Patent Application
20200208188
The present invention relates generally to the biotechnological field, and in particular to a DNA assembly method and its application.
DNA assembly is the basic enabling technology for synthetic biology and bioengineering. Currently, DNA assembly methods fall into two main categories: assembly strategies based on Restriction endonucleases (RE) and assembly strategies based on homologous fragments. The RE-based DNA assembly methods are the most widely used assembly methods.
In the development of RE-based DNA assembly methods, the BioBrick™ method (Shetty, R P, Endy, D., and Knight, T. F, Jr. (2008) Engineering BioBrick vectors from BioBrick parts, J Biol Eng 2, 5) is the first method that was developed and put into practical use. This method utilizes four Type IIP REs (e.g., EcoRI, XbaI, SpeI, and PstI) for the recyclable DNA fragment assembly, in which two Type IIP REs (e.g., XbaI and SpeI) are isocaudomers.
By adding the EcoRI/XbaI site and the SpeI/PstI site at the 5′ end of a DNA fragment, the BioBrick™ method ensures that the recombinant vector still remains singular EcoRI/XbaI site and the SpeI/PstI site to achieve recyclability of the enzymes and the vector, thereby integrating new fragments into the assembled DNA in a continuously cyclic (recursive) way. This recursive DNA assembly method can achieve multiple rounds of “design-build-test” cycle to solve bioengineering trial and error verification studies without adequate mastery of genetic, physiological, and metabolic mechanisms. Especially in metabolic engineering studies, multiple rounds of “design-build-test” cycle can gradually solve the problems such as key enzyme screening, metabolic bottleneck removal, metabolic flux optimization and metabolic pathway reconstruction, gradually increase the yield, transformation rate and production intensity of specific metabolites, and build high-performance engineered strain to meet the needs of industrial production.
The easy-to-use and recursive assembly of the BioBrick™ method has led to its widespread use, and a number of similar DNA assembly methods have been derived therefrom, such as BglBrick (Anderson, J. C., Dueber, J. E., Leguia, M., Wu, G. C., Goler, J. A., Arkin, A. P., and Keasling, J. D. (2010) BglBricks: A flexible standard for biological part assembly, J Biol Eng 4, 1), iBrick (Liu, J. K., Chen, W. H., Ren, S. X., Zhao, G. P., and Wang, J. (2014) iBrick: A New Standard for Iterative Assembly of Biological Parts with Homing Endonucleases, Plos One 9), C-Brick (Li, S. Y., Zhao, G. P., and Wang, J. (2016) C-Brick: A New Standard for Assembly of Biological Parts Using Cpfl, Acs Synth Biol 5, 1383-1388), and YaliBrick (Wong, L., Engel, J., Jin, E., Holdridge, B., and Xu, P. (2017) YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica, Metab Eng Commun 5, 68-77). However, these methods introduce extra base sequences (“SCAR sequences”) at the position where a pair of isocaudomers of the DNA fragment are fused. For example, the BioBrick™ method produces 8 bp SCAR sequences, and the BglBrick, C-Brick, CCTL, and YaliBrick methods will produce 6 bp SCAR sequences. The SCAR sequence between the spliced DNA fragments affects the integrity of the DNA sequence, the secondary structure of the mRNA, and the correct expression of the protein, increasing the difficulty of DNA sequence design and limiting its application in the need for SCAR-free assembly and precise assembly. In particular, genetic elements that affect the function of upstream and downstream sequences, such as enhancers, promoters, RBS, spacer sequences, coding sequences, and terminator sequences, are accurately assembled into open reading frames, gene loops, metabolic pathways, or metabolic modules. It is desired to solve the problem of SCAR sequences in BioBrick™ and its derived assembly methods and develop a SCAR-free recursive DNA assembly technique.
The present invention establishes a SCAR-free PS-Brick assembly method for the “SCAR” sequence problem in the above-described recursive DNA assembly method. The method simultaneously uses the Type IIP and Type IIS REs, and combines the characteristics of the PCR product to realize the SCAR-free, iterative and tandem repeat sequence assembly, and has the characteristics of cost effectiveness and ease of use. This method is used for metabolic engineering of threonine strains, including thrA site mutation screening and modular integration with thrB, thrC, elimination of metabolic bottlenecks, identification of core efflux genes, and assembly of CRISPR-sgRNA repeat alignment vectors for co-knockout of threonine catabolic pathway-related genes. Moreover, through several rounds of “design-build-test” cycle, the method builds an engineering strain with high accumulation of threonine, which proves the industrial applicability of the method.
The present invention provides a plasmid comprising single adjacent Type IIP and Type IIS RE recognition sites.
Preferably, according to the above-described plasmid, the Type IIP RE is a Type IIP RE which cleaves to produce sticky ends with two or more bases.
Preferably, according to the above-described plasmid, the Type IIS RE is a Type IIS RE which cleaves to produce single-base sticky ends; or a Type IIS RE which cleaves to produce blunt ends.
More preferably, according to the above-described plasmid, the Type IIS RE is BmrI, BciVI, HphI or MlyI.
The present invention further provides a DNA assembly method, comprising: (1) performing a single-ended ligation of a gene to be inserted (i.e., a template) into a DNA fragment containing adjacent Type IIP and Type IIS RE recognition sites to obtain a target gene; (2) cleaving the target gene using the corresponding Type IIP RE to obtain a donor DNA; (3) cleaving the above plasmid using the corresponding Type IIP and Type IIS REs to obtain an acceptor DNA, wherein the plasmid comprises the same Type IIP and Type IIS RE recognition sites as the target gene; and (4) ligating the donor DNA to the acceptor DNA.
Preferably, according to the above method, in the target gene, the Type IIP RE recognition site is outside the Type IIS RE recognition site.
Preferably, according to the above method, when the Type IIS RE is a Type IIS RE which cleaves to produce single-base sticky ends, the step (1) further comprises attaching an A base to the other end of the gene to be inserted.
Preferably, according to the above method, in step (3), the plasmid is first cleaved using a corresponding Type IIP RE to obtain a linearized plasmid, and the linearized plasmid is cleaved using a corresponding Type IIS RE.
The present invention further provides a recombinant strain constructed according to the above method.
Preferably, according to the above recombinant strain, the recombinant strain is a recombinant strain producing threonine, and has increased expression of aspartate kinase ThrA, homoserine kinase ThrB, threonine synthase ThrC, aspartate semialdehyde dehydrogenase Asd, and threonine efflux transporter RhtC as compared with the original strain and has reduced expression of threonine dehydrogenase Tdh and threonine dehydratase IlvA as compared with the original strain.
More preferably, the increase in expression is achieved by transforming a plasmid carrying the corresponding gene to the original strain, and the reduction in expression is achieved by knocking out the corresponding gene of the original strain.
Further preferably, the plasmid carrying the corresponding gene is constructed by the above method, and a vector for knocking out the corresponding gene is constructed by the above method.
The present application discloses a DNA assembly method based on Type IIP and Type IIS REs, i.e., PS-Brick. The method combines the properties of PCR products, Type IIP and Type IIS REs to achieve recursive cycling, SCAR-free and repeat sequence assembly. The PS-Brick assembly method is used for metabolic engineering breeding, which has industrial applicability: based on the advantage of seamless assembly of this method, the codon saturation mutagenesis and the precise splicing of the bicistronics are realized; based on the advantage of the tandem repeat fragment assembly of this method, the tandem CRISSPR sgRNA repeats with the same promoter and terminator are assembled; based on the cyclic iterative assembly characteristics of PS-Brick, the feedback inhibition of threonine biosynthesis is gradually eliminated, the metabolic bottleneck is eliminated, threonine efflux is strengthened, the threonine catabolism is inactivated, and the metabolic pathway of threonine is systematically optimized and transformed, and an engineering strain for efficient production of threonine is obtained. In addition, the PS-Brick assembly method has the advantages of simplicity, time saving and high efficiency, and has high practicability.
Compared with the existing DNA assembly technique, the novel design of the PS-Brick assembly method is mainly reflected in the following aspects:
(1) Existing RE-based DNA assembly methods (such as the BioBrick and Golden Gate assembly methods) use only one type of RE, i.e., Type IIP or Type IIS REs, respectively. The method of the present invention uses REs of both Type IIP and Type IIS, to achieve both recursive and non-marking advantages of the above two methods.
(2) The target gene hangs the adjacent RE recognition sites only at a single end, so that a blunt end or an A-binding sticky end at the other end of the target gene can be simultaneously utilized.
(3) A Type IIS RE producing blunt ends and a blunt-ended target gene, or a Type IIS RE producing single-base sticky ends and a target gene with A-binding sticky ends are used to achieve SCAR-free splicing of DNA fragments.
(4) A single pair of adjacent Type IIP and Type IIS RE sites form recyclable import sites for recursive cycling assembly.
The recognition and restriction sites of the Type IIP RE are of the same palindromic sequence. At present, Type IIP RE-based DNA assembly methods can only use four specific REs at the same time. For example, the BioBrick method can only use SpeI, PstI, XbaI and SpeI at the same time; BglBrick can only use EcoRI, BglII, BamHI, and XhoI at the same time; YaliBrick can only use SpeI, XbaI, NheI, and AvrII at the same time. The PS-Brick assembly technique can use any of the hundreds of Type IIP REs that produce sticky ends with two or more bases, greatly reducing site restriction and increasing the sequence design of the PS-Brick assembly method.
The restriction site of the Type IIS RE is outside the recognition site, and different Type IIS REs can produce sticky ends with 1-4 bases, respectively, and can also produce blunt ends. PS-Brick only uses Type IIS REs that produce blunt ends or single-base sticky ends. Currently, three single-base sticky end Type IIS REs (Bind, BciVI and HphI) and a blunt-ended Type IIS RE MlyI can be purchased through commercial channels.
In addition, primers for PCR amplification of donor DNA do not require special modifications (e.g., 5′port phosphorylation), thereby reducing the application cost of the present technique.
The embodiments of the present invention will be described in more detail in conjunction with the accompanying drawings and embodiments, in order to provide a better understanding of the embodiments of the present invention and the advantages thereof. However, the specific embodiments and examples described below are illustrative only and should be construed as limiting the present invention.
The present invention cites publications for the purpose of more clearly describing the present invention. These publications are hereby incorporated by reference in their entireties as if their full texts have been repeatedly described herein.
The order of execution of the steps in the method mentioned in the present invention is not limited to the order shown in the text of the present invention unless otherwise specified, that is, the order of execution of the steps may be changed, and between two steps, additional steps may be inserted as needed.
The “original strain” referred to in the present invention means the initial strain used in the genetic modification strategy of the present invention. The strain may be a naturally occurring strain, or may be a strain bred by mutagenesis or genetic engineering. In order to build an engineered strain for producing threonine, the original bacterium is preferably a strain capable of accumulating threonine.
The expression “increased expression of . . . ” as used in the present invention is intended to indicate that the expression of a protein encoded by the corresponding gene is increased. It can be achieved by overexpression of the corresponding gene, for example, by constructing a recombinant plasmid containing the gene, and then introducing the recombinant plasmid in the original strain; it can also be achieved by inserting the gene into the chromosome in the original strain. These methods are commonly used in the art and will not be described again. The vector used to construct the recombinant plasmid is not limited and may be any suitable plasmid, for example, pXMJ19.
The expression “reduced expression of . . . ” as used in the present invention is intended to indicate that the expression of a protein encoded by the corresponding gene is reduced. It can be achieved by inactivating the corresponding gene, and “inactivating” refers to a change in the corresponding engineered object, thereby achieving certain effects, including but not limited to, site-directed mutagenesis, insertional inactivation, and/or knockout.
The “ligation . . . recognition sites” referred to in the present invention can be introduced by PCR primers.
The target gene may be a PCR product having a prominent single “A” base at the 3′ end amplified by a DNA polymerase such as Taq, LA Taq or EX Taq, or a blunt-ended PCR product amplified by high-fidelity polymerases such as Q5, KAPA, KOD or Pfu.
The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. For the quantitative tests in the following examples, three replicate experiments are set, and the results are averaged. Unless otherwise specified in the following examples, the technical means used in the examples are conventional means well known to those skilled in the art and commercially available instruments and reagents, see “Molecular Cloning: A Laboratory Manual (3rd Edition)” (Science Press), “Microbiology Experiment (4th Edition)” (Higher Education Press), the manufacturer's instructions for the corresponding instruments and reagents, etc.
RE BciVI is purchased from Thermo Fisher Scientific, and other REs are purchased from New England Biolabs (NEB). Kapa hot start high-fidelity polymerase is purchased from Kapa Biosystems, Inc., and Ex-Taq DNA polymerase is purchased from TaKaRa-Bio.
The strains, plasmids and primer sequences (5′→3′) used in the examples are as follows:
E. coli DH5α
E. coli MG1655
Escherichia coli
GCGCCGGTTTTAGAGCT AGAAATAGC
GATGAAGACTAGTATTA TACCTAGGA
CCGCAGTTTTAGAGCTAGAAATAGC
GCCAAAGACTAGTATTA TACCTAGGA
As shown in
In order to verify the feasibility of this method, Type IIP RE SphI and two Type IIS REs BmrI (producing sticky ends of 1 nt base after cleavage) and MlyI (producing blunt ends after cleavage) are selected to study the application effect of different Type IIS REs. As shown in
(I) Construction of Original Vectors pOB Plasmid and pOM Plasmid for PS-Brick
Plasmid pUC19 (SEQ ID NO. 1) is used as a basic vector for verifying the PS-Brick assembly method. One BmrI site and three MlyI sites in the vector are removed through overlap extension PCR method (Ho, S N, Hunt, H D, Horton, R M, Pullen, J. K, and Pease, L. R (1989) Site-Directed Mutagenesis by Overlap Extension Using the Polymerase Chain-Reaction, Gene 77, 51-59). Specifically, two DNA fragments are first obtained through amplification using primer pairs UC709-F/UC1179-R and UC1179-F/UC1695-R, respectively, and the two DNA fragments are further spliced by overlap PCR using a primer pair UC709-F/UC1746-R. By using the splicing product as a large primer and the plasmid pUC19 as a template, one of the BmrI sites and three MlyI sites are mutated. Other two BmrI sites and MlyI sites in the multiple cloning site sequence on the plasmid pUC19 are removed by double-cleavage with SphI and NdeI to obtain a pUC19 vector backbone without BmrI and MlyI sites.
With the pSEVA237R vector (Martinez-Garcia, E., Aparicio, T., Goni-Moreno, A., Fraile, S., and de Lorenzo, V. (2015) SEVA 2.0: an update of the Standard European Vector Architecture for de-/re-construction of bacterial functionalities, Nucleic Acids Res 43, D1183-D1189) as a template, PCR amplification is carried out respectively using the same forward primer mC-F and different reverse primers mCB-R (carrying adjacent SphI/BmrI sites) and mCM-R (carrying adjacent SphI/MlyI sites) to obtain two different mCherry truncated fragments. The truncated site is located in the MlyI reverse recognition site “GACTC” (
The Kapa hot-start high-fidelity polymerase is used to carry out PCR reaction on the above two PCR fragments to be integrated. The reaction cycle is carried out at 95° C. for 3 min for one cycle, 98° C. for 20 s, 65° C. for 20 s, 72° C. for 30 s, totaling 27 cycles and finally at 72° C. for 1 min. The PCR products are purified and recovered, and the DNA concentration is determined using Nanodrop 2000c (Thermo Fisher Company).
The purified PCR products are double digested with SphI and NdeI and ligated to the above pUC19 vector backbone without BmrI and MlyI sites, respectively. By sequencing, the pOB plasmid with the SphI/MlyI adjacent sites and the pOM plasmid with the SphI/BmrI adjacent sites are obtained as PS-Brick original vectors (
(II) DNA Fragment Assembly
The PCR product FB amplified by Ex-Taq polymerase and the primer FB-R carrying the SphI/BmrI site and the primer FB-F is used as an insert for the TA clone (
The PCR reaction using Ex-Taq polymerase were as follows: pre-denaturation at 94° C. for 5 min, further at 94° C. for 30 s, at 54° C. for 30 s and at 72° C. for 30 s, totaling 27 cycles and extension at 72° C. for 5 min. After completion of PCR using Ex-Taq polymerase or Kapa hot-start high-fidelity polymerase, the PCR product is gel electrophoresed, and stripes of correct size are cut out, purified by column, and then digested with SphI.
PS-Brick original vectors pOB and pOM are digested with Bind and MlyI for 15 min, respectively. The linearized vectors are separated by gel electrophoresis. After column purification, the vectors are digested with SphI for the second time for 15 min, and then heat inactivated at 60° C. for 20 min and then column purified. After two digestions, the recognition site of Type IIS Bind or MlyI and half of the Type IIP SphI site are detached from the original vector backbone, and Bind produces a sticky end with 1-nt at one end of the vector (or MlyI produces a blunt end), and SphI produces a sticky end with 4-nt at the other end of the vector (
Enzyme digestion systems of all of the above REs are 50 μl, containing 20 units of enzyme and 1 μg of DNA, reacting at 37° C. The 10 μl of ligation reaction system containing 1 μL of T4 DNA ligase, 20 ng of linearized vector and 5-fold molar weight of inserted DNA fragment is incubated at 25° C. for 15 minutes, then placed on ice and transformed into 100 μL of homemade E. coli DH5a competent cells (the efficiency of transforming the pUC19 plasmid is (1.17±0.19)×106 CFU/μg DNA). Primers UC1-6 are used for PCR identification of transformant colonies.
The pOB and pOM vectors are single digested with Type IIS REs BmrI and MlyI, respectively. The digestion reaction system is as described in Example 1, and the reaction time lasts from 15 minutes to 180 minutes according to the reaction conditions of Time-Saver product, followed by electrophoresis to detect cleavage efficiency. As shown in
The BmrI-cleaved pOB and MlyI-cleaved pOM stripes are recovered with gel, further digested with SphI for 15-180 minutes, inactivated at 65° C., and recovered and ligated to SphSphI-digested PCR products FB and FM, respectively, and transformed into Escherichia coli. DH5a competent cells. The transformon units (CFUs) represent DNA assembly efficiency. Twenty clones are picked and sequenced, and the proportion of correctly assembled clones is used as the accuracy of the assembly method.
The SphI single-digested DNA fragments are ligated to the corresponding double-digested vectors under different enzyme digestion time conditions of SphI. The conversion and correct rate of PS-Brick ligation are determined by colony count and PCR identification of transformants. The results show that the conversion rates in the case of the SphI/BmrI combination and in the case of the SphI/MlyI combination both reach 104-105 cfu/μg DNA under different enzyme digestion time (
The above results indicate that the 15-minute digestion time is sufficient for the Type IIS and IIP REs used in the present example. Taking into account 30 minutes for each of two DNA recovery operations, 20 minutes for RE inactivation, 30 minutes for DNA ligation, 30 minutes for reversal and 40 minutes for resuscitation, PS-Brick's main experimental procedure can be completed in half a day (
The PS-Brick assembly technique is used to carry out the “design-build-test” cycle of multiple iterations, thus constructing the engineered strain producing threonine. Since gene expression in organisms and the regulation and interaction of signaling networks are very complex, and there is a lack of prior knowledge for predicting how a DNA assembly introduced into a cell can function, multiple versions of the construct need to be tested to obtain an optimal assembly plan. Multiple rounds of step-by-step “design-build-test” experiments require iterative DNA assembly methods.
The metabolic engineering strategy for constructing a threonine-engineered strain usually includes the following steps: releasing the feedback inhibition of the threonine operon, enhancing the threonine terminal synthesis pathway, removing the metabolic bottleneck, blocking the threonine catabolism, modifying the threonine transport system and enhancing cofactor regeneration (
(I) SCAR-Free Fusion of ThrA to Achieve Codon Saturation Mutagenesis
This example uses HindIII and MlyI as the Type IIP and IIS REs for PS-Brick assembly, respectively. Three MlyI sites on the plasmid pACYC184 (SEQ ID NO. 3) are mutated with the primer pairs AC3211-F/AC727-R and AC727-F/AC1143-R by overlap extension PCR and the other MlyI site located at the multiple cloning site is removed through double digestion with HindIII and NruI REs.
The truncated thrABC operon (SEQ ID NO. 4) is amplified using three pairs of primers TAB-F/TAB-R, TBC-F/TBC-R and TC-F/TC-R for overlapping PCR. The RE NruI site is designed to be outside the primer of thrC, and the adjacent HindIII and MlyI restriction sites are designed to be outside the truncation site of thrA (
The thrA insert for the next step is amplified using Kapa hot-start high-fidelity polymerase. The adjacent HindIII and MlyI restriction sites are designed to be outside the forward primer TA-F, and the 20 reverse primers TAAA-R respectively carry a codon sequence causing saturation mutagenesis in the 433th residue of ThrA. Twenty PCR products are digested with HindIII and ligated with HindIII and MlyI double-digested pOthr vectors, respectively, to obtain 20 pthrA433BC SCAR-free spliced saturated mutant vectors. These vectors contain the same HindIII/MlyI site for the next round of DNA assembly.
The obtained expression vectors containing 20 different thrA* mutant sequences are transferred into E. coli MG 1655. The shake flask fermentation and comparison are carried out for 12 hours, the threonine yields of the engineered strains overexpressing different mutants are quite different (
So far, thrA433BC described below refers to thrAGly433PheBC.
(II) Identification of Metabolic Bottlenecks of Threonine Biosynthesis
In order to identify the metabolic bottlenecks of threonine biosynthesis, a total of five key enzyme genes, i.e., aspA (SEQ ID NO. 5), aspC (SEQ ID NO. 5), ppc (SEQ ID NO. 7), asd (SEQ ID NO. 8) and pntAB (SEQ ID NO. 9) are selected and ligated to the thrAGly433Phe vector, respectively, as described above. The aspA gene is amplified using the primers aspA-F/aspA-R, and the aspC gene is amplified using the primers aspC1-F/aspC1-R and aspC2-F/aspC2-R; the ppc gene is amplified using the primers ppc1-F/ppc1-R, ppc2-F/ppc2-R, ppc3-F/ppc3-R and ppc4-F/R; the asd gene is amplified using the primers asd1-F/asd1-R and asd2-F/asd2-R; the pntAB gene is amplified using the primers pntA/B1-F/pntA/B1-R, pntA/B2-F/pntA/B2-R, pntA/B3-F/pntA/B3-R, pntA/B4-F/pntA/B4-R and pntA/B5-F/pntA/B5-R. The PCR products are respectively ligated to the vector pthrA433pheBC by the second round of PS-Brick reaction and transformed into DH5α competent cells, and the correct vector pACYC184-thrA433BC-ppc/aspA/aspC/asd/pntAB, is sequenced and transformed to E. coli MG655. After the shake flask fermentation for 12 h, the threonine yields of the strains carrying different vectors are determined. The results show that the engineered strain carrying the pACYC184-thrA433BC-asd vector has the highest yield of threonine, which is 56.7% higher than the control strain (E. coli MG1655/pACYC184-thrA433BC) (
(III) Screening of Threonine Efflux Transporter
Further, four threonine efflux transporter genes rhtA (SEQ ID NO. 12), rhtB (SEQ ID NO. 13), rhtC (SEQ ID NO. 14), yecC (SEQ ID NO. 15) are respectively assembled on the pACYC184-thrA433BC-asd vector. The rhtA gene is amplified using the primer rhtA-F/rhtA-R, the rhtB gene is amplified using the primer rhtB-F/rhtB-R, the rhtC gene is amplified using the primer rhtC-F/rhtC-R, and the yecC gene is amplified using the primer yec-F/yec-R. The PCR products are ligated to the vector pACYC184-thrA433 BC-asd by a third round of PS-Brick reaction to obtain four vectors pACYC184-thrA433 BC-asd-rhtA/rhtB/rhtC/yecC, respectively. Further, the primers T-F/TBCD-R and TBCD-F/BCD-R are used to splice the amplification promoter PT (SEQ ID NO. 10) and the bicistronic design element BCD1 (SEQ ID NO. 11), and the PCR products are ligated to the vectors pACYC184-thrA433 BC-asd-rhtA/rhtB/rhtC/yecC respectively by a fourth round of PS-Brick reaction to obtain pACYC184-thrA433 BC-asd-PTBCD1-rhtA/rhtB/rhtC/yecC (
(IV) Blocking of Catabolic Pathway of Threonine
In the above examples, the key gene for threonine synthesis is gradually integrated on the expression vector based on the RE MlyI blunt-ligated PS-Brick method. In this example, a CRISPR sgRNA repeat sequence for knocking out the threonine catabolic pathway gene is assembled based on the PS-Brick method using TA clone of BciVI restriction endonuclease.
The reported CRISPR-Cas9 gene editing system containing pCas9 and pTargetF vectors (Jiang, Y., Chen, B., Duan, C. L, Sun, B. B, Yang, J. J, and Yang, S. (2015) Multigene Editing in The Escherichia coli Genome via the CRISPR-Cas9 System, Appl Environ Microb 81, 2506-2514) is used to knock out the tdh gene (SEQ ID NO. 17) and the ilvA gene (SEQ ID NO. 16). The BciVI site in the pTargetF plasmid is mutated using the primer TGB-F/R for PCR and DpnI digestion and transformation. The donor DNA contains a homologous sequence of 500 base pairs at upstream and downstream of the target genes ilvA and tdh as a template for gene editing. Editing templates for the three target genes are spliced by overlap PCR using primers ilv1/2-F/R and tdh1/2-F/R. The adjacent HindIII-BciVI RE site is designed to be outside the primer ilv1-F, and the BamHI site is designed to be outside the primer tdh2-R. The donor DNA fragment and the pTargetF vector are double digested with REs BamHI and HindIII, and ligated to obtain a ptargetET vector containing a HindIII-BciVI RE site for assembly of the CRISPR sgRNA repeat fragment (
First, primers N20-tdh-F/R and N20-ilvA-F/R containing the N20 sequences of tdh and ilvA genes are respectively used for PCR amplification with pTargetF as a template, and the products are recovered and digested with DpnI. The enzyme-digested products are transfected into DH5a competent cells to obtain two vectors pTargetF-tdh and pTargetF-ilv respectively containing the N20 sequences of the tdh and ilvA genes. Next, EX Taq DNA polymerase and the primer sgRNA-F/R are used for PCR amplification with the pTargetF-tdh vector as a template to obtain an sgRNA fragment of the tdh gene (i.e., a fragment containing PJ23119, tdhN20, and sgRNA) (Jiang, Y., Chen, B., Duan, C. L, Sun, B. B, Yang, J. J, and Yang, S. (2015) Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Appl Environ Microb 81, 2506-2514). This fragment is single digested with HindIII and ligated to the HindIII/BciVI double-digested ptargetET vector to obtain a ptargetET-tdh vector. This vector contains the HindIII-BciVI site introduced by the primer sgRNA-F as an entrance site for the next round of PS-Brick assembly. EX Taq DNA polymerase and the primer sgRNA-F/R are then used for PCR amplification with the pTargetF-ilv vector as a template to obtain an sgRNA fragment of the ilvA gene. This fragment is single digested with HindIII and ligated to the HindIII/BciVI double-digested ptargetET-tdh vector to obtain the ptargetET-tdh-ilv vector (
The vector ptargetET-tdh-ilv containing two identical promoter and terminator sgRNA sequences is constructed by two rounds of PS-Brick assembly, and gene editing is then carried out according to the reference (Jiang, Y., Chen, B., Duan, C. L, Sun, B. B, Yang, J. J, and Yang, S. (2015) Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System, Appl Environ Microb 81, 2506-2514). The pTargetET-tdh-ilvA plasmid is transformed into the MG1655/pCas9 competent cells, and then the cells are applied to a double-resistant plate containing 50 mg/L kanamycin and 50 mg/L spectinomycin and cultured at 30° C. Transformants with the ilvA and tdh genes knocked out are identified using primer pairs ilvA-I-F/R and tdh-I-F/R, respectively. The transformants in which the tdh and ilvA genes are successfully knocked out are selected, and the ptargetET-tdh-ilvA and pCas plasmids are sequentially eliminated to obtain E. coli G1655ΔtdhΔilvA (
(V) Fermentation Test of Threonine Engineered Strain
In the above description, the shake flask fermentation test method is specifically as follows unless otherwise specified.
Shake Flask Fermentation Test:
1. The test strain is taken, streaked on a solid LB medium plate containing 34 mg/L chloramphenicol, and performed static culture at 37° C. for 12 hours.
2. After step 1, the lawn on the plate is picked up and inoculated into the slant of the LB medium, and performed static culture for 10-12 hours at 37° C.
3. After step 2, the lawn on the plate is picked up, inoculated into liquid LB medium, cultured at 37° C., shaken at 220 rpm for 12 hours to obtain a seed liquid.
4. After step 3, the seed liquid is inoculated into the fermentation medium according to an inoculum volume of 3%, and shaken at 220 rpm and 37° C.
One liter of fermentation medium includes: 80 g/L of MOPS, 20.0 g/L of glucose, 15.0 g/L of ammonium sulfate, 2.0 g/L of monopotassium phosphate, 2.0 g/L of magnesium sulfate heptahydrate, 2.0 g/L of yeast powder, 5 mL/L of a mixed solution of trace elements, and the balance of water.
One liter of the mixed solution of trace elements includes: 10 g/L of FeSO4.7H2O, 1.35 g/L of CaCl2, 2.25 g/L of ZnSO4.7H2O, 0.5 g/L of MnSO4.4H2O, 1 g/L of CuSO4.5H2O, 0.106 g/L (NH4)6Mo7O24. 4H2O, 0.23 g/L of Na2B4O7.10H2O, 0.48 g/L of CoCl2.6H2O, 10 mL/L of 35% HCl and the balance of water.
During the culture process, the pH of the reaction system is adjusted with ammonia water to be maintained at 6.8-7.0.
During the culture process, samples are taken every 3-4 hours, and the glucose content is measured using a biosensor analyzer SBA-40D. When the glucose content in the system is less than 5 g/L, glucose is added so that the glucose concentration in the system reaches 10 g/L.
After 24 hours of culture, samples are taken, centrifuged at 12000 g for 2 minutes, and the supernatant is taken to measure the concentration of L-threonine.
In the above description, the fermenter fermentation test method is specifically as follows unless otherwise specified.
Fermenter Fermentation Test:
The method of this example refers to the patent (application No. 201110279419.1), and the seed culture medium used is composed of water and solutes; the concentrations of the solutes in the medium are as follows: glucose 40 g/L, ammonium sulfate 15 g/L, monopotassium phosphate 2 g/L, magnesium sulfate 2 g/L, yeast powder 2 g/L, L-isoleucine 0.05 g/L, calcium carbonate 15 g/L, the mixed solution of trace elements 2 mL/L.
The fermentation initial medium used in this example is composed of water and solutes; the concentrations of the solutes in the medium are as follows: glucose 10 g/L, ammonium sulfate 10 g/L, monopotassium phosphate 2 g/L, magnesium sulfate 2 g/L, yeast powder 2 g/L, and the mixed solution of trace elements 2 mL/L.
The cell yield of E. coli MG1655 with L-isoleucine as substrate is determined by pre-experiment to be 107 g/g (i.e., 107 g dry weight of E. coli MG1655 cells generated from per gram of L-isoleucine).
1. Obtain Seed Liquid
(1) The test strain stored in a −80° C. cryotube is streaked into the LB medium plate, and incubated in a 37° C. incubator for 12 hours.
(2) The single colony of step (1) is transferred into a test tube containing 3 mL of liquid LB medium, and incubated in 37° C. shaker at 180 rpm for 12 hours.
(3) The bacterial solution of step (2) is transferred in a 500 mL shake flask containing 30 mL of seed culture medium according to an inoculum volume of 3% (volume ratio), and then incubated in a 37° C. shaker at 220 rpm for 12 hours to obtain a seed liquid (OD600=8.5).
2. The seed liquid of step 1 is inoculated into the fermentation initial medium in the fermenter according to an inoculum volume of 3% (volume ratio), and cultured until 3 g (dry weight) of cells is contained per liter of the fermentation liquid, and a total of 2.2 L of fermentation broth is obtained.
In the whole step 2: the fermentation temperature is controlled to be 37° C. by the heating jacket and the cooling water; air is introduced to supply dissolved oxygen, and if necessary, the mixture of oxygen and air is introduced in a ratio of 1:1 (volume ratio), the dissolved oxygen is controlled to be 50% by the rotation speed-dissolved oxygen signal cascade control; the addition of 25% (volume ratio) of ammonia water adjusts the pH and maintains it at 6.8.
3. Feeding is carried out by feed liquid A (L-isoleucine aqueous solution), and a constant speed programmable control pump built in the fermenter is controlled by BioCommand Plus biological process software to realize index feeding: input the following program: F=(μ·X0·V0·eμt)/(S·Yile/X), where F refers to index feeding rate, μ refers to the set specific growth rate, and X0 refers to the initial cell concentration (its value is 3 g·L−1), V0 is the initial fermentation liquid volume (its value is 2.2 L), e is a natural logarithm (its value is 2.718), t refers to the fermentation time, and S refers to the concentration of L-isoleucine in the feed liquid A (its value is 2 g/L), Yile/X refers to cell yield (its value is 107 g/g); the specific growth rate is set to be 0.16 h−1. The index feeding and fermentation are continued until the growth of the cells does not increase.
In the whole step 3: the fermentation temperature is controlled to be 37° C. by the heating jacket and the cooling water; air is introduced to supply dissolved oxygen, and if necessary, the mixture of oxygen and air is introduced in a ratio of 1:1 (volume ratio), the dissolved oxygen is controlled to be 50% by the rotation speed-dissolved oxygen signal cascade control; the addition of 25% (volume ratio) of ammonia water adjusts the pH and maintains it at 6.8.
During the culture, samples are taken every 3-4 hours, centrifuged at 12000 g for 2 minutes, and the supernatant is taken to measure the concentration of L-threonine.
(VI) Threonine HPLC Detection Method
Method for detecting L-threonine concentration: High-performance liquid phase method, optimized on the basis of the amino acid detection method in the reference (Amino Acids and Biological Resources, 2000, 22, 59-60); the specific method is as follows (2,4-dinitrofluorobenzene (FDBN) pre-column derivatization high-performance liquid phase method):
10 μL of the supernatant is added into a 2 mL centrifuge tube, 200 μL of 0.5 M NaHCO3 aqueous solution and 100 μL of 1% (volume ratio) FDBN-acetonitrile solution are then added, the reaction system is heated in a dark place in a 60° C. water bath for 60 min and then cooled to room temperature; next, 700 μL of 0.04 mol/L KH2PO4 aqueous solution (pH=7.2±0.05, pH is adjusted with 40 g/L KOH aqueous solution) is added and shaken to be uniform; the reaction system is rested for 15 min and then filtered; the filtrate is collected. The filtrate is used for loading and the sample amount is 15 μL.
The chromatography column is a C18 column (ZORBAX Eclipse XDB-C18, 4.6*150 mm, Agilent, USA); column temperature: 40° C.; UV detection wavelength: 360 nm; mobile phase A is 0.04 mol/L KH2PO4 aqueous solution (pH=7.2±0.05, the pH is adjusted with 40 g/100 mL of KOH aqueous solution), and mobile phase B is 55% by volume of acetonitrile aqueous solution, and the total flow rate of the mobile phase is 1 mL/min.
Elution process: at the start time of elution (0 min), the volume fraction of mobile phase A in the total flow of the mobile phase is 86%, and the volume fraction of mobile phase B in the total flow of the mobile phase is 14%; the elution process is divided into 4 stages. In each stage, the volume fractions of mobile phase A and mobile phase D in the total flow of the mobile phase both change linearly; at the end of the first stage (lasting 2 minutes from the start time), the volume fraction of mobile phase A in the total flow of the mobile phase is 88%, and the volume fraction of mobile phase B in the total flow of the mobile phase is 12%; at the end of the second stage (lasting 2 minutes from the end of the first stage), the volume fraction of the mobile phase A in the total flow of the mobile phase is 86%, and the volume fraction of the mobile phase B in the total flow of the mobile phase is 14%; at the end of the third stage (lasting 6 minutes from the end of the second stage), the volume fraction of the mobile phase A in the total flow of the mobile phase is 70%, and the volume fraction of the mobile phase B in the total flow of the mobile phase is 30%; at the end of the fourth stage (lasting 10 minutes from the end of the third stage), the volume fraction of the mobile phase A in the total flow of the mobile phase is 30%, and the volume fraction of the mobile phase B in the total flow of the mobile phase is 70%.
A standard curve is prepared using commercially available L-threonine as a standard (purchased from sigma, Cat. No. 8917) to calculate the threonine concentrations of the samples.
It should be noted that the above-described examples are merely illustrative of the invention and are not intended to limit the implementations. Other variations or modifications of the various forms may be made by those skilled in the art in light of the above description. There is no need and no way to exhaust all of the implementations. Obvious changes or variations resulting therefrom are still within the scope of the invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201811613376.4 | Dec 2018 | CN | national |
