CN115851472A - Gene knockout engineering bacterium for high yield of sophorolipid and construction method and application thereof - Google Patents
Gene knockout engineering bacterium for high yield of sophorolipid and construction method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a gene knockout engineering bacterium for high yield of sophorolipid and a construction method and application thereof, wherein the engineering bacterium takes bumblebee candida as an original strain, and replaces a gene sequence seq2 for regulating and controlling an lactonization reaction in a sophorolipid synthesis path with a gF-Hph-gR nucleotide sequence ， Wherein the gF-Hph-gR nucleotide sequence is SEQ ID No.1, the SEQ2 gene sequence is SEQ ID No.2The seq2 gene knockout box with the resistance screening marker is constructed by adopting a method of fusing pcr, and can be obtained within the shortest 1 day, so that a new way is provided for efficiently constructing the sophorolipid production engineering strain, the gene knockout strain with high sophorolipid yield provided by the invention also lays a foundation for large-scale industrial production of sophorolipid, and the gene knockout strain meets market demands, and has wide application prospects, economic values and social benefits.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a gene knockout engineering bacterium for high yield of sophorolipid and a construction method and application thereof.
Background
Sophorolipids synthesized by microorganisms are a mixture of a series of sophorolipid molecules, mainly produced by saccharomyces. The sophorolipid molecules are composed of two parts of hydrophilic group and lipophilic group, the hydrophilic group is sophorose formed by connecting two glucoses through beta-1, 2 glycosidic bond, the lipophilic group is saturated/unsaturated long-chain omega or omega-1 hydroxy fatty acid, and the two groups are connected through glycosidic bond. Sophorolipids have both hydrophilic and hydrophobic groups and possess the general characteristics of surfactants, such as: the surface tension, the foaming property, the emulsifying property and the like are reduced, and the surfactant has the characteristics of being utilized and degraded by microorganisms, and is a green biosurfactant which cannot cause pollution to the environment. In recent years, the application of sophorolipids as biosurfactants has received increasing attention. The sophorolipid has the advantages of no toxicity, high temperature resistance and good surface activity in a high-salt-concentration solution, so that the sophorolipid has great potential value in the field of detergents such as dish washing detergents, clothes detergents and the like. Sophorolipids are also used as penetration enhancers for cosmetics due to their strong skin-friendly properties.
Microorganisms producing sophorolipids are many, and currently, candida bombicola (starmerella abormbicola) of the yeast species is commonly used. The sophorolipid produced by microbial synthesis has two molecular structures of lactone type and acid type, and sophorolipid with different molecular structures has different performances in the aspects of antibacterial property and surface activity property.
The use of sophorolipids as a cleaning or cosmetic material makes it inevitable to compete with the relatively inexpensive chemically synthesized surfactants currently used. However, the wild strain of candida bombicola has low sophorolipid yield and high purification cost, and the large-scale production of sophorolipid has high cost, which is a main problem that the industrialized production and the wide application of sophorolipid are hindered. Therefore, an engineering bacterium for high yield of sophorolipid is urgently needed as one of the main means for reducing the production cost of sophorolipid.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a gene knockout engineering bacterium for high yield of sophorolipid, which can inhibit genes for regulating and controlling lactonization reaction in a sophorolipid synthesis path.
In order to solve the technical problems, the invention aims to realize the following technical scheme: providing a genetically knocked-out engineering bacterium for high yield of sophorolipid, wherein the engineering bacterium takes bumblebee candida utilis as an original strain, and replaces a gene sequence seq2 for regulating and controlling an lactonization reaction in a sophorolipid synthesis path with a gF-Hph-gR nucleotide sequence;
wherein the gF-Hph-gR nucleotide sequence is SEQ ID No.1, and the SEQ2 gene sequence is SEQ ID No.2.
In addition, the technical problem to be solved by the invention is to provide a construction method of the gene knockout engineering bacteria for high yield of sophorolipid.
In order to solve the technical problems, the invention aims to realize the following technical scheme: a construction method of a gene knockout engineering bacterium for high yield of sophorolipid is characterized by comprising the following steps:
s1, constructing a knockout box;
s2, preparing wild type bumblebee candida utilis competent cells.
Preferably, the step S1 includes the following steps:
a1, optimizing hygromycin resistance genes gF-Hph-gR with strong promoters and terminators;
a2, inserting a gF-Hph-gR nucleotide sequence into a pUC57 plasmid to construct a hygromycin screening marker plasmid pUHP;
a3, amplifying a seq2 gene sequence by using high-fidelity enzyme, taking the DNA of the bumblebee candida utilis genome as a template and SBLEdl F1/SBLEdlR1 as a template; amplifying upstream and downstream homologous arm sequences 5 'and 3' of seq2 gene by using primers SBLEDLF2/SBLEDLR2 and SBLEDLF4/SBLEDLR4 respectively by using the sequence of seq2 gene as a template;
a4, using hygromycin screening marker plasmid pUHP as a template, and using a primer SBLEDLF3/SBLEDLR3 to amplify to obtain a hygromycin high-efficiency expression cassette gF-Hph-gR with two ends respectively provided with upstream and downstream 20bp homologous sequences of seq2 gene;
a5, performing gel cutting purification to obtain 5 'flight and 3' flight of upstream and downstream homologous arms of seq2 gene, performing gel cutting purification, and carrying out hygromycin high-efficiency expression cassette gF-Hph-gR with homologous sequence;
a6, fusion pcr: and fusing the 3 purified genes to finally obtain the linear seq2 gene knockout box. Preferably, in step A2, the sequence of the primers SBLEDLF1/SBLEDLR1 is shown as SEQ ID No.5/SEQ ID No.6, the sequence of the primers SBLEDLF2/SBLEDLR2 is shown as SEQ ID No.7/SEQ ID No.8, and the sequence of the primers SBLEDLF4/SBLEDLR4 is shown as SEQ ID No.9/SEQ ID No.10 ， The sequence of the primers SBLEdlF3/SBLEdlR3 is shown in SEQ ID No.11/SEQ ID No.12 。
Preferably, the 5 'and 3' homologous sequences of the hygromycin gene linear expression cassette with homologous sequences are shown in SEQ ID No.3-1 and SEQ ID No.3-2, respectively.
Preferably, in step S2, the linear seq2 knockout cassette is transformed into competent cells, and the strain that normally grows on a YPD plate containing hygromycin is selected for YPD liquid culture to extract genomic DNA; using the extracted genomic DNA as a template and TaKaRaPremixTaq TM And (3) carrying out PCR amplification by using enzyme and SBLEdl F2/SBLEdlR4 as primers to obtain a DNA fragment.
Moreover, the invention also provides application of the gene knockout engineering bacteria for high yield of sophorolipid, in particular application in fermentation production of sophorolipid.
Preferably, the step of producing sophorolipid by fermentation is as follows:
b1, inoculating the engineering bacteria into a test tube filled with a 5mLYPD seed culture medium for culture;
b2, inoculating the seed liquid into a shake flask containing 50mLYPD culture medium according to the inoculation amount of 2% (v/v) for primary shake flask fermentation;
b3, wait for OD 600 =1.0, and inoculated in a 500mL shake flask containing 100mL of fermentation medium at an inoculum size of 5% (v/v) for 5 to 10 days.
Preferably, the YPD medium consists of: 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose.
Preferably, the fermentation medium comprises the following components: 1% (w/v) yeast extract, 4% (w/v) linoleic acid, 8% (w/v) glucose, 0.1% KH2PO4, 0.1% Na2HPO4.12H2O, 0.05% MgSO4.7H2O.
Preferably, the fermentation culture time is 5 days.
The commonly used method for obtaining the sophorolipid high-yield strain mainly comprises mutagenesis screening or operation of knocking out and over-expressing a plurality of genes, and has the problems of randomness and complex operation; and the method adopted for constructing the gene knockout box and the over-expression box mostly comprises the steps of connecting a pre-modified gene fragment with a linearized vector to obtain a recombinant plasmid, carrying out competent cell transformation on the recombinant plasmid, plating and culturing transformed competent cells, selecting a single colony, screening the successfully transformed single colony by using Colonypcr, then culturing the single colony, extracting the recombinant plasmid from the thallus, and finally obtaining the linear gene knockout/over-expression box by using the recombinant plasmid as a template and pcr amplification, wherein the time is 3 days or more. The method for obtaining the high-yield sophorolipid strain through single gene knockout provided by the invention adopts a method of fusing pcr to construct a seq2 gene knockout box with a resistance screening marker, can be obtained within 1 day shortest, improves the gene modification efficiency, and provides a new way for efficiently constructing and producing sophorolipid genetic engineering strains. The gene engineering strain constructed by the invention has the advantages that the total sophorolipid yield is improved by 39.8 percent compared with the wild strain, the production cost of sophorolipid is greatly reduced, particularly, the yield of acid sophorolipid is improved by 258.9 percent compared with the wild strain, and the acid sophorolipid can be widely applied to washing, oil extraction, skin care products, food and medicine industries due to the characteristics of high solubility, good foaming and emulsifying properties and the like, so the gene knockout strain lays a foundation for large-scale industrial production of sophorolipid and meeting market requirements, and has wide application prospect, economic value and social benefit.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic diagram of construction of seq2 knockout strain delta seq2 by homologous recombination using a fusion pcr method
FIG. 2 shows agarose gel electrophoresis results of 3 gene fragments for constructing seq2 knockout cassette and seq2 knockout cassette obtained by fusion pcr amplification
FIG. 3 shows the results of agarose gel electrophoresis of DNA fragments obtained by pcr amplification using genomic DNAs of a wild type strain and a. DELTA.seq 2 strain as templates and SBLEDLF2/SBLEDLR4 as primers, respectively
FIG. 4 is a graph showing the fermentation results of sophorolipid in fermentation broth of wild strain and Δ seq2 strain according to the present invention
FIG. 5 is a graph comparing the difference between the crude fermentation broth of the wild strain and the Δ seq2 strain in the examples of the present invention
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It will be understood that the terms "comprises" and/or "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 this 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", and the like are intended to indicate that the relative terms are not necessarily strictly required, but may have some deviation. For example: "substantially equal" does not mean absolute equality, but it is difficult to achieve absolute equality in actual production and operation, and certain deviations generally exist. Thus, in addition to absolute equality, "substantially equal" also includes the above-described case where there is some deviation. In this case, unless otherwise specified, terms such as "substantially", and the like are used in a similar manner to those described above.
The invention provides a high-yield sophorolipid gene knockout engineering bacterium, wherein the high-yield sophorolipid gene knockout engineering strain takes bumblebee candida (S.bombicola) as an initial strain, and replaces a gene sequence seq2 for regulating and controlling the lactonization reaction in a sophorolipid synthesis path with a gF-Hph-gR nucleotide sequence to realize the inhibition of the lactonization reaction; wherein the gF-Hph-gR nucleotide sequence is shown as SEQ ID No.1, and the SEQ2 gene sequence is shown as SEQ ID No. 2; the amino acid sequence coded by SEQ2 gene sequence is shown in SEQ ID No.4, compared with wild strains, the total sophorolipid yield is improved by more than 35%, wherein the acid sophorolipid yield is improved by more than 200%, and the high-yield strain is obtained.
The gene knockout engineering bacteria mutant strain with high sophorolipid yield constructed by the invention is preserved in the Guangdong province microorganism strain preservation center (No. 59 building 5 of Michelia Tokyo 100, guangdong province academy of sciences) at 25/8 in 2022, the preservation number is: GDMCC No. 62738, taxonomic name: starmerella globicola.
The construction method of the gene knockout engineering bacteria for high yield of sophorolipid comprises the following steps:
s1, constructing a knockout box: optimizing hygromycin resistance genes gF-Hph-gR with strong promoters and terminators to enable the hygromycin resistance genes gF-Hph-gR to be efficiently expressed in candida bombicola, inserting a gF-Hph-gR nucleotide sequence into a pUC57 plasmid, and constructing to obtain a hygromycin screening marker plasmid pUHP; using high fidelity enzyme Prime
The MaxDNAzolerase takes the genome DNA of the candida bombesia as a template, and takes SBLEDLF1/SBLEDLR1 as the template to amplify the seq gene sequence; then, using the obtained seq gene sequence as a template, and respectively amplifying the upstream and downstream homologous arm sequences of the seq2 gene by using primers SBLEDLF2/SBLEDLR2 and SBLEDLF4/SBLEDLR 4; using hygromycin screening marker plasmid pUHP as a template, and using a primer SBLEdl F3/SBLEdlR3 to amplify to obtain a hygromycin efficient expression cassette with 20bp homologous sequences at the upstream and downstream of seq2 gene at two ends respectively; purifying the obtained seq2 gene upstream and downstream homology arms and a hygromycin high-efficiency expression cassette with a homologous sequence by using TaKaRaMiniBESTAgaroseGelDNAextraction kit cutting glue; fusing the 3 purified genes by adopting a method of fusing pcr to finally obtain a linear seq2 gene knockout box; the nucleotide sequence of the SEQ2 gene is shown in SEQ ID No.2, and the 5 'end and 3' end homologous sequences of the hygromycin gene linear expression cassette with the homologous sequences are shown in SEQ ID No.3-1 and SEQ ID No.3-2 respectively.
S2, preparing a wild-type bumblebee candida (S.bombicola) competent cell by using a lithium acetate method, transforming a linear seq2 gene knockout box into the competent cell, selecting a strain which normally grows on a hygromycin-containing YPD plate, carrying out YPD liquid culture, and extracting genome DNA; using the extracted genomic DNA as a template and TaKaRaPremixTaq TM And (2) enzyme, using SBLEDLF2/SBLEDLR4 as a primer, amplifying the primer pcr to obtain a DNA fragment, and confirming that the amplified fragment is a seq2 gene knockout box sequence through DNA sequencing, namely, the success of seq2 gene knockout is demonstrated.
In addition, the gene knockout engineering bacterium for high yield of sophorolipid provided by the invention is applied to fermentation production of sophorolipid.
The method for producing sophorolipid by fermentation comprises the following steps: inoculating the gene knockout engineering bacteria into a test tube filled with 5mLYPD seed culture medium, culturing for 24-36h at 30 ℃ and the rotating speed of 200-250rpm, inoculating the seed liquid into a shake flask containing 50mLYPD culture medium according to the inoculum size of 2% (v/v) for primary shake flask fermentation, performing shake culture at 25-30 ℃ and 200-300rpm for 24-48 h, and waiting until OD is reached 600 =1.0, then inoculating into a 500mL shaking flask containing 100mL fermentation medium with the inoculation amount of 5% (v/v), and culturing for 5-10 days at the temperature of 25-30 ℃ and the rpm of 200-250; the content of total sophorolipid and lactone sophorolipid in the fermentation liquor is determined by adopting an anthrone method, the content of acid sophorolipid is obtained by subtracting the lactone sophorolipid from the content of the total sophorolipid, the residual glucose content in the fermentation liquor is determined by adopting a biosensor analyzer SBA-40E, and the used engineering bacteria and the fermentation production effect thereof are evaluated.
The YPD medium comprises the following components: 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose.
The fermentation medium comprises the following components: 1% (w/v) yeast extract, 4% (w/v) linoleic acid, 8% (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。
The culture conditions of the seed liquid are preferably: culturing at 30 deg.C and rotation speed of 200-250rpm for 24-30h.
The preferable conditions of the first-stage shake flask fermentation are as follows: inoculating the seed solution into 50mL of culture solution according to 2-4% (v/v), wherein the culture conditions are as follows: culturing at 30 deg.C and rotation speed of 200-300rpm for 36-48h.
The conditions for the shake flask fermentation described above are preferably: the primary shake flask fermentation broth was inoculated at 5% (v/v) into 100mL of the culture broth and cultured at 30 ℃ and 200-250rpm for 120 hours.
The analysis of fermentation products of the high-yield sophorolipid engineering strain and the wild strain comprises the following steps:
(1) And (3) fermenting the wild strains and the engineering strains in a fermentation culture medium, and sampling fermentation liquor after the fermentation is finished to determine and analyze residual glucose amount, total sophorolipid yield and lactone and acid sophorolipids.
(2) Determination of residual sugar content: the glucose concentration in the culture broth was determined using the biosensor analyzer SBA-40E.
Centrifuging the fermentation liquor to obtain supernatant, filtering the supernatant with a 0.45 μm filter membrane, diluting the filtrate to a proper amount, sampling 25 μ L, and injecting sample, wherein the residual glucose content (g/L) = n × m/100 (n is the reading of the instrument, and m is the dilution factor).
(3) The sophorolipid content in the fermentation liquor is determined by adopting an anthrone-sulfuric acid method.
Taking 500 mu L of fermentation liquor, adding 1mL of ethanol, shaking and uniformly mixing, 12,000r.min -1 Centrifuge for 10min. mu.L of the supernatant was added to an 8mL EP tube, and 980. Mu.L of distilled water (diluted 50-fold) was added. Measuring the total sugar content by anthrone-sulfuric acid method, removing residual glucose content in the fermentation liquid by using the total sophorolipid content as the total sugar content, and determining the total sophorolipid content according to the ratio between sophorolipid and glucose molecular weight, namely 1.91g of sophorolipid is equivalent to 1g of glucose.
Taking 500 mu L of fermentation liquid, adding 1mL of ethyl acetate, performing vortex oscillation and full extraction, 12,000r min- 1 Centrifuge for 10min. mu.L of the supernatant was added to an 8mL EP tube, and 380. Mu.L of distilled water (diluted 20-fold) was added. Taking the supernatant, adopting the OD value of the sophorolipid sample measured by an anthrone-sulfuric acid method at 620nm, calculating the glucose content from a glucose standard curve, and obtaining the lactone-type sophorolipid content according to the ratio of 1. Acid type sophorolipid content = total sophorolipid content-lactone type sophorolipid content.
The experimental detection shows that: after the high-yield sophorolipid engineering strain delta seq2 constructed by the method is fermented in a shake flask for 5 days, the total sophorolipid yield reaches 64.3g/L and is increased by 39.8 percent compared with a wild strain, and the acid sophorolipid yield reaches 50.6g/L and is increased by 258.9 percent compared with the wild strain. Because the acidic sophorolipid has high solubility and good foaming and emulsifying properties, the acidic sophorolipid can be widely applied to the industries of food, medicine, cosmetics, cleaning and the like, so that the delta seq2 engineering strain becomes an advantageous strain for producing sophorolipid, particularly acidic sophorolipid.
The following embodiments are described in detail
Example 1 construction of Gene knockout cassette with gF-Hph-gR resistance selection marker
(1) A nucleotide sequence fragment shown in SEQ ID NO.2 was obtained by PCR amplification using genomic DNA of Candida bombicola (S.bombicola) as a template and SBLEdl F1 and SBLEdlR1 as primers. Amplifying a fragment 5' of the upstream region by PCR by taking the gene fragment as a template and SBLEDLF2 and SBLEDLR2 as primers; in the same manner, the downstream region fragment of 3' flinking was obtained by PCR amplification using SBLEDLF4 and SBLEDLR4 as primers. And (3) carrying out PCR amplification by using the pUHP plasmid as a template and SBLEdlF3 and SBLEdl R3 as primers to obtain a gF-Hph-gR fragment.
(2) And finally, connecting the three fragments of the 5 'flaring fragment, the 3' flaring fragment and the gF-Hph-gR fragment by using SBLEDLF2 and SBLEDLR4 as primers through a fusion PCR reaction, thereby obtaining the linear knockout cassette for knocking out the gene fragment shown in SEQ ID NO: 2. The fusion pcr results are shown in figure 2.
The gene knockout box with the resistance screening marker is constructed by the method, and can be obtained within 1 day shortest.
EXAMPLE 2 production of knockout strains
A single colony of Candida bombicola (S.bombicola) was inoculated into a 250mL flask containing 25mL LYPD medium and cultured at 30 ℃ for 18 hours at 300 rpm.
Inoculating the above culture solution into a 250mL shake flask containing 50mLYPD medium in an amount of 2% (v/v), and culturing the cells at 30 deg.C and 120rpm until OD 600 The value is between 1 and 2.
Loading the bacterial liquid into a 50mL centrifuge tube, centrifuging for 5 minutes at 3000g and 4 ℃, collecting thalli, then suspending and precipitating by using 50mL sterile water cooled on ice, centrifuging for 5 minutes at 3000g and 4 ℃, and discarding supernatant; the pellet was resuspended in 50mL of ice-chilled sterile water and centrifuged.
The cell pellet was resuspended in 4mL of ice-cold 1M sterile sorbitol solution, centrifuged at 3000g at 4 ℃ for 5 minutes, and the supernatant was discarded.
The precipitate was suspended in 4mL of freshly prepared 0.1M lithium acetate solution (3500. Mu.5 water, 400. Mu.01M lithium acetate, 100. Mu.01M DTT) and left at room temperature for 15 minutes, after which the supernatant was discarded after centrifugation at 3000g and 4 ℃ for 5 minutes.
The cell pellet was suspended in 4mL of ice-cold 1M sterile sorbitol solution, centrifuged at 3000g at 4 ℃ for 5 minutes, and the supernatant was discarded.
The cells were suspended in 1M sorbitol solution, placed on ice and used as soon as possible.
The yeast suspension was aspirated to 50. Mu.0 into a centrifuge tube, and 2.53. Mu.g of a DNA solution for transformation (i.e., a knockout fragment of the nucleotide sequence shown by SEQ ID NO: 2) was added to the yeast suspension, mixed well, and placed on ice for precooling for 5 minutes.
The above mixture was also transferred to an electric tumbler with a gap of 0.2cm and placed on ice for 5 minutes. Subsequently, the mixture was pulsed for 5ms and 2.5kV using MicroPulser (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. The 100. Mu.0 mixture solution was applied to the selective medium and incubated at 30 ℃ for about 1 week. For the selection medium, an agar medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose and 500ppm hygromycin was used.
And performing liquid amplification culture on the grown single colony, and extracting genome DNA. And (3) respectively taking the wild type strains and the selected single colony genomic DNA as templates, taking SBLEDLF2/SBLEDLR4 as primers to carry out pcr amplification, detecting the amplified DNA fragment, and taking the single colony genomic DNA as the template to amplify to obtain the DNA fragment with the length consistent with the length of the constructed knockout box, thus proving that the target gene knockout engineering strain (delta seq2 strain) is obtained, and the result is shown in figure 3.
Example 3 Sophora lipid fermentation production of the Δ seq2 and wild type strains
Culturing the strain:
selecting single colony of the strain delta seq2 obtained in example 2 and its wild strain, inoculating into test tube containing 5mLYPD seed culture medium, culturing at 30 deg.C and 200-250rpm for 24-36h, and culturing seed liquid at 2% (v/v)Inoculating the inoculum size into a shake flask containing 50mLYPD culture medium, performing primary shake flask fermentation, performing shake culture at 25-30 deg.C and 200-300rpm for 24-48 hr until OD is reached 600 =1.0, then inoculating to a 500mL shake flask containing 100mL fermentation medium with an inoculum size of 5% (v/v), and culturing at 200-250rpm at 25-30 ℃ for 5-10 days.
The culture conditions of the seed liquid are preferably: culturing at 30 deg.C and rotation speed of 200-250rpm for 24-30h.
The preferable conditions of the first-stage shake flask fermentation are as follows: inoculating the seed solution into 50mL of culture solution according to 2-4% (v/v), wherein the culture conditions are as follows: culturing at 30 deg.C and rotation speed of 200-300rpm for 36-48h.
The conditions for the shake flask fermentation described above are preferably: the primary shake flask fermentation broth was inoculated at 5% (v/v) into 100mL of the culture broth and cultured at 30 ℃ and 200-250rpm for 120 hours.
Example 4 detection and evaluation of production amount of sophorolipid
After the culture was completed, the residual sugar content and sophorolipid content in the culture broth were measured. The total sophorolipid yield is more than or equal to 60g/L; 40-60 g/L; the strain with the yield of <40g/L is divided into a high-yield sophorolipid strain, a medium-yield sophorolipid strain and a low-yield sophorolipid strain, and the yield of the acid sophorolipid is more than or equal to 45g/L; 30-45 g/L; the sophorolipid accounts for 30g/L, accounts for more than 80 percent of the total sophorolipid, and is divided into three grades, namely high-acid-production sophorolipid, medium-acid-production sophorolipid and low-acid-production sophorolipid.
(1) Determination of residual sugar content: the glucose concentration in the culture broth was determined using the biosensor analyzer SBA-40E.
Centrifuging the fermentation liquor to obtain supernatant, filtering the supernatant with 0.45-heart filter membrane, diluting the filtrate to proper times, sampling 25 μ 5 sample, and introducing the fermentation liquor with residual glucose content (g/L) = n × m/100 (n is the instrument reading, and m is the dilution times).
(2) For the measurement of sophorolipid content: the total sugar content in the culture solution is determined by anthrone method, and then the sophorolipid content can be converted according to the ratio between the sophorolipid and the glucose molecular weight, namely 1.91g of sophorolipid is equivalent to 1g of glucose.
Total sophorolipid content: extracting with ethanol, measuring total sugar concentration in the supernatant, subtracting residual sugar content in the supernatant to obtain glucose content in sophorolipid, and converting to obtain total sophorolipid content.
(3) Lactone type sophorolipid content: extracting with ethyl acetate, measuring glucose content in lactone type sophorolipid in supernatant of ethyl acetate layer, and converting to obtain lactone type sophorolipid content.
Comparative example 1
A yeast strain only producing acid sophorolipid is constructed by knocking out an esterase encoding gene SBLE and a peroxidase membrane transporter encoding gene PXA1 in the yeast and excessively expressing a glucosyltransferase gene UGTB, and the yeast strain only producing the acid sophorolipid comprises the following components of glucose 132.0g/L, yeast powder 4.0g/L, sodium citrate 4.0g/L and KH 2 PO 4 1.0g/L，K 2 HPO 4 ·12H 2 O0.16g/L，MgSO 4 ·7H 2 O0.7g/L, rapeseed oil 37.5g/L, naCl0.5g/L, caCl 2 ·2H 2 When the cells are cultured in the fermentation culture solution of O0.27g/L for 9 days, the total yield of the acid sophorolipids is 44g/L. (from Zhang Jiang Rui, et al. Microbiological newspaper, 2019)
Comparative example 2, an engineered strain with high sophorolipid yield was obtained by knocking out leu3 transcription factor and zinc finger transcription factor ztf1 which respond to pH-related protein rlp gene and regulate branched chain amino acid synthesis in yeast, and its components were glucose 80.0g/L, yeast powder 3.0g/L, KH 2 PO 4 1.0g/L，Na 2 HPO 4 ·12H 2 O1.0g/L，MgSO 4 ·7H 2 O0.5g/L and rapeseed oil 80ml/L, cultured for 7 days, the total sophorolipid yield reaches 102g/L, but the acid sophorolipid accounts for about 53% lower.
The yield of the total sophorolipid and the acid sophorolipid of the delta seq2 strain is 64.3g/L and 50.6g/L respectively, and is improved by 39.8 percent and 258.9 percent respectively compared with 46.0g/L and 14.1g/L of the wild strain. The results are shown in FIGS. 4 and 5, the statistical results are shown in Table 1, and the yield evaluations are shown in Table 2.
Table 1: the result of the detection
Table 2: evaluation of yield
In conclusion, the invention successfully realizes the construction of the high-yield sophorolipid, in particular to the acid-type sophorolipid engineering strain. By adopting a fusion pcr method, the gene knockout box is constructed efficiently, the engineering strain (the candida utilis engineering strain with the genotype of delta seq 2) with obviously improved sophorolipid total yield and acid sophorolipid yield is obtained, an excellent and stable high-yield strain is provided for the industrial production of sophorolipid, and simultaneously, the theoretical research significance is higher. The acid sophorolipid has the characteristics of good dissolution, foaming, emulsification, cleaning and the like, has wide application prospect in the industries of household cleaning, personal care, food, medicine, oil extraction and the like, and has good practical application value.
While the invention has been described with reference to specific embodiments, the invention is not limited thereto, and various equivalent modifications and substitutions can be easily made by those skilled in the art within the technical scope of the invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A gene knockout engineering bacterium for high yield of sophorolipid is characterized in that: the engineering bacteria use bumblebee candida as an original strain, and a gF-Hph-gR nucleotide sequence replaces a gene sequence seq2 for regulating and controlling the lactonization reaction in the sophorolipid synthetic path;
wherein the gF-Hph-gR nucleotide sequence is SEQ ID No.1, and the SEQ2 gene sequence is SEQ ID No.2.
2. A construction method of a gene knockout engineering bacterium for high yield of sophorolipid is characterized by comprising the following steps:
s1, constructing a knockout box;
s2, preparing wild type bumblebee candida utilis competent cells and verifying a transformation result.
3. The method for constructing genetically knocked-out engineered bacterium with high sophorolipid yield according to claim 2, wherein the step S1 comprises the following steps:
a1, optimizing hygromycin resistance genes gF-Hph-gR with strong promoters and terminators;
a2, inserting a gF-Hph-gR nucleotide sequence into a pUC57 plasmid to construct a hygromycin screening marker plasmid pUHP;
a3, amplifying a seq2 gene sequence by using high-fidelity enzyme, taking the DNA of the bumblebee candida utilis genome as a template and SBLEdl F1/SBLEdl R1 as a template; amplifying upstream and downstream homologous arm sequences 5 'and 3' of seq2 gene by using primers SBLEdl F2/SBLEdl R2 and SBLEdl F4/SBLEdl R4 respectively by using the sequence of the seq2 gene as a template;
a4, using hygromycin screening marker plasmid pUHP as a template, and using a primer SBLEdl F3/SBLEdl R3 to amplify to obtain a hygromycin high-efficiency expression cassette gF-Hph-gR with two ends respectively provided with upstream and downstream 20bp homologous sequences of seq2 gene;
a5, performing gel cutting purification to obtain 5 'flight king and 3' flight king of upstream and downstream homologous arms of seq2 gene, and performing gel cutting purification to obtain a hygromycin efficient expression cassette gF-Hph-gR with a homologous sequence;
a6, fusion pcr: and fusing the 3 purified genes to finally obtain the linear seq2 gene knockout box.
4. The method for constructing a genetically knocked-out engineered bacterium capable of producing sophorolipids in high yield according to claim 3, wherein in step A2, the sequence of the primers SBLEdl F1/SBLEdl R1 is shown as SEQ ID No.5/SEQ ID No.6, the sequence of the primers SBLEdl F2/SBLEdl R2 is shown as SEQ ID No.7/SEQ ID No.8, the sequence of the primers SBLEdl F4/SBLEdl R4 is shown as SEQ ID No.9/SEQ ID No.10, and the sequence of the primers SBLEdl F3/SBLEdl R3 is shown as SEQ ID No.11/SEQ ID No. 12.
5. The method for constructing genetically knocked-out engineered bacterium for high yield of sophorolipid as claimed in claim 3, wherein: the 5 'end and 3' end homologous sequences of the hygromycin gene linear expression cassette with the homologous sequences are respectively shown as SEQ ID No.3-1 and SEQ ID No. 3-2.
6. The method for constructing a genetically-knocked-out engineered bacterium capable of producing sophorolipid at a high yield according to claim 2, wherein the genetically-knocked-out engineered bacterium comprises the following steps: in the step S2, the linear seq2 gene knockout box is transformed into competent cells, the transformed competent cells are spread on a YPD plate containing hygromycin for culture, colonies growing normally on the plate are selected for YPD liquid culture, and the colony genome DNA is extracted; and (3) judging a conversion result according to the length of a DNA fragment obtained by PCR amplification by using the extracted genome DNA as a template, using Premix Taq enzyme and SBLEdl F2/SBLEdl R4 as primers.
7. The application of the gene knockout engineering bacterium for producing sophorolipid at high yield is characterized in that the engineering bacterium is applied to fermentation production of sophorolipid.
8. The use of the genetically-knocked-out engineered bacterium for producing sophorolipid in high yield as claimed in claim 7, wherein the fermentation process for producing sophorolipid comprises the following steps:
b1, inoculating the engineering bacteria into a test tube filled with a 5mLYPD seed culture medium for culture;
b2, inoculating the seed liquid into a shake flask containing 50mLYPD culture medium according to the inoculation amount of 2% (v/v) for primary shake flask fermentation;
b3, waiting for OD 600 =1.0, and inoculated in a 500mL shake flask containing 100mL of fermentation medium at an inoculum size of 5% (v/v) for 5 to 10 days.
9. The use of the genetically-knocked-out engineered bacterium for producing sophorolipid in high yield according to claim 8, wherein the YPD medium comprises the following components: 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose.
10. The use of the genetically engineered bacterium with high sophorolipid yield as claimed in claim 8, wherein the fermentation medium comprises the following components: 1% (w/v) yeast extract, 4% (w/v) linoleic acid, 8% (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。
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| CN116555269A (en) * | 2023-06-28 | 2023-08-08 | 百葵锐(深圳)生物科技有限公司 | Bumblebee candida utilis inducible promoter and application thereof |
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| CN116555269B (en) * | 2023-06-28 | 2023-09-15 | 百葵锐(深圳)生物科技有限公司 | Bumblebee candida utilis inducible promoter and application thereof |
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