阅读说明：本技术 一种改进的生物技术生产l-色氨酸的方法 (Improved method for producing L-tryptophan by biotechnology ) 是由 曾安平 陈林 于 2018-11-12 设计创作，主要内容包括：本发明的目的之一在于通过基因突变细菌细胞,使之表达的吲哚-3-甘油磷酸合成酶IGPs相较于野生型IGPs,对抑制剂邻氨基苯甲酸的敏感度下降,甚至可以被氨基苯甲酸激活。(It is an object of the present invention to make it possible to express indole-3-glycerophosphate synthase IGPs with reduced sensitivity to the inhibitor anthranilic acid, or even to be activated by aminobenzoic acid, compared with wild-type IGPs, by genetically mutating the bacterial cells.)

1. A bacterial cell genetically mutated to express indole 3-glycerophosphate synthase IGPs, wherein the bacterial cell produces IGPs having reduced sensitivity to anthranilic acid inhibition as compared to wild-type IGPs.

2. The bacterial cell of claim 1, wherein the cell is mutated to produce:

a) mutants of bacterial IGPs which have reduced sensitivity to inhibition by anthranilic acid compared to IGPs produced by wild-type cells, or

b) A heterologous enzyme having the activity of IGPs, which enzyme has reduced inhibitory sensitivity to anthranilic acid compared to wild-type IGPs.

3. The bacterial cell of claim 2, wherein said mutant bacterial IGPs are homologous to the genetically mutated bacterial cell.

4. A bacterial cell according to any one of claims 2 to 3 wherein the IGPs produced by the cell have at least one amino acid mutation in the anthranilate binding site compared to wild-type IGPs, and wherein the mutated IGPs are active and have reduced sensitivity to inhibition by aminobenzoic acid.

5. The bacterial cell of any one of claims 2-4, wherein said mutated IGPs have the following characteristics:

a) compared with the sequence of SEQ ID NO. 1, serine is used for replacing alanine or glycine at the 60 th position, and/or isoleucine is used for replacing valine at the 8 th position, and/or leucine is used for replacing phenylalanine at the 188 th position, or serine is used for replacing glutamine at the 58 th position, proline is used for replacing valine at the 59 th position, serine is used for replacing phenylalanine at the 60 th position, and lysine is used for replacing glutamine at the 61 st position; or

b) Compared with the sequence of SEQ ID NO. 1, the mutant sequence has at least one amino acid in the 8 th to 188 th positions substituted by other amino acids, but the mutant still has the activity of IGPs, and compared with the IGPs expressed by wild strains, the mutant IGPs have reduced inhibition sensitivity to anthranilic acid.

6. The bacterial cell of any one of claims 2-5, wherein said mutated IGPs have one of SEQ ID NO 2-5 or SEQ ID NO 30.

7. The bacterial cell of any one of claims 1-6, wherein the bacterial cell is an E.

8. The bacterial cell of claim 2, wherein said heterologous enzyme having IGPs activity is an enzyme having an anthranilate synthase II domain derived from saccharomyces or aspergillus, said saccharomyces bacteria is saccharomyces cerevisiae, and said aspergillus bacteria is aspergillus niger.

9. An isolated or synthetic enzyme having an amino acid sequence selected from the group consisting of SEQ ID NO 2 to SEQ ID NO 5, and SEQ ID NO 30.

10. A method for producing L-tryptophan by using biotechnology, comprising the step of culturing the bacterial cell having the genetic mutation according to any one of claims 1 to 8 in a bioreactor.

11. The method according to claim 10, wherein the genetically mutated bacterial cell is E.coli.

12. Use of a bacterial cell according to any of claims 1 to 8 or an enzyme according to claim 9 for the production of L-tryptophan.

13. The use of claim 12, wherein L-tryptophan is produced industrially on a large scale using a bioreactor.

Technical Field

The invention relates to a method for producing tryptophan and derivatives thereof by using biotechnology.

Background

L-trp can also be used as a synthetic precursor [1] of various active secondary metabolites and antitumor drugs, such as syringin and deoxysyringin [2-4], which lays the foundation for high-value biotherapy based on L-trp.

In microorganisms, tryptophan is produced from chorismic acid and is the metabolic end product of the shikimic acid pathway. Starting from chorismate, tryptophan is biosynthesized via anthranilic Acid (ANT), phosphoribosylaminobenzoic acid (PRA or PA), carboxyphenylamino-deoxyribose-5-phosphate (CdRP), indole-3-glycerophosphate (IGP) and indole. The enzymes involved in the synthesis include anthranilate synthase (EC4.1.3.27) encoded by the trpE gene, anthranilate phosphoribosyltransferase (EC 2.4.1.28) encoded by the trpD gene, phosphoribosylaminobenzoate isomerase (EC 5.3.1.24, PRAi) and indole-3-glycerophosphate synthase (EC4.1.1.48, IGP) encoded by the trpC gene, and tryptophan synthase (EC 4.2.1.20) encoded by the trpB and trpA genes. These genes are clustered on the trp operon. TrpC (IGPs) have the activity of phosphoribosylaminobenzoate isomerase (PRAi) and indole-3-Glycerophosphate Synthase (IGPs).

Disclosure of Invention

It is an object of the present invention to reduce the sensitivity of the expressed indole-3-glycerophosphate synthase IGPs to the inhibitor anthranilate compared to wild-type IGPs by genetically mutating the bacterial cells.

The present inventors have discovered that microbial indole-3-Glycerophosphate Synthases (IGPs), such as EcIGPs or eIGPs from E.coli, have high sensitivity to the inhibitor anthranilic acid, this type of inhibition is also referred to as "feed forward inhibition" because anthranilic acid is an intermediate in the L-tryptophan pathway for chorismate synthesis and is synthesized prior to IGPs.

The term "heterologous" in this application is a meaning well known to those skilled in the art and denotes an element that is foreign, such as an enzyme or a protein. By "exogenous" is meant that the element is not present in the target cell, e.g., a cell or organism derived from a different genetic makeup, such as an organism of a different species.

The term "homologous" means that the enzyme or protein is a native enzyme or protein, i.e., the enzyme or protein is naturally present in the cell of interest, and is a corresponding heterologous enzyme or protein.

"expression" as used herein refers to the transformation of genetic information, such as the formation of a protein or nucleic acid based on the genetic information. In particular, it is intended that the term encompass the synthesis of proteins based on genetic information, including prior steps in the synthesis of proteins, such as the transcription of mRNA from a DNA template.

"bacterial cell genetically mutated to express indole-3-glycerophosphate synthetase" refers to a bacterial cell genetically engineered to express indole-3-glycerophosphate synthetase. The term "indole-3-glycerophosphate synthetase" (IGPs) is an enzyme having the activity of IGPs (EC 4.1.1.48) which has the activity of catalyzing the conversion of carboxyphenylamino-deoxyribose-5-phosphate (CdRP) to indole-3-glycerophosphate (IGP). The term includes activities which, in addition to IGPs activity, have one or more other activities, such as phosphoribosylaminobenzoic acid isomerase (PRAi) activity.

The term "reduced inhibitory sensitivity to anthranilate" means that the enzyme activity of the former is higher than that of the latter under the same anthranilate concentration and under the same test conditions (e.g., temperature, pH, salt concentration, etc.), here because of the same enzyme-catalyzed reaction.

"mutant" refers to a protein, such as an enzyme, whose amino acid sequence differs from that of the wild type. The term encompasses the presence of a difference in amino acid sequence compared to the wild type due to a mutation in the gene sequence encoding the protein.

"heterologous enzyme having IGPs activity" refers to a heterologous enzyme having indole-3-glycerophosphate synthase activity, which enzyme may also have one or more enzymatic activities, such as phosphoribosylaminobenzoic acid isomerase (PRAi) activity or anthranilate synthase activity.

"anthranilate synthase II domain" or "AS II domain" refers to component II of a multifunctional anthranilate synthase having transglutaminase activity. The process of catalyzing the formation of anthranilic acid from chorismate by anthranilate synthase can be catalyzed by component I of anthranilate synthase, which utilizes ammonia rather than glutamine, or by component II, which has transglutaminase activity.

The "anthranilate binding site" refers to a region of the enzyme, herein the region in which indole-3-glycerophosphate synthetase binds to anthranilic acid (2-aminobenzoic acid, CAS 118-92-3). In the present invention, a "region" is not limited to a portion of contiguous amino acids, but also includes several amino acid residues located at different positions in the enzyme but close to each other by spatial folding. An "anthranilate binding site" encompasses an amino acid residue that forms a temporary bond with anthranilate. And the term also encompasses amino acid residues adjacent to the amino acid residue forming the temporary bond with anthranilic acid. "adjacent" means 3, preferably two, most preferably 1 amino acid residues comprised to the left and/or right of the amino acid residue forming the temporary bond with anthranilic acid. The "anthranilate binding site" and "anthranilate binding domain" are meant to be the same herein.

Preferred embodiments of the invention: the bacterial cells with the mutated genes are used for expressing:

a) mutants of bacterial IGPs which have reduced sensitivity to inhibition by anthranilic acid compared to IGPs produced by wild-type cells, or

b) A heterologous enzyme having the activity of IGPs, which enzyme has reduced inhibitory sensitivity to anthranilic acid compared to wild-type IGPs.

The present inventors have found that bacterial IGPs have anthranilic acid binding sites and that, upon binding of anthranilic acid to IGPs, non-competitively inhibit the enzymatic conversion of phosphoribosylaminobenzoic acid (PRA or PA) to indole-3-glycerophosphate (IGP) via carboxyphenylamino-deoxyribose-5-phosphate (CdRP); the genetically engineered IGPs of the invention may exhibit reduced sensitivity to the inhibitor anthranilate. Accordingly, the present invention discloses mutants of bacterial IGPs that produce IGPs with reduced sensitivity to inhibition by p-aminobenzoate compared to IGPs produced by wild-type cells.

In a preferred embodiment, the mutant bacterial IGPs according to the invention are homologous to the genetically mutated bacterial cell.

Preferably. The genetically mutated bacterial cells of the invention are E.coli cells used to express the mutated IGPs.

Alternatively, the present invention produces enzymes having the activity of IGPs by heterologous expression of bacterial cell genes, which have reduced inhibitory sensitivity to anthranilic acid as compared to wild-type IGPs. The heterologous enzyme may be of bacterial or other origin, e.g.heterologous expression using yeast. The inventors have also found that IGPs derived from non-bacterial cells (e.g.Saccharomyces cerevisiae and Aspergillus) are also active, but are not sensitive to anthranilate and can even be activated. In one embodiment of the invention, bacterial cells that utilize genetic mutations express active heterologous IGPs, but the enzyme is not anthranilate-sensitive and (or) can be activated by anthranilate; for example, an enzyme derived from Saccharomyces cerevisiae or Aspergillus, preferably from Saccharomyces cerevisiae and Aspergillus niger, having an anthranilate synthase II domain.

In another preferred embodiment of the present invention, the mutant strain of IGPs is:

a) compared with the sequence of SEQ ID NO. 1, the mutation sites are: serine for alanine or glycine at position 60 and (or) isoleucine for valine at position 8 and (or) leucine for phenylalanine at position 188 or serine for glutamine at position 58, proline for valine at position 59, serine for phenylalanine at position 60 and lysine for glutamine at position 61; or

b) Compared with the sequence of SEQ ID NO. 1, the mutant sequence has at least one amino acid in the 8 th to 188 th positions substituted by other amino acids, but the mutant still has the activity of IGPs, and compared with the IGPs expressed by wild strains, the mutant IGPs have reduced inhibition sensitivity to anthranilic acid.

In a preferred embodiment of the invention, the mutant sequences of bacterial IGPs are any one of SEQ ID NO 2 to SEQ ID NO 5, or SEQ ID NO 30.

Preferably, the mutant bacterial cell is an escherichia coli cell.

SEQ ID NO 1 is a wild type Escherichia coli IGPs sequence (EcTrpC), SEQ ID NO 2 is an I8V mutant sequence, SEQ ID NO 3 is an S60A mutant sequence, SEQ ID NO 4 is an S60G mutant sequence, SEQ ID NO 5 is a L188F mutant sequence, SEQ ID NO 30 is an S58Q, P59V, S60F, K61Q mutant sequence, SEQ ID NO 6 is a wild type Saccharomyces cerevisiae IGPs sequence (AgTrpC), and SEQ ID NO 7 is a wild type Aspergillus niger IGPs sequence (AgTrpC).

In a second aspect, the invention also relates to an enzyme corresponding to SEQ ID NO 2-5 or SEQ ID NO 30 obtained by isolation or synthesis.

In a third aspect, the present invention also relates to a method for producing L-tryptophan by a biotechnological process comprising culturing mutated bacterial cells according to the method of the first aspect in a bioreactor,

preferably, the genetically mutant bacteria used in the method of the invention are E.coli.

In another aspect, the invention also relates to the use of the bacterial cells and enzymes obtained by the above method to produce L-tryptophan and to enable industrial production of L-tryptophan in a bioreactor.

The invention has the advantages that: by genetically mutating bacterial cells, the expressed indole-3-glycerophosphate synthetase IGPs have reduced sensitivity to the inhibitor anthranilate, and may even be activated by aminobenzoate, as compared to wild-type IGPs.

Drawings

FIG. 1 is a scheme showing the biosynthesis of L-trp in Escherichia coli as chorismate.

FIG. 2 is a graph of the effect of anthranilic acid on eIGPS activity; wherein (a) represents the inhibitory effect of anthranilic acid on the activity of eIGPs; (b) represents the kinetic curve of non-competitive inhibition.

Figure 3 is a graph of the effect of anthranilic acid on the activity of wild-type and mutant eIGPs.

FIG. 4 shows the gene types and fermentation results of four strains (S028/ptrpE (S40F), S028TC/ptrpE (S40F), S028/ptrc99A, and S028TC/ptrc 99A), wherein (a) is a growth curve, (b) is a glucose consumption curve, (c) is a dehydroshikimate accumulation curve, (d) is a shikimate accumulation curve, (e) is an ammonium ion consumption curve, (f) is an L-trp production curve, (g) is a L-tyr formation curve, (h) is a L-phe formation curve, and the results of the above experiment are obtained by adding 0.2mM IPTG during 3h of fermentation.

FIG. 5 is the rate of formation of Trp, anthranilic acid, Phe and Tyr during batch fermentation for strains S028/ptrpE (S40F) and S028TC/ptrpE (S40F); TP1 to TP6 correspond to fermentation times of 3 to 8.5, 8.5 to 14.5, 14.5 to 22.5, 22.5 to 27.5, 27.5 to 33 and 33 to 37.5 hours, respectively.

FIG. 6 shows the production rates of L-trp for strains S028/ptrc99A (black) and S028TC/ptrc99A (white) during shake flask fermentation, and TP1 to TP6 for fermentation times of 3 to 8.5, 8.5 to 14.5, 14.5 to 22.5, 22.5 to 27.5, 27.5 to 33, and 33 to 37.5 hours, respectively.

FIG. 7 is a graph of the feed forward regulation of indole glycerol phosphate synthase activity during TrpC synthesis by anthranilic acid. EcTrpC and TrpC of Escherichia coli are negatively and positively regulated by anthranilate, and ScTrpC and AgTrpC and TrpC of Saccharomyces cerevisiae and Aspergillus niger, respectively.

FIG. 8 is a graph showing the effect of anthranilic acid on the activity of ScTrpC and ScIGPs; wherein the ScIGPs are ScTrpCs without anthranilate synthase II domain.

FIGS. 9 to 11 are plasmid maps of pAgTrpC, pEcTrpC and pScTrpC, respectively.

FIG. 12 is a graph of the effect of anthranilic acid concentration on the viability of EcTrpC WT and TrpC QVFQ.

FIG. 13A shows the crystal structure alignment of indole phosphate synthetase from E.coli and M.tuberculosis (PDB _ ID:3T44, light grey, MtIGPS or mIGPs; PDB _ ID: 1PII, black, EcIGPS or eIGGPs); b is the indoxyl phosphate synthetase and anthranilic acid binding site comparison from escherichia coli and mycobacterium tuberculosis (light grey is mIGPs, black is eIGGPs); the residue sites marked for eIGPS binding to anthranilic acid (black columns); wherein ANT is anthranilic acid.

FIG. 14 is the binding site of anthranilic acid and mIGPs PDB _ ID:3T 44; igp 300(A) and BE 2273 (A) represent the indole-3-glycerophosphate and anthranilate ligands (or inhibitors; neither are residues nor the IGPs enzymes themselves, respectively, which are independent of the anthranilate binding site produced by IGPs.

Detailed Description

As shown in FIG. 1, L-trp is synthesized from chorismate, which is also a precursor of aromatic amino acids L-phenylalanine and L-tyrosine, in E.coli, chorismate synthesis L-trp is carried out under the action of enzymes encoded by five structural genes of TrpE, TrpD, TrpC, TrpB and TrpA, which constitute the trp operon, previous studies have shown that the trp operon is strictly regulated by a final product L-trp, including feedback inhibition, attenuation control and attenuation of the biosynthetic components of [7-10 ]. L-trp, and five enzymes are involved in catalytic reactions, which are TrpE, TrpD, TrpC, TrpA and TrpB, respectively, wherein TrpC is a bifunctional gene, and has phosphoribosyl-aminobenzoate synthase activity (PRAi) and indole-3-glycerophosphate synthase activity (IGRP) and indole-3-glycerophosphate synthase activity (PRRP). InP-7-anthranilate synthase (IGRP) and two anthranilate-phosphoribosyl-1-35-phosphoribosyl synthase (PREP), which are converted under the catalytic reactions of the aforementioned phosphoribosyl synthase (PREP, TrpP-11-7-1, 3548-aminobenzoate (IGP), and the following the catalytic reactions of the catalytic action of the enzyme, the enzyme activity of the enzyme which encodes anthranilate synthase (PRRP, the enzyme (PREP-phosphoribosyl transferase (IBP) and the enzyme (IBP) under the aforementioned phosphoribosyl transferase (IBP) and the aforementioned phosphoribosyl transferase (IBP) to convert the aforementioned phosphoribosyl transferase (IBP) to form.