CN110408603B

CN110408603B - A54145C1 mutant with changed fatty acyl selectivity as well as construction and application thereof

Info

Publication number: CN110408603B
Application number: CN201910708729.7A
Authority: CN
Inventors: 刘倩; 樊文杰; 冯雁; 赵娅瑾
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2019-08-01
Filing date: 2019-08-01
Publication date: 2022-09-23
Anticipated expiration: 2039-08-01
Also published as: CN110408603A

Abstract

The invention provides an A54145C1 mutant with changed fatty acyl selectivity, and construction and application thereof. Specifically, the invention provides a54145 initial condensation domain C1 mutant, the catalytic substrate selectivity of which can be changed. Wherein the substrate catalysis of the T16A, A350D, A386S mutants is biased towards short chain fatty acyl substrates; the A152G and V300L mutants tend to prefer long-chain fatty acyl substrates. The mutant breaks through substrate diversity catalysis of natural enzyme, so that the antibiotic (such as daptomycin) with improved activity and yield is obtained.

Description

A54145C1 mutant with changed fatty acyl selectivity as well as construction and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an A54145C1 mutant with changed fatty acyl selectivity, and construction and application thereof.

Background

The lipopeptide natural product has rich structure and bioactivity diversity, and the chemical structure of the lipopeptide natural product, namely the short oligopeptide (2-25 amino acids) skeleton is acylated and modified by N-terminal fatty acid. The natural products of lipopeptides are widely applied to the fields of agriculture, food, industry, livestock raising, medicine and the like, such as clinically important bacteriostatic medicament daptomycin, anticancer medicament bleomycin, immunosuppressant cyclosporin A and biosurfactant surfactin lipopeptide. The lipopeptide natural products currently isolated and found from naturally occurring organisms generally exist as a mixture of multicomponent homologs, one of the major differences being the N-terminal fatty acyl modifier group; and the structure-activity relationship of various lipopeptide natural products shows that different N-acyl modifications have obvious influence on the biological activity of the lipopeptide natural products. The lipopeptide antibiotic A54145 produced by Streptomyces fradiae NRRL18158 has a structure which is highly similar to that of daptomycin which is a clinically important medicament (as shown in figure 1), and has good antibacterial effect on staphylococcus, streptococcus, clostridium and enterococcus. Natural bacteria-produced A54145 is a multi-component N-fatty acyl homologue with a main component of ten-carbon (iC10 and nC10) fatty acid modification, and structure-activity relationship studies show that increased fatty acyl chain length generally leads to enhanced activity. Therefore, the system explores and modifies the fatty acyl modification of the natural product of the lipopeptide, improves the catalytic efficiency of a specific fatty acyl substrate, optimizes the composition of in vivo bioactive components, and has important value in the synthesis of the lipopeptide medicament at present. The method applies modern biotechnology to develop fatty acyl mutants with excellent catalytic activity, provides a new technology for deriving and customizing new active molecules of lipopeptides, enriches the evidence of excellent production of lipopeptide medicaments, and is a research hotspot concerned by scholars.

The biosynthesis of the fatty acyl modification of A54145 and the design research of corresponding enzyme molecules are yet to be disclosed. It is speculated that biosynthesis of such fatty acyl modifications is achieved by the involvement of multiple enzymes, first activated by fatty acids to load and condense with the first amino acid of the peptide backbone, followed by polypeptide elongation to finally produce the natural lipopeptide product. Among these, fatty acid activation has a broader selectivity for fatty acyl substrates, whereas the condensation step is catalyzed by the initial condensation domain C1, whose selectivity for fatty acyl substrates determines the type of final lipopeptide modification. The current research on C1 mainly aims at single point mutation of conserved catalytic residues, substrate selection ability evaluation and the like. Therefore, the research on the complex multi-enzyme catalytic system catalyzed by the initial condensation domain C1 particularly relates to the research and modification on the recognition of upstream fatty acyl substrates, and the customization of fatty acyl of the lipopeptide antibiotics can be hopefully realized by modifying key factors and hot residues related to the binding and selectivity of the fatty acyl substrates through the enzymatic design.

It is currently the mainstream method to locate the selective hot spot region and residues of enzyme substrates by analyzing the three-dimensional structure of enzymes and enzyme-substrate complexes. However, it is time-consuming, labor-consuming and expensive to analyze the protein structure, so that it is an effective means to analyze and simulate the enzyme catalysis process by using bioinformatics technology, and further guide the enzyme modification. Directed evolution is currently the mainstay of enzymatic engineering, but requires the construction of large libraries of mutants in which only a small fraction of the protein sequence is efficiently mutated. Therefore, more researches are also attempted to rapidly and efficiently locate specific regions affecting the protein function, such as substrate binding pockets, interaction interfaces and the like, and the aim of designing libraries with relatively small capacity and relatively high quality is achieved by means of enzymatic modification and assistance of bioinformatics means. There are many successful cases at present, and enzyme molecules designed rationally based on computer simulation are used to modify catalytic properties such as catalytic activity and stability.

There are a number of deficiencies in the prior art studies on the lipopeptide antibiotic a 54145. For example, the original species that naturally produces the lipopeptide antibiotic a54145 (e.g., Streptomyces fradiae NRRL18158) produces a multi-component product with a variety of different fatty acyl modifications, only those of a particular chain length having optimal antimicrobial activity. In addition, because lipopeptide antibiotic fatty acyl modification is a complex system of multi-enzyme catalysis, and relates to condensation of fatty acyl substrates and acyl substrates, large-scale random mutation library construction for high-throughput screening is difficult to realize. There are reports of single point mutation studies on only two conserved residues located in the catalytic center.

In conclusion, there is an urgent need in the art to develop new lipopeptide antibiotic A54145 mutants, especially lipopeptide antibiotic A54145 mutants with altered lipopeptide substrate binding and selectivity, and methods of construction and use thereof.

Disclosure of Invention

The invention aims to provide a lipopeptide antibiotic A54145 initial condensation domain C1 mutant and construction and application thereof.

Specifically, the invention aims to provide a mutant of an initial condensation domain C1 which is derived from Streptomyces fradiae NRRL18158, is used for lipopeptide antibiotic A54145 biosynthesis and has selectively changed fatty acyl substrates, a construction method thereof, and application of the C1 mutant in lipopeptide antibiotic biosynthesis.

In a first aspect of the invention, there is provided a mutant of the initial condensation domain C1, said mutant of C1 being useful for the biosynthesis of the lipopeptide antibiotic A54145,

and, the mutant has an altered catalytic substrate selectivity compared to the wild-type initial condensation domain C1.

In another preferred embodiment, the C1 mutant catalyzes the condensation of a fatty acyl substrate and an acyl substrate to synthesize the lipopeptide antibiotic A54145,

also, the C1 mutant tends to catalyze the condensation of short chain fatty acyl substrates, or of long chain fatty acyl substrates with aminoacyl substrates.

In another preferred embodiment, the acyl and aminoacyl substrates are intermediates in the synthesis of the lipopeptide antibiotic A54145

In another preferred embodiment, the "mutation" means that the mutant starting condensation domain C1 is mutated with respect to the wild-type starting condensation domain C1.

In another preferred embodiment, the amino acid sequence of the wild-type initiation condensation domain C1 is shown in SEQ ID NO. 2.

In another preferred embodiment, the nucleotide sequence encoding the wild-type initial condensation domain C1 is as shown in SEQ ID No. 1.

In another preferred embodiment, the lipopeptide antibiotic A54145 is derived from Streptomyces fradiae, preferably from Streptomyces fradiae NRRL 18158.

In another preferred embodiment, the initiation condensation domain C1 is the NRPS (non-ribosomal peptide synthetase) first module of S.fradiae NRRL 18158.

In another preferred embodiment, the "change in selectivity of catalytic substrate" means that the catalytic reaction is favored over short-chain fatty acyl substrates, and/or that the catalytic reaction is favored over long-chain fatty acyl substrates.

In another preferred embodiment, the "altered selectivity of catalytic substrate" comprises an increased catalytic activity for short chain fatty acyl substrates, and/or an increased catalytic activity for long chain fatty acyl substrates.

In another preferred embodiment, the "increased catalytic activity" refers to an increase in the catalytic activity (availability) of the C1 mutant for short chain fatty acyl substrates, and/or long chain fatty acyl substrates of at least 5%, preferably at least 10%, more preferably at least 30%, as compared to the wild-type starting condensation domain C1.

In another preferred embodiment, the short chain fatty acyl substrate is a fatty acyl substrate having less than 10C atoms.

In another preferred embodiment, the short chain fatty acyl substrate is a fatty acyl substrate having a C atom number greater than 10.

In another preferred embodiment, the short chain fatty acyl substrate is a shorter fatty acyl substrate relative to the primary natural substrates nC10 and iC10, preferably the short chain fatty acyl substrate is selected from the group consisting of: nC8 and nC 9.

In another preferred embodiment, the long chain fatty acyl substrate is selected from the group consisting of: nC12, nC14, iC12, aC12 and aC 13.

In another preferred embodiment, the mutant has 1-5 mutation sites, preferably 1-3 mutation sites, and more preferably 1 mutation site.

In another preferred embodiment, the initial condensation domain C1 mutant has at least 90%, preferably at least 95%, and more preferably at least 98% homology with the sequence shown in SEQ ID NO. 2.

In another preferred embodiment, the initial condensation domain C1 mutant is mutated at one or more sites selected from the group consisting of: the amino acid residue at the 16 th position, the amino acid residue at the 350 th position, the amino acid residue at the 386 th position, the amino acid residue at the 152 nd position, the amino acid residue at the 300 th position or the combination thereof, wherein the numbering of the amino acid residue adopts the numbering shown in SEQ ID NO. 2.

In another preferred embodiment, the 16 th amino acid residue of the initial condensation domain C1 mutant is mutated from T to A, V, L, I, preferably to a.

In another preferred embodiment, the 350 th amino acid residue of the initial condensation domain C1 mutant is mutated from a to D, E, preferably to D.

In another preferred embodiment, the 386 amino acid residue of the initial condensation domain C1 mutant is mutated from a to S, T, preferably to S.

In another preferred embodiment, the 152 nd amino acid residue of the initial condensation domain C1 mutant is mutated from a to G, P, A, preferably to G.

In another preferred embodiment, the 300 th amino acid residue of the initial condensation domain C1 mutant is mutated from V to L, I, V, M, A, F, preferably to L.

In another preferred embodiment, the initial condensation domain C1 mutant has one or more mutations selected from the group consisting of: T16A, A350D, A386S, A152G, V300L, or any combination of the foregoing mutations, wherein the numbering of the amino acid residues is as shown in SEQ ID No. 2.

In another preferred example, the initial condensation domain C1 mutant has a T16A, a350D, and/or a386S mutation, and the C1 mutant has increased catalytic activity for a short chain fatty acyl substrate.

In another preferred example, the initial condensation domain C1 mutant has an a152G, and/or V300L mutation, and the C1 mutant has increased catalytic activity for long chain fatty acyl substrates.

In another preferred embodiment, the C1 mutant has substantially the same amino acid as the wild-type initial condensation domain C1 except for amino acid residue 16, amino acid residue 350, amino acid residue 386, amino acid residue 152 and amino acid residue 300.

In another preferred embodiment, the substantial identity is at most 50 (preferably 1-20, more preferably 1-10) amino acids different, wherein the difference comprises amino acid substitution, deletion or addition, and the C1 mutant still has catalytic activity.

In a second aspect of the invention, there is provided a polynucleotide molecule encoding a C1 mutant according to the first aspect of the invention.

In another preferred embodiment, the polynucleotide is selected from the group consisting of: a DNA sequence, an RNA sequence, or a combination thereof.

In a third aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the second aspect of the invention.

In another preferred embodiment, the vector comprises a plasmid or a viral vector.

In another preferred embodiment, the plasmid comprises a pET-28a derived plasmid.

In a fourth aspect of the invention, there is provided a host cell comprising a vector or chromosome of the third aspect of the invention into which a nucleic acid molecule of the second aspect of the invention has been integrated.

In another preferred embodiment, the host cell is a prokaryotic cell, or a eukaryotic cell.

In another preferred embodiment, the prokaryotic cell is E.coli, preferably E.coli BL21(DE 3).

In a fifth aspect of the present invention, there is provided a method for preparing the C1 mutant according to the first aspect of the present invention, comprising the steps of:

(i) culturing the host cell of the fourth aspect of the invention under suitable conditions such that the mutant is expressed; and

(ii) isolating the C1 mutant.

In another preferred example, the temperature at which the host cell is cultured in step (i) is 20 ℃ to 40 ℃; preferably from 25 ℃ to 37 ℃, e.g. 37 ℃.

In a sixth aspect of the invention, there is provided an enzyme preparation comprising a C1 mutant according to the first aspect of the invention.

In a seventh aspect of the invention, there is provided a C1 mutant of the first aspect of the invention and an enzyme preparation of the sixth aspect of the invention for use in the preparation of an antibacterial medicament.

In another preferred embodiment, the C1 mutant is used for synthesizing the lipopeptide antibiotic A54145.

In another preferred embodiment, the C1 mutant can be used to break through substrate diversity catalysis of natural enzymes, thereby obtaining antibiotics (such as daptomycin) with improved activity and yield.

In another preferred embodiment, the mutants can be used for preparing novel antibiotics modified by specific artificial acyl substrates.

In an eighth aspect of the present invention, there is provided a method of screening for a mutant according to the first aspect of the present invention, comprising the steps of:

(a) providing a first recombinant strain for expressing a fatty acyl ligase dptE protein, a second recombinant strain for expressing an acyl carrier dptF protein, a third recombinant strain for expressing an amino carrier dptPCP1 protein and a fourth recombinant strain for expressing a to-be-detected initial condensation domain C1 mutant, thereby obtaining each protein expressed and purified in vitro;

(b) mixing the dptE protein expressed by the first recombinant strain and the dptF protein expressed by the second recombinant strain with fatty acid to carry out fatty acyl loading reaction, thereby obtaining a fatty acyl substrate;

(c) mixing the dptPCP1 protein expressed by the third recombinant strain with a synthetic substrate Trp-CoA to carry out aminoacyl loading reaction, thereby obtaining an aminoacyl substrate;

(d) and mixing the fatty acyl substrate and the aminoacyl substrate with an initial condensation domain C1 mutant to be detected, which is expressed by a fourth recombinant strain, performing catalytic condensation reaction, and detecting the utilization rate of the fatty acyl substrate and/or a reaction product in a reaction system, thereby screening and obtaining the C1 mutant with the changed selectivity of the catalytic substrate.

In another preferred embodiment, the detection reaction product comprises a detection fatty acyl substrate (FA-dptF), an aminoacyl substrate (Trp-dptPCP1) and a C1 condensation product (FA-Trp-dptPCP 1).

In another preferred example, the dptF protein and the dptPCP1 protein are holo-proteins for successfully modifying the Pquant arm (the host Escherichia coli BAP1 is Escherichia coli with genome-integrated phosphopantetheinyl transferase gene, and can be subjected to phosphopantetheinyl modification after expressing the target vector protein, namely, the Pquant arm of the dptF or the dptPCP1 is modified)

In a ninth aspect of the present invention, there is provided a reaction system comprising:

(a) the C1 mutant according to the first aspect of the invention; and

(b) a fatty acyl substrate.

In another preferred embodiment, the fatty acyl substrate comprises a long chain fatty acyl substrate, or a short chain fatty acyl substrate.

In another preferred embodiment, the fatty acyl substrate comprises a linear fatty acyl substrate, or a branched fatty acyl substrate.

In another preferred embodiment, said fatty acyl substrate comprises FA-dptF.

In another preferred example, the initial condensation domain C1 mutant has a T16A mutation or a350D mutation or a386S mutation or a152G mutation or a V300L mutation. The fatty acyl substrates are nC8, nC9, nC10, nC12, nC14, iC10, iC12, aC12 and aC 13.

In another preferred embodiment, the initial condensation domain C1 mutant has a T16A mutation, and/or a386S mutation, and the short chain fatty acyl substrate is nC 9.

In another preferred embodiment, the initial condensation domain C1 mutant has an a350D mutation and the short chain fatty acyl substrate is nC8, and/or nC 9.

In another preferred embodiment, the initial condensation domain C1 mutant has an a152G mutation and the long chain fatty acyl substrate is nC 12.

In another preferred embodiment, the mutant initiation condensation domain C1 has a V300L mutation and the long chain fatty acyl substrate is nC14, and/or iC 10.

In another preferred embodiment, the reaction system further comprises an aminoacyl substrate.

In another preferred embodiment, the aminoacyl substrate comprises Trp-dpppcp 1.

In another aspect of the present invention, there is also provided a rational design method for screening the C1 mutant according to the first aspect of the present invention, obtaining the A54145C1 three-dimensional structure by computer homology modeling, locating the active center binding to the substrate, then computationally modeling molecular docking with the substrate, locating the region of the substrate pocket that may have important effects on catalysis and substrate binding, and finally selecting the region around the acyl substrate of the docking pocket

Amino acids within the range serve as key candidate residues. In addition, a complex structure of the whole catalytic system of the dptF-A54145C1-dptPCP1 is obtained by calculating and simulating protein-protein molecular docking, and a protein interaction interface and key residues are predicted to be used as key candidate residues influencing catalysis.

In another aspect of the present invention, there is also provided a method for constructing a small intelligent mutation library for screening the C1 mutant according to the first aspect of the present invention, wherein rational mutation concept is adopted, and mutations are designed according to the size, polarity and hydrophobicity of the side chain residue selected from the substrate pocket and the interaction interface, so as to obtain the small intelligent mutation library with the changed size, polarity or hydrophobicity of the side chain residue.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. For reasons of space, they will not be discussed in detail.

Drawings

FIG. 1 shows the structure of the multicomponent products A21978C and A54145 of natural products. Wherein, the upper diagram is A21978C components, and the fatty acyl loaded by daptomycin is n-decanoyl; the lower panel shows the components A54145, which are structurally similar to daptomycin (especially component A54145B loaded with n-decanoyl).

FIG. 2 shows a diagram of the biocatalytic mechanism. Wherein, the R' group is a group connected with a loaded fatty acid chain carbonyl group, and the in vitro experiment constructed by the invention can detect the catalytic activity of the A54145 initial condensation domain C1 on different fatty acyl substrates, and compare the catalytic spectrum difference of wild type and mutant.

FIG. 3 shows the in vitro enzyme activity identification screening method, and a new product peak can be detected by HPLC by C1 catalyzed condensation reaction. The control reaction was a reaction in which mutant H143A with an inactive active center was added.

Figure 4 shows the characterization of the properties of the mutants. Among them, fig. 4A is the in vitro reaction result of mutant T16A, showing that its catalytic activity for the main natural substrates nC10 and iC10 is almost unchanged and catalytic activity for short chain fatty acyl substrate nC9 is increased, relative to Wild Type (WT); FIG. 4B is the in vitro reaction results of mutant A350D, showing that compared with WT, the catalytic activity for the main natural substrates nC10 and iC10 is almost unchanged, and the catalytic activity for the short-chain fatty acyl substrates nC8 and nC9 is improved; FIG. 4C is the in vitro reaction results of mutant A386S, showing that compared with WT, its catalytic activity for the main natural substrates nC10 and iC10 is significantly reduced, while for the short chain fatty acyl substrate nC9 is increased; FIG. 4D is the in vitro reaction results of mutant A152G, which shows that compared with WT, the catalytic activity for the main natural substrates nC10 and iC10 is obviously reduced, and the catalytic activity for the long-chain fatty acyl substrate nC12 is improved; fig. 4E is the in vitro reaction results of mutant V300L, showing that relative to WT, its catalytic activity for the main natural substrate nC10 is almost unchanged, while for iC10 and long chain fatty acyl substrate nC14 is increased.

FIG. 5 shows a schematic of the position of the mutation site. V300 is located at the dptF-A54145C1 interface, A386 and A152 are located at the substrate binding pocket near the catalytically active center; a350 and T16 are located at the dptPCP1-A54145C1 interface.

Detailed Description

The inventor of the invention has extensively and deeply studied, and through the identification and screening of the in vitro enzyme activity of the small intelligent mutation library, the initial condensation domain C1 mutant for A54145 biosynthesis is unexpectedly found to have changed catalytic substrate selectivity. Specifically, the present invention provides a mutant in which threonine at position 16 is substituted with alanine, a mutant in which alanine at position 152 is substituted with glycine, a mutant in which valine at position 300 is substituted with leucine, a mutant in which alanine at position 350 is substituted with aspartic acid, and a mutant in which alanine at position 386 is substituted with serine. Wherein, the substrates of three C1 mutants of T16A, A350D and A386S are inclined to short-chain fatty acyl substrates in catalysis; the C1 mutant A152G and V300L have a tendency to favor long-chain fatty acyl substrates. The present invention has been completed based on this finding.

Specifically, the invention focuses on mutating the A54145 initial condensation domain C1, and obtains an ideal mutant with changed substrate spectrum through molecular design and modification by an effective and feasible mutant construction method related to the patent, so as to generate new application and new functions. Because the in vitro catalytic system of the C1 structural domain is complex and needs to be realized by the participation of multiple enzymes, firstly, fatty acid is used for activating and loading dptF as a fatty acyl substrate, amino acid is used for activating and loading PCP as an aminoacyl substrate, and C1 catalyzes the condensation of the fatty acyl substrate and the aminoacyl substrate to complete the modification of the aminoacyl substrate by the fatty acyl substrate. Therefore, the substrate is difficult to prepare on a large scale, the high-throughput screening of the substrate is difficult to realize, and as the reaction mechanism involves multi-enzyme reaction, no direct structural data supports the rational modification of the C1 domain. The research carries out rational design by combining bioinformatics analysis, obtains mutants with obviously changed substrate spectra through a small amount of mutation, provides direction for modifying an initial condensation structural domain C1 and realizing loading of various types and non-natural fatty acyl substrates, is expected to realize high-efficiency loading of a certain specific fatty acyl substrate or non-natural fatty acyl substrate by changing the substrate spectra of lipopeptide antibiotics (such as daptomycin), and obtains a more high-efficiency medicament with improved in vitro activity.

Term(s)

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

Construction of mutants

The inventor of the application develops the research work of enzyme molecule design and evolution for a long time, provides the enzyme molecule rational evolution strategies of enzyme activity center stabilization, directional protein module assembly and the like, obtains an evolved enzyme library superior to natural enzymes, and is applied to the high-efficiency biosynthesis of important microbial drugs such as pseudo saccharides, glycosphingolipids, ginsenosides and the like. Based on an enzymatic design platform developed by the inventor, a small intelligent mutation library can be established, rare mutants can be obtained through mutation of a plurality of key residues, and powerful guarantee is provided for screening and obtaining mutants with excellent properties.

Specifically, the invention mainly aims at fatty acyl modification in the biosynthesis process of the lipopeptide antibiotics, and performs rational modification by using enzyme molecule design. By comprehensively utilizing technologies such as protein engineering, computer simulation and the like, a small intelligent mutation library of an A54145 initial condensation domain C1 derived from Streptomyces fradiae NRRL18158 is constructed; an in vitro activity screening method aiming at the initial condensation domain C1 of A54145 is established, a novel C1 mutant with obviously changed catalytic substrate selectivity is obtained, and ideas are provided for obtaining antibiotics (such as daptomycin) with improved activity and yield and obtaining novel antibiotics modified by specific artificial acyl substrates.

Mutant

As used herein, the terms "mutant", "C1 mutant" and "A54145C 1 mutant" have the same meaning and refer to the mutant of the initial condensation domain C1 with selectively altered fatty acyl group in the biosynthesis of A54145 provided in the first aspect of the present invention, respectively, the mutant in which the threonine at position 16 is replaced with alanine, the mutant in which the alanine at position 152 is replaced with glycine, the mutant in which the valine at position 300 is replaced with leucine, the mutant in which the alanine at position 350 is replaced with aspartic acid, and the mutant in which the alanine at position 386 is replaced with serine.

The design idea of the mutant of the invention is as follows:

sites 152 and 386 are located at the substrate pocket of the initial condensation domain C1, and mutations are designed with consideration of changing the size of the amino acid residue side chain group and changing the catalytic ability by reshaping the pocket.

16 site, 300 site and 350 site are positioned at a protein-protein interaction interface, and the mutation is designed by considering the polarity of residues and the residue type of the corresponding position of a daptomycin biosynthesis C1 structural domain and changing the binding of C1 and upstream and downstream carrier protein substrates.

Fatty acid chain structure

The fatty acid chains loaded by different lipopeptide antibiotics have differences in carbon chain length, linear/branched chain, degree of saturation, oxidation state, etc., which also contribute to their structural and activity diversity. For the lipopeptide antibiotics described in this patent, daptomycin is a minor component of the multi-component product A21978C produced by Streptomyces roseosporus, and A54145 is a multi-component product produced by Streptomyces fradiae.

Thus, this experiment explored the types of fatty acids that both A54145 and daptomycin can modify, and fatty acid products that are commercially available. Because of the difficulty in synthesizing branched fatty acids, they are currently purchased directly from Lardon, sweden, and methyl-modified branched fatty acids of various lengths are determined from the product catalog. The catalytic loading capacity of the initial condensation domain C1 for the following medium-long chain linear/branched saturated fatty acids was finally determined. Includes 5 straight chains: n-octanoid acid (nC8), n-nonanic acid (nC9), n-decanoic acid (nC10), n-docosanoic acid (nC12), n-tetradecanoic acid (nC 14); 4 branched chains: iso-decanoic acid (iC10), iso-dodecanoic acid (iC12), anteiso-dodecanoic acid (aC12), anteiso-triacanoic acid (aC 13).

Position of the mutation site

The positions of the mutation sites of the 5 mutants (T16A, A152G, V300L, A350D and A386S) of the invention on the protein are shown in 5. H143 is the active center A54145C 1. T16, A350 are located at the interaction interface of A54145C1 and dptPCP1, and V300 is located at the interaction interface of A54145C1 and dptF; a152 and A386 are located in the substrate binding pocket (the positions into which the fatty acyl chains extend after the condensation reaction is catalyzed by C1).

In vitro enzyme activity identification method

The invention provides an in vitro enzyme activity identification method of an A54145 initial condensation structural domain C1. During the biosynthesis of lipopeptide antibiotics such as A54145, daptomycin, etc., the initial condensation domain C1 is responsible for catalyzing the synthesis of a key fatty acyl unit, i.e., the condensation of a fatty acyl substrate with an acyl substrate (see FIG. 2). The invention selects escherichia coli BL21(DE3) as A54145C1 and fatty acyl ligase dptE protein expression strain; escherichia coli BAP1 is selected as an acyl carrier protein dptF and an amino carrier protein dptPCP1 protein expression strain to obtain holo-protein for successfully modifying 4' -phosphopantetheine (Pquant) arm. Establishing an in vitro multi-enzyme reaction and product detection system, loading fatty acid to generate a fatty acyl substrate by using a dptE-dptF enzyme in vitro reaction, loading tryptophan to generate an aminoacyl substrate by using a synthetic substrate Trp-CoA and a dptPCP1 in vitro reaction, carrying out a condensation reaction on A54145C1, and detecting a product by HPLC (as shown in figure 3). The in vitro reaction and detection system is applied to the verification of the mutant activity in a small intelligent mutation library.

Rational design method of mutant

The invention provides a rational design method for an A54145 initial condensation domain C1. Firstly obtaining A54145C1 three-dimensional structure through computer homologous modeling, positioning the active center combined with the substrate, secondly performing molecular docking with the substrate through computational simulation, positioning the substrate pocket region which can have important influence on catalysis and substrate combination, and finally selecting the surrounding acyl substrate of the docking pocket

Small intelligent mutation library

The invention provides a construction method of an A54145 initial condensation domain C1 small intelligent mutation library. A rational mutation concept is adopted, and according to a plurality of key residues selected in a substrate pocket and an interaction interface, the mutation is designed by combining the size, polarity and hydrophobicity of the side chain residue, so that a small intelligent mutation library with the changed size, polarity or hydrophobicity of the side chain residue is obtained.

Technical problem to be solved by the invention

The technical problem to be solved by the invention is to establish a rational design method of A54145 initial condensation domain C1, thereby obtaining a mutant catalyzing selective change of a substrate, and modifying to generate a specific acyl-modified lipopeptide antibiotic. Specifically, the technical problems solved by the invention include:

(a) the invention establishes a reaction and detection system of A54145 initial condensation structural domain C1 in vitro multi-enzyme catalysis.

(b) The invention constructs a small intelligent mutation library of A54145 initial condensation domain C1 from Streptomyces fradiae NRRL18158 by using protein rational design technology and computer simulation.

(c) The invention applies the in vitro enzyme activity identification method to the screening of a mutation library to obtain the A54145 mutant with the selectivity changed by the catalytic substrate.

The main advantages of the invention include:

(a) the invention establishes a rational design method based on the A54145 initial condensation domain C1. The method reasonably designs A54145C1 to obtain a small intelligent mutation library, solves the problem that the in vitro multi-enzyme complex reaction system is difficult to realize high-throughput screening, does not need large-scale random mutation with high intensity and low efficiency, greatly reduces the mutation space range, and increases the probability of obtaining forward mutation.

(b) The invention obtains a mutant of the A54145 initial condensation domain C1 with different fatty acyl substrate catalytic substrate selectivity change. Provides a direction for modifying the initial condensation structural domain C1 to realize loading of different and non-natural fatty acyl substrates, provides a new technology for derivation and customization of new active molecules of the lipopeptide, and enriches the demonstration of excellent production of the lipopeptide drug.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: conditions described in a Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Universal material

1. Primer synthesis: the primers used in the invention are prepared by limited engineering synthesis of Jinwei Zhi biotechnology.

2. Enzyme preparations such as DpnI enzyme used in the experiment were purchased from Thermo Scientific; PrimeStar Max was purchased from TAKARA; the plasmid extraction kit and the PCR product purification kit used were purchased from Axygen.

Example 1 establishment of screening method for in vitro enzyme Activity identification of the initial condensation Domain C1A 54145

In order to construct an in vitro enzyme activity identification screening method aiming at A54145C1, a series of works such as strain construction, multi-enzyme expression purification, construction of an in vitro multi-enzyme reaction system and the like are carried out. The specific operation is as follows:

1. construction of E.coli strains for enzyme expression and in vitro purification of enzymes

pET-28a (+) is used as a plasmid template, Escherichia coli BL21(DE3) is used as an A54145C1 and dppE protein expression strain, and Escherichia coli BAP1 is used as a dppF and dppPCP 1 protein expression strain, so that holo-protein for successfully modifying the Pquant arm is obtained. The histidine-tagged protein was purified by Ni-NTA gravity column. Buffer 50mM HEPES (pH 7.6), 0.5M NaCl,10mM MgCl ₂ The nickel column was then depurated using washing buffer containing 20mM, 50mM imidazole and finally eluted with elution buffer containing 250mM imidazole. The purified protein was verified by SDS-PAGE.

2. Construction of in vitro multiple enzyme reaction System

Based on the catalytic characteristics of A54145C1, fatty acid is loaded by using the in vitro reaction of a dptE-dptF enzyme, tryptophan is loaded by using the in vitro reaction of synthetic substrates Trp-CoA and dptPCP1, and then A54145C1 is added for condensation reaction. The dptPCP1 amino acid loading reaction system was 50mM HEPES (pH 7.6), 50mM NaCl,1mMmM MgCl ₂ 1mM DTT,1.25mM Trp-CoA, 100. mu.M dptPCP1, reacted at 25 ℃ for 0.5 h. The dptE-dptF fatty acid loading reaction system was 50mM HEPES (pH 7.6), 50mM NaCl,1mM MgCl ₂ 1mM ATP,1mM DTT,1mM fatty acid, 10. mu.M dptE, 100. mu.M dptF, reaction at 25 ℃ for 2 h. After completion of both reactions, the reaction systems were mixed in equal amounts, 10. mu. M A54145C1 was added, and reacted at 25 ℃ for 2 hours. The reaction was product-identified by HPLC and validated by TOF-MS.

HPLC detection was performed using a C4(Waters Corporation) reverse phase chromatography column, mobile phase A was ultrapure water (0.1% TFA), mobile phase C was acetonitrile (0.1% TFA), flow rate was 0.5mL/min, sample size was 20. mu.L, temperature was 40 ℃, and detection wavelength was 215 nm. The elution procedure was as follows:

TABLE 1 HPLC elution procedure

Example 2A 54145 design of Small Intelligent mutation library of the Start condensation Domain C1

1. Homologous modeling and molecular docking

Because A54145C1, dptF and dptPCP1 crystals which are not analyzed at present are not obtained, SWISS-MODEL online software is used for carrying out homologous modeling to obtain a three-dimensional protein structure. Then, the A54145C1 and the Ppar-C10 small molecule substrate are subjected to molecular docking by using Schrodinger9.2 software to obtain a protein-substrate complex structure. Protein-protein molecular docking of A54145C1 with dptF and dptPCP1 was performed using ZDCK software to obtain protein complex structures.

2. Construction of A54145C1 substrate pocket region mutant

According to the protein-substrate molecule docking result, the position near the fatty acyl chain docked into the substrate pocket is selected

The inner residues are used as key residues, and the mutation is designed by combining the size, polarity and hydrophobicity of the side chain residues to obtain a small intelligent mutation library with the changed size, polarity or hydrophobicity of the side chain residues.

3. Construction of A54145C1 interaction interface mutant.

According to the relative position of the protein in the protein complex, a plurality of key residues are positioned and selected on an A54145C1-dptF interaction interface and an A54145C1-dptPCP1 interaction interface respectively, and a small intelligent mutation library with the changed size, polarity or hydrophobicity of the side chain residue is obtained by combining the size, polarity and hydrophobicity of the side chain residue to design mutation.

Example 3 cloning, expression and purification of initial condensation Domain C1 mutant A54145

The A54145C1 mutant was expressed in E.coli BL21(DE3) by single point mutation using wild-type pET-28a (+) vector as a template and purified in vitro using Ni-NTA gravity column.

The full plasmid amplification system constructed by the mutant is as follows:

TABLE 2 Whole plasmid amplification reaction System (50. mu.L)

The PCR reaction program is: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 15s, annealing at 60 ℃ for 15s, and extension at 72 ℃ for 95s for 30 cycles; finally, extension is carried out for 5min at 72 ℃. After the amplification, the whole plasmid PCR product was detected by electrophoresis on a 1% agarose gel. After purification of the PCR product, template digestion was performed using DpnI enzyme, after which the plasmid was transformed into E.coli BL21(DE3), and single clones were picked for sequencing to verify that the recombinant clone construction was successful.

The mutant was transformed into E.coli BL21(DE3) for expression and purification.

EXAMPLE 4 characterization of the mutants

And carrying out in-vitro reaction and HPLC detection on the mutant. From the HPLC results, the relative activity of each mutant to the wild-type, catalyzing various chain length fatty acid substrates, was calculated and used to assess changes in its substrate profile.

The results showed that the catalytic substrate selectivity of 5 mutants was significantly changed from that of the wild type (FIG. 4). Compared with the wild type A54145C1, the catalytic activity of mutants T16A and A386S on nC9 substrates is improved, the catalytic activity of mutant A350D on nC8 and nC9 substrates is improved, and the substrates of the three mutants are catalyzed by short-chain fatty acyl substrates; while mutant A152G showed higher catalytic activity on nC12 substrate, V300L showed higher catalytic activity on nC14 substrate and natural iC10 substrate, with a tendency to favor long chain fatty acyl substrate.

All documents referred to herein are incorporated by reference into this application as if each had been individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

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ccggcggcgc cgaccacccc ggcgaccccg gcggcgccgg cggcgccgga tgagctgcgt 720

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Gly Ile Trp Thr Ala Gln Arg Leu Arg Gly Asp Asp Arg Leu Tyr Ala

20 25 30

Cys Gly Leu Phe Leu Glu Leu Asp His Val Val Glu Glu Val Leu Ser

35 40 45

Glu Ala Ile Arg Arg Ala Val Ala Asp Thr Glu Ala Leu Arg Thr Ala

50 55 60

Phe Arg Glu Asp Ala Asp Gly Ala Leu Glu Gln His Val Leu Ala Arg

65 70 75 80

Pro Pro Ser Thr Gln Thr Arg Leu Phe His Ala Asp Pro Ser Gly Gly

85 90 95

Thr Pro Ser Arg Ser Ala Ser Leu Asp Trp Met Asp Arg Gln Arg Ala

100 105 110

Gln Pro Trp Asp Leu Ala Ser Gly Asp Thr Cys Arg His Thr Leu Ile

115 120 125

Pro Leu Gly Gly Asp Arg Ser Leu Leu His Leu Arg Tyr His His Leu

130 135 140

Ala Leu Asp Gly Tyr Gly Ala Ala Leu Tyr Leu Asp Arg Leu Ala Ala

145 150 155 160

Val Tyr Arg Ala Leu Arg Thr Gly His Gln Pro Pro Pro Cys Ala Phe

165 170 175

Ala Pro Leu Ala Arg Leu Val Glu Glu Asp His Ala Tyr Arg Asn Ser

180 185 190

Ala Arg His Arg Ala Asp Ala Asn His Trp Arg Asp Arg Phe Ala Asp

195 200 205

Leu Pro Arg Pro Thr Ser Leu Ala Asp Ala Thr Thr Pro Ala Ala Pro

210 215 220

Thr Thr Pro Ala Thr Pro Ala Ala Pro Ala Ala Pro Asp Glu Leu Arg

225 230 235 240

Arg Thr Val Arg Leu Ser Ala Ala Arg Ser Ala Ala Leu Arg Arg Ala

245 250 255

Ser Asp Arg Ser Gly Arg Pro Trp Pro Val Tyr Ala Thr Ala Ala Val

260 265 270

Ala Ala Phe Leu Ser Arg Leu Ala Pro Gly Glu Glu Val Val Val Gly

275 280 285

Leu Pro Val Thr Ala Arg Val Thr Pro Ala Ala Val Arg Thr Pro Gly

290 295 300

Met Leu Ala Asn Val Val Pro Leu Arg Leu Pro Val Arg Gln Gly Met

305 310 315 320

Ser Thr Ala Glu Leu Leu Glu Leu Thr Ala Ala Glu Ile Ser Thr Thr

325 330 335

Leu Arg His Gln Arg His Arg Thr Glu Asp Ile Gly Arg Ala Leu Gly

340 345 350

Leu His Gly Ala Pro Pro Ala Thr Thr Leu Val Asn Val Met Ala Phe

355 360 365

Ala Pro Val Leu Asp Phe Gly Asp Cys Arg Ala Pro Val His Gln Leu

370 375 380

Ser Ala Gly Pro Val Glu Asp Leu Val Val Asn Leu Leu Gly Thr Pro

385 390 395 400

Gly Asp Gly Gly Glu Ser Asp Gly Thr Glu Leu Glu Ile Thr Val Ala

405 410 415

Ala Asn Pro Arg Leu His Ser Ala Asp Ala Val Ala Ser Leu Ala Ala

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Arg Leu Ala Glu Phe Leu Thr His Met Gly Gln Asp Ala Glu Ala Pro

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Leu Gly Arg Thr Arg Leu Leu Asp

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Claims

1. An initiation condensation domain C1 mutant, wherein the C1 mutant is useful for the biosynthesis of lipopeptide antibiotic a54145, and wherein the initiation condensation domain C1 mutant has a mutation selected from the group consisting of: T16A, A350D, A386S, A152G or V300L, wherein the numbering of the amino acid residues is that shown in SEQ ID NO.2,

and, the catalytic substrate selectivity of the C1 mutant was altered compared to the wild-type initial condensation domain C1.

2. The C1 mutant according to claim 1, wherein the C1 mutant catalyzes the condensation of a fatty acyl substrate and an acyl substrate to synthesize the lipopeptide antibiotic A54145,

also, the C1 mutant is prone to catalyze short chain fatty acyl substrates, or is prone to catalyze long chain fatty acyl substrates.

3. The C1 mutant according to claim 1, wherein the "altered selectivity of catalytic substrate" comprises increased catalytic activity for short chain fatty acyl substrates or increased catalytic activity for long chain fatty acyl substrates.

4. The C1 mutant according to claim 1, wherein the "mutation" is a mutation in the starting condensation domain C1 mutant relative to the wild-type starting condensation domain C1.

5. The C1 mutant according to claim 1, wherein the C1 mutant has a T16A, a350D, or a386S mutation and the C1 mutant has increased catalytic activity for short chain fatty acyl substrates.

6. The C1 mutant of claim 1, wherein the C1 mutant has an a152G, or V300L mutation and the C1 mutant has increased catalytic activity for a long chain fatty acyl substrate.

7. The C1 mutant according to claim 1, wherein the C1 mutant has the same amino acid residue as the wild-type initial condensation domain C1 except for amino acid residue 16, amino acid residue 350, amino acid residue 386, amino acid residue 152 and amino acid residue 300.

8. A nucleic acid molecule encoding the C1 mutant of claim 1.

9. A vector comprising the nucleic acid molecule of claim 8.

10. A host cell comprising the vector or chromosome of claim 9 integrated with the nucleic acid molecule of claim 8.

11. The host cell of claim 10, wherein the host cell is e.

12. A method of making the C1 mutant of claim 1, comprising the steps of:

(i) culturing the host cell of claim 10 under suitable conditions to express the mutant; and

(ii) isolating the C1 mutant.

13. An enzyme preparation comprising the C1 mutant of claim 1.

14. Use of the C1 mutant according to claim 1, for the preparation of antibacterial medicaments.

15. The method of screening for the C1 mutant of claim 1, comprising the steps of:

(d) and mixing the fatty acyl substrate and the aminoacyl substrate with an initial condensation domain C1 mutant to be detected, which is expressed by a fourth recombinant strain, performing catalytic condensation reaction, and detecting the utilization rate of the fatty acyl substrate and/or detecting a reaction product in a reaction system, thereby screening and obtaining the C1 mutant with the changed catalytic substrate selectivity.

16. A reaction system, comprising:

(a) the C1 mutant of claim 1; and

(b) a fatty acyl substrate.