CN117264791A - Kluyveromyces marxianus engineering strain for efficiently expressing exogenous proteins and application thereof - Google Patents
Kluyveromyces marxianus engineering strain for efficiently expressing exogenous proteins and application thereof Download PDFInfo
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- CN117264791A CN117264791A CN202310264215.3A CN202310264215A CN117264791A CN 117264791 A CN117264791 A CN 117264791A CN 202310264215 A CN202310264215 A CN 202310264215A CN 117264791 A CN117264791 A CN 117264791A
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a Kluyveromyces marxianus engineering strain for efficiently recombining and expressing exogenous proteins and application thereof. The Kluyveromyces marxianus engineering strain provided by the invention is prepared by taking Kluyveromyces marxianus FIM1 as a starting strain, adding 16 loxPSym sites on a chromosome of the FIM1, treating by Cre recombinase, and then screening at a high temperature and pressure of 46 ℃ to obtain a mutant strain, and marking the mutant strain as FDHY23; the mutant strain carries at least a mutation of the adenylate cyclase gene. The mutant strain FDHY23 can recombinantly express different exogenous proteins, the expression level is improved by 1.6-5.5 times, and the mutant strain still has the capability of expressing exogenous proteins at high temperature.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a Kluyveromyces marxianus engineering strain for efficiently recombining and expressing exogenous proteins and application thereof.
Background
Genetic mutation is an important means to increase the ability of host strains to recombinantly express foreign proteins. Genetic mutations can be produced by a variety of methods, including physico-chemical mutagenesis (Liu Y et al Microb Cell face.2018 Sep 14;17 (1): 144.), adaptive evolution (Huang CJ et al mol Biol Evol.2018 Aug 1;35 (8): 1823-1839.), genomic recombination (Wu L et al front bioengin Biotechnol.2022 Jan 11; 9:799756.), chromosomal rearrangement (Gowers GF et al Nat Commun.2020 Feb 13;11 (1): 868), and the like. The Cre-loxP system is a DNA recombination technology of specific sites of genome, and consists of Cre recombinase from P1 phage and 34bp loxP sequence. By introducing loxP sites into genomic DNA, the original 34bp loxP sequence is directional, and Cre recombinase can delete, insert, repeat, invert and translocate at specific sites according to the directionality of two loxP sequences, resulting in genetic alteration of genomic DNA (Sternberg N et al J Mol biol.1981Aug 25;150 (4): 467-86.). The loxPSym sequence is a variant of the loxP sequence, a 34bp sequence in full-symmetry palindromic, without orientation (Hoess RH et al nucleic Acids Res.1986Mar11; 14 (5): 2287-300.). The loxPsym sequence is introduced into genome, and under the action of Cre recombinase, the rearrangement type is random, so that more chromosome variation types can be generated. When using these genetic mutation methods, a large number of mutant strains are often generated, and rapid selection of high-yield strains therefrom has been a problem. The traditional screening method comprises enzyme activity measurement, fluorescence screening and the like. These methods are complicated to operate, time-consuming, and require a lot of manpower.
Recombinant high expression of foreign proteins in host cells often causes endoplasmic reticulum stress and oxidative stress in the host cells, and it is a key to high production of foreign proteins to increase host cell tolerance to oxidative stress (Raschmanov.a.H et al appl Microbiol Biotechnol.2021 Jun;105 (11): 4397-4414.Chen X et al.Metab Eng.2022 Jul;72:311-324.). The high temperature and the like can cause a series of stress reactions in cells, the high temperature is used as screening pressure, the cells need to obtain the capability of coping with the stress for survival, and the strain with improved tolerance also has the potential of high yield of the exogenous protein. The temperature and pressure are used for screening, and the screening method has the advantages of simplicity in operation, large screening amount, short time consumption and the like.
Kluyveromyces marxianus FIM1 is a host bacterium with food safety grade, and is preserved in China general microbiological culture Collection center (CGMCC) No.10621. Kluyveromyces marxianus FIM1 has good industrial application potential, and particularly, the Kluyveromyces marxianus engineering strain with high yield of exogenous proteins is a core technology for industrial production and application. The recombinant expression level of the exogenous protein is influenced by the genetic mutation of the host Kluyveromyces marxianus, and different mutation can produce different effects, so that the recombinant expression capacity of the Kluyveromyces marxianus for the exogenous protein is influenced.
Disclosure of Invention
The invention aims to provide a Kluyveromyces marxianus engineering strain capable of efficiently expressing exogenous proteins and application thereof.
The invention firstly provides a Kluyveromyces marxianus engineering strain for efficiently expressing exogenous proteins. The strain takes Kluyveromyces marxianus (Kluyveromyces marxianus) FIM1 preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.10621 and the preservation date of 2015.3.13 as a starting strain, adds 16 loxPSym sites on a chromosome of the FIM1, carries out Cre recombinase treatment, and then carries out screening under the high temperature pressure of 46 ℃ to obtain a mutant strain which is marked as FDHY23;
specifically, the gene URA3 on the FIM1 genome is deleted using conventional genome editing techniques; in the URA3 defective strain, 2 sites are respectively selected to integrate loxPSym sequences on 8 chromosomes of Kluyveromyces marxianus by utilizing a conventional gene editing technology, the obtained strain is named LHP1044, and the chromosomes of the strain contain 16 loxPSym sequences; the Cre recombinase gene is connected to a vector pFA6a-PanARS-PLAC4 (-1000) -KANMX6 by utilizing a conventional gene cloning technology to obtain a recombinant plasmid LHZ893 (SEQ ID No. 1); the recombinant plasmid LHZ893 is transformed into LHP1044 by a conventional lithium acetate transformation method of yeast, and is cultured and screened to obtain a mutant strain with obvious growth advantage at 46 ℃ under high temperature and pressure, and the strain is named FDHY23.
The Kluyveromyces marxianus FDHY23 mutant strain provided by the invention has obvious high-temperature growth advantage.
The Kluyveromyces marxianus FDHY23 mutant strain provided by the invention carries 94 mutations, 38 of which are SNP,56 of which are InDel, and the specific mutation conditions are shown in Table 1.
The Kluyveromyces marxianus FDHY23 mutant strain at least carries the mutation of an adenylate cyclase gene.
The Kluyveromyces marxianus FDHY23 mutant strain disclosed by the invention has CYR1 gene mutation, at least comprises, but is not limited to, point mutation, namely, the 1546 th amino acid is changed from asparagine (N) to lysine (K).
The mutation of the adenylate cyclase gene CYR1 can improve the recombinant expression level and high-temperature tolerance of the protein.
The Kluyveromyces marxianus (Kluyveromyces marxianus) mutant FDHY23 is preserved in China general microbiological culture Collection center (CGMCC) No.26724, and the preservation date is as follows: 2023.3.3.
the Kluyveromyces marxianus engineering strain FDHY23 constructed by the invention can be used for efficiently expressing exogenous proteins.
Experiments show that the Kluyveromyces marxianus FDHY23 recombinantly expresses different exogenous proteins, the expression level is improved by 1.6-5.5 times, and the Kluyveromyces marxianus FDHY has the capability of expressing exogenous proteins at high temperature.
The exogenous proteins specifically include, but are not limited to: feruloyl esterase (from yak stomach), feruloyl esterase (from Aspergillus niger), saccharifying enzyme (from Botrytis cinerea, adenylate budding), porcine circovirus-like particle protein, infectious bursal disease virus-like particle protein, ferritin, and leghemoglobin.
Drawings
FIG. 1 selection of high temperature resistant mutants of Kluyveromyces marxianus. Wherein (a) the growth of 24 Cre enzyme treated mutants was compared at 46 ℃. (b) Comparison of the temperature tolerance of mutant FDHY23 and of the starting strain LHP1044.
FIG. 2 analysis of the ability of Kluyveromyces marxianus mutant FDHY23 to grow at different temperatures. Wherein (a) the maximum biomass (OD) of the growth of strain FDHY23 and strain LHP1044 at 30 ℃, 46 ℃ and 47 DEG C 600 ). (b) Maximum growth rates of strain FDHY23 and strain LHP1044 at 30 ℃, 46 ℃ and 47 ℃.
FIG. 3 relative expression levels of different recombinant proteins expressed recombinantly by Kluyveromyces marxianus mutant FDHY23. Samples are taken after the engineering strain is fermented for 72 hours in a shaking bottle for enzyme activity measurement or SDS-PAGE analysis, and protein relative quantitative analysis is carried out on a gray-scale scanning SDS-PAGE protein band, wherein Control is the enzyme activity or gray-scale scanning value of different recombinant proteins expressed by the LHP1044 of the original strain and is defined as 1.EstE1 engineering strain FDHY23-Est1E has relative enzyme activity compared with feruloyl esterase (derived from yak stomach) of LHP1044-Est1E shake flask fermentation supernatant. AnfaeA is the relative enzyme activity of feruloyl esterase (from Aspergillus niger) of engineering strain FDHY23-AnfaeA compared with LHP1044-AnfaeA shake flask fermentation supernatant. Badgla engineering strain FDHY23-Badgla has relative enzyme activity compared with that of LHP1044-Badgla shake flask fermentation supernatant. PCV2 relative expression level (gray scale scan) of recombinant expression porcine circovirus-like particle protein by shake flask fermentation of engineering strain FDHY23-PCV2 compared with LHP1044-PCV2. HVP2 engineering strain FDHY23-HVP2 compared with LHP1044-HVP2, shake flask fermentation to recombinant express infectious bursal disease virus like particle protein. Fth1 engineering strain FDHY23-Fth1 compared with LHP1044-Fth1, shake flask fermentation and recombinant expression of ferritin (gray scanning). LBA relative expression level (gray scale scan) of recombinant expressed hemoglobin by engineering strain FDHY23-LBA versus LHP1044-LBA shake flask fermentation.
FIG. 4 comparison of recombinant expression of hemoglobin at high temperature 46℃of the starting strain LHP1044 and the mutant FDHY23. Protein expression level SDS-PAGE analysis of engineering strains FDHY23-LBA and LHP1044-LBA shake flask fermented to different time points (24 h,36h,48h,60h and 72 h) at 46 ℃.
FIG. 5 sequence comparison of the mutant part of the adenylate cyclase gene CYR1 of mutant FDHY23 relative to strain LHP 10444.
FIG. 6A-CYR 1 point mutation of the adenylate cyclase gene increases temperature tolerance and recombinant protein expression. LHP1044 is the starting strain, FDHY23 is the mutant strain obtained by screening, LHP1044-CYR1 N1546K The amino acid of gene CYR1 of LHP1044 is replaced by K at 1546 position by utilizing genome editing technology, and FDHY23-CYR1 is CYR1 in mutant strain FDHY23 by utilizing genome editing technology N1546K Is reverted to wild-type CYR1. Wherein (a) the CYR1 is point mutated N1546K Influence on temperature resistance. (b.) Point mutation CYR1 N1546K Effects on recombinant expressed proteins. Left diagram: recombinant LBA protein expressing engineering strain LHP1044-LBA and LHP1044-CYR1 N1546K LBA shake flask fermentation was performed for 72h, protein expression was analyzed by SDS-PAGE, LHP1044 fermentation was used as a blank. Right figure: relative quantitative analysis was performed by gray scanning the SDS-PAGE LBA protein bands of the left panel.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1
Screening of high temperature resistant mutant strains of Kluyveromyces marxianus
The strain FIM1 preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.10621 is taken as a starting strain, and the version number of a reference genome of the FIM1 is GCA_001854445.2. The gene URA3 on the genome was deleted using conventional genome editing techniques. In the URA3 deficient strain, 2 sites of integrated loxPSym sequences are selected on 8 chromosomes of Kluyveromyces marxianus respectively by utilizing a conventional gene editing technology, and 16 loxPSym sequences are integrated in total, so that the obtained strain is named as LHP1044. Specific positions of integration of the 16 loxppsym sequences on the chromosome are: 80654bp and 297580bp of chromosome 1, 706654bp and 885427bp of chromosome 2, 327216bp and 1226714bp of chromosome 3, 715829bp and 1172907bp of chromosome 4, 340152bp and 746775bp of chromosome 5, 130814bp and 446862bp of chromosome 6, 392737bp and 674407bp of chromosome 7, and 435906bp and 606918bp of chromosome 8. The Cre recombinase gene was ligated to the vector pFA6a-PanARS-PLAC4 (-1000) -KANMX6 using conventional gene cloning techniques to obtain a recombinant plasmid LHZ893 (SEQ ID No. 1). The recombinant plasmid LHZ893 was transformed into LHP1044 by a conventional lithium acetate transformation method using yeast, and the SD plate was coated and cultured at 30℃for 2-3 days. The transformants were collected, mixed and transferred to YPD liquid medium for 10-15 hours at 30℃and diluted to a suitable gradient for coating YPD solid plates, and placed in a 46℃incubator for culture, and 24 clones with growth advantage were selected (FIG. 1 a). These 24 clones were inoculated into 96-well plates, after incubation at 30℃for 4h, spotted onto YPD solid plates using a spike plate, and clone No. 23 of the 24 clones had a clear growth advantage under 46℃incubation (FIG. 1 a), which strain was designated FDHY23. The strain was subjected to a temperature tolerance test, the strain FDHY23 having an increased temperature tolerance at 46 ℃,47 ℃ and 48 ℃ relative to the starting strain LHP1044 (FIG. 1 b).
Example 2
Kluyveromyces marxianus mutant FDHY23 grows better than the original strain LHP1044 at high temperature
The mutant FDHY23 and the starting strain LHP1044 were inoculated into 3mL YPD liquid medium, respectively, cultured overnight at 30℃and then at an initial OD of 0.1 600 Transferring into 50mL YPD culture medium, sampling at 30deg.C, 46 deg.C and 47 deg.C every 6h or 12h, and measuring cell density OD 600 The values were sampled for 72 hours and the cells had entered the decay phase. Biomass and maximum growth rate of FDHY23 and LHP1044 were compared. The results show that at 30 ℃, the growth rate, biomass of the strains FDHY23 and LHP1044 during the growth phase were not significantly different (fig. 2). The maximum growth rate of the strain FDHY23 is obviously higher than that of the starting strain LHP1044 (figure 2) under the high temperature condition of 46 ℃ and 47 ℃, which shows that the growth capacity of the mutant strain FDHY23 under the high temperature condition is better than that of the starting strain, namely that the FDHY23 can grow rapidly under the high temperature condition.
Example 3
Kluyveromyces marxianus mutant FDHY23 improves the expression level of feruloyl esterase (derived from yak stomach)
The coding gene Est1E of the feruloyl esterase (GenBank: AXK 50449.1) from the stomach of the yak is cloned to an expression plasmid PUKDN132 by utilizing a conventional molecular cloning technology to obtain a recombinant expression plasmid PUKDN132-Est1E, and the recombinant expression plasmid PUKDN132-Est1E is respectively transformed to a strain FDHY23 and a strain LHP1044 to obtain engineering strains which are respectively named as FDHY23-Est1E and LHP1044-Est1E and are used for recombinant expression of the feruloyl esterase (from the stomach of the yak). The strains FDHY23-Est1E and LHP1044-Est1E are respectively inoculated into 50mL YD culture medium, and fermented and cultured at 30 ℃ for 72 hours, and the enzyme activity of feruloyl esterase (derived from yak stomach) is measured. The results show that the recombinant expression of the feruloyl esterase derived from the stomach of the yak by the engineering strain FDHY23-Est1E is 2.1 times that of the engineering strain LHP1044-Est1E (figure 3).
Example 4
Kluyveromyces marxianus mutant FDHY23 improves the expression level of feruloyl esterase (of Aspergillus niger origin)
Cloning the encoding gene AnfaeA of the feruloyl esterase (UniProtKB: O42807.1) from Aspergillus niger onto an expression plasmid PUKDN132 by utilizing a conventional molecular cloning technology to obtain a recombinant expression plasmid PUKDN132-AnfaeA, and respectively converting the recombinant expression plasmid PUKDN132-AnfaeA into a strain FDHY23 and a strain LHP1044 to obtain engineering strains which are respectively named as FDHY23-AnfaeA and LHP1044-AnfaeA and are used for recombinant expression of the feruloyl esterase (Aspergillus niger source). The strains FDHY23-AnfaeA and LHP1044-AnfaeA are respectively inoculated into 50mL YD culture medium, fermented and cultured for 72h at 30 ℃, and the enzyme activity of exogenous protein feruloyl esterase AnfaeA (from Aspergillus niger) is measured. The results show that the expression level of the feruloyl esterase derived from Aspergillus niger by the recombinant expression of the engineering strain FDHY23-AnfaeA is 1.7 times that of LHP1044-AnfaeA (figure 3).
Example 5
Kluyveromyces marxianus mutant FDHY23 improves expression level of saccharifying enzyme (derived from Botrytis cinerea)
The coding gene Badgla of the saccharifying enzyme (GenBank: CAA 86997.1) from Botrytis cinerea is cloned to a plasmid PUKDN132 by utilizing a conventional molecular cloning technology to obtain a recombinant expression plasmid PUKDN132-Badgla, and the recombinant expression saccharifying enzyme (derived from Botrytis cinerea) is obtained by respectively transforming a strain FDHY23 and a strain LHP1044, and engineering strains named FDHY23-Badgla and LHP1044-Badgla are obtained. The strains FDHY23-Badgla and LHP1044-Badgla are respectively inoculated into 50mL YD culture medium, and are fermented and cultured for 72 hours at 30 ℃ to determine the enzyme activity of the saccharifying enzyme. As a result, it was found that the recombinant expression level of the saccharifying enzyme derived from Botrytis cinerea gracilis in FDHY23-Badgla was 5.5 times that of HP1044-Badgla (FIG. 3).
Example 6
Kluyveromyces marxianus mutant FDHY23 improves expression level of porcine circovirus-like particle protein
The encoding gene PCV2 of the porcine circovirus capsid protein (GenBank: ABV 21950.1) is cloned on a plasmid PUKDN115 by utilizing a conventional molecular cloning technology to obtain a recombinant plasmid PUKDN115-PCV2, and the recombinant plasmid PUKDN115-PCV2 is respectively transformed into a strain FDHY23 and a strain LHP1044 to obtain engineering strains which are respectively named as FDHY23-PCV2 and LHP1044-PCV2 and are used for recombinant expression of the porcine circovirus-like particle protein. Strains FDHY23-PCV2 and LHP1044-PCV2 are respectively inoculated into 50mL of YD culture medium, fermentation culture is carried out for 72h at 30 ℃, the protein expression level is characterized by SDS-PAGE, the result shows that the expression level of porcine circovirus capsid protein PCV2 in FDHY23-PCV2 is obviously higher than that in LHP1044-PCV2, and gray level scanning shows that the expression level of porcine circovirus-like particle protein of FDHY23-PCV2 is 1.6 times that of LHP1044-PCV2 (figure 3).
Example 7
Kluyveromyces marxianus mutant FDHY23 improves the expression level of infectious bursal disease virus-like particle protein
The coding gene HVP2 of bursal disease virus capsid protein (GenBank: OK 167034) is cloned to plasmid PUKDN115 by utilizing the conventional molecular cloning technology to obtain recombinant plasmid PUKDN115-HVP2, which is respectively transformed to strain FDHY23 and strain LHP1044 to obtain engineering strains which are respectively named FDHY23-HVP2 and LHP1044-HVP2 for recombinant expression of infectious bursal disease virus-like particle protein. The strains FDHY23-HVP2 and LHP1044-HVP2 are respectively inoculated into 50mL YD culture medium, fermented and cultured for 72h at 30 ℃, and the protein expression quantity is characterized by SDS-PAGE, so that the result shows that the expression level of the FDHY23-HVP2 is obviously higher than that of the LHP1044-HVP2, and the gray level scanning shows that the protein expression quantity of infectious bursal disease virus-like particles of the FDHY23-HVP2 is 1.7 times that of the LHP1044-HVP2 (figure 3).
Example 8
Kluyveromyces marxianus mutant FDHY23 improves expression level of ferritin
The encoding gene FTH1 of ferritin (GenBank: NP-002023.2) is cloned on a plasmid PUKDN115 by utilizing a conventional molecular cloning technology to obtain a recombinant plasmid PUKDN115-Fth1, and the recombinant plasmid PUKDN115-Fth1 is respectively transformed into a strain FDHY23 and a strain LHP1044 to obtain engineering strains which are respectively named as FDHY23-Fth1 and LHP1044-Fth1 and are used for recombinant expression of ferritin. The engineering strains FDHY23-Fth1 and LHP1044-Fth1 are respectively inoculated into 50mL YD culture medium, fermented and cultured for 72h at 30 ℃, protein expression quantity is characterized by SDS-PAGE, the result shows that the expression level of the FDHY23-Fth1 is obviously higher than that of the LHP1044-Fth1, and gray level scanning shows that the ferritin expression level of the FDHY23-Fth1 is 2.9 times that of the LHP1044-Fth1 (figure 3).
Example 9
Kluyveromyces marxianus mutant FDHY23 can increase expression level of hemoglobin
The coding gene LBA of the leghemoglobin (NCBI Reference Sequence: NP-001235928.1) is cloned to a plasmid PUKDN115 by utilizing a conventional molecular cloning technology to obtain a recombinant plasmid PUKDN115-LBA, and the recombinant plasmid PUKDN115-LBA is respectively transformed to a strain FDHY23 and a strain LHP1044 to obtain engineering strains for recombinant expression of the hemoglobin, which are named as FDHY23-LBA and LHP1044-LBA respectively. Strains FDHY23-LBA and LHP1044-LBA were inoculated into 50mL of YD medium, respectively, and fermented and cultured at 30℃for 72 hours, and protein expression levels were characterized by SDS-PAGE, which showed that FDHY23-LBA expression levels were significantly higher than LHP1044-LBA, and grayscale scanning showed that FDHY23-LBA had 2.6 times the hemoglobin expression level of LHP1044-LBA (FIG. 3).
Example 10
The ability of the Kluyveromyces marxianus mutant FDHY23 to express protein at high temperature is better than that of the original strain LHP1044
The recombinant hemoglobin expression engineering strains FDHY23-LBA and LHP1044-LBA are inoculated into 50mL YD culture medium, fermented at 46 ℃ and cultured to different time points (24 h,36h,48h,60h and 72 h) for sampling, and protein expression level is characterized by SDS-PAGE. The results show that FDHY23-LBA can still express hemoglobin at 46℃and that control LHP1044-LBA hardly detects hemoglobin expression (FIG. 4).
Example 11
Kluyveromyces marxianus mutant FDHY23 carries CYR1 N1546K Mutation
The genomes of the strain FDHY23 and LHP1044 were subjected to second-generation DNA sequencing and comparative analysis, respectively, and it was found that the adenylate cyclase CYR1 gene of the mutant strain FDHY23 was subjected to a point mutation relative to the adenylate cyclase CYR1 gene of the strain LHP1044, namely that the 1546 th amino acid was changed from asparagine (N) to lysine (K) (FIG. 5).
Example 12
CYR1 mutation improves high temperature tolerance and exogenous protein expression quantity
The point mutation (N1546K) of the gene CYR1 is made in the original strain LHP1044 by using the conventional CRISPR technology, and the constructed strain is named LHP1044-CYR1 N1546K The method comprises the steps of carrying out a first treatment on the surface of the Meanwhile, the conventional CRISPR technology is utilized to mutate the CYR1 point in the high-temperature resistant mutant strain FDHY23The N1546K is subjected to back mutation, and the constructed strain is named as FDHY23-CYR1. LHP1044-CYR1 can be seen by dotting experiments N1546K Tolerance to high temperatures is significantly higher than LHP1044 and there is no significant difference in growth at 30 ℃. And when the CYR1 gene of the high temperature resistant strain FDHY23 is subjected to back mutation, the growth capacity of the FDHY23-CYR1 is obviously reduced under the high temperature condition of 46 ℃ (figure 6 a), and the result shows that the strain containing the CYR1 mutation has obviously higher high temperature tolerance capacity than that of a wild strain LHP1044.
The strain FDHY23 containing CYR1 mutation can improve the expression level of proteins such as hemoglobin, feruloyl esterase, virus-like particles and the like. In the presence of CYR1 N1546K In the mutant strain, the recombinant plasmid PUKDN115-LBA is transformed to obtain the engineering strain LHP1044-CYR1 N1546K -LBA; meanwhile, the wild strain LHP1044 also converts recombinant plasmid PUKDN115-LBA to obtain engineering strain LHP1044-LBA. The effect of wild type and mutant strains containing CYR1 mutations on protein expression levels was analyzed in comparison. Two engineering strains are respectively inoculated into 50mL YD culture medium, fermented and cultured for 72 hours at 30 ℃, and protein expression level is characterized by SDS-PAGE, so that the result shows that the strain containing CYR1 mutation has obviously higher capacity of expressing exogenous protein than that of wild strain LHP1044-LBA (figure 6 b)
In conclusion, the Kluyveromyces marxianus mutant strain FDHY23 can improve the expression quantity of exogenous proteins and the growth capacity at high temperature. Mutant strains of the adenylate cyclase gene CYR1, e.g.point mutant CYR1 N1546K The high temperature tolerance is improved, and the expression quantity of the exogenous protein is improved.
Example 13
Mutation carried by Kluyveromyces marxianus mutant strain FDHY23
The genomes of the strain FDHY23 and the LHP1044 are respectively subjected to second-generation DNA sequencing and comparison analysis, the strain FDHY23 has 38 SNP and 56 InDel relative to the original strain LHP1044, and the specific details are shown in a list 1.
TABLE 1 mutation of FDHY23 relative to LHP1044
Embodiments relate to materials and methods
Fermentation culture
The culture medium for basic growth of Kluyveromyces marxianus is YPD culture medium: 2% polypeptone, 2% glucose, 1% yeast extract, at 30℃and 220rpm. Culturing recombinant expression strain in shake flask, and selecting YD culture medium: 2% yeast extract, 4% glucose, at 30℃and 220rpm for 72 hours, and the expression of the foreign protein in K.marxinus was examined. SD solid medium for transformation coating: 6.7g/L yeast nitrogen source without amino acid, 20g/L glucose and 20g/L agar powder.
Plasmid transformed kluyveromyces marxianus strain
Selecting bacteria to 3mL YPD small test tube, shake culturing at 30deg.C for 18-19h; taking 1mL of bacterial liquid, centrifuging at 8000rpm, removing supernatant, washing with 1mL of sterile water, centrifuging at 8000rpm, and removing supernatant; washing with 1mL of 1 XDE/LiAc (0.1M/L LiAc,10mM/L Tris,1mM/L EDTA), centrifuging at 8000rpm, removing supernatant, repeating once; adding 10. Mu.L of ligation product (or 3-4. Mu.L of plasmid), 600. Mu.L of PEG solution (0.1M/L LiAc,10mM/L Tris,1mM/L EDTA,40%PEG 4000) and DTT (final concentration: 10 mmol); water bath at 30deg.C for 15min and water bath at 47 deg.C for 15min; the supernatant was removed immediately, 100. Mu.L of sterile water was added, the plates were plated after resuspension, and incubated in an oven at 30 ℃.
Kluyveromyces marxianus cell disruption
1mL of fresh recombinant yeast cells were collected by centrifugation at 8000rpm for 5min, followed by centrifugation with PBS buffer (137 mM NaCl, 2.7mM KCl, 10mM Na) 2 HPO 4 、1.8mM KH 2 PO 4 pH 7.4) was washed once and finally resuspended in 500. Mu.L of PBS buffer. Approximately 400. Mu.L of glass beads was added thereto. Cells were disrupted at 6m/s for 2min using a Bead-bed (FastPrep-24, MP, california, USA).
SDS-PAGE analysis of foreign protein expression level
Taking the supernatant of the fermentation liquid or the supernatant of cell disruption to carry out SDS-PAGE, and simultaneously quantifying the exogenous proteins by taking lactoglobulin with different concentrations as a standard. Lactoglobulin was prepared at concentrations of 500, 400, 300, 200 and 100mg/L, and SDS-PAGE analysis was performed after sample preparation. The intensity of the lactoglobulin bands separated on SDS-PAGE gel was analyzed by gray scale scanning using GenoSens analysis software to prepare a standard curve. And meanwhile, carrying out gray scanning analysis on the band intensity of the foreign protein separated on the SDS-PAGE gel by using GenoSens analysis software, and calculating the expression quantity of the foreign protein according to a standard curve.
Determination of feruloyl esterase enzyme Activity
The supernatant of the fermentation broth was taken up with 1 XPBST (8 g/L NaCl,1.42g/L Na) 2 HPO 4 ，0.2g/L KCl，0.27g/L KH 2 PO 4 Samples were diluted to appropriate concentrations at 2.5% triton-X-100, ph=6.4, 20 μl of enzyme solution to be tested was transferred to 96-well flat bottom plates, and then 180 μl of CNPF mixed reaction solution (10 μl of CNPF (MB 7591, meropenem organism) and 170 μl of 1×pbst) was added to each reaction system, and mixed well. The reaction system was placed in a 37℃incubator for a reaction for 20min, and the absorbance of the sample was rapidly measured by a BioTek Eon microplate reader at 410 nm. Calculating the enzyme activity according to an enzyme activity conversion formula:
enzyme activity (U/mL) = ((a) 410 -0.0521)×V 1 X d x 1000 x dilution)/(6.6764 x T x V) 2 )
Note that: v (V) 1 Represents the total volume (L), V 2 The volume (mL) of the enzyme solution added to the reaction system is shown, d is the dilution factor of the enzyme solution, and T is the reaction time (min).
Feruloyl esterase enzyme activity definition: under the reaction condition of 37 ℃ and pH=6.4, the enzyme quantity required for decomposing 1nmol of substrate CNPF or releasing 1nmol of colored group CNP in the reaction system is one enzyme activity unit (U).
Determination of saccharification enzyme Activity
The supernatant of the fermentation broth is taken, a sample is diluted to a proper concentration by using 20mM NaAc solution, 50uL of enzyme solution to be tested is taken, 500 uL of 1% soluble starch and 450 uL of 20mM NaAc solution are added, and the mixture is uniformly mixed. The reaction system is placed in a 60 ℃ incubator for standing reaction for 5min, 1mL of DNS solution is rapidly added to react in boiling water for 5min after the reaction is finished, 100 mu L of final reaction liquid is transferred to a 96-well flat bottom plate, and the absorbance value of a sample is rapidly detected by a BioTek Eon microplate reader under the condition of 540 nm. Calculating the enzyme activity according to an enzyme activity conversion formula:
concentration of reducing sugar produced by hydrolysis (mg/mL) = (A) 540 -0.0002)/1.7853
Enzyme activity (U/mL) = (amount of reducing sugar produced by hydrolysis×1000×dilution)/45
The definition of the enzymatic activity of the saccharifying enzyme is: the amount of enzyme required to hydrolyze 1. Mu. Mol of soluble starch substrate or release 1. Mu. Mol of reducing sugar per minute at 60℃under pH5.5 is one enzyme activity unit (U).
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Claims (9)
1. a Kluyveromyces marxianus engineering strain for efficiently expressing exogenous proteins is characterized in that Kluyveromyces marxianus FIM1 with a preservation number of CGMCC No.10621 is taken as an original strain, and a genome editing technology is utilized to delete a gene URA3 on the genome of the Kluyveromyces marxianus FIM 1; 2 sites are respectively selected to integrate loxPSym sequences on 8 chromosomes of the ura3 defective strain by utilizing a gene editing technology, so as to obtain a strain which is named as LHP1044, wherein the chromosomes of the strain contain 16 loxPSym sequences; the Cre recombinase gene is connected to a vector to obtain a recombinant plasmid LHZ893, the nucleotide sequence of which is shown as SEQ ID NO:1 is shown in the specification; after a recombinant plasmid LHZ893 is transformed into a strain LHP1044 by a yeast lithium acetate transformation method, a mutant strain with obvious growth advantages is obtained by screening under the high temperature pressure of 46 ℃, the mutant strain is named as FDHY23, the preservation number is CGMCCNo.26724, and the preservation date is as follows: 2023.3.3.
2. the kluyveromyces marxianus engineering strain for efficiently expressing exogenous proteins according to claim 1, wherein the specific positions of integration of the 16 loxppsym sequences on the chromosome are: 80654bp and 297580bp of chromosome 1, 706654bp and 885427bp of chromosome 2, 327216bp and 1226714bp of chromosome 3, 715829bp and 1172907bp of chromosome 4, 340152bp and 746775bp of chromosome 5, 130814bp and 446862bp of chromosome 6, 392737bp and 674407bp of chromosome 7, and 435906bp and 606918bp of chromosome 8; the reference gene version number of the Kluyveromyces marxianus FIM1 is GCA_001854445.2.
3. The kluyveromyces marxianus engineered strain of claim 1, wherein the strain FDHY23 carries 94 mutations, 38 of which are SNPs and 56 of which are InDel, the specific mutation being shown in table 1.
4. The kluyveromyces marxianus engineered strain of claim 1, wherein the mutant strain carries at least a mutation in an adenylate cyclase gene.
5. The kluyveromyces marxianus engineered strain of claim 4, wherein the adenylate cyclase gene CYR1 gene of strain FDHY23 is mutated relative to the CYR1 gene of strain LHP1044, comprising at least a point of mutation: the amino acid at position 1546 is changed from asparagine (N) to lysine (K).
6. Mutation of the CYR1 gene for improving the temperature resistance and protein expression level of the strain.
7. The use of a kluyveromyces marxianus engineering strain and CYR1 gene mutation according to any one of claims 1-6 for the efficient expression of exogenous proteins.
8. The use according to claim 7, wherein the foreign protein is feruloyl esterase, glucoamylase, porcine circovirus-like particle protein, infectious bursal virus-like particle protein, ferritin or leghemoglobin.
9. The use according to claim 7, characterized in that:
the coding genes Est1E of the ferulic acid esterase from the yak stomach are obtained through PCR amplification by a homologous recombination one-step method, and a PUKDN132 carrier is connected to construct a recombinant expression plasmid PUKDN132-Est1E, and the recombinant expression plasmid is converted into a bacterial strain FDHY23 by a lithium acetate conversion method to obtain an engineering bacterial strain FDHY23-Est1E of the ferulic acid esterase from the yak stomach;
connecting encoding genes AnfaeA and PUKDN132 vectors of the feruloyl esterase from the Aspergillus niger obtained by PCR amplification by a homologous recombination one-step method to construct recombinant expression plasmid PUKDN132-AnfaeA, and transforming the recombinant expression plasmid into a strain FDHY23 by a lithium acetate transformation method to obtain an engineering strain FDHY23-AnfaeA for efficiently recombining and expressing the feruloyl esterase from the Aspergillus niger;
the coding genes Badgla and the PUKDN132 vector of the saccharifying enzyme from Botrytis cinerea obtained by PCR amplification are connected by a homologous recombination one-step method to construct a recombinant expression plasmid PUKDN132-Badgla, and the recombinant expression plasmid is transformed into a strain FDHY23 by a lithium acetate transformation method to obtain an engineering strain FDHY23-Badgla for efficiently and recombinantly expressing the saccharifying enzyme from Botrytis cinerea;
connecting a coding gene PCV2 of a porcine circovirus capsid protein with a GenBank number of ABV21950.1 obtained by PCR amplification and a PUKDN115 vector by using a homologous recombination one-step method to construct a recombinant expression plasmid PUKDN132-PCV2, and transforming the recombinant expression plasmid into a bacterial strain FDHY23 by using a lithium acetate transformation method to obtain an engineering bacterial strain FDHY23-PCV2 for efficiently and recombinantly expressing porcine circovirus-like particle proteins;
the coding gene of the bursal disease virus capsid protein gene HVP2 with the GenBank number of OK167034 obtained by PCR amplification and a PUKDN115 vector are connected by a homologous recombination one-step method to construct a recombinant expression plasmid PUKDN132-HVP2, and the recombinant expression plasmid is transformed into a bacterial strain FDHY23 by a lithium acetate transformation method to obtain an engineering bacterial strain FDHY23-HVP2 with high efficiency recombinant expression of bursal disease virus capsid protein;
connecting the encoding gene FTH1 of ferritin with the GenBank number of NP 002023.2 obtained by PCR amplification and a PUKDN115 carrier by using a homologous recombination one-step method to construct a recombinant expression plasmid PUKDN132-Fth1, and transforming the recombinant expression plasmid into a bacterial strain FDHY23 by using a lithium acetate transformation method to obtain an engineering bacterial strain FDHY23-Fth1 for high-efficiency recombinant expression of ferritin;
or, the coding gene of the NCBI reference sequence No. NP 001235928.1 bean hemoglobin gene LBA obtained by PCR amplification and the PUKDN115 carrier are connected by a homologous recombination one-step method to construct a recombinant expression plasmid PUKDN132-LBA, and the recombinant expression plasmid is transformed into the strain FDHY23 by a lithium acetate transformation method to obtain the engineering strain FDHY23-LBA for efficiently recombining and expressing the hemoglobin.
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