WO2019072081A1 - Microorganism and method of increasing yield of hydrophobic compound from fermentation of microorganism - Google Patents

Abstract

The present invention provides a microorganism. The microorganism comprises the following features: over-expressing at least one selected from the following genes: PAH1, DGA1, OLE1, ACC1**, ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, and fabB; and silencing at least one selected from FLD1 and TGL3. The microorganism has the potential to synthesize a hydrophobic compound.

Description

Microorganisms and methods for increasing fermentation yield of microbial hydrophobic compounds

Priority information

The present application claims priority to and the benefit of the patent application No. PCT Application No.

Technical field

The invention relates to the field of biotechnology, and in particular to a method for increasing the yield of a hydrophobic product by lipid synthesis.

Background technique

Microbial fermentation production of compounds has become a very mature technology, such as Saccharomyces cerevisia as a mature food-safe strain that can be fermented on a large scale, with a clear genetic background, a mature genetic manipulation system, and a research and transformation of many food-grade products. The object is also a model strain in industrial production. The mature fermentation platform and surrounding industries have also facilitated the subsequent promotion of downstream products.

However, how to further improve the microbial fermentation products is still a key issue for researchers to solve.

Summary of the invention

This application was made by the inventors based on the discovery of the following problems and facts:

Most of the hydrophobic compounds are synthesized in microorganisms due to their low solubility, which leads to the inability to accumulate in large amounts. For example, lycopene is a typical hydrophobic compound. Due to its poor water solubility, the product is localized to the cell membrane after synthesis, which not only limits the limitation. The accumulation of the product is also toxic to the cells, the accumulation of the product is limited to a certain level, and the cell growth is also limited. This poses a huge obstacle to the scale-up production of engineered strains. The inventors unexpectedly found in the experiment that if the lipid content in the microorganism is increased, the yield of the hydrophobic compound in the microorganism is significantly increased. After further research by the inventors, it was found that the lipid content in the microorganisms is improved, which provides a bearing environment for the accumulation of hydrophobic products such as lycopene, astaxanthin, natamycin and spinosad in the body, and The toxicity of the hydrophobic product in the cell is reduced as little as possible, and the accumulation of the hydrophobic product is enhanced, and the effect of the accumulation of the hydrophobic product on the cell growth is effectively reduced.

Based on this, in the first aspect of the invention, the invention proposes a microorganism. According to an embodiment of the invention, the microorganism comprises: overexpression comprising at least one selected from the group consisting of: PAH1, DGA1, OLE1, ACC1**, ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB; and silencing comprising at least one selected from the group consisting of FLD1, TGL3, wherein the microorganism is a microorganism having the potential to synthesize a hydrophobic compound. According to the embodiment of the present invention, the yield of the hydrophobic compound of the microorganism can be increased by more than 10%, for example, the yield of lycopene is increased by more than 40%, the yield of natamycin is increased by 14%, and the yield of spinosad is increased by 20%. The yield of astaxanthin is increased by 50%, and the product is less toxic to cell growth.

According to an embodiment of the present invention, the microorganism may further include at least one of the following additional technical features:

According to an embodiment of the invention, the PAH1, DGA1, OLE1, ACC1** are derived from Saccharomyces cerevisiae, preferably, the ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D are derived from Streptomyces, Preferably, the EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB are derived from Escherichia coli.

According to an embodiment of the present invention, the amino acid sequence of the polypeptide encoded by the above gene is shown in SEQ ID NOs: 1, 6-27.

The inventors have found that genes derived from the above microorganisms are overexpressed to microorganisms having the potential to synthesize hydrophobic compounds, and can significantly promote the accumulation of hydrophobic compounds in microorganisms.

According to an embodiment of the present invention, the microorganism comprises at least one selected from the group consisting of yeast, Escherichia coli, actinomycetes, Bacillus subtilis, Corynebacterium glutamicum, Aspergillus niger, Aspergillus oryzae, Trichoderma viride, and Trichoderma reesei. One. The inventors have found that overexpression of at least one of the above genes and silencing in the above microorganisms includes at least one selected from the group consisting of FLD1 and TGL3, and the accumulation of hydrophobic compounds in the microorganisms is further significantly improved.

According to an embodiment of the present invention, the hydrophobic compound comprises at least one selected from the group consisting of lycopene, carotenes, astaxanthin, natamycin, spinosyn, polyketides, sesquiterpenes, triterpenes, and tetraterpenoids. one. The yield of the above hydrophobic compound in the microorganism according to the embodiment of the present invention is higher.

In a second aspect of the invention, the invention proposes a method of increasing the fermentation yield of a hydrophobic compound of microorganisms. According to an embodiment of the invention, the compound comprises: increasing the lipid content in the microorganism, wherein the microorganism is a microorganism having the potential to synthesize a hydrophobic compound. The inventors unexpectedly found in the experiment that increasing the lipid content in the microorganisms having the potential of synthetic hydrophobic compounds can significantly increase the fermentation yield of the hydrophobic compound of the microorganism, and the fermentation yield of the hydrophobic compound can be increased by more than 10%, such as lycopene. The yield increased by more than 40%, the production of natamycin increased by 14%, the production of spinosad increased by 20%, the yield of astaxanthin increased by 50%, and the product was less toxic to cell growth.

According to an embodiment of the present invention, the above method may further include at least one of the following additional technical features:

According to an embodiment of the invention, the lipid is a triglyceride. The inventors have found that increasing the content of triglyceride in microorganisms is more effective in promoting the fermentation yield of hydrophobic compounds.

According to an embodiment of the present invention, the increasing the lipid content in the microorganism is achieved by increasing at least one of a synthesis amount of the triglyceride, a size of the lipid droplets, and a content of the unsaturated fatty acid. Further, the fermentation yield of the hydrophobic compound is further increased.

According to an embodiment of the invention, the increasing the lipid content in the microorganism is by overexpression in the microorganism comprising at least one selected from the group consisting of: PAH1, DGA1, OLE1, ACC1**, ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB; and silencing comprising at least one selected from the group consisting of FLD1, TGL3. Through the above method, the lipid content in the microorganism can be effectively increased, thereby providing a bearing environment for the accumulation of the hydrophobic product in the body, and reducing the toxicity of the hydrophobic product in the cell as much as possible, thereby further increasing the accumulation of the hydrophobic product. At the same time, the effect of accumulation of hydrophobic products on cell growth can be further effectively reduced.

According to an embodiment of the present invention, the overexpression is achieved by introducing a construct into the microorganism, the construct comprising a gene to be overexpressed and a regulated expression promoter, the regulated expression promoter and the An operable linkage of the gene of interest. Further, the target gene can be overexpressed in a controlled manner, and the target gene is overexpressed in the cell growth phase, and the expression is stopped during the product accumulation phase.

According to an embodiment of the invention, the regulated expression promoter is pHXT1. pHXT1 is a glucose-controlled promoter, which enables more convenient and efficient controllable expression of the gene of interest.

According to an embodiment of the invention, the pHXT1 has the nucleotide sequence set forth in SEQ ID NO:28.

According to an embodiment of the present invention, the microorganism comprises at least one selected from the group consisting of yeast, Escherichia coli, actinomycetes, Bacillus subtilis, Corynebacterium glutamicum, Aspergillus niger, Aspergillus oryzae, Trichoderma viride, and Trichoderma reesei. one. The lipid content in the above microorganisms is increased, and the accumulation of hydrophobic compounds in the microorganisms is further significantly improved.

According to an embodiment of the present invention, the hydrophobic compound comprises a compound selected from the group consisting of lycopene, carotene, astaxanthin, natamycin, spinosyn, polyketone, sesquiterpene, triterpene, and tetraterpenoids. At least one. With the method according to an embodiment of the present invention, the yield of the above hydrophobic compound is higher.

In a third aspect of the invention, the invention proposes the use of the aforementioned microorganisms for increasing the fermentation yield of hydrophobic compounds. As described above, the microorganism according to the embodiment of the present invention has a property of producing a highly hydrophobic compound, and the microorganism according to the embodiment of the present invention can be effectively used in the fermentation production of a hydrophobic compound, and the yield of the hydrophobic compound is high and the toxicity to the microorganism is small.

It should be noted that the amino acid sequence of the polypeptide encoded by the gene disclosed in the present application means that any nucleotide sequence encoding a polypeptide having the amino acid sequence is within the protection scope of the present application.

It should be noted that the amino acid sequence of the polypeptide encoded by the gene disclosed in the present application means at least 70%, at least 75%, at least 80%, at least 85%, at least 90% with the disclosed amino acid sequence. a polypeptide that is at least 95% identical to at least 99% identical; or

Polypeptides having one or more amino acid substitutions, deletions, and/or additions to the disclosed amino acid sequences are within the scope of the present application.

The additional aspects and advantages of the invention will be set forth in part in the description which follows.

DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the results of a different degree of increase in the yield of lycopene producing bacteria overexpressing triglyceride than the original lycopene producing bacterium TM606 according to an embodiment of the present invention.

Detailed ways

The examples of the present invention are described in detail below, and are represented by high-yielding lycopene-producing Saccharomyces cerevisiae, high-yield astaxanthin-producing Escherichia coli, high-yield natamycin-producing Streptomyces, and high-seduce spinosad-producing bacteria. The embodiments described below are illustrative only and are not to be construed as limiting the invention. Modifications or substitutions of the methods, steps or conditions of the invention are intended to be included within the scope of the invention. Where the specific techniques or conditions are not indicated in the examples, they are carried out according to the techniques or conditions described in the literature in the art or in accordance with the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

In the following examples, the polypeptides encoded by the PAH1, DGA1, OLE1, ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB genes See the sequence listing for the amino acid sequence.

Example 1 Plasmid construction required for lycopene producing bacteria

The primers used in the examples are shown in Table 1.

Table 1: Primers used to construct lycopene engineering strains

The plasmids constructed in the examples and the fragments used in the plasmid construction were subjected to PCR amplification according to the corresponding primers and templates, as shown in Table 2.

Table 2: Plasmids constructed in this example

PCR reaction system: 30.5 μL H ₂ O, 10 μL 5×reaction buffer, 4 μL 2.5 mM dNTPs, 2 μL 10 mM forward primer, 2 μL 10 mM reverse primer, 1 μL template DNA (1-100 ng), 0.5 μL Phusion High-Fidelity DNA Polymerase . PCR reaction procedure: pre-denaturation at 98 ° C for 30 s; denaturation at 98 ° C for 10 s, annealing at 56 ° C for 30 s, extension at 72 ° C (30 s / K), 30 cycles; finally extending at 72 ° C for 10 min.

The plasmids constructed in the examples were all constructed by the yeast assembly method: different plasmids were assembled according to the fragments listed in Table 2.

The PCR fragment was cut into gels, and the volume used was calculated by using 300 ng per fragment.

According to the requirements of each plasmid, take the corresponding fragment, mix, calculate the volume, add 10% volume of 3M NaAc, 2% volume of 10mg/mL glycogen, mix, add 2 times volume of absolute ethanol and mix, -80 °C Place for 2 h, 13,000 rpm, centrifuge at 20 ° C for 20 min, discard the supernatant. Wash once with 500 μL of 70% ethanol, centrifuge at room temperature for 13 min at 13,200 rpm, discard the supernatant, allow to dry, add 4 μL of ddH ₂ O to re-dissolve, and set aside.

The treated mixed fragments were transformed into S. cerevisiae by a lithium acetate method, and the corresponding YPD-resistant plates were coated and cultured at 30 °C. (The colony grows in about 3 days). To grow the colony to a suitable size, pick a liquid medium with monoclonal to YPD resistance, incubate at 30 ° C, 220 rpm for 20 h, extract the plasmid, transform E. coli DH10B, and coat LB solid plate (Ampicillin resistance), 37 ° C After culturing for about 2 days, the monoclonal medium was picked up to LB-Amp liquid medium, and the plasmid was cultured at 37 ° C, 220 rpm for 16 h, and the enzyme was digested and verified.

Example 2 Construction of Saccharomyces Cerevisiae Lycopene Strain

Construction of Saccharomyces cerevisiae lycopene strain TM606, the construction method is as follows:

Expression of tHMG1 increased the conversion rate of MVA pathway; silencing GAL1,7,10 induced the production of lycopene by galactose; the construction method was to digest the plasmid pZY141 with NotI, and recover the target fragment, and integrate it into the yeast solution by the amount of 200 ng. In the Saccharomyces cerevisiae (CEN.PK2-1D) strain, the target strain was screened by using a plate containing the corresponding auxotrophy, and verified by PCR.

Expression of PaCrtB (SEQ ID NO: 3) derived from Ascomycota, TmCrtE (SEQ ID NO: 2) derived from Taxus chinensis, BtCrtI (SEQ ID NO: 4) heterologous from P. sphaericus Synthesize lycopene and adjust the copy number.

The construction method comprises the steps of: digesting the plasmid pZY151, pZY184, pZY196 with NotI, recovering the target fragment, and continuously integrating into the above strain by yeast transformation of lithium acetate, and screening with the plate containing the corresponding auxotrophy to obtain the target strain, and verifying by PCR.

The POS5 gene was expressed in the balance of the reducing power in the yeast; the plasmid pTM206 was digested with NotI, and the target fragment was recovered, transformed into the above strain, and the target strain was screened by the corresponding resistant plate, and verified by PCR.

Expression of ADH2, ACS6 (SEQ ID NO: 5), the ALD6 gene increases the supply of precursor material for the synthesis of lycopene.

The construction method comprises the following steps: digesting the plasmid pTM303, digesting with NotI, recovering the target fragment, transforming and integrating into the above strain, and screening with the corresponding resistant plate to obtain the target strain, and verifying by PCR.

Silencing Ypl062W, the Exg1 gene, regulates the yeast synthesis of the lycopene system as a whole. Construction method: The corresponding knockout cassette fragment was constructed by using the hygromycin resistance gene as a marker in the Ypl062W gene to be inactivated, and the corresponding knockout cassette fragment was constructed by using the G418 resistance gene as the marker of the Exg1 gene, and the fragment was passed through lithium acetate. Yeast transformation was integrated into the above engineered bacteria, screened with hygromycin-resistant plates, and verified by PCR.

When the strain needs to reuse the resistance screening marker, use the following method to discard the marker:

The plasmid to be deleted was transformed into pSH47 plasmid. After the bacteria grew out, multiple colonies were picked in 5 mL YPD medium at 30 ° C, cultured at 220 rpm overnight, centrifuged at 3,000 rpm for 5 min at room temperature, the supernatant was discarded, and 5 mL of glucose-free 1 was used. The galactose-containing YPDG medium was washed twice, and 5 mL of the medium was added to culture at 30 ° C, 220 rpm overnight, and a SC non-auxotrophic solid medium containing a final concentration of 1 g/L 5-fluoro-orotic acid was applied at 30 ° C. to cultivate. After the bacteria grow out, pick up the corresponding YPD solid plate that is coated with the monoclonal marker for verification.

Example 3 Construction of lycopene strains with high yield of lipid synthesis

The transformation strategy used in this embodiment is as follows:

(1) To increase the synthesis of triglyceride, the over-expression of PAH1 (SEQ ID NO: 8), DGA1 (SEQ ID NO: 9) gene can increase the downstream synthesis flux of triglyceride, and the constructed strain is TM6065. The construction method is as follows: :

The plasmid pTM705 was digested with NotI, and the target fragment was recovered. The recombinant plasmid was transformed into the TM606 strain by the lithium acetate method at a dose of 200 ng, and the resistant strain was screened by the plate containing the corresponding resistance, and verified by PCR.

(2) To increase the synthesis flux of triglyceride by overexpressing the ACC1** (SEQ ID NO: 1) gene with two mutation sites, and to construct the strain. TM6066, the construction method is as follows:

The plasmid pTM706 was digested with NotI, and the target fragment was recovered, transformed into TM606 strain by the above method, and screened with a plate containing the corresponding resistance to obtain a resistant strain, which was verified by PCR.

(3) Decreasing the degradation of triglyceride based on the improvement of triglyceride synthesis, by knocking out the TGL3 (SEQ ID NO: 11) gene construct strain TM6068 on the basis of strain TM6065, the construction method is as follows:

The plasmid pTM708 was digested with NotI, and the target fragment was recovered, transformed into TM606 strain, and screened with a plate containing the corresponding resistance to obtain a resistant strain, which was verified by PCR.

(4) To increase the size of intracellular lipid droplets, the FLD1 gene (SEQ ID NO: 10) was knocked out to promote lipid droplet polymerization, and the strain was constructed as TM701. The construction method was as follows:

The plasmid pTM701 was digested with NotI, and the target fragment was recovered, transformed into a TM606 strain, and screened with a plate containing the corresponding resistance to obtain a resistant strain, which was verified by PCR.

(5) To increase intracellular unsaturated fatty acids, by overexpressing the OLE1 (SEQ ID NO: 7) gene, the constructed strain was TM704, and the construction method was as follows:

The plasmid pTM704 was digested with NotI, and the target fragment was recovered, transformed into TM563 strain, and screened with a plate containing the corresponding resistance to obtain a resistant strain, which was verified by PCR.

(6) The method of combining the upstream and downstream synthesis of triglyceride, constructing strain TM60656 or TM60686, respectively, the plasmid pTM705/pTM708, pTM706 was digested with NotI, and the target fragment was recovered and integrated into TM606 strain, and then plated with corresponding resistance. The strains with resistance were screened and verified by PCR.

(7) Combining the upstream and downstream synthesis of intracellular triglycerides with the size of intracellular lipid droplets, the constructed strain was TM70156/TM70186, and the construction method was as follows:

The plasmid pTM705/pTM708 and pTM706 were digested with NotI, and the target fragment was recovered, transformed into TM701, and screened with a plate containing the corresponding resistance to obtain a resistant strain, which was verified by PCR.

(8) To increase the size of intracellular unsaturated fatty acid-binding lipid droplets, the strain was constructed as TM707, and the construction method was as follows:

The plasmid pTM707 was digested with NotI, and the target fragment was recovered, transformed into TM563 strain, and screened with a plate containing the corresponding resistance to obtain a resistant strain, which was verified by PCR.

(9) Combining to improve the upstream and downstream synthesis and intracellular unsaturated fatty acid content of intracellular triglyceride, the strain was constructed as TM70456/TM70486, and the construction method was as follows:

The plasmid pTM705/pTM708 and pTM706 were digested with NotI, and the target fragment was recovered, transformed into TM704 strain, and screened with the corresponding resistant plates to obtain resistant strains, which were verified by PCR.

(10) Combining the method of increasing the upstream and downstream synthesis of intracellular triglyceride, the content of intracellular unsaturated fatty acid, and increasing the size of lipid droplets, the strain was constructed as TM70756/TM70786, and the construction method was as follows:

The plasmid pTM705/pTM708 and pTM706 were digested with NotI, and the target fragment was recovered, transformed into TM707 strain, and screened with the corresponding resistant plate to obtain resistant strain, which was verified by PCR.

Example 4 lycopene engineering bacteria shake flask culture fermentation process

In the present embodiment, the inventors detailed the fermentation culture process of the partially engineered strain obtained in Example 3.

The shake flask fermentation was carried out by two-stage seed culture, and the recombinant strain on the plate was picked into a PA bottle containing 5 mL of YPD medium, and cultured at 30 ° C overnight (generally 14-18 h), the cells grew to logarithmic growth phase ( The OD ₆₀₀ was about 5-8), the first-stage seed liquid was obtained, and the strain was transferred to a 250 mL shake flask containing 50 mL of YPD medium at a 1% inoculum, and the second-stage seed liquid was obtained after shaking for about 14-18 hours. The final concentration of the cells was calculated as OD ₆₀₀ = 0.5 and the secondary seed solution was inoculated into a 500 mL shake flask containing 200 mL of fermentation medium YPD (containing 1% galactose), and shake flask fermentation was carried out at 30 ° C and 220 rpm. After 96 hours, the samples were stored in a -80 ° C refrigerator, and the lycopene production was to be measured.

Example 5 Product Extraction and Detection

In the present embodiment, the inventors extracted the product obtained after the fermentation treatment and detected the yield of lycopene.

The sample was taken out from the refrigerator and thawed. 500 μL of the fermentation broth was taken in a 15 mL centrifuge tube (precooled on ice), and the cells were collected by centrifugation at 5,000 rpm for 2 min at 4 ° C, and the supernatant was removed. Add 4 mL of acetone (HPLC grade), 0.2 g of glass beads, 1% antioxidant, shake for 5 min, ice bath sonication for 5-10 min, 5,000 rpm, centrifuge at 2 ° C for 2 min, transfer the supernatant to a 50 mL centrifuge tube; repeat the above extraction process and The extract was collected until the cells had no obvious yellow color; the collected extract was mixed, taken in 2 mL, centrifuged at 12,000 rpm for 10 min, and the supernatant was taken to a brown sample bottle for HPLC analysis.

Detection method: Lycopene detection was carried out by using quaternary HPLC. The detector was an ultraviolet detector with an absorption wavelength of 474 nm, the column was Agilent Zorbax C18 (150 mm * 4.6 mm * 5 μm), and mobile phase A (acetonitrile: water = 9: 1) and mobile phase B (methanol: isopropanol = 3:2) were analyzed as follows: 0-90% B (0-15 min), 90% B (15-30 min), 90%-0B (30-35 min) ), flow rate 1 mL / min.

The test results are shown in Fig. 1. It can be seen that the lycopene producing bacteria overexpressing the triglyceride has a different degree of improvement than the original lycopene producing strain TM606.

Example 6 Construction of a natamycin producing strain overexpressing triglyceride and fermentation

Natamycin is a odorless, odorless, low-dose and safe food preservative. It is a white to milky white odorless and odorless crystalline powder. It is a mechanism of action. It binds to the ergosterol and other sterol groups of the fungus, and inhibits the biosynthesis of ergosterol, which causes the cell membrane to be distorted, eventually leading to leakage and causing cell death. The surface treatment of the dough with natamycin in the baked food has a significant effect of prolonging the shelf life. Natamycin is slightly soluble in water and is hardly soluble in most organic solvents. The solubility in water at room temperature is 30-100 mg/L. When the pH is lower than 3 or higher than 9, the solubility is improved, but the stability of natamycin is lowered.

In the present example, the inventors constructed a plasmid as shown in Table 2 in Streptomyces natto. In a specific manner, the corresponding fragment derived from Streptomyces was obtained by PCR in Example 1, and the Gibson method (Daniel G) was used. .Gibson, Enzymatic Assembly of Overlapping DNA Fragments, Methods in Enzymology, Volume 498, 2011, Pages 349-361.) ligated to the ermE* promoter in vector pSET152 after ligation in the corresponding promoter, construction of each desired plasmid. The constructed plasmid was electrotransformed into E. coli ET12567/pUZ8002, and the transformed Escherichia coli was cultured at 37 °C in 2 ml of LB overnight, and 200 μl of the bacterial solution was transferred to 5 ml of LB for 37-degree culture. At the same time, heat shock and pre-emergence treatment of the Streptomyces natto spores, suspending the Streptomyces natto spores in 2 ml of TES buffer, 10 minutes in a 50 ° water bath, cooling to room temperature, adding an equal volume of spore pre-germination The medium was placed in a 37-degree shaker for 2.5 hours with the previously prepared Escherichia coli, and the E. coli was washed twice with LB medium, while collecting Streptomyces spores by centrifugation, and Escherichia coli and Streptomyces. The spores were mixed evenly, mixed with E. coli cells in an amount of 10 ⁸ : 10 ¹⁰ , and coated on a plate containing SFM medium, dried and placed in a 30-degree incubator for cultivation, and containing plates after 18 hours. A plate of 1 ml of sterile water-repellent with a final concentration of 8 mg/L abramycin was covered, and a white junction transferer was observed after culture for 3 days in a 30-degree incubator. For the preliminary verification of the mutant strain, the first primers V-apr-F (5'-GCTCATCGGTCAGCTTCTCA 3') and V-apr-R (5'-TCGCATTCTTCGCATCCC 3') were used to verify the presence of the Abra resistance gene, if The mutant strain contains the Abrarian resistance gene and a 726 bp PCR product should be obtained.

The original Streptomyces avermitilis J1002 and the newly constructed natrimycin producing bacteria overexpressing triglyceride were placed on SFM plates (soya cake powder 20g/L, mannitol 20g/L, agar 16g/L, tap water 1L, pH 7. 5) After 7 days of 30-degree culture, pick 4 pieces of a total of 3.2 cm ² of spore-containing agar pieces inoculated in a 250 ml shake flask containing 30 mL of COM medium (10 g/L corn starch, 10 g/L oat flour, 5 g/ L malt extract, 2 g / L yeast extract, 15 g / L agar, pH 7.2), cultured at 30 ° C, 220 rpm for 48 h, 3 mL of seed culture was transferred to 25 mL of NPM medium (50.0 g / L corn starch, 18.0 g /L soy flour, 10.0g / L yeast extract, 1.5g / L CaCO ₃ , pH 7.2) in a shaker at 30 ° C, 220rpm incubation for 120h.

0.5 mL of fermentation broth was centrifuged at 7,000 rpm for 5 minutes, the supernatant was removed, and 1.5 mL of methanolic glacial acetic acid solution (methanol: glacial acetic acid = 95:5, v/v) was added to mix and precipitate. After ultrasonication for 20 min, centrifugation at 7,000 rpm for 5 minutes, 50 μl was removed. The supernatant was added with 950 μl methanolic glacial acetic acid solution (methanol: glacial acetic acid = 95:5, v/v) for HPLC detection. The HPLC detection conditions were: analytical column Agilent ZORBAX SB-C18 (4.6×250 nm), flow rate 0.5 mL min ^{- 1} , UV 303nm detection. Mobile phase is methanol: water: glacial acetic acid = 60:40:5 (V / V / V).

The results are shown in Table 3. The newly constructed strains had different degrees of improvement in natamycin production than the original production strain J1002.

Table 3: Natamycin-related strain information

Example 7 Construction of a spinosyn production strain and fermentation of an overexpressing triglyceride

Spinosad, also known as spinosad, is a macrocyclic lactone-free and highly effective biocide extracted from the fermentation broth of Saccharopolyspora spinosa. Spinosad is a light gray solid crystal with a smell similar to slightly stale soil. The spinosad has low solubility in water and is easily soluble in organic solvents such as methanol, ethanol, nitrile, acetone, dimethyl sulfoxide, dimethylformamide and the like.

In the present example, the inventors passed each related gene in the spinosyn-producing bacterium, and the plasmid constructed in Example 6 was joined and transferred into the spinosad-producing bacterium according to a similar method. The newly constructed strains are shown in Table 3.

The original strain of S. spinosa was purchased from the China Microbial Strain Maintenance Center (CGMCC) and the strain number was CGMCC4.1365. The strain was rejuvenated using a rejuvenating medium (yeast extract 1 g/L, beef extract 1 g/L, Casein Acids Hydrolysate 2 g/L, glucose 10 g/L, agar 15 g/L, pH 7.3). After culturing for 3 days in the rejuvenating liquid medium, 1 mL of the culture was transferred to a 250 mL spring shake flask containing 30 mL of the fermented seed medium. Seed medium: TSB 30 g/L, yeast extract 3 g/L, beef extract 3 g/L, MgSO ₄ ·7H ₂ O 2 g/L, glucose 10 g/L, corn syrup 2.5 g/L, pH 7.0. The fermented seeds were cultured for 50 hours at 30 ° C in a shaker at 220 rpm and then inoculated into the fermentation medium. The fermentation medium was: glucose 40g/L, beef extract 10g/L, MgSO ₄ ·7H ₂ O 2g/L, NaCl 2g/L, soybean meal 2g/L, soluble starch 30g/L, CaCO ₃ 2.4g/ L, yeast extract 0.34 g/L, peptone 6.34 g/L, pH 7.2. The fermentation medium was a 250 mL spring shake flask with a liquid volume of 50 mL and a seed inoculum of 10%. The fermentation conditions were 30 ° C and the number of revolutions was 220 rpm.

The spinosyn (CAS No: 168316-95-8) standard was purchased from Sigma (product number: 33706), which contains both spinosyn A and spinosyn D, with an HPLC purity of 97%. The chromatographic analysis of the HPLC analysis used a mobile phase of 10% phase A (0.2% aqueous ammonium acetate solution) and 90% phase B (methanol); the column was DOINEX C18 column (3 μm, 4.6, 150 mm); flow rate 0.8 mL /min; using a PDA detector, the detection wavelength is 245 nm. The MS detector is LCQ FLEET (Thermo scientific). Summary of HPLC standard curve: Weigh 5mg of spinosyn standard, add 1mL HPLC grade methanol, dissolve and mix to obtain 5g/L mother liquor, serially dilute the standard and test by HPLC. 0.2 mL of the fermentation culture was taken, and a total volume of 0.8 mL of acetonitrile was successively added, and the mixture was vortexed and mixed for 2 minutes, and then placed in a refrigerator at 4 ° C overnight. After centrifugation at 12,000 rpm for 10 min, the supernatant was filtered through a 0.45 μm nylon membrane and placed in a brown HPLC vial for analysis.

The fermentation results are shown in Table 4. J1016-1 has different degrees of increase in spinosad production than CGMCC4.1365.

Table 4: Spinosad-related strain information

Example 8 Construction of Astaxanthin Producing Bacteria Overexpressing Triglycerides

Natural astaxanthin (also known as ketone carotenoid or astaxanthin) is currently the strongest natural antioxidant found in nature. Astaxanthin has superior antioxidant activity and is more than 100 times stronger than vitamin E. It is called “super vitamin E”. It can effectively remove oxygen free radicals in cells and is the only carotenoid that can pass the blood-brain barrier. It has the functions of enhancing immunity, relieving fatigue, enhancing cell regeneration, preventing cancer, treating cardiovascular diseases, reducing accumulation of aging cells, inhibiting obesity, protecting eyes and central nervous system, and resisting ultraviolet rays. In recent years, astaxanthin has been widely used in medicines, health products, cosmetics, food additives, aquaculture and other aspects. With the development of China's national economy, its demand is also increasing.

In the present example, the inventors in the plasmids pMH1, pFZ81 (Fayin Zhu, In vitro reconstitution of mevalonate pathway and targeted engineering of farnesene overproduction in Escherichia coli, Biotechnol. Bioeng. 2014; 111: 1396-1405.), and plasmid pFZ153 (Tian Ma, Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli, Biotechnol. J. 2015; 11(2): 228-237.) based on the construction of the plasmid as shown in Table 5, and E. coli MG1655 competent cells were transformed according to the plasmid combination shown in Table 6, and an Escherichia coli astaxanthin producing strain was constructed.

Specific examples of plasmid construction of this example Referring to Example 1, the corresponding gene was amplified by PCR using the Gibson method (Daniel G. Gibson, Enzymatic Assembly of Overlapping DNA Fragments, Methods in Enzymology, Volume 498, 2011, Pages 349-361. After the corresponding promoters are ligated in sequence, the respective required plasmids are constructed.

Table 5 Construction of Escherichia coli Astaxanthin Synthetic Plasmid

Table 6 Construction of Escherichia coli Astaxanthin Synthetic Strain

Strain	description	Yield (mg/L)
TM8011	pMH1, pFZ81, pFZ153	72
TM8012	pTM801, pFZ81, pFZ153	85
TM8013	pMH1, pTM802, pFZ153	90
TM8014	pTM801, pTM802, pFZ153	98
TM8015	pTM801, pTM803, pFZ153	110

After the strain is constructed, the shake flask fermentation is carried out as follows:

After transformation, the transformants were picked and cultured in LB medium containing 34 μg/mL chloramphenicol, 50 μg/mL kanamycin, and 100 μg/mL ampicillin at 37 ° C, 220 rpm overnight. 200 mL of an LB medium containing 34 μg/mL of chloramphenicol, 50 μg/mL of kanamycin, and 100 μg/mL of ampicillin was incubated at 30 ° C and 200 rpm at a rate of 1%. When the OD600 reached 0.7-0.9, the final concentration was 0.1 mM IPTG (isopropyl-β-D-thiogalactoside). After 15 hours of culture, 2 mL was sampled, centrifuged at 12,000 rpm for 3 min, the supernatant was removed, and 1 mL of extractant (V) was added. Acetone: V methanol = 4:1), after shaking the cells, the mixture was sonicated for 10 min, centrifuged at 13,000 rpm, 4 ° C for 10 min, and the supernatant was taken for high performance liquid chromatography (HPLC) detection according to the following method, and the operation was protected from light.

Quaternary HPLC (Thermo Fisher Ultimate 3000), column Agilent Zorbax C18 (4.6-mm x 150-mm x 5-μm). The chromatographic conditions were as follows: mobile phase A (acetonitrile: water = 9:1, V/V) and mobile phase B (methanol: isopropanol = 3:2, V/V). 0 min: 0% B, 15 min: 90% B, 30 min: 90% B, 35 min: 0% B. The flow rate was 1 mL/min. Column temperature: 25 ° C. Detector: UV detector with a detection wavelength of 474 nm.

The results are shown in Table 6. The engineered bacteria in the engineered strain had higher astaxanthin yield.

Example 9 Controlling Triglyceride Overexpression in Microorganisms

On the basis of Example 3, we replaced the promoter of the PAH1, DGA, ACC1, OLE1 gene with pHXT1 (SEQ ID NO: 28), which is characterized by a gene overexpressed under conditions of glucose. It can be expressed that in the absence of glucose, the relevant genes are not expressed. Therefore, through our fermentation control, the lycopene producing strain can accumulate triglycerides in the early stage of fermentation, and after the glucose is consumed, the triglycerides are no longer accumulated. In order to control the lipid content in the microorganisms, the microorganisms can use more substrate and energy to synthesize lycopene in the later stage of fermentation, thereby further increasing the production of lycopene. The specific construction method was carried out by referring to Example 1-2 and replacing the promoter. The strain details are shown in Table 7, and the fermentation and detection methods of Examples 4-5 were carried out. The results showed that the yield of lycopene was further improved by controlling the expression of triglyceride.

Table 7: Transformation information and yield of lycopene engineering strain

In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

While the embodiments of the present invention have been shown and described, the embodiments of the invention may The scope of the invention is defined by the claims and their equivalents.

Claims (11)

1. A microorganism characterized by comprising:

   Overexpression includes at least one selected from the group consisting of:

   PAH1, DGA1, OLE1, ACC1**, ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB;

   Silence includes at least one selected from the group consisting of FLD1 and TGL3.

   Wherein the microorganism is a microorganism having the potential to synthesize a hydrophobic compound.
2. The microorganism according to claim 1, wherein the PAH1, DGA1, OLE1, ACC1** are derived from Saccharomyces cerevisiae,

   Optionally, the ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D are derived from Streptomyces,

   Optionally, the EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB are derived from Escherichia coli,

   Optionally, the ACC1** encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:1,

   Optionally, the fabA encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:6,

   Optionally, the OLE1 encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:7,

   Optionally, the PAH1 encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:8,

   Optionally, the DGA1 encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:9,

   Optionally, the FLD1 encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 10,

   Optionally, the TGL3 encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:11,

   Optionally, the ACCA2 encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 12,

   Optionally, the ACCB encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 13,

   Optionally, the ACCE encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 14,

   Optionally, the DGAT encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 15,

   Optionally, the LPPβ encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:16,

   Optionally, the OLE1A encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:17,

   Optionally, the OLE1B encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:18,

   Optionally, the OLE1C encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:19,

   Optionally, the OLE1D encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:20,

   Optionally, the EcACCA encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 21,

   Optionally, the EcACCB encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:22,

   Optionally, the EcACCC encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:23,

   Optionally, the EcACCD encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 24,

   Optionally, the pgpB encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:25,

   Optionally, the atfA encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:26,

   Optionally, the fabB encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:27.
3. The microorganism according to claim 1, wherein the microorganism comprises a yeast selected from the group consisting of yeast, Escherichia coli, actinomycetes, Bacillus subtilis, Corynebacterium glutamicum, Aspergillus niger, Aspergillus oryzae, Trichoderma viride, and At least one of Trichoderma.
4. The microorganism according to claim 1, wherein the hydrophobic compound comprises a compound selected from the group consisting of lycopene, carotene, astaxanthin, natamycin, spinosyn, polyketone, sesquiterpene, triterpene And at least one of the tetraterpenoids.
5. A method for increasing fermentation yield of a hydrophobic compound of a microorganism, comprising: increasing a lipid content in a microorganism, wherein the microorganism is a microorganism having the potential to synthesize a hydrophobic compound.
6. The method of claim 5 wherein the lipid is a triglyceride.
7. The method according to claim 5, wherein said increasing the lipid content in the microorganism is achieved by increasing at least one of a synthesis amount of the triglyceride, a size of the lipid droplets, and a content of the unsaturated fatty acid.
8. The method according to claim 5, wherein said increasing the lipid content in the microorganism is by overexpression in said microorganism comprising at least one selected from the group consisting of: PAH1, DGA1, OLE1, ACC1**, ACCA2, ACCB, ACCE, DGAT, LPPβ, OLE1A, OLE1B, OLE1C, OLE1D, EcACCA, EcACCB, EcACCC, EcACCD, pgpB, atfA, fabA, fabB; and silencing comprising at least one selected from the group consisting of FLD1, TGL3,

   Wherein the ACC1** encodes a polypeptide having the amino acid sequence of SEQ ID NO: 1,

   The fabA encodes a polypeptide having the amino acid sequence of SEQ ID NO: 6,

   The OLE1 encodes a polypeptide having the amino acid sequence of SEQ ID NO: 7,

   The PAH1 encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:8,

   The DGA1 encodes a polypeptide having the amino acid sequence of SEQ ID NO: 9,

   The FLD1 encodes a polypeptide having the amino acid sequence of SEQ ID NO: 10,

   The TGL3 encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:11,

   The ACCA2 encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 12,

   The ACCB encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 13,

   The ACCE encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 14,

   The DGAT encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 15,

   The LPP encodes a polypeptide having the amino acid sequence of SEQ ID NO: 16,

   The OLE1A encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:17,

   The OLE1C encodes a polypeptide having the amino acid sequence of SEQ ID NO: 19,

   The OLE1D encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:20,

   The EcACCA encodes a polypeptide having the amino acid sequence of SEQ ID NO: 21,

   The EcACCB encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:22,

   The EcACCC encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:23,

   The EcACCD encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:24,

   The pgpB encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:25,

   The atfA encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:26,

   The fabB encodes a polypeptide having the amino acid sequence of SEQ ID NO:27.
9. The method according to claim 8, wherein said overexpression is achieved by introducing a construct into said microorganism, said construct comprising a gene to be overexpressed and a regulated expression promoter, said regulation An expression promoter is operably linked to the gene of interest,

   Preferably, the inducible promoter is pHXT1,

   Optionally, the PHXT1 has the nucleotide sequence set forth in SEQ ID NO:28.
10. The method according to claim 5, wherein the microorganism comprises a yeast selected from the group consisting of yeast, Escherichia coli, actinomycetes, Bacillus subtilis, Corynebacterium glutamicum, Aspergillus niger, Aspergillus oryzae, Trichoderma viride, and At least one of Trichoderma, etc.

    Optionally, the hydrophobic compound comprises at least one selected from the group consisting of lycopene, carotenes, astaxanthin, natamycin, spinosyn, polyketones, sesquiterpenes, triterpenoids, tetraterpenoids .
11. Use of the microorganism according to claims 1 to 4 for increasing the fermentation yield of a hydrophobic compound.

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