Engineered microorganisms and methods for producing aromatic amino acids

Abstract

This disclosure provides engineered microorganisms capable of bioproducing aromatic amino acids, which have reduced production of unwanted byproducts and / or increased yield or conversion rate of aromatic amino acids. Methods for preparing and using said engineered microorganisms are also provided, including methods for producing aromatic amino acids.

Description

Engineered microorganisms and methods for producing aromatic amino acids

[0001] Cross-references

[0002] This application is based on PCT patent application with application number PCT / CN2024 / 070048 and application date January 2, 2024, and claims priority of the PCT patent application. The entire content of the PCT patent application is hereby incorporated into this application by reference. Technical Field

[0003] The present disclosure provides engineered microorganisms capable of bioproducing aromatic amino acids with reduced production of unwanted byproducts and / or increased yield or conversion of aromatic amino acids. Methods of preparing and using the engineered microorganisms are also provided, including methods for producing aromatic amino acids. Background Art

[0004] Aromatic amino acids, including L-tryptophan (L-TRP), L-phenylalanine (L-PHE), and L-tyrosine (L-TYR), are essential amino acids with diverse applications in the food and medical industries, such as as additives to animal feed and food, and as components of many pharmaceuticals. Consequently, there is a high industrial demand for aromatic amino acids.

[0005] Aromatic amino acids are the end products of aromatic biosynthetic pathways, including the shikimate (SHK) pathway, which links central carbon metabolism (CCM) with the biosynthesis of chorismate (CHA). These pathways exist in bacteria and some eukaryotic organisms (e.g., fungi). However, such microorganisms generally do not naturally overproduce these compounds. To achieve production, it is necessary to functionally integrate heterologous pathways or genetically modify naturally occurring pathways.

[0006] Given the high demand for aromatic amino acids, there remains a need in the art for alternative and improved methods for the bioproduction of aromatic amino acids while reducing the production of unwanted by-products and / or increasing the efficiency of aromatic amino acid production.

[0007] SUMMARY OF THE INVENTION

[0008] According to a first aspect, there is provided an engineered microbial cell for producing aromatic amino acids, comprising:

[0009] (a) heterologous expression of an exogenous transketolase or a variant thereof, wherein the exogenous transketolase or variant thereof has a higher catalytic efficiency towards glyceraldehyde-3-phosphate (G3P) and / or fructose-6-phosphate (F6P) than towards erythrose-4-phosphate (E4P) and / or xylulose-5-phosphate (X5P); and

[0010] (b) Blocked or reduced flux through the pentose phosphate pathway.

[0011] In some embodiments, the higher catalytic efficiency comprises a lower Km and / or a higher kcat. In some embodiments, the higher catalytic efficiency comprises at least a lower Km.

[0012] In some embodiments, the ratio of the exogenous transketolase or variant thereof Km for G3P or F6P to the Km for E4P or X5P is less than 1:1, eg, less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.

[0013] In some embodiments, the exogenous transketolase or variant thereof further has the following characteristics: the Km for glyceraldehyde-3-phosphate (G3P) or sedoheptulose-7-phosphate (S7P) is lower than the Km for xylulose 5-phosphate (X5P) or ribose 5-phosphate (R5P); for example, the ratio of the Km for G3P or S7P to the Km for X5P or R5P is less than 1:1, for example, less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5.

[0014] In some embodiments, the exogenous transketolase is derived from Leishmania mexicana, Scheffersomyces stipites, Suhomyces tanzawaensis, Candida subhashii, Candida tenuis, Hyphopichia burtonii, Spathaspora passalidarum, Candida intermedia, or Pichia sorbitophila.

[0015] In some embodiments, the exogenous transketolase comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-12 or a functionally equivalent variant thereof.

[0016] In some embodiments, the above-mentioned engineered microbial cell comprises the oxidative stage of the pentose phosphate pathway that blocks or reduces.In some embodiments, the engineered microbial cell comprises the activity of the reduction of 6-phosphogluconate dehydrogenase (6-phosphogluconate dehydrogenase) and / or glucose-6-phosphate dehydrogenase (glucose-6-phosphate dehydrogenase), for example, by deleting, knocking out or striking low endogenous gnd gene or zwf gene or transforming or replacing endogenous gnd gene and / or zwf gene to realize to comprise rare codon.

[0017] In some embodiments, the engineered microbial cells further comprise enhanced flux through the nitrogen assimilation pathway, e.g., comprising increased activity of glutamate dehydrogenase and / or glutamine synthetase, decreased activity of glutamate synthase, or any combination thereof. In certain embodiments, the gltBD gene is deleted, knocked down, or knocked down. In other embodiments, the gdhA and glnA genes are overexpressed. In other embodiments, the gltBD gene is deleted, knocked out, or knocked down, and the gdhA and glnA genes are overexpressed.

[0018] In some embodiments, the engineered microbial cell comprises: (i) an exogenous transketolase having a ratio of Km for F6P to Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P of less than 1:1 (e.g., 1:4); and (ii) a deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene; optionally further comprising (iii) a deletion, knockout, or knockdown of an endogenous gltBD gene.

[0019] In some embodiments, the engineered microbial cells further comprise reduced endogenous transketolase activity, for example, by deleting, knocking out, or knocking down the endogenous tktA gene and / or tktB gene, or modifying or replacing the endogenous tktA gene and / or tktB gene to include rare codons.

[0020] In some embodiments, the engineered microbial cells further comprise reduced endogenous transaldolase activity, for example, by deleting, knocking out, or knocking down the endogenous talA gene and / or talB gene, or modifying or replacing the endogenous talA gene and / or talB gene to comprise rare codons.

[0021] In some embodiments, at least two or more optional modifications can be combined to obtain an engineered microbial cell with desired properties. Thus, in some embodiments, the engineered microbial cell comprises: (i) an exogenous transketolase having a Km for F6P to a Km for X5P ratio of less than 1:1 (e.g., 1:4), and a Km for S7P to a Km for X5P ratio of less than 1:1 (e.g., 1:4); and (ii) a deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene;

[0022] Optionally, the engineered microbial cell further comprises any of the following:

[0023] (iii) deletion, knockout, or knockdown of the endogenous gltBD gene;

[0024] (iv) deletion, knockout or knockdown of the endogenous tktA gene and / or tktB gene;

[0025] (v) deletion, knockout or knockdown of the endogenous talA gene and / or talB gene;

[0026] (vi) Any combination of (iii)-(v).

[0027] In some embodiments, any of the aforementioned engineered microbial cells described herein optionally further comprise one or more additional genetic modifications that promote or optimize the biosynthesis of aromatic amino acids.

[0028] In some embodiments, the engineered microbial cells optionally further comprise a blocked or weakened TCA cycle flux. In some embodiments, the engineered microbial cells comprise reduced activity of pyruvate dehydrogenase and / or citrate synthase, for example, by deleting, knocking out, or knocking down the lpd, aceF, aceE gene or gltA gene, or by modifying or replacing the endogenous lpd, aceF, aceE gene or gltA gene to include rare codons. In some embodiments, the engineered microbial cells comprise reduced activity of citrate synthase, for example, by modifying or replacing the endogenous gltA gene to include rare codons.

[0029] In some embodiments, above-mentioned engineered microorganism cell also comprises the oxaloacetic acid biosynthetic pathway of blocking or reducing.In some embodiments, described engineered microorganism cell comprises the activity of the phosphoenolpyruvate carboxylase (phosphoenolpyruvate carboxylase) that reduces, for example, by deleting, knocking out or striking low ppc or transforming or replacing endogenous ppc gene to realize to comprise rare codon.

[0030] In some embodiments, the engineered microbial cells further comprise an inactivated, blocked, or weakened naturally occurring phosphotransferase (PTS) transport system. In some embodiments, the engineered microbial cells comprise reduced activity of one or more PTS system proteins, for example, by deleting, knocking out, or knocking down one or more genes selected from ptsH, ptsI, crr, ptsG, or any combination thereof, or modifying or replacing endogenous ptsH, ptsI, crr, ptsG genes, or any combination thereof, to include rare codons.

[0031] In some embodiments, the engineered microbial cell further comprises an increased activity of at least one non-PTS sugar transporter, such as overexpression of an endogenous non-PTS sugar transporter or heterologous expression of an exogenous non-PTS sugar transporter, such as a glucose facilitator (Glf) (e.g., from Zymomonas mobilis). In some embodiments, the engineered microbial cell further comprises an increased activity of glucokinase, such as overexpression of an endogenous glk gene or heterologous expression of an exogenous glk gene (e.g., from Zymomonas mobilis). In some embodiments, the engineered microbial cell comprises: one or more gene deletions, knockouts, or knockdowns selected from ptsH, ptsI, crr, ptsG, or any combination thereof, or modification or replacement of endogenous ptsH, ptsI, crr, ptsG genes, or any combination thereof, to comprise rare codons; and overexpression of an endogenous glf gene or heterologous expression of an exogenous glk gene (e.g., from Zymomonas mobilis).

[0032] In some embodiments, the engineered microbial cells further comprise blocked or reduced acetic acid and / or lactic acid production pathways. In some embodiments, the engineered microbial cells comprise reduced activity of one or more enzymes selected from phosphate acetyltransferase, pyruvate dehydrogenase, lactate dehydrogenase, or any combination thereof; for example, by deleting, knocking out, or knocking down one or more genes selected from pta, poxB, ldhA, or any combination thereof, or modifying or replacing endogenous pta, poxB, ldhA, or any combination thereof to include rare codons.

[0033] In some embodiments, the engineered microbial cells further comprise an enhanced phosphoenolpyruvate (PEP) biosynthetic pathway. In certain embodiments, the engineered microbial cells comprise increased phosphoenolpyruvate synthase activity to enhance the conversion of pyruvate to PEP, for example, by overexpression of endogenous ppsA or heterologous expression of an exogenous ppsA gene. In other embodiments, the engineered microbial cell comprises increased activity of pyruvate carboxylase and PEP carboxykinase to enhance the conversion of pyruvate to PEP, for example, by overexpression of an endogenous or exogenous pyc gene and a combination of an endogenous or exogenous pck gene, for example, by overexpression of endogenous pyc and pck genes, heterologous expression of exogenous pyc and pck genes, or, by overexpression of an endogenous pck gene and heterologous expression of an exogenous pyc gene (e.g., from Cytobacillus firmus).

[0034] In some embodiments, at least two or more optional modifications can be combined to obtain an engineered microbial cell with desired properties. Thus, in some embodiments, the engineered microbial cell comprises:

[0035] (i) an exogenous transketolase having a ratio of Km for F6P to Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P of less than 1:1 (e.g., 1:4); and

[0036] (ii) deletion, knockout, or knockdown of the endogenous gnd gene and / or zwf gene;

[0037] Optionally, the engineered microbial cell further comprises any of the following:

[0038] (iii) deletion, knockout, or knockdown of the endogenous gltBD gene;

[0039] (iv) deletion, knockout or knockdown of the endogenous tktA gene and / or tktB gene;

[0040] (v) deletion, knockout or knockdown of the endogenous talA gene and / or talB gene;

[0041] (vi) the endogenous gltA gene was modified or replaced to include rare codons;

[0042] (vii) deletion, knockout, or knockdown of the endogenous ppc gene;

[0043] (viii) deletion, knockout, or knockdown of endogenous ptsH, ptsI, crr, ptsG, or any combination thereof;

[0044] (ix) overexpression of endogenous glf gene or glk gene or heterologous expression of exogenous glf gene or glk gene;

[0045] (x) deletion, knockout or knockdown of endogenous pta, poxB, ldhA or any combination thereof;

[0046] (xi) overexpression of endogenous ppsA, pyc, pck genes or any combination thereof or heterologous expression of exogenous ppsA, pyc, pck genes or any combination thereof;

[0047] Any combination of (xii)(iii)-(xi).

[0048] Any aforementioned engineered microbial cell described herein can be bacterium, fungi or yeast.In some embodiments, described engineered microbial cell is intestinal bacteria (Escherichia coli), Corynebacterium glutamicum (Corynebacterium glutamicum), subtilis (Bacillus subtilis) or Bacillus licheniformis (Bacillus licheniformi).

[0049] According to a second aspect, there is also provided use of any of the aforementioned engineered microbial cells described herein in the biosynthesis of aromatic amino acids.

[0050] According to a third aspect, a method for producing an aromatic amino acid is also provided, comprising culturing any of the aforementioned engineered microbial cells described herein in a culture medium. In some embodiments, the method further comprises recovering the aromatic amino acid from the culture.

[0051] According to the fourth aspect, there is also provided a method for reducing the production of by-products and / or increasing the yield or conversion rate of aromatic amino acids in aromatic amino acid biosynthesis, comprising introducing the genetic modification as defined in the first aspect into a microbial cell having an aromatic amino acid biosynthetic pathway.

[0052] Further features and advantages of certain embodiments of the present disclosure will become more apparent from the following description of the embodiments and the accompanying drawings, and from the claims.

[0053] BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1. Knockout of the gnd gene affects strain growth.

[0055] Figure 2. Introduction of exogenous tkt gene reduces byproduct accumulation.

[0056] Figure 3. Introduction of exogenous tkt mutants increases tryptophan production.

[0057] Figure 4. Introduction of exogenous tkt mutants and overexpression of ppsA increase tryptophan production.

[0058] Figure 5. Knockdown of gltBD increases tryptophan production.

[0059] Figure 6. Alterations in nitrogen cycling increase tryptophan conversion.

[0060] Figure 7. Combined modification of the nitrogen cycle and tkt strategies increases tryptophan production.

[0061] Figure 8. Combined modification of the carbon cycle, nitrogen cycle, and tkt strategies increases tryptophan conversion.

[0062] Figure 9. Combined modification of the tkt strategy and the TCA attenuation strategy increases tryptophan conversion efficiency.

[0063] Figure 10. Knockout of zwf increases tryptophan conversion.

[0064] Detailed Description of the Invention

[0065] definition

[0066] The genes disclosed in this disclosure are all known in the art and their sequences are available from public databases. The following exemplary sequences of genes and gene products are provided.

[0067] Abbreviations used herein have their conventional meanings in the fields of chemistry and biology. Some abbreviations used in this article are as follows: aromatic amino acid (AAA), tryptophan (TRP), phenylalanine (PHE), tyrosine (TYR), phosphoenolpyruvate (PEP), chorismate (CHA), pyruvate (PYR), glyceraldehyde-3-phosphate (G3P), fructose-6-phosphate (F6P), erythrose-4-phosphate (E4P), xylulose 5-phosphate (X5P), sedoheptulose-7-phosphate (S7P), ribose-5-phosphate (R5P), pentose phosphate pathway (HMP), tricarboxylic acid cycle (TCA), phosphoenolpyruvate:sugar phosphotransferase system (PTS).

[0068] In the present disclosure, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer in the range, and, where appropriate, its fraction. Unless otherwise indicated, the term "about" as used herein refers to ±10% (e.g., ±5%, ±2% or ±1%) of the indicated range or value. It should be understood that the terms "a" and "an" as used herein refer to "one or more / one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination thereof.

[0069] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In order to better understand the present disclosure, the definitions and explanations of the terms are provided below.

[0070] As used herein, the term "aromatic amino acid" refers to tryptophan (TRP), phenylalanine (PHE) and tyrosine (TYR). In general, amino acids synthesized by organisms can be in L-configuration. Therefore, in some embodiments of the present invention, tryptophan, phenylalanine and tyrosine are in L-configuration. In the examples herein, the advantages of the engineered microbial cells of the present invention are demonstrated using tryptophan as an example. However, it is readily understood that the engineered microbial cells of the present invention can also be used to produce other aromatic amino acids, such as phenylalanine (PHE) and tyrosine (TYR).

[0071] As used herein, the term "Km," also known as the Michaelis constant, refers to the substrate concentration at which the reaction rate is 50% of Vmax (the maximum rate of the reaction when all enzyme active sites are saturated with substrate). Km is a measure of the affinity of an enzyme for its substrate; the lower the Km value, the more efficiently the enzyme performs its function at lower substrate concentrations.

[0072] The term "kcat" is the turnover number, which describes the number of times each enzyme site converts substrate to product per unit time. The higher the kcat, the more substrate is turned over per second.

[0073] In certain embodiments, the Km and kcat of a transketolase can be measured as follows: in the forward reaction, the kinetic parameters of the transketolase are determined by detecting the conversion of D-glyceraldehyde-3-phosphate from D-xylulose-5-phosphate and its paired aldehyde acceptor using the auxiliary enzymes triosephosphate isomerase and glycerol-3-phosphate dehydrogenase (Sprenger GA, et al. Eur J Biochem. 1995; 230(2):525-532.); in the reverse reaction, the kinetic parameters of the transketolase are determined by detecting the conversion of D-xylulose-5-phosphate from D-sedoheptulose-7-phosphate and its paired aldehyde acceptor using sorbitol dehydrogenase. The transketolase-catalyzed reaction can be carried out at 25°C in 100 μl of a reaction solution containing 20 mM Tris (pH 8.0), 100 mM NaCl, 1 mM ThDP, 0.1 mM NADH, 1 U ml -1Glycerol-3-phosphate dehydrogenase and triosephosphate isomerase, or 1 U ml for the reverse reaction -1 Sorbitol dehydrogenase. NADH consumption was monitored continuously at 340 nm for 10 min using a Beckman DU-800 spectrophotometer (Beckman Coulter, Fullertron, CA, USA).

[0074] As used herein, the term "endogenous" refers to a nucleic acid or protein present in or expressed in the wild-type or parental microorganism from which the engineered microorganism disclosed herein is derived. For example, an endogenous gene is a gene naturally present in the wild-type or parental microorganism from which the engineered microorganism disclosed herein is derived. In one embodiment, the expression of an endogenous gene can be controlled by an exogenous regulatory element, such as an exogenous promoter.

[0075] As used herein, the term "exogenous" refers to nucleic acids or proteins derived from the outside of the engineered microorganisms disclosed herein. For example, exogenous genes or enzymes can be artificially or recombinantly produced and introduced into the engineered microorganisms disclosed herein or expressed therein. Exogenous genes or enzymes can also be separated from heterologous microorganisms and introduced into the engineered microorganisms disclosed herein or expressed therein. Exogenous nucleic acids can be adapted to be integrated into the genome of the engineered microorganisms disclosed herein or to maintain an extrachromosomal state in the engineered microorganisms disclosed herein, for example, in a plasmid.

[0076] As used herein, the term "heterologous" refers to a nucleic acid or protein that is not present in the wild type or parent microorganism from which the engineered microorganism disclosed herein is derived. For example, a heterologous gene or enzyme can be derived from a different strain or species and introduced into or expressed in an engineered microorganism disclosed herein. A heterologous gene or enzyme can be introduced into or expressed in an engineered microorganism disclosed herein in a form that it appears in a different strain or species. Alternatively, a heterologous gene or enzyme can be modified in some way, such as by codon-optimizing it for expression in an engineered microorganism disclosed herein or by engineering it to change function, such as reversing the direction of enzymatic activity or changing substrate specificity.

[0077] As used herein, the term "heterologous expression" involves the introduction of a gene encoding a protein of interest from one species or cell into another species or cell, thereby allowing the host cell to express the foreign protein.

[0078] As used herein, the term "microbe," "microorganism," or "microbial cell" means any organism that exists in microscopic cellular form, including within the categories of archaea, bacteria, or eukaryotes. Thus, the term is intended to encompass prokaryotic or eukaryotic cells or organisms of microscopic size, and includes all species of bacteria, archaea, and eubacteria, as well as eukaryotic microorganisms such as yeast and fungi.

[0079] As used herein, the term "host" refers to a cell or microorganism that can be genetically modified, for example, by mutation, use of exogenous nucleic acid molecules, by knockout, or a combination thereof, to improve the production of aromatic amino acids relative to an unmodified host cell. In certain embodiments, the host cell may optionally already possess other genetic modifications that confer other desired properties.

[0080] As used herein, the term "parent cell" refers to a cell having the same genetic background as the host cell disclosed herein, except that it does not comprise a specific genetic modification (e.g., a mutation or knockout of an endogenous nucleic acid, an introduction of an exogenous or heterologous nucleic acid, or a combination thereof), and serves as a starting point for introducing the genetic modification, resulting in the generation of a host cell disclosed herein. In addition, it is easy to understand that when describing the relative change (e.g., blocking, reducing, weakening, knocking down, enhancing, improving, increasing, etc.) of a substance such as a gene or protein or pathway, if no reference object is specified, the reference object can be a corresponding substance such as a gene or protein or pathway in a wild-type cell or parent cell.

[0081] As used herein, the term "overexpression" refers to a level of gene expression or gene product in a non-natural or recombinant microorganism that is higher than that found in the parent or wild-type microorganism when grown under the same conditions. In certain embodiments, overexpression can occur at the transcriptional level, translational level, or both, which can be due to altered regulatory control (e.g., use of a strong promoter) or an increase in copy number, or both.

[0082] As used herein, the terms "delete", "knockout" and "knockdown" have meanings that are generally understood by those skilled in the art. The term "delete" refers to the removal or destruction of a specific gene in the genome of an organism so that the gene cannot be normally expressed, transcribed or translated. The term "knockout" refers to the complete deletion or inactivation of the DNA sequence of the target gene from a cell or organism, blocking its transcription and translation processes, so that the target gene completely loses its function. Deletion or knockout of endogenous genes can be achieved by any gene editing system known in the art, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeats (CRISPR). The term "knockdown" refers to reducing the expression level of a target gene by gene silencing in a cell or organism, resulting in a temporary change in expression. Exemplary gene silencing methods include, but are not limited to, RNA interference (RNAi), small interfering RNA (siRNA) or short hairpin RNA (shRNA), which are used to inactivate the messenger RNA of a specific gene and effectively inhibit the expression of the gene.

[0083] As used herein, the term "rare codon" refers to a codon encoding an amino acid that is used less frequently during gene expression in a particular species or cell. For example, when expressing a foreign gene in E. coli, rare codons that are less frequently used by E. coli can reduce the availability of cognate tRNAs, thereby reducing the efficiency and level of protein expression. For a particular species or cell, those skilled in the art can easily determine rare codons using various analytical tools (e.g., bioinformatics tools).

[0084] As used herein, the term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows expression of a protein encoded by a polynucleotide inserted therein, the vector is referred to as an expression vector. A vector may have genetic material elements carried and expressed in a host cell by transformation, transduction or transfection into a host cell. Vectors are well known to those skilled in the art and include but are not limited to plasmids, bacteriophages, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector may include multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector may include an origin of replication.

[0085] As used herein, the term "homolog" describes those nucleotide sequences that have sequence similarity and encode polypeptides that share at least one functional characteristic (e.g., biochemical activity), or those polypeptides that have sequence similarity and simultaneously have at least one functional similarity (e.g., biochemical activity).

[0086] As used herein, the term "ortholog" refers to a homologous gene or protein that is functionally equivalent to a gene or protein referenced in another species. Orthologous sequences are homologous sequences in different species that are derived from the vertical descent of a single sequence from the last common ancestor, wherein the sequence and its primary function are conserved. Homologous sequences are sequences inherited by a common ancestor in two species. When referring to an amino acid or nucleotide / nucleic acid sequence from a given species, the term "ortholog" refers to the same amino acid or nucleotide / nucleic acid sequence from different species. It should be understood that when two sequences are closely related in terms of their sequence and their biological function by linear descent, they are orthologs to each other. Orthologs will typically have a high degree of sequence identity, but may not (and usually will not) have 100% sequence identity.

[0087] As used herein, the term "paralog" describes a homologous sequence derived from a sequence duplication event. Paralogous sequences generally belong to the same species, but this is not essential. Paralogs can be divided into in-paralogs (in-paralogs) (paralogs occurring after the speciation event) and out-paralogs (out-paralogs) (paralogs occurring before the speciation event). Outer paralogs between species are paralogs present in two organisms due to duplication before speciation. Outer paralogs in species are paired paralogs present in the same organism, but their duplication event occurs after speciation. Paralogs generally have identical or similar functions.

[0088] As used herein, the term "variant" is a polynucleotide or polypeptide that is different from a reference polynucleotide or polypeptide but retains basic properties. Typical variants of a polynucleotide are different from another reference polynucleotide in nucleotide sequence. Changes in the nucleotide sequence of a variant may or may not change the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Typical variants of a polypeptide are different from another reference polypeptide in amino acid sequence. Generally speaking, the differences are limited so that the sequences of the reference polypeptide and the variant are very similar overall and are identical in many regions. Variant and reference polypeptide may differ in amino acid sequence due to one or more substitutions, additions, or deletions in any combination. The variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or it may be an unknown naturally occurring variant. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art. Due to the retention of basic properties (or functional properties), variants are also referred to as functional variants or functionally equivalent variants in this article.

[0089] As used herein, the term "identity" refers to a measure of similarity between nucleotide sequences or amino acid sequences. In general, sequences are aligned to obtain a maximum match. "Identity" has a meaning well known in the art and can be calculated by a disclosed algorithm (e.g., BLAST).

[0090] Engineered microorganisms producing AAA

[0091] The present disclosure provides engineered biosynthetic pathways to facilitate or optimize the biosynthesis of aromatic amino acids.

[0092] According to some embodiments, the present disclosure improves the production efficiency of aromatic amino acids by at least (i) introducing an exogenous transketolase with specific substrate selectivity to produce erythrose 4-phosphate (E4P) independently of the pentose phosphate pathway and (ii) reducing the carbon flux into the oxidative stage of the pentose phosphate pathway.

[0093] In certain aspects, provided herein are engineered microbial cells that produce aromatic amino acids, comprising:

[0094] (a) heterologous expression of an exogenous transketolase, wherein the exogenous transketolase or a variant thereof has a higher catalytic efficiency towards glyceraldehyde-3-phosphate (G3P) and / or fructose-6-phosphate (F6P) than towards erythrose-4-phosphate (E4P) and / or xylulose-5-phosphate (X5P); and

[0095] (b) Blocked or reduced flux through the pentose phosphate pathway.

[0096] Transketolase

[0097] The introduction of an exogenous transketolase or a variant thereof as provided herein can control the directionality of the reaction from G3P+F6P to E4P+X5P independently of the pentose phosphate pathway. For example, by providing a transketolase having a higher catalytic efficiency for G3P and / or F6P than for E4P and / or X5P; or by providing a transketolase having a higher catalytic activity (i.e., transketolase activity) for G3P and / or F6P than for E4P and / or X5P, the reaction directionality can be controlled.

[0098] In some embodiments, the exogenous transketolase or variant thereof provided herein further controls the directionality of the reaction from G3P+S7P to X5P+R5P independently of the pentose phosphate pathway. The reaction directionality can be controlled, for example, by providing a transketolase having a higher catalytic efficiency for G3P and / or S7P than for X5P and / or R5P; or by providing a transketolase having a higher catalytic activity (i.e., transketolase activity) for G3P and / or S7P than for X5P and / or R5P.

[0099] In some embodiments, the exogenous transketolase or variant thereof has at least the following characteristics: the catalytic efficiency or transketolase activity on fructose-6-phosphate (F6P) is higher than that on xylulose-5-phosphate (X5P), and optionally also has the following characteristics: the catalytic efficiency or transketolase activity on sedoheptulose 7-phosphate (S7P) is higher than that on xylulose 5-phosphate (X5P).

[0100] I. Catalytic Activity

[0101] Transketolase catalyzes the reversible transfer of two-carbon (1,2-dihydroxyethyl) units from ketose phosphate to the C1 position of aldose phosphate, thereby providing a reversible link between the glycolytic and pentose phosphate pathways, along with the Schiff base-forming transaldolase. Transketolase acts on ketose phosphate (donor) and aldose phosphate (acceptor) substrates of varying carbon chain lengths (3-7 carbons) via two major, essentially reversible reactions:

[0102] In some embodiments, the exogenous transketolase or variant thereof has higher transketolase activity on glyceraldehyde-3-phosphate (G3P) and / or fructose-6-phosphate (F6P) than on erythrose-4-phosphate (E4P) and / or xylulose 5-phosphate (X5P).

[0103] In some embodiments, the exogenous transketolase or variant thereof has one or more of the following: (i) a transketolase activity on glyceraldehyde-3-phosphate (G3P) that is greater than the transketolase activity on erythrose-4-phosphate (E4P); (ii) a transketolase activity on glyceraldehyde-3-phosphate (G3P) that is greater than the transketolase activity on xylulose-5-phosphate (X5P); (iii) a transketolase activity on fructose-6-phosphate (F6P) that is greater than the transketolase activity on erythrose-4-phosphate (E4P); (iv) a transketolase activity on fructose-6-phosphate (F6P) that is greater than the transketolase activity on xylulose-5-phosphate (X5P); or any combination thereof.

[0104] In some embodiments, the exogenous transketolase and variants have at least the following characteristics: a transketolase activity on fructose-6-phosphate (F6P) is higher than a transketolase activity on xylulose-5-phosphate (X5P).

[0105] In some embodiments, the ratio of transketolase activity of the exogenous transketolase and variant on G3P or F6P to transketolase activity on E4P or X5P is greater than 1: 1. In some embodiments, the ratio is greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1.

[0106] In some embodiments, the exogenous transketolase and variants have one or more of the following: (i) a ratio of transketolase activity on G3P to transketolase activity on E4P greater than 1:1, e.g., greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1; (ii) a ratio of transketolase activity on G3P to transketolase activity on X5P greater than 1:1, e.g., greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1; , 4.5:1 or 5:1; (iii) the ratio of transketolase activity on F6P to transketolase activity on E4P is greater than 1:1, e.g., greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1; (iv) the ratio of transketolase activity on F6P to transketolase activity on X5P is greater than 1:1, e.g., greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1; or any combination thereof.

[0107] In some embodiments, the exogenous transketolase and variants have at least the following characteristics: the ratio of transketolase activity on F6P to transketolase activity on X5P is higher than 1:1, such as higher than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, such as higher than 1:4.

[0108] In some embodiments, the exogenous transketolase and variants described herein also have higher transketolase activity on glyceraldehyde-3-phosphate (G3P) or sedoheptulose-7-phosphate (S7P) than on xylulose-5-phosphate (X5P) or ribose-5-phosphate (R5P). In some embodiments, the exogenous transketolase and variants have one or more of the following: (a) a transketolase activity on glyceraldehyde-3-phosphate (G3P) that is greater than the transketolase activity on xylulose-5-phosphate (X5P); (b) a transketolase activity on glyceraldehyde-3-phosphate (G3P) that is greater than the transketolase activity on ribose-5-phosphate (R5P); (c) a transketolase activity on sedoheptulose-7-phosphate (S7P) that is greater than the transketolase activity on xylulose-5-phosphate (X5P); (d) a transketolase activity on sedoheptulose-7-phosphate (S7P) that is greater than the transketolase activity on ribose-5-phosphate (R5P); or any combination thereof.

[0109] In some embodiments, the ratio of transketolase activity of the exogenous transketolase and variant on G3P or S7P to transketolase activity on X5P or R5P is greater than 1: 1. In some embodiments, the ratio is greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1.

[0110] In some embodiments, the exogenous transketolase and variants have one or more of the following: (a) a ratio of transketolase activity on G3P to transketolase activity on X5P greater than 1:1, e.g., greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1; (b) a ratio of transketolase activity on G3P to transketolase activity on R5P greater than 1:1, e.g., greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1. 1, 4.5:1 or 5:1; (c) the ratio of transketolase activity on S7P to transketolase activity on X5P is greater than 1:1, for example, greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1; (d) the ratio of transketolase activity on S7P to transketolase activity on R5P is greater than 1:1, for example, greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1; or any combination thereof.

[0111] In some embodiments, the ratio of transketolase activity of the exogenous transketolase or variant thereof on S7P to transketolase activity on X5P is higher than 1:1, such as higher than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1, such as higher than 4:1.

[0112] In some embodiments, the exogenous transketolase and variants have at least the following characteristics: higher transketolase activity on fructose-6-phosphate (F6P) than on xylulose-5-phosphate (X5P), and optionally further have the following characteristics: higher transketolase activity on sedoheptulose-7-phosphate (S7P) than on xylulose-5-phosphate (X5P).

[0113] In some embodiments, the exogenous transketolase or variant thereof has a ratio of transketolase activity on F6P to transketolase activity on X5P that is higher than 1:1 (e.g., 4:1), and a ratio of transketolase activity on S7P to transketolase activity on X5P that is higher than 1:1 (e.g., 4:1).

[0114] II. Catalytic Efficiency

[0115] In some embodiments, the exogenous transketolase or variant thereof has a higher catalytic efficiency for glyceraldehyde 3-phosphate (G3P) and / or fructose 6-phosphate (F6P) than for erythrose 4-phosphate (E4P) and / or xylulose 5-phosphate (X5P).

[0116] The term "catalytic efficiency" as used herein may be given by any enzyme kinetic parameter known to those skilled in the art, such as Km, kcat and the kcat / Km ratio.

[0117] The Km value is inversely proportional to the affinity of the enzyme for its substrate and its catalytic efficiency. A high Km value corresponds to a low affinity for the substrate and, therefore, a low catalytic efficiency for the substrate. A low Km value for an enzyme corresponds to a high affinity for the substrate and, therefore, a high catalytic efficiency for the substrate.

[0118] The kcat value is positively correlated with the catalytic efficiency of an enzyme toward its substrate. A high kcat value corresponds to a high catalytic efficiency toward the substrate. A low kcat value for an enzyme corresponds to a low catalytic efficiency toward the substrate.

[0119] In some embodiments, the higher catalytic efficiency described herein comprises a lower Km and / or a higher kcat. In some embodiments, the higher catalytic efficiency described herein comprises at least a lower Km.

[0120] II-A. Km value

[0121] In some embodiments, the engineered microbial cells described herein comprise an exogenous transketolase or a variant thereof having one or more of the following: (i) a lower Km for glyceraldehyde-3-phosphate (G3P) than for erythrose-4-phosphate (E4P); (ii) a lower Km for glyceraldehyde-3-phosphate (G3P) than for xylulose-5-phosphate (X5P); (iii) a lower Km for fructose-6-phosphate (F6P) than for erythrose-4-phosphate (E4P); (iv) a lower Km for fructose-6-phosphate (F6P) than for xylulose-5-phosphate (X5P); or any combination thereof. By introducing an exogenous transketolase or a variant thereof having specific substrate selectivity, the reaction directionality from G3P+F6P to E4P+X5P can be controlled independently of the pentose phosphate pathway. In some embodiments, the variant can have a high structural similarity to the wild-type transketolase from which it is derived. In some embodiments, the variant may have at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity compared to the wild-type transketolase from which it is derived. In some embodiments, the variant may have one or more substitutions (e.g., one substitution) compared to the wild-type transketolase from which it is derived. The variant retains the specific substrate selectivity of the wild-type transketolase.

[0122] In some embodiments, the exogenous transketolase and variants have at least the following characteristics: a Km for fructose-6-phosphate (F6P) is lower than a Km for xylulose-5-phosphate (X5P).

[0123] In some embodiments, the ratio of the Km of the exogenous transketolase and variant for G3P or F6P to the Km for E4P or X5P is less than 1: 1. In some embodiments, the ratio is less than 1: 1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.

[0124] In some embodiments, the exogenous transketolase and variants have one or more of the following: (i) a ratio of Km for G3P to Km for E4P of less than 1:1, e.g., less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5; (ii) a ratio of Km for G3P to Km for X5P of less than 1:1, e.g., less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:4. , 1:4.5 or 1:5; (iii) the ratio of the Km for F6P to the Km for E4P is less than 1:1, for example, less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5; (iv) the ratio of the Km for F6P to the Km for X5P is less than 1:1, for example, less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5; or any combination thereof.

[0125] In some embodiments, the exogenous transketolase and variants have at least the following characteristics: the ratio of Km for F6P to Km for X5P is less than 1:1, such as less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, such as less than 1:4.

[0126] In some embodiments, the exogenous transketolase and variants described herein further have the following characteristics: the Km for glyceraldehyde-3-phosphate (G3P) or sedoheptulose-7-phosphate (S7P) is lower than the Km for xylulose-5-phosphate (X5P) or ribose-5-phosphate (R5P). In some embodiments, the exogenous transketolase and variant have one or more of the following: (a) a lower Km for glyceraldehyde-3-phosphate (G3P) than for xylulose-5-phosphate (X5P); (b) a lower Km for glyceraldehyde-3-phosphate (G3P) than for ribose-5-phosphate (R5P); (c) a lower Km for sedoheptulose-7-phosphate (S7P) than for xylulose-5-phosphate (X5P); (d) a lower Km for sedoheptulose-7-phosphate (S7P) than for ribose-5-phosphate (R5P); or any combination thereof. By introducing an exogenous transketolase or variant with specific substrate selectivity, the reaction directionality from G3P+S7P to X5P+R5P will be controlled independently of the pentose phosphate pathway.

[0127] In some embodiments, the ratio of the Km of the exogenous transketolase and variant for G3P or S7P to the Km for X5P or R5P is less than 1: 1. In some embodiments, the ratio is less than 1: 1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.

[0128] In some embodiments, the exogenous transketolase and variants have one or more of the following: (a) a ratio of Km for G3P to Km for X5P of less than 1:1, e.g., less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5; (b) a ratio of Km for G3P to Km for R5P of less than 1:1, e.g., less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. 4, 1:4.5 or 1:5; (c) the ratio of Km for S7P to Km for X5P is less than 1:1, for example, less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5; (d) the ratio of Km for S7P to Km for R5P is less than 1:1, for example, less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5; or any combination thereof.

[0129] In some embodiments, the ratio of the exogenous transketolase or variant thereof Km for S7P to Km for X5P is less than 1:1, such as less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, such as less than 1:4.

[0130] In some embodiments, the exogenous transketolase and variants have at least the following characteristics: the Km for fructose-6-phosphate (F6P) is lower than the Km for xylulose 5-phosphate (X5P), and optionally also have the following characteristics: the Km for sedoheptulose-7-phosphate (S7P) is lower than the Km for xylulose-5-phosphate (X5P).

[0131] In some embodiments, the exogenous transketolase or variant thereof has a ratio of Km for F6P to Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P of less than 1:1 (e.g., 1:4).

[0132] In some embodiments, the exogenous transketolase or variant thereof has a Km of about 0.02-0.04 mM (e.g., about 0.025-0.035 mM, such as about 0.029 mM) for F6P, a Km of about 0.02-0.04 mM (e.g., about 0.025-0.035 mM, such as about 0.031 mM) for S7P, and a Km of about 0.1-0.6 mM (e.g., about 0.1-0.55 mM, such as 0.145 mM) for X5P.

[0133] II-B. kcat value

[0134] In some embodiments, the exogenous transketolase or variant thereof has one or more of the following: (i) a higher kcat for glyceraldehyde-3-phosphate (G3P) than for erythrose-4-phosphate (E4P); (ii) a higher kcat for glyceraldehyde-3-phosphate (G3P) than for xylulose-5-phosphate (X5P); (iii) a higher kcat for fructose-6-phosphate (F6P) than for erythrose-4-phosphate (E4P); (iv) a higher kcat for fructose-6-phosphate (F6P) than for xylulose-5-phosphate (X5P); or any combination thereof.

[0135] In some embodiments, the exogenous transketolase and variants have at least the following characteristics: a kcat for fructose-6-phosphate (F6P) is higher than a kcat for xylulose-5-phosphate (X5P).

[0136] In some embodiments, the ratio of kcat for G3P or F6P to kcat for E4P or X5P of the exogenous transketolase and variant is greater than 1: 1. In some embodiments, the ratio is greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1.

[0137] In some embodiments, the exogenous transketolase and variants have one or more of the following: (i) a ratio of kcat for G3P to kcat for E4P greater than 1:1, e.g., greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1; (ii) a ratio of kcat for G3P to kcat for X5P greater than 1:1, e.g., greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1; , 4.5:1 or 5:1; (iii) the ratio of kcat for F6P to kcat for E4P is greater than 1:1, for example, greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1; (iv) the ratio of kcat for F6P to kcat for X5P is greater than 1:1, for example, greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1; or any combination thereof.

[0138] In some embodiments, the exogenous transketolase and variants have at least the following characteristics: the ratio of kcat for F6P to kcat for X5P is higher than 1:1, such as higher than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1, such as higher than 4:1.

[0139] In some embodiments, the exogenous transketolase and variants described herein further have the following characteristics: the kcat for glyceraldehyde-3-phosphate (G3P) or sedoheptulose-7-phosphate (S7P) is higher than the kcat for xylulose-5-phosphate (X5P) or ribose-5-phosphate (R5P). In some embodiments, the exogenous transketolase and variant have one or more of the following: (a) a higher kcat for glyceraldehyde-3-phosphate (G3P) than for xylulose-5-phosphate (X5P); (b) a higher kcat for glyceraldehyde-3-phosphate (G3P) than for ribose-5-phosphate (R5P); (c) a higher kcat for sedoheptulose-7-phosphate (S7P) than for xylulose-5-phosphate (X5P); (d) a higher kcat for sedoheptulose-7-phosphate (S7P) than for ribose-5-phosphate (R5P); or any combination thereof. By introducing an exogenous transketolase or variant with specific substrate selectivity, the reaction directionality from G3P+S7P to X5P+R5P will be controlled independently of the pentose phosphate pathway.

[0140] In some embodiments, the ratio of kcat of the exogenous transketolase and variant for G3P or S7P to kcat for X5P or R5P is greater than 1: 1. In some embodiments, the ratio is greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1.

[0141] In some embodiments, the exogenous transketolase and variants have one or more of the following: (a) a ratio of kcat for G3P to kcat for X5P greater than 1:1, e.g., greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1; (b) a ratio of kcat for G3P to kcat for R5P greater than 1:1, e.g., greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1. 1, 4.5:1 or 5:1; (c) the ratio of kcat for S7P to kcat for X5P is higher than 1:1, for example, higher than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1; (d) the ratio of kcat for S7P to kcat for R5P is higher than 1:1, for example, higher than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1; or any combination thereof.

[0142] In some embodiments, the exogenous transketolase or variant thereof has a ratio of kcat for S7P to kcat for X5P greater than 1:1, such as greater than 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, or 5:1, such as greater than 4:1.

[0143] In some embodiments, the exogenous transketolase and variants have at least the following characteristics: a kcat for fructose-6-phosphate (F6P) is higher than the kcat for xylulose 5-phosphate (X5P), and optionally also have the following characteristics: a kcat for sedoheptulose-7-phosphate (S7P) is higher than the kcat for xylulose-5-phosphate (X5P).

[0144] In some embodiments, the exogenous transketolase or variant thereof has a ratio of kcat for F6P to kcat for X5P greater than 1:1 (e.g., 4:1), and a ratio of kcat for S7P to kcat for X5P greater than 1:1 (e.g., 4:1).

[0145] III. Exemplary Transketolase

[0146] Exemplary sequences and sources of exogenous transketolase enzymes are provided below.

[0147] In some embodiments, the exogenous transketolase or variant thereof is derived from Leishmania mexicana, Scheffersomyces stipites, Debaryomyces hansenii, Suhomyces tanzawaensis, Candida subhashii, Candida tenuis, Hyphopichia burtonii, Spathaspora passalidarum, Candida intermedia, or Pichia sorbitophila.

[0148] In some embodiments, the exogenous transketolase is selected from the group consisting of: a Scheffersomyces stipites transketolase set forth in SEQ ID NO: 1 or a variant thereof. In some embodiments, the Scheffersomyces stipites transketolase set forth in SEQ ID NO: 1 has a Km for F6P of about 0.029 mM, a Km for S7P of about 0.031 mM, and a Km for X5P of about 0.145 mM.

[0149] In some embodiments, the exogenous transketolase is selected from the group consisting of: a Scheffersomyces stipites transketolase variant set forth in SEQ ID NO: 12. In some embodiments, the Scheffersomyces stipites transketolase variant set forth in SEQ ID NO: 12 has a Km for F6P of about 0.029 mM, a Km for S7P of about 0.031 mM, and a Km for X5P of about 0.517 mM.

[0150] In some embodiments, the exogenous transketolase is selected from the group consisting of: Leishmania mexicana transketolase shown in SEQ ID NO: 2 or a variant thereof.

[0151] In some embodiments, the exogenous transketolase is selected from the group consisting of: Debaryomyces hansenii transketolase shown in SEQ ID NO: 3 or a variant thereof.

[0152] In some embodiments, the exogenous transketolase is selected from the group consisting of: Suhomyces tanzawaensis transketolase shown in SEQ ID NO: 4 or a variant thereof.

[0153] In some embodiments, the exogenous transketolase is selected from the group consisting of: [Candida] subhashii transketolase shown in SEQ ID NO: 5 or a variant thereof.

[0154] In some embodiments, the exogenous transketolase is selected from the group consisting of: Candida tenuis transketolase shown in SEQ ID NO: 6 or a variant thereof.

[0155] In some embodiments, the exogenous transketolase is selected from the group consisting of: Hyphopichia burtonii transketolase shown in SEQ ID NO: 7 or a variant thereof.

[0156] In some embodiments, the exogenous transketolase is selected from the group consisting of: Spathaspora passalidarum transketolase shown in SEQ ID NO: 8 or a variant thereof.

[0157] In some embodiments, the exogenous transketolase is selected from the group consisting of: [Candida] intermedia transketolase shown in SEQ ID NO: 9 or a variant thereof.

[0158] In some embodiments, the exogenous transketolase is selected from the group consisting of: Pichia sorbitophila transketolase shown in SEQ ID NO: 10 or a variant thereof.

[0159] In some embodiments, the exogenous transketolase is selected from the group consisting of: Pichia sorbitophila transketolase shown in SEQ ID NO: 11 or a variant thereof.

[0160] In some embodiments, the exogenous transketolase comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-12.

[0161] Although exemplary sequences and sources of exogenous transketolase are provided herein, the present invention is not limited to these sequences and sources, and also encompasses variants. The term "variant" includes proteins whose sequences differ from the sequences of the reference proteins exemplified herein. For example, a variant has substantially the same specific substrate selectivity, catalytic activity, and / or catalytic efficiency as the reference exogenous transketolase exemplified herein. Such variants may be referred to herein as "functionally equivalent variants."

[0162] In some embodiments, the functionally equivalent variant has at least a Km value as defined in Section II-A above.

[0163] In some embodiments, functionally equivalent variants may have a high degree of structural similarity to the reference exogenous transketolase enzymes exemplified herein.

[0164] In some embodiments, functionally equivalent variants may have at least 90% (e.g., at least 91%, at least 92%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity compared to a reference transketolase exemplified herein.

[0165] In some embodiments, a functionally equivalent variant can have one or more substitutions (eg, one substitution) compared to a reference transketolase exemplified herein.

[0166] pentose phosphate pathway

[0167] Through engineering approaches microbial cells as herein described comprises the flux through pentose phosphate pathway that blocks or reduces.Pentose phosphate pathway (HMP) is the process that glucose-6-phosphate is decomposed into NADPH and pentose (5 carbon sugars) for downstream biological process.This approach has two different stages: oxidation stage and non-oxidation stage.The first stage is oxidation stage, wherein 6-phosphate glucose (glucose-6-phosphate) is converted into 5-phosphate ribulose (ribulose-5-phosphate).The second stage of this approach is the non-oxidation synthesis of pentose.

[0168] The phrase "blocking or reducing flux" means reducing the degree or flow of metabolic carbon through or to a desired pathway, pathway product, intermediate, or biologically derived compound. The reduction can be relative to a predetermined baseline (e.g., the level of a wild-type cell or parent cell) of the pathway product, intermediate, or biologically derived compound. For example, blocking or reducing flux through the pentose phosphate pathway can be achieved by attenuating a pentose phosphate pathway enzyme compared to a functional enzyme. As used herein, the term "pathway enzyme" refers to an enzyme that plays a role in a desired pathway that produces a naturally occurring molecule.

[0169] In some embodiments, the engineered microbial cell comprises a blocked or reduced oxidative stage of the pentose phosphate pathway, which can be achieved, for example, by reducing the activity of an enzyme involved in the oxidative stage.

[0170] In some embodiments, the engineered microbial cell comprises reduced activity of 6-phosphogluconate dehydrogenase and / or glucose-6-phosphate dehydrogenase, e.g., by deleting, knocking out, or knocking down an endogenous gene encoding the enzyme, or modifying or replacing an endogenous gene encoding the enzyme to include a rare codon.

[0171] In some embodiments, the endogenous gene encoding 6-phosphogluconate dehydrogenase is deleted, knocked out or struck low or is transformed or replaced to comprise rare codons, and the endogenous gene comprises a gnd gene or its species homologue (e.g., an orthologue or paralogue). In some embodiments where the microbial cell is Escherichia coli, the endogenous gnd gene is deleted, knocked out or struck low.

[0172] In some embodiments, the endogenous gene of the coding glucose-6-phosphate dehydrogenase is deleted, knocked out or struck low, and the endogenous gene includes zwf gene or its species homologue (for example straight to homologue or paralogue).In some embodiments that the microbial cell is Escherichia coli, endogenous zwf gene is deleted, knocked out or struck low.

[0173] Other genetic modifications (I)

[0174] In any of the aforementioned engineered microbial cells, one or more other genetic modifications that promote or optimize aromatic amino acid biosynthesis can also be introduced. For example, such genetic modifications include, but are not limited to: (i) enhanced flux through the nitrogen assimilation pathway, (ii) reduced endogenous transketolase activity, (iii) reduced endogenous transaldolase activity, and / or (iv) blocked or weakened flux through the TCA cycle.

[0175] I. Nitrogen assimilation pathway

[0176] In some embodiments, the engineered microbial cells as herein described may also comprise an enhanced flux through the nitrogen assimilation pathway. The phrase "enhanced flux" is intended to represent strengthening, increasing, or further improving the degree or flow of metabolic carbon through or arriving at the desired pathway, pathway product, intermediate, or biologically derived compound. The intensity, increase, or improvement may be with respect to a predetermined baseline (e.g., level of wild-type cell or parent cell) of a pathway product, intermediate, or biologically derived compound. For example, the enhanced flux through the nitrogen assimilation pathway may be achieved by overexpression of glutamate dehydrogenase and / or glutamine synthetase compared to a parent or wild-type microbial cell.

[0177] In some embodiments, the engineered microbial cell comprises one or more of: (i) increased activity of glutamate dehydrogenase and / or glutamine synthetase, (ii) decreased activity of glutamate synthase, or any combination thereof.

[0178] In some embodiments, the reduced activity of glutamate synthase can be achieved, for example, by deleting, knocking out, or knocking down an endogenous gene encoding glutamate synthase, or modifying or replacing an endogenous gene encoding glutamate synthase to include rare codons, wherein the endogenous gene includes a gltBD gene or a species homolog thereof (e.g., an ortholog or paralog). In some embodiments in which the microbial cell is Escherichia coli, the endogenous gltBD gene is deleted, knocked out, or knocked down.

[0179] In some embodiments, the increased activity of glutamate dehydrogenase and / or glutamine synthetase can be achieved, for example, by overexpression of endogenous genes encoding the enzymes, including gdhA and glnA genes or their species homologs (e.g., orthologs or paralogs). In some embodiments where the microbial cell is an Escherichia coli, the endogenous gdhA and glnA genes are overexpressed. In certain embodiments, overexpression can occur at the transcriptional level, translational level, or both, which can be due to altered regulatory control (e.g., use of a strong promoter) or an increase in copy number, or both.

[0180] In some embodiments where the microbial cell is E. coli, the endogenous gltBD gene is deleted, knocked out, or knocked down, and the endogenous gdhA and glnA genes are overexpressed.

[0181] II. Modification of endogenous transketolase

[0182] In some embodiments, the engineered microbial cells as described above further comprise reduced endogenous transketolase activity. Reduced activity can be achieved by any method known in the art, including but not limited to introducing gene disruption, such as complete gene deletion or knockout by gene editing systems, or knocking down by using oligonucleotides that bind to the gene encoding the enzyme or its mRNA, or by modifying or replacing endogenous genes to include rare codons, resulting in a temporary change in expression.

[0183] In some embodiments, the endogenous gene encoding transketolase is deleted, knocked out or struck low, or is modified or replaced to include rare codons. The endogenous gene encoding transketolase includes tktA gene and / or tktB gene or its species homolog (e.g., ortholog or paralog). In some embodiments where the microbial cell is Escherichia coli, the endogenous tktA gene is deleted, knocked out or struck low, or the endogenous tktA gene is modified or replaced to include rare codons. In some embodiments where the microbial cell is Escherichia coli, the endogenous tktB gene is deleted, knocked out or struck low, or the endogenous tktB gene is modified or replaced to include rare codons. In some embodiments where the microbial cell is Escherichia coli, the endogenous tktA and tktB genes are deleted, knocked out or struck low, or the endogenous tktA and tktB genes are modified or replaced to include rare codons.

[0184] In some embodiments, the engineered microbial cell comprises: (i) an exogenous transketolase having a ratio of Km for F6P to Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P of less than 1:1 (e.g., 1:4); and (ii) a deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene; optionally further comprising (iii) a deletion, knockout, or knockdown of an endogenous tktA gene and / or tktB gene.

[0185] III. Modification of endogenous transaldolase

[0186] In some embodiments, the engineered microbial cells described herein further comprise reduced endogenous transaldolase activity. Reduced activity can be achieved by any method known in the art, including but not limited to introducing gene disruption, such as complete gene deletion or knockout by a gene editing system, or by knocking down using oligonucleotides that bind to the gene encoding the enzyme or its mRNA, resulting in a temporary change in expression.

[0187] In some embodiments, the endogenous gene encoding transaldolase is deleted, knocked out or strikes low or the endogenous gene encoding transaldolase is transformed or replaced to comprise rare codons. The endogenous gene encoding transaldolase comprises talA gene and / or talB gene or its species homologue (for example orthologue or paralogue). In some embodiments that microbial cell is colibacillary, endogenous talA gene is deleted, knocked out or strikes low. In some embodiments that microbial cell is colibacillary, endogenous talB gene is deleted, knocked out or strikes low. In some embodiments that microbial cell is colibacillary, endogenous talA and talB genes are deleted, knocked out or strikes low.

[0188] In some embodiments, the engineered microbial cells described above comprise: (i) an exogenous transketolase having a ratio of Km for F6P to Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P of less than 1:1 (e.g., 1:4); and (ii) deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene; optionally further comprising (iii) deletion, knockout, or knockdown of an endogenous talA gene and / or talB gene.

[0189] IV. TCA Cycle

[0190] In some embodiments, the engineered microbial cells described herein comprise a blocked or weakened TCA cycle flux. The tricarboxylic acid cycle (TCA) is a series of biochemical reactions that produces energy in the form of ATP by the oxidation of acetyl-CoA derived from carbohydrates, fats, and proteins.

[0191] The phrase "blocked or weakened flux" means a reduction in the degree or flow of metabolic carbon through or to a desired pathway, pathway product, intermediate, or bioderived compound. The reduction can be relative to a predetermined baseline (e.g., the level of a wild-type cell or parent cell) of the pathway product, intermediate, or bioderived compound. For example, a blocked or weakened flux through the TCA pathway can be achieved by weakening a TCA pathway enzyme compared to a functional enzyme. As used herein, the term "TCA pathway enzyme" refers to an enzyme that functions in the TCA pathway.

[0192] In some embodiments, the engineered microbial cell comprises reduced acetyl-CoA flux into the TCA cycle, for example, by reducing the activity of pyruvate dehydrogenase and / or citrate synthase.

[0193] In some embodiments, the engineered microbial cell comprises reduced activity of pyruvate dehydrogenase and / or citrate synthase, e.g., by deleting, knocking out, or knocking down endogenous genes encoding pyruvate dehydrogenase and / or citrate synthase, or modifying or replacing endogenous genes encoding pyruvate dehydrogenase and / or citrate synthase to include rare codons.

[0194] In some embodiments, the endogenous gene encoding pyruvate dehydrogenase is deleted, knocked out or struck low, or the endogenous gene encoding pyruvate dehydrogenase is modified or replaced to include rare codons. The endogenous gene includes, for example, lpd, aceF, aceE genes or their species homologs (e.g., orthologs or paralogs). In some embodiments where the microbial cell is Escherichia coli, one or more genes selected from lpd, aceF, aceE are deleted, knocked out or struck low.

[0195] In some embodiments, the endogenous gene encoding citrate synthase is deleted, knocked out or knocked down, or the endogenous gene encoding citrate synthase is modified or replaced to include rare codons. The endogenous gene includes, for example, the gltA gene or a species homolog thereof (e.g., an ortholog or paralog). In some embodiments in which the microbial cell is Escherichia coli, the endogenous gltA gene is modified or replaced to include rare codons.

[0196] The codon preferences used by different species or their cells to encode certain amino acids may be different. Those codons that are most frequently utilized are called optimal codons, while those that are rarely utilized are called rare codons or codons with low utilization rates. Therefore, in this article, the term "rare codon" refers to the codons of the amino acids that are less frequently used in the gene expression process of a particular species or cell. For example, when expressing foreign genes in E. coli, the rare codons that are less used by E. coli can reduce the availability of cognate tRNA, thereby reducing the expression efficiency and level of the protein. For a particular species or cell, those skilled in the art can easily determine rare codons using various analytical tools (e.g., bioinformatics tools).

[0197] In some embodiments, the engineered microbial cell comprises: (i) an exogenous transketolase having a Km for F6P to a Km for X5P ratio of less than 1:1 (e.g., 1:4), and a Km for S7P to a Km for X5P ratio of less than 1:1 (e.g., 1:4); and (ii) a deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene; optionally, the engineered microbial cell further comprises (iii) modification or replacement of an endogenous gltA gene to include a rare codon.

[0198] Other genetic modifications (II)

[0199] In any of the aforementioned engineered microbial cells, one or more other genetic modifications that promote or optimize aromatic amino acid biosynthesis can also be introduced. For example, strategies that allow for higher PEP availability for aromatic amino acid biosynthesis can be employed, including, for example, (i) limiting the carbon flux from phosphoenolpyruvate (PEP) to oxaloacetate, (ii) replacing the glucose transport and phosphorylation capacity of the phosphotransferase (PTS) transport system with alternative enzymes, and / or (iii) improving the recycling of pyruvate (PYR) to PEP. For example, methods that reduce the accumulation of acetate and / or lactate can also be employed to overcome inhibition of microbial growth.

[0200] I. Oxaloacetate biosynthetic pathway

[0201] In some embodiments, the engineered microbial cell as described above further comprises a blocked or reduced oxaloacetate biosynthetic pathway to limit carbon flow from phosphoenolpyruvate (PEP) to oxaloacetate.

[0202] In some embodiments, described engineered microbial cell comprises the activity of the phosphoenolpyruvate carboxylase of minimizing.In some embodiments, the activity of the phosphoenolpyruvate carboxylase of minimizing can for example realize to comprise rare codon by deleting, knocking out or striking the endogenous gene of low coding phosphoenolpyruvate carboxylase or transformation or replacement coding phosphoenolpyruvate carboxylase, described endogenous gene comprises ppc gene or its species homologue (for example, directly to homologue or paralog).In microbial cell is colibacillary some embodiments, endogenous ppc gene is deleted, knocks out or strikes low.

[0203] In some embodiments, the engineered microbial cell comprises: (i) an exogenous transketolase having a Km for F6P to a Km for X5P ratio of less than 1:1 (e.g., 1:4), and a Km for S7P to a Km for X5P ratio of less than 1:1 (e.g., 1:4); and (ii) a deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene; optionally, the engineered microbial cell further comprises (iii) a deletion, knockout, or knockdown of an endogenous ppc gene.

[0204] II.PTS Transshipment System

[0205] The phosphotransferase (PTS) transport system is widely present in bacteria, fungi and some archaea. It is the main system for the transport and phosphorylation of glucose from the periplasmic space to the cytoplasmic environment, where one PEP molecule is consumed and converted into pyruvate (PYR). In order to prevent PEP from being lost to the PTS system and to increase the availability of PEP in the cell, the naturally occurring PTS system can be disabled. Therefore, in order to maintain the ability of engineered microbial cells to use a certain sugar (such as glucose) as a carbon source and energy source, it is necessary to express or overexpress an import protein that is independent of phosphoenolpyruvate:sugar phosphotransferase. In addition, the expression or overexpression of a kinase that can phosphorylate the certain sugar may also be required, or reduced and / or weakened, to respectively achieve the metabolism of the sugar or its accumulation in a non-phosphorylated form in the cell.

[0206] In some embodiments, the engineered microbial cells described above further comprise an inactivated naturally occurring phosphotransferase (PTS) transport system. In some embodiments, the inactivated naturally occurring PTS transport system comprises reduced activity of one or more PTS system proteins. In some embodiments, the PTS system proteins comprise soluble non-sugar-specific protein component enzyme I (EI, ptsI) and histidine protein (HPr, ptsH), soluble glucose-specific enzyme IIA Glc (crr) and membrane-integrated glucose-specific permease IICB Glc (ptsG).

[0207] In some embodiments, the PTS transport system is disabled by deleting, knocking out, or knocking down one or more genes selected from ptsH, ptsI, crr, ptsG, or species homologs thereof (e.g., orthologs or paralogs), or any combination thereof. In some embodiments where the microbial cell is E. coli, one or more genes selected from ptsH, ptsI, crr, ptsG are deleted, knocked out, or knocked down.

[0208] In some embodiments, the PTS transport system is disabled by deleting, knocking out, or knocking down the ptsI and / or ptsG genes or their species homologs (e.g., orthologs or paralogs). In some embodiments where the microbial cell is E. coli, the ptsI and / or ptsG genes are deleted, removed, or knocked down.

[0209] In some embodiments, the engineered microbial cell comprises: (i) an exogenous transketolase having a ratio of Km for F6P to Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P of less than 1:1 (e.g., 1:4); and (ii) a deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene; optionally, the engineered microbial cell further comprises (iii) a deletion, knockout, or knockdown of endogenous ptsH, ptsI, crr, ptsG, or any combination thereof.

[0210] In some embodiments, the engineered microbial cell further comprises an increased activity of at least one non-PTS sugar transporter. Suitable non-PEP-PTS transporters capable of transferring monosaccharides (glucose and / or fructose and / or galactose and / or N-acetylglucosamine and / or N-acetylneuraminic acid and / or fucose) into microbial cells are known to those skilled in the art, such as Glf from Zymomonas mobilis.

[0211] In some embodiments, increased activity is achieved by overexpression of an endogenous non-PTS sugar transporter.

[0212] In some embodiments, increased activity is achieved by heterologous expression of an exogenous non-PTS sugar transporter, such as the glucose-facilitating protein (Glf) of Zymomonas mobilis.

[0213] In some embodiments, the through engineering approaches microbial cell also comprises the activity of the increase of glucokinase.In some embodiments, the activity of the increase of glucokinase can be realized by the overexpression of the endogenous gene of for example encoding enzyme, and the endogenous gene comprises glk gene or its species homologue (for example, orthologue or paralogue), or realizes by the heterologous expression of exogenous glk gene (for example from zymomonas mobilis).In some embodiments that microbial cell is in escherichia coli, endogenous glk gene is overexpressed, or introduces exogenous glk gene (for example, from zymomonas mobilis).

[0214] In some embodiments, the engineered microbial cell comprises: (i) an exogenous transketolase having a Km for F6P to a Km for X5P ratio of less than 1:1 (e.g., 1:4), and a Km for S7P to a Km for X5P ratio of less than 1:1 (e.g., 1:4); and (ii) a deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene; optionally, the engineered microbial cell further comprises (iii) overexpression of an endogenous glf gene or glk gene or heterologous expression of an exogenous glf gene or glk gene.

[0215] In some embodiments, the engineered microbial cell comprises an inactivated naturally occurring phosphotransferase (PTS) transport system; and increased activity of at least one non-PTS sugar transporter. In some embodiments where the microbial cell is E. coli, one or more genes selected from ptsH, ptsI, crr, ptsG, or any combination thereof are deleted, knocked out, or knocked down, or endogenous ptsH, ptsI, crr, ptsG genes, or any combination thereof are modified or replaced to include rare codons; and overexpression of an endogenous glf gene or heterologous expression of an exogenous glk gene (e.g., from Zymomonas mobilis).

[0216] In some embodiments, the engineered microbial cell comprises: (i) an exogenous transketolase having a Km for F6P to a Km for X5P ratio of less than 1:1 (e.g., 1:4), and a Km for S7P to a Km for X5P ratio of less than 1:1 (e.g., 1:4); and (ii) a deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene; optionally, the engineered microbial cell further comprises (iii) overexpression of an endogenous glf gene or glk gene or heterologous expression of an exogenous glf gene or glk gene, and a deletion, knockout, or knockdown of endogenous ptsH, ptsI, crr, ptsG, or any combination thereof.

[0217] III. Acetic and / or lactic acid production pathways

[0218] The accumulation of acetic acid and / or lactic acid leads to an inhibitory effect on microbial growth and thus limits the production efficiency of aromatic amino acids. Therefore, reducing the accumulation of acetic acid and / or lactic acid will be beneficial to the production of aromatic amino acids.

[0219] In some embodiments, the engineered microbial cells described above further comprise a blocked or reduced acetate and / or lactate production pathway.

[0220] In some embodiments, the engineered microbial cell comprises the activity of the reduction of one or more enzymes selected from phosphate acetyltransferase, pyruvate dehydrogenase, lactate dehydrogenase or any combination thereof. In some embodiments, the activity of reduction can be achieved by, for example, deleting, knocking out or striking down one or more endogenous genes encoding one or more enzymes, the endogenous genes comprising pta, poxB, ldhA genes or their species homologs (e.g., orthologs or paralogs). In some embodiments where the microbial cell is Escherichia coli, one or more genes selected from pta, poxB and / or ldhA are deleted, knocked out or struck down.

[0221] In some embodiments, the engineered microbial cell comprises: (i) an exogenous transketolase having a Km for F6P to a Km for X5P ratio of less than 1:1 (e.g., 1:4), and a Km for S7P to a Km for X5P ratio of less than 1:1 (e.g., 1:4); and (ii) a deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene; optionally, the engineered microbial cell further comprises (iii) a deletion, knockout, or knockdown of endogenous pta, poxB, ldhA, or any combination thereof.

[0222] IV. Recirculation to PEP

[0223] The flux of carbon to phosphoenolpyruvate (PEP) can be enhanced to increase the availability of PEP for aromatic amino acid biosynthesis.

[0224] In some embodiments, the engineered microbial cell described above further comprises an enhanced phosphoenolpyruvate (PEP) biosynthetic pathway.

[0225] In some embodiments, the enhanced PEP biosynthetic pathway includes increased phosphoenolpyruvate synthase activity to enhance the conversion of pyruvate to PEP. For example, increased activity can be achieved by overexpression of an endogenous gene encoding the enzyme or heterologous expression of an exogenous gene encoding the enzyme, the endogenous gene including a ppsA gene or a species homolog thereof (e.g., an ortholog or paralog). In some embodiments where the microbial cell is E. coli, the endogenous ppsA gene is overexpressed. In some embodiments where the microbial cell is E. coli, the exogenous ppsA gene is heterologously expressed.

[0226] In some embodiments, the enhanced PEP biosynthetic pathway includes the activity of the increase of pyruvate carboxylase and PEP carboxykinase to enhance the conversion from pyruvic acid to PEP.The activity increased can for example be realized by the overexpression of endogenous or exogenous pyc gene and endogenous or exogenous pck gene combination, for example, by the overexpression of the endogenous gene of encoding enzyme, described endogenous gene includes pyc and pck gene or its species homologue (for example orthologue or paralogue), or by the heterologous expression of exogenous pyc and pck gene (for example from the exogenous pyc gene of cystic bacillus firmus).For example, in some embodiments, the activity increased can be realized by the overexpression of the endogenous pyc gene of encoding pyruvate carboxylase or its species homologue (for example orthologue or paralogue), or by the heterologous expression of exogenous pyc gene (for example from the exogenous pyc gene of cystic bacillus firmus). In some embodiments, the increased activity can be achieved by overexpression of an endogenous pck gene encoding a PEP carboxykinase or its species homologue (e.g., a direct homologue or a paralogue), or by heterologous expression of an exogenous pck gene. In some embodiments, the microbial cell is an Escherichia coli cell, endogenous pyc and pck genes are overexpressed. In some embodiments, the microbial cell is an Escherichia coli cell, endogenous pck gene is overexpressed and an exogenous pyc gene (e.g., from Bacillus firmus) is introduced.

[0227] In some embodiments, the engineered microbial cell comprises: (i) an exogenous transketolase having a Km for F6P to a Km for X5P ratio of less than 1:1 (e.g., 1:4), and a Km for S7P to a Km for X5P ratio of less than 1:1 (e.g., 1:4); and (ii) a deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene; optionally, the engineered microbial cell further comprises (iii) overexpression of endogenous ppsA, pyc, pck genes, or any combination thereof, or heterologous expression of exogenous ppsA, pyc, pck genes, or any combination thereof.

[0228] Exemplary Engineered Microorganisms

[0229] Exemplary embodiments of engineered microbial cells provided herein include, but are not limited to, the following.

[0230] In some embodiments, provided herein is an engineered microbial cell, and at least two or more optional modifications can be combined to obtain an engineered microbial cell with desired properties.

[0231] In some embodiments, provided herein is an engineered microbial cell comprising:

[0232] (i) an exogenous transketolase having a ratio of Km for F6P to Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P of less than 1:1 (e.g., 1:4); and

[0233] (ii) deletion, knockout, or knockdown of the endogenous gnd gene or zwf gene;

[0234] Optionally, the engineered microbial cell further comprises any of the following:

[0235] (iii) deletion, knockout, or knockdown of the endogenous gltBD gene;

[0236] (iv) deletion, knockout or knockdown of the endogenous tktA gene and / or tktB gene;

[0237] (v) deletion, knockout or knockdown of the endogenous talA gene and / or talB gene;

[0238] (vi) Any combination of (iii)-(v).

[0239] In some embodiments, provided herein is an engineered microbial cell comprising:

[0240] (i) an exogenous transketolase having a ratio of Km for F6P to Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P of less than 1:1 (e.g., 1:4); and

[0241] (ii) deletion, knockout, or knockdown of the endogenous gnd gene and / or zwf gene;

[0242] Optionally, the engineered microbial cell further comprises any of the following:

[0243] (iii) deletion, knockout, or knockdown of the endogenous gltBD gene;

[0244] (iv) deletion, knockout or knockdown of the endogenous tktA gene and / or tktB gene;

[0245] (v) deletion, knockout or knockdown of the endogenous talA gene and / or talB gene;

[0246] (vi) the endogenous gltA gene was modified or replaced to include rare codons;

[0247] (vii) deletion, knockout, or knockdown of the endogenous ppc gene;

[0248] (viii) deletion, knockout, or knockdown of endogenous ptsH, ptsI, crr, ptsG, or any combination thereof;

[0249] (ix) overexpression of endogenous glf gene or glk gene or heterologous expression of exogenous glf gene or glk gene;

[0250] (x) deletion, knockout or knockdown of endogenous pta, poxB, ldhA or any combination thereof;

[0251] (xi) overexpression of endogenous ppsA, pyc, pck genes or any combination thereof or heterologous expression of exogenous ppsA, pyc, pck genes or any combination thereof;

[0252] Any combination of (xii)(iii)-(xi).

[0253] In some embodiments, provided herein is an engineered microbial cell comprising:

[0254] (i) an exogenous transketolase having a ratio of Km for F6P to Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P of less than 1:1 (e.g., 1:4); and

[0255] (ii) deletion, knockout, or knockdown of the endogenous gnd gene and / or zwf gene;

[0256] (iii) deletion, knockout or knockdown of the endogenous talA gene and / or talB gene;

[0257] Optionally, the engineered microbial cell further comprises any of the following:

[0258] (iv) deletion, knockout, or knockdown of the endogenous gltBD gene;

[0259] (v) deletion, knockout or knockdown of the endogenous tktA gene and / or tktB gene;

[0260] (vi) the endogenous gltA gene was modified or replaced to include rare codons;

[0261] (vii) deletion, knockout, or knockdown of the endogenous ppc gene;

[0262] (viii) deletion, knockout, or knockdown of endogenous ptsH, ptsI, crr, ptsG, or any combination thereof;

[0263] (ix) overexpression of endogenous glf gene or glk gene or heterologous expression of exogenous glf gene or glk gene;

[0264] (x) deletion, knockout or knockdown of endogenous pta, poxB, ldhA or any combination thereof;

[0265] (xi) overexpression of endogenous ppsA, pyc, pck genes or any combination thereof or heterologous expression of exogenous ppsA, pyc, pck genes or any combination thereof;

[0266] Any combination of (xii)(iv)-(xi).

[0267] In some embodiments, provided herein is an engineered microbial cell comprising:

[0268] (i) an exogenous transketolase having a ratio of Km for F6P to Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P of less than 1:1 (e.g., 1:4); and

[0269] (ii) deletion, knockout, or knockdown of the endogenous gnd gene and / or zwf gene;

[0270] (iii) deletion, knockout, or knockdown of the endogenous gltBD gene;

[0271] Optionally, the engineered microbial cell further comprises any of the following:

[0272] (iv) deletion, knockout or knockdown of the endogenous tktA gene and / or tktB gene;

[0273] (v) deletion, knockout or knockdown of the endogenous talA gene and / or talB gene;

[0274] (vi) the endogenous gltA gene was modified or replaced to include rare codons;

[0275] (vii) deletion, knockout, or knockdown of the endogenous ppc gene;

[0276] (viii) deletion, knockout, or knockdown of endogenous ptsH, ptsI, crr, ptsG, or any combination thereof;

[0277] (ix) overexpression of endogenous glf gene or glk gene or heterologous expression of exogenous glf gene or glk gene;

[0278] (x) deletion, knockout or knockdown of endogenous pta, poxB, ldhA or any combination thereof;

[0279] (xi) overexpression of endogenous ppsA, pyc, pck genes or any combination thereof or heterologous expression of exogenous ppsA, pyc, pck genes or any combination thereof;

[0280] Any combination of (xii)(iv)-(xi).

[0281] In some embodiments, provided herein is an engineered microbial cell comprising:

[0282] (i) an exogenous transketolase having a ratio of Km for F6P to Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P of less than 1:1 (e.g., 1:4); and

[0283] (ii) deletion, knockout, or knockdown of the endogenous gnd gene and / or zwf gene;

[0284] (iii) deletion, knockout, or knockdown of the endogenous gltBD gene;

[0285] (iv) deletion, knockout or knockdown of the endogenous tktA gene and / or tktB gene;

[0286] (v) deletion, knockout or knockdown of the endogenous talA gene and / or talB gene;

[0287] Optionally, the engineered microbial cell further comprises any of the following:

[0288] (vi) the endogenous gltA gene was modified or replaced to include rare codons;

[0289] (vii) deletion, knockout, or knockdown of the endogenous ppc gene;

[0290] (viii) deletion, knockout, or knockdown of endogenous ptsH, ptsI, crr, ptsG, or any combination thereof;

[0291] (ix) overexpression of endogenous glf gene or glk gene or heterologous expression of exogenous glf gene or glk gene;

[0292] (x) deletion, knockout or knockdown of endogenous pta, poxB, ldhA or any combination thereof;

[0293] (xi) overexpression of endogenous ppsA, pyc, pck genes or any combination thereof or heterologous expression of exogenous ppsA, pyc, pck genes or any combination thereof;

[0294] (xii) Any combination of (vi)-(xi).

[0295] In some embodiments, provided herein is an engineered microbial cell comprising:

[0296] (i) an exogenous transketolase having a ratio of its Km for F6P to its Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of its Km for S7P to its Km for X5P of less than 1:1 (e.g., 1:4);

[0297] (ii) deletion, knockout, or knockdown of the endogenous gnd gene;

[0298] (iii) deletion, knockout, or knockdown of the endogenous gltBD gene;

[0299] (iv) deletion, knockout, or knockdown of the endogenous tktA and tktB genes;

[0300] (v) deletion, knockout or knockdown of the endogenous talA and talB genes; and

[0301] (vi) Overexpression of endogenous ppsA gene or heterologous expression of exogenous ppsA gene.

[0302] Optionally, the engineered microbial cell further comprises any of the following:

[0303] (vii) the endogenous gltA gene was modified or replaced to contain rare codons;

[0304] (viii) deletion, knockout, or knockdown of the endogenous ppc gene;

[0305] (ix) deletion, knockout or knockdown of endogenous ptsH, ptsI, crr, ptsG or any combination thereof;

[0306] (x) overexpression of endogenous glf gene or glk gene or heterologous expression of exogenous glf gene or glk gene;

[0307] (xi) deletion, knockout or knockdown of endogenous pta, poxB, ldhA or any combination thereof;

[0308] (xii) overexpression of endogenous pyc or pck genes or heterologous expression of exogenous pyc or pck genes;

[0309] Any combination of (xiii)(vii)-(xii).

[0310] In some embodiments, provided herein is an engineered microbial cell comprising:

[0311] (i) an exogenous transketolase having a ratio of its Km for F6P to its Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of its Km for S7P to its Km for X5P of less than 1:1 (e.g., 1:4);

[0312] (ii) deletion, knockout, or knockdown of the endogenous gnd gene;

[0313] (iii) deletion, knockout, or knockdown of the endogenous gltBD gene;

[0314] (iv) deletion, knockout, or knockdown of the endogenous tktA and tktB genes;

[0315] (v) deletion, knockout, or knockdown of the endogenous talA and talB genes;

[0316] (vi) the endogenous gltA gene is modified or replaced to contain a rare codon; and

[0317] (vii) Overexpression of endogenous ppsA gene or heterologous expression of exogenous ppsA gene.

[0318] Optionally, the engineered microbial cell further comprises any of the following:

[0319] (viii) deletion, knockout, or knockdown of the endogenous ppc gene;

[0320] (ix) deletion, knockout or knockdown of endogenous ptsH, ptsI, crr, ptsG or any combination thereof;

[0321] (x) overexpression of endogenous glf gene or glk gene or heterologous expression of exogenous glf gene or glk gene;

[0322] (xi) deletion, knockout or knockdown of endogenous pta, poxB, ldhA or any combination thereof;

[0323] (xii) overexpression of endogenous pyc or pck genes or heterologous expression of exogenous pyc or pck genes;

[0324] Any combination of (xiii)(viii)-(xii).

[0325] In some embodiments, provided herein is an engineered microbial cell comprising:

[0326] (i) an exogenous transketolase having a ratio of its Km for F6P to its Km for X5P of less than 1:1 (e.g., 1:4), and a ratio of its Km for S7P to its Km for X5P of less than 1:1 (e.g., 1:4);

[0327] (ii) deletion, knockout, or knockdown of endogenous gnd and zwf genes;

[0328] (iii) deletion, knockout, or knockdown of the endogenous gltBD gene;

[0329] (iv) deletion, knockout, or knockdown of the endogenous tktA and tktB genes;

[0330] (v) deletion, knockout or knockdown of the endogenous talA and talB genes; and

[0331] (vi) Overexpression of endogenous ppsA gene or heterologous expression of exogenous ppsA gene.

[0332] Optionally, the engineered microbial cell further comprises any of the following:

[0333] (vii) the endogenous gltA gene was modified or replaced to contain rare codons;

[0334] (viii) deletion, knockout, or knockdown of the endogenous ppc gene;

[0335] (ix) deletion, knockout or knockdown of endogenous ptsH, ptsI, crr, ptsG or any combination thereof;

[0336] (x) overexpression of endogenous glf gene or glk gene or heterologous expression of exogenous glf gene or glk gene;

[0337] (xi) deletion, knockout or knockdown of endogenous pta, poxB, ldhA or any combination thereof;

[0338] (xii) overexpression of endogenous pyc or pck genes or heterologous expression of exogenous pyc or pck genes;

[0339] Any combination of (xiii)(vii)-(xii).

[0340] Additionally, exemplary embodiments of engineered microbial cells provided herein include:

[0341] Embodiment 1. An engineered microbial cell comprising: (i) heterologous expression of an exogenous transketolase or a variant thereof as described herein; and (ii) deletion, knockout, or knockdown of an endogenous gene encoding 6-phosphogluconate dehydrogenase (e.g., gnd) or glucose-6-phosphate dehydrogenase (e.g., zwf).

[0342] Embodiment 2. The engineered microbial cell of embodiment 1, wherein the exogenous transketolase or a variant thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-12 or a functionally equivalent variant thereof.

[0343] Embodiment 3. The engineered microbial cell of embodiment 1 or 2, wherein item (ii) comprises deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene or a species homolog thereof (e.g., an ortholog or paralog). In some cases where the microbial cell is Escherichia coli, the endogenous gnd gene or zwf gene is deleted or knocked out.

[0344] Embodiment 4. The engineered microbial cell of any one of embodiments 1-3, optionally further comprising reduced endogenous transketolase activity, for example, by deleting, knocking out, or knocking down the tktA gene.

[0345] Embodiment 5. An engineered microbial cell comprising (i) heterologous expression of an exogenous transketolase or a variant thereof as described herein; and (ii) overexpression of an endogenous phosphoenolpyruvate synthase (e.g., ppsA) or heterologous expression of an exogenous phosphoenolpyruvate synthase (e.g., ppsA).

[0346] Embodiment 6. The engineered microbial cell of embodiment 5, wherein the exogenous transketolase or a variant thereof comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-12 or a functionally equivalent variant thereof.

[0347] Embodiment 7. The engineered microbial cell of embodiment 5 or 6, comprising overexpression of an endogenous gene encoding phosphoenolpyruvate synthase or heterologous expression of an exogenous gene encoding phosphoenolpyruvate synthase, wherein the endogenous gene comprises a ppsA gene or a species homolog thereof (e.g., an ortholog or paralog). In some cases where the microbial cell is E. coli, the endogenous ppsA gene is overexpressed. In some cases where the microbial cell is E. coli, the exogenous ppsA gene is heterologously expressed.

[0348] Embodiment 8. An engineered microbial cell comprising (i) deletion, knockout, or knockdown of an endogenous gltBD gene or a species homolog thereof (e.g., an ortholog or paralog), and (ii) overexpression of gdhA and glnA genes or species homologs thereof (e.g., an ortholog or paralog). In some cases where the microbial cell is Escherichia coli, the endogenous gltBD gene is deleted, knocked out, or knockdown, and the endogenous gdhA and glnA genes are overexpressed.

[0349] Embodiment 9. An engineered microbial cell comprising: (i) heterologous expression of an exogenous transketolase or a variant thereof as described herein; (ii) deletion, knockout, or knockdown of an endogenous gene encoding 6-phosphogluconate dehydrogenase (e.g., gnd) or glucose-6-phosphate dehydrogenase (e.g., zwf); and (iii) deletion, knockout, or knockdown of an endogenous gene encoding glutamate synthase (e.g., gltBD).

[0350] Embodiment 10. The engineered microbial cell of embodiment 9, wherein the exogenous transketolase or variant thereof comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1-12, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto.

[0351] Embodiment 11. The engineered microbial cell of embodiment 9 or 10, wherein item (ii) comprises deletion, knockout, or knockdown of an endogenous gnd gene or zwf gene or a species homolog thereof (e.g., an ortholog or paralog). In some cases where the microbial cell is E. coli, the endogenous gnd gene or zwf gene (e.g., gnd gene) is deleted or knocked out.

[0352] Embodiment 12. The engineered microbial cell of any one of embodiments 9-11, wherein item (iii) comprises a deletion, knockout, or knockdown of an endogenous gltBD gene or a species homolog thereof (e.g., an ortholog or paralog). In some cases where the microbial cell is E. coli, the endogenous gltBD gene is deleted or knocked out.

[0353] Embodiment 13. An engineered microbial cell comprising: (i) heterologous expression of an exogenous transketolase or a variant thereof as described herein; (ii) deletion, knockout, or knockdown of an endogenous gene encoding 6-phosphogluconate dehydrogenase (e.g., gnd); (iii) deletion, knockout, or knockdown of an endogenous gene encoding glutamate synthase (e.g., gltBD); (iv) overexpression of an endogenous gene encoding phosphoenolpyruvate synthase (e.g., ppsA) or heterologous expression of an exogenous gene encoding an exogenous phosphoenolpyruvate synthase (e.g., ppsA); (v) deletion, knockout, or knockdown of endogenous genes encoding transketolase (e.g., tktA and tktB); (vi) deletion, knockout, or knockdown of endogenous genes encoding transaldolase (e.g., talA and talB genes).

[0354] Embodiment 14. The engineered microbial cell of embodiment 13, wherein the exogenous transketolase or variant thereof comprises the amino acid sequence of any one of SEQ ID NOs: 1-12, or a sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity thereto.

[0355] Embodiment 15. The engineered microbial cell of embodiment 13 or 14, wherein item (ii) comprises a deletion, knockout, or knockdown of an endogenous gnd gene or a species homolog thereof (e.g., an ortholog or paralog). In some cases where the microbial cell is E. coli, the endogenous gnd gene is deleted or knocked out.

[0356] Embodiment 16. The engineered microbial cell of any one of embodiments 13-15, wherein item (iii) comprises a deletion, knockout, or knockdown of an endogenous gltBD gene or a species homolog thereof (e.g., an ortholog or paralog). In some cases where the microbial cell is E. coli, the endogenous gltBD gene is deleted or knocked out.

[0357] Embodiment 17. The engineered microbial cell of embodiment 13, further comprising an endogenous gene encoding citrate synthase (eg, gltA) that is modified or replaced to include a rare codon.

[0358] Embodiment 18. The engineered microbial cell of embodiment 17, comprising an endogenous gltA gene or a species homolog thereof (e.g., an ortholog or paralog) modified or replaced to include a rare codon. In some cases where the microbial cell is E. coli, the endogenous gltA gene is modified or replaced to include a rare codon.

[0359] Embodiment 19. The engineered microbial cell of Example 13, further comprising a deletion, knockout, or knockdown of an endogenous gene encoding glucose-6-phosphate dehydrogenase (eg, zwf).

[0360] Embodiment 20. The engineered microbial cell of embodiment 19, further comprising a deletion, knockout, or lowing of an endogenous zwf gene or its species homolog (e.g., an ortholog or paralog). In some cases where the microbial cell is Escherichia coli, the endogenous zwf gene is deleted or knocked out.

[0361] terminal pathway

[0362] The three AAAs, tryptophan (TRP), phenylalanine (PHE), and tyrosine (TYR), are synthesized through the shikimate pathway and subsequent branched-chain aromatic amino acid biosynthesis pathway, with chorismate (CHA) as the last common precursor in the terminal branch.

[0363] In some embodiments, any of the aforementioned engineered microbial cells further comprise expression (e.g., heterologous expression) of genes required for the TRP branch, PHE branch, or TYR branch to provide an aromatic amino acid biosynthetic pathway.

[0364] The TRP branch requires the following genes to encode the enzymes required to synthesize TRP from CHA: trpA, trpB, trpC, trpD and trpE or feedback-resistant variants thereof (e.g., trpE(fbr)) or species homologs thereof (e.g., orthologs or paralogs). In some embodiments where the target product is a TRP, the engineered microbial cells disclosed herein further comprise overexpression of trpA, trpB, trpC, trpD and trpE or feedback-resistant mutants thereof (e.g., trpE(fbr)) or species homologs thereof (e.g., orthologs or paralogs). In some embodiments where the microbial cell is E. coli, the engineered microbial cells comprise overexpression of trpA, trpB, trpC, trpD and trpE or feedback-resistant mutants thereof (e.g., trpE(fbr)). In some embodiments, overexpression of serA and aroG or feedback-resistant mutants thereof (e.g., aroG(fbr)) is also comprised.

[0365] The PHE branch requires the following genes to encode enzymes required for the synthesis of PHE from CHA: pheA, tyrB, or their species homologs (e.g., orthologs or paralogs). In some embodiments where the target product is PHE, the engineered microbial cells disclosed herein further comprise overexpression of pheA, tyrB, or their species homologs (e.g., orthologs or paralogs). In some embodiments where the microbial cell is E. coli, the engineered microbial cells comprise overexpression of pheA, tyrB.

[0366] The TYR branch requires the following genes to encode the enzymes required for the synthesis of TYR from CHA: tyrA, tyrB, or their species homologs (e.g., orthologs or paralogs). In some embodiments where the target product is TYR, the engineered microbial cells disclosed herein further comprise overexpression of tyrA, tyrB, or their species homologs (e.g., orthologs or paralogs). In some embodiments where the microbial cell is E. coli, the engineered microbial cell comprises overexpression of tyrA, tyrB.

[0367] Also provided herein are methods for reducing the production of byproducts and / or increasing the yield or conversion rate of aromatic amino acids in aromatic amino acid biosynthesis, comprising introducing the genetic modifications contained in any of the aforementioned engineered microbial cells into a microbial cell that naturally has an aromatic amino acid biosynthetic pathway.

[0368] In some embodiments, the genetic modification comprises at least: (i) introduction of an exogenous transketolase having a Km for F6P and / or G3P to a Km for E4P and / or X5P ratio of less than 1:1 (e.g., 1:4), and a Km for S7P and / or G3P to a Km for dX5P and / or R5P ratio of less than 1:1 (e.g., 1:4); and (ii) deletion, knockout, or knockdown of an endogenous gene encoding 6-phosphogluconate dehydrogenase or glucose-6-phosphate dehydrogenase; optionally further comprising (iii) deletion, knockout, or knockdown of an endogenous gene encoding glutamate synthase. In some embodiments, the genetic modification described in (i) at least includes: introducing an exogenous transketolase, whose ratio of Km for F6P to Km for X5P is less than 1:1 (e.g., 1:4), and the ratio of Km for S7P to Km for X5P is less than 1:1 (e.g., 1:4).

[0369] microbial cells

[0370] The present disclosure provides the genetic modification that promotes or optimizes AAA biosynthesis.It will be appreciated by those skilled in the art that the genetic modification exemplified herein is described with reference to suitable host organisms such as Escherichia coli and corresponding metabolic reactions thereof.However, considering the complete genome sequencing of a variety of organisms and the high technology level in the field of genomics, those skilled in the art will be able to easily apply the teaching and guidance provided herein to substantially all other organisms.For example, the Escherichia coli metabolic changes exemplified herein can be easily applied to other species by incorporating the same or similar encoding nucleic acids from species other than the reference species through the displacement of species homologues (for example, straight homologues or paralogs).

[0371] Straight homologues and paralogues gene displacement can be determined by methods well known to those skilled in the art. For example, algorithms such as Align, BLAST or ClustalW can be adopted to determine sequence identity or the similarity between two comparison sequences, and to determine the presence or significance of breaches in the sequence, which can be assigned weights or scores. Based on this type of similarity, those skilled in the art can determine whether similarity is sufficiently high to show that the protein is relevant through the evolution of a common ancestor.

[0372] In some embodiments, the host microorganism can be selected from, and the engineered microbial cells can be generated in, for example, bacteria, yeast, fungi, or any of a variety of other microorganisms suitable for fermentation processes, particularly aromatic amino acid biosynthesis.

[0373] In some embodiments, the host microorganism naturally has an aromatic amino acid biosynthetic pathway. AAA biosynthesis is highly conserved within microorganisms, including the shikimic acid (SHK) pathway, which links the biosynthesis of central carbon metabolism (CCM) with chorismate (CHA). AAA biosynthesis begins with the condensation of phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). This achieves the formation of 3-deoxy-D-arabinoheptone 7-phosphate (DAHP), the first metabolite of the seven-step shikimic acid pathway for the production of chorismate. Chorismate is a precursor of all three standard AAAs and a key branching point for secondary aromatic metabolites.

[0374] In some embodiments, the host microorganism is a microorganism suitable for industrial fermentation.

[0375] Exemplary bacteria include species selected from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Bacillus licheniformis.

[0376] E. coli is a particularly useful host organism because it is a well-characterized microbial organism suitable for genetic engineering.It will be appreciated that any suitable microbial host organism can be used to introduce metabolic and / or genetic modifications to produce a desired product.

[0377] In some embodiments, the engineered microbial cell as described above is a bacterium, a fungus, or a yeast. In some embodiments, the engineered microbial cell as described above is Escherichia coli.

[0378] Methods for making engineered microorganisms

[0379] The engineered microbial cells disclosed herein can be produced by any genetic engineering method known to those skilled in the art without limitation.

[0380] The expression of enzyme in host cell can be realized by introducing nucleic acid into host cell, and described nucleic acid is included in the nucleotide sequence of encoding enzyme under the control of regulatory element, and this regulatory element allows expression in host cell.In some embodiments, nucleic acid is extrachromosomal plasmid.In other embodiments, nucleic acid is chromosomal integration vector, and it can be integrated nucleotide sequence into the chromosome of host cell.Exemplary techniques of nucleic acid encoding these enzymes are introduced into host cell include but not limited to spheroplasting (spheroplasting), electroporation, PEG 1000 mediation conversion and lithium acetate or lithium chloride mediation conversion.

[0381] The overexpression of enzyme in host cell can be realized for example by providing multiple copies of gene or by changing promoter region to provide stronger expression.The copy number of enzyme in host cell can be changed by modifying the transcription of the gene encoding this enzyme.This can be realized for example by modifying the copy number of the nucleotide sequence encoding enzyme (for example, by using the expression vector comprising the higher or lower copy number of this nucleotide sequence, or by introducing the extra copy of this nucleotide sequence into the genome of host cell), or by increasing the promoter operably connected with nucleotide sequence or the intensity of operon.

[0382] In some embodiments, the nucleic acid used to genetically modify the host cell comprises one or more selectable markers, such as antibiotic resistance markers, which can be used to select transformed host cells and to exert selective pressure on the host cell to maintain the exogenous DNA.

[0383] Inhibition of enzymatic activity in the host cell can be achieved, for example, by deleting or destroying the nucleotide sequence encoding the enzyme in the host cell genome. In some embodiments, the activity reduced can be achieved by introducing gene disruption. The term "gene disruption" encompasses any genetic alteration that inactivates the gene product of coding. Genetic alteration can be, for example, deletion of the entire gene or knocking out, transcription or translation of the required regulatory sequence, deletion of a part for the gene to cause a truncated gene product or by any various mutation strategies that inactivate the gene product of coding.

[0384] In certain embodiments, a particularly useful gene disruption method can be complete gene deletion or knockout, because it reduces or eliminates the occurrence of genetic reversal in the engineered microorganisms of the present disclosure. Deletion or knockout of endogenous genes can be achieved by any gene editing system known in the art, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR).

[0385] In other embodiments, endogenous genes can also be knocked down by gene silencing, for example, by using oligonucleotides that bind to the gene encoding it or its mRNA, resulting in a temporary change in expression. Exemplary gene silencing methods include, but are not limited to, RNA interference (RNAi), small interfering RNA (siRNA) or short hairpin RNA (shRNA), which are used to inactivate the messenger RNA of a specific gene and effectively inhibit the expression of the gene.

[0386] In other embodiments, the enzyme activity can be inhibited by modifying or replacing endogenous genes to include rare codons. For example, when expressing exogenous genes in E. coli, rare codons that are rarely used by E. coli will reduce the availability of cognate tRNAs, thereby reducing the efficiency and level of protein expression. For specific species or cells, those skilled in the art can easily determine rare codons using various analytical tools (e.g., bioinformatics tools).

[0387] Method for producing AAA

[0388] In a further aspect, provided are methods or uses of producing AAA using the engineered microbial cells described herein.

[0389] In some embodiments, provided herein are methods for producing aromatic amino acids, comprising culturing any of the aforementioned engineered microbial cells disclosed herein in a culture medium. In some embodiments, the method further comprises recovering the aromatic amino acid from the culture. In some embodiments, the culture is continued for a time sufficient to produce AAA.

[0390] The method generally relates to cultivating host cells in a suitable culture medium comprising a carbon source under suitable conditions. Suitable conditions and suitable culture medium for cultivating microorganisms are well known in the art. In some embodiments, the carbon source is a monosaccharide (simple sugar), a disaccharide, a polysaccharide, a non-fermentable carbon source, or one or more combinations thereof. The non-limiting examples of suitable monosaccharides include glucose, galactose, mannose, fructose, ribose, and combinations thereof. The non-limiting examples of suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. The non-limiting examples of suitable polysaccharides include starch, glycogen, cellulose, chitin, and combinations thereof. The non-limiting examples of suitable non-fermentable carbon sources include acetate and glycerol. In some embodiments, suitable culture medium is supplemented with one or more other reagents, such as inducing compounds (for example, when one or more nucleotide sequences of the coding gene product are under the control of an inducible promoter), repressive compounds (for example, when one or more nucleotide sequences of the coding gene product are under the control of a repressible promoter), or selection agents (for example, for selecting the antibiotic comprising the genetically modified microorganism).

[0391] In some embodiments, the culture medium is suitable for culturing E. coli.

[0392] In some embodiments, the culture medium may comprise LB medium supplemented with peptone, yeast extract and NaCl. In some embodiments, the culture medium may comprise LB medium supplemented with 10 g / l peptone, 5 g / l yeast extract and 10 g / l NaCl.

[0393] The engineered microbial cells disclosed herein can produce increased amounts of AAA and / or decreased amounts of byproducts compared to a host cell (e.g., a wild-type or parental host cell) that does not comprise the genetic modifications of the engineered microbial cells disclosed herein but is otherwise genetically identical.

[0394] In some embodiments, the increase is by at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or at least about 2-fold (e.g., about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold) as measured under the same culture conditions, e.g., in grams per liter of cell culture, milligrams per gram of dry cell weight, per unit volume of cell culture, per unit dry cell weight, per unit volume of cell culture per unit time, or per unit dry cell weight per unit time.

[0395] In some embodiments, the amount of reduction is at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or at least about 2-fold (e.g., about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold) as measured under the same culture conditions, e.g., in grams per liter of cell culture, milligrams per gram of dry cell weight, per unit volume of cell culture, per unit dry cell weight, per unit volume of cell culture per unit time, or per unit dry cell weight per unit time.

[0396] In some embodiments, wherein the engineered microbial cell comprises an exogenous transketolase having a ratio of Km for G3P and / or F6P to Km for E4P and / or X5P of less than 1:1 (e.g., less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5), and a ratio of Km for G3P and / or S7P to Km for X5P and / or R5P of less than 1:1 (e.g., less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5), the engineered microbial cell produces increased amounts of AAA and / or decreased amounts of side products compared to: (a) a host cell that does not comprise an exogenous transketolase but is otherwise genetically identical; or (b) a host cell that comprises overexpression of an endogenous transketolase but is otherwise genetically identical.

[0397] Also provided is the use of any of the foregoing engineered microbial cells disclosed herein in the biosynthesis of aromatic amino acids. Example

[0398] The examples provided below are for illustrative purposes only and are not intended to be limiting unless otherwise stated. Therefore, the present invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations that become apparent as a result of the teachings provided herein.

[0399] Example 1. Construction of tryptophan biosynthesis pathway vector

[0400] 1. Acquisition of genes encoding tryptophan metabolic pathways

[0401] The major tryptophan biosynthesis pathway genes, trpA, trpB, trpC, trpD, and trpE, were all derived from the Escherichia coli K-12 genome (NCBI Reference Sequence: NC_000913.3). PCR amplification was performed using primers F1 and F2 using the genome as a template. The resulting PCR product was named E. coli-trp (6866 bp).

[0402] The tryptophan biosynthesis pathway genes together with the tac promoter and RBS sequence were integrated into the backbone plasmid pUC19 (Addgene, #50005) using the ClonExpress MultiS One-Step Cloning Kit (Vazyme, Jiangsu, China) to obtain the tryptophan pathway exogenous expression vector pUC-TrpEDCBA.

[0403] To increase the amount of tryptophan precursors, aroG and serA need to be overexpressed. Using the E. coli K-12 genome as a template, PCR amplification was performed using primers F2 and R2 to obtain a PCR product named E. coli-aroG (1061 bp). PCR amplification was performed using primers F3 and R3 to obtain a PCR product named E. coli-serA (1328 bp).

[0404] The aroG and serA sequences, along with the tac promoter and RBS sequences, were ligated into the pUC19 backbone plasmid using the ClonExpress MultiS One-Step Cloning Kit (Vazyme, Jiangsu, China). The ligation product was chemically transformed into competent JM109 cells (a commercial strain). Positive clones were selected, plasmids were extracted, and sequencing was performed. The resulting recombinant plasmid was designated pTrp-GA vector.

[0405] 2. Construction of the tryptophan biosynthesis pathway vector pTrp-F

[0406] Gene expression vectors pUC-TrpEDCBA and pTrp-GA were used as templates, and primers F4 and R4, and F5 and R5 were used for amplification to obtain gene sequences with the tac promoter and RBS, respectively. pRGD-TcR (Addgene, #74110) was used as a template, and primers F6 and R6 were used for amplification to obtain the linearized vector.

[0407] The TrpEDBCA sequence, which carries the tac promoter, and the aroG and serA sequences, which also carry the tac promoter, were ligated into the backbone plasmid pRGD-TcR using the ClonExpress MultiS One-Step Cloning Kit (Vazyme, Jiangsu, China). The ligation products were chemically transformed into JM109 competent cells, and positive clones were selected, and the plasmids were extracted and sequenced. The resulting recombinant plasmid was designated pTrp-F vector.

[0408] The obtained vector pTrp-F was transformed into the wild-type strain M000, and the recombinant strain was named M000-Trp.

[0409] Example 2. Knockout of gnd gene

[0410] 2.1 Construction of the pSC101-gnd-KO vector

[0411] Primers gnd-HA1-F and gnd-HA1-R were used to amplify the upstream homology arm of the gnd gene, and primers gnd-HA2-F and gnd-HA2-R were used to amplify the downstream homology arm. Overlap PCR was then performed using the upstream and downstream homology arms as templates with primers gnd-HA1-F and gnd-HA2-R. The resulting fragment was named the gnd-HA fragment.

[0412] pSC101-PBAD-sgRNA (Huang, C., et al., Efficient long fragment editing technique enables large-scale and scarless bacterial genome engineering. Applied Microbiology and Biotechnology, 2020.104(18): p.7943-7956.) was used as a template and primers gnd-backbone-F and gnd-backbone-R were used to amplify the corresponding sgRNA linearized vector fragment.

[0413] The gnd-HA fragment was ligated with the linearized vector fragment using the ClonExpress MultiS One-Step Cloning Kit (Vazyme, Jiangsu, China). Chemical transformation was then performed into competent JM109 cells. Positive clones were selected, and plasmids were extracted and sequenced. The resulting recombinant plasmid was named pSC101-gnd-KO.

[0414] 2.2 Knockout of the gnd gene

[0415] The λ-Red-mediated CRISPR-Cas9 method was used to perform gene knockout in the wild-type Escherichia coli K12 strain (M000, maintained in the laboratory), and the gnd gene in the M000 genome was deleted to generate the mutant strain M001.

[0416] The p15A-PBAD-Cas9-PT5-Redγβα vector (Huang, C., et al., 2020), expressing λ-Red and the CRISPR-Cas9 system, was first transformed into competent cells of strain M000, which were then prepared as chemically competent cells. The gnd knockout vector pSC101-gnd-KO was then transformed into competent M000 cells containing the p15A-PBAD-Cas9-PT5-Redγβα vector, and gene editing was performed after single colonies were grown.

[0417] A single colony was inoculated in LB and allowed to recover for 2 hours. Kanamycin and ampicillin were then added to final concentrations of 50 μg / L and 100 μg / L, respectively, along with IPTG at a final concentration of 1 mM. The culture was then incubated for 1 hour. 1 M L-arabinose was then added to a final concentration of 200 mM and incubated for 3 hours. One μl of the culture was then plated on LB agar plates containing Amp, Kan, and L-arabinose and incubated overnight at 30°C. PCR was performed using primers gnd-verification-F and gnd-verification-R to verify successful knockout of the gnd gene. The knockout strain was designated strain M001.

[0418] 2.3 Construction of fermentation strain M001-Trp

[0419] The tryptophan biosynthesis pathway vector pTrp-F was transformed into competent cells of strain M001 to obtain a recombinant strain, named M001-Trp, and into competent cells of a wild-type strain to obtain a control strain.

[0420] 2.4 Growth of recombinant strain M001-Trp

[0421] The bacterial liquid with OD600 = 1 was inoculated at 1% into 200 ml seed medium (LB medium supplemented with 10 g / l peptone, 5 g / l yeast extract and 10 g / l NaCl) containing tetracycline with a final concentration of 20 μl / mL. When OD600 reached 4-5, the seed liquid was transferred to a 2 L fermentor medium and the growth of the strain was monitored in real time.

[0422] As shown in Figure 1 , knockout of the gnd gene affected the growth of the strain.

[0423] Example 3. Knockout of endogenous tktA

[0424] 3.1 Construction of pSC101-tktA-KO vector

[0425] The upstream homology arm of the endogenous tktA gene was amplified using primers tktA-HA1-F and tktA-HA1-R, and the downstream homology arm was amplified using primers tktA-HA2-F and tktA-HA2-R. Overlapping PCR was then performed using the upstream and downstream homology arms as templates with primers tktA-HA1-F and tktA-HA2-R. The resulting fragment was designated the tktA-HA fragment.

[0426] pSC101-PBAD-sgRNA was used as a template and primers tktA-backbone-F and tktA-backbone-R were used to amplify the linearized vector fragment containing the corresponding sgRNA.

[0427] The tktA-HA fragment was ligated with the linearized vector fragment using the ClonExpress MultiS One-Step Cloning Kit (Vazyme, Jiangsu, China). Chemical transformation was then performed into competent JM109 cells. Positive clones were selected, and plasmids were extracted and sequenced. The resulting recombinant plasmid was named pSC101-tktA-KO.

[0428] 3.2 Knockout of the endogenous tktA gene

[0429] The strain M001 was knocked out using the λ-Red-mediated CRISPR-Cas9 method to delete the tktA gene in the M001 genome and generate the mutant strain M002.

[0430] First, the p15A-PBAD-Cas9-PT5-Redγβα vector expressing λ-Red and the CRISPR-Cas9 system was transformed into M001 competent cells, and colonies were grown and inoculated to prepare chemically competent cells. The tktA knockout vector pSC101-tktA-KO was then transformed into M001 competent cells containing the p15A-PBAD-Cas9-PT5-Redγβα vector, and gene editing was performed after single colonies were grown.

[0431] A single colony was inoculated in LB and allowed to recover for 2 hours. Kanamycin and ampicillin were then added to final concentrations of 50 μg / L and 100 μg / L, respectively, along with IPTG to a final concentration of 1 mM. The cells were cultured for 1 hour. 1 M L-arabinose was then added to a final concentration of 200 mM and cultured for 3 hours. One μl of the culture was plated onto LB agar plates containing Amp, Kan, and L-arabinose and incubated overnight at 30°C. PCR was performed using primers tktA-verification-F and tktA-verification-R to verify successful knockout of the tktA gene. The knockout strain was designated strain M002.

[0432] Example 4. Overexpression of exogenous tkt

[0433] 4.1 Insertion of exogenous wild-type tkt gene

[0434] The mutant strain M003 was generated by inserting the gene tkt (SEQ ID NO: 1) encoding exogenous wild-type TKT into strain M000 using the λ-Red-mediated CRISPR-Cas9 method.

[0435] First, the tkt fragment was obtained using primers tkt-F and tkt-R and a synthetic gene vector containing the gene encoding exogenous transketolase (SEQ ID NO: 1) as a template. Then, using pSC101-tktA-KO as a template, primers tkt-backbone-F and tkt-backbone-R were used to amplify a linearized plasmid. The tkt fragment was ligated to the linearized plasmid using the ClonExpress MultiS One-Step Cloning Kit (Vazyme, Jiangsu, China). The plasmid was then chemically transformed into competent JM109 cells. Positive clones were selected, and the plasmids were extracted and sequenced. The resulting recombinant plasmid was named pSC101-tkt-In.

[0436] The p15A-PBAD-Cas9-PT5-Redγβα vector expressing λ-Red and the CRISPR-Cas9 system was transformed into M000 competent cells, and colonies were grown and inoculated to prepare chemically competent cells. The pSC101-tkt-In vector, used for tkt genome insertion, was transformed into M000 competent cells containing the p15A-PBAD-Cas9-PT5-Redγβα vector, and gene editing was performed after single colonies were grown.

[0437] A single colony was inoculated in LB and allowed to recover for 2 hours. Kanamycin and ampicillin were then added to final concentrations of 50 μg / L and 100 μg / L, respectively, along with IPTG to a final concentration of 1 mM. The culture was then incubated for 1 hour. 1 M L-arabinose was then added to a final concentration of 200 mM and incubated for 3 hours. One μl of the culture was plated onto LB agar plates containing Amp, Kan, and L-arabinose and incubated overnight at 30°C. PCR was performed using primers tktA-verification-F and tktA-verification-R to verify successful insertion of the tkt gene into the genome. The recombinant strain was designated strain M003.

[0438] 4.2 Construction of fermentation strains M000-Trp and M003-Trp

[0439] The tryptophan biosynthesis pathway vector pTrp-F was transformed into competent cells of strains M000 and M003 to obtain recombinant strains M000-Trp and M003-Trp, respectively. The control strain was a wild-type strain containing the pTrp-F plasmid.

[0440] 4.3 Tryptophan production by recombinant strains M000-Trp and M003-Trp

[0441] Tryptophan synthesis was carried out as follows: a bacterial solution with an OD600 of 1 was inoculated at 1% into 200 ml of seed culture medium containing tetracycline at a final concentration of 20 μl / mL. When the OD600 reached 4-5, the seed culture was transferred to a 2 L fermentation tank medium. After 40 hours of fermentation, the amount of tryptophan in the fermentation broth was measured.

[0442] As shown in Figure 2, strain M000-Trp accumulated a significant amount of organic acids during fermentation, including 0.42 g / L citric acid, 1.86 g / L succinic acid, 0.78 g / L malic acid, and 0.9 g / L acetic acid. Strain M003-Trp accumulated fewer organic acids, with no detectable citric acid, only 0.3 g / L succinic acid, 0.48 g / L malic acid, and 0.24 g / L acetic acid.

[0443] Example 5. Comparison between exogenous wild-type tkt gene and tkt mutant

[0444] 5.1 Construction of pSC101-tkt-R356L-In vector

[0445] Using the pSC101-tkt-In vector as a template, PCR amplification with primers R356L-F and R356L-R yielded a fragment of the correct size, designated tkt-R356L. This fragment was chemically transformed into competent JM109 cells, and a single colony was isolated, plasmid extracted, and sequenced. The resulting recombinant plasmid was named pSC101-tkt-R356L-In.

[0446] 5.2 Insertion of exogenous tkt gene

[0447] Gene insertion was performed by inserting the exogenous wild-type tkt gene and tkt mutant (tkt-R356L, SEQ ID NO: 12) into strain M001 using the λ-Red-mediated CRISPR-Cas9 method to generate mutant strains M004 and M005, respectively.

[0448] First, the p15A-PBAD-Cas9-PT5-Redγβα vector expressing λ-Red and the CRISPR-Cas9 system was transformed into M001 competent cells, and colonies were grown and plated to prepare chemically competent cells. The pSC101-tktA-In and pSC101-tktA-R356L-In vectors were transformed into M001 competent cells containing the p15A-PBAD-Cas9-PT5-Redγβα vector, and gene editing was performed after single colonies were grown.

[0449] A single colony was inoculated into LB and allowed to recover for 2 hours. Kanamycin and ampicillin were then added to final concentrations of 50 μg / L and 100 μg / L, respectively, along with IPTG to a final concentration of 1 mM. The cells were then cultured for 1 hour. 1 M L-arabinose was then added to a final concentration of 200 mM and cultured for 3 hours. One μl of the culture was plated onto LB agar plates containing Amp, Kan, and L-arabinose and incubated overnight at 30°C. PCR was performed using the verification primers tkt-verification-F and tkt-verification-R to verify successful insertion of the tkt gene into the genome. The recombinant strains were designated strains M004 and M005.

[0450] 5.3 Construction of M004-Trp and M005-Trp fermentation strains

[0451] The tryptophan biosynthesis pathway vector pTrp-F was transformed into competent cells of strains M004 and M005, respectively, to obtain recombinant strains, which were named M004-Trp and M005-Trp.

[0452] 5.4 Tryptophan Production by Recombinant Strains M004-Trp and M005-Trp

[0453] Tryptophan synthesis was carried out as follows: 1% of the bacterial solution with OD600 = 1 was inoculated into 200 ml of seed culture medium containing tetracycline at a final concentration of 20 μl / mL, and then the seed solution was transferred to a 2 L fermentation tank culture medium when OD600 reached 4-5 and fermented for 40 hours.

[0454] As shown in Figure 3, the recombinant strain M004-Trp expressing wild-type tkt produced approximately 28 g / L of tryptophan, while the strain M005-Trp expressing the mutant tkt-R356L produced approximately 32 g / L of tryptophan. These results indicate that the mutant tkt-R356L has a higher tryptophan production potential than wild-type tkt.

[0455] Example 6. Conversion of pyruvate to PEP

[0456] 6.1 Construction of ptkt-wt and ptkt-wt-ppsA vectors expressing tkt and ppsA genes

[0457] The vector with p15A replicon and spectinomycin resistance was used as a template and primers vector-F1 and vector-R1 were used to perform PCR to obtain the linearized vector.

[0458] The tkt-wt fragment (containing the gene encoding exogenous transketolase (SEQ ID NO: 1)) was ligated with the linearized plasmid vector using the ClonExpress MultiS one-step cloning kit (Vazyme, Jiangsu, China). The tkt-wt fragment was then chemically transformed into competent JM109 cells. Positive clones were selected, and the plasmids were extracted and sequenced. The resulting recombinant plasmid was named ptktA-wt.

[0459] Using the ptkt-wt vector as a template, PCR was performed using primers vector-tkt-F1 and vector-tkt-R1 to obtain a linearized vector. The ppsA fragment was then ligated into the linearized plasmid using the ClonExpress MultiS One-Step Cloning Kit (Vazyme, Jiangsu, China). Chemical transformation was then performed into competent JM109 cells. Positive clones were selected and the plasmid was transferred into competent JM109 cells. The resulting recombinant plasmid was extracted and sequenced, and was named ptktA-wt-ppsA.

[0460] 6.2 Construction of recombinant strains M006 and M007

[0461] The obtained vectors ptkt-wt and ptkt-wt-ppsA were transformed into the M000 strain to obtain recombinant strains M006 and M007, respectively.

[0462] 6.3 Construction of M006-Trp and M007-Trp fermentation strains

[0463] The tryptophan fermentation pathway vector pTrp-F was transformed into competent cells of strains M006 and M007 to obtain recombinant strains, which were named M006-Trp and M007-Trp, respectively.

[0464] 6.4 Tryptophan Production in Recombinant Strains M006-Trp and M007-Trp

[0465] Tryptophan synthesis was carried out as follows: a bacterial solution with an OD600 of 1 was inoculated at 1% into 200 ml of seed culture medium containing tetracycline at a final concentration of 20 μl / mL. When the OD600 reached 4-5, the seed culture was transferred to a 2 L fermentation tank medium. After 40 hours of fermentation, the amount of tryptophan in the fermentation broth was measured.

[0466] As shown in Figure 4, overexpression of ppsA significantly increased tryptophan production from 31.9 g / L to 38.15 g / L. Therefore, overexpression of the ppsA gene to convert pyruvate into phosphoenolpyruvate can enhance tryptophan production.

[0467] Example 7. Modification of the nitrogen (N) cycle

[0468] 7.1 Construction of knockout vector pSC101-gltBD-KO

[0469] The upstream homology arm of the gltBD gene was amplified using primers gltBD-HA1-F and gltBD-HA1-R, and the downstream homology arm was amplified using primers gltBD-HA2-F and gltBD-HA2-R. Overlap PCR was then performed using the upstream and downstream homology arms as templates with primers gltBD-HA1-F and gltBD-HA2-R. The resulting fragment was designated the gltBD-HA fragment.

[0470] pSC101-PBAD-sgRNA was used as a template and amplified using primers gltBD-sgRNA-F and gltBD-sgRNA-R. The resulting fragment was named gltBD-sgRNA. The linearized plasmid vector was amplified using primers gltBD-backbone-F and gltBD-backbone-R.

[0471] The gltBD-HA fragment and gltBD-sgRNA fragment were ligated with the linearized plasmid vector using the ClonExpress MultiS one-step cloning kit (Vazyme, Jiangsu, China). The fragments were then chemically transformed into competent JM109 cells. Positive clones were picked and plasmids were extracted to obtain the correct recombinant plasmid, which was named pSC101-gltBD-KO.

[0472] 7.2 Knockout of the gltBD gene

[0473] The wild-type strain was subjected to gene knockout using the λ-Red-mediated CRISPR-Cas9 method to generate the mutant strain M014.

[0474] First, the p15A-PBAD-Cas9-PT5-Redγβα vector expressing λ-Red and the CRISPR-Cas9 system was transformed into competent cells of the M000 strain. Colonies were grown and inoculated to prepare chemically competent cells. The gltBD knockout vector, pSC101-gltBD-KO, was then transformed into the M000 strain containing the p15A-PBAD-Cas9-PT5-Redγβα vector. After single colonies were grown, gene editing was performed.

[0475] A single colony was inoculated into LB solution and allowed to recover for 2 hours. Then, 50 μg / L kanamycin, 100 μg / L ampicillin, and IPTG were added to a final concentration of 1 mM and cultured for 1 hour. Then, L-arabinose was added to a final concentration of 200 mM and cultured for 3 hours. 1 μl of the culture was coated onto a low-salt LB agar plate containing Amp, Kan, and L-arabinose and incubated overnight at 30°C. PCR was performed using the verification primers gltBD-verification-F and gltBD-verification-R to verify that the gltBD gene had been successfully knocked out. The gltBD knockout strain was designated M014.

[0476] 7.3 Construction of M014-Trp fermentation strain

[0477] The tryptophan fermentation pathway vector pTrp-F was transformed into competent cells of strain M014 to obtain a recombinant strain labeled M014-Trp.

[0478] 7.4 Tryptophan Production by Recombinant Strain M014-Trp

[0479] Tryptophan synthesis was carried out as follows: a bacterial solution with an OD600 of 1 was inoculated at 1% into 200 ml of seed culture medium containing tetracycline at a final concentration of 20 μl / mL. When the OD600 reached 4-5, the seed culture was transferred to a 2 L fermentation tank medium. After 40 hours of fermentation, the amount of tryptophan in the fermentation broth was measured.

[0480] As shown in Figure 5, knockout of gltBD increased tryptophan production from 32.11 g / L to 36.51 g / L. Therefore, modification of the N cycle can promote the increase in tryptophan production.

[0481] Example 8. Enhanced Nitrogen (N) Cycle

[0482] 8.1 Acquisition of glnA and gdhA Genes

[0483] The glnA gene of E. coli was obtained from the genome of E. coli K-12. PCR amplification was performed using the genome of the strain as a template using primers glnA-F1 and glnA-R1. The resulting correct PCR product was named E. coli-glnA (1478 bp).

[0484] The gdhA gene of E. coli was obtained from the genome of E. coli K-12. PCR amplification was performed using primers gdhA-F1 and gdhA-R1, using the genome of the strain as a template. The resulting correct PCR product was named E. coli-gdhA (1413 bp).

[0485] Overlapping PCR was performed using primers glnA-F1 and gdhA-R1, and the resulting fragment was named glnA-gdhA (2870 bp).

[0486] 8.2 Construction of the expression vector pglnA-gdhA

[0487] The vector with p15A replicon and spectinomycin resistance was used as a template and primers vector-F1 and vector-R1 were used for PCR to obtain the linearized vector.

[0488] The glnA-gdhA fragment was then ligated with the linearized plasmid vector using the ClonExpress MultiS one-step cloning kit (Vazyme, Jiangsu, China) and chemically transformed into competent cells JM109. Positive clones were picked, and the plasmids were extracted for sequencing. The resulting correct recombinant plasmid was named pglnA-gdhA.

[0489] 8.3 Construction of recombinant strains overexpressing glnA and gdhA genes

[0490] The obtained plasmid pglnA-gdhA was transformed into the strain M014-Trp obtained in Example 7 by chemical transformation to obtain the tryptophan-producing strain M015-Trp, in which gltBD was knocked out and glnA and gdhA were overexpressed.

[0491] 8.4 Production of M014-Trp and M015-Trp Strains

[0492] Tryptophan synthesis was performed as follows: a 1% inoculation of a bacterial suspension with an OD600 of 1 was performed into 200 mL of seed culture medium containing tetracycline at a final concentration of 20 μl / mL. When the OD600 reached 4-5, the seed culture was transferred to a 2 L fermentor. After 36 hours of fermentation, the tryptophan content in the fermentation broth was measured. The conversion rate was calculated using the formula: [Tryptophan production] g / [Glucose added] g × 100%.

[0493] As shown in FIG6 , the transformation efficiency of strain M014-Trp was 16.7%, and the transformation efficiency of strain M015-Trp was 18.93%, indicating that the combination of overexpression of glnA and gdhA and knockout of gltBD can improve the transformation efficiency.

[0494] Example 9. Combined modification of nitrogen cycle and tkt strategy

[0495] 9.1 Construction of strain M016

[0496] Mutant strain M016 was generated by knocking out the gnd gene from strain M014 using the λ-Red-mediated CRISPR-Cas9 method.

[0497] First, the vector p15A-PBAD-Cas9-PT5-Redγβα (expressing λ-Red and the CRISPR-Cas9 system) was transformed into M014 competent cells, which were then prepared as chemically competent cells. The vector pSC101-gnd-KO for gnd knockout was then transformed into M014 competent cells containing the vector p15A-PBAD-Cas9-PT5-Redγβα, and gene editing was performed after single colonies were grown.

[0498] A single colony was inoculated in LB and allowed to recover for 2 hours. Kanamycin and ampicillin were then added to final concentrations of 50 μg / L and 100 μg / L, respectively, along with IPTG at a final concentration of 1 mM. The cells were then incubated for 1 hour. Subsequently, 1 M L-arabinose was added to a final concentration of 200 mM and incubated for 3 hours. One μl of this culture was plated onto LB agar plates containing Amp, Kan, and L-arabinose and incubated overnight at 30°C. PCR was performed using primers gnd-verification-F and gnd-verification-R to verify successful knockout of the gnd gene. The recombinant strain was designated strain M016.

[0499] 9.2 Construction of strain M017

[0500] First, the p15A-PBAD-Cas9-PT5-Redγβα vector (expressing λ-Red and the CRISPR-Cas9 system) was transformed into M016 competent cells, which were then prepared as chemically competent cells. The vector pSC101-tktA-R356L-In for tkt insertion was transformed into M016 competent cells containing the p15A-PBAD-Cas9-PT5-Redγβα vector, and gene editing was performed after single colonies were grown.

[0501] A single colony was inoculated in LB and allowed to recover for 2 hours. Kanamycin and ampicillin were then added to final concentrations of 50 μg / L and 100 μg / L, respectively, along with IPTG at a final concentration of 1 mM. The culture was then incubated for 1 hour. 1 M L-arabinose was then added to a final concentration of 200 mM and incubated for 3 hours. One μl of the culture was then plated on LB agar plates containing ampicillin, kanamycin, and L-arabinose and incubated overnight at 30°C. PCR was performed using primers tkt-verification-F and tkt-verification-R to verify successful insertion of the tkt gene into the genome. The recombinant strain was designated strain M017.

[0502] 9.3 Construction of M017-Trp fermentation strain

[0503] The tryptophan fermentation pathway vector pTrp-F was transformed into competent cells of strain M017 to obtain a recombinant strain labeled M017-Trp.

[0504] 9.4 Tryptophan Production by Recombinant Strain M017-Trp

[0505] Tryptophan synthesis was carried out using tryptophan-producing strains M000-Trp and M017-Trp according to the following steps: a bacterial solution with an OD600 of 1 was inoculated at 1% into 200 ml of seed culture medium containing tetracycline at a final concentration of 20 μl / mL. When the OD600 reached 4-5, the seed culture was transferred to a 2 L fermentor medium. After 40 hours of fermentation, the amount of tryptophan in the fermentation broth was measured, and the L-tryptophan yield per OD was calculated.

[0506] As shown in Figure 7 , after 40 h of fermentation, the Trp yield of the control strain M000-Trp was 0.48 g / L / OD, and the Trp yield of the strain M017-Trp was 0.51 g / L / OD, indicating that the combined modification of knocking out gltBD, knocking out gnd, and inserting exogenous tktA-R356L effectively enhanced the production of tryptophan.

[0507] Example 10. Combined modification of carbon cycle, nitrogen cycle and tkt strategy

[0508] 10.1 Construction of M018 strain:

[0509] The mutant strain M018 was generated by inserting the exogenous wild-type TKT gene tkt and phosphoenolpyruvate synthase ppsA into strain M002 using the λ-Red-mediated CRISPR-Cas9 method.

[0510] First, the upstream homology arm of the endogenous tktA gene was amplified using primers tkt-ppsA-HA1-F and tkt-ppsA-HA1-R, the downstream homology arm was amplified using primers tkt-ppsA-HA2-F and tkt-ppsA-HA2-R, the exogenous tkt gene fragment was amplified using primers tkt-F and tkt-R, and the ppsA gene fragment was amplified using primers ppsA-F and ppsA-R. Overlapping PCR was then performed using primers tkt-ppsA-HA1-F and tkt-ppsA-HA2-R, using the upstream and downstream homology arms, the exogenous tkt gene fragment, and the ppsA gene fragment as templates. The resulting fragment was designated the tkt-ppsA-HA fragment. pSC101-PBAD-sgRNA was used as a template and amplified using primers tkt-ppsA-backbone-F and tkt-ppsA-backbone-R to obtain a linearized vector fragment containing the corresponding sgRNA. The tkt-ppsA-HA fragment was ligated to the linearized vector fragment using the ClonExpress MultiS One-Step Cloning Kit (Vazyme, Jiangsu, China). The fragment was then chemically transformed into competent JM109 cells. Positive clones were selected, and the plasmids were extracted and sequenced. The resulting recombinant plasmid was named pSC101-tkt-ppsA-In.

[0511] The p15A-PBAD-Cas9-PT5-Redγβα vector expressing λ-Red and the CRISPR-Cas9 system was transformed into M002 competent cells, and colonies were grown and plated to prepare chemically competent cells. The vector pSC101-tkt-ppsA-In, used to insert tkt-ppsA into the genome, was transformed into M002 competent cells containing the p15A-PBAD-Cas9-PT5-Redγβα vector, and gene editing was performed after single colonies were grown.

[0512] A single colony was inoculated in LB and allowed to recover for 2 hours. Kanamycin and ampicillin were then added to final concentrations of 50 μg / L and 100 μg / L, respectively, along with IPTG at a final concentration of 1 mM. The culture was then incubated for 1 hour. 1 M L-arabinose was then added to a final concentration of 200 mM and incubated for 3 hours. One μl of the culture was plated onto LB agar plates containing Amp, Kan, and L-arabinose and incubated overnight at 30°C. PCR was performed using primers tkt-ppsA-verification-F and tkt-ppsA-verification-R to verify successful genome insertion of the tkt and ppsA genes. The recombinant strain was designated strain M018.

[0513] 10.2 Construction of strain M019

[0514] Mutant strain M019 was generated by knocking out the gltBD gene from strain M018 using the λ-Red-mediated CRISPR-Cas9 method. The vector p15A-PBAD-Cas9-PT5-Redγβα (expressing both λ-Red and the CRISPR-Cas9 system) was transformed into M018 competent cells, which were then prepared as chemically competent cells. The gltBD knockout vector pSC101-gltBD-KO was then transformed into M018 competent cells containing the vector p15A-PBAD-Cas9-PT5-Redγβα, and gene editing was performed after single colonies were grown.

[0515] A single colony was inoculated in LB and allowed to recover for 2 hours. Kanamycin and ampicillin were then added to final concentrations of 50 μg / L and 100 μg / L, respectively, along with IPTG at a final concentration of 1 mM. The culture was then incubated for 1 hour. 1 M L-arabinose was then added to a final concentration of 200 mM and incubated for 3 hours. One μl of this culture was plated onto LB agar plates containing Amp, Kan, and L-arabinose and incubated overnight at 30°C. PCR was performed using primers gltBD-verification-F and gltBD-verification-R to verify successful knockout of the gltBD gene. The recombinant strain was designated strain M019.

[0516] 10.3 Construction of M020 strain

[0517] Mutant strain M020 was generated by knocking out the talA-tktB genes from strain M019 using the λ-Red-mediated CRISPR-Cas9 method.

[0518] First, the upstream homology arm of the endogenous tktB-talA gene was amplified using primers tktB-talA-HA1-F and tktB-talA-HA1-R, and the downstream homology arm was amplified using primers tktB-talA-HA2-F and tktB-talA-HA2-R. Overlapping PCR was then performed using the upstream and downstream homology arms as templates with primers tktB-talA-HA1-F and tktB-talA-HA2-R. The resulting fragment was designated the tktB-talA-HA fragment. pSC101-PBAD-sgRNA was used as a template to amplify a linearized vector fragment containing the corresponding sgRNA using primers tktB-talA-backbone-F and tktB-talA-backbone-R. The tktB-talA-HA fragment was ligated with the linearized vector fragment using the ClonExpress MultiS One-Step Cloning Kit (Vazyme, Jiangsu, China). Chemical transformation was then performed into competent JM109 cells. Positive clones were selected, and plasmids were extracted and sequenced. The resulting recombinant plasmid was named pSC101-tktB-talA-KO.

[0519] The p15A-PBAD-Cas9-PT5-Redγβα vector (expressing λ-Red and the CRISPR-Cas9 system) was transformed into M019 competent cells, which were then prepared as chemically competent cells. The pSC101-tktB-talA-KO vector for talA-tktB knockout was transformed into M018 competent cells containing the p15A-PBAD-Cas9-PT5-Redγβα vector, and gene editing was performed after single colonies were grown.

[0520] A single colony was inoculated in LB and allowed to recover for 2 hours. Kanamycin and ampicillin were then added to final concentrations of 50 μg / L and 100 μg / L, respectively, along with IPTG at a final concentration of 1 mM. The culture was then incubated for 1 hour. Subsequently, 1 M L-arabinose was added to a final concentration of 200 mM and incubated for 3 hours. One μl of this culture was plated onto LB agar plates containing Amp, Kan, and L-arabinose and incubated overnight at 30°C. PCR was performed using primers talA-tktB-verification-F and talA-tktB-verification-R to verify successful knockout of the talA-tktB gene. The recombinant strain was designated strain M020.

[0521] 10.4 Construction of strain M021

[0522] The mutant strain M021 was generated by knocking out the talB gene from strain M020 using the λ-Red-mediated CRISPR-Cas9 method.

[0523] First, primers talB-HA1-F and talB-HA1-R were used to amplify the upstream homology arm of the endogenous talB gene, and primers talB-HA2-F and talB-HA2-R were used to amplify the downstream homology arm. Then, using the upstream and downstream homology arms as templates, overlapping PCR was performed using primers talB-HA1-F and talB-HA2-R, and the resulting fragment was named talB-HA fragment. pSC101-PBAD-sgRNA was used as a template to obtain a linearized vector fragment containing the corresponding sgRNA by amplification using primers talB-backbone-F and talB-backbone-R. The talB-HA fragment was connected to the linearized vector fragment using the ClonExpress MultiS one-step cloning kit (Vazyme, Jiangsu, China), and chemical transformation was used to transform into competent cells JM109, positive clones were picked, and plasmids were extracted for sequencing. The resulting correct recombinant plasmid was named pSC101-talB-KO.

[0524] The p15A-PBAD-Cas9-PT5-Redγβα vector (expressing λ-Red and the CRISPR-Cas9 system) was transformed into M020 competent cells, which were then prepared as chemically competent cells. The talB knockout vector pSC101-talB-KO was transformed into M020 competent cells containing the p15A-PBAD-Cas9-PT5-Redγβα vector, and gene editing was performed after single colonies were grown.

[0525] A single colony was inoculated in LB and allowed to recover for 2 hours. Kanamycin and ampicillin were then added to final concentrations of 50 μg / L and 100 μg / L, respectively, along with IPTG at a final concentration of 1 mM. The culture was then incubated for 1 hour. 1 M L-arabinose was then added to a final concentration of 200 mM and incubated for 3 hours. One μl of this culture was plated onto LB agar plates containing Amp, Kan, and L-arabinose and incubated overnight at 30°C. PCR was performed using primers talB-verification-F and talB-verification-R to verify successful knockout of the talB gene. The recombinant strain was designated strain M021.

[0526] 10.5 Construction of M021-Trp Fermentation Strain

[0527] The tryptophan fermentation pathway vector pTrp-F was transformed into competent cells of strain M021 to obtain a recombinant strain labeled M021-Trp.

[0528] 10.6 Tryptophan Production by Recombinant Strain M021-Trp

[0529] Tryptophan synthesis was carried out using tryptophan-producing strains M000-Trp and M021-Trp according to the following steps: a bacterial solution with an OD600 of 1 was inoculated at 1% into 200 ml of seed culture medium containing tetracycline at a final concentration of 20 μl / mL. When the OD600 reached 4-5, the seed solution was transferred to a 2 L fermentor. After 40 hours of fermentation, the amount of tryptophan in the fermentation broth was measured, and the L-tryptophan yield per OD was calculated.

[0530] As shown in Figure 8, after 40 hours of fermentation, the Trp conversion rate of the control strain M000-Trp was 15.2%, and the Trp conversion rate of the strain M021-Trp was 17.2%, indicating that the combined modification of overexpressing ppsA, knocking out gltBD, knocking out gnd, knocking out endogenous tkt and introducing exogenous tkt, and knocking out tal effectively enhanced the production of tryptophan.

[0531] Example 11. Combination modification of the tkt strategy and weakening of the TCA cycle

[0532] 11.1 Construction of strain M022

[0533] The mutant strain M022 was generated by replacing rare codons in the gltA gene in strain M021 using the λ-Red-mediated CRISPR-Cas9 method.

[0534] First, the upstream homology arm of the endogenous gltA gene was amplified using primers gltA-HA1-F and gltA-HA1-R, the downstream homology arm was amplified using primers gltA-HA2-F and gltA-HA2-R, and the gltA gene fragment with the rare codon replaced was amplified using primers gltA-F and gltA-R. Overlapping PCR was then performed using the upstream and downstream homology arms and the gltA gene fragment as templates using primers gltA-HA1-F and gltA-HA2-R. The resulting fragment was designated the gltA-HA fragment. pSC101-PBAD-sgRNA was used as a template and amplified using primers gltA-backbone-F and gltA-backbone-R to obtain a linearized vector fragment containing the corresponding sgRNA. The gltA-HA fragment was ligated to the linearized vector fragment using the ClonExpress MultiS One-Step Cloning Kit (Vazyme, Jiangsu, China). The gltA-HA fragment was then chemically transformed into competent JM109 cells. Positive clones were selected, and plasmids were extracted for sequencing. The obtained correct recombinant plasmid was named pSC101-gltA-In.

[0535] The vector p15A-PBAD-Cas9-PT5-Redγβα (expressing λ-Red and the CRISPR-Cas9 system) was transformed into M021 competent cells, which were then prepared as chemically competent cells. The vector pSC101-gltA-In, used to replace gltA, was transformed into M021 competent cells containing the vector p15A-PBAD-Cas9-PT5-Redγβα, and gene editing was performed after single colonies were grown.

[0536] A single colony was inoculated in LB and allowed to recover for 2 hours. Kanamycin and ampicillin were then added to final concentrations of 50 μg / L and 100 μg / L, respectively, along with IPTG at a final concentration of 1 mM. The cells were then incubated for 1 hour. 1 M L-arabinose was then added to a final concentration of 200 mM and incubated for 3 hours. One μl of this culture was plated onto LB agar plates containing Amp, Kan, and L-arabinose and incubated overnight at 30°C. PCR using primers gltA-verification-F and gltA-verification-R, followed by sequencing, verified the successful replacement of the gltA gene. The recombinant strain was designated strain M022.

[0537] 11.2 Construction of M022-Trp Fermentation Strain

[0538] The tryptophan fermentation pathway vector pTrp-F was transformed into competent cells of strain M022 to obtain a recombinant strain labeled M022-Trp.

[0539] 11.3 Tryptophan Production by Recombinant Strain M022-Trp

[0540] Tryptophan synthesis was carried out using tryptophan-producing strains M021-Trp and M022-Trp according to the following steps: a bacterial solution with an OD600 of 1 was inoculated at 1% into 200 ml of seed culture medium containing tetracycline at a final concentration of 20 μl / mL. When the OD600 reached 4-5, the seed solution was transferred to a 2 L fermentor. The amount of tryptophan in the fermentation broth was measured after 40 hours of fermentation.

[0541] As shown in Figure 9, after 40 h of fermentation, the Trp conversion rate of the control strain M021-Trp was 17.2%, and the Trp conversion rate of the strain M022-Trp was 17.8%, indicating that the combined modification of the tkt strategy and the weakened TCA cycle effectively enhanced the production of tryptophan.

[0542] Example 12. Knockout of the zwf gene

[0543] 12.1 Construction of strain M023

[0544] Mutant strain M023 was generated by knocking out the zwf gene from strain M021 using the λ-Red-mediated CRISPR-Cas9 method.

[0545] First, the upstream homology arm of the endogenous zwf gene was amplified using primers zwf-HA1-F and zwf-HA1-R, and the downstream homology arm was amplified using primers zwf-HA2-F and zwf-HA2-R. Then, overlapping PCR was performed using the upstream and downstream homology arms as templates using primers zwf-HA1-F and zwf-HA2-R. The resulting fragment was named the zwf-HA fragment. pSC101-PBAD-sgRNA was used as a template and a linearized vector fragment containing the corresponding sgRNA was obtained by amplification using primers zwf-backbone-F and zwf-backbone-R. The zwf-HA fragment and the linearized vector fragment were ligated using the ClonExpress MultiS One-Step Cloning Kit (Vazyme, Jiangsu, China) and chemically transformed into competent cells JM109. Positive clones were picked, and the plasmids were extracted and sequenced. The resulting correctly recombinant plasmid was named pSC101-zwf-KO.

[0546] The vector p15A-PBAD-Cas9-PT5-Redγβα (expressing λ-Red and the CRISPR-Cas9 system) was transformed into M021 competent cells, which were then prepared as chemically competent cells. The zwf knockout vector pSC101-zwf-KO was transformed into M021 competent cells containing the vector p15A-PBAD-Cas9-PT5-Redγβα, and gene editing was performed after single colonies were grown.

[0547] A single colony was inoculated in LB and allowed to recover for 2 hours. Kanamycin and ampicillin were then added to final concentrations of 50 μg / L and 100 μg / L, respectively, along with IPTG at a final concentration of 1 mM. The culture was then incubated for 1 hour. 1 M L-arabinose was then added to a final concentration of 200 mM and incubated for 3 hours. One μl of the culture was plated onto LB agar plates containing Amp, Kan, and L-arabinose and incubated overnight at 30°C. PCR was performed using primers zwf-verification-F and zwf-verification-R to verify successful knockout of the zwf gene. The recombinant strain was designated strain M023.

[0548] 12.2 Construction of M023-Trp Fermentation Strain

[0549] The tryptophan fermentation pathway vector pTrp-F was transformed into competent cells of strain M023 to obtain a recombinant strain labeled M023-Trp.

[0550] 12.3 Tryptophan Production by Recombinant Strain M023-Trp

[0551] Tryptophan synthesis was carried out using tryptophan-producing strains M021-Trp and M023-Trp according to the following steps: a bacterial solution with an OD600 of 1 was inoculated at 1% into 200 ml of seed culture medium containing tetracycline at a final concentration of 20 μl / mL. When the OD600 reached 4-5, the seed solution was transferred to a 2 L fermentor. The amount of tryptophan in the fermentation broth was measured after 40 hours of fermentation.

[0552] As shown in FIG10 , after 40 hours of fermentation, the Trp conversion rate of the control strain M021-Trp was 17.2%, and the Trp conversion rate of the strain M023-Trp was 17.9%, indicating that knocking out zwf effectively enhanced the production of tryptophan.

[0553] sequence

Claims

1. An engineered microbial cell for producing aromatic amino acids, comprising: (a) heterologous expression of an exogenous transketolase or a variant thereof, wherein the exogenous transketolase or a variant thereof has a higher catalytic efficiency (e.g., lower Km and / or higher kcat) or higher transketolase activity towards glyceraldehyde-3-phosphate (G3P) and / or fructose-6-phosphate (F6P) than towards erythrose-4-phosphate (E4P) and / or xylulose-5-phosphate (X5P); and (b) blocked or reduced flux through the pentose phosphate pathway.

2. The engineered microbial cell of claim 1, wherein the ratio of the Km of the exogenous transketolase or a variant thereof for G3P or F6P to the Km for E4P or X5P is less than 1:1, e.g., less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:

5.

3. The engineered microbial cell of claim 1 or 2, wherein the exogenous transketolase or a variant thereof further has the following characteristics: the Km for glyceraldehyde-3-phosphate (G3P) or sedoheptulose-7-phosphate (S7P) is lower than the Km for xylulose 5-phosphate (X5P) or ribose-5-phosphate (R5P); e.g., the ratio of the Km for G3P or S7P to the Km for X5P or R5P is less than 1:1, e.g., less than 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:

5.

4. The engineered microbial cell of any one of claims 1 to 3, wherein the exogenous transketolase is derived from Leishmania mexicana, Scheffersomyces stipites, Suhomyces tanzawaensis, [Candida] subhashii, Candida tenuis, Hyphopichia burtonii, Spathaspora passalidarum, [Candida] intermedia or Pichia sorbitophila; preferably, the exogenous transketolase comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-12 or a functionally equivalent variant thereof.

5. The engineered microbial cell of any one of claims 1 to 4, which comprises a blocked or reduced oxidative phase of the pentose phosphate pathway.

6. The engineered microbial cell of claim 5, which comprises reduced activity of 6-phosphogluconate dehydrogenase and / or glucose-6-phosphate dehydrogenase, e.g., by deletion, knockout or knockdown of the gnd gene and / or the zwf gene, or by engineering or replacing the endogenous gnd gene and / or zwf gene to contain rare codons.

7. The engineered microbial cell of any one of claims 1 to 6, which further comprises enhanced flux through the nitrogen assimilation pathway, for example, comprising increased activity of glutamate dehydrogenase and / or glutamine synthetase, decreased activity of glutamate synthase, or any combination thereof.

8. The engineered microbial cell of claim 7, wherein the gltBD gene is deleted, knocked out, or knocked down.

9. The engineered microbial cell of claim 7 or 8, which comprises overexpression of the gdhA and / or glnA gene.

10. The engineered microbial cell of any one of claims 1 to 9, which comprises: (i) an exogenous transketolase, the ratio of the Km for F6P to the Km for X5P of which is less than 1:1 (e.g., 1:4), and the ratio of the Km for S7P to the Km for X5P of which is less than 1:1 (e.g., 1:4); and (ii) deletion, knockout, or knockdown of the endogenous gnd gene or zwf gene; optionally further comprising (iii) deletion, knockout, or knockdown of the endogenous gltBD gene.

11. The engineered microbial cell of any one of claims 1 to 10, which further comprises decreased endogenous transketolase activity, for example, achieved by deleting, knocking out, or knocking down the endogenous tktA gene and / or tktB gene, or by engineering or replacing the endogenous tktA gene and / or tktB gene to contain rare codons.

12. The engineered microbial cell of any one of claims 1 to 11, which further comprises decreased endogenous transaldolase activity, for example, achieved by deleting, knocking out, or knocking down the endogenous talA gene and / or talB gene, or by engineering or replacing the endogenous talA gene and / or talB gene to contain rare codons.

13. The engineered microbial cell of any one of claims 1 to 12, which further comprises blocked or weakened flux of the TCA cycle.

14. The engineered microbial cell of claim 13, which comprises decreased activity of pyruvate dehydrogenase and / or citrate synthase, for example, achieved by deleting, knocking out, or knocking down the lpd, aceF, aceE gene or the gltA gene, or by engineering or replacing the lpd, aceF, aceE gene or the gltA gene to contain rare codons, preferably, achieved by engineering or replacing the endogenous gltA gene to contain rare codons.

15. The engineered microbial cell of any one of claims 1 to 14, which further comprises blocked or reduced oxaloacetate biosynthesis pathway.

16. The engineered microbial cell of claim 15, which comprises decreased phosphoenolpyruvate carboxylase activity, for example, achieved by deleting, knocking out, or knocking down ppc, or by engineering or replacing the endogenous ppc gene to contain rare codons.

17. The engineered microbial cell of any one of claims 1 to 16, wherein the naturally occurring phosphotransferase (PTS) transport system is inactivated.

18. The engineered microbial cell of claim 17, which comprises a reduced activity of one or more PTS system proteins, for example, achieved by deleting, knocking out, or knocking down one or more genes selected from ptsH, ptsI, crr, ptsG, or any combination thereof, or by engineering or replacing the endogenous ptsH, ptsI, crr, ptsG genes or any combination thereof to contain rare codons.

19. The engineered microbial cell of claim 17 or 18, which further comprises an increased activity of at least one non-PTS sugar transporter, for example, overexpression of an endogenous non-PTS sugar transporter or heterologous expression of an exogenous non-PTS sugar transporter (such as the glucose facilitator protein (Glf) of Zymomonas mobilis).

20. The engineered microbial cell of claim 19, which further comprises an increased activity of glucokinase, for example, overexpression of an endogenous glk gene or heterologous expression of an exogenous glk gene (such as from Zymomonas mobilis).

21. The engineered microbial cell of any one of claims 1 to 20, which further comprises a blocked or reduced acetic acid and / or lactic acid production pathway.

22. The engineered microbial cell of claim 21, which comprises a reduced activity of one or more enzymes selected from phosphotransacetylase, pyruvate dehydrogenase, lactate dehydrogenase, or any combination thereof; for example, achieved by deleting, knocking out, or knocking down one or more genes selected from pta, poxB, ldhA, or any combination thereof, or by engineering or replacing pta, poxB, ldhA, or any combination thereof to contain rare codons.

23. The engineered microbial cell of any one of claims 1 to 22, which further comprises an enhanced phosphoenolpyruvate (PEP) biosynthesis pathway.

24. The engineered microbial cell of claim 23, which comprises an increased activity of phosphoenolpyruvate synthase to enhance the conversion from pyruvate to PEP, for example, achieved by overexpression of an endogenous ppsA gene or heterologous expression of an exogenous ppsA gene.

25. The engineered microbial cell of claim 23, which comprises an increased activity of pyruvate carboxylase and PEP carboxykinase to enhance the conversion from pyruvate to PEP, for example, achieved by overexpression of a combination of both an endogenous or exogenous pyc gene and an endogenous or exogenous pck gene, for example, by overexpression of endogenous pyc and pck genes or heterologous expression of exogenous pyc and pck genes, or by overexpression of an endogenous pck gene and heterologous expression of an exogenous pyc gene (such as from Cytobacillus firmus).

26. The engineered microbial cell of any one of claims 1 to 25, which comprises: (i) An exogenous transketolase with a ratio of the Km for F6P to the Km for X5P less than 1:1 (e.g., 1:4), and a ratio of the Km for S7P to the Km for X5P less than 1:1 (e.g., 1:4); and (ii) deletion, knockout, or knockdown of the endogenous gnd gene and / or zwf gene; Optionally, the above-engineered microbial cell further comprises any one of the following: (iii) Deletion, knockout, or knockdown of the endogenous gltBD gene; (iv) Deletion, knockout, or knockdown of the endogenous tktA gene and / or tktB gene; (v) Deletion, knockout, or knockdown of the endogenous talA gene and / or talB gene; (vi) The endogenous gltA gene is engineered or replaced to contain rare codons; (vii) Deletion, knockout, or knockdown of the endogenous ppc gene; (viii) Deletion, knockout, or knockdown of the endogenous ptsH, ptsI, crr, ptsG, or any combination thereof; (ix) Overexpression of the endogenous glf gene or glk gene or heterologous expression of an exogenous glf gene or glk gene; (x) Deletion, knockout, or knockdown of the endogenous pta, poxB, ldhA, or any combination thereof; (xi) Overexpression of the endogenous ppsA, pyc, pck gene, or any combination thereof, or heterologous expression of an exogenous ppsA, pyc, pck gene, or any combination thereof; (xii) Any combination of (iii)-(xi).

27. The engineered microbial cell of any one of claims 1 to 26, which comprises: (i) An exogenous transketolase with a ratio of the Km for F6P to the Km for X5P less than 1:1 (e.g., 1:4), and a ratio of the Km for S7P to the Km for X5P less than 1:1 (e.g., 1:4); (ii) deletion, knockout, or knockdown of the endogenous gnd gene and / or zwf gene; and (iii) deletion, knockout, or knockdown of the endogenous gltBD gene; Optionally, the above-engineered microbial cell further comprises any one of the following: (iv) Deletion, knockout, or knockdown of the endogenous tktA gene and / or tktB gene; (v) Deletion, knockout, or knockdown of the endogenous talA gene and / or talB gene; (vi) The endogenous gltA gene is engineered or replaced to contain rare codons; (vii) Deletion, knockout, or knockdown of the endogenous ppc gene; (viii) Deletion, knockout, or knockdown of the endogenous ptsH, ptsI, crr, ptsG, or any combination thereof; (ix) Overexpression of the endogenous glf gene or glk gene or heterologous expression of an exogenous glf gene or glk gene; (x) Deletion, knockout, or knockdown of the endogenous pta, poxB, ldhA, or any combination thereof; (xi) Overexpression of the endogenous ppsA, pyc, pck gene, or any combination thereof, or heterologous expression of an exogenous ppsA, pyc, pck gene, or any combination thereof; (xii) Any combination of (iv)-(xi).

28. The engineered microbial cell of any one of claims 1 to 27, which comprises: (i) An exogenous transketolase having a ratio of Km for F6P to Km for X5P less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P less than 1:1 (e.g., 1:4); (ii) deletion, knockout or knockdown of the endogenous gnd gene and / or zwf gene; (iii) deletion, knockout or knockdown of the endogenous gltBD gene; (iv) deletion, knockout or knockdown of the endogenous tktA gene and / or tktB gene; and (v) deletion, knockout or knockdown of the endogenous talA gene and / or talB gene; Optionally, the engineered microbial cell further comprises any one of the following: (vi) The endogenous gltA gene is engineered or replaced to contain rare codons; (vii) Deletion, knockout or knockdown of the endogenous ppc gene; (viii) Deletion, knockout or knockdown of the endogenous ptsH, ptsI, crr, ptsG or any combination thereof; (ix) Overexpression of the endogenous glf gene or glk gene or heterologous expression of an exogenous glf gene or glk gene; (x) Deletion, knockout or knockdown of the endogenous pta, poxB, ldhA or any combination thereof; (xi) Overexpression of the endogenous ppsA, pyc, pck gene or any combination thereof or heterologous expression of an exogenous ppsA, pyc, pck gene or any combination thereof; (xii) Any combination of (vi)-(xi).

29. The engineered microbial cell of claim 1, comprising: (1) (i) An exogenous transketolase having a ratio of Km for F6P to Km for X5P less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P less than 1:1 (e.g., 1:4); and (ii) deletion, knockout or knockdown of the endogenous gnd gene; (2) (i) An exogenous transketolase having a ratio of Km for F6P to Km for X5P less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P less than 1:1 (e.g., 1:4); (ii) deletion, knockout or knockdown of the endogenous gnd gene; and (iii) deletion, knockout or knockdown of the endogenous talA gene and talB gene; (3) (i) An exogenous transketolase having a ratio of Km for F6P to Km for X5P less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P less than 1:1 (e.g., 1:4); (ii) deletion, knockout or knockdown of the endogenous gnd gene; and (iii) deletion, knockout or knockdown of the endogenous gltBD gene; (4) (i) An exogenous transketolase having a ratio of Km for F6P to Km for X5P less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P less than 1:1 (e.g., 1:4); (ii) deletion, knockout or knockdown of the endogenous gnd gene; (iii) deletion, knockout or knockdown of the endogenous gltBD gene; (iv) deletion, knockout or knockdown of the endogenous tktA gene and tktB gene; (v) deletion, knockout or knockdown of the endogenous talA gene and talB gene; and (vi) overexpression of the endogenous ppsA gene; (5) (i) An exogenous transketolase having a ratio of Km for F6P to Km for X5P less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P less than 1:1 (e.g., 1:4); (ii) deletion, knockout or knockdown of the endogenous gnd gene; (iii) deletion, knockout or knockdown of the endogenous gltBD gene; (iv) deletion, knockout or knockdown of the endogenous tktA gene and tktB gene; (v) deletion, knockout or knockdown of the endogenous talA gene and talB gene; (vi) the endogenous gltA gene is engineered or replaced to contain rare codons; and (vii) overexpression of the endogenous ppsA gene; or (6) (i) An exogenous transketolase having a ratio of Km for F6P to Km for X5P less than 1:1 (e.g., 1:4), and a ratio of Km for S7P to Km for X5P less than 1:1 (e.g., 1:4); (ii) deletion, knockout or knockdown of the endogenous gnd gene and zwf gene; (iii) deletion, knockout or knockdown of the endogenous gltBD gene; (iv) deletion, knockout or knockdown of the endogenous tktA gene and tktB gene; (v) deletion, knockout or knockdown of the endogenous talA gene and talB gene; and (vi) overexpression of the endogenous ppsA gene.

30. The engineered microbial cell of claim 29, further comprising any one of the following: (i) Deletion, knockout or knockdown of the endogenous ppc gene; (ii) Deletion, knockout or knockdown of the endogenous ptsH, ptsI, crr, ptsG or any combination thereof; (iiii) Overexpression of the endogenous glf gene or glk gene or heterologous expression of an exogenous glf gene or glk gene; (iv) Deletion, knockout or knockdown of the endogenous pta, poxB, ldhA or any combination thereof; (v) Overexpression of the endogenous pyc, pck gene or any combination thereof or heterologous expression of an exogenous pyc, pck gene or any combination thereof; (vi) Any combination of (i)-(v).

31. The engineered microbial cell of any one of claims 1 to 30, which is a bacterium, fungus or yeast.

32. An engineered microbial cell according to any one of claims 1 to 30, which is Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis or Bacillus licheniformis.

33. Use of an engineered microbial cell according to any one of claims 1 to 32 in the biosynthesis of aromatic amino acids.

34. A method for producing an aromatic amino acid, comprising culturing an engineered microbial cell according to any one of claims 1 to 32 in a culture medium.

35. The method of claim 34, which further comprises recovering the aromatic amino acid from the culture.

36. A method for reducing the production of by-products and / or increasing the yield or conversion rate of an aromatic amino acid in the biosynthesis of an aromatic amino acid, which comprises introducing a genetic modification as defined in any one of claims 1 to 30 into a microbial cell having an aromatic amino acid biosynthesis pathway.