CN113736675B - Method for improving xylose conversion capability of saccharomyces cerevisiae - Google Patents
Method for improving xylose conversion capability of saccharomyces cerevisiae Download PDFInfo
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- CN113736675B CN113736675B CN202010475833.9A CN202010475833A CN113736675B CN 113736675 B CN113736675 B CN 113736675B CN 202010475833 A CN202010475833 A CN 202010475833A CN 113736675 B CN113736675 B CN 113736675B
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Abstract
The invention discloses a method for improving the xylose conversion capability of saccharomyces cerevisiae, which comprises the following steps: taking Saccharomyces cerevisiae with the capability of converting xylose into ethanol as a chassis cell, and enabling a coding gene of cysteine desulphurase NFS1 in a genome of the Saccharomyces cerevisiae to generate I492N mutation; integrating at least one heterologous gene encoding xylose isomerase in the genome. The method can directly obtain the saccharomyces cerevisiae for rapidly fermenting xylose to generate ethanol without depending on domestication, and has wide application prospect.
Description
Technical Field
The invention belongs to the field of genetic engineering, and relates to a method for improving the xylose conversion capability of saccharomyces cerevisiae, and saccharomyces cerevisiae genetic engineering bacteria for producing ethanol by metabolizing xylose and application thereof.
Background
Biomass is the only renewable energy source containing carbon in nature, and the development of green bio-production to produce various chemicals including fuel ethanol is the best utilization way. Fifteen departments such as the development and reform commission of the 9 th 2017 country jointly print 'the implementation scheme for expanding the production of the biofuel ethanol and popularizing and using the ethanol gasoline for vehicles', and the biofuel ethanol industry of China is on a great opportunity for further development. In particular, the fermentation production technology of the second-generation fuel ethanol using lignocellulose as the raw material has the advantages of 'no grain competition with people, no land competition with grain', ecological environment improvement, economic development promotion in agricultural rural areas and the like compared with the first-generation fuel ethanol (using starch base or glycosyl as the raw material), and is an important direction of the future fuel ethanol development.
The production of the second-generation fuel ethanol needs to pretreat and enzymatically hydrolyze lignocellulose raw materials to release monosaccharide components, wherein 38% -50% of the lignocellulose raw materials are glucose, and 23% -32% of the lignocellulose raw materials are xylose for microbial fermentation utilization. Saccharomyces cerevisiae is considered an ideal cellulose ethanol production strain because of its efficient glucose-converting ethanol production capacity and its greater tolerance to inhibitors relative to bacteria (produced during pretreatment of the feedstock). However, saccharomyces cerevisiae cannot naturally utilize xylose, and if the xylose metabolism capability is given to the Saccharomyces cerevisiae, the utilization rate of raw materials can be improved, so that the economy of cellulosic ethanol is further improved.
Saccharomyces cerevisiae is first required to metabolize xylose by introducing heterologous pathway genes to convert xylose into xylulose that can be utilized by itself, and there are two pathways for converting xylose into xylulose in nature, see FIG. 1: one is the redox pathway present in certain fungi, xylose reductase and xylitol dehydrogenase catalyze the conversion of xylose to xylulose. However, the preference of the two enzyme cofactors is different, which finally results in a large accumulation of xylitol as an intermediate product, and seriously affects the progress of the downstream pathway reaction. The other is an isomerization pathway existing in certain bacteria, xylose is converted into xylulose through one-step catalysis of Xylose Isomerase (XI), the problem of unbalanced cofactor is avoided, and the method is a potential saccharomyces cerevisiae Xylose utilization pathway.
The level of XI activity directly affects the rate at which yeast metabolizes xylose. Proven effective and widely used is XI from the genus Runnum (Piromyces sp.E2). In addition, by matching with overexpression endogenous Xylulokinase (XK) and non-oxidized pentose phosphate pathway (Pentose Phosphate Pathway, PPP for short) genes RPE1, RKI1, TAL1 and TKL1, and knocking out GRE3 genes (the gene coding protein can convert xylose into xylitol with strong inhibition effect on XI), the yeast strain with preliminary xylose metabolism capability can be obtained. Continuous passage on xylose culture medium, i.e. adaptive evolution (domestication), can greatly improve xylose utilization capacity of the strain (see table 1).
Although domestication can greatly enhance the phenotype of the strain, the molecular targets behind are not completely clear. The strains before and after acclimation are subjected to comparative histology analysis to obtain differential molecular targets, the strains reversely after acclimation or the strains before acclimation are subjected to forward reversion to corresponding mutation to verify the effectiveness of the targets, and a plurality of molecular targets favorable for fermenting xylose by saccharomyces cerevisiae are obtained, wherein Kim, S.R. the mutation of an alkaline phosphatase-encoding gene PHO13 in 3 acclimated strains is found, the inactivation of the gene can reduce the expression level of GRE3 and reduce the accumulation of XI inhibitor xylitol, and the deletion of the gene directly before acclimation has promotion effect on xylose utilization (Kim, S.R., et al., PLoS One,2013.8 (2): p.e 57048.). Bao Xiao the teaching problem group found that mutations or deletions in the gene ASK10 encoding the stress response modifier could up-regulate the genes HSP26, SSA1 and HSP104 encoding chaperone proteins, promote correct folding of the XI protein, enhance enzyme activity and ultimately improve xylose fermentation capacity of the strain (Hou, j., et al, meta Eng, 2016.38:p.241-250.). In addition, HOG1 (encoding mitotic activator protein kinase) gene, cAMP-PKA pathway inhibitor gene IRA2 (encoding GTPase activator protein) and MAPKKK signal pathway kinase encoding gene SSK2 involved in yeast intracellular stress response all prove to have promotion effect on fermenting xylose by yeast strains.
The two parts to be improved exist in the work, firstly, the strain used in the previous study is a laboratory strain, the gene operation is dependent on plasmids, and the problems of low strain robustness and the like exist; based on the molecular targets, only partial strain phenotypes can be restored, in other words, the molecular targets obtained by directly integrating the strains before domestication are difficult to reach the domestication level, namely, rational reconstruction cannot be realized. In general, a xylose isomerase expression gene is introduced on the basis of an original saccharomyces cerevisiae strain, a pentose phosphate pathway gene is overexpressed, an aldose reductase gene GRE3 is knocked out, a basic strain with weak xylose metabolism capability can be obtained, and the method is a well-accepted practice in constructing xylose yeast. At present, only the strain modified by the molecules is continuously passaged (domesticated) in a culture medium with xylose as the sole carbon source, so that the xylose yeast strain with the industrial application level can be obtained. No method is available to realize construction of industrial-grade Saccharomyces cerevisiae strains without dependence on domestication and through complete molecular operation. The existence of the problems limits the development of cellulose ethanol xylose yeast strains and severely restricts the green production of fuel ethanol.
Disclosure of Invention
Based on the above-mentioned shortcomings, this classThe industrial diploid saccharomyces cerevisiae CCTCC M94055 is selected as an initial strain in the early stage of the subject group, psXyl A genes are integrated, GRE3 is knocked out, non-oxidized pentose phosphate pathway and xylulokinase genes are overexpressed, a strain CIBTS0573 is obtained, continuous passage is carried out for a long time in a xylose culture medium, and finally a strain CIBTS0735 which is used for metabolizing 80g/l glucose and 40g/l xylose completely in 20 hours is obtained, and the xylose consumption rate is up to 2.5g g -1 l -1 Is the highest value reported at present and is considered to be a xylose yeast strain with industrial application prospect (Diao, L., et al, construction of fast xylose-fermenting yeast based on industrial ethanol-producing diploid Saccharomyces cerevisiae by rational design and adaptive evaluation.BMC Biotechnol, 2013.13:p.110.). Further genomic sequencing analysis found that the XylA copy number increased to 52 in the acclimatized strain CIBTS 0735. Besides that there are unreported NFS1 I492N (encoding cysteine desulphurase, important structural protein synthesized by Saccharomyces cerevisiae mitochondrial Fe-S cluster, mutation of isoleucine 492 to asparagine) mutation. Iterative integration of 50 copies of XyleA in pre-acclimated strain CIBTS0573 with the simultaneous introduction of NFS1 I492N Mutation, the phenotype of the obtained strain CIBTS0573-50-N is equal to that of the domesticated strain CIBTS 0735. The method is characterized in that the construction of the quick-fermentation xylose yeast strain with phenotype comparable to domestication is realized for the first time through molecular operation without depending on domestication. That is, by such an operation, we can construct a strain of Saccharomyces cerevisiae that achieves rapid fermentation of xylose to the level of industrial application. The important discovery forms the basis of the invention, has important significance for obtaining saccharomyces cerevisiae engineering bacteria with higher capability of converting xylose into ethanol, and has wide application prospect. Specifically, the invention comprises the following technical scheme:
a method for improving the xylose conversion capability of saccharomyces cerevisiae, comprising the following steps:
A. the coded gene of cysteine desulphurase NFS1 in the genome of Saccharomyces cerevisiae with the capability of converting xylose into ethanol is subjected to I492N mutation NFS1 by taking the Saccharomyces cerevisiae as a chassis cell I492N The cytoplasmic iron ion content is improved by inhibiting synthesis of iron-sulfur clusters (Fe-S clusters) in mitochondria;
B. at least one heterologous gene encoding xylose isomerase is integrated in the genome, preferably multiple copies of the heterologous gene are integrated.
The Saccharomyces cerevisiae described in step A above is preferably a diploid Saccharomyces cerevisiae.
Preferably, the Saccharomyces cerevisiae described in step A is an industrial diploid Saccharomyces cerevisiae strain CIBTS0573 or CIBTS0735 reported in literature Diao, L., et al, BMC Biotechnol,2013.13:p.110. Or patent literature CN 201811024423.1.
In one embodiment, the above method further comprises the steps of: C. and the gene ISU1 of the iron-sulfur cluster synthesis pathway is knocked out, so that the normal assembly of the iron-sulfur cluster is inhibited.
Preferably, the method further comprises the steps of: D. inactivating more than one gene selected from the group consisting of: CCC1, YFH1, ISU2, SSQ1, GRX5, GRX3/4, FRA1/2, TYW1, YAP5; and/or to enhance the gene AFT1/2.
Optionally, the above method further comprises the step of knocking out the gene CCC1 and/or GRE3, inhibiting the synthesis of iron-sulfur clusters in mitochondria.
Step B above is the integration of more than 26, preferably more than 30, preferably more than 34 copies of the heterologous gene into the genome. The heterologous gene is for example the xylose isomerase gene xylA (or PsXyl A gene) from Rumex rumens (Piromyces sp.E2) with the nucleotide sequence SEQ ID NO:1, see document Diao, L.et al, BMC Biotechnol,2013.13:p.110. The heterologous gene can also be a Niu Liuwei metagenome-derived xylose isomerase gene (RuXyl A for short), and the nucleotide sequence is SEQ ID NO. 2.
According to a second aspect of the present invention, there is provided a genetically engineered bacterium constructed according to the above-described method.
According to a third aspect of the invention, there is provided the use of the genetically engineered bacterium described above in the production of ethanol by fermentation. In the fermentation process, the carbon source in the fermentation medium comprises xylose and/or xylan.
Preferably, iron ions such as ferrous ions are added to the fermentation medium.
The invention directly integrates high in a strain of diploid saccharomyces cerevisiae fermenting xyloseCopy xylose isomerase gene XyleA or RuxylA, mutant NFS1 I492N The industrial diploid saccharomyces cerevisiae engineering strain is constructed, and the method can directly obtain the saccharomyces cerevisiae for rapidly fermenting xylose to generate ethanol without domestication, so that the industrial application prospect is wide.
Drawings
FIG. 1 is a diagram of the metabolic pathway of ethanol production by Saccharomyces cerevisiae fermentation xylose in nature. Wherein XK: xylulokinase; PDC: a pyruvate decarboxylase; ADH: an alcohol dehydrogenase; XR: xylose reductase; XDH: xylitol dehydrogenase.
FIG. 2 is a bar graph of copy number of XylA gene of pre-acclimatized strain CIBTS0573 versus post-acclimatized CIBTS 0735.
FIG. 3 is a schematic diagram of the mechanism of amplification of XYLA gene during strain acclimation.
FIG. 4 shows the growth of OD in YPX40 medium after knockout of CIBTS0735 high copy XyleA 600 Graph of values.
FIG. 5 is NFS1 in CIBTS0735 I492N After reversion of the mutation to wild type the strain grows OD in YPX40 medium 600 Graph of values.
FIG. 6 growth OD in YPX40 Medium for strains iteratively integrated with different XylA copy numbers in CIBTS0573 600 Graph of values.
FIG. 7 is a diagram containing NFS1 I492N Mutant different copies of XyleA strains grown OD in YPX40 Medium 600 Graph of values.
FIG. 8 is a chart of three NFS1 strains of different XYLA copy numbers I492N Graph of xylose consumption time of mutant strain in YPX40 medium.
FIG. 9 is a chart of three NFS1 strains with different XYLA copy numbers I492N Time profile of ethanol production of mutant strains in YPX40 medium.
FIG. 10 shows the OD growth in YPX40 medium of different strains before and after knocking out ISU1 gene 600 Graph of values.
FIG. 11 is a graph showing the residual xylose content of the strain CIBTS0573-26-N in a medium supplemented with different levels of ferric ions.
FIG. 12 is a graph showing comparison of intracellular iron ion concentrations of different strains.
FIG. 13 is a structural map of pSCm-N20-V3 plasmid.
FIG. 14 is a scatter plot of OD 24 hours of xylose fermentation for different strains.
FIG. 15 shows the growth of OD in YPX40 medium for NFS1 Gene mutant and wild-type NFS1 Gene Strain cloned with high copy number RuXyl A, respectively 600 Graph of values.
Detailed Description
The inventor finds that XylA copy number in the domesticated strain genome is amplified to 52 by comparing genome analysis on an industrial diploid saccharomyces cerevisiae strain which is domesticated and obtained and rapidly ferments xylose, and NFS1 generates I492N homozygous mutation. Therefore, the strain is subjected to mutation before domestication, the xylose fermenting capacity of the obtained strain is equal to that of the domesticated strain, a genetic mechanism of the xylose metabolism capacity of the strain obtained through domestication is disclosed, and the saccharomyces cerevisiae strain rational reconstruction of the fermentable xylose is realized.
The invention firstly carries out comparative genome analysis on a Saccharomyces cerevisiae strain CIBTS0735 (see the literature Diao, L., et al, BMC Biotechnol,2013.13: p.110. Or patent literature CN 201811024423.1) obtained by early domestication and a starting strain CIBTS0573, and finds that 21 genes are subjected to SNP (single nucleotide variation) change, 23 genes are subjected to InDel (insertion or deletion) change, and the copy number of XyleA of an introduced xylose isomerase gene derived from the ruminococcus is increased to 52. The PCR sequencing of 21 SNPs and 23 InDel sites again verifies that 10 SNP sites and 5 InDel sites confirm the change. Analysis of the possible links between altered gene function and xylose metabolism we speculate that the gene NFS1 encoding cysteine desulphurase is most likely to cooperate with high copy XylA to promote high speed fermentation of xylose by the strain.
We first verify NFS1 I492N Mutations and high copy xylA were necessary for fermenting xylose by the domesticated strain CIBTS 0735. According to XyleA amplification mechanism, cre enzyme expression plasmid is introduced into CIBTS0735, xyleA 'loop out' among a plurality of loxP sites is induced, high-copy XyleA is knocked out in one step, and the growth of the obtained strain in a 40g/L xylose culture medium (YPX 40) is greatly reducedWeak, indicating that high copy XylA is necessary for the strain to ferment xylose. At the same time we will be NFS1 in CIBTS0735 I492N The mutation reverted to wild type, 52 copies of XyleA were retained, and the resulting strain showed a similar significant decrease in growth on YPX40 medium, indicating NFS1 I492N Mutations are also necessary.
To further verify the contribution of the above two targets to the xylose fermentation by the strain, we tried to introduce these two mutations in the pre-acclimated strain CIBTS0573, testing whether rational reconstitution of the fast fermenting xylose yeast strain could be achieved. Iterative integration of xylA copy number directly at specific sites of chromosome CIBTS0573 to 50, found that with increasing xylA copy number, the OD value of the strain in YPX40 increased, but there was a larger gap from the acclimatized strain CIBTS 0735. When NFS1 is introduced I492N After mutation, the growth capacity of the strain is greatly improved, in particular, the strain containing 42 and 50 copies of XylA is in NFS1 I492N In the presence of mutation, the xylose utilization capacity and ethanol production capacity completely level the domesticated strain CIBTS0735, and the result shows that the XyleA and the NFS1 are high in copy number I492N Mutation is the condition of CIBTS0573 to obtain high speed xylose fermenting capacity, so that xylose yeast strain with phenotype comparable to that of domesticated strain may be reconstructed through molecular operation without dependence on domestication.
We found in the study that in addition to the rumen-derived xylose isomerase gene PsXyleA gene (SEQ ID NO: 1), other species-derived xylose isomerase genes such as the Niu Liuwei metagenome-derived xylose isomerase gene RuXyleA (SEQ ID NO: 2) also have good xylose metabolism promoting effects in the NFS1 mutant strain.
Gene NFS1 influences the synthesis of iron-sulfur clusters in saccharomyces cerevisiae mitochondria, mutation of the gene inhibits the normal assembly of the iron-sulfur clusters, and we knock out gene ISU1 which is the synthesis pathway of the iron-sulfur clusters, and find that the growth condition of a xylose culture medium of the strain is greatly improved, so that the xylose metabolism capability of the strain can be greatly improved by inhibiting the synthesis of the iron-sulfur clusters. The mutation or inactivation of NFS1 and ISU1 genes inhibits the synthesis of Fe-S clusters, which are closely related to cellular iron ion metabolism, so that further foothold is possible to increase cytoplasmic iron ion content. The determination shows that the above gene change can greatly improve the iron ion content of cells, and in addition, the iron ion can be directly added into the culture medium to obviously improve the xylose utilization capacity of the strain, and the removal of the iron ion from the culture medium can greatly reduce the xylose utilization capacity of the strain. The results clearly show that molecular operations, such as NFS1 mutation or ISU1 gene inactivation, which can improve the cytoplasmic iron ion content, can be combined with chromosome integration of high copy xylose isomerase genes, such as 34 copies and above, so that the quick fermentation xylose saccharomyces cerevisiae strain reaching the industrial application level can be directly obtained.
The terms "xylose yeast strain", "(xylose-fermenting) s.cerevisiae", "diploid s.cerevisiae" are used herein in the same sense and refer to s.cerevisiae used for the conversion of xylose to ethanol, such as strain CIBTS0735 after acclimation, strain CIBTS0573 before acclimation.
In this context, for the sake of simplicity of description, a certain protein, such as cysteine desulphurase NFS1, is sometimes used in combination with the name of its coding gene (DNA) NFS1, it being understood by the person skilled in the art that they represent different substances in the different descriptive contexts. Those skilled in the art will readily understand their meaning depending on the context and context. For example, for NFS1, when used to describe a cysteine desulphurase function or class, reference is made to a protein; when described as a gene, it refers to the gene encoding the NFS 1. Obviously, the gene mutation NFS1 I492N Refers to a mutation in a gene encoding an I492N mutant of NFS 1.
In the examples, for convenience of description, the genetic alterations in a strain such as CIBTS0735 are abbreviated as suffix, such as CIBTS0735-52-W indicates that strain CIBTS0735 contains 52 copies of XylA in its genome and NFS1 is not mutated (wild type W); CIBTS0735-2-N indicates that the strain CIBTS0735 contains 2 copies of XyleA in its genome and NFS1 is mutated to NFS1 I492N And so on.
The present invention will be described in further detail with reference to specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The amounts, amounts and concentrations of various substances are referred to herein, wherein the percentages refer to percentages by mass unless otherwise specified.
Examples
Materials and methods
The molecular biology experiments herein include plasmid construction, enzyme digestion, competent cell preparation, transformation, etc., and are mainly performed by referring to "molecular cloning experiment guidelines (third edition), J.Sam Broker, D.W. Lassel, huang Peitang, et al, science Press, beijing, 2002). For example, competent cell transformation methods and competent preparation methods were carried out according to chapter 1, page 96 of the guidelines for molecular cloning experiments (third edition). The specific experimental conditions can be determined by simple experiments, if necessary.
The main culture medium comprises:
LB medium: 5g/L yeast extract, 10g/L tryptone, 10g/L sodium chloride. (LB solid medium additionally added with 20g/L agar powder.).
YPD20 medium: 10g/L yeast extract, 20g/L tryptone, 20g/L glucose.
YPX40 medium: 10g/L yeast extract, 20g/L tryptone, 40g/L xylose.
YPD20X10 medium: 10g/L yeast extract, 20g/L tryptone, 20g/L glucose, 10g/L xylose.
YPGal medium: 10g/L yeast extract, 20g/L tryptone, 20g/L galactose.
YP80D40X medium: 10g/L yeast extract, 20g/L tryptone, 80g/L glucose, 40g/L xylose.
Example 1: genomic analysis of pre-xylose acclimation CIBTS0573 versus post-acclimation CIBTS0735 strain
The experimental method comprises the following steps:
the strain genome was extracted, sequenced by the company pesenuo, and analyzed for Single Nucleotide Polymorphisms (SNPs), base insertions or deletions (indels).
The experimental steps are as follows:
diploid Saccharomyces cerevisiae strains CIBTS0573 and CIBTS0735 were deposited by the subject group and were available to any entity or individual for use in validating the present invention, but were not allowed by the subject group to be used for other purposes, including scientific research and teaching.
Strains CIBTS0573 and CIBTS0735 were grown in YPD20 liquid medium to logarithmic phase, cells were collected, and genome was extracted and submitted to Parkino for genome sequencing. Briefly, strain CCTCC M94055, CIBTS0573 and CIBTS0735 400bp paired-end genomic libraries were constructed and sequenced using an Illumina Hiseq2500 machine. The resulting high quality sequencing data was pasted back to the reference genome using the bwa 0.7.12 command (Li, h., aligning sequence reads, clone sequences and assembly contigs with BWA-mem.2013.1303.). SNPs and InDels were identified based on the comparison using the VarScan 2.4.0 command (Koboldt, D.C., et al, varScan 2:somatic mutation and copy number alteration discovery in cancer by exome sequencing.Genome Res,2012.22 (3): p.568-76.). And (3) carrying out PCR amplification sequencing on SNPs and InDels obtained by genome sequencing to further confirm.
Experimental results:
compared with the strain CIBTS0573 before domestication, the domesticated strain CIBTS0735 has the advantages that SNPs are generated in 21 genes and InDels are changed in 23 genes through genome sequencing analysis. The sequencing results are subjected to PCR amplification sequencing again to verify that the confirmation of 10 SNP loci and 5 InDel loci is changed, and the details are shown in Table 1.
TABLE 1 comparative genomic analysis results of strains before and after acclimation
a: SNP changes in brackets indicate changes in the homozygous/heterozygous situation, e.g., NFS1 gene is homozygous and the others are heterozygous; the absence of brackets after SNPs indicates that the mutation occurred in the non-coding region of the gene; only RNQ1 gene locus changes in InDel occur in the coding region.
Example 2: copy number variation of XylA Gene of CIBTS0573 before xylose acclimation and CIBTS0735 Strain after acclimation
The experimental method comprises the following steps:
the copy number changes of the XylA gene at both CIBTS0573 and CIBTS0735 strains were determined using fluorescence quantification PCR (real time PCR) techniques.
The experimental steps are as follows:
yeast DNA was extracted using the Tian Gen Yeast genome extraction kit, and the extracted DNA concentration was determined using the NanoDrop ND-1000 (Thermo Fisher Scientific). The gene copy number was calculated using the following formula: n (N) Target /N Reference ＝(2 CT(Reference) /2 CT(Target) ) X 2. In which ACT1 is used as an internal gene, and the host is diploid, the copy number is calculated by multiplying it by 2.
Experimental results:
the copy number was precisely determined using Real-time PCR, as shown in FIG. 2, and it was found that in the acclimatized strain CIBTS0735, the XylA gene copy number reached 52, which was significantly improved compared to the 2 copies before acclimatization.
Example 3: reduced xylose metabolism ability of the strain after knocking out high copy XyleA in acclimatized strain CIBTS0735
The experimental method comprises the following steps:
the xylose metabolizing ability of the resulting strain was determined based on the Cre-loxP technique, which knocked out 50 copies of XylA in the CIBTS0735 genome at one time, according to the XylA amplification mechanism during acclimation (see fig. 3).
The experimental steps are as follows:
(1) The Cre recombinase expression plasmid is introduced into the CIBTS0735 strain, the transformant is cultured in YPGal culture medium to induce XYLA between loxP sites to loop out, and finally the mutant CIBTS0735-2-N with only two copies of XYLA reserved is obtained.
(2) The strain CIBTS0735-2-N was tested for its ability to metabolize xylose in YPX40 medium. Specifically, colonies on the plates or glycerol tubes frozen at-80℃were inoculated into 3ml YPD20X10 medium for activation culture at 30℃and 240rpm. 200 μl of the culture medium was transferred to a new YPD20X10 tube for culture to prepare secondary seeds. Cells were collected by centrifugation at 12000rpm for 3min, washed 1 time with sterile water, and inoculated into YPX40 fermentation medium at an inoculum size of 0.5g/l, at 30℃and fermented at 240rpm. Wherein the conversion reference between strain OD value and dry weight (Diao, L., et al, BMC Biotechnol,2013.13 (1): p.110.), briefly 1OD 600 =0.63 g DCW/L. Samples were analyzed at specific time points during fermentation.
(3) Cell growth was measured using a Beckman Coulter DU 730.730 spectrophotometer at 600nm. Xylose and ethanol concentrations were determined using Agilent 1200HPLC, with the specific conditions being a column Bio-Rad HPX-87H, differential detector, column temperature 65 ℃, mobile phase 5mM dilute sulfuric acid, flow rate 0.6ml/min.
Experimental results:
OD of three strains CIBTS0573, CIBTS0735 and CIBTS0735-2-N grown in YPX40 Medium 600 The values are listed in table 2.
TABLE 2 OD of three strains grown in YPX40 Medium 600 Value of
After knocking out the high copy XYLA of CIBTS0735, the growth condition of the obtained strain CIBTS0735-2-N in a xylose culture medium is obviously deteriorated, which indicates that the metabolic capacity of xylose is greatly reduced. See fig. 4.
Example 4: NFS1 in CIBTS0735 I492N Reduced xylose metabolism in the strain after reversion to wild type
The experimental method comprises the following steps:
gene NFS1 in CIBTS0735 genome based on CRISPR technology I492N The mutation reverts to wild type NFS1 and the resulting strain is tested for xylose fermentation ability. The 492 th asparagine (N) codon is AAC, the wild type is isoleucine (I), and the codon is ATC, so that the asparagine can be reverted to isoleucine by replacing the base AAC with ATC.
The experimental steps are as follows:
cas9 protein expression plasmid pHCas9-Nours (see Wang, X., et al, biotechnol bioeng.2019,116 (2): 283-293.) was introduced into CIBTS0735, and the gRNA expression plasmid pSCm-gRNA-NFS1mu and corresponding homology arms were returned to NFS1 I492N Methods for mutagenesis to wild-type, plasmid and fragment transfer into Saccharomyces cerevisiae cells can be referred to (Gietz, R.D. and R.H. Schiestl. Nat. Protoc,2007,2 (1): p.31-4.).
(1) Construction of the gRNA expression plasmid pSCm-gRNA-NFS1 mu: the pSCm-N20-V3 plasmid was digested with restriction enzyme BsaI, and the 5984bp fragment was recovered from the gel as a backbone for use. Synthesizing N20 (492N) -F and N20 (492N) -R primers, and annealing the two primers to form a double-stranded sequence, wherein the reaction system is as follows:
the temperature is kept at 95 ℃ for 5 minutes, then reduced by 5-10 ℃ per minute, and the temperature is kept at 16 ℃ for 10 minutes. The obtained product is diluted 10 times and then subjected to ligation reaction with a 5984bp skeleton fragment by using a T4 library kit at 16 ℃, then is chemically transformed into DH5 alpha competent cells, an LB solid plate containing 100 mu g/mL ampicillin is coated, the mixture is cultured overnight at 37 ℃ until transformants appear, colony PCR verification is carried out on the transformants by using primers c-SCm-gRNA-F/N20 (492N) -R, and 288bp bands can be amplified from positive transformants; transformants verified to be correct by colony PCR were inoculated into LB tubes containing 100. Mu.g/mL ampicillin, incubated overnight at 37℃and extracted from the plasmid to confirm correct transformants, and the sequencing primer was c-SCm-gRNA-F. The map structure of plasmid pSCm-N20-V3 is shown in FIG. 13.
(2) Homology arm construction for site-directed change: the genome of CIBTS0573 strain is used as a template, a 560bp fragment is amplified by a primer pair NFS1 (1513-1535) -F/NFS1-dn (2044-2072) -R, and gel is recovered as a homology arm.
(3) Introduction of plasmids and fragments into host cells and identification of transformants: pHCas9-Nours plasmid was introduced into CIBTS0735, coated with YPD20 plates containing 200. Mu.g/mL of nourseothricin and incubated at 30 ℃; picking out the grown transformant to prepare competent cells, introducing pSCm-gRNA-NFS1mu and the homology arm fragment into the prepared competent cells, coating YPD20 plates containing 200 mu g/mL of nociceptin and 400 mu g/mL of hygromycin, and culturing at 30 ℃; several transformants were randomly picked and colony PCR amplified using the primers NFS1 (1513-1535) -F/NFS1-dn (2044-2072) -R, the products were sequenced, the sequencing primer was NFS1 (1513-1535) -F, and the success of the mutation was verified.
PCR primer sequence listing
| Primer(s) | Sequence (5 '-3') |
| N20(492N)-F | GATCTTGACTTAAACTCCAACAAA |
| N20(492N)-R | AAACTTTGTTGGAGTTTAAGTCAA |
| c-SCm-gRNA-F | GAAAAGATAATGTATGATTA |
| NFS1(1513-1535)-F | ATCGTTATCCAGGGTGTGTTAAC |
| NFS1-dn(2044-2072)-R | TTTATCAAGAAGGAGAAAAAGGAGGATGT |
(4) Picking up the recovered transformant CIBTS0735-52-W, and verifying the xylose fermentation capacity.
Experimental results:
OD of three strains CIBTS0573, CIBTS0735 and CIBTS0735-52-W grown in YPX40 Medium 600 The values are listed in table 3.
TABLE 3 OD of three strains grown in YPX40 Medium 600 Value of
NFS1 in CIBTS0735 I492N After the mutation reverts to the wild type, the obtained strain CIBTS0735-52-W obviously worsens in the growth condition of the xylose culture medium, which shows that the xylose metabolism capability is greatly reduced. See fig. 5.
Example 5: iterative integration of high copy XyleA in Pre-acclimated Strain CIBTS0573
The experimental method comprises the following steps:
to further verify the contribution of high copy XylA to the xylose fermentation of the strain, the high copy XylA was iteratively integrated in pre-acclimated strain CIBTS0573 and the resulting strain was tested for growth in YPX40 medium.
The experimental steps are as follows:
the growth of the test strains in YPX40 medium was tested by site-directed integration of 10, 18, 26, 34, 42, 50 copies of XyleA at the loci XII-1, XI-3, XII-5, X-2, X-3, XI-2, XII-3, X-4, L36, L37, L39 and L44 of the chromosome XII-1, XI-3, XII-5, X-2, X-3, X-39 and L44 containing 2 copies of XyleA strain CIBTS 0573.
(1) 8 copies of the PsXYLA gene were integrated at XII-5 and X-2 sites: psXYLA expression plasmids pXII-5-2PsXI and pX-2-2PsXI, respectively, with homology arms at XII-5 site and X-2 site, were first constructed.
Construction of pX-2-2PsXI plasmid: the plasmid pX-2-RuXI (constructed and preserved by the subject group, which can be obtained by any unit or individual for verification of the present invention, but which is not allowed to be used for other purposes by the subject group, including scientific research and teaching) is used as a template, the primer pair pX-PsXI-F1/pX-PsXI-R1 is amplified by 3286bp, and the gel is recovered as a backbone; the strain CIBTS0573 containing PsXYLA expression frame is used as a template, a primer pair PsXI-TPI-R1/PsXI-TPI-F1 is amplified for 1356bp, and gel is recovered as a fragment; the above skeleton and fragment are recombined in vitro by using a vazyme multis recombination kit at 37 ℃, then are chemically transformed into Top10 competent cells, LB plates containing 100 mug/mL ampicillin are coated, and the mixture is cultured overnight at 37 ℃ until transformants are obtained; transformants were randomly picked up, inoculated into LB tubes containing 100. Mu.g/mL ampicillin, incubated overnight at 37℃and plasmids were extracted, digested with ScaI and the correct plasmid was maintained for further use, and the resulting plasmid was designated as pX-2-PsXI. Using pX-2-PsXI as template, using primer TPI-RuXI-F2/TPI-RuXI-R2 to amplify 2297bp, recovering gel, using EcoRI and EcoRV enzyme to cut, cleaning and recovering as fragment; the plasmid pX-2-PsXI was digested with EcoRI and EcoRV, and 4551bp was recovered as a backbone from the gel; after the above-mentioned scaffold and fragment were ligated in vitro with Solution I kit at 16℃and then chemically transformed into Top10 competent cells, LB plates containing 100. Mu.g/mL ampicillin were spread, the transformants were cultured overnight at 37℃in an incubator until there were transformants, several transformants were randomly picked up, and the transformants were inoculated into LB tubes containing 100. Mu.g/mL ampicillin, cultured overnight at 30℃and the plasmids were extracted, and digested with ScaI to confirm that the correct plasmid was designated as pX-2-2PsXI. Xho1 is digested, cleaned and recycled for standby.
Construction of pXII-5-2PsXI plasmid: amplifying the downstream homology arm by using CIBTS0573 strain as a template, amplifying the downstream homology arm by using primers XII-5D-F/XII-5D-R and XII-5U-F/XII-5U-R, recovering gel, performing overlap-PCR (abbreviated as OE-PCR) by using the primers XII-5D-F/XII-5U-R to obtain 459bp, and performing enzyme digestion by using PvuII, recovering gel as a fragment; the pX-2-2PsXI plasmid was digested with PvuII, and the 6440bp fragment was recovered as a backbone (hereinafter abbreviated as PsXI backbone) from the gel. The PsXI backbone and the above fragments were recombined in vitro using a vazyme Multis recombination kit at 37℃and chemically transformed into Top10 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, and incubated overnight in an incubator at 37℃until transformants were obtained. Several transformants were randomly picked, colony PCR verified with primers Uc-sq-R/XII-5D-F, the positive transformants were 470bp long, and plasmids were extracted from the correct transformants by amplification, designated pXII-5-2PsXI. Xho1 is digested, cleaned and recycled for standby.
gRNA plasmid pYES2-XII-5-X-2-gRNA-hyg, targeting both XII-5 and X-2 sites, see Wang, X., et al 4Biotechnol Bioeng.2019,116 (2): 283-293.
pHCas9-Nours was plasmid-transferred into CIBTS0573 strain, then pXII-5-2PsXI and pX-2-2PsXI fragments recovered after pYES2-XII-5-X-2-gRNA-hyg plasmid and Xho1 were introduced. Transformants were screened using a double-antibody panel of North silk fibroin plus hygromycin, and the resulting transformants were subjected to verification of XII-5 site integration using primers Uc-sq-R/XII-5up-F, XII-5up-F/XII-5dn-R and X-2 site integration using primers Uc-sq-R/X-2up-F, X-2up-F/X-2 dn-R. Ensuring that the two sites together integrate 8 copies of PsXYLA. The resulting strain was designated CIBTS0573-10-W.
(2) 8 copies of the PsXYLA gene were integrated at XII-1 and XI-3 sites: psXYLA expression plasmids pXII-1-2PsXI and pXI-3-2PsXI, respectively, with homology arms at XII-1 site and XI-3 site were first constructed.
Construction of pXII-1-2PsXI plasmid: the strain CIBTS0573 is used as a template, the primers XII-1D-F/XII-1D-R and XII-1U-F/XII-1U-R are used for amplifying the downstream homology arms respectively, the primer XII-1D-F/XII-1U-R is used for OE-PCR amplification to obtain 341bp fragments, and the fragments are digested with PvuII and recovered by gel. The PsXI backbone and the above fragments were recombined in vitro with a vazyme Multis recombination kit at 37℃and chemically transformed into Top10 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, and incubated overnight in an incubator at 37℃until transformants were obtained. Several transformants were randomly picked and colony PCR verified with primers Uc-sq-R/XII-1D-F, positive transformants were 381bp in length. The plasmid was extracted from the correct transformant by amplification and designated pXII-1-2PsXI. Xho1 is digested, cleaned and recycled for standby.
Construction of pXI-3-2PsXI plasmid: the strain CIBTS0573 is used as a template, the primers XI-3D-F/XI-3D-R and XI-3U-F/XI-3U-R are used for amplifying the upstream homology arms respectively, the primers XI-3D-F/XI-3U-R are used for carrying out OE-PCR to obtain 359bp fragments, pvuII is used for enzyme digestion, and gel recovery is used as the fragments. The PsXI backbone and the above fragments were recombined in vitro with a vazyme Multis recombination kit at 37℃and chemically transformed into Top10 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, and incubated overnight in an incubator at 37℃until transformants were obtained. Several transformants were randomly picked and colony PCR verified with primers Uc-sq-R/XI-3D-F, positive transformants were 393bp in length. Plasmids were extracted from the correct transformants by amplification and designated pXI-3-2PsXI. Xho1 is digested, cleaned and recycled for standby.
gRNA plasmids pYES2-XII-1-XI-3-gRNA-hyg targeting positions XII-1 and XI-3 see Wang, X., et al Unraveling the genetic basis of fast l-arabinose consumption on top of recombinant xylose-fermenting Saccharomyces cerevisiae.Biotechnol bioeng.2019,116 (2): 283-293.
pHCas9-Nours was plasmid transformed into CIBTS0573-10-W strain, followed by pYES2-XII-1-XI-3-gRNA-hyg plasmid and pXII-1-2PsXI and pXI-3-2PsXI fragments recovered after Xho1 cleavage. Transformants were screened using a North-wire-plus-hygromycin double-antibody plate, and the resulting transformants were verified for XII-1 site integration using the primers Uc-sq-R/XII-1up-F, XII-1up-F/XII-1dn-R and for XI-3 site integration using the primers Uc-sq-R/XI-3up-F, XI-3up-F/XI-3 dn-R. Ensuring that the two sites together integrate 8 copies of PsXYLA. The resulting strain was designated CIBTS0573-18-W.
(3) 8 copies of the PsXYLA gene were integrated at XII-3 and X-4 sites: psXYLA expression plasmids pXII-3-2PsXI and pX-4-2PsXI, respectively, with homology arms at XII-3 site and X-4 site, were first constructed.
Construction of pXII-3-2PsXI plasmid: the strain CIBTS0573 is used as a template, the primers XII-3D-F/XII-3D-R and XII-3U-F/XII-3U-R are used for amplifying the downstream homology arms respectively, the primer XII-3D-F/XII-3U-R is used for OE-PCR amplification to obtain 418bp fragments, and the 418bp fragments are digested with PvuII and recovered by gel as fragments. The PsXI backbone and the above fragments were recombined in vitro with a vazyme Multis recombination kit at 37℃and chemically transformed into Top10 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, and incubated overnight in an incubator at 37℃until transformants were obtained. Several transformants were randomly picked and colony PCR verified with primers Uc-sq-R/XII-3D-F, positive transformants were 452bp in length. The plasmid was extracted from the correct transformant by amplification and designated pXII-3-2 PsXI. Xho1 is digested, cleaned and recycled for standby.
Construction of pX-4-2PsXI plasmid: the strain CIBTS0573 is used as a template, the primers X-4D-F/X-4D-R and X-4U-F/X-4U-R are used for respectively amplifying the upstream homology arms, the primer X-4D-F/X-4U-R is used for carrying out OE-PCR amplification to obtain 442bp fragments, and the fragments are cut by PvuII and recovered by gel to be used as fragments. The PsXI backbone and the above fragments were recombined in vitro with a vazyme Multis recombination kit at 37℃and chemically transformed into Top10 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, and incubated overnight in an incubator at 37℃until transformants were obtained. Randomly picking a plurality of transformants, and carrying out colony PCR verification by using a primer Uc-sq-R/X-4D-F, wherein the length of a positive transformant is 476bp. Plasmids were extracted from the correct transformants by amplification and designated pX-4-2 PsXI. Xho1 is digested, cleaned and recycled for standby.
gRNA plasmid pYES2-XII-3-X-4-gRNA-hyg targeting sites XII-3 and X-4 see Wang, X., et al, biotechnol bioeng.2019,116 (2): 283-293.
pHCas9-Nours was plasmid transformed into CIBTS0573-10-W strain, followed by pYES2-XII-3-X-4-gRNA-hyg plasmid and pXII-3-2PsXI and pX-4-2PsXI fragments recovered after Xho1 cleavage. Transformants were screened using a double-antibody panel of North silk fibroin plus hygromycin, and the resulting transformants were subjected to verification of XII-3 site integration using primers Uc-sq-R/XII-3up-F, XII-3up-F/XII-3dn-R and X-4 site integration using primers Uc-sq-R/X-4up-F, X-4up-F/X-4 dn-R. Ensuring that the two sites together integrate 8 copies of PsXYLA. The resulting strain was designated CIBTS0573-26-W.
(4) Integration of 8 copies of the PsXYLA gene at the X-3 and XI-2 sites: psXYLA expression plasmids pX-3-2PsXI and pXI-2-2PsXI were first constructed with homology arms to the X-3 site and XI-2 site, respectively.
Construction of pX-3-2PsXI plasmid: the bacterial strain CIBTS0573 is used as a template, the primers X-3D-F/X-3D-R and X-3U-F/X-3U-R are used for respectively amplifying the upstream homology arms, the primers X-3D-F/X-3U-R are used for carrying out OE-PCR amplification to obtain a 412bp fragment, and the fragment is cut by PvuII and recovered by gel to be used as a fragment. The PsXI backbone and the above fragments were recombined in vitro with a vazyme Multis recombination kit at 37℃and chemically transformed into Top10 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, and incubated overnight in an incubator at 37℃until transformants were obtained. Several transformants were randomly picked and colony PCR verified with primers Uc-sq-R/X-3D-F, positive transformants were 446bp in length. Plasmids were extracted from the correct transformants by amplification and designated pX-3-2 PsXI. Xho1 is digested, cleaned and recycled for standby.
Construction of pXI-2-2PsXI plasmid: the strain CIBTS0573 is used as a template, the primers XI-2D-F/XI-2D-R and XI-2U-F/XI-2U-R are used for amplifying the upstream homology arms respectively, the primers XI-2D-F/XI-2U-R are used for carrying out OE-PCR amplification to obtain a 408bp fragment, and the 408bp fragment is digested with PvuII and recovered by gel to be used as the fragment. The PsXI backbone and the above fragments were recombined in vitro with a vazyme Multis recombination kit at 37℃and chemically transformed into Top10 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, and incubated overnight in an incubator at 37℃until transformants were obtained. Randomly picking a plurality of transformants, and carrying out colony PCR verification by using a primer Uc-sq-R/X-4D-F, wherein the length of a positive transformant is 442bp. Plasmids were extracted from the correct transformants by amplification and designated pXI-2-2PsXI. Xho1 is digested, cleaned and recycled for standby.
The gRNA plasmid pYES2-X-3-XI-2-gRNA-hyg targeting the X-3 and XI-2 sites is described in Wang, X., et al, biotechnol bioeng.2019,116 (2): 283-293.
pHCas9-Nours was plasmid transformed into CIBTS0573-26-W strain, followed by pYES2-X-3-XI-2-gRNA-hyg plasmid and pX-3-2PsXI and pXI-2-2PsXI fragments recovered after Xho1 cleavage. Transformants were screened using a North-wire-and-hygromycin-double-antibody plate, and the resulting transformants were subjected to verification of X-3 site integration using primers Uc-sq-R/X-3up-F, X-3up-F/X-3dn-R and XI-2 site integration using primers Uc-sq-R/XI-2up-F, XI-2up-F/XI-2 dn-R. Ensuring that the two sites together integrate 8 copies of PsXYLA. The resulting strain was designated CIBTS0573-34-W.
(5) Integration of 8 copies of the PsXYLA gene at the L39 and L44 sites: first, psXYLA expression plasmids pL39-2PsXI and pL44-2PsXI were constructed, which bear homology arms for the L39 site and the L44 site, respectively.
Construction of pL39-2PsXI plasmid: the strain CIBTS0573 is used as a template, the primers L39D-F/L39D-R and L39U-F/L39U-R are used for respectively amplifying the upstream homology arms, the primers L39D-F/L39U-R are used for carrying out OE-PCR amplification to obtain 468bp fragments, and the fragments are digested with PvuII and recovered by gel to be used as fragments. The PsXI backbone and the above fragments were recombined in vitro with a vazyme Multis recombination kit at 37℃and chemically transformed into Top10 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, and incubated overnight in an incubator at 37℃until transformants were obtained. Several transformants were randomly picked and colony PCR verified with primers Uc-sq-R/L39D-F, positive transformants were 497bp in length. Plasmid was extracted from the correct transformant by amplification and designated pL39-2PsXI. Xho1 is digested, cleaned and recycled for standby.
Construction of pL44-2PsXI plasmid: the strain CIBTS0573 is used as a template, the primers L44D-F/L44D-R and L44U-F/L44U-R are used for respectively amplifying the upstream homology arms, the primers L44D-F/L44U-R are used for OE-PCR amplification to obtain 509bp fragments, and the fragments are digested with PvuII and recovered by gel as fragments. The PsXI backbone and the above fragments were recombined in vitro with a vazyme Multis recombination kit at 37℃and chemically transformed into Top10 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, and incubated overnight in an incubator at 37℃until transformants were obtained. Randomly picking a plurality of transformants, and carrying out colony PCR verification by using a primer Uc-sq-R/L44D-F, wherein the length of a positive transformant is 517bp. Plasmid was extracted from the correct transformant by amplification and designated pL44-2PsXI. Xho1 is digested, cleaned and recycled for standby.
Construction of pSCM-L39-L44-gRNA-hyg plasmid: with plasmid pSCm-N20-V3 (amp) r ) As a template, bsaI was used for cleavage, and 5984bp was recovered from the gel as a scaffold (hereinafter abbreviated as gRNA scaffold). First, construct pSCM-L39-gRNA-hyg plasmid: the two synthesized N20 oligo primers are annealed to form double-chain sequences, and the system is as follows: deionized water 35. Mu.L, T4 library buffer 5. Mu.L, primer N20-L39-F5. Mu.L (20. Mu.M), N20-L39-R5. Mu.L (20. Mu.M). The temperature is kept at 95 ℃ for 5min, the temperature is reduced by 10 ℃ per minute, and the temperature is kept at 16 ℃ for 10min. The annealed product was diluted 10-fold as a fragment. The gRNA skeleton and the fragment are connected in vitro by using T4 ligase at 16 ℃ and then are chemically transformed into DH5 alpha competent cells, LB plates containing 100 mug/mL ampicillin are coated, the transformants are cultured overnight at 37 ℃ and randomly picked up, and colony PCR verification is carried out by using a primer c-SCm-gRNA-F/N20-L39-R, and the length is 288bp. The plasmid was extracted from the correct transformant by amplification and designated pSCM-L39-gRNA-hyg. The gel was digested with SacI and BamHI, and 6008bp was recovered as L39-gRNA backbone for use. Then construct pSCM-L44-gRNA-hyg plasmid: the two synthesized N20 oligo primers are annealed to form double-chain sequences, and the system is as follows: deionized water 35. Mu.L, T4 library buffer 5. Mu.L, primer N20-L44-F5. Mu.L (20. Mu.M), N20-L44-R5. Mu.L (20. Mu.M). The temperature is kept at 95 ℃ for 5min, the temperature is reduced by 10 ℃ per minute, and the temperature is kept at 16 ℃ for 10min. The annealed product was diluted 10-fold as a fragment. The gRNA skeleton and the fragment are connected in vitro by using T4 ligase at 16 ℃ and then are chemically transformed into DH5 alpha competent cells, LB plates containing 100 mug/mL ampicillin are coated, the transformants are cultured overnight at 37 ℃ and randomly picked up, and colony PCR verification is carried out by using a primer c-SCm-gRNA-F/N20-L44-R, and the length is 288bp. The plasmid was extracted from the correct transformant by amplification and designated pSCM-L44-gRNA-hyg. The pSCM-L44-gRNA-hyg plasmid is used as a template, a primer BamH1-F/SacI-R is amplified to obtain 388bp, gel is recovered, sacI and BamHI are used for enzyme cutting of the fragments, and the fragments are cleaned and recovered to be used as L44-gRNA fragments for standby. The L39-gRNA backbone was combined with L44-g RNA fragments were ligated in vitro at 16℃using Solution I kit, chemically transformed into Top10 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, incubated overnight at 37℃in an incubator until transformants were present, randomly picked up several transformants, and colony PCR verified with primer BamHI-F/L44-R, 293bp in length. The plasmid was extracted from the correct transformant by amplification and designated pSCM-L39-L44-gRNA-hyg.
pHCas9-Nours was plasmid transformed into CIBTS0573-34-W strain, followed by pSCM-L39-L44-gRNA-hyg plasmid and pL39-2PsXI and pL44-2PsXI fragments recovered after Xho1 cleavage. Transformants were screened using a North-silk fibroin plus hygromycin double antibody plate, and the resulting transformants were verified for L39 site integration using primers Uc-sq-R/L39-id-F, L39-id-F/L39-id-R and L44 site integration using primers Uc-sq-R/L44-id-F, L-id-F/L44-id-R. Ensuring that the two sites together integrate 8 copies of PsXYLA. The resulting strain was designated CIBTS0573-42-W.
(6) Integration of 8 copies of the PsXYLA gene at the L36 and L37 sites: first, psXYLA expression plasmids pL36-2PsXI and pL37-2PsXI were constructed, which bear homology arms for the L36 site and the L37 site, respectively.
Construction of pL36-2PsXI plasmid: the strain CIBTS0573 is used as a template, the primers L36D-F/L36D-R and L36U-F/L36U-R are used for respectively amplifying the upstream homology arms, the primers L36D-F/L36U-R are used for OE-PCR amplification to obtain 455bp fragments, and the fragments are digested with PvuII and recovered by gel as fragments. The PsXI backbone and the above fragments were recombined in vitro with a vazyme Multis recombination kit at 37℃and chemically transformed into Top10 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, and incubated overnight in an incubator at 37℃until transformants were obtained. Several transformants were randomly picked and colony PCR verified with primers Uc-sq-R/L36D-F, positive transformants were 462bp in length. Plasmid was extracted from the correct transformant by amplification and designated pL36-2PsXI. Xho1 is digested, cleaned and recycled for standby.
Construction of pL37-2PsXI plasmid: the strain CIBTS0573 is used as a template, the primers L37D-F/L37D-R and L37U-F/L37U-R are used for respectively amplifying the upstream homology arms, the primers L37D-F/L37U-R are used for OE-PCR amplification to obtain 445bp fragments, and the fragments are digested with PvuII and recovered by gel to be used as fragments. The PsXI backbone and the above fragments were recombined in vitro with a vazyme Multis recombination kit at 37℃and chemically transformed into Top10 competent cells, plated with LB plates containing 100. Mu.g/mL ampicillin, and incubated overnight in an incubator at 37℃until transformants were obtained. Randomly picking a plurality of transformants, and carrying out colony PCR verification by using a primer Uc-sq-R/L37D-F, wherein the length of a positive transformant is 452bp. Plasmid was extracted from the correct transformant by amplification and designated pL37-2PsXI. Xho1 is digested, cleaned and recycled for standby.
Construction of pSCM-L36-L37-gRNA-hyg plasmid: first, construct pSCM-L36-gRNA-hyg plasmid: the two synthesized N20 oligo primers are annealed to form double-chain sequences, and the system is as follows: deionized water 35. Mu.L, T4 library buffer 5. Mu.L, primer N20-L36-F5. Mu.L (20. Mu.M), N20-L36-R5. Mu.L (20. Mu.M). The temperature is kept at 95 ℃ for 5min, the temperature is reduced by 10 ℃ per minute, and the temperature is kept at 16 ℃ for 10min. The annealed product was diluted 10-fold as a fragment. The gRNA skeleton and the fragment are connected in vitro by using T4 ligase at 16 ℃ and then are chemically transformed into DH5 alpha competent cells, LB plates containing 100 mug/mL ampicillin are coated, the transformants are cultured overnight at 37 ℃ and randomly picked up, and colony PCR verification is carried out by using a primer c-SCm-gRNA-F/N20-L36-R, and the length is 288bp. The plasmid was extracted from the correct transformant by amplification and designated pSCM-L36-gRNA-hyg. The gel was digested with SacI and BamHI, and 6008bp was recovered as L36-gRNA scaffold for use. Then construct pSCM-L37-gRNA-hyg plasmid: the two synthesized N20 oligo primers are annealed to form double-chain sequences, and the system is as follows: deionized water 35. Mu.L, T4 library buffer 5. Mu.L, primer N20-L37-F5. Mu.L (20. Mu.M), N20-L37-R5. Mu.L (20. Mu.M). The temperature is kept at 95 ℃ for 5min, the temperature is reduced by 10 ℃ per minute, and the temperature is kept at 16 ℃ for 10min. The annealed product was diluted 10-fold as a fragment. The gRNA skeleton and the fragment are connected in vitro by using T4 ligase at 16 ℃ and then are chemically transformed into DH5 alpha competent cells, LB plates containing 100 mug/mL ampicillin are coated, the transformants are cultured overnight at 37 ℃ and randomly picked up, and colony PCR verification is carried out by using a primer c-SCm-gRNA-F/N20-L37-R, and the length is 288bp. The plasmid was extracted from the correct transformant by amplification and designated pSCM-L37-gRNA-hyg. The pSCM-L37-gRNA-hyg plasmid is used as a template, a primer BamH1-F/SacI-R is amplified to obtain 388bp, gel is recovered, sacI and BamHI are used for enzyme cutting of the fragments, and the fragments are cleaned and recovered to be used as L37-gRNA fragments for standby. The L36-gRNA skeleton and the L37-gRNA fragment are connected in vitro by using a Solution I kit at 16 ℃, are chemically transformed into Top10 competent cells, are coated with LB plates containing 100 mug/mL ampicillin, are cultured overnight in a 37 ℃ incubator until transformants are present, randomly pick up a plurality of transformants, and are subjected to colony PCR verification by using a primer BamHI-F/L37-R, and have a length of 293bp. The plasmid was extracted from the correct transformant by amplification and designated pSCM-L36-L37-gRNA-hyg.
pHCas9-Nours was plasmid transformed into CIBTS0573-42-W strain, followed by pSCM-L36-L37-gRNA-hyg plasmid and pL36-2PsXI and pL37-2PsXI fragments recovered after Xho1 cleavage. Transformants were screened using a North-silk fibroin plus hygromycin double antibody plate, and the resulting transformants were verified for L36 site integration using primers Uc-sq-R/L36-id-F, L-id-F/L36-id-R and L37 site integration using primers Uc-sq-R/L37-id-F, L-id-F/L37-id-R. Ensuring that the two sites together integrate 8 copies of PsXYLA. The resulting strain was designated CIBTS0573-50-W.
PCR primer sequence listing
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Experimental results:
OD of strains containing different XylA copy numbers grown in YPX40 Medium 600 The values are shown in fig. 6.
As can be seen from the figure, the XyleA copy number is gradually supplemented back to 50 in the CIBTS0573 genome, and the strain is subjected to OD (OD) in xylose culture medium along with the increase of the XyleA copy number 600 The value gradually increases, which shows that the copy number has a good promotion effect on the xylose utilization of the strain. But the OD value did not increase further by 34 copies and at this time the strain grew significantly weaker than the acclimatized strain CIBTS0735, indicating additional limitationsFactors restrict the xylose utilization of the strain.
Example 6: introduction of NFS1 into Pre-acclimatized Strain CIBTS0573 I492N Mutating, iteratively integrating high copy XylA
The experimental method comprises the following steps:
mutation of isoleucine at 492 rd position of cysteine desulphurase NFS1 in pre-acclimatized strain CIBTS0573 to asparagine while iterative integration of high copy XyleA, test of the resulting strain for OD growth in YPX40 Medium 600 Values.
The experimental steps are as follows:
mutation of the gene encoding xylose isomerase NFS1 in the genome of the CIBTS0573 strain based on CRISPR technology, mutation of isoleucine at position 492 of NFS1 to asparagine plasmid and fragment transfer into Saccharomyces cerevisiae cell reference (Gietz, R.D.and R.H.Schiestl. Nat Protoc,2007,2 (1): p.31-4.).
(1) Construction of the gRNA expression plasmid pSCm-gRNA-NFS1 wt: the pSCm-N20-V3 plasmid was digested with restriction enzyme BsaI, and the 5984bp fragment was recovered from the gel as a backbone for use. Synthesizing the gRNA-492I-F and the gRNA-492I-R primers, and annealing the two primers to form a double-stranded sequence, wherein the reaction system is as follows:
the temperature is kept at 95 ℃ for 5 minutes, then reduced by 5-10 ℃ every minute, and the temperature is kept at 16 ℃ for 10 minutes. The obtained product is diluted 10 times and then subjected to ligation reaction with a 5984bp skeleton fragment by using a T4 library kit at 16 ℃, then is chemically transformed into DH5 alpha competent cells, an LB solid plate containing 100 mu g/mL ampicillin is coated, the mixture is cultured overnight at 30 ℃ until transformants appear, colony PCR verification is carried out on the transformants by using primers c-SCm-gRNA-F/gRNA-492I-R, and 288bp bands can be amplified from positive transformants; transformants verified to be correct by colony PCR were inoculated into LB tubes containing 100. Mu.g/mL ampicillin, incubated overnight at 30℃and extracted from plasmid to confirm correct transformants by sequencing with the primers c-SCm-gRNA-F.
(2) Homology arm construction for site-directed change: the genome of CIBTS0735 strain is used as a template, a 560bp fragment is amplified by a primer pair NFS1 (1513-1535) -F/NFS1-dn (2044-2072) -R, and gel is recovered as a homology arm.
(3) Introduction of plasmids and fragments into host cells and identification of transformants: pHCas9-Nours plasmid was introduced into CIBTS0573, coated with YPD20 plates containing 200. Mu.g/mL of nourseothricin and incubated at 30 ℃; picking out the grown transformant to prepare competent cells, introducing pSCm-gRNA-NFS1wt and the homology arm fragment into the prepared competent cells, coating YPD20 plates containing 200 mug/mL of nociceptin and 400 mug/mL of hygromycin, and culturing at 30 ℃; several transformants were randomly picked and colony PCR amplified using the primers NFS1 (1513-1535) -F/NFS1-dn (2044-2072) -R, the products were sequenced, the sequencing primer was NFS1 (1513-1535) -F, and the success of the mutation was verified.
PCR primer sequence listing
| Primer(s) | Sequence (5 '-3') |
| gRNA-492I-F | GATCTCAATGACCTGACCATTTGA |
| gRNA-492I-R | AAACTCAAATGGTCAGGTCATTGA |
(2) Based on the strain obtained in step (1), 10, 18, 26, 34, 42, 50 copies of XYLA were site-directed integrated on the chromosomes XII-1, XI-3, XII-5, X-2, X-3, XI-2, XII-3, X-4, L36, L37, L39 and L44 in the same manner as in example 5, and the strain was tested for growth in YPX40 medium.
Experimental results:
bacteria containing different XYLA copy numbers after gene NFS1 mutationOD of strain grown in YPX40 Medium 600 The values are shown in fig. 7.
The results in FIG. 7 clearly show that the OD of the mutant strain grown in YPX40 medium gradually increased with increasing XylA copy number. In particular, at 42 and 50 copies, the strain completely reached growth performance comparable to that of the acclimatized strain CIBTS0735, indicating that NFS1 was introduced based on CIBTS0573, the strain prior to acclimatization I492N The mutant, superimposed with high copy XyleA, can directly obtain the reconstructed strain with growth comparable to CIBTS0735 without depending on domestication.
FIG. 14 is a comprehensive comparison of strains CIBTS0573 and NFS 1-bearing strains prior to acclimation I492N As a result of iteratively integrating multiple copies of PsXylA based on the mutant pre-acclimatized strain, the xylose metabolism ability was represented by the strain growth OD600 value in 24 hours YPX40 medium, which can be seen intuitively in the presence of NFS1 I492N In the mutant CIBTS0573 strain, when 34 copies were integrated iteratively, the strain grew OD 600 The values were essentially the same as the acclimatized strain CIBTS0735 (black filled circles) and reached 50 copies, which had reached the acclimatized strain level completely. The chromosome of the strain integrates high-copy PsXyl A before domestication, and NFS1 capable of improving the cytoplasmic iron ion content is introduced I492N Mutation, i.e., direct construction of xylose yeast strains that achieve industrial-grade levels of use, was unexpected before.
Example 7: NFS1 with three different XYLA copy numbers I492N Sugar consumption and alcohol production test of mutant strain in YPX40 culture medium
The experimental method comprises the following steps:
the xylose consumption and ethanol production of the three recombinant CIBTS0573-34-N, CIBTS0573-42-N, CIBTS0573-50-N strains in YPX40 medium were compared in detail.
The experimental steps are as follows:
the second seed solution of the test strain is prepared and inoculated into YPX40 culture medium according to 0.5g/L dry weight, and the residual xylose and the concentration of ethanol produced in the culture medium are measured by periodic sampling liquid chromatography.
Experimental results:
as shown in fig. 8, in NFS1 I492N When the XylA copy number reaches 42 in the presence of mutation, the xylose metabolism ability of the strain is almost the sameIs identical to the domesticated plant. The ethanol curves shown in FIG. 9 show similar results, with strains CIBTS0573-42-N and CIBTS0573-50-N containing 42 and 50 copies of XyleA eventually producing the same ethanol content as the domesticated strain CIBTS0735 and peaking earlier than CIBTS 0735. Except for OD which characterizes growth 600 The values, xylose consumption and ethanol production clearly indicate that NFS1 was introduced on the pre-acclimated strain CIBTS0573 I492N Mutations and copying XyleA by 42 or more, and can realize the rational reconstruction of the saccharomyces cerevisiae strain for rapidly fermenting xylose without depending on domestication.
Example 8: knocking out ISU1 gene also promotes strain to ferment xylose
The experimental method comprises the following steps:
the ISU1 gene was knocked out from CIBTS0573 strain containing 34 copies of XyleA, and the growth of the resulting strain in YPX40 medium was determined.
The experimental steps are as follows:
(1) The CRISPR technique was used to knock out the ISU1 gene from the CIBTS0573 strain containing 34 copies of XylA. Specific knockout phase Guan Zhili was constructed as reference example 4.
(2) Picking up transformant which is verified to be knocked out successfully, testing YPX40 culture medium growth condition, sampling periodically and measuring OD 600 Values.
Experimental results:
strain containing 34 copies of XyleA knocks out OD grown in YPX40 Medium before and after ISU1 Gene 600 The values are listed in table 4.
TABLE 4 OD of different strains grown in YPX40 Medium before and after knocking out ISU1 Gene 600 Value of
The gene NFS1 codes cysteine desulphurase and supplies the sulfur element of the iron-sulfur cluster, so NFS1 is a key gene for the synthesis of the iron-sulfur cluster. The experiment is also selected fromThe ISU1 gene of the iron-sulfur cluster synthesis pathway is tested for the effect on xylose fermentation by the strain. As shown in FIG. 10, after knocking out ISU1 gene based on 34 copies of XyleA, the strain grew significantly better in YPX40 medium with the effect of leveling NFS1 I492N Mutation. The result shows that the inhibition of iron-sulfur cluster synthesis is the reason for the improvement of xylose fermenting capability of the strain.
Example 9: gene mutant NFS1 I492N Can increase the intracellular iron ion concentration of Saccharomyces cerevisiae
The experimental method comprises the following steps:
determination of Gene mutation NFS1 Using atomic absorption Spectrophotometer I492N And intracellular iron ion concentration of the unmutated strain.
The experimental steps are as follows:
(1) Culturing and collecting the bacterial cells: the test bacteria were inoculated into 20mL of YP80D40X medium at an initial concentration of 0.5g dry weight/L, and cultured by anaerobic fermentation at 30℃and 180 rpm. Fermentation was stopped at 16.5h, the fermentation broth was collected into a 50mL centrifuge tube, centrifuged at 5000rpm for 2min and the supernatant was discarded and washed twice with sterile deionized water. Subpackaging the thalli into 3 separation tubes, centrifuging at 1,2000rpm for 2min, discarding supernatant, opening a cover of the centrifuge tube, placing the centrifuge tube into a breathable box, placing the box into an oven at 80 ℃ for 8-10h, and drying. Judging the drying standard: taking out and weighing every 3.5h, weighing, and taking out and weighing twice continuously, wherein the weight difference is less than 0.3mg, and then, taking the dried product as the drying product.
(2) Nitric acid treatment: the dried thalli is transferred into a 50mL centrifuge tube by placing the sterile 50mL centrifuge tube on a balance, zeroing, and recording the dry weight after the balance indication is stable. A10% nitric acid solution was taken, nitric acid was added in a ratio of 0.1g of dry bacteria to 5mL of 10% nitric acid, and the mass of nitric acid added was recorded. Two glass beads washed by ultrasonic are added. And vibrating the centrifuge tube, uniformly mixing the thalli, and observing that the bottom is free of blocky thalli, namely uniformly mixing. Placing the centrifuge tube in a water bath shaker at 90 ℃ for 3 hours to release intracellular metal ions; the sample was dispensed, and the supernatant was collected by centrifugation at 5000rpm for 2min in the 50mL centrifuge tube and dispensed into a 2mL centrifuge tube. Centrifuging at 1,2000rpm for 2min, collecting supernatant, and packaging into 1.5mL centrifuge tube according to each 1.5 mL. Note that the split charging needs to use an inlet gun head, so that the sample is ensured to be clean and free from other impurity ions.
(3) Atomic spectrophotometry detects iron concentration: preparing 0.4, 1.0, 1.6, 2.2 and 2.8mg/L of Fe/HCl with the concentration of 1g/L serving as a standard substance by using a national secondary standard sample for drawing a standard curve; in addition, it is known to refer to powder FeSO 4 -7H 2 O preparing a Fe sample with the concentration of 1.7mg/L, and comparing the two standard substances with each other; opening an atomic absorption spectrophotometer, and operating through SOLAAR software, wherein the flow is based on instrument description; and calculating the measured iron concentration, dry weight and nitric acid volume to obtain the intracellular iron concentration of the strain.
Experimental results:
gene mutant NFS1 I492N Intracellular iron ion concentrations of the strains CIBTS0573-2-N, CIBTS0573-34-N, CIBTS0735, the NFS1 wild type strain CIBTS0573, and the CIBTS0735-52-W strain are shown in Table 6 and FIG. 12.
TABLE 6 NFS1 I492N And NFS1 wt Influence on intracellular iron ion concentration value on bacterial strain
| Strain | Intracellular iron ion concentration ug/g DCW of strain |
| CIBTS0573 | 59.65±16.08 |
| CIBTS0573-2-N | 140.20±2.70 |
| CIBTS0573-34-N | 131.66±5.80 |
| CIBTS0735 | 129.16±9.62 |
| CIBTS0735-52-W | 50.29±3.96 |
FIG. 12 shows a gene mutant NFS1 I492N Influence on intracellular iron ion concentration of the strain. As can be seen from fig. 12, NFS1 I492N The intracellular iron concentration of the strains CIBTS0573-2-N and CIBTS0573-34-N was 1.3 times higher than that of the non-mutant strain CIBTS0573 of NFS1, and after the back mutation of NFS1 of CIBTS0735 to wild type, the intracellular iron concentration of the obtained strain CIBTS0735-52-W was reduced to 50.29. Mu.g/g DCW. The above experiments clearly show that the gene mutation NFS1 I492N Can increase the intracellular iron ion concentration of Saccharomyces cerevisiae. Namely, NFS1 mutation and ISU1 knockout which change Fe-S cluster synthesis are used for promoting the strain to ferment xylose by increasing the iron ion content of cells.
Example 10: direct iron ion addition of culture medium to promote strain to ferment xylose
The experimental method comprises the following steps:
the effect on the metabolic capacity of CIBTS0573-26-N xylose was tested by adding different concentrations of ferrous ion or iron chelator directly to YPX40 medium.
The experimental steps are as follows:
(1) The test strain is a strain CIBTS0573-26-N integrated with 26 copies of PsXYLA;
(2) Iron ion chelator ferrozine (phenanthrazine), defined as "iron-deficient" medium, was added to YPX40 medium at a final concentration of 1mM, and 1% strength ferrous ammonium sulfate, defined as "iron-rich" medium, was added to YPX40 medium;
(3) The bacterial strain CIBTS0573-26-N was tested for xylose metabolism in the "iron deficiency" and "iron rich" media, and the effect of iron ions on xylose fermentation by the bacterial strain was analyzed.
Experimental results:
the xylose consumption of the strain CIBTS0573-26-N in the above medium is shown in Table 5.
TABLE 5 xylose utilization values of CIBTS0573-26-N strain in "iron deficiency" and "iron rich" media
FIG. 11 shows the effect of the iron ion content of the medium on xylose fermentation by the strain CIBTS 0573-26-N. As can be seen from FIG. 11, a certain content of Fe was added to the medium 2+ The xylose utilization of the strain can be obviously improved, and compared with a control, the xylose of 40g/l can be consumed in 36 hours; however, after the ferrozine serving as an iron ion chelating agent is added into the culture medium, the xylose fermentation rate of the strain is obviously slowed, and the experiment shows that the addition of iron ions into the culture medium has a certain promotion effect on xylose fermentation of the strain, and the genetic operation for changing the content of cytoplasmic iron ions is verified again, so that the rapid xylose fermentation of the strain can be realized by matching with high-copy XyleA.
Example 11: NFS1 in multiple copies of RuXYLA Strain I492N Mutations promote xylose utilization
The experimental method comprises the following steps:
the effect of NFS1 (I493N) mutation on the background of different xylose isomerase strains was tested, NFS1 (I492N) mutation was tested on strain xylose medium growth in strains containing high copy Niu Liuwei metagenome derived xylose isomerase expression genes (abbreviated as RuXylA).
The experimental steps are as follows:
(1) Construction of high copy RuXylA-containing strains: the CCTCC M94055 strain is used as a parent strain, which is also a parent strain of the patent related to strain CIBTS0573 (see Diao, l., et al, BMC Biotechnol,2013.13: p.110. Or patent document CN 201811024423.1). Firstly 2 copies of RuXylA are integrated at the chromosome delta site, secondly XKS1 gene and pentose phosphate pathway gene are overexpressed, and specific methods of operation can be referred to in the literature Diao, l., et al, BMC Biotechnol,2013.13:p.110. The resulting strain was named CIBTS0933.
(2) The 32 copies of RuXYLA were iteratively integrated at the CIBTS0933 strain chromosomes XII-5, X-2, X-3, XI-2, XII-3, X-4, XII-1, XI-3 sites, and the specific integration method was the same as in example 5, and the resulting strain was named CIBTS0933-34-W.
(3) The wild-type NFS1 on chromosome CIBTS0933-34-W was mutated to I492N and the resulting strain was designated CIBTS0933-34-N as described in example 6.
(4) 3 transformants were selected from each of CIBTS0933-34-W and CIBTS0933-34-N, and strain growth OD was measured in YPX40 medium at a certain time interval 600 Values.
Experimental results:
strains CIBTS0933-34-W and CIBTS0933-34-NCIBTS0573-26-N grown OD in YPX40 Medium 600 The values are listed in table 6.
TABLE 6 CIBTS0933-34-W and CIBTS0933-34-N in YPX40 Medium OD 600 Value of
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FIG. 15 shows the growth of strains with 34 copies of RuXYLA, NFS1 gene mutant or NFS1 gene wild type, respectively, in YPX40 medium. The result shows that the growth of the mutant strain of the NFS1 is obviously better than that of the wild type of the NFS1, indicating that the NFS1 I492N Mutation significantly improved xylose utilization capacity of RuXyl A strain, indicating NFS1 I492N The locus not only acts on PsXyl A background strains, but also has good promotion effect on xylose isomerase genes other than PsXyl A, such as RuXyl A.
As can be seen from the above experimental results, the present invention mutates NFS1 by directly integrating high copy number xylose isomerase gene XYLA in diploid Saccharomyces cerevisiae I492N The ISU1 gene is knocked out, so that the saccharomyces cerevisiae for rapidly fermenting xylose to generate ethanol can be directly obtained without depending on domestication, a genetic mechanism of the strain for obtaining xylose metabolism capability through domestication is disclosed, and meanwhile, the rational reconstruction of the saccharomyces cerevisiae strain capable of fermenting xylose is realized, so that the method has an industrial application prospect.
It should be noted that the listing or discussion of a prior-published document in this specification should not be taken as an acknowledgement that the document is prior art or common general knowledge.
Sequence listing
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<120> a method for improving xylose conversion ability of Saccharomyces cerevisiae
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Claims (8)
1. A method for improving the xylose conversion capability of saccharomyces cerevisiae, comprising the following steps:
A. the coded gene of cysteine desulphurase NFS1 in the genome of Saccharomyces cerevisiae with the capability of converting xylose into ethanol is subjected to I492N mutation by taking the Saccharomyces cerevisiae as chassis cellNFS1 I492N ；
B. Integrating at least one heterologous gene encoding xylose isomerase in the genome, said heterologous gene being rumen chytrid @, and Piromyces sp, E2) derived xylose isomerase genesXylAThe nucleotide sequence is SEQ ID NO. 1; or Niu Liuwei metagenome-derived xylose isomerase geneRuXylAThe nucleotide sequence is SEQ ID NO. 2,
wherein the Saccharomyces cerevisiae in the step A is an industrial diploid Saccharomyces cerevisiae strain CIBTS0573 or CCTCC M94055.
2. The method of claim 1, wherein the saccharomyces cerevisiae in step a is a diploid saccharomyces cerevisiae.
3. A method for improving the xylose conversion capability of saccharomyces cerevisiae, which is characterized by comprising the following steps:
(1) Integrating at least one heterologous gene encoding xylose isomerase in the genome, said heterologous gene being rumen chytrid @, andPiromyces sp, E2) derived xylose isomerase genesXylAThe nucleotide sequence is SEQ ID NO. 1; or Niu Liuwei metagenome-derived xylose isomerase geneRuXylAThe nucleotide sequence is SEQ ID NO. 2;
(2) Gene for knocking out iron-sulfur cluster synthesis pathwayISU1Inhibit the normal assembly of the iron-sulfur cluster,
wherein the saccharomyces cerevisiae is an industrial diploid saccharomyces cerevisiae strain CIBTS0573 or CCTCC M94055.
4. The method of claim 1, wherein step B is the integration of 26 copies of the heterologous gene in the genome.
5. The method of claim 1, wherein step B is the integration of 30 copies of the gene in the genomeXylAOr alternativelyRuXylA。
6. The method of claim 1, wherein step B is the integration of 34 copies of the gene in the genomeXylAOr alternativelyRuXylA。
7. A genetically engineered bacterium constructed according to the method of any one of claims 1-6.
8. The use of the genetically engineered bacterium of claim 7 in the production of ethanol by fermentation.
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| EP1577396A1 (en) * | 2004-03-16 | 2005-09-21 | Ajinomoto Co., Inc. | Method for producing L-amino acids by fermentation using bacteria having enhanced expression of xylose utilization genes |
| CN108064269A (en) * | 2014-08-11 | 2018-05-22 | 拉勒曼德匈牙利流动管理有限责任公司 | Improve the mutation in iron-sulphur cluster protein of xylose utilization |
| CN110872596A (en) * | 2018-09-04 | 2020-03-10 | 中国科学院上海生命科学研究院 | Construction method of saccharomyces cerevisiae for co-utilizing xylose and arabinose |
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| EP1577396A1 (en) * | 2004-03-16 | 2005-09-21 | Ajinomoto Co., Inc. | Method for producing L-amino acids by fermentation using bacteria having enhanced expression of xylose utilization genes |
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