CN117551565A

CN117551565A - Engineering strain for high-yield medium-short chain lactone type sophorolipid and construction method and application thereof

Info

Publication number: CN117551565A
Application number: CN202311580035.2A
Authority: CN
Inventors: 张迷敏; 刘杰; 叶灿彬; 张利萍; 陈伟哲; 陈玉娥
Original assignee: Guangzhou Liby Enterprise Group Co Ltd
Current assignee: Guangzhou Liby Enterprise Group Co Ltd
Priority date: 2023-11-23
Filing date: 2023-11-23
Publication date: 2024-02-13

Abstract

The invention provides an engineering strain for high-yield medium-short chain lactone type sophorolipid, a construction method and application thereof, and relates to the technical field of genetic engineering. The original strain of the engineering strain is the bumblebee candida (Starmerella bombicola), and the FADH gene locus of the bumblebee candida is inserted with the lactonase SBLE gene and the sophorolipid transferase MDR gene so that the FADH gene is knocked out. The engineering strain can ferment and produce medium-short chain lactone type sophorolipid with high yield, and the medium-short chain lactone type sophorolipid (C6-C12) has the biological function activities of antibacterial, antitumor and the like of the medium-short chain sophorolipid and has the surface activities of better solubility, emulsifying and the like of the medium-short chain sophorolipid, so that the engineering strain has wide application prospect and economic value. The engineering strain and the application of the engineering strain constructed by the invention provide an effective way for the short-chain lactone type sophorolipid in large-scale production.

Description

Engineering strain for high-yield medium-short chain lactone type sophorolipid and construction method and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to an engineering strain for high-yield medium-short chain lactone type sophorolipid, a construction method and application thereof.

Background

The sophorolipid synthesized by microorganism is a mixture composed of a series of sophorolipid molecules, sophorose formed by connecting beta-1, 2 glycosidic bond is used as hydrophilic group, saturated/unsaturated long-chain omega or omega-1 hydroxy fatty acid is used as lipophilic group, and the two groups are connected through glycosidic bond to form the biosurfactant with hydrophilic group and hydrophobic group and surfactant. The sophorolipid has the advantages of reducing the surface tension, foaming and emulsifying, keeping good surface activity in high-temperature high-salt concentration solution, and being capable of being utilized and degraded by microorganisms. Therefore, sophorolipids have great potential value as biosurfactants in the industries of daily chemical washing, home care, beauty and skin care and the like, and have been receiving more and more attention for several years.

However, currently, sophorolipids have many problems in the fields of industrial production and application, mainly including: 1. the biosynthesis yield of sophorolipids is also low, and as a detergent or cosmetic material, the cost and price are high compared to the relatively inexpensive chemically synthesized surfactants currently used; 2. the chemical structure of the components of the sophorolipid product obtained by fermentation at present is mainly sophorolipid molecules which take C18 and C16 as hydrophobic groups, and the surface activity and the biological activity of the sophorolipid molecules still have certain limitations.

Sophorolipids of different molecular structures have different performances in terms of surface activity properties and biological activities, while sophorolipids of medium short chain lactones are well represented in terms of both surface activities such as foamability, emulsifying property and solubility, and biological activities such as inhibiting cancer cell growth, antiviral and bacteriostatic properties. However, the research on the high-efficiency biosynthesis technology and application of the short-chain lactone type sophorolipid is very few at present, and the development of the application range and economic value of the short-chain lactone type sophorolipid is severely limited. Therefore, development of engineering bacteria of sophorolipids with high-yield medium-short-chain lactone type chemical structures is urgently needed so as to research the relationship between the special structures and functions and improve the application value of the sophorolipids.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide an engineering strain for high-yield medium-short chain lactone type sophorolipids, so as to relieve the problem of low yield of medium-short chain lactone type sophorolipids. The invention also aims at providing a construction method and application of the engineering strain for high-yield medium-short chain lactone type sophorolipids.

In order to solve the technical problems, the invention adopts the following technical scheme:

in a first aspect, an engineering strain for high-yield medium-short chain lactone type sophorolipid is provided, an original strain of the engineering strain is a bumblebee candida (Starmerella bombicola), and a formaldehyde dehydrogenase FADH gene locus of the bumblebee candida is inserted into a lactonase SBLE gene and a sophorolipid transporter MDR gene, so that the FADH gene is knocked out.

In a second aspect, there is also provided a method for constructing an engineered strain of high-yield medium-short-chain lactone-type sophorolipids of the first aspect, comprising: the vector containing the insert comprising the SBLE gene and the MDR gene was constructed and then introduced into the starting strain.

In a third aspect, there is also provided a method for producing sophorolipid by fermentation, comprising producing sophorolipid using the engineered strain fermentation substrate of the first aspect.

In a fourth aspect, there is also provided the use of the engineered strain of high-yielding medium-short chain lactone-type sophorolipids of the first aspect, or the construction method of the second aspect, or the fermentation production method of sophorolipids of the third aspect, in the preparation of a surfactant.

Compared with the prior art, the invention has the following beneficial effects:

the chemical structure of sophorolipids in nature is mainly composed of a fatty acid chain of 16-18 carbons in length and one sophorose molecule. The utilization rate of the microorganism to the medium and short chain fatty acid is very low, so the amount of the medium and short chain sophorolipids with 6-12 carbon synthetic hydrophobic groups is very small. The lactone type sophorolipid has the general characteristics of a surfactant and special biological activities such as anti-tumor, antibacterial and anti-inflammatory, but has low solubility, so that the application of the lactone type sophorolipid in industries such as washing, food, medicine and the like is limited. For sophorolipid molecules, the hydrophilicity can be improved by reducing the length of the carbon chain of the hydrophobic group. Therefore, the high-yield medium-chain lactone type sophorolipid engineering bacteria constructed by the invention and the application thereof in fermenting and synthesizing sophorolipid can improve the yield of the medium-chain lactone type sophorolipid by microorganism fermentation and synthesis, and solve the problems of low solubility, limited application and the like of the existing lactone type sophorolipid.

The engineering strain of the high-yield medium-short chain lactone type sophorolipid uses the candida bumblebee as an original strain, and inserts an SBLE gene with a strong promoter for regulating esterification reaction and an MDR gene for regulating sophorolipid transport into a FADH gene locus in a fatty acid beta-oxidation metabolic regulation path, so that the FADH gene knockout and the over-expression of the SBLE and the MDR genes are realized. When ethyl laurate (8%), n-heptanoic acid (8%) and n-hexanoic acid (8%) are used as hydrophobic substrates, the amount of sophorolipid synthesized by the engineering strain is obviously higher than that of a wild strain, and under the condition of fermentation culture of 250ml of culture medium, the total sophorolipid synthesized by the engineering strain is respectively as follows: 8.42g, 24.89g, 21.00g, wild strains were: 5.62g, 15.51g, 13.66g; the total sophorolipid yield of the engineering strain is respectively improved by 49.8%, 60.5% and 53.7% compared with the wild strain, the lactone type sophorolipid accounts for 45-60% of the engineering strain, and the wild strain accounts for 20-30%. Because the medium-short chain sophorolipid has a short chain compared with long chain sophorolipid hydrophobic groups, the solubility and the emulsifying performance are relatively enhanced, and the internal fat type sophorolipid has better anti-tumor, antibacterial and antiviral functional activities compared with acid type sophorolipid, the internal fat type sophorolipid can be widely applied to industries such as cleaning, cosmetics, foods, medicines and the like, so that the engineering strain becomes an dominant strain for producing sophorolipid, particularly medium-short chain lactone type sophorolipid.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of the construction of ΔFADH of example 1 ^- SBLE ⁺ MDR ⁺ Schematic diagram of engineering strain;

FIG. 2 shows the effect of using different promoters on the transcription level of the overexpressed gene in effect example 1;

FIG. 3 is a graph showing the results of fermentation synthesis of total sophorolipids using ethyl laurate, n-heptanoic acid and n-hexanoic acid as hydrophobic substrates for the engineering strain and the wild strain of example 1, respectively;

FIG. 4 is a graph showing the results of fermentation synthesis of lactone type sophorolipids using ethyl laurate, n-heptanoic acid and n-hexanoic acid as hydrophobic substrates for the engineering strain and the wild strain of example 1, respectively;

FIG. 5 is an engineering strain and ΔFADH of example 1 ^- Strain, ΔFADH ^- SBLE ⁺ Strain, ΔFADH ^- MDR ⁺ When the strains respectively take ethyl laurate as a hydrophobic substrate, the yield of the total sophorolipids synthesized by fermentation is compared;

FIG. 6 is an engineering strain and ΔFADH of example 1 ^- Strain, ΔFADH ^- SBLE ⁺ Strain, ΔFADH ^- MDR ⁺ When the strains respectively take ethyl laurate as a hydrophobic substrate, the yield of the fermented synthetic lactone type sophorolipid is compared;

FIG. 7 shows the UPLC-MS results of fermentation synthesis of sophorolipids using ethyl laurate as a hydrophobic substrate for the engineering strain and the wild strain of example 1.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be understood that the terms "comprises" and "comprising," when used in this specification and the appended claims, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

It is also to be understood that the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

It should be further understood that the term "and/or" as used in the present specification and the appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes such combinations.

Furthermore, the terms "substantially," "essentially," and the like, are intended to be limited to the precise form disclosed herein and are not necessarily intended to be limiting. For example: the term "substantially equal" does not merely mean absolute equal, but is difficult to achieve absolute equal during actual production and operation, and generally has a certain deviation. Thus, in addition to absolute equality, "approximately equal to" includes the above-described case where there is a certain deviation. In other cases, the terms "substantially", "essentially" and the like are used in a similar manner to those described above unless otherwise indicated.

In a first aspect, an engineering strain of high-yield medium-short chain lactone type sophorolipid is provided, the engineering strain takes candida bumblebee (Starmerella bombicola) as an original strain, the engineering strain inserts a lactonase gene (SBLE) for regulating esterification reaction and a sophorolipid transporter gene (MDR) for regulating sophorolipid transport at a formaldehyde dehydrogenase gene (FADH) site in a fatty acid beta-oxidative metabolic pathway regulated by candida bumblebee, so that the knockout of the FADH gene and the overexpression of the SBLE gene and the MDR gene are realized, and the genotype is delta FADH ^- SBLE ⁺ MDR ⁺ . When fatty acid/fatty acid ester with the carbon chain length of 6-12 is used as a hydrophobic substrate, compared with a wild strain, the total sophorolipid yield of the engineering strain is improved by more than 40%, wherein the proportion of lactone type sophorolipid accounts for more than 40% of the total sophorolipid content.

In an alternative embodiment, the starting strain candida bumblebee (s.bobnicola) is candida bumblebee ATCC22214 strain.

In an alternative embodiment, the nucleotide sequence of the FADH gene is shown in SEQ ID NO. 1.

In alternative embodiments, the SBLE gene in the engineered strain is regulated by a strong promoter.

In an alternative embodiment, the strong promoter that modulates the SBLE gene is selected from TubP or AcP.

In an alternative embodiment, the SBLE gene is regulated by a strong promoter TubP and a terminator TubT.

In an alternative embodiment, the nucleotide sequence of the SBLE gene is shown in SEQ ID NO. 2.

In alternative embodiments, the MDR gene in the engineered strain is regulated by a strong promoter.

In alternative embodiments, the strong promoter that modulates the MDR gene is selected from TubP or AcP.

In alternative embodiments, the MDR gene is regulated by a strong promoter, acP, and a terminator, acT.

In an alternative embodiment, the nucleotide sequence of the MDR gene is shown in SEQ ID NO. 3.

In an alternative embodiment, the nucleotide sequence of the strong promoter AcP is shown in SEQ No. 4.

In an alternative embodiment, the nucleotide sequence of the strong promoter TubP is shown in SEQ NO. 5.

In an alternative embodiment, the nucleotide sequence of terminator AcT is shown in SEQ NO. 6.

In an alternative embodiment, the nucleotide sequence of terminator TubT is shown in SEQ NO. 7.

In alternative embodiments, the FADH gene locus insert further comprises a selectable marker.

In alternative embodiments, the selectable marker comprises a resistance gene, and exemplary resistance genes include a hygromycin resistance gene.

In alternative embodiments, the resistance gene is expressed under the control of a strong promoter and terminator from candida.

In an alternative embodiment, the FADH gene locus insert comprises, in order from the 5 'end to the 3' end, an SBLE gene, a selectable marker gene, and an MDR gene.

In an alternative embodiment, the FADH gene locus insert comprises, in order from the 5 'end to the 3' end, an SBLE gene, a hygromycin resistance gene and an MDR gene, the SBLE gene being regulated by a strong promoter TubP and a terminator TubT, the hygromycin resistance gene being regulated by a candida strong promoter and a terminator, the MDR gene being regulated by a strong promoter AcP and a terminator AcT.

In an alternative embodiment, the fragments with homology arms, including SBLE gene fragment, MDR gene fragment, resistance gene fragment, promoter fragment and terminator fragment, are obtained separately and then assembled into inserts by fusion PCR.

In an alternative embodiment, the insert has an upstream homology arm and a downstream homology arm homologous to the FADH gene, and the SBLE gene and the MDR gene are inserted into the FADH gene site by homologous recombination.

In an alternative embodiment, the vector containing the insert is introduced into the S.bumpy competent cells using electrotransformation.

In an alternative embodiment, the vector containing the insert for introducing into the competent cells of candida bumblebee growth is linear DNA.

In some embodiments, constructing a vector containing an insert comprises the steps of:

a1, obtaining SBLE gene fragment, MDR gene fragment, promoter fragment, terminator fragment, and hygromycin resistance gene fragment (P-Hph-T) with candida strong promoter and terminator, wherein the preferred promoters are AcP and TubP, and the terminators are AcT and TubT.

A2, PCR was performed using the fragments of A1 as templates, and TubP and AcP fragments (fTubP and fAcP) having homology arms, tubT and AcT fragments (fTubT and fAcT) having homology arms, SBLE fragment (fSBLE) having homology arms, MDR fragment (fMDR) having homology arms, and P-Hph-T fragment (fP-Hph-T) having homology arms were amplified, respectively.

A3, fusing the fTubP fragment, the fSBLE fragment and the fTubT fragment amplified in the A2 through fusion PCR to obtain a TubP-SBLE-TubT fragment, and fusing the fAcP fragment, the fMDR fragment and the fAcT fragment to obtain an AcP-MDR-AcT fragment.

And A4, fusing the fP-Hph-T gene fragment with the homology arm in A2, the TubP-SBLE-TubT fragment in A3 and the AcP-MDR-AcT fragment by fusion PCR to obtain pSBLET-pHpht-pMDRt fragment, and further amplifying the obtained pSBLET-pHpht-pMDRt by PCR reaction.

A5, obtaining FADHP and FADHT of the upstream and downstream fragments of the FADH gene; and (3) PCR amplifying the fFADHP and the fFADHT with homology arms by using the FADHP and the FADHT as templates respectively.

A6, amplifying the pSBLET-pHpht-pMDRt fragment in A4, the fFADHP fragment and the fFADHT fragment in A5 by Two-step fusion PCR to obtain the FADH-pSBLET-pHpht-pMDRt-FADH linear target DNA vector.

And A7, carrying out single-stranded treatment on the FADH-pSBLet-pHpht-pMDRt-FADH linear DNA vector in the A6 to obtain a linear single-stranded DNA vector for electric transformation.

In some specific embodiments, the bumblebee candida utilis competent cells and the electrotransformation are prepared as follows:

preparing competent cells of wild bumblebee candida, electrically transforming FADH-pSBLet-pHpht-pMDRt-FADH linear DNA vectors into competent cells, coating the transformation solution on a YPD plate containing 500ppm hygromycin, selecting strains which normally grow on the YPD plate containing hygromycin, carrying out YPD liquid culture, and extracting genome DNA; the extracted genome DNA is used as a template, taKaRa Premix TaqTM enzyme and FADHPF, FADHTR are used as primers to carry out PCR reaction, and the amplified DNA fragment is detected by agarose gel electrophoresis.

In an alternative embodiment, a C6-C12 fatty acid or a C6-C12 fatty acid ester is used as the hydrophobic substrate. Exemplary substrates include, but are not limited to, one or more of lauric acid, ethyl laurate, n-heptanoic acid, and n-hexanoic acid.

In alternative embodiments, the substrate is present in the fermentation medium in an amount of 6 to 10% w/v, such as, but not limited to, 6, 7, 8, 9 or 10% w/v.

In an alternative embodiment, the fermentation medium further comprises yeast powder, glucose, KH ₂ PO ₄ 、Na ₂ HPO ₄ ·12H ₂ O and MgSO ₄ ·7H ₂ O。

In an alternative embodiment, the fermentation medium contains 6-10% w/v of fermentation substrate, 0.3% w/v of yeast powder, 0.3% w/v of glucose, KH ₂ PO ₄ 0.1％w/v、Na ₂ HPO ₄ ·12H ₂ O0.1% w/v and MgSO ₄ ·7H ₂ O 0.05％w/v。

In an alternative embodiment, the fermentation medium contains ethyl laurate or lauric acid 6-10% w/v, yeast powder 0.3% w/v, glucose% w/v, KH ₂ PO ₄ 0.1％w/v、Na ₂ HPO ₄ ·12H ₂ O0.1% w/v and MgSO ₄ ·7H ₂ O 0.05％w/v。

In an alternative embodiment, the fermentation medium contains 6 to 10% w/v of n-heptanoic acid or n-hexanoic acid, 0.3% w/v of yeast powder, 0.3% w/v of glucose, KH ₂ PO ₄ 0.1％w/v、Na ₂ HPO ₄ ·12H ₂ O0.1% w/v and MgSO ₄ ·7H ₂ O 0.05％w/v。

In an alternative embodiment, the sophorolipid fermentation production method further comprises expanding the seed solution of the engineering strain, and then inoculating the expanded bacterial solution (od600=1.0) to the fermentation medium at 5%v/v.

In an alternative embodiment, the culturing conditions for fermentation include culturing at 30℃for 5-10 days at 200-250 rpm.

In some specific embodiments, the steps of fermentatively producing sophorolipids are as follows:

b1, inoculating the engineering strain into a test tube filled with 5mL YPD seed culture medium for culture.

B2, inoculating the seed solution into a shake flask containing 50ml of YPD medium according to an inoculum size of 2% v/v, and performing primary shake flask fermentation.

B3, after OD600 = 1.0, is inoculated in an inoculum size of 5% v/v into a 500ml shake flask containing 250ml of fermentation medium for 5-10 days, preferably 5 days.

The YPD medium comprises the following components: 1% w/v yeast extract, 2% w/v peptone and 2% w/v glucose.

The invention is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the invention in any way.

Example 1

Example 1 provides an engineering strain (hereinafter also referred to as engineering strain or engineering strain) for high-yield medium-short chain lactone type sophorolipid, wherein the engineering strain is prepared by taking a bumblebee candida ATCC22214 strain as an original strain and adopting the following method:

the construction of the bumblebee candida FADH-pSBLet-pHpht-pMDRt-FADH linear DNA vector is shown in figure 1.

A1, extracting genome of the candida bumblebee, and taking genome DNA as a template, carrying out PCR amplification on cDNA sequence of SBLE gene, cDNA sequence of MDR gene, promoter sequence and terminator sequence which are used for amplification, and cutting gelatin to refine each gene segment to synthesize hygromycin resistance gene sequence (P-Hph-T) with strong promoter and terminator of candida bumblebee; preferred promoters are TubP and AcP, and terminators are TubT and AcT. The primer sequences used are shown below:

TABLE 1

A2, PCR was performed using the fragment purified in A1 as a template to amplify TubP (fTubP) and AcP (fAcP) sequences having homology arms, tubT (fTubT) and AcT (fAcT) sequences having homology arms, SBLE (fSBLE) sequences having homology arms, MDR (fMDR) sequences having homology arms, and P-Hph-T (fP-Hph-T) sequences having homology arms, respectively. The primer sequences used are shown below:

TABLE 2

A3, fusing the fTubP fragment, the fSBLE fragment and the fTubT fragment amplified in the A2 through fusion PCR to obtain a TubP-SBLE-TubT fragment, fusing the fAcP fragment, the fMDR fragment and the fAcT fragment to obtain an AcP-MDR-AcT fragment, and cutting and purifying the fused fragment.

And A4, fusing the fP-Hph-T gene fragment with the homology arm in A2, the TubP-SBLE-TubT fragment in A3 and the AcP-MDR-AcT fragment by fusion PCR to obtain pSBLET-pHpht-pMDRt fragment, and further amplifying the obtained pSBLET-pHpht-pMDRt by PCR reaction, and cutting and refining.

A5, PCR amplifying the FADH-P and FADH-T of the up and downstream sequences of the FADH gene by taking the genome DNA of the bumblebee candida as a template; and (3) respectively using FADH-P and FADH-T as templates, amplifying fFADH-P and fFADH-T with homology arms by PCR, and cutting and refining. The primer sequences used are shown below:

TABLE 3 Table 3

A6, amplifying the pSBLET-pHpht-pMDRt fragment in A4, the fFADH-P and the fFADH-T fragment in A5 by Two-step fusion PCR to obtain the FADH-pSBLET-pHpht-pMDRt-FADH linear target DNA vector.

A7, carrying out single-stranded treatment on the FADH-pSBLet-pHpht-pMDRt-FADH linear DNA vector in the A6 to obtain a linear single-stranded DNA vector (the nucleotide sequence is shown as SEQ ID NO. 8) for electric transformation.

And (II) preparing and electroconverting the bumblebee candida growing competent cells.

A single colony of Candida buminosa (S.bobnicola) was inoculated into a 250ml shake flask containing 25ml of YPD medium and cultured at 30℃and 300rpm for 18 hours.

The culture broth from the previous step was inoculated in an amount of 2% v/v into 250ml shake flasks containing 50ml YPD medium and the cells were cultured at 30℃and 120rpm until the OD600 value was between 1 and 2.

Loading the bacterial liquid into a 50ml centrifuge tube, centrifuging at 3000g and 4 ℃ for 5 minutes to collect bacterial bodies, then re-suspending and precipitating with 50ml of sterile water cooled on ice, centrifuging at 3000g and 4 ℃ for 5 minutes, and discarding the supernatant; the pellet was resuspended in 50ml of ice-cooled sterile water and centrifuged.

The cell pellet was resuspended in 4ml ice-cold 1M sterile sorbitol solution, centrifuged at 3000g at 4℃for 5min and the supernatant discarded.

The precipitate was suspended with 4ml of an freshly prepared 0.1M lithium acetate solution (3500. Mu.l of water, 400. Mu.l of 1M lithium acetate, 200. Mu.l of 1M DTT), and after 15 minutes at room temperature, centrifuged at 3000g for 5 minutes at 4℃and the supernatant was discarded.

The cells were suspended with 1M sorbitol solution, placed on ice and used as soon as possible.

50. Mu.l of the yeast suspension was pipetted into a centrifuge tube, 2.5mg of the DNA solution for transformation (i.e., single-stranded DNA of the nucleotide sequence shown in SEQ ID NO: 4) was added to the yeast suspension and mixed well, and the mixture was placed on ice for pre-cooling for 5 minutes.

The above mixture was transferred to an electric rotating cup with a gap of 0.2cm and left on ice for 5 minutes. Subsequently, a pulse of 5ms and 2.5kV was applied to the mixture using a Micro Pulser (Bio-Rad).

The electric beaker was removed, ice-cooled 1M sorbitol was immediately added, gently mixed and transferred to a 1.5ml centrifuge tube, and the mixture was allowed to stand at 30℃for 1 hour. 200. Mu.l of the mixture solution was spread on a selective medium and incubated at 30℃for about 1 week. For the selection medium, agar medium containing 1% w/v yeast extract, 2% w/v peptone, 2% w/v glucose and 500ppm hygromycin was used.

The single colony which has grown is subjected to liquid amplification culture, and then genomic DNA is extracted. And (3) taking the genome DNA as a template, carrying out PCR amplification by using the primer in the step (A6), and detecting the amplified DNA fragment by agarose gel electrophoresis, wherein the length and the size are consistent with those of the constructed vector, thus proving that the target engineering strain is obtained.

Example 2

Example 2 provides a recombinant bumblebee candida, which is different from example 1 only in that a FADH-noble-pHpht-mdr-FADH linear DNA vector is constructed by adopting a native promoter and a terminator, and the vector construction method is the same as that of example 1, and is different from that of example 1 in that: unlike the TubP and AcP promoters and TubT and AcT terminators of example 1, in example 2, about 500bp upstream and downstream of the SBLE cDNA was used as the promoter and terminator, respectively, and about 500bp upstream and downstream of the MDR cDNA was used as the promoter and terminator; transferring the constructed vector into bumblebee candida by an electrotransformation method, inoculating the transformation solution into a YPD plate containing hygromycin, and screening positive clones.

Example 3

A fermentation production method of sophorolipid is provided, which takes ethyl laurate, n-heptanoic acid or n-caproic acid as a hydrophobic substrate and uses the engineering strain of the example 1 for fermentation production.

The single colony of the engineering strain obtained in example 1 was picked up and inoculated into a test tube containing 5mL of YPD seed medium, the rotation speed was 200250rpm, the culture was 2436 hours, the seed solution was inoculated into a shake flask containing 50mL of YPD medium according to the inoculum size of 2% v/v, primary shake flask fermentation was performed, shaking culture was performed at 200-300rpm for 24-48 hours at 25-30℃until OD600 = 1.0, and then inoculated into a 500mL shake flask containing 200mL of fermentation medium according to the inoculum size of 5% v/v, and the culture was performed at 200250rpm for 5-10 days at 25-30 ℃.

The culture conditions of the seed liquid are as follows: culturing at 30deg.C and 200-250rpm for 24-30 hr.

The primary shaking flask fermentation conditions are as follows: seed solution was inoculated into 50ml of culture medium at 2-4% v/v under the following conditions: culturing at 30 deg.C and 200-300rpm for 36-48 hr.

The conditions of the shake flask fermentation are as follows: the primary shake flask broth was inoculated into 250ml of culture medium at 5% v/v and incubated at 30℃for 120 hours at 200-250 rpm.

The fermentation medium comprises the following components: 0.3% w/v yeast powder, 8% w/v ethyl laurate or n-heptanoic acid or n-hexanoic acid, 10% w/v glucose, 0.1% KH ₂ PO ₄ 、0.1％ Na ₂ HPO ₄ ·12H ₂ O、0.05％ MgSO ₄ ·7H ₂ O。

Comparative example 1

ΔFADH ^- The strain (bumblebee candida strain only knocking out FADH gene) is constructed by the following steps:

a1, taking FADHP and FADHT in the embodiment 1 as templates, and amplifying f2FADHP and f2FADHT with homology arms by PCR; using hygromycin resistance gene sequence (P-Hph-T) as template, PCR amplifying f2P-Hph-T with homology arm, cutting gel and refining, the primer sequence is as follows:

TABLE 4 Table 4

A2, fusing and amplifying the f2FADHP, the f2P-Hph-T and the f2FADHT fragments by PCR to obtain the FADH ^- Gene knockout box, cutting glue and refining. The method for electrotransformation and engineering bacteria verification is the same as in example 1, except that the DNA solution for transformation is FADH ^- Gene knockout cassette sequence solution.

Comparative example 2

ΔFADH ^- SBLE ⁺ The strain (SBLE gene is inserted into FADH gene to knock out FADH gene) is constructed as follows:

b1, using FADHP and FADHT in example 1 as a model for PCR amplification of f3FADHP and f3FADHT with homology arms; PCR amplification of f2SBLE with homology arms using TubP-SBLE-TubT as template in example 1; PCR amplification of f3P-Hph-T with homology arm by using hygromycin resistance gene sequence (P-Hph-T) as template, and gel cutting and refining, the primer sequences are as follows:

TABLE 5

B2, fusion and amplification of f3FADHP, f2SBLE, f3P-Hph-T and f3FADHT fragments by PCR to obtain delta FADH ^- SBLE ⁺ Linear carrierCutting and refining; the electrotransformation and engineering bacteria verification method is the same as in example 1, except that the transformation DNA solution is ΔFADH ^- SBLE ⁺ Linear carrier solution.

Comparative example 3

ΔFADH ^- MDR ⁺ (insertion of MDR gene into FADH gene to knock out FADH gene), the construction method is as follows:

b1, using FADHP and FADHT in example 1 as a model for PCR amplification of f4FADHP and f4FADHT with homology arms; PCR amplification of f2MDR with homology arms using AcP-MDR-AcT as template in example 1; PCR amplification of f4P-Hph-T with homology arm by using hygromycin resistance gene sequence (P-Hph-T) as template, and gel cutting and refining, the primer sequences are as follows:

TABLE 6

B2, fusion and amplification of f4FADHP, f4P-Hph-T, f MDR and f4FADHT fragments by PCR to obtain delta FADH ^- MDR ⁺ The method for linear vector, gel cutting and refining, electrotransformation and engineering bacteria verification is the same as example 1, except that the DNA solution for transformation is delta FADH ^- MDR ⁺ Linear carrier solution.

Comparative example 4

MEF-2 knock-out strain (Inge N.A Van Boeagrt, knocking out the MFE-2gene of Candida bombicola leads to improved medium-chain sophorolipid production, FEMS Yeast Res,2009, (9) 610-617) MEF-2 knock-out strain construction methods are described in the literature.

Comparative example 5

A fermentation production method of sophorolipid was provided, which was different from example 3 only in that ΔFADH provided in comparative example 1 was used ^- And (5) fermenting and producing the strain.

Comparative example 6

A fermentation production method of sophorolipid was provided, which was different from example 3 only in that ΔFADH provided in comparative example 2 was used ^- SBLE ⁺ And (5) fermenting and producing the strain.

Comparative example 7

A fermentation production method of sophorolipid was provided, which was different from example 3 only in that ΔFADH provided in comparative example 3 was used ^- MDR ⁺ And (5) fermenting and producing the strain.

Comparative example 8

A method of producing sophorolipid by fermentation is provided which differs from example 3 only in that MEF-2 knock-out strain provided in comparative example 4 is used for fermentative production.

Comparative example 9

A fermentation production method of sophorolipid was provided, which was different from example 3 only in that the wild-type strain was used for fermentation production.

Effect example 1

And detecting transcription levels of SBLE genes and MDR genes.

After the wild strain and the engineering strain obtained in examples 1 and 2 were inoculated onto YPD plates for activation, an inoculating loop was selected respectively in a test tube of 5mL YPD liquid medium, and cultured for 16-24 hours, and after the OD600 = 1.0, the strain was inoculated into 100mL fermentation medium at an inoculum size of 5% v/v, and cultured at 30℃for 4 days at 200-250 rpm. The cells were collected by centrifugation and cultured for 4 days, and RiboPure was used ^TM Total RNA was extracted using RNA purification kit (Thermo Fisher Scientific, america). Using PrimeScript with the extracted RNA as a template ^TM RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, japan) kit for reverse transcription synthesis of cDNA, PCR determination of transcription amounts of SBLE gene and MDR gene in different samples by using cDNA as template, and 10 ⁵ Correcting the copy number of GAPDH as a standard, detecting the transcription level of the over-expressed target gene in each sample, and performing PCR reaction by the following procedures:

TABLE 7

The results are shown in FIG. 2, and demonstrate that the transcription level of SBLE gene is significantly higher than that of the native promoter and terminator at 4 days of fermentation culture using TubP and TubT as the promoter and terminator of SBLE gene; the promoter and terminator of the MDR gene are adopted as the AcP and AcT, and the transcription level of the MDR gene is obviously higher than that of the promoter and terminator of the MDR gene by adopting the promoter and terminator of the MDR gene at the 4 th day of fermentation culture.

Effect example 2

Detection and evaluation of sophorolipid production, and after completion of the culture, the residual sugar content and sophorolipid content in the culture solution were measured.

(1) Determination of residual sugar content: glucose concentration in the culture broth was determined using a biosensing analyzer SBA-40E.

The fermentation broth was centrifuged to collect the supernatant, the supernatant was filtered through a 0.45 μm filter, the filtrate was diluted to a suitable multiple, 25 μl of sample was taken, and the residual glucose content of the broth (g/L) =n×m/200 (n is the instrument reading and m is the dilution).

(2) Determination of sophorolipid content: the total sugar content in the culture solution is determined by adopting an anthrone-sulfuric acid method, and the specific steps are as follows: the total sugar concentration in the supernatant is measured by ethanol extraction, the residual sugar content in the supernatant is subtracted to obtain the glucose content in the sophorolipid, and the total sophorolipid content is obtained after conversion. The method comprises the following specific steps: taking 500 mu L of fermentation liquor, adding 1mL of ethanol, shaking and mixing uniformly, and centrifuging for 10min at 12,000 r.min < -1 >. mu.L of the supernatant was taken and added to an 8mL EP tube, and 980. Mu.L of distilled water (50-fold dilution) was added. The total sugar content was measured by the anthrone-sulfuric acid method, and the total sophorolipid content was determined by removing the residual glucose content from the fermentation broth as the total sugar content, and determining the total sophorolipid content based on the ratio between the sophorolipid and the glucose molecular weight, i.e., 1.91g of sophorolipid corresponds to 1g of glucose.

(3) Lactone type sophorolipid content: and (3) extracting by ethyl acetate, measuring the glucose content in the lactone type sophorolipid in the supernatant of the ethyl acetate layer, and converting to obtain the lactone type sophorolipid content. The method comprises the following specific steps: taking 500 mu L of fermentation liquor, adding 1mL of ethyl acetate, fully extracting by vortex oscillation, and centrifuging for 10min at 12,000 r.min < -1 >. mu.L of the supernatant was taken and added to an 8mL EP tube, and 380. Mu.L of distilled water (20-fold dilution) was added. Taking the supernatant, determining the OD value of a sophorolipid sample at 620nm by adopting an anthrone-sulfuric acid method, determining the glucose content from a glucose standard curve, and obtaining the lactone type sophorolipid content according to a ratio of 1:1.91.

The results of the sophorolipid fermentation synthesis of the engineering strain (example 3) and the wild-type strain (comparative example 9) using ethyl laurate, n-heptanoic acid and n-hexanoic acid as hydrophobic substrates, respectively, are shown in FIGS. 3 and 4.

The ethyl laurate is used as a hydrophobic substrate, 250ml of fermentation culture is adopted, the total amount of sophorolipid produced by the engineering strain of the example 1 is 8.42g, which is improved by 49.8% compared with 5.62g of wild strain, and the lactone ratio is improved from 28% to 57%;

the n-heptanoic acid is used as a hydrophobic substrate, 250ml of fermentation culture is adopted, the total amount of sophorolipid produced by the engineering strain of the example 1 is 24.89g, which is improved by 60.5% compared with 15.51g of wild bacteria, and the lactone ratio is improved from 24% to 45%;

the total amount of sophorolipids produced by the engineering strain of the example 1 is 21.00g by taking n-caproic acid as a hydrophobic substrate and fermenting and culturing with 250ml, which is improved by 53.7% compared with 13.66g of wild strain, and the lactone ratio is improved from 20% to 52%.

The results of the fermentation methods of example 3 and comparative examples 5 to 9 for sophorolipid production using ethyl laurate as a hydrophobic substrate are shown in FIGS. 5 and 6, and the sophorolipid production ability evaluation is shown in Table 8.

The results of FIGS. 5 and 6 show that the engineering strain constructed in example 1 is superior to FADH gene single-knock-out strain (delta FADH-strain), MFE-2gene single-knock-out strain, delta FADH in terms of total sophorolipid yield and lactone sophorolipid ratio ^- SBLE ⁺ Strains and Δfadh ^- MDR ⁺ Strains.

According to the results of FIG. 5, the yield of sophorolipid is equal to or higher than 30g/L, the yield of sophorolipid is 25-30g/L, the yield of sophorolipid is middle-yield strain, and the yield of sophorolipid is less than 25g/L, the yield of sophorolipid is low-yield strain; the lactone-type ratio is > 40% high and < 40% low.

TABLE 8

Effect example 3

UPLC-MS analysis of the synthetic sophorolipid fraction with the engineering strain and the wild strain constructed in example 1 using ethyl laurate as hydrophobic substrate, respectively.

The crude sophorolipids were filtered through a 0.22 μm organic filter, and the sophorolipids were sampled using UPLC-MS with a column of BEH C18 (250 mm. Times.4.6 mm, USA). Chromatographic conditions: the mobile phase was water and acetonitrile at a flow rate of 0.3mL/min. The UPLC procedure is as follows: 015min, acetonitrile up to 60% from 40%; 1530min, acetonitrile slowly rising from 60% to 70%;3040min, acetonitrile was raised from 70% to 90% and left for 5min. The sample volume was 5. Mu.L, the detection temperature was 35℃and the wavelength detected by the detector was 207nm. The mass spectrum conditions are as follows: ion source: ESI, spray voltage: 2000V, ion source temperature: 120 ℃.

As a result, as shown in FIG. 7, the wild strain contained more acid-type sophorolipid fraction, while the content (ratio) of the acid-type sophorolipid fraction of the engineering strain was significantly reduced. The engineering strain constructed in example 1 performs over-expression genetic manipulation on a lactonase (SBLE) gene in a bacterial cell, and the synthesized acid type sophorolipid is catalyzed into the lactone type sophorolipid more under the action of lactonase.

In conclusion, the invention realizes the construction of the short-chain lactone type sophorolipid engineering strain in high yield. Under the fermentation culture condition of taking ethyl laurate, n-heptanoic acid and n-caproic acid as hydrophobic substrates, the total yield of sophorolipid is higher than that of a wild strain, and the ratio of lactone type sophorolipid is higher than that of the wild strain. The invention provides excellent stable high-yield strain for the industrial production of medium-short chain lactone type sophorolipid, and has higher theoretical research significance and practical application value.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. The engineering strain is characterized in that an original strain of the engineering strain is a bumblebee candida (Starmerella bombicola), and a formaldehyde dehydrogenase FADH gene locus of the bumblebee candida is inserted into a lactonase SBLE gene and a sophorolipid transporter MDR gene, so that the FADH gene is knocked out.

2. The engineered strain of claim 1, wherein the SBLE gene and MDR gene are expressed under the control of a strong promoter;

preferably, the strong promoter comprises TubP and/or AcP;

preferably, the FADH gene locus insert further comprises a selectable marker gene;

preferably, the selectable marker comprises a resistance gene, further preferably a hygromycin resistance gene.

3. The engineering strain according to claim 1, wherein the nucleotide sequence of the SBLE gene is shown in SEQ ID No. 2; and/or the nucleotide sequence of the MDR gene is shown as SEQ ID NO. 3.

4. An engineered strain according to any one of claims 1 to 3, wherein the FADH gene locus insert comprises, in order from 5 'to 3', an SBLE gene, a selectable marker gene, and an MDR gene;

preferably, the FADH gene locus insert comprises an SBLE gene, a hygromycin resistance gene and an MDR gene from the 5 'end to the 3' end in sequence, wherein the SBLE gene is regulated and expressed by a strong promoter TubP and a terminator TubT, the hygromycin resistance gene is regulated and expressed by a candida strong promoter and a terminator, and the MDR gene is regulated and expressed by a strong promoter AcP and a terminator AcT.

5. The method for constructing an engineering strain of high-yield medium-short chain lactone-type sophorolipids according to any of claims 1 to 4, comprising constructing a vector containing the insert, and then introducing the vector into the starting strain; the inserts include SBLE genes and MDR genes.

6. The method according to claim 5, wherein the insert has an upstream homology arm and a downstream homology arm homologous to the FADH gene, and the SBLE gene and the MDR gene are inserted into the FADH gene site by homologous recombination.

7. The method according to claim 5, wherein the vector containing the insert is introduced into the competent cells of Candida buminosa by electrotransformation;

preferably, the vector is linear DNA.

8. A process for producing sophorolipid by fermentation, which comprises fermenting a substrate with the engineering strain according to any one of claims 1 to 4.

9. The method for producing sophorolipid according to claim 8, wherein the substrate comprises a C6-C12 fatty acid or a C6-C12 fatty acid ester;

preferably, the substrate comprises one or more of lauric acid, ethyl laurate, n-heptanoic acid and n-hexanoic acid;

preferably, the substrate content in the fermentation medium is 6-10% w/v;

preferably, the fermentation medium contains 6-10% of fermentation substrate, 0.3% of yeast powder, 0.3% of glucose and KH ₂ PO ₄ 0.1％w/v、Na ₂ HPO ₄ ·12H ₂ O0.1% w/v and MgSO ₄ ·7H ₂ O 0.05％w/v。

10. Use of the engineered strain of high-yielding medium-short-chain lactone-type sophorolipids according to any one of claims 1 to 4, or the construction method according to any one of claims 5 to 7, or the sophorolipid fermentation production method according to claim 8 or 9, for the preparation of a surfactant.