LU101172B1 - Method for in vivo screening of engineered enzyme variants - Google Patents

Abstract

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.

Description

PAT 1716 LU ei LU101172

METHOD FOR IN VIVO SCREENING OF ENGINEERED ENZYME VARIANTS

DESCRIPTION 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 al., 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.1b). It has been widely and successfully used for improving the performance of many enzymes (Chen et al., 201 1a; 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. | I.

PAT 1716 LU ID LU101172 Recently, the CRISPR/Cas9 technology for genome editing has made great advances and received large attention (Cho et al., 2018; Donohoue et al., 2017; Jakociünas et al., 2015; Zhang ct al., 2018). Among others, it is applied for the engineering of microbial production strains (Jakotiünas 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). It is an object of the invention to provide a reliable method for in vivo screening of engineered enzyme variants, which enables a comparatively fast and simple identification of enzyme variants suitable for use in bioprocesses.

To solve the above problem the invention provides 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, (i1i) 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, | ¢. Determining the growth of the cell, and ‘ d.

Determining the synthesis of the reporter molecule.

PAT 1716 LU 3 LU101172 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 usefull 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.

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 (A-red) recombineering system. The | lamda-red recombineering system consist 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 |

PAT 1716 LU 4 LU101172 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, c.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 VE. 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

PAT 1716 LU Sn LU101172 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 erRNA, thus forming an erRNA:tracrRNA duplex.

The term “CRISPR guide RNA”, also “CRISPR gRNA” or “gRNA”, refers to a | erRNA: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. |

PAT 1716 LU 6 LU101172 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 DHAP (3-deoxy-D-arabino-hept-2-ulosonate-7-phosphate) from phosphoenolpyruvate and : D-erythrose 4-phosphate. The term “AroG” relates a DAHP synthase isoform from E. coli ! which is feedback-inhibited by the amino acid phenylalanine (Phe). 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 ;

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PAT 1716 LU _7- LU101172 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 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 contro! 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 chemicalty 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 |

PAT 1716 LU —8_ LU101172 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 thaCAB operon, with eGFP fused upstream of tnaC. eGFP will only be expressed in the presence of tryptophan.

The production of eGFP can be determined qualitatively or quantitatively by measuring fluorescence.

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. 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. [ fo

PAT 1716 LU _9_ LU101172 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 (A-red) recombineering system, comprising the components Exo, Beta and Gam. The lambda-red recombincering system is ; used for mediating homologous recombination in order to insert the donor gene into the genome | of the cell. The A-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 à-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.

PAT 1716 LU —10— LU101172 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 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.

The invention also relates to mutated DAHP synthase (AroG) variants, 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.

In preferred embodiments, the mutated DAHP synthase (AroG) has one of the sequences of SEQ ID NO: 2, 3 or 4. These embodiments of mutated DAHP synthases (AroGs) of the invention, denoted here as AroGP*P7A (SEQ ID NO: 2), AroGP°--P7P (SEQ ID NO: 3), and AroGP°P-P"! (SEQ ID NO: 4), have been identified using the above-described screening method of the invention, 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®'** known from the prior art in the presence of 40 mM Phe. The replacement of AroG*150F with the best-performing newly identified AroGP°S-P74 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 DHAP | 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 tryptophan. For this purpose, the bacterial cell, which preferably is an E. coli cell, is preferably grown in a suitable medium in a bioreactor. ;

PAT 1716 LU “11 = LU101172 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 (AroG™) 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™ with good resistance to Phe can produce enough AAAs and sustain cell growth. These strains were further characterized using the strength of fluorescent signal (medium fluorescent unit, MFU) of a Trp biosensor (Prac-TnaC-eGFP) representing the productivity of Trp.

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PAT 1716 LU —12— LU101172 Figure 4. Comparison of the growth and fluorescence of the two strains WS003 and WS004 generated by introducing aroG"" and aroG*"® 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”°XP7X 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"T and its variants AroGS!80F, AroG”°1-P7F, AroGP°P-P7 and AroGPSS PTA (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 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. 1A). 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 |

PAT 1716 LU _13— LU101172 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 plamids 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. 1B). Cell growth and the synthesis of the reporter molecule are determined.

By carrying out the above-decribed 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). Exampies

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 ]

PAT 1716 LU —]4_ LU101172 (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 S028), the parent strain of S028 (Chen and Zeng, 2017) in which the temperature-sensitive A- red recombineering system had been removed from the chromosome, was used as starting strain. In the strain S028}, the DAHP synthase activity is offered only by the Phe-resistant AroG*'#F, To apply the CRISPR/Cas9 technique to mediate genome editing, the plasmid pCas (Jiang et al, 2015) was introduced into S028A, resulting in the strain S028AC (Table 1). Table 1. Main strains and plasmids used in this study Strains/Plasmids Characteristics Sources strains ~~ S028 DY330 rpsL (StrR) Amtr AtnaA AtnaB AA AaroF Chen and Zeng, AaroG AaroH::Pi3)19-mpsLotac-(aroG31¥0F_ ger A H3M4A/N364A) 2017 Ptre-trpES40FDCBA S028A S028 strain with A-red recombination system SO28AC S028A containing the plasmid pCas9 WS001 S028AC AtrpR::Ptac-tnaC-eGFP (Trp sensor) WS002 WS001 A(P123119-psL1ac-2r0GS 8): (P1231 10-rpst em“ CmR) WS003 WS002 ACmR::aroG"" without pCas | WS004 WS002 ACmR::aroGS13%F without pCas WS005 WS002 ACmR::aroGP°S-D7A without pCas S028GM1 $028 Aaro©!50F.;anoGP6S-D7A

PAT 1716 LU _—15— LU101172 Topl0 F- merA A(mrr-hsdRMS-merBC) 480lacZAMI5 Invitrogen AlacX74 nupG recAl araD139 A(ara-leu)7697 galE15 galK 16 rpsL{StrR) endA 1 A- Plasmids pCas expressing Cas9 protein and offering sgRNA for hang 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 pN20-trpR pTagAmpR trpR-sgRNA*® pCm-aroG"T pTagAmpR cm®-sgRNA AcmP::P123119-spst-cmr-aroGWT pCm-aroG3"¥F pTagAmpR cmR-sgRNA AcmP::P23119-psL-emR- aroGS180F pCm-aroGP*P™ pTagAmpR cm®-sgRNA Acm®::P123110mpstcmk- aroGP6X-D7X pCm-aroGP°9-PTA pTagAmpR cmR202-sgRNA AemR::P123119-rps1-tac- aro(GP6S-D7A pET22b(+) pET vector pET-aroG"" pET22b inserted with aroGY" gene pET-aroG®'8E pET22b inserted with aroGS'S gene pET-aroGP°l-P7P PET22b inserted with aroGP°--P7P gene pET-aroGP"P7" pET22b inserted with aroGP°P-P7! gene pET-aroGP°$-P7A pET22b inserted with aroGPS-P74 gene : pJLC AmpR, PMB], P123119-rpsL-mpsL-CMR [ * trpR-sgRNA, sgRNA with an N20 sequence for targeting the tpR locus. 7 | To construct plasmids for protein expression and purification, the encoding gene of aroG" was | isolated from the strain E. coli DY330 (Yu et al., 2000) with primers aroG-His-Hindill and | Xbal-serA (Table 2). It was then inserted into the vector pET22b(+) at the sites Hindill and | Xbal to generate the plasmid pET-AroGWT.

The plasmids pET-AroGS!5F, pET-AroGPt6-D7A {

PAT 1716 LU —156— LU101172 PET-AroGPL-P7P and pET-AroGP°P-P71 were generated by using mutagenic primers (Table 2) to amplify the whole plasmid pET-AroG"T,

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 Top10 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,GorT.K =K =GorT. [seen re pTargR actagtattatacctaggactgagctagetgtcaag AroG-N20 gctcagtectaggtataatactagtCAGGAAGCAGTGCGGCGC ACgttttagagctagaaatagcaagttaaaataagectagtecg TrpR-N20 gctcagtectaggtataatactagtTCAGGTTTAACAACGGTAA 7 Agtittagagctagaaatagcaagttaaaataagectagtecg Cm-N20 gctcagtectaggtataatactagtlT GATGAACCTGAATCGCCA : Ggttttagagctagaaatagcaagttaaaataaggctagtecg Cm-N202 gctcagtcctaggtataatactagtGCTGATGCCGCTGGCGATT | Cgttttagagctagaaatagcaagttaaaataaggetagtecg : no

PAT 1716 LU 17 LU101172 D67X-F ggaacagacatgaattatcagaacNNKNNK ttacgcatcaaagaaatcaa 18 a ee aroG-His- ceccegaagetttcattagtgetgetpetegigptggeccgcgacgcecttttac 24

A NN Tsen-trpR-IF ggataaaccgacgttgatgagcgccacggaatggggacgtegttactgatecg 29

TE Tsen-trpR-IR teaatcgettttcagcaacacctcttccagecactggegeagetegacgggegeg 30 ; up-aroH-out ggggcgtigptotaaagattattgcectecaceetgtacgggtgagggcgtagag 31 ; Cm-delG-R gtatcticccagectatgcaggcategetgatectettaccgtaggccagcacct 32 : Notl-pTac-aroG agtgcageggcegctettgacaattaatcategectegtataatetgtaggggaa 33 |

PAT 1716 LU 18 LU101172 u-rpsLp-tac tigtetgaggacettttatlacgtgtttacgaagcaaaagctaaaaccaggagctat 35 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 (Hang 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 aroG5!5° gene with the antibiotic resistance gene Cm®. 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 P123119-ps1-CMR was amplified from the plasmid pJLC with the primers up-aroH-out and Cm-delG-R. Construction of the plasmid libraries pCm-aroG™ | 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 pN20CMR using primers pN20VRCm and pN20VFSerA (Table 2). Meanwhile, the DNA fragments F-CmR, 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-C1/Cm-C2, WaroG-FF/aroG-Fus-R, and serA-Fus-F/serA-Fus-R, respectively. Then four | fragments (V-N20CmR, F-CmR, F-aroG, and F-serA) were fused together to construct the final | plasmid pCm-aroG”" by using In-Fusion HD Cloning kits (Clontech® Laboratories, Inc.). The |

PAT 1716 LU 19 LU101172 plasmids of pCm-aroG*!*F and pCm-aroG™ were constructed using a corresponding pair of mutagenic primers (Table 2) to amplify the whole plasmid pCm-aroGYT, Construction of the strain S028GM1 In order to replace the promoter of P123119-rpsL-cmr-aroGP°S-P7A by the same one as S028 (Ppay10- psL-tac-ar0GS!8%), the plasmid pN202-CmR was firstly constructed as the same as other sgRNA plasmids but with primers pTargR and CmR-N202. The donor DNA fragment P123119-rpsL-tac- aroG"°$-PTA was obtained from two round PCR. The first round is for adding the tac promoter to the aroGP°S-DAT gene by amplifying the plasmid pET-AroGP6S-PA7 with the primers Notl- 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-CmR and the fragment P23; 19-mpst-tac-AF0OGP°S-P7A were co- transformed into the strain WS005 with the plasmid pCas for generating the strain S028GMI.

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 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 0.2em cuvette at 2.5 | kV, and the cells were suspended in 1 m! SOB medium and recovered for 2 hours before [ plating. Plates were incubated more than 24 hours at 30 °C. After 24 hours cultivation, one | positive single colony was inoculated in LB medium with 30 pg/ml kanamycin, 100 pg/mL | ampicillin, and 10 mM L-arabinose. After overnight cultivation, the cell culture was inoculated | into the agar palte with 30 pg/ml kanamycin, 100 pg/mL ampicillin, and 10 mM L-arabinose.

PAT 1716 LU _20— LU101172 After 24 hours cultivation, the 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”" (Table 1). After digestion of the template DNA with Dpnl, the PCR products were then transformed into chemically competent E. coli Top10 cells, the reaction products were suspended in SOB medium. After incubation at 37 °C for 1 hour, ali 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: pCm-aroGP°*D7X (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 I (FM-IT) at 30 °C for 24 h. FM-II was nearly the same as reported previously (Gu et al., 2012), but contained 0.5 g/L instead of 5 g/L of ; MgSO4-7H20 and had 30 g/L initial glucose. Additionally, 12 g/L K:HPO* (for buffering pH), | mM Phe and 0.1 mM IPTG were added. The mutants giving higher medium fluorescent (MFU) were selected for sequencing. ;

2.5 Method for measurement of fluorescent 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 {

PAT 1716 LU _91- LU101172 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 (MFT 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 al, 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 AroGfbr 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 1s 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 uM MnS04, 600 uM 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 À recombination | system integrated in the strain E. coli DY 330 can cause temperature-sensitive cell growth at ;

PAT 1716 LU _29- LU101172 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 À 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, Jülich, 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 S028A (Chen and Zeng, 2017) were first knocked out (Table 1). Deleting the aroG®'**F 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 al., 2016). The Trp biosensor is composed of tnaC, which encodes the leader sequence of the tnaCAB operon (Bischoff et al, 2014; Gong et al., 2001); the eGFP protein was fused to the upstream of tnaC (Fang et al., 2016). Specifically, the strain WS001 (Table 1) was first constructed based on the Trp producer SO028AC (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 aroG3'*F gene with the antibiotic resistance gene CmŸ which hi

PAT 1716 LU _23_ LU101172 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*!8% (Ger et al., 1994) into the chromosome of the strain WS002 at the locus of the Cm? gene using the CRISPR/Cas9 technique with the plasmids pCm-aroG¥T and pCm-aroGS'#F, 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 Lac] 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 aroGS'® 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"" 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"T 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 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. |

PAT 1716 LU — 74 — LU101172

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 AroGP6*- 7X 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 AroGP°*P7X 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 AroGP°S-P74, AroGP6L-D7P AroGPSP-PT AroGPSF-P7Y, AroGPSY-P7C, and AroGPSFPL, with the number of occurring being 7, 6, 4, 1, 1, and 1, respectively (Table 3). We then carried out fermentations in FM-I 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 AroGS!80F variant. It was found that the strains carrying the variants AroGP°6-P7A, AroGPSE-PIP, or AroGPSP-P7 which had the higher frequency of occurrence in the 20 candidates (Table 3), had also higher Trp productivity (Fig. 3). Especially the first two mutants had much higher productivity than that of the reference | strain (AroG®'8"F), 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 AroGP66-P7A, ; AroGP’®L-D7V and AroGP°P-P71 have a higher inhibitor tolerance than the variant AroG$!®F under | the test conditions. To provide more direct evidence, enzyme assay was done with purified | protein of these variants. |

PAT 1716 LU _95_ LU101172 Table 3. Comparison of fermentation results with E. coli strains containing the AroG"T, AroGS!8%F, and AroG™ variants grown on FM-II with 25 mM Phe.

Mutated Number of DCW Trp es cee | wo |b

EL CC |S | aos as

CREME [oro || wows | oo 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™ 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 AroGP®G-D7A AroGP6L-D7P and AroGP6P-D7F 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 AroG5'50F all of the three variants AroGP¢S- DTA AraGP®L-D7P and AroGP°P-P71 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, AroGP°$-P74 variant has the highest

PAT 1716 LU _26— LU101172 specific activity, which is nearly twice as high as that of the variant AroGP°P-P"1 regardless of the Phe concentration. It’s also remarkably higher than that of the positive control AroGS!8%F,

3.4 Improvement of the chorismate pathway and Trp biosynthesis To explore the impact of the best variant AroGP°S-P7A on strain development for the biosynthesis of aromatic amino acids, the variant AroG*'"*F in our previously constructed Trp- producing strain S028 was substituted with AroGP°S-P74, generating the strain S028GMI (see above). The capacity of Trp production of S028GM 1 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 S028GM 1 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 S028GM 1 (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.

Te) during the whole fermentation. In addition, during the fermentations, the strain S028GM 1 | 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 S028GMI 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 AroGP6S-D74 and AroG*'8%, These results clearly showed that the variant AroGP°S-P7A is more efficient for the chorismate pathway towards bio-production of aromatic amino acids and their derivatives. |

4. Conclusion |

PAT 1716 LU _27— LU101172 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 (AroGP6S-D7A, AroGP6L-P7P and AroGP6F- D") were revealed to be more resistant to Phe than the Phe-resistant variant AroGS'*F reported so far in literature. The replacement of AroG*'9°F with AroGP°S-P7A 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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Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., Yang, S., 2015. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl. Environ. Microbiol. 81, 2506-

2514.

Kim, S. C., Min, B. E., Hwang, H. G., Seo, S. W., Jung, G. Y., 2015. Pathway optimization by re-design of untranslated regions for L-tyrosine production in Escherichia coli. Sci. Rep. 5,

13853. [ Lee, 5. Y., Kim, H. U., 2015. Systems strategies for developing industrial microbial strains. | Nat. Biotechnol. 33, 1061-1072. Liao, J. C., Mi, L., Pontrelli, S., Luo, S., 2016. Fuelling the future: microbial engineering for the production of sustainable biofuels. Nat. Rev. Microbiol. 14, 288-304. ;

PAT 1716 LU _3]- LU101172 Lin, S., Meng, X., Jiang, J., Pang, D., Jones, G., OuYang, H., Ren, L., 2012. Site-directed mutagenesis and over expression of aroG gene of Escherichia coli K-12. Int. J. Biol. Macromol.

51, 915-919, Lu, J., Tang, J., Liu, Y., Zhu, X., Zhang, T., Zhang, X., 2012. Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization. Appl. Microbiol.

Biotechnol. 93, 2455-2462.

Luz, J. A, Hans, E., Zeng, A. P., 2014. Automated fast filtration and on-filter quenching improve the intracellular metabolite analysis of microorganisms. Eng. Life Sci. 14, 135-142, McCandliss, R. J., Poling, M., Herrmann, K., 1978. 3-Deoxy-D-arabino-heptulosonate 7- phosphate synthase. Purification and molecular characterization of the phenylalanine-sensitive isoenzyme from Escherichia coli. J. Biol. Chem. 253, 4259-4265.

Mora-Villalobos, J. A., Zeng, A. P., 2017. Protein and pathway engineering for the biosynthesis of 5-hydroxytryptophan in Escherichia coli. Eng. Life Sci. 17, 892-899.

Nagaraja, P., Yathirajan, H. S., Vasantha, R. A, 2003. Highly sensitive reaction of tryptophan with p-phenylenediamine. Anal. Biochem. 312, 157-161.

Ogino, T., Garner, C., Markley, J. L., Herrmann, K. M., 1982. Biosynthesis of aromatic | compounds: 13C NMR spectroscopy of whole Escherichia coli cells. Proc. Natl, Acad. Sci. ; USA 79, 5828-5832. | Park, J., Bae, S., Kim, J.-S., 2015. Cas-Designer: a web-based tool for choice of CRISPR-Cas9 | target sites. Bioinformatics. 31, 4014-4016. Rees, H. A, Komor, A. C., Yeh, W.-H., Caetano-Lopes, J., Warman, M., Edge, A. S., Liu, D. | R., 2017. Improving the DNA specificity and applicability of base editing through protein . engineering and protein delivery. Nat. Commun. 8, 15790. :

PAT 1716 LU _32- LU101172 Ren, Kun Xu, David Jay Segal, Zhang, Z., 2018. strategies for the enrichment and selection of genetically modified cells. Trends Biotechnol. doi: 10.1016/j.tibtech.2018.07.017.

Ren, C., Xu, K., Liu, Z., Shen, J., Han, F., Chen, Z., Zhang, Z., 2015. Dual-reporter surrogate systems for efficient enrichment of genetically modified cells. Cell Mol. Life Sci. 72, 2763-

2772.

Schoner, R., Herrmann, K. M., 1976. 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase.

Purification, properties, and kinetics of the tyrosine-sensitive isoenzyme from Escherichia coli.

J. Biol. Chem. 251, 5440-5447.

Schuster, A., Erasimus, H., Fritah, S., Nazarov, P. V., van Dyck, E., Niclou, S. P., Golebiewska, A., 2018. RNAi/CRISPR Screens; from a Pool to a Valid Hit. Trends Biotechnol. doi:

10.1016/j.tibtech.2018.08.002.

Sprenger, G. A., 2006. Aromatic amino acids. Amino Acid Biosynthesis- Pathways, Regulation and Metabolic Engineering. Springer, pp. 93-127.

Sun, M. G., Seo, M.-H., Nim, S., Corbi-Verge, C., Kim, P. M., 2016. Protein engineering by highly parallel screening of computationally designed variants. Sci. Adv. 2, e1600692.

Wu, W. B., Guo, X. L., Zhang, M. L., Huang, Q. G., Qi, F., Huang, J. Z., 2018. Enhancement of l-phenylalanine production in Escherichia coli by heterologous expression of Vitreoscilla hemoglobin. Biotechnol. Appl. Biochem. 65, 476-483.

Yu, D,, Ellis, H. M,, Lee, E.-C., Jenkins, N. A, Copeland, N. G., 2000. An efficient recombination system for chromosome engineering in Escherichia coli. Proc. Natl. Acad. Sci. USA 97, 5978-5983, Zhang, J., Zong, W., Hong, W., Zhang, Z.-T., Wang, Y., 2018. Exploiting endogenous | CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer | the strain for high-level butanol production. Met. Eng. 47, 49-59, |

PAT 1716 LU _33_ LU101172 Zhou, L.-B., Zeng, A.-P., 2015. Exploring lysine riboswitch for metabolic flux control and improvement of L-lysine synthesis in Corynebacterium glutamicum. ACS Synth. Biol. 4, 729-

734.

Zhu, X., Zhao, D., Qiu, H., Fan, F., Man, S., Bi, C., Zhang, X., 2017. The CRISPR/Cas9- facilitated multiplex pathway optimization (CFPO) technique and its application to improve the Escherichia coli xylose utilization pathway. Met. Eng. 43, 37-45. Zurawski, G., Gunsalus, R., Brown, K., Yanofsky, C., 1981. Structure and regulation of aroH, the structural gene for the tryptophan-repressible 3-deoxy-D-arabino-heptulosonic acid-7- phosphate synthetase of Escherichia coli. J. Mol. Biol. 145, 47-73.

PAT 1716 LU — 34 — LU101172

SEQUENCE LISTING <110> Technische Universität Hamburg <120> Method for in vivo screening of engineered enzyme variants <130> PAT 1716 LU <160> 35 <170> BiSSAP 1.3.6 <210> 1 <21li> 350 <212> PRT <213> Artificial Sequence . <220> <223> AroG mutant general sequence <220> <221> UNSURE <222> 6..7 <223> X = any amino acid <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 : : Ala Asn Thr Val Ala His Ala Arg Lys Ala Ile His Lys Ile Leu Lys : 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

PAT 1716 LU — LU101172 Lys Pro Arg Thr Thy Vai 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 130 335 140 Leu Asp Met Ile Thr Pro Gin Tyr Leu Ala Asp Leu Met Ser Trp Gly 145 150 155 160 Ala Ile Gly Ala Arg Thr Tkr Glu Ser Gin 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 380 185 190 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 Vai 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 <21G> 2 <211> 350 <212> PRT <213> Artificial Sequence

PAT 1716 LU —36 — LU101172 <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

Ala Asn Thr Val Ala His Ala Arg Lys Ala Ile His Lys Ile Leu Lys 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 115 320 125 Leu Leu Leu Asp Ile Asn Asp Ser Gly Leu Pro Ala Ala Giy 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 Gin 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 Glan “Wo

PAT 1716 LU _37_ LU101172 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 Gin Asn Leu Pro Leu Arg Ile Lys Glu Ile Lys Glu Leu 1 5 LC 15 Leu Pro Pro Val Ala Leu Leu Glu Lys Phe Pro Ala Thr Giu Asn Ala

Ala Asn Thr Val Ala His Ala Arg Lys Ala Tle His Lys Ile Leu Lys 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 Vai 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 Gin Tyr Leu Ala Asp Leu Met Ser Trp Gly Wwe

PAT 1716 LU _38_ LU101172 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 i Ë

PAT 1716 LU _39_ LU101172

Ala Asn Thr Val Ala His Ala Arg Lys Ala Ile His Lys Ile Leu Lys 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 SC 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 Giy 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 Iie 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 Giy Lys Ser Ile Thr Asp Ala Cys Ile Gly Trp Glu Asp Thr Asp Ala 325 330 335 di

PAT 1716 LU 40 — LU101172

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 tgagectaget gtcaag 36 <210> 6 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Primer AroG-N20 <400> 6 gctcagtect aggtataata ctagtcagga agcagtqgcgg cgcacgtttt agagctagaa 60 atagcaagtt aaaataaggc tagtccg 87 <210> 7 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Primer TrpR-N20

PAT 1716 LU _ 41 — LU101172 <400> 7 getcagtecoct aggtataata ctagttcagg tttaacaacyg gtaaagtttt agagctagaa 60 atagcaagtt aaaataagge tagtccg 87 <210> 8 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Primer Cm-N20 <400> 8 gctcagtcect aggtataata ctagttgatg aacchtgaatc gecaggtttt agagctagaa 60 atagcaagtt aaaataaggc tagtccg 87 <210> 9 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Primer Cm-N202 <400> 9 gctcagteet aggtataata ctagtgctga tgccgetgge gattegtttt agagctagaa 60 atagcaagtt aaaataaggc tagtccy 87 <210> 10 <211> 26

PAT 1716 LU —42 = LU101172

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer serA-Fus-F

<400> 10 cctacggtaa gagcatcacec gatace 26

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer serA-Fus-R

<400> 11 gccecaattge gtaccaatat gaccg 25

<210> 12 ; <211> 30 <212> DNA ] <213> Artificial Sequence ] <220> <223> Primer Cm-C1 <400> 12 Î gatataccac cgttgatata teoccaatgge 30 <210> 13 <211> 25

PAT 1716 LU

— 43 — LU101172 <212> DNA <213> Artificial Sequence <220> <223> Primer Cm-C2 <400> 13 ttacatcagc accttgtcge cttge 25 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer pN20VRCm <400> 14 caacggtggt atataaaaaa gcaccgactce gatgce 36 <210> 15 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Primer pN20VFSeraA <400> 15 ggtacgcaat tgggetegag gtgaagacga aagggectce 39 <210> 16 <21l> 53 y

PAT 1716 LU

— 44 — LU101172 <212> DNA <213> Artificial Sequence <220> <223> Primer WaroG-FF <400> 16 ggcgacaagy tgctgatota atattgcatt cactaagata agtatggcaa cac 53 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Primer aroG-fus-R <400> 17 ggcatcggtg atgctettac 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, €, G or T" <220> nf

PAT 1716 LU _45_ LU101172 <221> misc feature <222> 28..29 <223> /note="n = A, C, G or T" <400> 18 ggaacagaca tgaattatca gaacnnknnk ttacgcatca aagaaatcaa agagttactt 60 cetceetgteq € 71 <210= 19 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer D67X-R <400> 19 gttctgataa ttcatgtctg ttecagtgtt gcc 33 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer aroG S180F F <400> 20 gcatcaggge ttttttgtec ggteggcttce 30 <210> 21 <211> 30 <212> DNA

PAT 1716 LU — 46 — LU101172

<213> Artificial Sequence

<220>

<223> Primer aroG S180F R

<400> 21 gaagcecgacc ggacaaaaaa gcecectgatgo 30

<210> 22

<211> 23

<2i2> DNA

<213> Artificial Sequence

<220>

<223> Primer pTagCl

<400> 22 ttgagtgagec tgataccget cgc 23

<210> 23

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer XbaI-serA

<400> 23 gagcggataa caatteocect C 21

<210> 24

<211> 62

<212> DNA Wo

PAT 1716 LU _47_ LU101172 <213> Artificial Sequence <220> <223> Primer aroG-His-HindIII <400> 24 cgccggaage tttcattagt ggtggtggtg gtggtggecc gegacgeget tttactgcat 60 te 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 ctetttgatt tcectttgatgc gtaaaggcaa gttcetgataa ttcatatgta tatctcc 57 <21G> 27

PAT 1716 LU — 48 — LU101172

<211> 57

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer D67M-PIR

<400> 27 ctetttgatt tcttbtgatge gtaaaatagg gttctgataa ttcatatgta tatctec 57

<210> 28

<211> 57

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer D67M-GAR

<400> 28 ctctttgatt tctttgatgc gtaaagececc gttetgataa ttcatatgta tatctec 57

<210> 29

<211> 84

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer Tsen-trpR-IF

<400> 29 ggataaaceg acgttgatga gcgccacgga atggggacgt cgttactgat cogcacaget 60 gttgacaatt aatcatccgg ctcg 84 “woo

PAT 1716 LU

— 49 — LU101172 <210> 30 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> Primer Tsen-trpR-IR <400> 30 tcaatcgett ttcagcaaca cetottocag ccactggegc agctcgacgyg gogeggettt 60 cttttacttg tacagctcgt ccatgece 87 <210> 31 <211> 80 <212> DNA <213> Artificial Sequence <220> <223> Primer up-aroH-out <400> 31 ggggcgttgg tgtaaagatt attgcectca ceoctgtacqg gtgagggegt agagagatta 60 cqcqgeocgct tctagagttg 80 <210> 32 <211> 72 <212> DNA <213> Artificial Sequence <220> <223> Primer Cm-delG-R v

PAT 1716 LU — 50 — LU101172 <400> 32 gtatcttcce agccotatgca ggcatcgatg atgeotottac cgtaggecag cacctgaagt 60 cageceoccata cg 72 <210> 33 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Primer Not!-pTac-aroG <400> 33 agtgcagcgg ceactgttga caattaatca tcggectegta taatgtgtag gggaattgtg 60 agcggataac 70 <210> 34 <2il> 36 <212> DNA <213> Artificial Sequence <220> <223> Primer aroG-spel <400> 34 tgcggcacta gtttattacc cgeogacgeoge ttttac 36 <210> 35 <211> 85 <212> DNA <213> Artificial Sequence

PAT 1716 LU — 5] — LU101172 <220> <223> Primer u-rpsLp-tac <400> 35 ttgtgtgagg acgttttatt acgtgtttac gaagcaaaag ctaaaaccag gagctattta 60 ctgttgacaa ttaatcatcg gctco 85

Claims (16)

| PAT 1716 LU -52- LU101172 CLAIMS

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

2. The method according to claim 1, wherein the Cas protein is a class II Cas protein, preferably a Cas9 protein.

3. The method according to claim 1 or 2, wherein the cell is further genetically engineered to include an expressible recombineering system, the recombineering system preferably being a lambda-red recombineering system, preferably under the control of an inducible promoter.

4. The method according to claim 3, wherein the donor DNA that is biotechnologically introduced into the cell comprises a donor gene coding for a variant of the enzyme necessary nr

PAT 1716 LU —33- LU101172 for the synthesis of the organic compound, and flanking sequences homologous to the target gene on the chromosome.

5. The method according to any of the preceding claims, wherein the cell is a microbial cell, preferably a bacterial cell, further preferred an enterobacterial cell, and especially preferred an Escherichia coli cell.

6. The method according to any of the preceding claims, wherein a plurality of cells as defined in claim 1 is complemented, and wherin each cell is complemented with another enzyme variant.

7. The method according to any of the preceding claims, wherein the organic compound is an essential amino acid.

8. The method according to any of the preceding claims, wherein the cell is being genetically engineered to be auxotrophic for the organic compound.

9. The method according to any of the preceding claims, wherein the gene coding for the Cas protein is contained in a first plasmid introduced into the cell, and preferably is under the control of a constitutive promoter, and wherein the CRISPR guide RNA is contained in a second plasmid introduced into the cell.

10. The method according to any of the preceding claims, wherein the donor DNA is a dsDNA, preferably contained in a plasmid.

11. The method according to claim 9, wherein the donor DNA is contained in the second plasmid together with the CRISPR guide RNA.

12. A DHAP 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.

PAT 1716 LU — 54 — LU101172

13. The DHAP synthase according to claim 12 having the sequence of one of SEQ ID NO: 2, 3 or 4.

14. A bacterial cell being genetically engineered to express a DHAP synthase according to one of claims 12 or 13.

15. The bacterial cell according to claim 14, wherein the bacterial cell is an E. coli cell.

16. Use of a bacterial cell according to one of claims 14 or 15 for the production of tryptophan.

y