CN113677795B - Novel DAHP synthetase - Google Patents

Novel DAHP synthetase Download PDF

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CN113677795B
CN113677795B CN202080027938.6A CN202080027938A CN113677795B CN 113677795 B CN113677795 B CN 113677795B CN 202080027938 A CN202080027938 A CN 202080027938A CN 113677795 B CN113677795 B CN 113677795B
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曾安平
陈林
陈民良
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Zeng Anping
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Abstract

The present invention provides a mutant DAHP synthase that suffers less feedback inhibition from phenylalanine than the wild-type enzyme and is suitable for biotechnological production of aromatic amino acids (e.g., tryptophan).

Description

Novel DAHP synthetase
Technical Field
The present invention relates to a novel DAHP synthase. Furthermore, the invention relates to a method for screening engineered enzyme mutants in vivo.
Background
Microorganisms have been extensively engineered to produce value-added compounds from renewable feedstocks (Becker et al, 2015; lee and Kim, 2015; liao et al, 2016). For this reason, engineering of biosynthetic pathways plays a vital role. Several strategies based on gene expression and enzyme concentration regulation have been developed (Alper et al, 2005; blazeck and Alper, 2013; hwang et al, 2018; zhou and Zeng, 2015). However, they cannot overcome some of the inherent limitations associated with the enzymes themselves. For example, regulatory mechanisms of enzyme activity, such as feedback inhibition or feedforward inhibition (Chen et al, 2018; zurawski et al, 1981), limit the specific activity or substrate specificity of enzymes (Mora-Villalobos and Zeng, 2017). In this regard, the construction of efficient pathways inevitably requires protein engineering (Chen et al, 2013; chen et al, 20110 b; der et al, 1994; forest et al, 2012).
In general, protein engineering consists of three distinct steps: (i) construction of a gene mutant library; (ii) Screening of the library and (iii) further characterization of candidate enzyme mutants (Boville et al, 2018; buller et al, 2018) (FIG. 1 b). It has been widely and successfully used to improve the performance of many enzymes (Chen et al, 20111 a; rees et al, 2017; grand et al, 2016). However, due to some limiting factors, mutant strains containing enzyme mutants obtained from conventional protein engineering methods may be phenotypically difficult to distinguish, which not only makes it very difficult and time-consuming to screen and identify the desired enzyme mutants (Ren et al, 2018), but also makes it difficult to find the enzyme mutants that perform best among the significantly improved enzyme mutants (Ren et al, 2015). Furthermore, the subsequent in vitro and in vivo characterization of the enzyme mutants determined by screening to perform optimally may not be relevant to the actual biological production process in which the host microorganism is used. In view of this, methods for identifying enzyme mutants having improved properties and suitable for use in practical biological production processes have received attention.
Recently, CRISPR/Cas9 technology for genome editing has been greatly advanced and has received extensive attention (Cho et al, 2018; donohoue et al, 2017; Et al, 2015; zhang et al, 2018). Wherein it is applied to engineering of microorganism producing strains (/ >) Et al, 2015; jiang et al, 2015; schuster et al, 2018). Because of its simplicity and efficiency, CRISPR/Cas9 is a suitable genome editing tool for rapid and efficient integration of gene mutants of target enzymes into the chromosome of production strains (Guo et al, 2018).
L-tryptophan is an essential amino acid and is widely used in medicine, food industry, animal feed industry (Becker et al, 2015), and as a precursor for other products (Huccetogullari et al, 2019; noda and Kondo, 2017; vargas-Tah et al, 2015; wendisch, 2014). Therefore, it is of great interest to be able to produce tryptophan in a simple and cost-effective manner. The production of tryptophan by fermentative biotechnology, such as with E.coli (E.coli), has become an important source of this amino acid. However, biosynthesis of aromatic amino acids such as tryptophan still needs to be optimized.
Disclosure of Invention
It is an object of the present invention to provide an improved biotechnological process for the production of aromatic amino acids, in particular tryptophan.
In order to achieve the above object, the present invention provides a mutant DAHP synthase (aroG) having the sequence of SEQ ID NO. 1, wherein the amino acids at positions 6 and 7 are not simultaneously aspartic acid.
The DAHP synthase of the invention is not or significantly less feedback inhibited by phenylalanine (Phe) than the wild-type enzyme and shows a higher specific enzyme activity in the presence of 40mM Phe than the reference mutant AroG S180F known in the prior art.
The term "engineered enzyme mutant" refers to an enzyme whose amino acid sequence has been biotechnologically altered compared to the wild-type enzyme. In engineered enzyme mutants, one or more amino acids at a given position in the wild-type enzyme may be substituted, e.g., with other amino acids, either purposefully or randomly. The term "mutant enzyme" may also be used for the enzyme mutant.
The term "auxotroph of an organic compound" refers to the inability of a cell or organism to synthesize the particular organic compound required for its growth.
The term "recombinant engineering system" (also referred to as "recombination system") relates to one or more components of a homologous recombination system, i.e. a recombination-mediated genetic engineering system, which is necessary for homologous recombination in vivo. One example of a recombinant engineering system is a lamda-red (lambda-red) recombinant engineering system. The lambda-red recombination engineering system consists of three components, the proteins Exo, beta and Gam, that are necessary for dsDNA homologous recombination. Another example of a recombinant engineering system is the Rec E/T system, consisting of proteins Rec E and Rec T.
The term "expressible" in relation to a coding DNA sequence means that the DNA sequence can be transcribed into RNA transcripts and translated into the corresponding protein products under the control of an inducible or constitutive promoter. For example, in the case of a recombinant engineering system or gene, this means that components of the recombinant engineering system or gene can be transcribed into RNA transcripts under the control of an inducible promoter or a constitutive promoter and translated into the corresponding protein components of the recombinant engineering system or gene. With respect to non-coding DNA sequences, the term is understood to mean that RNA transcripts are formed from the DNA sequences under the control of an inducible promoter or a constitutive promoter. In this context, the term "expressible CRISPR guide RNA" relates to a DNA sequence encoding a CRISPR guide RNA, i.e. a DNA sequence whose transcription results in an RNA transcript into a CRISPR guide RNA.
The term "genetically engineered synthesis of a reporter in the presence of an organic compound" refers to synthesis of a reporter in the presence of an organic compound. A "reporter" is a molecule that is easily detected, such as a visualized molecule. For example, the reporter molecule may be a fluorescent molecule. The synthesis of the reporter molecule may depend directly or indirectly on the presence of the organic compound. An example of a reporter molecule is Green Fluorescent Protein (GFP) or a mutant thereof, such as enhanced green fluorescent protein (eGFP).
The term "Cas protein" refers to a CRISPR-associated protein. CRISPR is an abbreviation of the term "regularly spaced clustered short palindromic repeats" used to denote prokaryotic DNA fragments comprising palindromic repeats, separated by variable spaced DNA fragments. The term includes the family of proteins that are part of the CRISPR-Cas system, which fall into two classes (class 1 and class 2) and several types (I to VI) and subtypes. Class 1 includes types I, III and IV, and class 2 includes types II, V, and VI. Cas-type is generally characterized by a characteristic protein. For example, cas9 is a type CasII signature protein. See, for example, makarova et al, 2015; konin et al, 2017; haft et al, 2005, wright et al, 2016. Cas proteins have nuclease activity and use CRISPR RNAS (crRNAs) to direct Cas nuclease components to the target nucleic acid molecule to be cleaved.
The term "Cas9 protein" or "Cas9" refers to CRISPR-associated protein 9.Cas9 is an RNA-guided DNA endonuclease, e.g., from streptococcus pyogenes. The term "Cas9" as used herein also includes all Cas9 orthologs as well as recombinants, i.e., engineered mutants thereof. Cas9 nucleases are active when forming a complex with a guide RNA, which may consist of two separate RNA molecules, a tracrRNA and a crRNA, or a single stranded RNA molecule consisting of a tracrRNA and a crRNA fused together. The specificity of the nuclease is determined by the base complementary pairing of a partial crRNA sequence with the target DNA sequence. By specifying the targeting sequence of the crRNA, the CRISPR-Cas9 system can be directed to the appropriate target site ("pre-spacer"). A further requirement of Cas 9-mediated DNA targeting is the presence of short (e.g., 2-6 base pairs) and conserved pre-spacer adjacent motifs (PAMs) near, e.g., near downstream of, the target site. PAM sequences can be varied from Cas protein to Cas protein and can also be modulated by engineering Cas proteins.
The term "crRNA" refers to CRISPR RNA, meaning an RNA molecule that is capable of being complementary to a tracrRNA. The crRNA confers target specificity to a Cas protein, such as Cas 9. RNA: RNA duplex consisting of crRNA and tracrRNA can be fused together to form a single stranded RNA molecule, also known as guide RNA (gRNA), and bind to Cas protein. In bacterial CRISPR loci, crrnas are located in the CRISPR repeat/spacer region, consisting of a spacer complementary to the target gene and a repeat complementary to the tracrRNA.
The term "tracrRNA" refers to trans-activating RNA, a small trans-coding RNA that is partially complementary to and base-paired with crRNA, thereby forming crRNA: tracrRNA duplex.
The term "CRISPR guide RNA", also known as "CRISPR GRNA" or "gRNA", refers to a crRNA: tracrRNA duplex. crRNA and tracrRNA may or may not be bound to single stranded RNA molecules. Thus, the term "CRISPR guide RNA" includes the term "CRISPR single stranded guide RNA" (sgRNA).
The term "base pairing" or "hybridization" refers to the formation of a duplex by base pairing between complementary nucleic acid molecules, for example between two RNA or DNA molecules or between one RNA and one DNA molecule.
The term "genomic DNA" is used herein as meaning chromosomal DNA, and does not refer to extrachromosomal DNA such as plasmid DNA. The term "genome" as used herein also refers to genomic genetic material, i.e., genetic material on a chromosome, rather than genetic material on, for example, a plasmid.
The term "culturing cells in a growth medium suitable for cell growth in the absence of an organic compound" refers to culturing cells in a solid, liquid or semi-solid medium in the absence of an organic compound, but in the absence of an organic compound, containing nutrients that support cell growth. Thus, if the cells are able to synthesize organic compounds, the cells are able to grow on the medium, otherwise they will not. The growth medium may be a basal medium containing essential nutrients required for cell growth.
The term "determining the growth of a cell" relates to a qualitative and/or quantitative determination of a growth parameter, such as growth rate, growth efficiency (yield coefficient) or maximum biomass, preferably growth rate. Growth efficiency is related to the relationship between the amount of carbon source consumed and biomass produced.
The term "assaying synthesis of a reporter" relates to qualitative and/or quantitative assays of synthesis of a reporter. This may include, for example, measuring fluorescence of a fluorescent reporter. In particular, the term relates to determining the intensity of a signal, such as fluorescence, generated by a reporter molecule.
The term "DAHP synthase" relates to the synthesis of DAHP (3-deoxy-D-arabino-heptulo-2-onic acid-7-phosphate) from phosphoenolpyruvate and D-erythrose 4-phosphate by phospho-2-dehydro-3-deoxyheptuloaldolase (EC 2.5.1.54; also 3-deoxy-D-arabinoheptulo-7-phosphate). The term "AroG" relates to an isoform of DAHP synthase derived from e.coli that is feedback-inhibited by the amino acid phenylalanine (Phe), and may also be used herein as a synonym for the term "DAHP synthase".
The term "aromatic amino acid" relates to amino acids having an aromatic ring, in particular phenylalanine, tryptophan and tyrosine.
In a preferred embodiment, the mutated DAHP synthase (AroG) has the amino acid sequence of SEQ ID NO: 2. SEQ ID NO:3 or SEQ ID NO:4, and a sequence of one of the following. The embodiments of the mutant DAHP synthetases (AroGs) of the invention, represented herein as AroG D6G-D7A(SEQ ID NO:2)、AroGD6L-D7P (SEQ ID NO: 3) and AroG D6P-D7I (SEQ ID NO: 4), have been identified using novel screening methods as described below, using the growth rate and signal strength of the biosensors as a standard. The enzymes of the invention are not or significantly reduced by phenylalanine feedback inhibition compared to the wild-type enzyme and exhibit a higher specific enzyme activity in the presence of 40mM phenylalanine (Phe) than the reference mutant AroG S180F known in the prior art. In a simple fed-batch fermentation, aroG S180F was replaced with the newly identified AroG D6G-D7A that performed best among tryptophan (Trp) -producing strains, with an increase in Trp yield of 38.5% (24.03.+ -. 1.02g/L at 36 h).
The invention also relates to bacterial cells genetically engineered to express the DAHP synthase of the invention. Preferably, the bacterial cell is an E.coli cell.
In another aspect, the invention relates to the use of said bacterial cells for the production of aromatic amino acids, in particular tryptophan. For this purpose, the bacterial cells are preferably E.coli cells, preferably grown in a suitable medium in a bioreactor.
The invention also relates to a method for biosynthesis of an aromatic amino acid comprising the step of culturing the bacterial cell of the invention in a medium and under conditions suitable for growth of the bacterial cell.
In a preferred embodiment, the method of the invention further comprises the step of isolating the aromatic amino acids produced by the bacterial cells from the culture medium.
In a preferred embodiment of the method, the growing bacterial cell is an E.coli cell. The aromatic amino acid produced is preferably tryptophan.
The invention also relates to a method for screening engineering enzyme mutants in vivo, which comprises
A. supplementing cells with
(I) Due to the lack of functional genes encoding enzymes necessary for the synthesis of organic compounds, resulting in auxotrophy for the organic compounds,
(Ii) Genetically engineered to comprise an expressible gene encoding a Cas9 protein,
(Iii) Genetically engineered to include an expressible CRISPR guide RNA configured to base pair with a target sequence at a target site on a cell chromosome and direct a Cas9 protein to the target sequence,
(Iv) Genetically engineered to synthesize reporter molecules in the presence of organic compounds, and
(V) Genetically engineered to comprise a DNA sequence comprising a target sequence of a CRISPR guide RNA, a PAM sequence and an expressible target gene at a target site on a chromosome,
Introducing into the cell by biotechnology a donor DNA comprising a donor gene encoding a mutant of an enzyme required for the synthesis of the organic compound,
B. culturing the cells in the absence of an organic compound in a growth medium suitable for cell growth, said growth medium being free of an organic compound,
C. determining the growth of cells, and
D. the synthesis of the reporter is determined.
The method of the invention combines CRISPR/Cas-promoted direct chromosomal target genetic engineering with growth coupling and biosensor-guided in vivo screening and characterization of protein mutants. By measuring the cell growth conditions, such as growth rate, and the signal intensity of the biosensor, such as a reporter molecule, in combination with the method, improved enzyme mutants that perform best can be reliably identified. Thus, the method is particularly useful for protein engineering and pathway optimization.
The methods of the invention may be abbreviated herein as the term "CGSSC" for CRISPR/Cas 9-promoted target genetic engineering coupled with growth and sensor-guided in vivo screening and characterization.
The above-exemplified DAHP synthetases of the present invention have been identified using the above-described screening methods, and using the growth rate and signal intensity of the biosensor as criteria.
In the methods of the invention, better performing or optimal enzyme mutants can be identified among the improved enzyme mutants by measuring the growth of the cells and the synthesis of the reporter molecule. The results of cell growth and reporter synthesis for the different enzyme mutants were used to identify the enzyme mutants that performed best. For example, a first cell expressing a first enzyme mutant grows better, e.g., has a higher growth rate or higher growth yield, and produces more reporter molecules indicative of the production of an organic compound than a second cell expressing a second enzyme mutant. For example, if the reporter is a fluorescent molecule, the amount of reporter produced can be detected by fluorescence. Particularly preferably, the growth rate, growth yield or growth efficiency of the reporter molecule, and the productivity, yield or production efficiency are employed. Cells expressing one enzyme mutant may be identified as performing better or optimally than cells expressing another enzyme mutant with relatively better growth yields and higher specific fluorescence intensities at comparable physiological phases, e.g., at the end of the logarithmic growth phase. Growth yield can be determined, for example, by measuring the OD600 at the end of the logarithmic growth phase, i.e. its optical density at 600nm in a spectrophotometer.
The methods of the invention utilize cells genetically engineered in a specific manner to allow for efficient in vivo screening and characterization of enzyme mutants. The organic compounds, e.g., essential amino acids, of the cells that are essential for cell growth are auxotrophs. The cells may be naturally auxotrophic for the organic compound, but are preferably cells genetically engineered to be auxotrophic for the organic compound.
The cells are genetically engineered to contain a gene for a Cas protein. Cas proteins, such as Cas9 proteins, are used to allow efficient insertion of nucleic acids encoding enzyme mutants into the cell genome. Preferably, the gene encoding the Cas protein is contained in a plasmid, although Cas proteins may also be incorporated into the cell genome. The Cas protein may be under the control of a constitutive promoter or an inducible promoter. Preferably, the Cas protein is under the control of a constitutive promoter.
The CRISPR guide RNA is preferably introduced into a plasmid separate from the plasmid carrying the Cas protein (see Jiang et al, 2015), i.e. the Cas protein is introduced on a first plasmid and the CRISPR guide RNA is introduced on a second plasmid. Further preferably, the CRISPR guide RNA is introduced into a cell together with the donor DNA. For this purpose, the gRNA and the donor DNA may be contained in the same plasmid. The gRNA may be under the control of a constitutive or inducible promoter, and the gRNA is preferably under the control of a constitutive promoter. The gRNA is capable of base pairing with a target sequence of the target site on the chromosome, into which the donor DNA (i.e., DNA comprising the gene encoding the enzyme mutant) is to be inserted, and directs the Cas protein to the target site. Although the guide RNA may be a duplex of two separate RNA molecules, namely crRNA and tracrrRNA, in the method of the invention, it is preferred to use a single-stranded RNA molecule consisting of crRNA and tracrrRNA, namely monoguide RNA (sgRNA). Whether or not fused into a single stranded molecule, the gRNA consisting of crRNA and tracrRNA may be synthetic RNA, e.g., shorter synthetic RNA than naturally occurring gRNA. The synthetic gRNA may be chemically modified, for example, to stabilize the gRNA by replacing nucleotides naturally occurring in the gRNA with nucleotides not naturally occurring in the gRNA (see, for example, WO 2016/100951 A2).
The cell also includes a target gene located at a target site on the chromosome, the target gene being a placeholder for insertion of the enzyme mutant DNA into the genome. For example, the target gene may be an antibiotic resistance gene. For example, the target gene may be inserted into the site of the wild-type enzyme gene such that the wild-type enzyme gene is disrupted or replaced by the target gene. The target site further comprises a DNA sequence comprising a target sequence of the CRISPR guide RNA, i.e. a sequence (base pairs) to which the CRISPR guide RNA can hybridize, and a PAM sequence such that the Cas protein cleaves the target sequence.
In addition, the cells are genetically engineered to synthesize a reporter molecule based on the presence of a functional gene encoding an enzyme required for the synthesis of an organic compound. For example, the cells may be engineered so that a reporter gene is preferably included on the gene, the expression of which is dependent on the presence or absence of the organic compound. An example of such a reporter gene is tnaC, the tnaC encodes the leader sequence of the tnaCAB operon, upstream of the tnaC, fused to eGFP. The eGFP can be expressed in the presence of tryptophan. The eGFP production may be determined qualitatively or quantitatively by measuring fluorescence, preferably fluorescence intensity. The production of the reporter molecule is preferably related, e.g. linearly related, to the production of the organic compound. One skilled in the art can readily design a reporting system suitable for detecting the presence of another organic compound in a cell (Xu et al, 2014, fang et al, 2016).
In the method of the invention, the donor DNA is introduced into the cells by biotechnological means, for example by electroporation. The donor DNA comprises a donor gene encoding the enzyme mutant inserted into the cell. The donor gene is introduced into the cell such that the donor gene can be constitutively expressed or induced to be expressed. In the method of the present invention, recombinant cells expressing the enzyme mutants necessary for the synthesis of the organic compounds are thus prepared. The donor DNA is preferably double-stranded linear or circular DNA (dsDNA). Preferably, the donor DNA is inserted into a plasmid. In a preferred embodiment of the method of the invention, the plasmid further carries a gRNA for directing the Cas protein to the target site. The donor DNA comprises the donor gene, which can be introduced into the cell by methods such as electroporation, and which can be inserted into a target site of the cell genome by CRISPR/Cas-facilitated recombination. The donor gene may be flanked by "homology arms", i.e., sequences homologous to the target site on the chromosome for homologous recombination.
In a preferred embodiment of the method of the invention, the Cas protein is a class II Cas protein, preferably a Cas9 protein. The Cas protein may be a recombinant Cas protein, preferably a recombinant Cas protein of type II, e.g. a recombinant Cas9 protein. The gene of the Cas protein is preferably contained in a plasmid and is preferably under the control of a constitutive promoter.
The cells may be further genetically engineered to include genes of a recombinant engineering system. In certain embodiments of the methods of the invention, it may be advantageous to genetically engineer the cells to comprise a recombinant engineering system and a gene for a Cas protein. The genes of the Cas protein and the recombinant engineering system may be contained in the same or different plasmids. However, it is also possible for the recombination engineering system, in particular in the case of the lambda-red recombination engineering system, to be inserted into the genome. Preferably, the gene encoding Cas protein and the gene encoding recombinant engineering system are contained in the same plasmid. The Cas protein gene may also be integrated into the cell genome. The recombinant engineering system is preferably under the control of an inducible promoter, such as a temperature sensitive promoter. In one embodiment of the method of the invention, a genetically engineered cell is used which also comprises a recombinant engineering system gene, which is preferably flanked by "homology arms", i.e. sequences homologous to the target site on the chromosome, in order to be able to mediate homologous recombination by the recombinant engineering system. Thus, the donor DNA introduced into the cell by biotechnology contains a donor gene encoding an enzyme mutant necessary for the synthesis of an organic compound, and flanking sequences homologous to the target gene on the chromosome.
In a preferred embodiment of the method of the invention, the recombinant engineering system comprising components Exo, beta and Gam is used if the recombinant engineering system comprised in the cell used in the method is a lambda-red (lambda-red) recombinant engineering system. The lambda-red recombination engineering system is used to mediate homologous recombination to insert the donor gene into the cell genome. The lambda-red recombinant engineering system is preferably integrated in the genome. However, the recombinant engineering system may also be arranged in a plasmid, e.g. a plasmid carrying a Cas protein gene. Preferably, the lambda-red recombinant engineering system is expressed under the control of an inducible promoter, such as a temperature sensitive promoter.
The cells used in the method of the present invention are preferably microbial cells, more preferably bacterial cells, still more preferably E.coli cells, particularly preferably E.coli cells.
The method of the invention can be used for in vivo screening and characterization of enzyme mutant libraries. To this end, a plurality of cells as defined above are complemented with different enzyme mutants. Cells of the supplemented different enzyme mutants were grown in the absence of organic compounds and the growth of cells and synthesis of reporter molecules were assayed to identify enzyme mutants with superior properties compared to the wild-type enzyme. The enzyme mutants that perform optimally can be identified, for example, with the growth rate and signal intensity (e.g., fluorescence intensity) of the reporter molecule as parameters.
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Hereinafter, the present invention is described by way of the drawings and examples for illustrative purposes only.
FIG. 1 is a simplified schematic diagram of an embodiment of the method of the present invention. A: the organic compound auxotrophic cells are complementary to donor DNA that contains functional genes necessary for the synthesis of the organic compound. B: the donor gene is introduced into complementing cells of the cell genome by CRISPR/Cas 9-assisted recombination engineering.
Figure 2 shows a comparison of conventional screening and characterization methods in rational protein engineering (b, dashed line) with CRISPR/Cas 9-promoted engineering and growth coupling and sensor-directed in vivo screening and characterization (CGSSC) methods (a, solid line). In the progressive process of rational protein engineering, first, rational or semi-rational design changes are made to the gene of interest using knowledge based on protein structure and function or results of bioinformatics and modeling. High throughput screening and ex vivo characterization to identify optimal mutants is a laborious process. In CGSSC, the combined steps of growth coupled in vivo screening and sensor-guided in vivo characterization of enzyme mutants CRISPR/Cas 9-facilitated engineering make protein engineering more efficient and accurate.
FIG. 3 shows the design and implementation of the method of the invention in the screening and characterization of feedback-resistant AroG (AroGfbr) enzyme mutants. In E.coli, DAHP synthetases (aroG, aroF, and aroH) are key rate limiting enzymes in the Aromatic Amino Acid (AAA) biosynthetic pathway. An AAA auxotroph strain (strain WS 002) was constructed by disrupting DAHP synthase and used as a platform for screening aroG gene mutants, which were individually integrated into the chromosome of e.coli using the CRISPR/Cas9 system. At high concentrations of Phe, only strains expressing AroG fbr with good resistance to Phe were able to produce sufficient AAA and maintain cell growth. These strains were further characterized by the fluorescence signal intensity (moderate fluorescence units, MFU) of tryptophan biosensor (P tac -TnaC-eGFP) representing tryptophan productivity.
FIG. 4 shows a comparison of growth and fluorescence of two WS003 and WS004 strains produced by introducing aroG WT and aroG S180F, respectively, into the chromosome of WS002 strain under different growth conditions. The left panel shows the complex medium (LB-agar); the middle panel is M9-agar medium (without any amino acids); the right panel shows M9-agar medium containing 25mM Phe.
In FIG. 5 (a) the key residues of the E.coli AroG Phe binding site. (b) Fluorescence induction of AroG D6X-D7X mutants on reduced M9 agar (without Tyr and Trp) with addition of 25mM Phe and 0.1mM IPTG.
FIG. 6 shows the effect of Phe on the activity of aroG WT enzyme and its mutants aroG S180F、AroGD6L-D7P、AroGD6P-D7I and aroG D6G-D7A. (a) specific activity; (b) related Activity. Results were from three independent experiments.
FIG. 7 shows fed-batch fermentation results for strains S028 (circular) and S028GM1 (square). (a) cell growth; (b) glucose concentration; (c) tryptophan production; (d) Total productivity, (e) rate of tryptophan (qTrp) formation and (f) accumulation of intermediate shikimic acid (SA, open circles or open squares) and dehydroshikimic acid (DSA, filled circles or filled squares). All results are based on two independent fermentations.
FIG. 8 is a map of plasmid pCm-aroG. The donor DNA contained in the plasmid consisted of a portion of the CmR gene, the entire wild-type aroG gene and a portion of the serum (serA) gene.
FIG. 9 shows the amino acid sequences (single letter codes) of the DAHP synthetases of the invention (FIG. 9 a) and preferred embodiments thereof (FIGS. 9 b-c). X=any amino acid, but not both at the same time aspartic acid (D).
Detailed Description
Fig. 1 shows an embodiment of the method of the invention in a simplified and schematic manner. In the method of the present invention, a cell, preferably a bacterial cell such as E.coli, which is auxotrophic for an organic compound due to the lack of a functional gene encoding an enzyme required for the synthesis of the organic compound is supplemented by biotechnologically introducing a donor DNA comprising the donor gene encoding an enzyme required for the synthesis of the organic compound into the cell (FIG. 1A). The donor gene is introduced into the cell genome by CRISPR/Cas-assisted recombination engineering. To this end, in this embodiment, the cells are genetically engineered to include (a) an expressible recombinant engineering system; (b) an expressible gene encoding a Cas protein, herein Cas9; (c) An expressible CRISPR guide RNA (sgRNA) configured to base pair with a target sequence ("protospacer") of a target site on a cell chromosome and guide a Cas protein to the target sequence; and (d) a target site on the chromosome comprising a target sequence of a CRISPR guide RNA ("protospacer"), a PAM sequence and a DNA sequence that can express a target gene (e.g., a gene that confers resistance to an antibiotic). The cells are further genetically engineered to synthesize a reporter molecule in the presence of an organic compound. The donor gene introduced into the cell is flanked by sequences homologous to a target gene on the chromosome. The recombination engineering system (herein lambda-red recombination engineering system) and the gene of the Cas protein are placed on the same plasmid. However, the genes encoding the recombinant engineering system and Cas protein may also be placed on a single plasmid or in the genome. In the method of the present invention, the recombinant engineering system is not necessarily present.
Expressing the donor gene introduced into the chromosome of the cell, thereby producing a functional enzyme, which is necessary for the synthesis of the organic compound B from the precursor a, for which the cell is auxotrophic. The organic compound synthesizes and in the presence thereof forms a reporter molecule (fig. 1B). Cell growth and reporter synthesis were measured.
By performing the procedure described above with different donor genes encoding different enzyme mutants, the library of enzyme mutants can be tested to identify the enzyme mutants that perform best (see FIG. 2).
Examples
1. Introduction to the invention
3-Deoxy-D-arabinoheptulose 7-phosphate (DAHP) synthase is a key enzyme for the efficient biosynthesis of Aromatic Amino Acids (AAA) by engineered microorganisms: tryptophan (Trp), phenylalanine (Phe) and tyrosine (Tyr) (Chen and Zeng, 2017; kim et al, 2015; wu et al, 2018). It is strongly feedback inhibited by the final product (Ogino et al, 1982; sprenger, 2006). For example, in E.coli, all wild-type DAHP synthetases encoded by the genes aroG, aroF and aroH are feedback inhibited by Phe, tyr and Trp, respectively (McCandliss et al, 1978; schoner and Herrmann, 1976). Thus, engineering of anti-feedback DAHP synthetases is essential for the construction of efficient pathways for the production of AAA and its derivatives (Sprenger, 2006). The method of the invention, also known as "CGSSC", is used to engineer and screen anti-feedback DAHP synthetase mutants to improve the branching pathway for tryptophan production in E.coli.
2. Materials and methods
2.1 Strain and plasmid
Table 1 lists the strains and plasmids used in this study. Trp producing strain S028λ was used as the starting strain, wherein the temperature sensitive lambda-red recombinant engineering system was deleted on the chromosome of the parent strain of S028 (Chen and Zeng, 2017). In strain S028λ, DAHP synthase activity was provided only by AroGS F against phenylalanine. To mediate genome editing using CRISPR/Cas9 technology, plasmid pCas (Jiang et al, 2015) was inserted into S028λ, resulting in strain S028λc (table 1).
TABLE 1 Main strains and plasmids used herein
a TrpR-sgRNA, sgRNA with the N20 sequence, was used to target the trpR site.
To construct plasmids for protein expression and purification, the aroG WT -encoding gene was isolated from E.coli DY330 strain (Yu et al, 2000) using aroG-His-HindIII and XbaI-serA primers (Table 2). It was then inserted into vector pET22b (+) at HindIII and XbaI positions to generate plasmid pET-aroG WT. Plasmids pET-aroG S180F、pET-aroGD6G-D7A、pET-aroGD6L-D7P and pET-aroG D6P-D7I were generated by amplifying the entire plasmid pET-aroG WT using the mutagenesis primer (Table 2).
2.2 Molecular biology work
2.2.1 Construction of sgRNA plasmids and donor DNA
To construct plasmids expressing one-way guide RNAs (sgrnas), the present invention amplified pTagAmpR backbones by PCR using a set of primers (table 2) (fig. 8). With the aid of a network-based tool Cas-Designer (Bae et al, 2014; park et al, 2015), a 20bp spacer sequence specific for the target gene was selected and synthesized within the primer (Table 2, uppercase letters show). The PCR product was then directly transformed into E.coli Top10 competent cells to obtain the desired sgRNA plasmid. To construct the sgRNA plasmid containing the donor DNA, two 300-500bp homology arms and the DNA fragment for substitution were amplified separately and then fused together by PCR. After gel purification, the target PCR product was inserted into the desired sgRNA plasmid.
TABLE 2 primers used herein
The gRNA sequence is indicated in uppercase letters; n= A, C, G or T; k=g or T
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Construction of plasmids and strains
Construction of WS001 and WS002 strains
To avoid instability caused by the use of excessive plasmids, trp-biosensors were integrated into the chromosome of trpR gene locus, thereby giving WS001 strain. To this end, plasmid pN20-trpR was constructed from plasmid pTagAmpR (FIG. 8), pTagAmpR plasmid was derived from plasmid pTargetF (Jiang et al, 2015), and spectinomycin resistance was converted to ampicillin resistance, and primers pTargR and TrpR-N20 (Table 2) were used to express gRNA targeting the trpR gene. The donor DNA fragment Trp-Sensor was amplified from plasmid pSentrp (Square et al 2016) using primers Tsen-trpR-IF and Tsen-trpR-IR.
To construct an Aromatic Amino Acid (AAA) -auxotrophic strain (WS 002), the present invention removes the unique DAHP synthase of strain WS001 by replacing aroG S180F gene with antibiotic resistance gene Cm R. In this regard, plasmid pN20-aroG was first constructed from plasmid pTagAmpR using primers pTargR and aroG-N20 (Table 2) for expression of the aroG gene-targeted gRNA. The donor DNA fragment P J23119-rpsL-CmR was amplified from plasmid pJLC using primers up-aroH-out and Cm-delG-R.
Construction of pCm-aroG fbr plasmid library
To construct pCm-aroG (FIG. 8), the present invention uses primers pTargR and Cm-N20 (Table 2) to construct plasmid pN20-Cm R in the same manner as plasmid pN20-aroG was constructed. The DNA fragment V-N20CmR was amplified from plasmid pN20-Cm R using primers pN20VRCm and pN20VFSerA (Table 2). Meanwhile, DNA fragments F-Cm R, F-aroG and F-serA were generated from plasmid pJLC (Table 1), the genomic DNA of E.coli W3110 and plasmid strp015A (Chen and Zeng, 2017), respectively. The primer pairs are Cm-C1/Cm-C2, waroG-FF/aroG-Fus-R and serA-Fus-F/serA-Fus-R, respectively. Then the four fragments (V-N20 CmR, F-Cm R, F-aroG and F-serA) were fused together by using the fusion HD cloning kit (In-Fusion HD Cloning kits)Laboratories, inc.) the final plasmid pCm-aroG WT was constructed. Plasmids pCm-aroG S180F and pCm-aroG fbr were constructed using the corresponding pair of mutagenesis primers (Table 2) to amplify the entire plasmid pCm-aroG WT.
Construction of Strain S028GM1
To replace the promoter of P J23119-rpsL-cmR-aroGD6G-D7A with the same promoter as S028 (P J23119-rpsL-tac-aroGS180F), the same plasmid pN202-Cm R as the other sgRNA plasmids was first constructed, but with primers pTargR and CmR-N202. The donor DNA fragment P J23119-rpsL-tac-aroGD6G-D7A was obtained by two rounds of PCR. The first round was to add the tac promoter to aroG D6G-DA7 gene by amplifying plasmid pET-aroG D6G-DA7 with primers NotI-pTac-aroG and aroG-speI. The second round was by flanking the upstream homology arm with the PCR product of the first round as template using primers u-rpsLp-tac and aroG-fus-R. Plasmid pN202-Cm R and fragment P J23119-rpsL-tac-aroGD6G-D7A were co-transformed with plasmid pCas into strain WS005 to produce strain S028GM1.
2.2.1 Genome editing Using CRISPR/Cas9 technology
For integration of the target donor DNA fragment into the genome, the present invention uses a host strain carrying pCas expression vectors. Transformation was performed by electroporation and was modified slightly according to the protocol reported by Chen and Zeng (2017). Specifically, to prepare electroporation competent cells, overnight cultures (grown at 30 ℃) of strains carrying pCas were inoculated (2%, v/v) into 10mL fresh SOB medium containing 30 μg/mL kanamycin. After growth to an OD600 of about 0.4, the cells were transferred and incubated with shaking at 42 ℃ for 15 minutes, and then immediately placed on ice for 10 minutes. Thereafter, the cells were collected by centrifugation at 4℃and washed three times with pre-chilled 10% glycerol or distilled water. Competent cells were resuspended in 400 μl of pre-chilled 10% glycerol, 200 μl for each reaction. The corresponding sgRNA plasmids eluted in water (plus donor dsDNA if necessary) were mixed with competent cells for transformation. Electroporation was performed in 0.2cm cuvettes at 2.5kV, cells were suspended in 1mL of SOB medium and recovered at 30℃for 2 hours, followed by plating. Plates were incubated at 30℃for more than 24 hours. Transformants were identified by colony PCR and DNA sequencing.
2.4 Construction and screening of AroG mutant libraries
To demonstrate the effectiveness of the methods of the invention, the methods of the invention were applied to engineering anti-Phe AroG. The present study selected two residues (Asp 6 and Asp 7) of the Phe binding site of AroG (PDB ID:1 KFL) for saturation mutagenesis (NNK). Saturation mutagenesis was introduced with primers (Table 2) by amplifying the entire plasmid pCm-aroG WT (Table 1). After digestion of the template DNA with DpnI, the PCR product was transformed into chemically active E.coli Top10 cells, and the reaction product was suspended in SOB medium. After incubation at 37℃for 1 hour, all cells were transferred to 10mL fresh LB medium containing 100. Mu.g/mL ampicillin, and incubated at 37℃for 8-10 hours. Plasmids were extracted from these cultures and eluted in water as AroG mutant libraries: pCm-aroG D6X-D7X (FIG. 8, supra).
The AroG mutant library was transferred to WS002 strain (containing pCas) and after incubation in SOB medium at 30 ℃ for 2h, the cells were washed three times with M9 medium (without any amino acids). Cells were dispersed on M9 agar medium containing 25mM Phe and 0.1mM IPTG for selection. After incubation at 30 ℃ for more than 24 hours, transformants with larger size and stronger fluorescent signal were picked and re-examined by streaking on the same medium. Finally, candidate mutant strains were tested by incubation in 5mL fermentation medium II (FM-II) at 30℃for 24 hours. FM-II is nearly identical to that previously reported (Gu et al 2012) but contains 0.5g/L MgSO 4·7H2 O instead of 5g/L and has an initial glucose concentration of 30g/L. In addition, 12g/L K 2HPO4 (for pH buffering), 25mM Phe and 0.1mM IPTG were added. Mutants that produced stronger Moderate Fluorescence (MFU) were selected for sequencing.
2.5 Method for measuring fluorescence intensity
Mutants of the chromosomal Trp sensor containing the reported eGFP protein cultured in LB medium were collected by centrifugation and washed three times with M9 medium, respectively, to remove LB medium. Then, each mutant was inoculated with the same amount of cells into 10mL of fresh M9 medium containing 25mM Phe in a 50mL conical tube, and after culturing for 10 hours, the cells were subjected to fluorescence analysis by a flow cytometer. For this, each culture was first washed three times with PBS buffer and diluted 100-fold, and then monitored for eGFP fluorescence (MFI. Gtoreq.10000 events) using a flow cytometer (CytoFLEX, beckmann coulter) at an excitation wavelength of 488 nm. All data were processed with Beckman Flow software and positive signals were isolated from instrument and water sample background using electronic gating. For fluorescence intensity, the Moderate Fluorescence Units (MFU) for each culture were calculated.
2.6 Protein expression and purification
For the expression of the proteins, the corresponding plasmids (see section 2.1) were transformed into E.coli BL21, respectively, as host cells. The protein expression and purification procedure was slightly different from that reported previously (Chen et al, 2018). Purified proteins were quantified by pre-analysis in a Bio-Rad laboratory (Heracles, calif.) using bovine serum albumin as a standard according to the Coomassie Brilliant blue method.
2.7 Enzyme assay
The in vitro enzymatic kinetics of AroG fbr mutants were carried out as described in the literature with only minor modifications (Schoner and hermmann, 1976). The enzyme activity was measured by monitoring the disappearance of PEP by absorbance at 232nm, and the specific activity was calculated by measuring absorbance at 232nm in a cuvette according to the standard curve of PEP (not shown). To investigate the effect of Phe on the activity of the AroG mutants, the activity was measured in the presence of different concentrations (from 0 to 40 mM) of Phe. The reaction mixture contained 10mM ditripropane (Bis-tris propane) (BTP, pH 7.0), 50. Mu.M MnSO 4, 600. Mu.M PEP, 500. Mu. M E4P and 25. Mu.g purified enzyme, in a total volume of 0.2mL, placed in a test tube at 25℃with or without inhibitor. The mixture (without PEP and E4P) and the substrate (PEP and E4P) were equilibrated to the reaction temperature, respectively, and the reaction was started by adding the substrate (PEP and E4P).
2.8 Fermentation conditions
For fed-batch fermentation in a bioreactor, pre-culture and seed culture were performed under the same conditions as previously reported (Gu et al 2012). Since the lambda recombination system integrated in the E.coli DY330 strain can grow temperature-sensitive cells at 42℃it is removed from each DY 330-derived strain before use in fermentation. For this purpose, the removal procedure of the lambda recombination system was carried out according to the method reported by Chen and Zeng (2017). The fermentation was performed in a highly instrumented and automated 4-parallel tank 1.5L bioreactor system (DASSIP parallel bioreactor system of You Lixi, germany) with an initial working volume of 500mL. Unless otherwise indicated, the fermentation medium, feed solution and fermentation conditions in the bioreactor were the same as previously reported (Chen and Zeng, 2017).
2.9 Analytical methods
Glucose, 3-dehydroshikimic acid (DSA) and Shikimic Acid (SA) were quantified using high performance liquid chromatography as described previously (Bommareddy et al, 2014; luz et al, 2014). Tryptophan was measured by sensitive spectrophotometry (Nagaraja et al, 2003).
3. Results and discussion
3.1 Proof of concept of the method of the invention (CGSSC method)
To verify the concept, the present invention first constructed a screening strain that contains a tryptophan biosensor and lacks the DAHP synthase activity for screening the DAHP synthase mutants. For this, aroH and aroF genes in the previously developed tryptophan-producing E.coli strain S028λ (Chen and Zeng, 2017) were first knocked out (Table 1). Deletion of aroG S180F gene from this mutant resulted in auxotrophy of the strain for Aromatic Amino Acid (AAA). Thus, the growth of the mutant was related to the DAHP synthase activity at the time of its reintroduction (fig. 3). In principle, an engineered synthetase with higher activity should lead to faster accumulation of Trp, which in turn stimulates Trp biosensor-regulated expression of the reporter gene (Fang et al 2016).
The Trp biosensor consists of tnaC, tnaC encodes the leader sequence of the tnaCAB operon (Bischoff et al, 2014; gong et al, 2001); the eGFP protein was fused upstream of tnaC (Fang et al, 2016). Specifically, after replacing trpR gene with Trp biosensor using CRISP/Cas9 system, strain WS001 was first constructed based on Trp-producing strain S028λc (Chen and Zeng, 2017) (table 1). Then, we removed the unique DAHP synthase of strain WS001 by replacing aroGS F gene with antibiotic resistance gene CmR, which provided an sgRNA target for CRISPR/Cas9 system in further genome editing, yielding strain WS002 (table 1, fig. 3). As expected, strain WS002 was unable to grow in M9 medium without the addition of any of the aromatic amino acids Phe, tyr and Trp (data not shown). Then, it was tested whether the strain having the feedback-resistant DAHP synthase exhibited a different behavior from the strain having the wild-type DAHP synthase in terms of cell growth and reporter gene expression in a specific medium containing a high concentration of the enzyme inhibitor. For this purpose, the present invention uses CRISPR/Cas9 technology to introduce the wild-type aroG gene and the anti-feedback gene aroG S180F (Ger et al, 1994) into the Cm R gene locus on the strain WS002 chromosome using plasmids pCm-aroG WT and pCm-aroG S180F, respectively. The recombinants were plated on M9 agar medium containing 25mM Phe and no Tyr and Trp. The medium was also supplemented with 0.1mM IPTG, one reason for releasing the trp biosynthetic pathway, as it was regulated by LacI modulators, and the other reason for inducing the expression of sgRNA, thereby directing the cleavage of donor plasmid by Cas9, from which the gene of interest can also be expressed.
The results showed that many recombinants with strong fluorescent signals were grown under the above conditions after the aroGS F gene was introduced into the host (FIG. 4). Several colonies were selected for further identification in the present invention. These colonies all demonstrated the same mutation S180F. These recombinants were named WS004. Under the same conditions, no colonies of the host integrated with plasmid pCm-aroG WT were observed (fig. 4). It is speculated that the activity of wild-type AroG is severely inhibited by Phe and cannot support cell growth. However, it is also possible that the efficiency of the recombinant engineering is too low. To exclude the latter possibility, recombinants carrying aroG WT gene were also grown on LB agar medium containing IPTG. From the complex medium, a plurality of colonies (FIG. 4) were obtained, which were confirmed to be positive by colony PCR and designated WS003. Strains WS003 and WS004 were re-examined on M9 agar medium with and without 25mM Phe (FIG. 4). Strain WS003 was found to grow on media without Phe, but no growth was observed on media with Phe (fig. 4). As expected, the growth of strain WS004 did not show significant differences in medium with and without Phe. These results indicate that CGSSC is a method that helps to engineer enzymes with the desired properties (e.g., higher activity and higher inhibitor tolerance). CGSSC was used hereinafter to obtain AroG mutants with higher tolerance to Phe.
3.2CGSSC use in screening for Phe-resistant AroG
To demonstrate the effectiveness of the CGSSC method established above in obtaining more resistant AroG enzyme mutants, a library of AroG mutations was first generated.
For this purpose, a semi-rational strategy was employed, which exploits information from the crystal structure of AroG complexed with the inhibitor Phe (PDB: 1 KFL) (fig. 5 a). Residues D6 and D7 involved in Phe binding were selected as targets for saturation mutagenesis. Then, CGSSC was used for screening. As shown in FIG. 5b, for the AroG D6X-D7X mutant, about 100 colonies of different sizes and different fluorescent signal intensities were obtained on the aforementioned screening medium after about 30 hours of growth.
After the first round of screening, 30 AroG D6X-D7X mutant colonies were screened for relatively large volumes and high fluorescent signal intensity and re-identified on the screening medium. After confirming the phenotype, the mutated aroG gene was isolated from the 20 candidate genes for sequencing. Sequencing results showed that only 6 different AroG mutants (table 3), aroG D6G-D7A、AroGD6L-D7P、AroGD6P-D7I、AroGD6F-D7V、AroGD6V-D7C and AroG D6F-D7L, were present in the 20 candidate genes at 7, 6, 4, 1 and 1 (table 3), respectively. Then, fermentation was performed with the strains carrying these 6 recombinants in 50mL conical tubes containing FM-II medium, and they were compared with wild-type strain WS003 and strain WS004 with AroG S180F mutant. Strains carrying mutants AroG D6G-D7A、AroGD6L-D7P or AroG D6P-D7I, which appeared more frequently in the 20 candidate strains (table 3), were found to also have higher Trp productivity (fig. 3, see also fig. 9 for sequences). In particular, the yields of the first two mutants were much higher than the reference strain (AroG S180F). In addition, the relationship between tryptophan productivity and fluorescence signal intensity was also studied. Strains with stronger fluorescent signals also have higher tryptophan productivity (fig. 3). These results indicate that under the test conditions, the mutants AroG D6G-D7A、AroGD6L-D7V and AroG D6P-D7I have higher inhibitor tolerance than the mutant AroG S180F. To provide more direct evidence, enzyme assays were performed with purified proteins of these mutants.
TABLE 3 comparison of fermentation results of E.coli strains containing AroG WT、AroGS180F and AroG fbr mutants cultured on FM-II containing 25mM Phe DCW: dry cell weight
a The number of mutants refers to all 20 candidate genes tested; n.d. indicates no detection. Mean ± standard deviation was based on three independent experiments.
3.3 In vitro characterization of selected AroGfbr mutants
To examine whether the higher Trp productivity and stronger fluorescent signal observed in the strain was due to an increase in Phe tolerance of the corresponding AroG mutant, we studied the inhibitory behavior of mutants AroG D6G-D7A、AroGD6L-D7P and AroG D6P-D7I.
As shown in fig. 6, all mutants were significantly less sensitive to the inhibitor Phe, whereas the wild-type AroG was extremely sensitive. In the presence of 0.5mM Phe, the wild-type AroG was almost completely inactive, while all mutants remained more than 80% active under the same conditions. When the Phe concentration was higher than 10mM, both the three mutants AroG D6G-D7A、AroGD6L-D7P and AroG D6P-D7I were less sensitive to Phe than AroG S180F (FIG. 6 b). In the presence of more than 20mM Phe, they also have higher specific activity (FIG. 6 a). These results explain why strains with these three mutants exhibited better yields of Trp than the positive control when cultured in medium containing very high Phe concentrations (table 3). As shown in fig. 6a, the specific activity differs significantly between the three mutants resulting from the substitution of identical residues D6 and D7. Among them, aroG D6G-D7A mutant had the highest specific activity, which was almost twice as high as that of mutant AroG D6P-D7I regardless of Phe concentration. And also significantly higher than the positive control AroG S180F.
3.4 Improvements in the chorismate pathway and tryptophan biosynthesis
To explore the effect of the optimal mutant AroG D6G-D7A on the development of the aromatic amino acid biosynthetic strain, mutant AroG S180F in the previously constructed Trp-producing strain S028 was replaced with mutant AroG D6G-D7A, resulting in strain S028GM1 (see above). The tryptophan-producing capacity of the S028GM1 strain and the reference strain S028 was compared by performing a simple fed-batch fermentation in a bioreactor.
As shown in fig. 7a and 7c, strain S028GM1 produced significantly higher Trp than the reference strain during the delay period (about 10 h) to the end of fermentation. At the end of the fermentation (37 h), strain S028GM1 produced Trp 24.03.+ -. 1.02g/L, 38.50% higher than strain S028 (17.35.+ -. 1.16 g/L) (FIG. 7 c). The glucose concentration of both strains was controlled at nearly the same level throughout the fermentation. It was found that at the beginning of the exponential growth phase, the growth rate of strain S028GM1 (0.211 h 1) was slightly faster than that of strain S028 (0.184 h-1, FIG. 7 a). Enhancement of DAHP synthase activity clearly promotes an increase in growth rate to some extent. Higher biomass formation rates could reasonably allow higher productivity for strain S028GM1 (fig. 7 d). However, the major factor in the increase in Trp production was the direct enhancement of DAHP synthase activity, since the Trp formation rate of strain S028GM1 was significantly higher than that of strain S028 throughout the fermentation (fig. 7 e). Furthermore, during fermentation, strain S028GM1 accumulated higher amounts of intermediates SA and DSA of the chorismate pathway than the reference strain S028 (fig. 7 f). At the end of the fermentation, strain S028GM1 accumulated about 36% more of both intermediates than was produced by strain S028. This suggests that due to the difference between mutants AroG D6G-D7A and AroG S180F, more metabolic flux was redirected to the branch pathway in strain S028GM1 than in the reference strain. These results clearly demonstrate that mutant AroG D6G-D7A is more effective in biologically producing the branching pathway of aromatic amino acids and derivatives thereof.
4. Conclusion(s)
It has been shown that the method of the invention combines CRISPR/Cas 9-promoted engineering with growth coupling and sensor-guided in vivo screening and characterization (CGSSC), particularly for engineering and screening of enzyme mutants. Using methods of engineering and screening for 3-deoxy-D-arabinoheptulose 7-phosphate synthase (AroG), aroG mutants can be identified that are more resistant to Phe and thus more suitable for the biosynthesis of aromatic amino acids such as Trp. Based on the two mutation points selected by the structural information, the new mutants (AroG D6G-D7A、AroGD6L-D7P and AroG D6P-D7I) were found to be more resistant to Phe than the Phe resistant mutant AroG S180F reported in the literature. In the previously engineered tryptophan-producing E.coli strain (S028), replacement of AroG S180F with AroG D6G-D7A significantly increased tryptophan production by 38.05% in a simple fed-batch fermentation. Since the method of the invention is based on integrating the gene of an enzyme mutant into the chromosome, e.g. the above-mentioned gene mutant encoding AroG, it can also be used to optimize the expression level of an engineered enzyme in a strain, i.e. by constructing the corresponding gene using different promoters and/or ribosome binding sites. It is worth mentioning that CRISPR/Cas9 technology is efficient and allows multiple genome editing. Thus, the methods of the invention can be used to address multiple targets that require simultaneous modulation of multiple genes, for example for synthesis of metabolites that require multiple precursors, such as tryptophan.
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Sequence listing
<110> University of hamburger industry
<120> Novel DAHP synthase
<130> PAT 1716 WO
<150> LU101172
<151> 2019-04-10
<150> DE102019109423.2
<151> 2019-04-10
<160> 35
<170> BiSSAP 1.3.6
<210> 1
<211> 350
<212> PRT
<213> Artificial sequence
<220>
<223> AroG mutant conventional sequence
<220>
<221> Site
<222> 6..7
<223> Xaa = any amino acid, but not both at the same time Asp
<400> 1
Met Asn Tyr Gln Asn Xaa Xaa Leu Arg Ile Lys Glu Ile Lys Glu Leu
1 5 10 15
Leu Pro Pro Val Ala Leu Leu Glu Lys Phe Pro Ala Thr Glu Asn Ala
20 25 30
Ala Asn Thr Val Ala His Ala Arg Lys Ala Ile His Lys Ile Leu Lys
35 40 45
Gly Asn Asp Asp Arg Leu Leu Val Val Ile Gly Pro Cys Ser Ile His
50 55 60
Asp Pro Val Ala Ala Lys Glu Tyr Ala Thr Arg Leu Leu Ala Leu Arg
65 70 75 80
Glu Glu Leu Lys Asp Glu Leu Glu Ile Val Met Arg Val Tyr Phe Glu
85 90 95
Lys Pro Arg Thr Thr Val Gly Trp Lys Gly Leu Ile Asn Asp Pro His
100 105 110
Met Asp Asn Ser Phe Gln Ile Asn Asp Gly Leu Arg Ile Ala Arg Lys
115 120 125
Leu Leu Leu Asp Ile Asn Asp Ser Gly Leu Pro Ala Ala Gly Glu Phe
130 135 140
Leu Asp Met Ile Thr Pro Gln Tyr Leu Ala Asp Leu Met Ser Trp Gly
145 150 155 160
Ala Ile Gly Ala Arg Thr Thr Glu Ser Gln Val His Arg Glu Leu Ala
165 170 175
Ser Gly Leu Ser Cys Pro Val Gly Phe Lys Asn Gly Thr Asp Gly Thr
180 185 190
Ile Lys Val Ala Ile Asp Ala Ile Asn Ala Ala Gly Ala Pro His Cys
195 200 205
Phe Leu Ser Val Thr Lys Trp Gly His Ser Ala Ile Val Asn Thr Ser
210 215 220
Gly Asn Gly Asp Cys His Ile Ile Leu Arg Gly Gly Lys Glu Pro Asn
225 230 235 240
Tyr Ser Ala Lys His Val Ala Glu Val Lys Glu Gly Leu Asn Lys Ala
245 250 255
Gly Leu Pro Ala Gln Val Met Ile Asp Phe Ser His Ala Asn Ser Ser
260 265 270
Lys Gln Phe Lys Lys Gln Met Asp Val Cys Ala Asp Val Cys Gln Gln
275 280 285
Ile Ala Gly Gly Glu Lys Ala Ile Ile Gly Val Met Val Glu Ser His
290 295 300
Leu Val Glu Gly Asn Gln Ser Leu Glu Ser Gly Glu Pro Leu Ala Tyr
305 310 315 320
Gly Lys Ser Ile Thr Asp Ala Cys Ile Gly Trp Glu Asp Thr Asp Ala
325 330 335
Leu Leu Arg Gln Leu Ala Asn Ala Val Lys Ala Arg Arg Gly
340 345 350
<210> 2
<211> 350
<212> PRT
<213> Artificial sequence
<220>
<223> AroG mutant D6G-D7A
<400> 2
Met Asn Tyr Gln Asn Gly Ala Leu Arg Ile Lys Glu Ile Lys Glu Leu
1 5 10 15
Leu Pro Pro Val Ala Leu Leu Glu Lys Phe Pro Ala Thr Glu Asn Ala
20 25 30
Ala Asn Thr Val Ala His Ala Arg Lys Ala Ile His Lys Ile Leu Lys
35 40 45
Gly Asn Asp Asp Arg Leu Leu Val Val Ile Gly Pro Cys Ser Ile His
50 55 60
Asp Pro Val Ala Ala Lys Glu Tyr Ala Thr Arg Leu Leu Ala Leu Arg
65 70 75 80
Glu Glu Leu Lys Asp Glu Leu Glu Ile Val Met Arg Val Tyr Phe Glu
85 90 95
Lys Pro Arg Thr Thr Val Gly Trp Lys Gly Leu Ile Asn Asp Pro His
100 105 110
Met Asp Asn Ser Phe Gln Ile Asn Asp Gly Leu Arg Ile Ala Arg Lys
115 120 125
Leu Leu Leu Asp Ile Asn Asp Ser Gly Leu Pro Ala Ala Gly Glu Phe
130 135 140
Leu Asp Met Ile Thr Pro Gln Tyr Leu Ala Asp Leu Met Ser Trp Gly
145 150 155 160
Ala Ile Gly Ala Arg Thr Thr Glu Ser Gln Val His Arg Glu Leu Ala
165 170 175
Ser Gly Leu Ser Cys Pro Val Gly Phe Lys Asn Gly Thr Asp Gly Thr
180 185 190
Ile Lys Val Ala Ile Asp Ala Ile Asn Ala Ala Gly Ala Pro His Cys
195 200 205
Phe Leu Ser Val Thr Lys Trp Gly His Ser Ala Ile Val Asn Thr Ser
210 215 220
Gly Asn Gly Asp Cys His Ile Ile Leu Arg Gly Gly Lys Glu Pro Asn
225 230 235 240
Tyr Ser Ala Lys His Val Ala Glu Val Lys Glu Gly Leu Asn Lys Ala
245 250 255
Gly Leu Pro Ala Gln Val Met Ile Asp Phe Ser His Ala Asn Ser Ser
260 265 270
Lys Gln Phe Lys Lys Gln Met Asp Val Cys Ala Asp Val Cys Gln Gln
275 280 285
Ile Ala Gly Gly Glu Lys Ala Ile Ile Gly Val Met Val Glu Ser His
290 295 300
Leu Val Glu Gly Asn Gln Ser Leu Glu Ser Gly Glu Pro Leu Ala Tyr
305 310 315 320
Gly Lys Ser Ile Thr Asp Ala Cys Ile Gly Trp Glu Asp Thr Asp Ala
325 330 335
Leu Leu Arg Gln Leu Ala Asn Ala Val Lys Ala Arg Arg Gly
340 345 350
<210> 3
<211> 350
<212> PRT
<213> Artificial sequence
<220>
<223> AroG mutant D6L-D7P
<400> 3
Met Asn Tyr Gln Asn Leu Pro Leu Arg Ile Lys Glu Ile Lys Glu Leu
1 5 10 15
Leu Pro Pro Val Ala Leu Leu Glu Lys Phe Pro Ala Thr Glu Asn Ala
20 25 30
Ala Asn Thr Val Ala His Ala Arg Lys Ala Ile His Lys Ile Leu Lys
35 40 45
Gly Asn Asp Asp Arg Leu Leu Val Val Ile Gly Pro Cys Ser Ile His
50 55 60
Asp Pro Val Ala Ala Lys Glu Tyr Ala Thr Arg Leu Leu Ala Leu Arg
65 70 75 80
Glu Glu Leu Lys Asp Glu Leu Glu Ile Val Met Arg Val Tyr Phe Glu
85 90 95
Lys Pro Arg Thr Thr Val Gly Trp Lys Gly Leu Ile Asn Asp Pro His
100 105 110
Met Asp Asn Ser Phe Gln Ile Asn Asp Gly Leu Arg Ile Ala Arg Lys
115 120 125
Leu Leu Leu Asp Ile Asn Asp Ser Gly Leu Pro Ala Ala Gly Glu Phe
130 135 140
Leu Asp Met Ile Thr Pro Gln Tyr Leu Ala Asp Leu Met Ser Trp Gly
145 150 155 160
Ala Ile Gly Ala Arg Thr Thr Glu Ser Gln Val His Arg Glu Leu Ala
165 170 175
Ser Gly Leu Ser Cys Pro Val Gly Phe Lys Asn Gly Thr Asp Gly Thr
180 185 190
Ile Lys Val Ala Ile Asp Ala Ile Asn Ala Ala Gly Ala Pro His Cys
195 200 205
Phe Leu Ser Val Thr Lys Trp Gly His Ser Ala Ile Val Asn Thr Ser
210 215 220
Gly Asn Gly Asp Cys His Ile Ile Leu Arg Gly Gly Lys Glu Pro Asn
225 230 235 240
Tyr Ser Ala Lys His Val Ala Glu Val Lys Glu Gly Leu Asn Lys Ala
245 250 255
Gly Leu Pro Ala Gln Val Met Ile Asp Phe Ser His Ala Asn Ser Ser
260 265 270
Lys Gln Phe Lys Lys Gln Met Asp Val Cys Ala Asp Val Cys Gln Gln
275 280 285
Ile Ala Gly Gly Glu Lys Ala Ile Ile Gly Val Met Val Glu Ser His
290 295 300
Leu Val Glu Gly Asn Gln Ser Leu Glu Ser Gly Glu Pro Leu Ala Tyr
305 310 315 320
Gly Lys Ser Ile Thr Asp Ala Cys Ile Gly Trp Glu Asp Thr Asp Ala
325 330 335
Leu Leu Arg Gln Leu Ala Asn Ala Val Lys Ala Arg Arg Gly
340 345 350
<210> 4
<211> 350
<212> PRT
<213> Artificial sequence
<220>
<223> AroG mutant D6P-D7I
<400> 4
Met Asn Tyr Gln Asn Pro Ile Leu Arg Ile Lys Glu Ile Lys Glu Leu
1 5 10 15
Leu Pro Pro Val Ala Leu Leu Glu Lys Phe Pro Ala Thr Glu Asn Ala
20 25 30
Ala Asn Thr Val Ala His Ala Arg Lys Ala Ile His Lys Ile Leu Lys
35 40 45
Gly Asn Asp Asp Arg Leu Leu Val Val Ile Gly Pro Cys Ser Ile His
50 55 60
Asp Pro Val Ala Ala Lys Glu Tyr Ala Thr Arg Leu Leu Ala Leu Arg
65 70 75 80
Glu Glu Leu Lys Asp Glu Leu Glu Ile Val Met Arg Val Tyr Phe Glu
85 90 95
Lys Pro Arg Thr Thr Val Gly Trp Lys Gly Leu Ile Asn Asp Pro His
100 105 110
Met Asp Asn Ser Phe Gln Ile Asn Asp Gly Leu Arg Ile Ala Arg Lys
115 120 125
Leu Leu Leu Asp Ile Asn Asp Ser Gly Leu Pro Ala Ala Gly Glu Phe
130 135 140
Leu Asp Met Ile Thr Pro Gln Tyr Leu Ala Asp Leu Met Ser Trp Gly
145 150 155 160
Ala Ile Gly Ala Arg Thr Thr Glu Ser Gln Val His Arg Glu Leu Ala
165 170 175
Ser Gly Leu Ser Cys Pro Val Gly Phe Lys Asn Gly Thr Asp Gly Thr
180 185 190
Ile Lys Val Ala Ile Asp Ala Ile Asn Ala Ala Gly Ala Pro His Cys
195 200 205
Phe Leu Ser Val Thr Lys Trp Gly His Ser Ala Ile Val Asn Thr Ser
210 215 220
Gly Asn Gly Asp Cys His Ile Ile Leu Arg Gly Gly Lys Glu Pro Asn
225 230 235 240
Tyr Ser Ala Lys His Val Ala Glu Val Lys Glu Gly Leu Asn Lys Ala
245 250 255
Gly Leu Pro Ala Gln Val Met Ile Asp Phe Ser His Ala Asn Ser Ser
260 265 270
Lys Gln Phe Lys Lys Gln Met Asp Val Cys Ala Asp Val Cys Gln Gln
275 280 285
Ile Ala Gly Gly Glu Lys Ala Ile Ile Gly Val Met Val Glu Ser His
290 295 300
Leu Val Glu Gly Asn Gln Ser Leu Glu Ser Gly Glu Pro Leu Ala Tyr
305 310 315 320
Gly Lys Ser Ile Thr Asp Ala Cys Ile Gly Trp Glu Asp Thr Asp Ala
325 330 335
Leu Leu Arg Gln Leu Ala Asn Ala Val Lys Ala Arg Arg Gly
340 345 350
<210> 5
<211> 36
<212> DNA
<213> Artificial sequence
<220>
<223> Primer pTargR
<400> 5
actagtatta tacctaggac tgagctagct gtcaag 36
<210> 6
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> Primer AroG-N20
<400> 6
gctcagtcct aggtataata ctagtcagga agcagtgcgg cgcacgtttt agagctagaa 60
atagcaagtt aaaataaggc tagtccg 87
<210> 7
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> Primer TrpR-N20
<400> 7
gctcagtcct aggtataata ctagttcagg tttaacaacg gtaaagtttt agagctagaa 60
atagcaagtt aaaataaggc tagtccg 87
<210> 8
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> Primer Cm-N20
<400> 8
gctcagtcct aggtataata ctagttgatg aacctgaatc gccaggtttt agagctagaa 60
atagcaagtt aaaataaggc tagtccg 87
<210> 9
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> Primer Cm-N202
<400> 9
gctcagtcct aggtataata ctagtgctga tgccgctggc gattcgtttt agagctagaa 60
atagcaagtt aaaataaggc tagtccg 87
<210> 10
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223> Primer serA-Fus-F
<400> 10
cctacggtaa gagcatcacc gatgcc 26
<210> 11
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> Primer serA-Fus-R
<400> 11
gcccaattgc gtaccaatat gaccg 25
<210> 12
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> Primer Cm-C1
<400> 12
gatataccac cgttgatata tcccaatggc 30
<210> 13
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> Primer Cm-C2
<400> 13
ttacatcagc accttgtcgc cttgc 25
<210> 14
<211> 36
<212> DNA
<213> Artificial sequence
<220>
<223> Primer pN20VRCm
<400> 14
caacggtggt atataaaaaa gcaccgactc ggtgcc 36
<210> 15
<211> 39
<212> DNA
<213> Artificial sequence
<220>
<223> Primer pN20VFSerA
<400> 15
ggtacgcaat tgggctcgag gtgaagacga aagggcctc 39
<210> 16
<211> 53
<212> DNA
<213> Artificial sequence
<220>
<223> Primer WaroG-FF
<400> 16
ggcgacaagg tgctgatgta atattgcatt cactaagata agtatggcaa cac 53
<210> 17
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223> Primer aroG-fus-R
<400> 17
ggcatcggtg atgctcttac cgtagg 26
<210> 18
<211> 71
<212> DNA
<213> Artificial sequence
<220>
<223> Primer D67X-F
<220>
<221> misc_feature
<222> 25..26
<223 >/Note= "n=a, C, G or T"
<220>
<221> misc_feature
<222> 28..29
<223 >/Note= "n=a, C, G or T"
<400> 18
ggaacagaca tgaattatca gaacnnknnk ttacgcatca aagaaatcaa agagttactt 60
cctcctgtcg c 71
<210> 19
<211> 33
<212> DNA
<213> Artificial sequence
<220>
<223> Primer D67X-R
<400> 19
gttctgataa ttcatgtctg ttccagtgtt gcc 33
<210> 20
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> Primer aroG_S180F_F
<400> 20
gcatcagggc ttttttgtcc ggtcggcttc 30
<210> 21
<211> 30
<212> DNA
<213> Artificial sequence
<220>
<223> Primer aroG_S180F_R
<400> 21
gaagccgacc ggacaaaaaa gccctgatgc 30
<210> 22
<211> 23
<212> DNA
<213> Artificial sequence
<220>
<223> Primer pTagC1
<400> 22
ttgagtgagc tgataccgct cgc 23
<210> 23
<211> 21
<212> DNA
<213> Artificial sequence
<220>
<223> Primer XbaI-serA
<400> 23
gagcggataa caattcccct c 21
<210> 24
<211> 62
<212> DNA
<213> Artificial sequence
<220>
<223> Primer aroG-His-HindIII
<400> 24
cgccggaagc tttcattagt ggtggtggtg gtggtggccc gcgacgcgct tttactgcat 60
tc 62
<210> 25
<211> 32
<212> DNA
<213> Artificial sequence
<220>
<223> Primer D67M-F
<400> 25
ttacgcatca aagaaatcaa agacttactt cc 32
<210> 26
<211> 57
<212> DNA
<213> Artificial sequence
<220>
<223> Primer D67M-LPR
<400> 26
ctctttgatt tctttgatgc gtaaaggcaa gttctgataa ttcatatgta tatctcc 57
<210> 27
<211> 57
<212> DNA
<213> Artificial sequence
<220>
<223> Primer D67M-PIR
<400> 27
ctctttgatt tctttgatgc gtaaaatagg gttctgataa ttcatatgta tatctcc 57
<210> 28
<211> 57
<212> DNA
<213> Artificial sequence
<220>
<223> Primer D67M-GAR
<400> 28
ctctttgatt tctttgatgc gtaaagcccc gttctgataa ttcatatgta tatctcc 57
<210> 29
<211> 84
<212> DNA
<213> Artificial sequence
<220>
<223> Primer Tsen-trpR-IF
<400> 29
ggataaaccg acgttgatga gcgccacgga atggggacgt cgttactgat ccgcacggct 60
gttgacaatt aatcatccgg ctcg 84
<210> 30
<211> 87
<212> DNA
<213> Artificial sequence
<220>
<223> Primer Tsen-trpR-IR
<400> 30
tcaatcgctt ttcagcaaca cctcttccag ccactggcgc agctcgacgg gcgcggcttt 60
cttttacttg tacagctcgt ccatgcc 87
<210> 31
<211> 80
<212> DNA
<213> Artificial sequence
<220>
<223> Primer up-aroH-out
<400> 31
ggggcgttgg tgtaaagatt attgccctca ccctgtacgg gtgagggcgt agagagatta 60
cgcggccgct tctagagttg 80
<210> 32
<211> 72
<212> DNA
<213> Artificial sequence
<220>
<223> Primer Cm-delG-R
<400> 32
gtatcttccc agcctatgca ggcatcggtg atgctcttac cgtaggccag cacctgaagt 60
cagccccata cg 72
<210> 33
<211> 70
<212> DNA
<213> Artificial sequence
<220>
<223> Primer NotI-pTac-aroG
<400> 33
agtgcagcgg ccgctgttga caattaatca tcggctcgta taatgtgtag gggaattgtg 60
agcggataac 70
<210> 34
<211> 36
<212> DNA
<213> Artificial sequence
<220>
<223> Primer aroG-speI
<400> 34
tgcggcacta gtttattacc cgcgacgcgc ttttac 36
<210> 35
<211> 85
<212> DNA
<213> Artificial sequence
<220>
<223> Primer u-rpsLp-tac
<400> 35
ttgtgtgagg acgttttatt acgtgtttac gaagcaaaag ctaaaaccag gagctattta 60
ctgttgacaa ttaatcatcg gctcg 85

Claims (5)

1. A DAHP synthetase is any one of the sequences of SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4.
2. A bacterial cell genetically engineered to express the DAHP synthase of claim 1.
3. The bacterial cell of claim 2, wherein the bacterial cell is an e.
4. Use of a bacterial cell according to claim 2 or 3 for the production of tryptophan.
5. A method of biosynthesis of tryptophan comprising the step of culturing the bacterial cell of claim 2 or 3 in a medium and under conditions suitable for culturing the bacterial cell.
CN202080027938.6A 2019-04-10 2020-04-09 Novel DAHP synthetase Active CN113677795B (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
LU101172A LU101172B1 (en) 2019-04-10 2019-04-10 Method for in vivo screening of engineered enzyme variants
LULU101172 2019-04-10
DE102019109423.2 2019-04-10
DE102019109423 2019-04-10
PCT/EP2020/060260 WO2020208191A1 (en) 2019-04-10 2020-04-09 Novel dhap synthase

Publications (2)

Publication Number Publication Date
CN113677795A CN113677795A (en) 2021-11-19
CN113677795B true CN113677795B (en) 2024-04-23

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Country Status (3)

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