WO2020208191A1 - Novel dhap synthase - Google Patents

Abstract

The invention provides a mutated DHPA synthase that is less feedback-inhibited by phenylalanine than the wildtype enzyme and useful in the biotechnological production of aromatic amino acids like tryptophan.

Description

NOVEL DAHP SYNTHASE

The invention relates to a novel DAHP synthase. Further, the invention relates to a method for in vivo screening of engineered enzyme variants.

Microbes have been extensively engineered to produce value-added compounds from renewable feedstock (Becker et al, 2015; Lee and Kim, 2015; Liao et ak, 2016). For this purpose, the engineering of biosynthetic pathways plays a crucial role. Several strategies based on the adjustment of gene expression and enzyme concentrations have been developed (Alper et al., 2005; Blazeck and Alper, 2013; Hwang et al., 2018; Zhou and Zeng, 2015). However, they are not able to overcome some inherent limitations associated with enzymes themselves. For instance, regulation mechanisms of enzyme activities such as feed-back or feed-forward inhibitions (Chen et al., 2018; Zurawski et al., 1981) restrict the specific activity or substrate specificity of enzymes (Mora- Villalobos and Zeng, 2017). In this regard, the construction of an efficient pathway inevitably requires protein engineering (Chen et al., 2013; Chen et al., 2011b; Ger et al., 1994; Lin et al., 2012).

Normally, protein engineering consists of three distinct steps: (i) construction of a gene variant library; (ii) screening of the library and (iii) further characterization of candidate enzyme variants (Boville et al., 2018; Buller et al., 2018) (Fig. lb). It has been widely and successfully used for improving the performance of many enzymes (Chen et al., 201 la; Rees et al., 2017;

Sun et al., 2016). However, due to several limiting factors, mutant strains harboring enzyme variants obtained from conventional protein engineering approaches may be phenotypically hardly distinguishable, making screening and characterization of desired enzyme variants not only rather challenging and time consuming (Ren et al., 2018), but also make it hard to identify the best performer among apparently improved enzyme variants (Ren et al., 2015). Further, the subsequent in vitro and in vivo characterizations of the best performer identified from screening may not be relevant to a real bioproduction process using the host microorganism. For these reasons, it is of great interest to have a method for identifying enzyme variants having improved properties and being suitable for a real bioproduction process. Recently, the CR1SPR/Cas9 technology for genome editing has made great advances and received large attention (Cho et al., 2018; Donohoue et al., 2017; Jakociunas et al., 2015; Zhang et al., 2018). Among others, it is applied for the engineering of microbial production strains (Jakociunas et al., 2015; Jiang et al., 2015; Schuster et al., 2018). Due to its simplicity and efficiency, CRISPR/Cas9 is a suitable genome editing tool for quickly and effectively integrating gene variants of a target enzyme into the chromosome of a production strain (Guo et al., 2018).

L-Tryptophan is an essential amino acid that is widely used in medicine, food industry, animal feed industry (Becker et al., 2015), and also as a precursor for other products (Huccetogullari et al., 2019; Noda and Kondo, 2017; Vargas-Tah et al., 2015; Wendisch, 2014). It is therefore of great interest to be able to produce Tryptophan in a simple and cost-efficient manner.

Biotechnological production of Tryptophan via fermentation, for example with Escherichia coli ( E . coli), has become an important source for this amino acid. However, there is still a need for optimizing biosynthesis of aromatic amino acids like tryptophan.

It is an object of the invention to provide a means for improving the biotechnological production of aromatic amino acids, in particular tryptophan.

In order to solve the object, the invention provides a mutated DAHP synthase (AroG) variant, having the sequence of SEQ ID NO: 1, with the proviso that the amino acids at positions 6 and 7 are not both aspartic acid.

The DAHP synthase of the invention is not or significantly less feedback-inhibited by phenylalanine than the wildtype enzyme and exhibits a higher specific enzyme activity than that of a reference variant AroG^sl80F known from the prior art in the presence of 40 mM Phe.

The term“engineered enzyme variants” means enzymes the amino acid sequences of which have been biotechnologically altered in comparison to the wild type enzyme. In an engineered enzyme variant, one or more amino acids at given positions in the wild type enzyme may, for example, purposefully or randomly be replaced by other amino acids. The term“mutated enzymes” may also be used for such enzyme variants. The term“auxotroph for an organic compound” relates to the inability of a cell or organism to synthesize a particular organic compound required for its growth.

The term“recombineering system” (also“recombination system”) relates to the component or the components of a homologous recombination systems, i.e. a system for recombination- mediated genetic engineering, which are necessary for homologous recombination in vivo. An example of a recombineering system is the lamda-red (l-red) recombineering system. The lamda-red recombineering system consists of three components necessary for homologous recombination of dsDNA, namely the proteins Exo, Beta and Gam. Another example for a recombineering system is the Rec E/T system, composed of the proteins Rec E and Rec T.

The term“expressible” in relation to a coding DNA sequence means that the DNA sequence can be transcribed under the control of an inducible or constitutive promoter into an RNA transcript and translated into the corresponding protein product. In relation to a recombineering system or a gene, for example, this means that the components of the recombineering system or the gene can be transcribed under the control of an inducible or constitutive promoter into an RNA transcript and translated into the corresponding protein components of the recombineering system or the gene. In relation to a noncoding DNA sequence the term is to be understood as meaning the formation of an RNA transcript from the DNA sequence under control of an inducible or 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 the transcription of which leads to a RNA transcript being a CRISPR guide RNA.

The term“genetically engineered to synthesize a reporter molecule in the presence of the organic compound” means that a reporter molecule is synthesized in the presence of the organic compound. A“reporter molecule” is a molecule that can easily be detected, e.g. visualized. A reporter molecule can, for example, be a fluorescent molecule. The synthesis of the reporter molecule may directly or indirectly depend on the presence of the organic compound. An example of a reporter molecule is green fluorescent protein (GFP) or variants thereof, e.g. eGFP. The term“Cas protein” refers to a CRISPR associated protein. CRISPR is an abbreviation for the term“Clustered Regularly Interspaced Short Palindromic Repeats” used to denote sections of prokaryotic DNA containing palindromic repeat sequences, interspaced by segments of variable spacer DNA. The term encompasses a family of proteins being part of the CRISPR- Cas system, which is divided in two classes (class 1 and 2) and several types (I to VI) and subtypes. Class 1 encompasses types I, III, and IV, class 2 types II, V, and VI. The Cas types are often characterized by a signature protein. Cas9, for example, is a signature protein for Cas type II. See, e.g., Makarova et al. 2015; Koonin et al. 2017; Haft et al. 2005, Wright et al. 2016. Cas proteins have nuclease activity and use CRISPR RNAs (crRNAs) to guide a Cas nuclease component to the target nucleic acid molecule to be cleaved.

The term“Cas9 protein” or„Cas9“ refers to the CRISPR associated protein 9. Cas9 is an RNA- guided DNA endonuclease enzyme, e.g. from Streptococcus pyogenes. The term„Cas9“ as used herein also encompasses all Cas9 orthologs and also recombinant, i.e. engineered variants thereof. The Cas9 nuclease is active when it forms a complex with a guide RNA, which can be composed of two separate RNA molecules, the tracrRNA and the crRNA, or a single RNA molecule, consisting of tracrRNA and crRNA fused together. Part of the crRNA sequence defines the specificity of the nuclease by complementary base pairing with the target DNA sequence. By specifying the targeting sequence of the crRNA it is possible to direct the

CRISPR-Cas9 system to the appropriate target site (“protospacer”). A further requirement for Cas9-mediated DNA targeting is the presence of a short (e.g. 2-6 base pairs) and conserved protospacer adjacent motif (PAM) in the immediate vicinity of the target site, e.g. immediately downstream of the target site. The PAM sequence differs for different Cas proteins and can also be adapted by engineering the Cas protein.

The term“crRNA” refers to CRISPR RNA, and refers to an RNA molecule being able to complement with a tracrRNA. The crRNA confers target specificity to a Cas protein, e.g. Cas9. The RNA:RNA duplex composed of crRNA and tracrRNA, which may be fused together to a single RNA molecule, is also called guide RNA (gRNA) and binds to the Cas protein. In bacterial CRISPR loci, the crRNA is found in the CRISPR repeat/spacer section and consists of the spacer, which is complementary to the target, and the repeat that complements with the tracrRNA. The term“tracrRNA” refers to trans-activating RNA, a small trans-encoded RNA that is partially complementary to and base pairs with crRNA, thus forming an crRNA:tracrRNA duplex.

The term“CRISPR guide RNA”, also“CRISPR gRNA” or“gRNA”, refers to a

crRNA:tracrRNA duplex. crRNA and tracrRNA may or may not be fused to a single RNA molecule. The term“CRISPR guide RNA” thus encompasses the term“CRISPR single guide RNA” (sgRNA).

The terms“base-pair” or“hybridize” relate to the formation of a duplex by base pairing between complementary nucleic acid molecules, e.g. between two RNA or DNA molecules or an RNA and a DNA molecule.

The term“genomic DNA” is used herein synonymously to chromosomal DNA and does not refer to extrachromosomal DNA like plasmid DNA. The term“genome” as used herein does also refer to genomic genetic material, i.e. genetic material on the chromosome and not to genetic material on plasmids, for example.

The term“growing the cell, in the absence of the organic compound, in a growth medium suitable for growing the cell” means growing the cell in a solid, liquid or semi-solid medium lacking the organic compound, but containing nutrients supporting growth of the cell in the absence of the organic compound. The cell will thus be able to grow on the medium if it is able to synthesize the organic compound and will otherwise not be able to grow. The growth medium may be a minimal medium containing the minimum nutrients possible for growth of the cell.

The term“Determining the growth of the cell” relates to the qualitative and/or quantitative determination of growth parameter, e.g. growth rate, growth efficiency (yield coefficient), or maximum biomass, preferably to the growth rate. Growth efficiency relates to the relation between the amount of carbon source consumed and the biomass produced. The term“Determining the synthesis of the reporter molecule” relates to the qualitative and/or quantitative determination of the synthesis of the reporter molecule. This may, for example, include the measurement of the fluorescence of a fluorescent reporter molecule. In particular, the term relates to the determination of the strength of the signal generated by the reporter molecule, e.g. the fluorescence intensity.

The term“DAHP synthase” relates to a phospho-2-dehydro-3-deoxyheptonate aldolase (EC 2.5.1.54; also 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) catalyzing the synthesis of DAHP (3-deoxy-D-arabino-hept-2-ulosonate-7-phosphate) from phosphoenolpyruvate and D-erythrose 4-phosphate. The term“AroG” relates to a DAHP synthase isoform from E. coli which is feedback-inhibited by the amino acid phenylalanine (Phe), and may also be used here synonymously for the term“DAHP synthase”.

The term“aromatic amino acid” relates to an amino acid having an aromatic ring, in particular to the amino acids phenylalanine, tryptophan, and tyrosine.

In preferred embodiments, the mutated DAHP synthase (AroG) has one of the sequences of SEQ ID NO: 2, 3 or 4. These embodiments of a mutated DAHP synthase (AroGs) of the invention, denoted here as AroG^{D6G D7A} (SEQ ID NO: 2), AroG^{D6L D7P} (SEQ ID NO: 3), and AroG^{D6P D71} (SEQ ID NO: 4), have been identified using a novel screening method, described below, using the growth rate and the signal strength of the biosensor as criteria. The enzymes of the invention are not or significantly less feedback-inhibited by phenylalanine than the wildtype enzyme and exhibits higher specific enzyme activities than that of a reference variant AroG^sl80F known from the prior art in the presence of 40 mM Phe. The replacement of AroG^sl80F with the best-performing newly identified AroG^{D6G D7A} in a Trp-producing strain significantly improved the Trp production by 38.5% (24.03 ± 1.02 g/L at 36h) in a simple fed-batch fermentation.

The invention further relates to a bacterial cell being genetically engineered to express a DAHP synthase of the invention. Preferably, the bacterial cell is an E. coli cell.

In a still further aspect the invention relates to the use of a bacterial cell of the invention for the production of an aromatic amino acid, in particular tryptophan. For this purpose, the bacterial cell, which preferably is an E. coli cell, is preferably grown in a suitable medium in a bioreactor.

The invention further relates to a method for the biosynthesis of an aromatic amino acid, comprising the step of growing a bacterial cell according to the invention in a medium and under conditions suitable for growing the bacterial cell.

In a preferred embodiment the method of the invention further comprises the step of isolating the aromatic amino acid produced by the bacterial cell from the medium.

In a preferred embodiment of the method the bacterial cell grown is an E. coli cell. The aromatic acid produced is preferably tryptophan.

The invention also relates to a method for in vivo screening of engineered enzyme variants, comprising

a. Complementing a cell, the cell

(i) being auxotrophic for an organic compound due to lack of a functional gene encoding an enzyme necessary for the synthesis of the organic compound,

(ii) being genetically engineered to include an expressible gene coding for a Cas9 protein,

(iii) being genetically engineered to include an expressible CRISPR guide RNA being configured to base-pair to a target sequence at a target site on the chromosome of the cell and to guide the Cas9 protein to the target sequence,

(iv) being genetically engineered to synthesize a reporter molecule in the presence of the organic compound, and

(v) being genetically engineered to include, at the target site on the chromosome, a DNA sequence comprising the target sequence for the CRISPR guide RNA, a PAM sequence and an expressible target gene,

by biotechnologically introducing into the cell a donor DNA comprising a donor gene coding for a variant of the enzyme necessary for the synthesis of the organic compound,

b. Growing the cell, in the absence of the organic compound, in a growth medium suitable for growing the cell, the growth medium lacking the organic compound,

c. Determining the growth of the cell, and d. Determining the synthesis of the reporter molecule.

The method of the invention combines CRISPR/Cas-facilitated engineering of target gene(s) directly on the chromosome with growth-coupled and biosensor-guided in vivo screening and characterization of protein variants. By determining cell growth, e.g. the growth rate, and a biosensor, e.g. the signal strength of the reporter molecule, in combination the method enables reliable identification of the best performer among the improved enzyme variants. The method is thus particularly useful for protein engineering and pathway optimization.

The method of the invention may be abbreviated herein with the term“CGSSC” for

CRISPR/Cas9-facilitated engineering of target gene(s) with growth-coupled and sensor-guided in vivo screening and characterization.

Using the above screening method, and using the growth rate and the signal strength of the biosensor as criteria, for example, the DAHP synthetase of the invention described above has been identified. in the method of the invention a better or best performer among improved enzyme variants can be identified by determining the growth of the cell and the synthesis of the reporter molecule. The results as regards cell growth and synthesis of the reporter molecule for different enzyme variants are taken to identify the best performing enzyme variants. As an example, a first cell expressing a first enzyme variant and growing better, e.g. having a higher growth rate or higher growth yield, and producing a higher amount of the reporter molecule, indicating the production of the organic compound, than a second cell expressing a second enzyme variant, is considered to perform better than the second cell. The amount of the reporter molecule produced can, for example, be detected by measuring fluorescence in case the reporter molecule is a fluorescent molecule. It is particular preferred to take the growth rate, growth yield or growth efficiency and the production rate, production yield, or yield efficiency of the reporter molecule. A cell expressing one enzyme variant and having, compared to a cell expressing another enzyme variant, a relatively better growth yield and a higher specific fluorescence intensity produced at a comparable physiological stage, e.g. at the end of the log growth phase, can, for example, be determined as a better or best performing cell. Growth yield may, for example, be measured by measuring the Oϋboo, i.e. the optical density measured in a spectrophotometer at 600 nm, at the end of the logarithmic growth phase.

The method of the invention makes use of a cell, which is genetically engineered in a specific manner in order to allow the efficient in vivo screening and characterization of enzyme variants. The cell is auxotrophic for an organic compound necessary for growth of the cell, e.g. an essential amino acid. The cell can be naturally auxotrophic for the organic compound, but it is preferred that the cell is genetically engineered to render it auxotrophic for the organic compound.

The cell is genetically engineered to include genes for a Cas protein. The use of a Cas protein, e.g. a Cas9 protein, allows for an efficient insertion of nucleic acids coding for enzyme variants into the genome of the cell. It is preferred that the gene encoding the Cas protein is contained in a plasmid, although it is also possible to incorporate the Cas protein in the genome of the cell. The Cas protein may be put under the control of a constitutive or an inducible promoter. It is preferred that the Cas protein is under the control of a constitutive promoter.

The CRISPR guide RNA is preferably introduced on 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 on a second plasmid. It is further preferred that the CRISPR guide RNA is introduced into the cell together with the donor DNA. For this purpose, the gRNA and the donor DNA may be contained in the same plasmid. The gRNA can be under the control of a constitutive or inducible promoter, and is preferably under the control of a constitutive promoter. The gRNA is able to base-pair to a target sequence at the target site on the chromosome into which the donor DNA, i.e. the DNA comprising the gene encoding an enzyme variant, shall be introduced, and to guide the Cas protein to the target site. Although the guide RNA can be a duplex of two separate RNA molecules, i.e. crRNA and tracrRNA, it is preferred to use a single RNA molecule composed of the crRNA and the tracrRNA, i.e. a single guide RNA (sgRNA), in the method of the invention. The gRNA composed of crRNA and tracrRNA, fused to a single molecule or not, can be a synthetic RNA, which is, for example, shorter than naturally occurring gRNA. The synthetic gRNA may be chemically modified, e.g. by replacing nucleotides naturally occurring in gRNA with nucleotides not naturally occurring in gRNA, e.g. in order to stabilize the gRNA (see, for example, WO 2016/100951 A2).

The cell further includes a target gene at a target site on the chromosome as a placeholder for the enzyme variant DNA to be inserted into the genome. The target gene may, for example, be an antibiotic resistance gene. The target gene can, for example, be introduced at the site of the gene for the wildtype enzyme, such that the gene for the wildtype enzyme is disrupted by or replaced with the target gene. The target site also contains a DNA sequence comprising a target sequence for the CRISPR guide RNA, i.e. a sequence to which the CRISPR guide RNA can hybridize (base-pair), and a PAM sequence in order to allow the Cas protein to cut the target sequence.

Further, the cell is genetically engineered to synthesize a reporter molecule depending on the presence of a functional gene encoding an enzyme necessary for the synthesis of the organic compound. As an example, the cell may be engineered to include, preferably genomically, a reporter gene the expression of which depends on the presence of the organic compound. An example of such a reporter gene is tnaC , encoding the leader sequence of the tnaCAB operon, with eGFP fused upstream of tnaC. eGFP will be expressed in the presence of tryptophan. The production of eGFP can be determined qualitatively or quantitatively, preferably quantitatively, by measuring fluorescence, preferably fluorescence intensity. The production of the reporter molecule preferably correlates, for example linearly, with the production of the organic compound. A reporter system suitable for detecting the presence of another organic compound in a cell can readily be designed by the skilled person (Xu et al. 2014, Fang et al. 2016).

In the method of the invention a donor DNA is introduced into the cell by biotechnological means, e.g. via electroporation. The donor DNA comprises a donor gene coding for a variant of the enzyme to be inserted into the cell. The donor gene is introduced into the cell such that the donor gene can be expressed constitutively or inducibly. In the method of the invention, a recombinant cell is thus generated expressing an enzyme variant necessary for the synthesis of the organic compound. The donor DNA preferably is a double-stranded linear or circular DNA (dsDNA). Preferably, the donor DNA is inserted in a plasmid. In a preferred embodiment of the method of the invention, the plasmid also carries the gRNA for guiding the Cas protein to the target site. The donor DNA comprising the donor gene can be introduced into the cell, e.g. via electroporation, and the donor gene can be inserted into the genome of the cell at the target site via 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 type II Cas protein, e.g. a recombinant Cas9 protein. The gene for the Cas protein is preferably contained in a plasmid, and is preferably under the control of a constitutive promoter.

The cell can further be genetically engineered to also include genes for a recombineering system. In certain embodiments of the method of the invention, it can be favourable to have the cell genetically be engineered to include both the genes for a recombineering system and a Cas protein. The genes for the Cas protein and the recombineering system may both be contained in the same or separate plasmids. It is however, also possible that the recombineering system, in particular in case of a lamda-red recombineering system, is inserted in the genome. It is preferred to have the gene encoding the Cas protein and the genes coding for the

recombineering system in the same plasmid. It is also possible to incorporate the Cas protein in the genome of the cell. The recombineering system is preferably under the control of an inducible promoter, e.g. a temperature-sensitive promoter. In an embodiment of the method of the invention using a cell being genetically engineered to also include genes for a

recombineering system, the donor gene is preferably flanked by“homology arms”, i.e.

sequences homologous to the target site on the chromosome in order to enable homologous recombination mediated by the recombineering system. The donor DNA that is

biotechnologically introduced into the cell thus comprises a donor gene coding for a variant of the enzyme necessary for the synthesis of the organic compound, and flanking sequences homologous to the target gene on the chromosome.

In a preferred embodiment of the method of the invention, the recombineering system, if included in the cell used in the method, is a lambda-red (l-red) recombineering system, comprising the components Exo, Beta and Gam. The lambda-red recombineering system is used for mediating homologous recombination in order to insert the donor gene into the genome of the cell. The l-red recombineering system is preferably incorparted in the genome. It is however, also possible to arrange the recombineering system in a plasmid, e.g. the plasmid carrying the gene for the Cas protein. It is preferred that the l-red recombineering system is expressed under the control of an inducible promoter, e.g. a temperature-sensitive promoter.

The cell used in the method of the invention is preferably a microbial cell, further preferred a bacterial cell, further preferred an enterobacterial cell, and especially preferred an Escherichia coli cell.

The method of the invention can be used for the in vivo screening and characterization of a library of enzyme variants. For this purpose, a plurality of cells as defined above is

complemented with different enzyme variants. The cells differing in the enzyme variant they are complemented with are grown in the absence of the organic compound, and the growth of the cells and the synthesis of the reporter molecule is determined in order to identify an enzyme variant with superior properties compared to the wild-type enzyme. The best performing enzyme variant(s) can be identified by taking, for example, the growth rate and the signal strength of the reporter molecule, e.g. the fluorescence intensity, as parameters.

In the following, the invention is described by way of the attached figures and examples for illustration purposes only.

Figure 1. Simplified schematic representation of an embodiment of the method of the invention. A. Complementation of a cell being auxotrophic for an organic compound with a donor DNA comprising a functional gene necessary for the synthesis of the organic compound. B.

Complemented cell with donor gene introduced into the genome of the cell via CRISPR/Cas9- assisted recombineering.

Figure 2. CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening and characterization (CGSSC) approach (a, solid line) compared to the conventional screening and characterization approach (b, dotted line) in rational protein engineering. In the step-by-step process of rational protein engineering, knowledge based on protein structure and function or results derived from bioinformatics and modeling are first used to make rationally or semi-rationally designed changes to the gene of interest. High-throughput screening and first in vitro and then in vivo characterizations to identify the best mutant is a laborious process. In CGSSC, the integrated steps of CRISPR/Cas9-facilitated engineering of enzyme variants with growth-coupled in vivo screening and sensor-guided in vivo characterization make protein engineering more efficient and precise.

Figure 3. Design and implementation of the method of the invention in screening and characterization of feedback-resistant AroG (AroG⁰”) enzyme variants. In E. coli, DAHP synthase (AroG, AroF, and AroH) is a key rate-limiting enzyme of the pathways for aromatic amino acids (AAAs) biosynthesis. An AAAs-auxotrophic strain (strain WS002) was constructed by disrupting the DAHP synthase and used as a platform for screening aroG gene variants, which were individually integrated into the chromosome of E. coli using the

CRISPR/Cas9 system. In the presence of a high Phe concentration, only strains that express AroG^fbr with good resistance to Phe can produce enough AAAs and sustain cell growth. These strains were further characterized using the strength of fluorescence signal (medium fluorescent unit, MFU) of a Trp biosensor (P_tac-TnaC-eGFP) representing the productivity of Trp.

Figure 4. Comparison of the growth and fluorescence of the two strains WS003 and WS004 generated by introducing aroG^WT and aroG^sl80F into the chromosome of the strain WS002, respectively, under different growth conditions. Left, complex medium (LB-agar); middle, M9- agar (without any amino acids); right, M9-agar with 25 mM Phe.

Figure 5. (a) Key residues involved in the Phe binding sites of AroG from E. coli. (b) The fluorescence induction for AroG^{D6X D7X} variants on reduced M9-agar (without Tyr and Trp) with the addition of 25 mM Phe and 0.1 mM IPTG.

Figure 6. Effect of Phe on the activities of the enzyme AroG^WT and its variants AroG^sl80F, AroG^{D6L D7P}, AroG^{D6P D71} and AroG^{D6G D7A}. (a) Specific activities; (b) Relative activities.

Results were derived from three independent experiments. Figure 7. Fed-batch fermentation results of the strains S028 (circle) and S028GM1 (square) (a) Cell growth; (b) Glucose concentration; (c) Trp production; (d) Overall productivity, (e) Formation rate of Trp (qTrp), and (f) Accumulation of the intermediates shikimate (SA, open circle or square) and dehydroshikimate (DSA, solid circle or square). All results are based on two independent fermentations.

Figure 8. Map of the plasmid pCm-aroG. The donor DNA contained in this plasmid is composed of a part of CmR gene, the whole wildtype aroG gene, and a part of serA gene.

Figure 9. Amino acid sequences (in one-letter code) of the DAHP synthase of the invention (Fig. 9a) and preferred embodiments (Fig. 9b-c) thereof. X = any amino acid, but not both aspartic acid (D).

Figure 1 shows in a simplified and schematic way an embodiment of the method of the invention. In the method of the invention, a cell, preferably a bacterial cell like an E. coli cell, which is auxotrophic for an organic compound due to lack of a functional gene encoding an enzyme necessary for the synthesis of the organic compound, is complemented by

biotechnologically introducing into the cell a donor DNA comprising a donor gene coding for an enzyme necessary for the synthesis of the organic compound (Fig. 1 A). The donor gene is introduced into the genome of the cell via CRISPR/Cas-assisted recombineering. For this purpose the cell is, in this embodiment, genetically engineered to include (a) an expressible recombineering system, (b) an expressible gene coding for a Cas protein, here Cas9, (c) an expressible CRISPR guide RNA (single guide RNA, sgRNA) being configured to base-pair to a target sequence (“protospacer”) at a target site on the chromosome of the cell and to guide the Cas protein to the target sequence, and, (d) at the target site on the chromosome, a DNA sequence comprising the target sequence (“protospacer”) for the CRISPR guide RNA, a PAM sequence and an expressible target gene, for example a gene conferring resistance against an antibiotic. The cell is further genetically engineered to synthesize a reporter molecule in the presence of the organic compound. The donor gene introduced into the cell is flanked by sequences homologous to the target gene on the chromosome. The genes for the

recombineering system, here a lambda-red recombineering system, and the Cas protein, are placed on the same plasmid. It is, however, also possible to place the genes encoding the recombineering system and the Cas protein on individual plasmids or in the genome. The presence of the recombineering system is not necessary in the method of the invention.

The donor gene introduced into the chromosome of the cell is expressed, such that a functional enzyme is produced, which is necessary for synthesizing the organic compound B, for which the cell was auxotrophic, from a precursor A. The organic compound is synthesized and a reporter molecule is formed in its presence (Fig. IB). Cell growth and the synthesis of the reporter molecule are determined.

By carrying out the above-described procedure with different donor genes coding for different enzyme variants, a library of enzyme variants can be tested in order to identify the best performing enzyme variant (see Fig. 2).

Examples

1. Introduction

3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase is a key enzyme for engineering microbes for efficient biosynthesis of aromatic amino acids (AAAs): Trp, phenylalanine (Phe) and tyrosine (Tyr) (Chen and Zeng, 2017; Kim et al., 2015; Wu et al., 2018). It is under strong feedback inhibition by the end products (Ogino et al., 1982; Sprenger, 2006). For instance, in E. coli, all the wild-types of DAHP synthase, encoded by the genes aroG , aroF and aroH , are subject to feedback inhibition by Phe, Tyr, and Trp, respectively (McCandliss et al., 1978; Schoner and Herrmann, 1976). Therefore, the engineering of feedback-resistant DAHP synthase is indispensable to the construction of efficient pathways for producing AAAs and their derivatives (Sprenger, 2006). The method of the invention, also denoted“CGSSC”, was used for engineering and screening feedback-resistant DAHP synthase variants in order to improve the chorismate pathway for Trp production in E. coli.

2. Materials and methods

2.1 Strains and plasmids The strains and plasmids used in this study are listed in Table 1. The Trp producing strain S028Z, the parent strain of S028 (Chen and Zeng, 2017) in which the temperature-sensitive l- red recombineering system had been removed from the chromosome, was used as starting strain. In the strain S028Z, the DAHP synthase activity is offered only by the Phe-resistant AroG^sl80F. To apply the CRISPR/Cas9 technique to mediate genome editing, the plasmid pCas (Jiang et al., 2015) was introduced into S028Z, resulting in the strain S028ZC (Table 1).

Table 1. Main strains and plasmids used in this study

Strains/Plasmids Characteristics Sources

Strains

S028 DY330 rpsL ( StrR ) Amtr AtnaA AtnaB Al AaroF Chen and Zeng,

AaroG aroH::P _{23ii9-rPsL-tac}-(aroG^sl80F-serA^H344A/N364A) 2017

Ptrc-trpE^S40FDCBA

S028Z S028 strain with l-red recombination system

S028ZC S028Z containing the plasmid pCas9

WS001 S028ZC AtrpR: :Ptac-tnaC-eGFP (Trp sensor)

WS002 aroG^SI80F) : : (P J23119-rpsL-cmR~

WS003

without pCas

WS004 WS002 ACnPy.aroG⁵¹⁸^ without pCas

WS005 WS002 ACnf- : :aroG^{D6G D7A} without pCas

S028GM1 S028 Aaro^G180F : :aroG^D6G~D7A

Top 10 F- mcrA A(mrr-hsdRMS-mcrBC) §80lacZAM15 Invitrogen

AlacX74 nupG recAl araD139 A{ara-leu)7697

galE15 galK16 rpsL(StrR ) endAl l-

Plasmids

pCas expressing Cas9 protein and offering sgRNA for Jiang et al., 2015 removing donor plasmid

pTargetF plasmid for expressing sgRNA or with offering donor Jiang et al., 2015

DNA, Spectinomycin resistance pTagAmpR plasmid for expressing sgRNA or with offering donor

DNA, Ampicillin resistance

pET vector

pET22b inserted with aroG^WT gene

pET22b inserted with aroG^sl80F gene

pET22b inserted with aroG^{D6L D7P} gene

pET22b inserted with aroG^{D6P D71} gene

pET22b inserted with aroG^{D6G D7A} gene

pJLC AmpR, PMB 1 , P J23119-r_pSL-r_pSL-Cm^R

a trpR-sgRNA, sgRNA with an N20 sequence for targeting the trpR locus.

To construct plasmids for protein expression and purification, the encoding gene of aroG^WT was isolated from the strain E. coli DY330 (Yu et ah, 2000) with primers aroG-His-Hindlll and Xbal-serA (Table 2). It was then inserted into the vector pET22b(+) at the sites Hindlll and Xbal to generate the plasmid pKT-aroG^WT. The plasmids pET -aroG^sl80F, pET -aroG^{D6G D7A}, pET -aroG^{D6L D7P} , and pET -aroG^/>6/’~/>7/ were generated by using mutagenic primers (Table 2) to amplify the whole plasmid pET -aroG^WT.

2.2 Molecular Biology Work

2.2.1 sgRNA plasmid and donor DNA construction

To construct the plasmid expressing single guide RNA (sgRNA), a set of primers (Table 2) was used to amplify the pTagAmpR backbone (Fig. 8) through PCR. The 20bp spacer sequence specific for the target gene was selected with the aid of the web-based tool Cas-Designer (Bae et al., 2014; Park et al., 2015) and was synthesized within the primers (Table 2, shown in capital letter). The PCR products were then transformed into E.coli ToplO competent cells directly to obtain the desired sgRNA plasmids. To construct sgRNA plasmids with donor DNAs, two 300- 500 bp homologous arms and the DNA fragment for substitution were separately amplified and then fused together by PCR. After gel-purification, the target PCR products were inserted into the desired sgRNA plasmids.

Table 2. Primers used in this study. gRNA sequences in capital letters. N = A, C, G or T. K = G or T.

Plasmids and strains construction

Construction of the strain WS001 and WS002

In order to avoid causing instability by using too many plasmids, the Trp-biosensor was integrated into the chromosome at the locus of the trpR gene, resulting the strain of WS001. To do so, the plasmid pN20 -trpR was constructed from the plasmid pTagAmpR (Fig.8), which is derived from the plasmid pTargetF (Jiang et al., 2015) with the change of spectinomycin resistance to ampicillin resistance, with the primers pTargR and TrpR-N20 (Table 2) for expressing gRNA targeting the trpR gene. The donor DNA fragment Trp-Sensor was amplified from the plasmid pSentrp (Fang et al., 2016) with the primers Tsen-trpR-IF and Tsen-trpR-IR.

In order to construct aromatic amino acids (AAA)-auxotrophic strain (WS002), we removed the sole DAHP synthase of strain WS001 by replacing the aroG^sl80F gene with the antibiotic resistance gene Cn^. In this regard, the plasmid pN20 -aroG was constructed at first from the plasmid pTagAmpR plasmid with the primers pTargR and AroG-N20 (Table 2) for expressing gRNA targeting the aroG gene. The donor DNA fragment P j23U9-r_PsL-Cm^R was amplified from the plasmid pJLC with the primers up-aroH-out and Cm-delG-R.

Construction of the plasmid libraries pCm -aroG^fl>r

In order to construct pCm -aroG (Fig. 8), we constructed the plasmid pN20-CmR at first with the primers pTargR and Cm-N20 (Table 2) in the same way as constructing the plasmid pN20- aroG. The DNA fragment V-N20CmR was amplified from the plasmid pN20 -CnP using primers pN20VRCm and pN20VFSerA (Table 2). Meanwhile, the DNA fragments F -CnP, F- aroG , and F -serA were generated from the plasmid pJLC (Table 1), the genome DNA of E. coli W3110, and the plasmid strp015A (Chen and Zeng, 2017), respectively. The primer pairs were Cm-Cl/Cm-C2 , WaroG-FF/aroG-Fus-R, and serA-Fus-F/serA-Fus-R, respectively. Then four fragments (^' V-N20CmR , F-Cm^R, F-aroG , and F -serA) were fused together to construct the final plasmid pCrs\-aroG^WT by using In-Fusion HD Cloning kits (Clontech® Laboratories, Inc.). The plasmids of pCm -aroG^sl80F and pCm -aroG^fl>r were constructed using a corresponding pair of mutagenic primers (Table 2) to amplify the whole plasmid pCm -aroG^WT.

Construction of the strain S028GM1

In order to replace the promoter oi Fj23U9-rpsL-cmR-aroG^D6G~D7A by the same one as S028 (P J23ii9- rpsL-tac-oroG^sl80F ), the plasmid pN202-C ^? was firstly constructed as the same as other sgRNA plasmids but with primers pTargR and CmR-N202. The donor DNA fragment P j23U9-r_PsL-tac- aroG^D6G~D7A was obtained from two round PCR. The first round is for adding the tac promoter to the aroG^{D6G~DA 7} gene by amplifying the plasmid pET-AroG^{D6G DA7} with the primers NotF pTac-aroG and aroG-spel. The second round is for flanking the upstream homologous arm by using the primers u-rpsLp-tac and aroG-fus-R and using the PCR product from the first round as the template. The plasmid pN202-Cm^/< and the fragment P j23ii9-r_PsL-tac-aroG^{D6G D7A} were co transformed into the strain WS005 with the plasmid pCas for generating the strain S028GM1.

2.2.1 Genome editing with CRISPR/Cas9 technique

To integrate target donor DNA fragment into the genome, host strain harbouring pCas expression vector was used. Transformation with electroporation method was performed following the protocol reported by Chen and Zeng (2017) with minor modifications. To prepare electroporation-competent cells, specifically, an overnight culture (grown at 30 °C) of the strain harboring pCas was inoculated (2%, vol./vol.) into 10 ml fresh SOB medium containing 30 pg/ml kanamycin. After growing to OD600 around 0.4, the cells were transferred into 42 °C and grown for 15 min with shaking and then put on ice immediately for 10 min. After that, the cells were harvested by centrifugation at 4 °C and washed three times with precooled 10% glycerol or distilled water. Competent cells were re-suspended in 400 pL precooled 10% glycerol and divided into 200 pL for each reaction. The corresponding sgRNA plasmid (if required, plus donor dsDNA) eluted in water was mixed with the competent cells for transformation. The electroporation was done in 0.2 cm cuvette at 2.5 kV, and the cells were suspended in 1 ml SOB medium and recovered at 30 °C for 2 hours before plating. Plates were incubated more than 24 hours at 30 °C. Transformants were identified by colony PCR and DNA sequencing.

2.4 AroG variant library construction and screening

To demonstrate the efficiency of the method of the invention, we applied it to engineer Phe- resistant AroG. Two residues (Asp6 and Asp7) involved in the Phe-binding site of AroG (PDB ID: 1KFL) were chosen for saturated mutagenesis (NNK) in this study. The saturation mutagenesis was introduced with primers (Table 2) by amplifying the whole plasmid pCm- aroG^WT (Table 1). After digestion of the template DNA with Dpnl, the PCR products were then transformed into chemically competent A. coli ToplO cells, the reaction products were suspended in SOB medium. After incubation at 37 °C for 1 hour, all the cells were transferred into 10 ml fresh LB medium with 100 pg/ml ampicillin and cultivated for 8-10 hours at 37 °C. The plasmids were extracted from these cultivations and eluted in water as AroG variant library: p C m -ar ( ^l>6''^{~l> 7L} (Fig.8, see above). The AroG variant library was transferred into the WS002 strain (containing pCas) and the cells were then washed with M9 medium (without any amino acids) three times after 2h incubation at 30 °C in SOB medium. The cells were spread onto M9 agar medium containing 25 mM Phe and 0.1 mM IPTG for screening. After incubation at 30 °C for more than 24 hours,

transformants with bigger size and stronger fluorescent signal were picked out and re-checked by streaking them on the same medium. Finally, the candidate mutant strains were tested by cultivation in 5 ml fermentation medium II (FM-II) at 30 °C for 24 h. FM-II was nearly the same as reported previously (Gu et ak, 2012), but contained 0.5 g/L instead of 5 g/L of MgSCL 7H₂O and had 30 g/L initial glucose. Additionally, 12 g/L K₂HPO₄ (for buffering pH), 25 mM Phe and 0.1 mM IPTG were added. The mutants giving higher medium fluorescence (MFU) were selected for sequencing.

2.5 Method for measurement of fluorescence intensities

The mutants containing chromosomal Trp sensor with reporter eGFP protein cultured the LB medium were harvested by centrifugation and individually washed three times with the M9 medium to remove the LB medium. Afterwards, each mutant was inoculated with the same amount of cells into 10 mL fresh M9 medium with 25 mM Phe in 50 mL conical tubes, and after a cultivation of 10 h cells were subjected to fluorescence analysis using flow cytometry.

To this end, each culture was first washed three times using PBS buffer and diluted 100-fold and then eGFP fluorescence was monitored (MFI of > 10,000 events) using a flow cytometer (CytoFLEX, Beckman Coulter) at an excitation wavelength of 488 nm. All data were processed with the Beckman Flow software, and electronic gating was used to separate positive signals from instrument and water sample background. For fluorescent intensities, medium

fluorescence unit (MFU) was calculated for each culture.

2.6 Protein expression and purification

For protein expression, the corresponding plasmids (see Section 2.1) were transformed into E. coli BL21 as host cells, respectively. Protein expression and purification procedures were slightly modified from the one reported previously (Chen et ak, 2018). The quantification of purified protein was performed according to Bradford’s method using bovine serum albumin as standard and a prefabricated assay from Bio-Rad Laboratories (Hercules, CA). 2.7 Enzyme assay

In vitro enzyme kinetics of AroG^fbr mutants was performed as described in literature with minor modification (Schoner and Herrmann, 1976). The enzyme activity was measured by monitoring the disappearance of PEP via absorbance at 232 nm, and the calculation of specific activity is according to the standard curve of PEP with the absorbance at 232 nm measured in cuvette (not shown). To investigate the effect of Phe on the activity of AroG variants, the activities were measured in the presence of different concentrations (from 0 to 40 mM) of Phe. The complete reaction mixture contained 10 mM Bis-tris propane (BTP, pH 7.0), 50 mM MnSCE, 600 mM PEP, 500 pM E4P, and 25 pg purified enzyme in a total volume of 0.2 mL in cuvette at 25°C with or without inhibitor. The mixture (without PEP and E4P) and the substrates (PEP and E4P) were equilibrated to reaction temperature, separately, and the reaction was started by adding the substrates (PEP and E4P).

2.8 Fermentation conditions

For fed-batch fermentations in a bioreactor, the pre-culture and seed culture were performed under the same conditions as reported previously (Gu et al., 2012). Because the l recombination system integrated in the strain E. coli DY330 can cause temperature-sensitive cell growth at 42 °C, it was removed from each strain derived from DY330 before it was used for

fermentation. For this purpose, the procedure for removal of l recombination system was performed according to the method reported by Chen and Zeng (2017). Fermentations were carried out in a highly instrumented and automated 4-paralell 1.5 L bioreactors system

(DASGIP Parallel Bioreactor System, Mich, Germany) with an initial working volume of 500 mL. If not indicated otherwise, the fermentation medium in bioreactor, feeding solution, and fermentation conditions were the same as reported previously (Chen and Zeng, 2017).

2.9 Analytical methods

The quantification of glucose, 3-dehydroshikimate (DSA), and shikimate (SA) was done using HPLC as reported previously (Bommareddy et al., 2014; Luz et al., 2014). The determination of Trp was carried out by using a sensitive spectrophotometric method (Nagaraja et al., 2003).

3. Results and discussion 3.1 Proof-of-concept of the method of the invention (CGSSC method)

For proof-of-concept, we first constructed a screening strain which contains a Trp biosensor and lacks DAHP synthase activity for screening variants of DAHP synthase. To this end, the genes aroH and aroF in our previously developed Trp-producing E.coli strain S028k (Chen and Zeng, 2017) were first knocked out (Table 1). Deleting the aroG^sl80F gene from this mutant made the strain auxotrophic for aromatic amino acids (AAAs). Consequently, the growth of the mutant is linked to the DAHP synthase activity upon its re-introduction (Fig. 3). In principle, an engineered DAHP synthase with a higher activity should lead to a faster accumulation of Trp, which in turn stimulates the expression of a report gene regulated by the Trp biosensor (Fang et ak, 2016).

The Trp biosensor is composed of tnaC , which encodes the leader sequence of the tnaCAB operon (Bischoff et ak, 2014; Gong et ak, 2001); the eGFP protein was fused to the upstream of tnaC (Fang et ak, 2016). Specifically, the strain WS001 (Table 1) was first constructed based on the Trp producer S028kC (Chen and Zeng, 2017) after replacement of the trpR gene with the Trp biosensor using the CRISP/Cas9 system. Then, we removed the sole DAHP synthase of the strain WS001 by replacing the aroG^sl80F gene with the antibiotic resistance gene CnP which offers sgRNA targets for the CRISPR/Cas9 system in further genome-editing, generating the strain WS002 (Table 1, Fig. 3). As expected, the strain WS002 is not able to grow in the M9 medium without the addition of any of the aromatic amino acids Phe, Tyr, and Trp (data not shown). Then, we tested if the strain with a feedback resistant DAHP synthase will behave differently from the one having the wild-type DAHP synthase in terms of cell growth and expression of the report gene in a defined medium containing a high concentration of enzyme inhibitor. To this end, we introduced the wild-type aroG gene and the feedback-resistant gene aroG^sl80F (Ger et ak, 1994) into the chromosome of the strain WS002 at the locus of the ( 'm^R gene using the CRISPR/Cas9 technique with the plasmids pCm -aroG^WT and pCm -aroG^sl80F, respectively. The recombinants were spread on M9-agar medium containing 25 mM Phe without Tyr and Trp. 0.1 mM IPTG was also supplemented into the medium for the following reasons. One is to release the trp biosynthetic pathway since it’s regulated by Lacl regulator, and the other is to induce the expression of sgRNA which guides Cas9 to cut the donor plasmids from which the gene of interest can also be expressed. The results showed that, after introduce of the aroG^sl80F gene into the host, many recombinants with strong fluorescent signal grow up under the conditions mentioned above (Fig. 4). We have selected several colonies for further characterization. It turned out that these colonies all have the same mutation S180F. These recombinants were designated as WS004. No colony was observed for the host integrated with the plasmid pCm -aroG^WT under the same conditions (Fig. 4). Presumably the activity of the wild-type AroG is seriously inhibited by Phe and it could not support the growth of cells. It could be, however, also possible that the recombineering efficiency is too low. To eliminate the latter possibility, the recombinants with the aroG^WT gene were also grown on LB-agar medium with IPTG. From the complex medium, we obtained many colonies (Fig. 4), which were positively confirmed by colony PCR and designated as WS003. The strains WS003 and WS004 were re-checked on M9-agar medium with and without 25 mM Phe (Fig. 4). The strain WS003 was found to grow on the medium without Phe but no growth was observed on the medium with Phe (Fig. 4). As expected, the growth of the strain WS004 did not show notable difference on the media with or without Phe. These results suggested that CGSSC is an approach useful to facilitate engineering of enzymes with desired performance such as higher activity and higher inhibitor tolerance. It is used in the following to obtain AroG variants with further improved tolerance against Phe.

3.2 CGSSC for screening Phe-resistant AroG

To demonstrate the usefulness of the CGSSC method established above for obtaining more resistant AroG enzyme variants, a mutation library of AroG was first generated.

For this purpose, a semi-rational strategy was adopted which makes use of information from the crystal structure of AroG complexed with its inhibitor Phe (PDB: 1KFL) (Fig. 5a). The residues D6 and D7 involved in the binding of Phe were selected as targets to perform saturation mutagenesis. They were then screened with the CGSSC. As shown in Fig. 5b, for the AroG^{D6X D7X} variants, there were around 100 colonies with different size and different strength of fluorescent signal obtained on the previously mentioned screening medium after grown for about 30 hours.

After the first round screening, 30 colonies of the AroG^{D6X D7X} variants, which have relatively bigger size and higher fluorescence signal, were selected and re-checked on the screening medium. After confirmation of the phenotypes, the mutated aroG genes from 20 candidates were isolated for sequencing. The sequencing results showed that there are only 6 different AroG variants among the 20 candidates (Table 3). They are AroG^{D6G D7A}, AroG^{D6L D7P},

AroG^{D6P D71}, AroG^{D6F D7V}, AroG^{D6V D7C}, and AroG^{D6F D7L}, with the number of occurring being 7, 6, 4, 1, 1, and 1, respectively (Table 3). We then carried out fermentations in FM-II medium in 50 mL conical tubes with the strains carrying these 6 recombinants and compared them with the wild-type strain WS003 and the strain WS004 has AroG^sl80F variant. It was found that the strains carrying the variants AroG^{D6G D7A}, AroG^{D6L D7P}, or AroG^{D6P D71}, which had the higher frequency of occurrence in the 20 candidates (Table 3), had also higher Trp productivity (Fig. 3, for sequences see also Fig. 9). Especially the first two mutants had much higher productivity than that of the reference strain (AroG^sl80F). In addition, we investigated the relationship between the Trp productivity and the strength of fluorescence signal. The strain which has a stronger fluorescence signal also has a higher Trp productivity (Fig. 3). These results suggest that the variants AroG^{D6G D7A}, AroG^{D6L D7V} and AroG^{D6P D71} have a higher inhibitor tolerance than the variant AroG^sl80F under the test conditions. To provide more direct evidence, enzyme assay was done with purified protein of these variants.

Table 3. Comparison of fermentation results with E. coli strains containing the AroG^WT, AroG^sl80F, and AroG^ variants grown on FM-II with 25 mM Phe. DCW: dry cell weight.

a Number of variants refers to all the examined 20 candidates; N. D., not detected. The average value ± standard deviation is based on three independent experiments. 3.3 Characterization of selected AroG^fbr variants in vitro

To examine if the higher Trp productivity and stronger fluorescence signal observed in the strains is due to improved Phe-tolerance of the corresponding AroG variants we investigated the inhibition behavior of the variants AroG^{D6G D7A}, AroG^{D6L D7P}, and AroG^{D6P D71}.

As shown in Fig. 6, all the variants are significantly less sensitive to the inhibitor Phe, while the wild-type AroG is extremely sensitive to it. In the presence of 0.5 mM Phe, the wild-type AroG almost completely lost its activity, whereas all the variants remained more than 80% of their activities under the same conditions. Compared to AroG^sl80F, all of the three variants AroG^{D6G D7A}, AroG^{D6L D7P}, and AroG^{D6P D71} have weaker sensitivities to Phe when the concentration of Phe was higher than 10 mM (Fig. 6b). They also had higher specific activities in the presence of more than 20 mM Phe (Fig. 6a). These results explain why the strains with these three variants performed better than the positive control in terms of the Trp production when they were cultivated in medium containing a very high Phe concentration (Table 3). As shown in Fig. 6a, the specific activities are significantly different between these three variants generated by the substitution of the same residues D6 and D7. Among them, AroG^{D6G D7A} variant has the highest specific activity, which is nearly twice as high as that of the variant AroG^{D6P D71} regardless of the Phe concentration. It’s also remarkably higher than that of the positive control AroG^sl80F.

3.4 Improvement of the chorismate pathway and Trp biosynthesis

To explore the impact of the best variant AroG^{D6G D7A} on strain development for the

biosynthesis of aromatic amino acids, the variant AroG^sl80F in our previously constructed Trp- producing strain S028 was substituted with AroG^{D6G D7A}, generating the strain S028GM1 (see above). The capacity of Trp production of S028GM1 strain was compared to that of the reference strain S028 by carrying out simple fed-batch fermentations in bioreactors.

As shown in Fig. 7a and 7c, the strain S028GM1 produced a significant higher amount of Trp than the reference strain after the lag phase (about 10 h) till the end of the fermentation. At the end of the fermentation (37h), the strain S028GM1 produced 24.03 ± 1.02 g/L, which is 38.50 % higher than that (17.35 ± 1.16 g/L) of the strain S028 (Fig. 7c). The concentrations of glucose were controlled at nearly the same level for both strains during the whole fermentations. It was found that the strain S028GM1 had a slightly faster growth rate (0.211 h ¹) than the strain S028 (0.184 h ¹, Fig. 7a) at the beginning of the exponential growth phase. The enhanced DAHP synthase activity has obviously contributed to the higher growth rate to certain extent. A higher biomass formation rate could reasonably result in a higher productivity for the strain S028GM1 (Fig. 7d). However, its higher Trp production can be considered to be mainly due to the enhanced DAHP synthase activity directly, because the specific Trp formation rate of the strain S028GM1 was remarkably higher than that of the strain S028 (Fig. 7e) during the whole fermentation. In addition, during the fermentations, the strain S028GM1 accumulated higher amounts of the intermediates SA and DSA (Fig. 7f) of the chorismate pathway than the reference strain S028. Both of the intermediates accumulated by the strain S028GM1 are about 36% higher than those produced by S028 at the end of fermentation. It suggests that more metabolic flux was redirected into the chorismate pathway in the strain S028GM1 than in the reference strain due to difference between the variants AroG^{D6G D7A} and AroG^sl80F. These results clearly showed that the variant AroG^{D6G D7A} is more efficient for the chorismate pathway towards bio-production of aromatic amino acids and their derivatives.

4. Conclusion

It has been shown, that the method of the invention, integrating CRISPR/Cas9-facilitated engineering with growth-coupled and sensor-guided in vivo screening and characterization (CGSSC), is particularly useful for engineering and screening of enzyme variants. Using the method for engineering and screening 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (AroG), AroG variants could be identified, which are more resistant to Phe and thus more suitable for the biosynthesis of aromatic amino acids like Trp. With two selected mutation points based on structural information new variants (AroG^{D6G D7A}, AroG^{D6L D7P}, and AroG^{D6P D71}) were revealed to be more resistant to Phe than the Phe-resistant variant AroG^sl80F reported so far in literature. The replacement of AroG^sl80F with AroG^{D6G D7A} in a previously engineered Trp producing E. coli strain (S028) remarkably increased the Trp production by 38.05 % in a simple fed-batch fermentation. Since the method of the invention is based on an integration of the genes of the enzyme variants, e.g. AroG-encoding gene variants as described above, into the chromosome, it can also be simultaneously used to optimize the expression level of the engineered enzyme in the strain, i.e. by constructing the corresponding gene using different promoters and/or ribosome binding sites. It is to mention that the high efficiency of CRISPR/Cas9 technology allows multiplex genome editing. The method of the invention can thus be used to address a wide range of targets that require a simultaneous modulation of multiple genes, e.g. for the synthesis of metabolites like Trp requiring multiple precursors.

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Claims

1. A DAHP synthase having the sequence of SEQ ID NO: 1, with the proviso that the amino acids at positions 6 and 7 are not both aspartic acid.

2. The DAHP synthase according to claim 1 having one of the sequences according to SEQ ID NO: 2, 3 or 4.

3. A bacterial cell being genetically engineered to express a DAHP synthase according to one of claims 1 or 2.

4. The bacterial cell according to claim 3, wherein the bacterial cell is an E. coli cell.

5. Use of a bacterial cell according to one of claims 3 or 4 for the production of an aromatic amino acid.

6. Use according to claim 5, wherein the aromatic amino acid is tryptophan.

7. Method for the biosynthesis of an aromatic amino acid, comprising the step of growing a bacterial cell according to one of claims 3 or 4 in a medium and under conditions suitable for growing the bacterial cell.

8. Method according to claim 7, wherein the bacterial cell grown is an E. coli cell.

9. Method according to one of claims 7 or 8, wherein the aromatic acid is tryptophan.