme digestion PCR product and plasmid pPIC 3.5K, utilize ligase to join the two, get final product The plasmid pPIC 3.5K_A was obtained.

The gene sequence of LovA is shown in SEQ ID NO:7.

4. Construction of plasmid pAZh_DF

Using plasmid pAG 32 (obtained from California Institute of Technology, USA) as a template, with the corresponding primers in Table 1, the hygromycin gene (Hyg) resistance expression cassette fragment was obtained by PCR amplification, and the plasmid pPIC was digested with BamH I and Bgl II enzymes ZB and PCR products were joined by ligase to obtain plasmid pAZh.

Use BamH I and BglII double enzymes to digest the plasmids constructed in "1" above, which contain the LovD and LovF expression cassettes respectively, and use BamH1 single enzyme to digest the plasmid pAZh, and use ligase to digest the LovD and LovF expression cassettes head and tail. The plasmid pAZh_DF can be obtained by linking and integrating into the plasmid pAZh.

Example 2. Preparation of target recombinant yeast

1. PCR verification of recombinant strains

Each expression plasmid was electrotransformed into Pichia GS 115. After recovery, it was spread on YPD solid medium with resistance. After culturing for 3 days, fresh colonies were picked and cultured in YPD liquid medium, and extracted using a yeast genome extraction kit. The genome of each transformant was identified by cloning PCR reaction to identify the integration of the polyketide synthase gene into the genome of Pichia pastoris.

Cloning PCR reaction conditions:

(1) Initial denaturation at 95°C for 5 minutes;

(2) Denaturation at 95°C for 30s, annealing at 50°C for 30s, extension at 72°C for 1 min/kb, and cyclic reaction 30 times;

(3) The final extension is 72°C for 7min.

2. Obtainment of recombinant bacteria containing 5 kinds of foreign genes

After the plasmids pPIC Z_BCGN and pPIC 3.5K_A were linearized and recovered, they were transformed into Pichia pastoris by electroporation, and the recombinants were screened using YPD solid culture with resistance to bleomycin and geneticin, and five kinds of transformed into 5 species were obtained. Pichia pastoris genetically engineered bacteria with foreign genes (LovB, LovC, LovG, NpgA, LovA genes).

3. Obtainment of recombinant bacteria containing 7 foreign genes

The recombinant yeast containing 5 kinds of exogenous genes obtained in the aforementioned 2 was prepared into a competent state, the linearized plasmid pAZh_DF was electro-transformed into the competent state, and a solid YPD medium with hygromycin resistance was used to screen the recombinants, Pichia genetically engineered bacteria transformed into 7 foreign genes (LovB, LovC, LovG, NpgA, LovA, LovD and LovF) were obtained.

Embodiment 3, recombinant engineering bacteria 5L tank high-density fermentation process and product quantitative analysis

1. Shake flask (250mL conical flask) horizontal fermentation

Recombinant bacteria were cultured to logarithmic phase in liquid YPD medium at 30°C and rotating speed of 200r/min, and the cells were collected and washed twice with sterile water, and then transferred to a 250mL conical flask containing 100mL of MM liquid medium. , cultured at 30°C for 72-96h at a speed of 200r/min. 0.5% (v/v) methanol was added to the liquid medium every 24 h during the fermentation.

2. 5L fermentation tank fermentation

The upper tank basal medium BSM (the formula has been commercialized by Invitrogen) was used as the medium, and the fermentation process was divided into batch stage and feeding stage. Flow feed. During this period, the stirring speed in the fermentation tank is controlled at 1000 rpm; the oxygen supply is provided to ensure that the dissolved oxygen in the fermentation broth is between 30% and 60%; the ammonia water feed is connected to maintain the pH of the fermentation broth at about 5.0. In the initial stage of fermentation, glycerol was used as carbon source to promote cell growth, and glycerol feed was provided until the cell growth reached 250 g/L wet weight, then glycerol feed was stopped and switched to methanol feed, and the methanol flow acceleration rate was gradually increased to It remained stable after 16 mL/h, and the fermentation ended after 72 h of methanol feeding.

Example 4. Extraction and identification of recombinant strain fermentation products

The recombinant strain after the aforementioned verification was cultured to log phase in YPD liquid medium, and 100 OD (Note: OD value is the yeast concentration unit, 1 OD is about 5× ^{10 7} yeast cells. The OD value is determined by UV spectrophotometer at 600nm Wavelength measured) bacterial cells, washed twice with sterile water and transferred to 100 mL of MM liquid medium, the temperature was 30 ° C, the rotation speed was 200 r/min for induction fermentation culture, the time was 72-96 h, and 0.5% was added every 24 h. of anhydrous methanol. From the fermentation broth after fermentation, take 20 mL of the same volume of ethyl acetate to extract the sample three times. The upper ethyl acetate organic phase was collected, distilled and then dissolved in 2 mL of methanol and diluted ten times for high performance liquid chromatography (HPLC) analysis.

High performance liquid chromatography analysis was performed by reversed high performance liquid chromatography Agilent 1100, the chromatographic column used a C18 column, the column temperature was 30° C., and the ultraviolet detection wavelength was 238 nm, and the elution conditions were as shown in Table 2.

Table 2, target product HPLC mobile phase conditions

Figure 1B shows the high-performance liquid identification diagram of the fermentation product of the recombinant bacteria containing 5 exogenous genes, Figure 1A is the high-performance liquid chromatogram of the negative control, and Figure 1C is the high-performance liquid chromatogram of the standard product. It can be seen from the figure that the recombinant yeast strain can produce monacolin J.

Figure 2B shows the high-performance liquid chromatographic identification diagram of the fermentation product of the recombinant bacteria containing 7 exogenous genes, Figure 2A is the high-performance liquid chromatogram of the negative control, and Figure 2C is the high-performance liquid chromatogram of the standard product. It can be seen from the figure that the recombinant yeast strain can produce lovastatin.

Example 5. Cellular biomass and product yield during fermentation

Fermentation was carried out according to the 5L fermentor fermentation culture method of Example 3. During the fermentation, samples were taken at 12 h intervals to determine the cell biomass and the amount of monacolin J product.

The results are shown in Figure 3. It can be seen from Figure 3A that with the increase of fermentation time, the cell biomass gradually increased, and the production of monacolin J also gradually increased, reaching the highest value at about 100 hours of fermentation, with a yield of about 215 mg /L.

It can be seen from Fig. 3B that the biomass of cells gradually increased with the increase of fermentation time in the initial stage, and no increase after about 32 hours of fermentation. Lovastatin was produced after about 23 hours of fermentation, and its yield gradually increased, reaching the highest value at about 60-65 hours of fermentation, with a yield of 180-185 mg/L.

Embodiment 6. Identification of genetic stability of recombinant engineered bacteria

The recombinant yeasts containing 5 kinds of exogenous genes and the recombinant yeasts containing 7 kinds of exogenous genes obtained above were inoculated into a 250mL conical flask containing 100mL liquid medium, and cultured in a shaker flask (30°C, 200 rpm), as described in Example 3. In addition, subculture was carried out for 10 generations, and the monacolin J production and lovastatin production of each generation of recombinant strains were determined to determine the genetic stability of the recombinant strains.

The results are shown in Figure 4. Each generation of strains can stably produce monacolin J and lovastatin, and with the increase of the number of passages, the product yield also slightly increases. The yield of Colin J was about 30 mg/L; the yield of lovastatin of the passaged strain was about 20 mg/L.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (6)

1. A method for producing lovastatin, the method comprising:

(1) providing engineering yeast strain, in which the expression cassettes of exogenous gene of following group are transformed, including L ovB, L ovC, L ovG, L ovA, NpgA, L ovD and L ovF, and making said engineering yeast strain be pichia pastoris, and

(2) and (2) fermenting and culturing the yeast engineering bacteria in the step (1) to generate a lovastatin product, wherein the fermentation process is divided into a batch phase and a feed phase, the feed phase comprises glycerol feed in the early stage and methanol flow feed in the middle and later stages, the glycerol is used as a carbon source to promote the growth of the thalli in the initial fermentation stage, the glycerol feed is stopped until the thalli grow to 250 g/L wet weight, and the glycerol feed is switched to the methanol feed.

2. the method of claim 1, wherein the methanol feed is carried out by gradually increasing the methanol feed rate to 16m L/h and then maintaining the steady state, and adding 0.5% of anhydrous methanol every 24 h.

3. The process as claimed in claim 1, wherein the rotational speed of the stirrer in the fermenter during the fermentation is 1000 rpm.

4. The method of claim 1, wherein the fermentation broth has dissolved oxygen of between 30% and 60%.

5. The method of claim 1, wherein the broth pH is maintained at 5.0 by feeding with aqueous ammonia.

6. The method of claim 1, wherein the preparation of the engineered yeast strain comprises:

after plasmids pPIC Z _ BCGN and pPIC 3.5K _ A are recovered by linearization, the plasmids are sequentially transformed into pichia pastoris competence by electricity, YPD solid culture with resistance to bleomycin and geneticin is used for screening recombinants, and pichia pastoris genetic engineering bacteria transformed into genes of L ovB, L ovC, L ovG, NpgA and L ovA are obtained;

wherein, the plasmid pPIC Z _ BCGN contains L ovB, L ovC, L ovG and NpgA genes, the plasmid pPIC 3.5K _ A contains L ovA genes, and the plasmid pAZH _ DF contains L ovD and L ovF genes.

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