CN115948264A - Genetically engineered bacterium for producing 3-hydroxypropionic acid and application thereof - Google Patents
Genetically engineered bacterium for producing 3-hydroxypropionic acid and application thereof Download PDFInfo
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- CN115948264A CN115948264A CN202210113919.6A CN202210113919A CN115948264A CN 115948264 A CN115948264 A CN 115948264A CN 202210113919 A CN202210113919 A CN 202210113919A CN 115948264 A CN115948264 A CN 115948264A
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- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention belongs to the technical field of metabolic engineering and synthetic biology, and relates to a genetic engineering bacterium for producing 3-hydroxypropionic acid and application thereof. The saccharomyces cerevisiae recombinant strain is characterized in that an exogenous modified malonyl coenzyme A reductase gene MCR is positioned in a saccharomyces cerevisiae mitochondria, and is expressed on a chromosome by two copies, and an endogenous mitochondria NADP specific isocitrate dehydrogenase encoding gene IDP1 and an endogenous mitochondria NADH kinase encoding gene POS5 are overexpressed. The recombinant saccharomyces cerevisiae strain with high yield of 3-HP is constructed through the transformation, the yield of the recombinant saccharomyces cerevisiae strain reaches 5.2g/L in shake flask fermentation, and the yield of the recombinant saccharomyces cerevisiae strain reaches 36.3g/L in 5L fermentation tank through fed-batch fermentation.
Description
Technical Field
The invention belongs to the technical field of metabolic engineering and synthetic biology, and relates to a genetic engineering bacterium for producing 3-hydroxypropionic acid and application thereof.
Background
3-hydroxypropionic acid (3-HP) is an attractive platform chemical, and can be used as a precursor for synthesizing compounds such as acrylic acid, acrylamide and the like, so that the sustainable production of materials such as super absorbent polymers, plastics, paints and the like is possible, and the application prospect is wide. The conventional chemical synthesis method for producing 3-HP has the problems of environmental friendliness of raw materials, high cost and the like, so that the biosynthesis method using microbial fermentation has more advantages for sustainable development.
The 3-HP natural synthetic pathway in microorganisms is present in a small number of thermophilic archaea and bacteria and is involved in the autotrophic carbon fixation cycle. The 3-HP biosynthesis methods that have been reported so far have shown that 3-HP can be produced by introducing exogenous pathways into other microorganisms from several intermediates, including glycerol, malonyl-CoA and beta-alanine, among others. Among them, many studies have been conducted on the malonyl-coa pathway, in contrast to which the glycerol pathway is somewhat limited due to the requirement for supply of coenzyme B12, and is mainly used in bacterial hosts. The malonyl-coa pathway has been applied to many microbial cell factories, such as escherichia coli, saccharomyces cerevisiae, etc., and is synthesized by using acetyl-coa, an intermediate metabolite obtained by metabolizing glucose or other carbon sources, as a precursor, catalytically converting it into malonyl-coa by acetyl-coa carboxylase (ACC), and then catalytically synthesizing 3-HP by malonyl-coa reductase (MCR).
Saccharomyces cerevisiae, a well-studied model eukaryotic microorganism, has a wide technical platform in both systems biology and synthetic biology, has been applied to cell factories of various biochemicals based on its advantages such as robustness to industrial conditions, high-density fermentation capability, insensitivity to phage contamination, and so on, and has made some progress in producing 3-HP using it at present, but still has a great room for improvement. On the basis, because the physicochemical environments of various subcellular organelles in the saccharomyces cerevisiae are different, various chemicals produced by utilizing different organelles are developed based on the characteristic. In particular, the intragranular environment within yeast has a higher redox potential and higher acetyl-coa concentration relative to the cytoplasm, and may be more advantageous for 3-HP synthesis.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a genetic engineering bacterium for producing 3-hydroxypropionic acid, which utilizes a malonyl coenzyme A approach, introduces malonyl coenzyme A reductase (MCR) with high enzyme activity from an external source, locates the malonyl coenzyme A reductase to mitochondria, and simultaneously increases the supply of a cofactor NADPH so as to achieve the purpose of increasing the yield of 3-HP.
Therefore, the invention provides a genetic engineering bacterium for producing 3-hydroxypropionic acid.
According to an embodiment of the first aspect of the invention, the genetically engineered bacterium producing 3-hydroxypropionic acid is a recombinant saccharomyces cerevisiae, the genome of which contains an exogenous modified malonyl-coa reductase gene MCR.
Specifically, the recombinant saccharomyces cerevisiae takes wild-type saccharomyces cerevisiae as a host bacterium, locates the exogenous modified malonyl-coenzyme A reductase gene MCR in saccharomyces cerevisiae mitochondria, and expresses the gene MCR on a chromosome in two copies.
In some embodiments of the invention, the MCR gene is divided into two sections, namely MCR-N and MCR-C, and the MCR-C is subjected to point mutation transformation through N940V, K1106W, S R.
In some further embodiments of the invention, MCR-N is expressed from the promoter PGK1p and MCR-C is expressed from the promoter TDH3p, and each gene is ligated at its 5' end with the mitochondrial localization sequence CAT2m, and a copy is inserted at chromosome XI-3 and X-4 after MCR-N is ligated to MCR-C.
For example, in some embodiments of the invention, malonyl-coenzyme A reductase (MCR) from Chloroflexus aurantiaca is first introduced using Saccharomyces cerevisiae as a host strain. In the invention, MCR-N and mutated MCR-C are expressed in saccharomyces cerevisiae in high intensity, and a mitochondrion positioning sequence is added at the N end. In the specific embodiment of the invention, N940V, K1106W, S R of MCR-C is subjected to point mutation and is expressed by a TDH3p promoter, and MCR-N is expressed by a PGK1p promoter. On the basis, a mitochondrial localization signal sequence CAT2m is added to the N ends of MCR-N and MCR-C respectively. And inserting a copy of the MCR gene after modification into XI-3 site and X-4 site of a saccharomyces cerevisiae genome respectively.
In the invention, the nucleotide sequence of the mitochondrion localization sequence CAT2m is shown as SEQ No. 1.
In the invention, the nucleotide sequence of the MCR-N is shown as SEQ No. 2.
In the invention, the nucleotide sequence of MCR-C (marked as MCR-mC) which is subjected to point mutation transformation of N940V, K1106W, S R is shown as SEQ No. 3.
In the invention, the nucleotide sequence of the MCR-C (without mutation point modification) is shown as SEQ No. 46.
In the invention, the nucleotide sequence of the promoter TDH3p is shown in SEQ No. 4.
According to an embodiment of the second aspect of the invention, the genetically engineered bacterium producing 3-hydroxypropionic acid also overexpresses the endogenous mitochondrial NADP-specific isocitrate dehydrogenase-encoding gene IDP1 and the mitochondrial NADH kinase-encoding gene POS5.
In some embodiments of the invention, mitochondrial NADP specific isocitrate dehydrogenase encoding gene IDP1 overexpression is achieved by replacing its original promoter on the chromosome with the promoter TEF1 p.
In the invention, the nucleotide sequence of the promoter TEF1p is shown as SEQ No. 5.
In other embodiments of the invention, overexpression of the mitochondrial NADH kinase coding gene POS5 is achieved by replacing its promoter with the TDH3p promoter and introducing it into the strain in the form of a high copy plasmid.
For example, in some specific examples of the present invention, on the basis of the recombinant yeast strain obtained in the embodiment of the first aspect, the mitochondrial NADP-specific isocitrate dehydrogenase-encoding gene IDP1 and the mitochondrial NADH kinase-encoding gene POS5 are overexpressed. Wherein, the promoter region of IDP1 is replaced by a strong promoter TEF1p on a saccharomyces cerevisiae genome for overexpression, and in addition, a high copy plasmid which uses a promoter TDH3p to carry out overexpression is introduced into a recombinant yeast strain.
In the present invention, the nucleotide sequence of the mitochondrial NADP specific isocitrate dehydrogenase encoding gene IDP1 is shown in SEQ No. 44.
In the invention, the nucleotide sequence of the mitochondrial NADH kinase coding gene POS5 is shown as SEQ No. 45.
In the invention, the saccharomyces cerevisiae used as the host cell is saccharomyces cerevisiae CEN 113-5D (MATa ura3-52 HIS3 LEU2 TRP1 MAL2-8c SUC2) (Biovector plasmid vector strain cell protein antibody gene collection-NTCC type culture collection).
The invention also provides application of the genetically engineered bacteria of the embodiments of the first aspect and the second aspect in synthesizing 3-hydroxypropionic acid.
In some embodiments of the invention, the application comprises subjecting the genetically engineered bacteria to fermentation culture to produce 3-hydroxypropionic acid.
In some preferred embodiments of the present invention, the shake flask fermentation culture conditions are: the temperature is 30 ℃, the rotation speed of a shaking table is 200rpm, and the composition of the culture medium is 5g/L (NH) 4 ) 2 SO 4 、3g/L KH 2 PO 4 、0.5g/L MgSO 4 ·7H 2 O, trace metal elements, vitamins and 20g/L glucose.
In some particularly preferred embodiments of the present invention, the shake flask fermentation culture conditions are: the temperature is 30 ℃, the rotation speed of a shaking table is 200rpm, and the composition of the culture medium is 5g/L (NH) 4 ) 2 SO 4 、3g/L KH 2 PO 4 、0.5g/L MgSO 4 ·7H 2 O, trace metal elements (15 mg/L EDTA, 4.5mg/L ZnSO) 4 ·7H 2 O、0.3mg/L CoCl 2 ·6H 2 O、1mg/L MnCl 2 ·4H 2 O、0.3mg/L CuSO 4 ·5H 2 O、4.5mg/L CaCl 2 ·2H 2 O、3mg/L FeSO 4 ·7H 2 O、0.4mg/L NaMoO 4 ·2H 2 O、1mg/L H 3 BO 4 0.1mg/L KI), vitamins (0.05 mg/L biotin, 1mg/L calcium pantothenate, 1mg/L nicotinic acid, 25mg/L inositol, 1mg/L thiamine hydrochloride, 1mg/L pyridoxine hydrochloride, 0.2mg/L p-aminobenzoic acid), and 20g/L glucose.
In other further preferred embodiments of the invention, the fermenter is fed batchThe fermentation culture conditions are as follows: the pH was maintained at 5, the temperature at 30 ℃, the dissolved oxygen limit at 30%, the aeration at 0.5VVm, the stirring set at 400-1200rpm, and the feed medium composition at 67.5g/L (NH) 4 ) 2 SO 4 、27g/L KH 2 PO 4 、4.5g/L MgSO 4 ·7H 2 O, trace metal elements, vitamins and 300g/L glucose.
In some particularly preferred embodiments of the invention, the fed-batch fermentation culture conditions are: the pH was maintained at 5, the temperature at 30 ℃, the DO limit at 30%, the aeration at 0.5VVm, the agitation set at 400-1200rpm, and the feed medium composition at 67.5g/L (NH) 4 ) 2 SO 4 、27g/L KH 2 PO 4 、4.5g/L MgSO 4 ·7H 2 O, trace metal elements (135 mg/L EDTA, 40.5mg/L ZnSO) 4 ·7H 2 O、2.7mg/L CoCl 2 ·6H 2 O、9mg/L MnCl 2 ·4H 2 O、2.7mg/L CuSO 4 ·5H 2 O、40.5mg/L CaCl 2 ·2H 2 O、27mg/L FeSO 4 ·7H 2 O、3.6mg/L NaMoO 4 ·2H 2 O、9mg/L H 3 BO 4 0.9mg/L KI), vitamins (0.45 mg/L biotin, 9mg/L calcium pantothenate, 9mg/L nicotinic acid, 225mg/L inositol, 9mg/L thiamine hydrochloride, 9mg/L pyridoxine hydrochloride, 1.8mg/L p-aminobenzoic acid), 300g/L glucose.
The invention takes a saccharomyces cerevisiae strain as a host strain, and inserts two copies of PGK1p-MCR-N and TDH3p-MCR-mC (MCR-C after the point mutation transformation) expressed by a strong promoter with mitochondrial signal peptide into the genome of the saccharomyces cerevisiae strain to obtain a 3-HP production strain; on the basis of the transformation, an IDP1 original promoter is replaced by TEF1p, an over-expressed TDH3p-POS5 high-copy plasmid is introduced, and the yield of 3-HP is further improved by increasing the NADPH content in a mitochondrial environment, so that the high-efficiency sustainable microbial fermentation production of 3-HP is realized.
Drawings
The invention is described in further detail below with reference to the attached drawing figures:
FIG. 1 shows the metabolic pathway of recombinant Saccharomyces cerevisiae for the synthesis of 3-hydroxypropionic acid;
FIG. 2 is a map of a fragment of the gene MCR inserted on the genome;
FIG. 3 is a map of the promoter of IDP1 gene replaced on the genome as TEF1p promoter fragment;
FIG. 4 is a plasmid pPOS5 map of POS5 in which TDH3p is overexpressed;
FIG. 5 is a bar graph of 3-hydroxypropionic acid production in shake flasks of recombinant strains of Saccharomyces cerevisiae in various embodiments of the invention.
Detailed Description
In order that the invention may be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
Where a range of values is provided, it is understood that each intervening value, to the extent that there is no stated or intervening value in that stated range, to the extent that there is no such intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a specified range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.
I. Term(s) for
The term "expression" as used herein refers to the expression of a gene in a metabolic pathway, and refers to the expression of its background promoter.
The term "overexpression" as used herein means that a strong promoter stronger than its background promoter is inserted in front of a gene to promote the expression of the gene.
The term "MCR-mC" in the invention refers to MCR-C which is transformed by N940V, K1106W, S R point mutation.
Symbol used in the present invention ": : "Gene insertion, accordingly, A: : b indicates the insertion of the B gene into the A locus.
Embodiments of
The current strains of Saccharomyces cerevisiae that utilize the 3-HP pathway of malonyl-CoA are based on cytoplasmic expression. Although cytoplasm has the advantages of large space, more intermediate metabolites, no need of transmembrane and the like, the cytoplasm has certain limitations and can be limited by transcriptional level regulation, cytoplasmic physicochemical environment, loss of intermediate products of competitive metabolic pathways, metabolite toxicity and the like. Organelles all have unique physicochemical environments (e.g., pH and redox potential), enzymes, metabolites, and cofactors, transferring metabolic pathways into smaller organelles, local concentrations of substrate and enzyme may increase reaction rates and yield, while potentially reducing intermediary metabolite toxicity and inhibiting byproduct production. The relocation of key genes in metabolic pathways in different environments in cells is an effective metabolic engineering strategy. Mitochondria are the locus of the tricarboxylic acid cycle, amino acid biosynthesis, various cofactors and metabolites. Mitochondrial matrices can provide higher pH, lower oxygen concentration, and higher redox potential than cytoplasmic matrices. Under aerobic conditions, glucose is converted into pyruvic acid, enters mitochondria to synthesize a large amount of acetyl coenzyme A, and in addition, the saccharomyces cerevisiae has the limitation that the acetyl coenzyme A cannot be directly shuttled across the membrane, so that the content of the acetyl coenzyme A in the mitochondria is higher than that in a cytoplasmic matrix. Also, since 3-HP is a compound synthesized using acetyl-CoA precursor, the present inventors considered that localization of the malonyl-CoA pathway to mitochondria might increase 3-HP production.
As shown in FIG. 1, the malonyl-CoA pathway for 3-HP synthesis is largely divided into two parts: a first part, glucose is converted to acetyl-coa by the glycolytic pathway, which is then converted to malonyl-coa by the action of acetyl-coa decarboxylase; the second part, the conversion of malonyl-CoA to 3-HP, is catalyzed by malonyl-CoA reductase (MCR). The conversion of malonyl-CoA into 3-HP is a key step in the production of 3-HP via the malonyl-CoA pathway in s.cerevisiae, and the present inventors have achieved the transfer of the key reaction of malonyl-CoA into 3-HP from the cytoplasm to the mitochondria by fusing the malonyl-CoA reductase MCR to the mitochondrial localization signal sequence CAT2m, thereby localizing the MCR to the mitochondria.
Researches show that the refining activity of MCR can be obviously improved after the MCR functionality is split into MCR-N and MCR-C. In an embodiment of the first aspect, the resolved MCR is integrated into the yeast genome as shown in figure 2. Firstly, introducing a mutation N940V, K1106W, S R into MCR-C (marked as MCR-mC), and connecting a saccharomyces cerevisiae strong promoter TDH3p and a terminator CYC1t to the MCR-mC before and after connecting the promoter and the terminator for expression in saccharomyces cerevisiae. In addition, a saccharomyces cerevisiae strong promoter PGK1p and a terminator ADH1t are connected to the front and back of MCR-N for expression in saccharomyces cerevisiae; in addition, a mitochondrial signal sequence CAT2m is connected to the N ends of MCR-N and MCR-mC respectively for localization in mitochondria. And respectively inserting the recombined MCRs into XI-3 and X-4 sites of a genome of the saccharomyces cerevisiae to obtain a recombined saccharomyces cerevisiae strain with two MCR copies, and naming the recombined saccharomyces cerevisiae strain as NmC2.
In the mitochondria of s.cerevisiae, NADPH is mainly derived from two pathways: firstly, NADH is catalyzed and reacted by mitochondrial NADH kinase Pos5p to synthesize NADPH, and secondly, mitochondrial NAD + NAD catalyzed first by Pos5p + Synthesis of NADP by kinase reaction + Then NADP + By mitochondrial NADP + The dependent dehydrogenase catalyzes a reaction to synthesize NADPH, and enzymes catalyzing this reaction include mitochondrial NADP-specific isocitrate dehydrogenase Idp1p, mitochondrial aldehyde dehydrogenase Ald4p, and the like.
In the synthesis of 3-HP via the malonyl-CoA pathway, the key step in the conversion of malonyl-CoA to 3-HP requires that NADPH, a cofactor, be involved in the catalysis of each molecule of 3-HP synthesis. Therefore, in order to further increase the amount of 3-HP synthesis, in an embodiment of the second aspect, the present inventors modified the strain NmC2 constructed in an embodiment of the first aspect to increase the amount of mitochondrial NADPH synthesis. The present inventors have found that over-expression of POS5 and IDP1 can achieve this goal. Specifically, a sequence 36bp ahead of an open reading frame of the IDP1 gene is replaced by a saccharomyces cerevisiae strong promoter TEF1p on a saccharomyces cerevisiae genome to realize the overexpression of the IDP1. In addition, a high copy plasmid of the Saccharomyces cerevisiae endogenous POS5 gene overexpressed by a strong promoter TDH3p is constructed and is transferred into a yeast strain.
The yeast chromosome is modified by using a CRISPR/Cas9 system, and the modification comprises the insertion of an exogenous gene and the replacement of a promoter of an endogenous gene. For this purpose, recombinant plasmids containing the Cas9 protein and gRNAs need to be constructed, and those used for integration of MCR include pCas9-XI-3.3 and pCas9-X-4 (Zhang Y, et al. Expression a cytotoxic viral gene expression in Microbial Cell genes [ J ]. Microbiological Cell industries, 2020, 19 (1)). For the promoter replacement of IDP1 on chromosome, the used recombinant plasmid is pCas9_ IDP1p, the construction process of the recombinant plasmid involves the assembly of a vector and a template plasmid, including pCas9-LacZ plasmid and pST1.G.Ura3 plasmid, by a plasmid construction method of GoldenGate, and the prediction and selection of the involved gRNAs are performed by the websites https: // www.atum.bio/catalog/vectors/grna-design. For the POS5 overexpression plasmid pPOS5, the vector plasmid used for construction was pwgg 1.
In the invention, a golden gate method is adopted for plasmid construction. Firstly, preparing a golden gate reaction system, adding a plasmid vector according to 17ng/kb, adding a DNA fragment according to the mol ratio of 1: 1 to the vector, adding 0.4 mu l T DNA ligase, 2 mu l T ligase buffer solution and 1.6 mu l BsaI restriction enzyme, and finally supplementing the mixture to 20 mu l with water. The reaction system was reacted according to the following procedure: 30min at 37 ℃; 10min at 37 ℃; 5min at 16 ℃; returning to the step 2, and performing 15 cycles; 30min at 16 ℃; 30min at 37 ℃; 5min at 80 ℃; keeping at 4 ℃. After the reaction, 4. Mu.l of the mixture was taken out and subjected to large intestine transformation.
In the invention, all yeast strains are constructed by adopting an electric shock transformation method. The specific method for electrotransformation competence preparation is as follows: firstly, the yeast on the plate is single bacteriumInoculating to 5mL YPD medium, and culturing in 50mL centrifuge tube at 30 deg.C and 200rpm overnight; initial OD of 0.3 in 250mL shake flasks 600 The pre-cultured cells were inoculated into 50mL of YPD medium and cultured at 30 ℃ to OD 600 1.2-1.6; centrifuging at 4 ℃ and 3000rpm for 3 minutes, and collecting the yeast cells in a 50mL centrifuge tube; washed once with 20ml of pre-cooled sorbitol; the cells were resuspended in 1M sorbitol (16mL) and added with 2mL of 10 XTE solution and 2mL of 10 XLiOAc solution; shaking the tube at 30 deg.C for 30min, adding 200 μ l1M DTT into the mixture, and shaking at 30 deg.C for 15 min; centrifuging at 4 ℃ and 3000rpm for 3 minutes, collecting bacteria, discarding supernatant, and washing twice with 20ml of precooled sorbitol; after re-centrifugation, sucking all liquid as much as possible, and then suspending the yeast cell sediment in 180. Mu.l of 1M sorbitol; the cell suspension was dispensed in 50. Mu.l per tube.
The electrotransformation steps are as follows: for gene insertion or promoter replacement on the chromosome, 100ng of the pCas9 plasmid and about 2000ng of the donor DNA fragment were gently mixed well with competent cells, and electric shock was performed. After electroporation the cells were cultured in 3mL YPD and 3mL1M sorbitol mixture for 3-5 hours. The cells were then washed once with water and resuspended in 1ml of sterile water. Wherein 100 μ l is directly coated on SC-URA plate, and the rest liquid is transferred to SC-URA liquid culture medium for further culture for 24 hours and then coated again. Verification was performed by colony PCR and sequencing. In addition, transformation of the plasmid into yeast cells according to the present invention was also carried out by an electroporation method, in which the plasmid and competent cells were mixed uniformly, followed by electric shock, transferred to a mixture of 3mL YPD and 3mL1M sorbitol for 1 hour, and then screened on SC-URA plates.
The culture medium used for culturing, constructing and screening the bacterial strain comprises a YPD culture medium and an SC-URA culture medium. The culture medium used for fermentation is Delft culture medium. The composition of the culture medium is as follows: YPD medium comprises 10g/L yeast extract, 20g/L peptone, and 20g/L glucose; SC-URA medium comprises 5g/L ammonium sulfate, 1.7g/L YNB (without ammonium sulfate and amino acid), 1.914g/L amino acid mixture (without uracil), 20g/L glucose, pH is adjusted to 6 with NaOH; delft medium included 5g/L (NH) 4 ) 2 SO 4 、3g/L KH 2 PO 4 、0.5g/L MgSO 4 ·7H 2 O, trace metal elements (15 mg/L EDTA, 4.5mg/L ZnSO) 4 ·7H 2 O、0.3mg/L CoCl 2 ·6H 2 O、1mg/L MnCl 2 ·4H 2 O、0.3mg/L CuSO 4 ·5H 2 O、4.5mg/L CaCl 2 ·2H 2 O、3mg/L FeSO 4 ·7H 2 O、0.4mg/L NaMoO 4 ·2H 2 O、1mg/L H 3 BO 4 0.1mg/L KI), vitamins (0.05 mg/L biotin, 1mg/L calcium pantothenate, 1mg/L nicotinic acid, 25mg/L inositol, 1mg/L thiamine hydrochloride, 1mg/L pyridoxine hydrochloride, 0.2mg/L p-aminobenzoic acid), 20g/L glucose, pH was adjusted to 6.5 with KOH.
In the present invention, the 3-HP concentration was measured by high performance liquid chromatography HPLC (Shimadzu LC-20 AT) using an Aminex HPX-87H (Bio-Rad) column and a UV detector AT 210 nm. 1mL of the fermentation solution was filtered through a 0.22mm pore size aqueous membrane into a liquid phase vial as a test sample. The mobile phase was 0.5mM H 2 SO 4 The column oven temperature was set to 65 ℃ and the detection time for each sample was set to 20 minutes. Preparing 3-HP standard substances with different concentration gradients, filtering into a clean liquid phase vial, and obtaining a standard curve of peak area and concentration according to a sample detection program. And after HPLC detection, substituting the samples into a standard curve to calculate the concentration of the 3-HP in each sample.
Example III
The present invention will be specifically described below with reference to specific examples. The experimental procedures described below are, unless otherwise specified, conventional laboratory procedures. The experimental materials described below can be obtained commercially or by conventional methods unless otherwise specified.
Example 1: transformation of MCR and positioning of brewing yeast mitochondria
First, the mitochondrially localized MCR plasmid pMCR1 was constructed. Taking pUGG1 as a vector, adding a CAT2m mitochondrial localization sequence at the 5' end of an MCR gene, wherein a promoter is TDH3p, and a terminator is CYC1t.
Using a yeast genome as a template to amplify to obtain a TDH3p fragment, and adding a CAT2m sequence to a primer to connect the fragment, wherein the sequence of the primer used for amplification is as follows:
P124：aaaGGTCTCaATCTTCATTATCAATACTGCCATTTCAAAGAATACG
P125：aaaGGTCTCagagagagttctcgaatgacagatcctcatTTTGTTTGTTTATGTGTGTTTATTCGAAAC
in addition, PCR was performed using pYC1 (Chen Y, et al. Coupled acquired precordial predictor and co-factor supplied enhanced 3-hydroxypropinic acid production in Saccharomyces cerevisiae [ J ]. Metabolic Engineering,2014, 22.) as a template to obtain MCR and CYC1t fragments, and the sequences of primers used for amplification were:
P126：AGGTCTCATCTCAAACTTAAAGGATCTTCCGATAACGTCAAGGAGAGCAAGTGGTACAGGTAGATTAGCAGG
P127：AAAGGTCTCACCTAGGTTCTTCAGCTCTGGC
P128：aaaGGTCTCatagGgacccacacgaaagacaac
P129：aaaGGTCTCaCGACcttcgagcgtcccaaaacc
the 3 DNA fragments obtained by PCR amplification and the vector pUGG1 are connected by a Golden Gate method to construct a plasmid pMCR1.
On the basis, the separated pMCR-N and pMCR-C plasmids with mitochondria positioned are respectively constructed.
The pMCR-N construction process comprises the steps of firstly carrying out PCR amplification by taking PYC1 as a template to respectively obtain a promoter PGK1p and a terminator ADH1t fragment, wherein the amplified primer sequences are as follows:
P140：aaaGGTCTCacagatcttatcgtcgtcatc
P141：aaaGGTCTCaATCTcctggaagtaccttcaaag
P144：aaaGGTCTCatgagctcttaattaacaattcttcg
P145：aaaGGTCTCaCGACcagctggataaaggcg
secondly, using PMCR1 as a template to amplify a segment of CAT2m connected with MCR-N, wherein the sequence of the used primer is as follows:
P142：aaaGGTCTCatctgatgaggatctgtcattcgag
P143：aaaGGTCTCactcagatgttggctggtatgttc
the fragments were sequentially ligated by the Golden Gate method using pUGG1 as the vector to obtain plasmid pMCR-N.
The pMCR-C construction process is that two sections of TDH3p-CAT2m and MCR-C-CYC1t are respectively subjected to PCR amplification by taking pMCR1 as a template, and the sequences of the used primers are as follows:
P124：aaaGGTCTCaATCTTCATTATCAATACTGCCATTTCAAAGAATACG
P139：aaaGGTCTCacggaTGCTCTCCTTGACGTTATC
P138：aaaGGTCTCatccgctaccactggtg
P129：aaaGGTCTCaCGACcttcgagcgtcccaaaacc
the fragments were sequentially ligated by the Golden Gate method using pUGG1 as the vector to obtain plasmid pMCR-C.
Based on pMCR-C, the modification of partial amino acid mutation by means of Golden Gate comprises N940V, K1106W, S R, and the plasmid pMCR-mC is obtained. Wherein MCR-mC is divided into three sections and is obtained by PCR amplification by taking pMCR-C as a template, and the used primers are respectively:
P124：aaaGGTCTCaATCTTCATTATCAATACTGCCATTTCAAAGAATACG
P134：aaaGGTCTCacaactctgtcagctaagtaatagacag
P135：aaaGGTCTCagttgtttccggtgaaacttttc
P146：aaaGGTCTCaagacaaagcaatccatctggcgactctgaag
P137：aaaGGTCTCagtctgatggtgctcgattagcattggtaacccc
P129：aaaGGTCTCaCGACcttcgagcgtcccaaaacc
inserting MCR-N and MCR-mC into XI-3 site of yeast chromosome at the same time, cas9 plasmid pCas9_ XI-3.3 with gRNA of XI-3 site and MCR donor DNA fragment with 50bp homology arm at both sides of XI-3.3 position need to be transformed into Saccharomyces cerevisiae cell at the same time.
Wherein, the MCR-N donor DNA is obtained by taking pMCR-N as a template for amplification, and the primer sequence is as follows:
XI-3.3-MCR_N-up：
ACTGATTAGTTTTCCGTTTTAGGATATTGACGCCAAGCGTGCGTCTGATTCTGGAAGTACCTTCAAAGAATGGG
P155：
cggAACGGTCGACcagctggataaaggcgcgccaaacgacctaggaattggagcgacctcatgctatacctg additionally, MCR-C donor DNA was obtained by amplification using pMCR-mC as template, primer sequence:
XI-3.3-CaMCR-dw：
CAGCCCAATCCTCAAAAATAAAATGGCCCTTCCAAAATTAGAGACATATCcttcgagcgtcccaaaacc
P156：
caattcctaggtcgtttggcgcgcctttatccagctgGTCGACCGTTccgTCATTATCAATACTGCCATTTCAAAGAATACG
preparing competent cells of Saccharomyces cerevisiae strain CEN.PK 113-5D, simultaneously electrotransfering the donor DNA and the plasmid into the cells, and screening to obtain a strain with single copy integration of MCR-N and MCR-mC (shown in figure 2).
Similarly, using the above recombinant strain as a background strain, a copy of MCR-N and MCR-mC (as shown in FIG. 2) is inserted at chromosome X-4, and the resulting recombinant strain is named as NmC2. The Cas9 plasmid used in the method is pCas9_ X-4, two donor DNA fragments are obtained by PCR amplification and purification, and the amplification primer sequences are as follows:
X-4-MCR-N-up：
CCAACTACCAAGGTTGTTGAGGGAACACTGGGGCAATAGGCTGTCGCCATCTGGAAGTACCTTCAAAGAATGGG
P155：
cggAACGGTCGACcagctggataaaggcgcgccaaacgacctaggaattggagcgacctcatgctatacctg
X-4-CaMCR-dw：
CAGACATCAGACATACTATTGTAATTCAAAAAAAAAAAGCGAATCTTCCCcttcgagcgtcccaaaacc
P156：
caattcctaggtcgtttggcgcgcctttatccagctgGTCGACCGTTccgTCATTATCAATACTGCCATTTCAAAGAATACG
example 2: construction of high-yield 3-HP strains
On the basis of the recombinant strain NmC2 constructed in example 1, the metabolic pathway thereof was further modified to increase the supply amount of NADPH, including two strategies of up-regulating the IDP1 gene and the POS5 gene.
As shown in fig. 3, the up-regulation of IDP1 is achieved by replacing the promoter of IDP1 on chromosome with strong promoter TEF1p, and gene manipulation is performed using CRISPR/Cas9 system. The Cas9 plasmid with the gRNA in IDP1p needs to be constructed first. The plasmid pCas9-LacZ is taken as a vector, pST1.G. Ura3 plasmid is taken as a template, and a fragment with gRNA is obtained by PCR and is assembled by a plasmid construction method of GoldenGate.
primers used for construction of pCas9_ IDP1p include:
IDP1p_g：
aaaGGTCTCtgatcTGTGAGACAACAGACGCACAGTTTTAGAGCTAGAAATAGCAAGTTA
Ura3_Bsa1：
aaaGGTCTCaaaacACACAGGGTAATAACTGATATAATTAAATTGAAGC
in addition, donor DNA of the TEF1p for replacement is obtained by PCR amplification by taking a yeast genome as a template, and amplification primers are as follows:
IDP1_TEF1p-f2：
CCATATAAAGAGGGACTCATATGATCTTATGAAATCTTCCTTCAAGCAATTTCAAAATGTTTCTACTCCTTTTTTAC
IDP1_TEF1p-r：
AAAGCAGCAAGGCGAGAGGTGGAAAATAATCTTCTAGATAACATACTCATCTAGAAAACTTAGATTAGATTGCTATG
as shown in FIG. 4, the up-regulation of POS5 was achieved by constructing a high copy number plasmid with POS5 over-expressed. pPOS5 was also constructed by the method of Golden Gate. Taking pUGG1 as a vector and yeast genome as a template, and respectively obtaining a fragment of a strong promoter TDH3p and a POS5 gene fragment by PCR amplification, wherein the used primer sequences are as follows:
TDH3p-GG-r：AAAGGTCTCATTTGTTTGTTTATGTGTGTTTATTCG
TDH3p-GG-f：AAAGGTCTCAATCTACCTCGAGATAAAAAACACGC
POS5t-GG-r：AAAGGTCTCACGACACGAAACATAGTCTCTCTCCA
POS5-GG-f：AAAGGTCTCACAAAATGTTTGTCAGGGTTAAATTGAAT
on the basis of the NmC2 strain, the replacement IDP1 promoter is a TEF1p promoter, and the obtained new recombinant bacterium group is named as NmC2IDP 1. On the basis of the NmC2 strain, pPOS5 plasmid is transformed into the strain, and the obtained new recombinant bacterium group is named as NmC2+ pPOS5. On the basis of the NmC2IDP1 strain, pPOS5 plasmid is transformed into the strain, and the obtained new recombinant bacterium group is named as NmC2IDP1+ pPOS5.
Example 3: 3-HP fermentation production of recombinant strains
The fermentation yield of the recombinant strain was first tested by shake flask fermentation. The empty plasmid pUGG1 was transformed in the NmC2 strain to provide the URA3 deletion in the background strain. And streaking the saccharomyces cerevisiae recombinant strain with URA3 marker plasmid on an SC-URA solid culture medium to obtain a monoclonal, and inoculating the monoclonal into an SC-URA liquid culture medium for activation to obtain a seed solution. Three clones were picked for each strain as replicates for fermentation experiments. The activated seed liquid is processed according to the initial OD 600 0.1 was inoculated into Delft medium and cultured in a shaking flask at 30 ℃ with shaking at 200rpm.
After the fermentation is finished, 1ml of fermentation liquor is taken, centrifuged, and then the supernatant is taken, filtered by a 0.22 mu m filter membrane and then subjected to high performance liquid chromatography to detect the concentration of the 3-HP. The detection was carried out at a flow rate of 0.5ml/min with a 0.5mM dilute sulfuric acid solution as a mobile phase and a column oven set at 65 ℃. The peak-off time of the 3-HP standard was 15.5min at 210nm of the UV detector.
As shown in FIG. 5, the yield of 3-HP by shake flask fermentation of each recombinant strain obtained after the test was as follows: the NmC2+ pUGG1 reaches 3.8g/L, the NmC2+ pPOS5 reaches 4.8g/L, and the NmC2IDP1+ pPOS5 reaches 5.2g/L. The results of shake flask fermentation experiments show that the introduction of mitochondrially positioned MCR in Saccharomyces cerevisiae can significantly improve the yield of 3-HP, and on the basis, the increase of the supply of NADPH in mitochondria by over-expressing POS5 and IDP1 can further promote the synthesis of 3-HP.
And (3) performing fed-batch fermentation on the recombinant yeast strain with high 3-HP yield in a 5L fermentation tank. The composition of the culture medium for batch fermentation is 5g/L (NH) 4 ) 2 SO 4 、14.4g/L KH 2 PO 4 、0.5g/L MgSO 4 ·7H 2 O, trace metal elements, vitamins and 20g/L glucose, and the composition in the supplemented medium comprises 67.5g/L (NH) 4 ) 2 SO 4 、27g/L KH 2 PO 4 、4.5g/L MgSO 4 ·7H 2 O, trace metal elements, vitamins and 300g/L glucose.
Similarly, single clones were inoculated into SC-URA liquid medium and activated by shaking culture in shake tubes as primary seedsAt OD 600 When reaching 1-2, the first-stage seed liquid is transferred to a Delft liquid culture medium to be cultured in a shake flask to be used as a second-stage seed liquid, and the second-stage seed liquid is cultured in OD 600 1-2 times with initial OD 600 =0.1 was inoculated into the fermenter. The temperature in the tank was set at 30 ℃ and the pH was maintained at 5. The dissolved oxygen limit was 30%, aeration was maintained at 0.5VVm, and agitation was set at 400-1200rpm. Stirring was maintained at 400rpm when the DO value was > 30%, and after the DO value decreased to 30% or less, stirring was increased to maintain 30% DO. Feeding was started when the glucose was depleted in the tank. Under fed-batch culture, the yield of the 3-hydroxypropionic acid is measured by HPLC to reach 36.3g/L after the NmC2IDP1+ pPOS5 strain is fermented for 86 hours.
The 3-hydroxy propionic acid gene engineering bacteria are subcultured for 10 generations, the strain is stable in character, and no back mutation occurs.
It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.
Sequence listing
<110> Beijing university of chemical industry
<120> genetically engineered bacterium for producing 3-hydroxypropionic acid and application thereof
<130> RB2102811-FF
<160> 46
<170> SIPOSequenceListing 1.0
<210> 1
<211> 66
<212> DNA
<213> (mitochondrial localization sequence CAT2 m)
<400> 1
atgaggatct gtcattcgag aactctctca aacttaaagg atcttccgat aacgtcaagg 60
agagca 66
<210> 2
<211> 1650
<212> DNA
<213> （MCR-N）
<400> 2
agtggtacag gtagattagc aggtaaaata gcattgataa caggtggtgc cggtaacata 60
ggttccgaat taacaagaag atttttggca gaaggtgcca ccgttattat ctctggtaga 120
aacagagcaa agttaactgc cttggctgaa agaatgcaag cagaagccgg tgtccctgct 180
aagagaattg atttggaagt tatggatggt tctgacccag tcgctgtaag agcaggtatt 240
gaagccatag tagctagaca tggtcaaatc gatatcttgg ttaacaacgc aggttcagct 300
ggtgcacaaa gaagattggc tgaaattcct ttaactgaag cagaattggg tccaggtgcc 360
gaagaaacat tacatgcatc cattgccaat ttgttgggta tgggttggca tttgatgaga 420
atagctgcac cacacatgcc tgttggtagt gcagttataa acgtctccac catcttcagt 480
agagctgaat attacggtag aattccttat gttactccaa aagccgcttt aaatgcattg 540
tctcaattag cagccagaga attaggtgct agaggtatta gagttaacac catatttcca 600
ggtcctatcg aatcagatag aattagaact gtcttccaaa gaatggatca attaaagggt 660
agacctgaag gtgacacagc tcatcacttt ttaaacacca tgagattgtg tagagcaaac 720
gatcaaggtg ccttggaaag aagatttcca tctgtaggtg acgttgctga cgctgcagtc 780
ttcttagcct ccgctgaaag tgccgctttg tcaggtgaaa ctattgaagt tacacatggt 840
atggaattgc cagcctgctc tgaaacatca ttgttagcaa gaaccgattt gagaactatt 900
gacgcttctg gtagaactac attgatctgt gctggtgacc aaattgaaga agtcatggct 960
ttgacaggca tgttaagaac ctgcggttct gaagtaatca ttggttttag atcagcagcc 1020
gctttagctc aattcgaaca agcagttaat gaatcaagaa gattggcagg tgccgatttt 1080
acaccaccta tagctttacc attagatcca agagatccag caaccatcga tgccgtattc 1140
gactggggtg ctggtgaaaa tacaggtggt atacatgcag ccgttatctt accagctacc 1200
tctcacgaac cagcaccttg tgtcatagaa gtagatgacg aaagagtttt gaacttctta 1260
gctgatgaaa tcacaggtac cattgtcata gcttccagat tagcaagata ttggcaaagt 1320
caaagattga ctcctggtgc tagagcaaga ggtccaagag taatcttttt gtctaatggt 1380
gctgatcaaa acggtaacgt ttacggtaga attcaatcag ctgcaatagg tcaattaatc 1440
agagtttgga gacatgaagc tgaattggat taccaaagag catctgccgc tggtgaccac 1500
gtcttaccac ctgtatgggc caatcaaatt gttagatttg ctaacagatc cttggaaggt 1560
ttagaattcg cctgtgcttg gactgctcaa ttgttgcata gtcaaagaca catcaacgaa 1620
atcacattga acataccagc caacatctga 1650
<210> 3
<211> 2013
<212> DNA
<213> （MCR-mC）
<400> 3
tccgctacca ctggtgctag atctgcatca gttggttggg ctgaaagttt gatcggtttg 60
catttgggta aagtcgcatt gatcacaggt ggttccgccg gtatcggtgg tcaaattggt 120
agattgttag cattaagtgg tgccagagtt atgttggcag ccagagatag acataagtta 180
gaacaaatgc aagctatgat tcaatcagaa ttggcagaag ttggttacac tgatgttgaa 240
gacagagtcc acatagctcc aggttgcgat gtctcttcag aagcccaatt ggctgactta 300
gtagaaagaa ctttgtctgc tttcggtaca gttgattatt tgattaataa cgcaggtata 360
gccggtgtag aagaaatggt tatagatatg cctgttgaag gttggagaca tacattgttc 420
gcaaatttga tctccaacta cagtttgatg agaaagttgg ctccattaat gaaaaagcaa 480
ggttccggtt acatattgaa cgtttccagt tacttcggtg gtgaaaaaga tgctgcaata 540
ccatatccta acagagctga ctacgcagtc tctaaggcag gtcaaagagc aatggccgaa 600
gtatttgcta gattcttagg tcctgaaatc caaattaatg ctattgcacc aggtcctgtt 660
gaaggtgaca gattaagagg tactggtgaa agaccaggtt tgtttgccag aagagctaga 720
ttgatcttgg aaaataagag attgaacgaa ttacatgccg ctttgattgc agccgctaga 780
acagatgaaa gatctatgca cgaattagta gaattgttgt tgcctaatga cgttgcagcc 840
ttggaacaaa accctgctgc accaactgcc ttgagagaat tagctagaag attcagatct 900
gaaggtgacc cagccgcttc ttcatccagt gcattgttga acagatcaat agcagccaag 960
ttattggcta gattacataa cggtggttat gttttgcctg ctgatatttt tgcaaatttg 1020
cctaacccac ctgacccatt tttcacaaga gcccaaattg atagagaagc tagaaaggtt 1080
agagacggta tcatgggcat gttgtacttg caaagaatgc caaccgaatt tgatgttgcc 1140
atggctactg tctattactt agctgacaga gttgtttccg gtgaaacttt tcatcctagt 1200
ggtggtttga gatatgaaag aactccaaca ggtggtgaat tgttcggttt accatctcct 1260
gaaagattgg ctgaattagt cggttcaaca gtatacttaa taggtgaaca tttgaccgaa 1320
cacttaaatt tgttggcaag agcctatttg gaaagatacg gtgcaagaca agttgtcatg 1380
attgttgaaa ccgaaactgg tgctgaaaca atgagaagat tattgcatga tcacgttgaa 1440
gctggtagat tgatgaccat tgttgctggt gaccaaatag aagctgcaat cgaccaagct 1500
attactagat atggtagacc aggtcctgta gtttgtactc cttttagacc attacctaca 1560
gttccattgg tcggtagaaa agattctgac tggtcaacag ttttatcaga agcagaattt 1620
gccgaattat gcgaacatca attgactcat cacttcagag tcgccagatg gattgctttg 1680
tctgatggtg ctcgattagc attggtaacc ccagaaacaa ccgctacttc cactacagaa 1740
caattcgctt tggcaaactt catcaagacc actttgcatg ccttcacagc taccattggt 1800
gtagaaagtg aaagaactgc tcaaagaata ttaatcaacc aagttgattt gacaagaaga 1860
gccagagctg aagaacctag ggacccacac gaaagacaac aagaattaga aagattcatt 1920
gaagcagtat tgttggttac tgccccattg ccaccagaag cagacacaag atacgcaggt 1980
agaatccaca gaggtagagc cattacagtc taa 2013
<210> 4
<211> 694
<212> DNA
<213> (promoter TDH3 p)
<400> 4
ataaaaaaca cgctttttca gttcgagttt atcattatca atactgccat ttcaaagaat 60
acgtaaataa ttaatagtag tgattttcct aactttattt agtcaaaaaa ttagcctttt 120
aattctgctg taacccgtac atgcccaaaa tagggggcgg gttacacaga atatataaca 180
tcgtaggtgt ctgggtgaac agtttattcc tggcatccac taaatataat ggagcccgct 240
ttttaagctg gcatccagaa aaaaaaagaa tcccagcacc aaaatattgt tttcttcacc 300
aaccatcagt tcataggtcc attctcttag cgcaactaca gagaacaggg gcacaaacag 360
gcaaaaaacg ggcacaacct caatggagtg atgcaacctg cctggagtaa atgatgacac 420
aaggcaattg acccacgcat gtatctatct cattttctta caccttctat taccttctgc 480
tctctctgat ttggaaaaag ctgaaaaaaa aggttgaaac cagttccctg aaattattcc 540
cctacttgac taataagtat ataaagacgg taggtattga ttgtaattct gtaaatctat 600
ttcttaaact tcttaaattc tacttttata gttagtcttt tttttagttt taaaacacca 660
agaacttagt ttcgaataaa cacacataaa caaa 694
<210> 5
<211> 402
<212> DNA
<213> (promoter TEF1 p)
<400> 5
ttcaaaatgt ttctactcct tttttactct tccagatttt ctcggactcc gcgcatcgcc 60
gtaccacttc aaaacaccca agcacagcat actaaatttc ccctctttct tcctctaggg 120
tgtcgttaat tacccgtact aaaggtttgg aaaagaaaaa agagaccgcc tcgtttcttt 180
ttcttcgtcg aaaaaggcaa taaaaatttt tatcacgttt ctttttcttg aaaatttttt 240
tttttgattt ttttctcttt cgatgacctc ccattgatat ttaagttaat aaacggtctt 300
caatttctca agtttcagtt tcatttttct tgttctatta caactttttt tacttcttgc 360
tcattagaaa gaaagcatag caatctaatc taagttttct ag 402
<210> 6
<211> 46
<212> DNA
<213> (primer P124)
<400> 6
aaaggtctca atcttcatta tcaatactgc catttcaaag aatacg 46
<210> 7
<211> 69
<212> DNA
<213> (primer P125)
<400> 7
aaaggtctca gagagagttc tcgaatgaca gatcctcatt ttgtttgttt atgtgtgttt 60
attcgaaac 69
<210> 8
<211> 72
<212> DNA
<213> (primer P126)
<400> 8
aggtctcatc tcaaacttaa aggatcttcc gataacgtca aggagagcaa gtggtacagg 60
tagattagca gg 72
<210> 9
<211> 31
<212> DNA
<213> (primer P127)
<400> 9
aaaggtctca cctaggttct tcagctctgg c 31
<210> 10
<211> 33
<212> DNA
<213> (primer P128)
<400> 10
aaaggtctca tagggaccca cacgaaagac aac 33
<210> 11
<211> 33
<212> DNA
<213> (primer P129)
<400> 11
aaaggtctca cgaccttcga gcgtcccaaa acc 33
<210> 12
<211> 30
<212> DNA
<213> (primer P140)
<400> 12
aaaggtctca cagatcttat cgtcgtcatc 30
<210> 13
<211> 33
<212> DNA
<213> (primer P141)
<400> 13
aaaggtctca atctcctgga agtaccttca aag 33
<210> 14
<211> 35
<212> DNA
<213> (primer P144)
<400> 14
aaaggtctca tgagctctta attaacaatt cttcg 35
<210> 15
<211> 30
<212> DNA
<213> (primer P145)
<400> 15
aaaggtctca cgaccagctg gataaaggcg 30
<210> 16
<211> 34
<212> DNA
<213> (primer P142)
<400> 16
aaaggtctca tctgatgagg atctgtcatt cgag 34
<210> 17
<211> 33
<212> DNA
<213> (primer P143)
<400> 17
aaaggtctca ctcagatgtt ggctggtatg ttc 33
<210> 18
<211> 46
<212> DNA
<213> (primer P124)
<400> 18
aaaggtctca atcttcatta tcaatactgc catttcaaag aatacg 46
<210> 19
<211> 33
<212> DNA
<213> (primer P139)
<400> 19
aaaggtctca cggatgctct ccttgacgtt atc 33
<210> 20
<211> 26
<212> DNA
<213> (primer P138)
<400> 20
aaaggtctca tccgctacca ctggtg 26
<210> 21
<211> 33
<212> DNA
<213> (primer P129)
<400> 21
aaaggtctca cgaccttcga gcgtcccaaa acc 33
<210> 22
<211> 46
<212> DNA
<213> (primer P124)
<400> 22
aaaggtctca atcttcatta tcaatactgc catttcaaag aatacg 46
<210> 23
<211> 37
<212> DNA
<213> (primer P134)
<400> 23
aaaggtctca caactctgtc agctaagtaa tagacag 37
<210> 24
<211> 32
<212> DNA
<213> (primer P135)
<400> 24
aaaggtctca gttgtttccg gtgaaacttt tc 32
<210> 25
<211> 41
<212> DNA
<213> (primer P146)
<400> 25
aaaggtctca agacaaagca atccatctgg cgactctgaa g 41
<210> 26
<211> 43
<212> DNA
<213> (primer P137)
<400> 26
aaaggtctca gtctgatggt gctcgattag cattggtaac ccc 43
<210> 27
<211> 33
<212> DNA
<213> (primer P129)
<400> 27
aaaggtctca cgaccttcga gcgtcccaaa acc 33
<210> 28
<211> 74
<212> DNA
<213> (primer XI-3.3-MCR-N-up)
<400> 28
actgattagt tttccgtttt aggatattga cgccaagcgt gcgtctgatt ctggaagtac 60
cttcaaagaa tggg 74
<210> 29
<211> 72
<212> DNA
<213> (primer P155)
<400> 29
cggaacggtc gaccagctgg ataaaggcgc gccaaacgac ctaggaattg gagcgacctc 60
atgctatacc tg 72
<210> 30
<211> 69
<212> DNA
<213> (primer XI-3.3-CaMCR-dw)
<400> 30
cagcccaatc ctcaaaaata aaatggccct tccaaaatta gagacatatc cttcgagcgt 60
cccaaaacc 69
<210> 31
<211> 82
<212> DNA
<213> (primer P156)
<400> 31
caattcctag gtcgtttggc gcgcctttat ccagctggtc gaccgttccg tcattatcaa 60
tactgccatt tcaaagaata cg 82
<210> 32
<211> 74
<212> DNA
<213> (primer X-4-MCR-N-up)
<400> 32
ccaactacca aggttgttga gggaacactg gggcaatagg ctgtcgccat ctggaagtac 60
cttcaaagaa tggg 74
<210> 33
<211> 72
<212> DNA
<213> (primer P155)
<400> 33
cggaacggtc gaccagctgg ataaaggcgc gccaaacgac ctaggaattg gagcgacctc 60
atgctatacc tg 72
<210> 34
<211> 69
<212> DNA
<213> (primer X-4-CaMCR-dw)
<400> 34
cagacatcag acatactatt gtaattcaaa aaaaaaaagc gaatcttccc cttcgagcgt 60
cccaaaacc 69
<210> 35
<211> 82
<212> DNA
<213> (primer P156)
<400> 35
caattcctag gtcgtttggc gcgcctttat ccagctggtc gaccgttccg tcattatcaa 60
tactgccatt tcaaagaata cg 82
<210> 36
<211> 60
<212> DNA
<213> (primer IDP1p _ g)
<400> 36
aaaggtctct gatctgtgag acaacagacg cacagtttta gagctagaaa tagcaagtta 60
<210> 37
<211> 49
<212> DNA
<213> (primer Ura3_ Bsa 1)
<400> 37
aaaggtctca aaacacacag ggtaataact gatataatta aattgaagc 49
<210> 38
<211> 77
<212> DNA
<213> (primer IDP1_ TEF1p-f 2)
<400> 38
ccatataaag agggactcat atgatcttat gaaatcttcc ttcaagcaat ttcaaaatgt 60
ttctactcct tttttac 77
<210> 39
<211> 77
<212> DNA
<213> (primer IDP1_ TEF1 p-r)
<400> 39
aaagcagcaa ggcgagaggt ggaaaataat cttctagata acatactcat ctagaaaact 60
tagattagat tgctatg 77
<210> 40
<211> 36
<212> DNA
<213> (primer TDH3 p-GG-r)
<400> 40
aaaggtctca tttgtttgtt tatgtgtgtt tattcg 36
<210> 41
<211> 35
<212> DNA
<213> (primer TDH3 p-GG-f)
<400> 41
aaaggtctca atctacctcg agataaaaaa cacgc 35
<210> 42
<211> 35
<212> DNA
<213> (primer POS5 t-GG-r)
<400> 42
aaaggtctca cgacacgaaa catagtctct ctcca 35
<210> 43
<211> 38
<212> DNA
<213> (primer POS 5-GG-f)
<400> 43
aaaggtctca caaaatgttt gtcagggtta aattgaat 38
<210> 44
<211> 1287
<212> DNA
<213> (mitochondrial NADP specific isocitrate dehydrogenase encoding gene IDP 1)
<400> 44
atgagtatgt tatctagaag attattttcc acctctcgcc ttgctgcttt cagtaagatt 60
aaggtcaaac aacccgttgt cgagttggac ggtgatgaaa tgacccgtat catttgggat 120
aagatcaaga agaaattgat tctaccctac ttggacgtag atttgaagta ctacgactta 180
tctgtcgaat ctcgtgacgc cacctccgac aagattactc aggatgctgc tgaggcgatc 240
aagaagtatg gtgttggtat caaatgtgcc accatcactc ctgatgaagc tcgtgtgaag 300
gaattcaacc tgcacaagat gtggaaatct cctaatggta ccatcagaaa cattctcggc 360
ggtacagtgt tcagagagcc cattgtgatt cctagaattc ctagactggt cccacgttgg 420
gaaaaaccaa tcattattgg aagacacgcc cacggtgatc aatataaagc tacggacaca 480
ctgatcccag gcccaggatc tttggaactg gtctacaagc catccgaccc tacgactgct 540
caaccacaaa ctttgaaagt gtatgactac aagggcagtg gtgtggccat ggccatgtac 600
aatactgacg aatccatcga agggtttgct cattcgtctt tcaagctggc cattgacaaa 660
aagctaaatc ttttcttgtc aaccaagaac actattttga agaaatatga cggtcggttc 720
aaagacattt tccaagaagt ttatgaagct caatataaat ccaaattcga acaactaggg 780
atccactatg aacaccgttt aattgatgat atggtcgctc aaatgataaa atctaaaggt 840
ggctttatca tggcgctaaa gaactatgac ggtgatgtcc aatctgacat cgtcgctcaa 900
ggatttggct ccttaggttt gatgacttct atcttagtta caccagacgg taaaactttc 960
gaaagtgaag ctgctcatgg taccgtgaca agacattata gaaagtacca aaagggtgaa 1020
gaaacttcta caaactccat tgcatccatt ttcgcgtggt cgagaggtct attgaagaga 1080
ggtgaattgg acaatactcc tgctttgtgt aaatttgcca atattttgga atccgccact 1140
ttgaacacag ttcagcaaga cggtatcatg acgaaggact tggctttggc ttgcggtaac 1200
aacgaaagat ctgcttatgt taccacagaa gaatttttgg atgccgttga aaaaagacta 1260
caaaaagaaa tcaagtcgat cgagtaa 1287
<210> 45
<211> 1245
<212> DNA
<213> (mitochondrial NADH kinase coding gene POS 5)
<400> 45
atgtttgtca gggttaaatt gaataaacca gtaaaatggt ataggttcta tagtacgttg 60
gattcacatt ccctaaagtt acagagcggc tcgaagtttg taaaaataaa gccagtaaat 120
aacttgagga gtagttcatc agcagatttc gtgtccccac caaattccaa attacaatct 180
ttaatctggc agaacccttt acaaaatgtt tatataacta aaaaaccatg gactccatcc 240
acaagagaag cgatggttga attcataact catttacatg agtcataccc cgaggtgaac 300
gtcattgttc aacccgatgt ggcagaagaa atttcccagg atttcaaatc tcctttggag 360
aatgatccca accgacctca tatactttat actggtcctg aacaagatat cgtaaacaga 420
acagacttat tggtgacatt gggaggtgat gggactattt tacacggcgt atcaatgttc 480
ggaaatacgc aagttcctcc ggttttagca tttgctctgg gcactctggg ctttctatca 540
ccgtttgatt ttaaggagca taaaaaggtc tttcaggaag taatcagctc tagagccaaa 600
tgtttgcata gaacacggct agaatgtcat ttgaaaaaaa aggatagcaa ctcatctatt 660
gtgacccatg ctatgaatga catattctta cataggggta attcccctca tctcactaac 720
ctggacattt tcattgatgg ggaatttttg acaagaacga cagcagatgg tgttgcattg 780
gccactccaa cgggttccac agcatattca ttatcagcag gtggatctat tgtttcccca 840
ttagtccctg ctattttaat gacaccaatt tgtcctcgct ctttgtcatt ccgaccactg 900
attttgcctc attcatccca cattaggata aagataggtt ccaaattgaa ccaaaaacca 960
gtcaacagtg tggtaaaact ttctgttgat ggtattcctc aacaggattt agatgttggt 1020
gatgaaattt atgttataaa tgaggtcggc actatataca tagatggtac tcagcttccg 1080
acgacaagaa aaactgaaaa tgactttaat aattcaaaaa agcctaaaag gtcagggatt 1140
tattgtgtcg ccaagaccga gaatgactgg attagaggaa tcaatgaact tttaggattc 1200
aattctagct ttaggctgac caagagacag actgataatg attaa 1245
<210> 46
<211> 2013
<212> DNA
<213> （MCR-C）
<400> 46
tccgctacca ctggtgctag atctgcatca gttggttggg ctgaaagttt gatcggtttg 60
catttgggta aagtcgcatt gatcacaggt ggttccgccg gtatcggtgg tcaaattggt 120
agattgttag cattaagtgg tgccagagtt atgttggcag ccagagatag acataagtta 180
gaacaaatgc aagctatgat tcaatcagaa ttggcagaag ttggttacac tgatgttgaa 240
gacagagtcc acatagctcc aggttgcgat gtctcttcag aagcccaatt ggctgactta 300
gtagaaagaa ctttgtctgc tttcggtaca gttgattatt tgattaataa cgcaggtata 360
gccggtgtag aagaaatggt tatagatatg cctgttgaag gttggagaca tacattgttc 420
gcaaatttga tctccaacta cagtttgatg agaaagttgg ctccattaat gaaaaagcaa 480
ggttccggtt acatattgaa cgtttccagt tacttcggtg gtgaaaaaga tgctgcaata 540
ccatatccta acagagctga ctacgcagtc tctaaggcag gtcaaagagc aatggccgaa 600
gtatttgcta gattcttagg tcctgaaatc caaattaatg ctattgcacc aggtcctgtt 660
gaaggtgaca gattaagagg tactggtgaa agaccaggtt tgtttgccag aagagctaga 720
ttgatcttgg aaaataagag attgaacgaa ttacatgccg ctttgattgc agccgctaga 780
acagatgaaa gatctatgca cgaattagta gaattgttgt tgcctaatga cgttgcagcc 840
ttggaacaaa accctgctgc accaactgcc ttgagagaat tagctagaag attcagatct 900
gaaggtgacc cagccgcttc ttcatccagt gcattgttga acagatcaat agcagccaag 960
ttattggcta gattacataa cggtggttat gttttgcctg ctgatatttt tgcaaatttg 1020
cctaacccac ctgacccatt tttcacaaga gcccaaattg atagagaagc tagaaaggtt 1080
agagacggta tcatgggcat gttgtacttg caaagaatgc caaccgaatt tgatgttgcc 1140
atggctactg tctattactt agctgacaga aatgtttccg gtgaaacttt tcatcctagt 1200
ggtggtttga gatatgaaag aactccaaca ggtggtgaat tgttcggttt accatctcct 1260
gaaagattgg ctgaattagt cggttcaaca gtatacttaa taggtgaaca tttgaccgaa 1320
cacttaaatt tgttggcaag agcctatttg gaaagatacg gtgcaagaca agttgtcatg 1380
attgttgaaa ccgaaactgg tgctgaaaca atgagaagat tattgcatga tcacgttgaa 1440
gctggtagat tgatgaccat tgttgctggt gaccaaatag aagctgcaat cgaccaagct 1500
attactagat atggtagacc aggtcctgta gtttgtactc cttttagacc attacctaca 1560
gttccattgg tcggtagaaa agattctgac tggtcaacag ttttatcaga agcagaattt 1620
gccgaattat gcgaacatca attgactcat cacttcagag tcgccagaaa gattgctttg 1680
tctgatggtg cttcattagc attggtaacc ccagaaacaa ccgctacttc cactacagaa 1740
caattcgctt tggcaaactt catcaagacc actttgcatg ccttcacagc taccattggt 1800
gtagaaagtg aaagaactgc tcaaagaata ttaatcaacc aagttgattt gacaagaaga 1860
gccagagctg aagaacctag ggacccacac gaaagacaac aagaattaga aagattcatt 1920
gaagcagtat tgttggttac tgccccattg ccaccagaag cagacacaag atacgcaggt 1980
agaatccaca gaggtagagc cattacagtc taa 2013
Claims (10)
1. A genetically engineered bacterium for producing 3-hydroxypropionic acid is a recombinant saccharomyces cerevisiae with a genome containing an exogenous modified malonyl coenzyme A reductase gene MCR.
2. The genetically engineered bacterium of claim 1, wherein the recombinant Saccharomyces cerevisiae uses wild-type Saccharomyces cerevisiae as a host bacterium, and locates exogenous modified malonyl-CoA reductase gene MCR in Saccharomyces cerevisiae mitochondria and expresses it as two copies on a chromosome.
3. The genetically engineered bacterium of claim 2, wherein the MCR gene is split into two segments, namely MCR-N and MCR-C, and the MCR-C is subjected to point mutation transformation through N940V, K1106W, S R.
4. The genetically engineered bacterium of claim 3, wherein MCR-N is expressed by a promoter PGK1p, MCR-C is expressed by a promoter TDH3p, and each segment of gene is linked at its 5' end to a mitochondrial localization sequence CAT2m, and a copy is inserted into each of chromosome XI-3 and X-4 after MCR-N is linked to MCR-C.
5. The genetically engineered bacterium of any one of claims 1 to 4, wherein endogenous mitochondrial NADP-specific isocitrate dehydrogenase-encoding gene IDP1 and mitochondrial NADH kinase-encoding gene POS5 are also overexpressed in the genetically engineered bacterium.
6. The genetically engineered bacterium of claim 5, wherein overexpression of the mitochondrial NADP-specific isocitrate dehydrogenase encoding gene IDP1 is achieved by replacing its original promoter on the chromosome with the promoter TEF1 p; and/or, the mitochondrial NADH kinase coding gene POS5 is over-expressed by replacing the promoter thereof with TDH3p promoter and introducing the promoter into the strain in the form of high-copy plasmid.
7. The use of the genetically engineered bacterium of any one of claims 1 to 6 in the synthesis of 3-hydroxypropionic acid.
8. The use of claim 7, wherein the use comprises subjecting the genetically engineered bacteria to fermentation culture to produce 3-hydroxypropionic acid.
9. The use of claim 8, wherein the shake flask fermentation culture conditions are: the temperature is 30 ℃, the rotation speed of a shaking table is 200rpm, and the composition of the culture medium is 5g/L (NH) 4 ) 2 SO 4 、3g/L KH 2 PO 4 、0.5g/LMgSO 4 ·7H 2 O, trace metal elements, vitamins and 20g/L glucose.
10. The use of claim 8, wherein the fermenter fed-batch fermentation culture conditions are: the pH was maintained at 5, the temperature at 30 ℃, the dissolved oxygen limit at 30%, the aeration at 0.5VVm, the stirring setting at 400-1200rpm, and the feed medium composition at 67.5g/L (NH) 4 ) 2 SO 4 、27g/LKH 2 PO 4 、4.5g/L MgSO 4 ·7H 2 O, trace metal elements, vitamins and 300g/L glucose.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690774A (en) * | 2011-03-24 | 2012-09-26 | 三星电子株式会社 | Method of producing 3-hydroxypropionic acid using malonic semialdehyde reducing pathway |
| CN103805641A (en) * | 2014-01-24 | 2014-05-21 | 武汉大学 | Method for producing 3-hydroxypropionic acid, acrylic acid and propanoic acid by immobilizing carbon dioxide in microorganisms |
| CN108866117A (en) * | 2018-07-05 | 2018-11-23 | 青岛农业大学 | It is a kind of to utilize the method for photosynthetic bacteria synthesis 3- hydracrylic acid and its corresponding recombinant cell and application |
| CN116987606A (en) * | 2023-07-27 | 2023-11-03 | 北京化工大学 | Genetically engineered bacterium for producing 3-hydroxy propionic acid by carbon neutralization and application thereof |
-
2022
- 2022-01-29 CN CN202210113919.6A patent/CN115948264A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102690774A (en) * | 2011-03-24 | 2012-09-26 | 三星电子株式会社 | Method of producing 3-hydroxypropionic acid using malonic semialdehyde reducing pathway |
| CN103805641A (en) * | 2014-01-24 | 2014-05-21 | 武汉大学 | Method for producing 3-hydroxypropionic acid, acrylic acid and propanoic acid by immobilizing carbon dioxide in microorganisms |
| CN108866117A (en) * | 2018-07-05 | 2018-11-23 | 青岛农业大学 | It is a kind of to utilize the method for photosynthetic bacteria synthesis 3- hydracrylic acid and its corresponding recombinant cell and application |
| CN116987606A (en) * | 2023-07-27 | 2023-11-03 | 北京化工大学 | Genetically engineered bacterium for producing 3-hydroxy propionic acid by carbon neutralization and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| CHELLADURAI RATHNASINGH等: "Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains", JOURNAL OF BIOTECHNOLOGY, vol. 157, 23 June 2011 (2011-06-23), pages 633 * |
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