Application of knocking out Da2d gene or Da2d gene and hpB gene in reducing the foaming performance of erythritol fermentation

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to a method for knocking out Da2d Gene or Da2d Genes and hpB The invention provides a method for reducing the foam production performance of erythritol fermentation by knocking out the gene in the strain. Da2d Gene or knockout strains Da2d Genes and hpB The application of gene in reducing the foam production performance of bacterial strain fermentation, the Da2d The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1. hpB The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO. 2. Da2d Genes and hpB Genes related to foam production, knockout Da2d Gene knockout and co-knockout in Yarrowia lipolytica Da2d and hpB The knockout gene can significantly reduce the foam-producing performance of the strain. At the same time, the strain after knockout retains the ability to efficiently synthesize erythritol, which is beneficial to the production of erythritol.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to a method for knocking out Da2d Gene or Da2d Genes and hpB Application of genes in reducing the foaming performance of erythritol fermentation. Background Art

[0002] Yarrowia lipolytica ( Yarrowia lipolytica ) can utilize microbial fermentation to produce erythritol, and this method has unique advantages in terms of fermentation yield and strain safety. However, the Yarrowia lipolytica strains currently used at home and abroad will produce a large amount of foam during the fermentation process. The foam produced by fermentation will not only reduce the effective utilization rate of the fermentation tank, but also easily overflow the tank and cause bacterial contamination, causing losses to the company. In order to reduce the formation of foam, it is necessary to add a defoaming agent to the fermentation broth, but the use of a defoaming agent will not only increase the cost and increase the difficulty of product separation and extraction, but also inhibit cell growth. At present, the cause of erythritol foam formation is unknown, and there are few reports on related genes and related functional strains that inhibit foam production. Therefore, the research and development of new strains of Yarrowia lipolytica with no-foam or low-foam fermentation is of great significance to improving the fermentation efficiency of erythritol and reducing fermentation costs. Summary of the invention

[0003] In order to solve the above problems, the present invention provides a method for knocking out Da2d Gene or Da2d Genes and hpB The invention discloses an application of a gene in reducing the foam production performance of erythritol fermentation. Da2d Genes and hpB Genes related to foam production, knockout Da2d Gene knockout in Yarrowia lipolytica Da2d and hpB Genes can significantly reduce the foam-producing performance of the strains.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a knockout strain Da2d Gene or knockout strains Da2d Genes and hpB The application of gene in reducing the foam production performance of bacterial strain fermentation, the Da2d The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1. hpB The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; the bacterial species includes Yarrowia lipolytica.

[0006] Preferably, theDa2d The nucleotide sequence of the gene is shown in SEQ ID NO.3. hpB The nucleotide sequence of the gene is shown in SEQ ID NO.4.

[0007] Preferably, the knockout Da2d The knockout component of the gene is the first knockout component; knocking out the gene in the strain Da2d Genes and hpB The knockout component of the gene consists of the first knockout component and the second knockout component; the nucleotide sequence of the first knockout component is shown in SEQ ID NO.14, and the nucleotide sequence of the second knockout component is shown in SEQ ID NO.24.

[0008] Preferably, the first kit for constructing the first knockout component includes a first primer set, and the second kit for constructing the second knockout component includes a second primer set; the first primer set includes a first primer pair, a second primer pair and a third primer pair, the nucleotide sequence of the first primer pair is shown in SEQ ID NO.5 and SEQ ID NO.6, the nucleotide sequence of the second primer pair is shown in SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of the third primer pair is shown in SEQ ID NO.9 and SEQ ID NO.10; the second primer set includes a fourth primer pair, a fifth primer pair and a sixth primer pair, the nucleotide sequence of the fourth primer pair is shown in SEQ ID NO.15 and SEQ ID NO.16, the nucleotide sequence of the fifth primer pair is shown in SEQ ID NO.17 and SEQ ID NO.18, and the nucleotide sequence of the sixth primer pair is shown in SEQ ID NO.19 and SEQ ID NO.20.

[0009] Preferably, the first kit further comprises Yarrowia lipolytica and pGAPZαA vector; the second kit further comprises Yarrowia lipolytica and pBC-hygro vector.

[0010] Preferably, the method for constructing the first knockout component comprises the following steps:

[0011] Using the genome of Yarrowia lipolytica as a template, PCR amplification was performed using the first primer pair to obtain Da2d The upstream fragment of the gene;

[0012] Using the genome of Yarrowia lipolytica as a template, PCR amplification was performed using the second primer pair to obtain Da2d Downstream fragment of the gene;

[0013] Using the pGAPZαA vector as a template, PCR amplification was performed using the third primer pair to obtain zeoresistance fragment;

[0014] Using the overlap extension PCR method, the Da2d The upstream fragment of the gene Da2d The downstream fragment of the gene and zeo The resistant fragments were connected to obtain the first knockout assembly.

[0015] Preferably, the method for constructing the second knockout component comprises the following steps:

[0016] Using the genome of Yarrowia lipolytica as a template, PCR amplification was performed using the fourth primer pair to obtain hpB The upstream fragment of the gene;

[0017] Using the genome of Yarrowia lipolytica as a template, PCR amplification was performed using the fifth primer pair to obtain hpB Downstream fragment of the gene;

[0018] Using the pBC-hygro vector as a template, PCR amplification was performed using the sixth primer pair to obtain Hyg resistance fragment;

[0019] Using the overlap extension PCR method, the hpB The upstream fragment of the gene hpB The downstream fragment of the gene and Hyg The resistance fragments were connected to obtain the second knockout component.

[0020] Preferably, the knockout Da2d The genetic method comprises the following steps:

[0021] Transforming the first knockout component into Yarrowia lipolytica to obtain a first transformant;

[0022] The first transformant was spread on a plate containing bleomycin, and the colonies obtained by culture were knockout Da2d Genes of Yarrowia lipolytica.

[0023] Preferably, the knockout Da2d Genes and hpB The genetic method comprises the following steps:

[0024] The second knockout component is transferred into the knockout Da2d The second transformant was obtained in Yarrowia lipolytica containing the gene;

[0025] The second transformant was spread on a plate containing hygromycin, and the colonies obtained by culture were knockout Da2d Genes and hpB Genes of Yarrowia lipolytica.

[0026] Beneficial effects:

[0027] The present invention provides a knockout strain Da2d Gene or knockout strains Da2d Genes and hpB The application of gene in reducing the foam production performance of bacterial strain fermentation, the Da2d The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1. hpB The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; the bacterial species includes Yarrowia lipolytica. Da2d Genes and hpB Genes related to foam production, knockout Da2d Gene knockout in Yarrowia lipolytica Da2d and hpB The knockout gene can significantly reduce the foam production performance of the strain, which is significantly reduced by 15%~75% compared with the original strain, and improves the fermentation efficiency. At the same time, the knocked-out strain retains the performance of efficiently synthesizing erythritol, which is beneficial to the production of erythritol. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0029] Figure 1 Erythritol production of different strains. DETAILED DESCRIPTION

[0030] The present invention provides a knockout strain Da2d Gene or knockout strains Da2d Genes and hpB The invention relates to an application of a gene in reducing the foam production performance of a strain, wherein the strain comprises Yarrowia lipolytica, and the Da2d The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1. hpB The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2, and is as follows:

[0031] SEQ ID NO.1:

[0032] MVSSAATSALPISAPYTFYPQARVPAPKKLVGLNAALEAQKNPEFEVKPEIFKEFSLPDGVAIVTGGNSGIGLEYSVCLAELGATVYCLDMPETPSEEFLACQSYVKRMPGNASLVFKRADVTDEETMNSLFQNIAETHGKIDVVIANAGVLGPRASCNEYPADWFRKVMDVNVTGVFITAQAASRQMIATKTSGSIIVTASMSGSIVNRDMPWCAYNASKAAAAHLVKSMAAELGQFEIRVNSISPGHIQTAMTDVCLDAEPGLGNQWAFQNPMGRLGGVSELRGVCAYLASSASSYTTGSDILVCGGHHVW;

[0033] SEQ ID NO.2:

[0034]

[0035] As an embodiment, the strain is Yarrowia lipolytica. As another embodiment, the Yarrowia lipolytica of the present invention is Yarrowia lipolytica CICC 1457, which is purchased from China Industrial Microbiological Culture Collection Center, and the strain collection number is CICC 1457.

[0036] As an implementation mode, the Da2d The nucleotide sequence of the gene is shown in SEQ ID NO.3. hpB The nucleotide sequence of the gene is shown in SEQ ID NO.4, and is as follows:

[0037] SEQ ID NO.3:5'-atggtttcttcagccgctacttctgctctgcccatctcggcaccctacaccttctaccctcaggctcgagttcctgcccccaagaagctcgttggactcaatgctgctctggaggcccagaagaaccccgagttcgaggtgaagcccgagatctttaaggagttctctctgcccgacggtgttgccattgtcaccggaggaaactccggtattggtcttgagtactcagtctgcctcgccgagctcggtgccactgtctactgtcttgacatgcccgagactccctctgaggagttcctggcttgccagtcctacgttaagcgaatgcccggcaacgcctctctggtcttcaagcgagccgacgtcactgacgaggagactatgaactccctcttccagaacattgccgagacccacggcaagattgacgttgtcatcgctaacgccggtgtgcttggacctcgagcctcttgcaacgagtaccccgctgactggttccgaaaggtcatggacgtcaacgtcaccggtgtctttatcaccgcccaggccgcctctcgacagatgattgccaccaagacttctggttctatcattgtcaccgcctccatgtccggctccattgtcaaccgagacatgccctggtgcgcctacaacgcctccaaggccgctgctgctcatcttgtcaagtccatggctgctgagctcggccagtttgagattcgagtcaactccatctcccccggtcacatccagactgctatgactgacgtctgtcttgacgctgagcccggtcttggtaaccagtgggccttccagaaccccatgggccgacttggaggtgtctccgagcttcgaggagtctgcgcctaccttgcatcttccgcctcctcctacaccaccggctctgacattcttgtctgcggtggccaccacgtctggtaa-3';

[0038]

[0039] The present invention finds Da2d Genes and hpB Genes related to foam production, knockout Da2d Gene knockout in Yarrowia lipolytica Da2d and hpB The gene can significantly reduce the foam-producing performance of the strain while retaining the ability to efficiently synthesize erythritol, which is of great significance for the fermentation synthesis of erythritol and also provides technical support for the foam-free or low-foam fermentation of other strains.

[0040] As an embodiment, knocking out Da2d The knockout component of the gene is the first knockout component; knocking out the gene in the strain Da2d Genes and hpB The knockout component of the gene consists of the first knockout component and the second knockout component; the nucleotide sequence of the first knockout component is shown in SEQ ID NO.14, and the nucleotide sequence of the second knockout component is shown in SEQ ID NO.24. The first knockout component of the present invention can be used to knock out the gene in Yarrowia lipolytica through the principle of homologous recombination. Da2d Replace part of the gene sequence to destroy the function of the gene, and first knock out the zeo The resistance fragment (shown in SEQ ID NO.13) can be used to screen the recombinant strain by bleomycin when constructing the recombinant strain, thereby obtaining a recombinant strain with significantly reduced foaming performance during the fermentation process, thereby simplifying the screening method; the second knockout component of the present invention can be used to knock out the recombinant strain in Yarrowia lipolytica by the principle of homologous recombination. hpB The partial sequence of the gene is replaced, destroying the function of the gene, and the second knockout component Hyg The resistance fragment (shown in SEQ ID NO.23) can be screened by hygromycin when constructing a recombinant strain to obtain a recombinant strain with significantly reduced foaming performance during fermentation, thereby simplifying the screening method. When the knockout component is the first knockout component and the second knockout component, the working principle and screening method are the same as above and will not be repeated here.

[0041] As an embodiment, a first kit for constructing the first knockout component includes a first primer set, and a second kit for constructing the second knockout component includes a second primer set; the first primer set includes a first primer pair, a second primer pair and a third primer pair, the nucleotide sequence of the first primer pair is shown in SEQ ID NO.5 and SEQ ID NO.6, the nucleotide sequence of the second primer pair is shown in SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of the third primer pair is shown in SEQ ID NO.9 and SEQ ID NO.10; the second primer set includes a fourth primer pair, a fifth primer pair and a sixth primer pair, the nucleotide sequence of the fourth primer pair is shown in SEQ ID NO.15 and SEQ ID NO.16, the nucleotide sequence of the fifth primer pair is shown in SEQ ID NO.17 and SEQ ID NO.18, and the nucleotide sequence of the sixth primer pair is shown in SEQ ID NO.19 and SEQ ID NO.20.

[0042] As an embodiment, the first kit further comprises Yarrowia lipolytica and pGAPZαA vector; the second kit further comprises Yarrowia lipolytica and pBC-hygro vector.

[0043] As an embodiment, the method for constructing the first knockout component comprises the following steps:

[0044] Using the genome of Yarrowia lipolytica as a template, PCR amplification was performed using the first primer pair to obtain Da2d The upstream fragment of the gene;

[0045] Using the genome of Yarrowia lipolytica as a template, PCR amplification was performed using the second primer pair to obtain Da2d Downstream fragment of the gene;

[0046] Using the pGAPZαA vector as a template, PCR amplification was performed using the third primer pair to obtain zeo resistance fragment;

[0047] Using the overlap extension PCR method, the Da2d The upstream fragment of the gene Da2d The downstream fragment of the gene and zeo The resistant fragments were connected to obtain the first knockout assembly.

[0048] As an embodiment, the method for constructing the second knockout component comprises the following steps:

[0049] Using the genome of Yarrowia lipolytica as a template, PCR amplification was performed using the fourth primer pair to obtain hpB The upstream fragment of the gene;

[0050] Using the genome of Yarrowia lipolytica as a template, PCR amplification was performed using the fifth primer pair to obtain hpB Downstream fragment of the gene;

[0051] Using the pBC-hygro vector as a template, PCR amplification was performed using the sixth primer pair to obtain Hyg resistance fragment;

[0052] Using the overlap extension PCR method, the hpB The upstream fragment of the gene hpB The downstream fragment of the gene and Hyg The resistance fragments were connected to obtain the second knockout component.

[0053] As an embodiment, knocking out Da2d The genetic method comprises the following steps:

[0054] Transforming the first knockout component into Yarrowia lipolytica to obtain a first transformant;

[0055] The first transformant was spread on a plate containing bleomycin, and the colonies obtained by culture were knockout Da2d Genes of Yarrowia lipolytica.

[0056] As another embodiment, knock out the strain Da2d Genes and hpB The genetic method comprises the following steps:

[0057] The second knockout component is transferred into the knockout Da2d The second transformant was obtained in Yarrowia lipolytica containing the gene;

[0058] The second transformant was spread on a plate containing hygromycin, and the colonies obtained by culture were knockout Da2d Genes and hpB Genes of Yarrowia lipolytica.

[0059] Based on the above advantages, the present invention preferably also provides the knockout Da2d The Yarrowia lipolytica or the knockout of the above-mentioned technical scheme Da2d Genes and hpB Application of Yarrowia lipolytica to the production of erythritol.

[0060] The present invention also preferably provides a method for knocking out Da2d The Yarrowia lipolytica or the knockout of the above-mentioned technical scheme Da2d Genes and hpB The method for producing erythritol by fermentation of Yarrowia lipolytica containing a gene comprises the following steps:

[0061] (1) Knock out the above technical solution Da2d The Yarrowia lipolytica or the knockout of the above-mentioned technical scheme Da2d Genes and hpB The Yarrowia lipolytica with the gene was inoculated into liquid culture medium and cultured until DO 600 =1.5~2, to obtain seed solution;

[0062] (2) The seed solution prepared in the above step (1) is inoculated into an erythritol fermentation medium at a volume fraction of 8% to 10%, and fermented to obtain erythritol.

[0063] As an embodiment, the composition of the liquid culture medium is 18-20 g / L glucose, 18-20 g / L peptone, 8-10 g / L yeast powder and the balance water; the culture temperature for preparing the seed liquid is 28-30°C.

[0064] As an embodiment, the composition of the erythritol fermentation medium is: 240-250 g / L glucose, 4-5 g / L yeast extract powder, 8-10 g / L peptone and the balance water; the fermentation culture temperature is 28-30°C, and the culture is for more than 30 hours.

[0065] In order to further illustrate the present invention, the following is a method provided by the present invention by knocking out Da2d Gene or Da2d Genes and hpB The application of genes in reducing the foaming performance of erythritol fermentation is described in detail, but they should not be understood as limiting the scope of protection of the present invention.

[0066] Example 1

[0067] The inventors studied the low-foaming strain Yarrowia lipolytica ( Yarrowia lipolytica ) The genome sequencing analysis of Q0010 revealed for the first time that the low-foaming strain Yarrowia lipolytica Q0010 Da2d There is a mutation in the gene, in order to verify Da2d The effect of genes on foam production was studied. Da2d Gene sequence analysis and cloning.

[0068] The inventors compared the Yarrowia lipolytica Q0010 before mutation Da2d Sequence and Yarrowia lipolytica CICC1457 (purchased from China Industrial Microbiological Culture Collection Center, with the culture collection number CICC 1457) Da2d The gene has a similarity of 100%, as shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by it is shown in SEQ NO.1.

[0069] The inventors have studied the Da2d Gene knockout to determine Da2d The relationship between genes and foam synthesis. The inventors found through experiments that Da2d The foaming performance of the recombinant Yarrowia lipolytica obtained after gene knockout was significantly reduced (reduced by 15% relative to CICC1457). Knocking out the Da2d gene in the strain can reduce the foaming performance of the strain. The construction method of the recombinant Yarrowia lipolytica is as follows:

[0070] Gene knockout components were constructed using gene fusion technology, and the genome of Yarrowia lipolytica CICC 1457 was used as a template to design amplification primers and amplify the gene by PCR reaction. Da2d The upstream and downstream fragments of the gene; using the pGAPZαA vector (purchased from Wuhan Miaoling Biotechnology Co., Ltd.) as a template, the amplification primers were designed and amplified by PCR reaction zeo Resistance fragments.

[0071] Da2d The PCR reaction system for the upstream and downstream fragments of the gene was: 2 μL yeast genomic DNA, 1 μL upstream primer, 1 μL downstream primer, 2 μL dNTP, 5 μL MgCl2, 5 μL LA Taq polymerase buffer, 0.5 μL LA Taq polymerase and 3.5 μL ddH2O3. Da2d The PCR reaction program of the gene was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 30 cycles; and extension at 72°C for 5 min.

[0072] zeo The PCR reaction system of the fragment was: 2 μL pGAPZαA vector DNA, 1 μL upstream primer, 1 μL downstream primer, 2 μL dNTP, 5 μL MgCl2, 5 μL LA Taq polymerase buffer, 0.5 μL LA Taq polymerase and 33.5 μL ddH2O. zeo The PCR reaction program of the fragment was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1.5 min, 30 cycles; and extension at 72°C for 5 min.

[0073] Amplification Da2d The primers for the upstream fragment of the gene are:

[0074] Da2d-UF: 5'-atggtttcttcagccgctac-3', SEQ ID NO.5;

[0075] Da2d-UR: 5'-ccgcacaaacgaaggtctggaagaggggagttcata-3', SEQ ID NO.6;

[0076] Amplification Da2d The primers for the downstream fragment of the gene are:

[0077] Da2d-DF: 5'-aaggctttaatttgcccaggccgcctctcgacaga-3', SEQ ID NO.7;

[0078] Da2d-DR: 5'-ttaccagacgtggtggccac-3', SEQ ID NO.8;

[0079] Amplification zeo The primers are:

[0080] zeo-F: 5'-ccagaccttcgtttgtgcggatccc-3', SEQ ID NO.9;

[0081] zeo-R: 5'-ggcctgggcaaattaaagccttcgagc-3', SEQ ID NO. 10.

[0082] Da2d The upstream fragment sequence of the gene is shown in SEQ ID NO.11. Da2d The downstream fragment sequence of the gene is shown in SEQ ID NO.12. zeo The sequence of the resistance fragment is shown in SEQ ID NO.13, which is as follows:

[0083] SEQ ID NO.11:5'-atggtttcttcagccgctacttctgctctgcccatctcggcaccctacaccttctaccctcaggctcgagttcctgcccccaagaagctcgttggactcaatgctgctctggaggcccagaagaaccccgagttcgaggtgaagcccgagatctttaaggagttctctctgcccgacggtgttgccattgtcaccggaggaaactccggtattggtcttgagtactcagtctgcctcgccgagctcggtgccactgtctactgtcttgacatgcccgagactccctctgaggagttcctggcttgccagtcctacgttaagcgaatgcccggcaacgcctctctggtcttcaagcgagccgacgtcactgacgaggagactatgaactccctcttccaga-3';

[0084] SEQ ID NO.12:5'-ccaggccgcctctcgacagatgattgccaccaagacttctggttctatcattgtcaccgcctccatgtccggctccattgtcaaccgagacatgccctggtgcgcctacaacgcctccaaggccgctgctgctcatcttgtcaagtccatggctgctgagctcggccagtttgagattcgagtcaactccatctcccccggtcacatccagactgctatgactgacgtctgtcttgacgctgagcccggtcttggtaaccagtgggccttccagaaccccatgggccgacttggaggtgtctccgagcttcgaggagtctgcgcctaccttgcatcttccgcctcctcctacaccaccggctctgacattcttgtctgcggtggccaccacgtctggtaa-3';

[0085]

[0086] FastPure Gel DNA Extraction Mini Kit Da2d Gene upstream fragments, downstream fragments and zeo The fragments were recovered by gel extraction and then cloned by overlap extension PCR. Da2d The upstream and downstream fragments of the gene were connected to zeo Upstream and downstream of gene expression elements, PCR reaction system: 2μL Da2d Gene upstream fragment, Da2d Gene downstream fragments and zeo The gel recovery product mixture of the fragment, 1 μL of upstream and downstream primers (SEQ ID NO.5 and SEQ ID NO.8), 2 μL of dNTP, 5 μL of MgCl2, 5 μL of LA Taq polymerase buffer, 0.5 μL of LA Taq polymerase and 29.5 μL of ddH2O; Da2d Gene upstream fragment, Da2d Gene downstream fragments and zeo The molar ratio of the fragments was 1:1:1. PCR cycle conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 2 min, 30 cycles; 72°C extension for 5 min. Da2d Gene knockout components, Da2d The gene knockout component is Da2d The fragment formed by the fusion of the upstream and downstream fragments of the gene and the resistance screening gene, Da2d The gene knockout component structure is: Da2d Upstream zeo Gene- Da2d Downstream, Da2d

[0087] Refer to the lithium acetate conversion method in Wang Yuancai's paper [Wang Yuancai. Metabolic engineering of Saccharomyces cerevisiae rTCA pathway and C4-dicarboxylic acid transport system to produce L-malic acid [D]. Jiangnan University, 2016.] Da2d The gene knockout component is transferred into the Yarrowia lipolytica CICC 1457 strain. After the gene knockout component enters the bacteria, the Yarrowia lipolytica CICC 1457 strain can be transformed into the Yarrowia lipolytica CICC 1457 strain through homologous recombination. Da2d The partial sequence of the gene was replaced to destroy the function of the gene. The transformants were screened on YPD plates containing 600 μg / mL zeocin (bleomycin) and obtained. Da2d The Yarrowia lipolytica strain in which the gene is knocked out is the recombinant yeast strain DS-10. Da2d Knockout components can also be introduced into Yarrowia lipolytica strains by electroporation.

[0088] Example 2

[0089] The inventors sequenced and analyzed the genome of the low-foaming strain Yarrowia lipolytica Q008 preserved in the laboratory and found that hpB There is a mutation in the gene, in order to verify hpB The effect of genes on foam production was studied. hpB Gene sequence analysis and cloning.

[0090] The inventors compared the hpB sequence before mutation in Yarrowia lipolytica Q008 and that in Yarrowia lipolytica CICC 1457. hpB The gene has a similarity of 100%, as shown in SEQ ID NO.4, and the amino acid sequence of the protein encoded by it is shown in SEQ NO.2.

[0091] The inventors have studied the hpB Gene knockout to determine hpB The relationship between genes and foam synthesis. The inventors found through experiments that hpB The foaming capacity of the recombinant Yarrowia lipolytica obtained by knocking out the gene was significantly reduced (reduced by 53% compared with CICC1457). hpB The gene can reduce the foaming performance of the strain. The construction method of the recombinant Yarrowia lipolytica is as follows:

[0092] Gene knockout components were constructed using gene fusion technology, and the genome of Yarrowia lipolytica CICC 1457 was used as a template to design amplification primers and amplify the gene by PCR reaction. hpBThe upstream and downstream fragments of the gene; using the pBC-hygro vector (purchased from Aonogen) as a template, the amplification primers were designed and amplified by PCR reaction Hyg Resistance fragments.

[0093] hpB The PCR reaction system for the upstream and downstream fragments of the gene was: 2 μL yeast genomic DNA, 2 μL upstream and downstream primers, 25 μL Phanta enzyme and 19 μL ddH2O. PCR cycle conditions: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1.5 min, 30 cycles; 72°C extension for 10 min.

[0094] Hyg The fragment PCR reaction system was: 2 μL pBC-hygro plasmid DNA, 2 μL upstream and downstream primers, 25 μL Phanta enzyme and 19 μL ddH2O. PCR cycle conditions: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1.5 min, 30 cycles; 72°C extension for 10 min.

[0095] In the PCR reaction, amplification hpB The primers upstream of the gene are:

[0096] hpB-UF: 5'-atgcttcccaaggctattct-3', SEQ ID NO.15;

[0097] hpB-UR: 5'-gcagcaacttcaaaggcgtcagtaaagaacttgtc-3', SEQ ID NO. 16;

[0098] Amplification hpB The primers downstream of the gene are:

[0099] hpB-DF: 5'-tcaccagcccctggggaactcccggacccgccgga-3', SEQ ID NO. 17;

[0100] hpB-DR: 5'-ttacatcagaagagggataa-3', SEQ ID NO. 18;

[0101] Amplification Hyg The primers are:

[0102] Hyg-F: 5'-acgcctttgaagttgctgcaagct-3', SEQ ID NO.19;

[0103] Hyg-R: 5'-gagttccccaggggctggtgacggaa-3', SEQ ID NO. 20.

[0104] hpB The upstream fragment sequence of the gene is shown in SEQ ID NO.21. hpB The downstream fragment sequence of the gene is shown in SEQ ID NO.22. Hyg The sequence of the resistance fragment is shown in SEQ ID NO.23, and is as follows:

[0105] SEQ ID NO.21: 5'-atgcttcccaaggctattctattgggcctcctggtccctctagccagcgccgatgttagtagtcacaacttcatcgtcgacaatatcgcctctgccgcctccaatgccattgtgaacaa cagagtgtccggataccccgaggctagtgcccagtccgaggtcggagaccttctgtcccacatcatgggcaaccagggcctctacaatatcttcgtctctgccattggcattgttctgaacggaggtt tcaatgctgacaatttccctccctttgtcaatgtcattgaaagcgccctcaattcgtacaagcagaccccagacttcaccactgcctcccagattgtgtcgcagtacaagtccaactactacgttacc ccagcctactctgctgccatctcaaccaacaccttctacgtcggtctactgactcctggtgtcaccaacgccgcgtctactaactcggtggcctccgcttctggacaagttctttactgacgc-3';

[0106] SEQ ID NO.22:5'-gaactcccggacccgccggaaagcctggtgcccctgctcagcctggtgctcctgctcagtctggccaccccgcccagcccaatactccaggtgctgccggaactcccggacccgccggaaagcctggtgcccctgctcagcctggtgcccctgctcagtctggccaccccgcccagccccatactccaggtgctgccggaactcccggacccgccggaaagcctggtgcccctgctcagtctgacgctcctgcgaaacctcaggcccctgctcaggccggcacccctggaaagccgggttctcctggagctcctgctccttctcagtctcccgcccaggtcggtactcccggcaagcctggtgttgccgctggacagccccaggctcccgctcaagctcccgcctcttctcagtttggtgttgctcaggccaacggcgccgctactacaggacctgccattctcactacactcttggctcttatccctcttctgatgtaa-3';

[0107]

[0108] FastPure Gel DNA Extraction Mini Kit hpB-based Due to the upstream fragment, downstream fragment and Hyg The fragments were recovered by gel extraction and then cloned by overlap extension PCR. hpB The upstream and downstream fragments of the gene were connected to Hyg Upstream and downstream of the gene expression element, PCR reaction system: 2μL hpB Gene upstream fragment, hpB Gene downstream fragments and Hyg The gel recovery product mixture of the fragment, 2 μL of upstream and downstream primers (SEQ ID NO.15 and SEQ ID NO.20), 25 μL of Phanta enzyme and 19 μL of ddH2O; the gel recovery product mixture hpB Gene upstream fragment, hpB Gene downstream fragments and Hyg The molar ratio of the fragments was 1:1:1. PCR cycle conditions: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, 30 cycles; 72°C extension for 10 min. hpB Gene knockout components, hpB The gene knockout component is hpB The fragment formed by the fusion of the upstream and downstream fragments of the gene and the resistance screening gene, hpB The gene knockout component structure is: hpB Upstream Hyg Gene- hpB Downstream, hpB

[0109] Referring to the lithium acetate conversion method in Wang Yuancai's paper, hpB The gene knockout component is transferred into the Yarrowia lipolytica CICC1457 strain. After the gene knockout component enters the bacteria, the Yarrowia lipolytica CICC 1457 strain can be transformed into the Yarrowia lipolytica CICC 1457 strain through homologous recombination. hpB The transformants were screened on YPD plates containing 500 μg / mL Hyg (hygromycin) and obtained hpB The Yarrowia lipolytica strain in which the gene is knocked out is the recombinant yeast strain DS-20. The hpB gene knockout component can also be transformed into the Yarrowia lipolytica strain by electroporation.

[0110] Example 3

[0111] Based on the findings in Example 1 and Example 2, the inventors conducted a Da2d Gene and hpB Gene knockout was performed to determine Da2d Genes and hpB Interrelationship between genes and foam synthesis.

[0112] The recombinant yeast strain DS-10 obtained in Example 1 was used as the starting strain, and the recombinant yeast strain DS-10 obtained in Example 2 was transformed into hpB The gene knockout component is transferred into the starting strain. After entering the bacteria, the gene knockout component can be used to knock out the recombinant yeast strain DS-10 through homologous recombination. hpB The transformants were screened on YPD plates containing hygromycin (concentration of 400 μg / mL) and bleomycin (concentration of 500 μg / mL) to obtain Da2d and hpB The gene co-knockout Yarrowia lipolytica strain is the recombinant yeast strain DS-30. The Da2d and hpB gene knockout components can also be transformed into the Yarrowia lipolytica strain by electroporation.

[0113] Example 4

[0114] A shake flask fermentation foaming test of a recombinant Yarrowia lipolytica strain for reducing fermentation foam is as follows:

[0115] The original strain Yarrowia lipolytica CICC 1457 and the recombinant strains in Example 1 (DS-10), Example 2 (DS-20) and Example 3 (DS-30) were inoculated into 250 mL shaking flasks containing 50 mL of liquid culture medium, the medium composition of which was: 20 g / L glucose, 20 g / L peptone, 10 g / L yeast powder and the rest of water; cultured at 30°C for 24 hours to obtain seed liquid, and the seed liquid was inoculated at a volume fraction of 10% into 50 mL of liquid fermentation medium, the fermentation medium composition of which was: 250 g / L glucose, 5 g / L yeast extract powder, 10 g / L peptone and the rest of distilled water. Cultured in a shaking table at 30°C for 30 hours, and observed the foaming situation.

[0116] After 30 hours of cultivation of the recombinant strain in a 250 mL shake flask, the original strain produced more foam, while the recombinant strain produced less foam. The foam of the original strain disappeared after standing for 35 minutes, while the foam produced by the recombinant strain disappeared completely after standing for 10 to 15 minutes. The foaming performance was calculated based on the foam disappearance time. The foaming performance of the recombinant strain DS-10 was significantly reduced by 15% compared with the original strain, the foaming performance of the recombinant strain DS-20 was significantly reduced by 53% compared with the original strain, and the foaming performance of the recombinant strain DS-30 was significantly reduced by 75% compared with the original strain.

[0117] The present invention finds Da2d and hpB Genes are linked to bubble formation. Da2d The gene is involved in the conversion between pentose and glucuronic acid in the metabolism of Yarrowia lipolytica, and to a certain extent in the conversion between fructose and mannitol. Da2d The gene can reduce the foam produced by fermentation of bacteria, providing some technical support for the cause of erythritol foam. Da2d and hpB Gene, which can significantly reduce the performance of the strain in producing foam while retaining the performance of efficiently synthesizing erythritol (see Figure 1 ); The present invention constructs Da2d Knockout strain DS-10 and Da2d Genes and hpB The gene co-knockout recombinant strain DS-30 has significant improvements in reducing foam and is beneficial to the production of erythritol.

[0118] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Knockout strains Da2D Gene or knockout strains Da2D Genes and HbB The application of gene in reducing the foam production performance of bacterial strain fermentation, the Da2D The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

1. HbB The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2; the bacterial species includes Yarrowia lipolytica ( Yarrowia lipolytica ).

2. The use according to claim 1, characterized in that: Said Da2D The nucleotide sequence of the gene is shown in SEQ ID NO.

3. HbB The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

3. The use according to claim 1, characterized in that: Knock out the strain Da2D The knockout component of the gene is the first knockout component; knocking out the gene in the strain Da2D Genes and HbB The knockout component of the gene consists of the first knockout component and the second knockout component; the nucleotide sequence of the first knockout component is shown in SEQ ID NO.14, and the nucleotide sequence of the second knockout component is shown in SEQ ID NO.

24.

4. The use according to claim 3, characterized in that: The first kit for constructing the first knockout component includes a first primer set, and the second kit for constructing the second knockout component includes a second primer set; the first primer set includes a first primer pair, a second primer pair and a third primer pair, the nucleotide sequence of the first primer pair is shown in SEQ ID NO.5 and SEQ ID NO.6, the nucleotide sequence of the second primer pair is shown in SEQ ID NO.7 and SEQ ID NO.8, and the nucleotide sequence of the third primer pair is shown in SEQ ID NO.9 and SEQ ID NO.10; the second primer set includes a fourth primer pair, a fifth primer pair and a sixth primer pair, the nucleotide sequence of the fourth primer pair is shown in SEQ ID NO.15 and SEQ ID NO.16, the nucleotide sequence of the fifth primer pair is shown in SEQ ID NO.17 and SEQ ID NO.18, and the nucleotide sequence of the sixth primer pair is shown in SEQ ID NO.19 and SEQ ID NO.

20.

5. The use according to claim 4, characterized in that: The first kit also includes Yarrowia lipolytica and pGAPZαA vector; the second kit also includes Yarrowia lipolytica and pBC-hygro vector.

6. The use according to claim 4, characterized in that: The method for constructing the first knockout component comprises the following steps: Using the genome of Yarrowia lipolytica as a template, PCR amplification was performed using the first primer pair to obtain Da2D The upstream fragment of the gene; Using the genome of Yarrowia lipolytica as a template, PCR amplification was performed using the second primer pair to obtain Da2D Downstream fragment of the gene; Using the pGAPZαA vector as a template, PCR amplification was performed using the third primer pair to obtain zeo resistance fragment; Using the overlap extension PCR method, the Da2D The upstream fragment of the gene Da2D The downstream fragment of the gene and zeo The resistant fragments were connected to obtain the first knockout assembly.

7. The use according to claim 4, characterized in that: The method for constructing the second knockout component comprises the following steps: Using the genome of Yarrowia lipolytica as a template, PCR amplification was performed using the fourth primer pair to obtain HbB The upstream fragment of the gene; Using the genome of Yarrowia lipolytica as a template, PCR amplification was performed using the fifth primer pair to obtain HbB Downstream fragment of the gene; Using the pBC-hygro vector as a template, PCR amplification was performed using the sixth primer pair to obtain Hyg resistance fragment; Using the overlap extension PCR method, the HbB The upstream fragment of the gene HbB The downstream fragment of the gene and Hyg The resistance fragments were connected to obtain the second knockout component.

8. The use according to any one of claims 3 to 7, characterized in that: Knock out the strain Da2D The genetic method comprises the following steps: Transforming the first knockout component into Yarrowia lipolytica to obtain a first transformant; The first transformant was spread on a plate containing bleomycin, and the colonies obtained by culture were knockout Da2D Genes of Yarrowia lipolytica.

9. The use according to claim 8, characterized in that: Knock out the strain Da2D Genes and HbB The genetic method comprises the following steps: The second knockout component is transferred into the knockout Da2D The second transformant was obtained in Yarrowia lipolytica containing the gene; The second transformant was spread on a plate containing hygromycin, and the colonies obtained by culture were knockout Da2D Genes and HbB Genes of Yarrowia lipolytica.

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