CN112522218A

CN112522218A - Key exchange structural domain for controlling lipopeptide chain length change and mutant and application thereof

Info

Publication number: CN112522218A
Application number: CN202011422022.9A
Authority: CN
Inventors: 卞小莹; 钟林; 张娜; 陈汉娜; 武大雷; 张友明
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2021-03-19
Anticipated expiration: 2040-12-08
Also published as: CN112522218B

Abstract

The invention discloses a key exchange structure domain for controlling the change of lipopeptide lipid chain length, wherein the key exchange structure domain refers to a Cs-AL structure domain after the Cs-AL structure domain of rhizopus amide and the Cs-AL structure domain of rhizopus peptide are exchanged, or refers to a Cs-AL structure domain after the Cs-AL structure domain of sliding rod bacteriocin is replaced by the Cs-AL structure domain of rhizopus peptide, or refers to a Cs-AL structure domain after the Cs-AL structure domain of sliding rod bacteriocin is replaced by the Cs-AL structure domain of sliding rod bacteriocin. Experiments prove that for rhizopus amide, rzmaCsholoA mutant can directionally obtain a derivative with the length of C8 lipid chain, for rhizopus peptide, holoacsrzmA mutant can directionally obtain a derivative with the length of C2 lipid chain, and two mutants of the slipsin, glbACsholA and glbACsglpa, can respectively obtain derivatives with the lengths of C8 and C10 lipid chains. The invention is indicated to have great application value in guiding the modification of lipopeptide which has medicinal prospect and even has been already prepared.

Description

Key exchange structural domain for controlling lipopeptide chain length change and mutant and application thereof

Technical Field

The invention relates to a key structural domain for controlling lipopeptide fat chain length change, a mutant and application thereof, belonging to the technical field of biology.

Background

Non-ribosomal peptides are important natural products of microorganisms, which have a wide range of biological activities, such as antibacterial, antitumor, immunosuppressive, etc. The gene cluster of non-ribosomal peptides is mainly synthesized into peptide chains of different lengths by an iterative type module structure (module), and the module can be further subdivided into domains (domains), mainly comprising: condensation domains (C domains), Adenylation domains (A domains), Thiolation domains (T domains). These three domains form a basic module and a plurality of modules form a giant non-ribosomal proteasome, wherein the initial thiolated domain, after activation by phosphopantetheinyl transferase, is converted into an active state form with a phosphopantetheinyl long arm, which then serves as a "robotic arm" within or between modules for substrate transfer. Within a module, it transfers a downstream substrate-adenylated amino acid activated by an adenylation domain to a condensation domain and subsequently undergoes condensation with an upstream substrate, which includes an intermediate synthesized from the previous module or an initial substrate (initial condensation domain) synthesized from a related gene, such as a fatty acid or the like. The condensation product is then transferred by the long arm to the next module as an upstream substrate for the next module for the next condensation cycle. Finally, complete non-ribosomal peptide products are gradually synthesized from multiple modules in sequence.

Lipopeptide compounds are a very important class of non-ribosomal peptide compounds, and already prepared medicines such as daptomycin (daptomycin) still play an important role in anti-infection. In most cases, the synthetic gene cluster is responsible for condensing downstream adenylylated amino acids with upstream initiation substrates in the form of Acyl Carrier Proteins (ACPs) or acyl CoA from an initiation Condensation domain (Cs domain) during the initiation phase. Research shows that the fatty acid chain introduced in the initial stage plays a very critical role in the biological activity of many lipopeptides, and the length of the fatty acid chain is closely related to the stability and the activity of natural products. And biological studies related to their fatty acid chain derivatization are almost blank. Currently, the most suitable products for lipopeptide lipid chain engineering are chemical semisynthesis and precursor feeding, such as daptomycin, the most suitable product of its native gene cluster is not daptomycin of decacarbonyl length, but the product named A21978C, A21978C1 with mainly anteiso-undecanoyl, A21978C2 with iso-dodecanoyl and A21978C3 with trans-isotridecanoyl. Research shows that when the length of the lipid chain at the N end of the product exceeds C11, the product has high toxicity to human bodies (16311632, 15907192). Daptomycin is obtained by a semisynthesis mode at first, and after the daptomycin is applied to clinic, people can produce saturated decaalkanoyl daptomycin with low toxicity and high activity by substrate feeding and host replacement, and even so, the yield of daptomycin is still not superior to other byproducts. Therefore, the method can search for a key exchange domain for directionally modifying the lipid chain length of the lipopeptide, or can provide great benefits for modifying potential lipopeptides and even lipopeptides which are already prepared, such as a lipoyl substrate of daptomycin during biosynthesis. On the other hand, until now, no cocrystallization structure of the Cs domain and its fatty acyl substrate has been reported, so that it is difficult to control the substrate specificity of the Cs domain in a targeted manner and further to engineer lipopeptides in a targeted manner by rational engineering.

In view of the above, there is a strong need in the art to provide a key domain for controlling the change of lipopeptide lipid chain length, and mutants and applications thereof, so as to realize the development of novel lipopeptide antibiotics or the further optimization of pharmaceutical lipopeptides.

Disclosure of Invention

Aiming at the defects of the prior art, the problem to be solved by the invention is to provide a key exchange domain for controlling the change of the lipopeptide chain length, and a mutant and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions.

The key exchange structural domain for controlling the change of the lipopeptide lipid chain length is an initial condensation structural domain and a boundary of the initial condensation structural domain and an adjacent adenylylation structural domain, wherein the initial condensation structural domain is named as a Cs structural domain, and the adenylylation structural domain is named as an A structural domain; the boundary name of the Cs structural domain and the A structural domain is a Cs-A linker region; wherein the Cs domain is a domain responsible for catalyzing the condensation of an initiating fatty acyl substrate with a first acyl substrate during lipopeptide biosynthesis, the Cs domain being specific for a lipopeptide's lipid chain and mediating initiation of lipopeptide biosynthesis; the A domain refers to an aminoacyl substrate that catalyzes the formation of an upstream Cs domain from a free amino acid, the aminoacyl substrate being an aminoacyl-peptide carrier protein; the exchange structure domain is a Cs structure domain and a Cs-A linker region and is named as a Cs-AL structure domain;

the method is characterized in that:

the key exchange structural domain for controlling the change of the lipopeptide lipid chain length is a Cs-AL structural domain after the Cs-AL structural domain of the rhizopus amide and the Cs-AL structural domain of the rhizopus peptide are exchanged; the Cs-AL domain of the rhizopus amide refers to a Cs domain and a Cs-A linker region which contain key amino acid sites Q36, Y138 and R148 for controlling the specificity of a fatty acyl substrate, and the amino acid sequence of the Cs-AL domain of the rhizopus amide is shown as SEQ NO. 1; the Cs-AL domain of the rhizopus peptide refers to a Cs domain and a Cs-A linker region which contain key amino acid sites Q37, Y139 and A149 for controlling the specificity of a fatty acyl substrate, and the amino acid sequence of the Cs-AL domain of the rhizopus peptide is shown in SEQ NO. 2; wherein the rhizopus amide is abbreviated as rzmA, the rhizopus peptide is abbreviated as holoA, and the biosynthesis gene clusters thereof are all derived from a bacterium Parabrukholderia rhizoxinica HKI 454;

or, the key exchange domain for controlling lipopeptide lipid chain length change is a Cs-AL domain after the Cs-AL domain of the glidescin is replaced by the Cs-AL domain of the rhizopus peptide; the Cs-AL domain of the sliding bar rhzomorph refers to a Cs domain containing key amino acid positions G36, G139 and L149 for controlling the specificity of a fatty acyl substrate and a Cs-A linker region, and the amino acid sequence of the Cs-AL domain of the sliding bar rhzomorph is shown as SEQ NO. 3; the Cs-AL domain of the rhizopus peptide refers to a Cs domain and a Cs-A linker region which contain key amino acid sites Q37, Y139 and A149 for controlling the specificity of a fatty acyl substrate, and the amino acid sequence of the Cs-AL domain of the rhizopus peptide is shown in SEQ NO. 2; wherein said SLIPIDIN is abbreviated glbA and its biosynthetic gene cluster is derived from the bacterium Schlegellella brevitalea DSM 7029;

or, the key exchange domain for controlling lipopeptide lipid chain length change is a Cs-AL domain after the Cs-AL domain of the slipperine is replaced by the Cs-AL domain of the slipperine; the Cs-AL domain of the sliding bar rhzomorph refers to a Cs domain containing key amino acid positions G36, G139 and L149 for controlling the specificity of a fatty acyl substrate and a Cs-A linker region, and the amino acid sequence of the Cs-AL domain of the sliding bar rhzomorph is shown as SEQ NO. 3; the Cs-AL domain of the peptide of the sliding rod is a Cs domain and a Cs-A linker region which contain key amino acid positions A51, M154 and V164 for controlling the specificity of the fatty acyl substrate, and the amino acid sequence of the Cs-AL domain of the peptide of the sliding rod is shown as SEQ NO. 4; wherein said slipvercin is abbreviated glbA and the gramicidin is abbreviated glpA, and the biosynthetic gene clusters are derived from the bacterium Schlegellella brevitalea DSM 7029.

The invention provides a mutant obtained by exchanging Cs-AL domains by using the key exchange domain for controlling the change of the lipopeptide lipid chain length, which is characterized in that: the rhizopus amide biosynthesis gene cluster mutant with the rhizopus peptide Cs-AL structural domain obtained after the Cs-AL structural domain of the rhizopus amide and the Cs-AL structural domain of the rhizopus peptide are exchanged is named rzmaCholA; the mutant of the rhizopus peptide biosynthetic gene cluster with the Cs-AL domain of the rhizopus amide is obtained after the Cs-AL domain of the rhizopus peptide is replaced by the Cs-AL domain of the rhizopus amide and is named as holoacsrzmA; the mutant of the slipperine biosynthetic gene cluster with the rhizopus peptide Cs-AL structural domain, which is obtained after the Cs-AL structural domain of the slipperine is replaced by the Cs-AL structural domain of the rhizopus peptide, is named as glbACsholA; the mutant of the biotransformation gene cluster of the sliding rod rhzomorph with the Cs-AL structural domain of the sliding rod rhzomorph is obtained after the Cs-AL structural domain of the sliding rod rhzomorph is replaced by the Cs-AL structural domain of the sliding rod rhzomorph and is named as glbACsglpa; the amino acid sequence of the rhizopus amide Cs-AL structural domain is shown as SEQ NO.1, the amino acid sequence of the rhizopus peptide Cs-AL structural domain is shown as SEQ NO.2, the amino acid sequence of the Cs-AL structural domain of the slipperine is shown as SEQ NO.3, and the amino acid sequence of the Cs-AL structural domain of the gramicidin is shown as SEQ NO. 4.

The mutant of the invention is applied to the preparation of lipopeptide derivatives with different lipid chain lengths.

Wherein: mutant rzmaCsyla catalyzes the synthesis of rzmA derivatives of C8 lipid chain length in a heterologous host Schlegellella brevitaea DSM 7029; the mutant holoacsrzma catalyzes the synthesis of C2 lipid chain length holoa derivatives in a heterologous host schlegellella brevitalea DSM 7029; the mutant glbACsholA catalyzes the synthesis of a C8 lipid chain length glbA derivative in the homologous host Schlegellella brevitalea DSM 7029; the mutant glbACsglpa catalyzes the synthesis of the C10 lipid chain length glbA derivative in the homologous host Schlegellella brevitalea DSM 7029.

The invention provides a key exchange structure domain for controlling the change of lipopeptide lipid chain length, namely, the Cs-AL structure domain of lipopeptide is exchanged for directionally transforming the lipopeptide lipid chain length of lipopeptide, thereby providing technical support for transforming potential lipopeptide, increasing the activity of the potential lipopeptide, reducing the toxic and side effects of the potential lipopeptide, promoting the clinical patent medicine of the lipopeptide or guiding the further optimization of the patent medicine lipopeptide, such as the yield optimization of daptomycin. In the embodiment of the invention, by utilizing the exchange of the Cs-AL region, the C2 acyl group of the lipopeptide rzmA is successfully prolonged to the length of the C8 acyl group, the C8 acyl group of the holoA is shortened to the length of the C2 acyl group, and the glbA is directionally transformed from the original C12-2,4 unsaturated double bond acyl group into derivatives of C8 saturated acyl groups and C10 saturated acyl groups.

The invention has the following beneficial effects and obvious advantages: 1) the invention utilizes the Cs-AL structural domain for exchange, has little influence on the yield of the derivative, and even improves the yield; 2) the invention is suitable for bacteria of different species; 3) the invention provides a basis for the reconstruction of non-ribosomal lipopeptide lipid chains and provides a reference and a technical method for the further optimization of subsequent patent drug compounds such as daptomycin and the further development and reconstruction of potential drugs.

Drawings

FIG. 1a shows the products of Rhizopus amide (Rhizommidea), both the original products and the derivatives thereof resulting from mutation, wherein 1a (C2-rzmA) is the original product resulting from rzmA of the original gene cluster, and 1d (C8-rzmA) is the product resulting from the mutant; b is a rhizopus peptide (Holrhizoxin) product, including an original product and a derivative product generated by mutation, wherein 2b (C8-holoA) is the original product generated by holoA of an original gene cluster, and 2a (C2-holoA) is the product generated by the mutant; and C is an LC-MS detection result, and is a derivative C8-rzmA generated by the mutant rzmaCholA, a wild-type rzmA product C2-rzmA, a derivative C2-holA generated by the holoacsrzmA, and a wild-type holoA product C8-holA from top to bottom respectively.

FIG. 2: a is the original product of the slipsin gene cluster (glbA gene cluster); b is the glbA derivative product produced by the mutant; and C is the LC-MS detection result, and the derivatives C10-glbA generated by the mutant glbACsglpA, C8-glbA generated by the glbACsjolA and C12-2,4 delta-glbA of the wild type glbA are respectively obtained from top to bottom.

Detailed Description

The present invention will be described in detail with reference to the following detailed drawings and examples. The following examples are only preferred embodiments of the present invention, and it should be noted that the following descriptions are only for explaining the present invention and not for limiting the present invention in any form, and any simple modifications, equivalent changes and modifications made to the embodiments according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.

In the following examples, materials, reagents and the like used were obtained commercially unless otherwise specified. Experimental methods without specific conditions being noted, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the recommendations of the reagent manufacturers.

All procedures involving molecular manipulation in E.coli, such as direct cloning, mutagenesis, plasmid construction, etc., use Red/ET recombinant engineering techniques. The method comprises an ExoCET direct cloning technology, a ccdB reverse screening technology, wire loop recombination, wire line recombination and the like. Specific implementation procedures are described in Wang H, Li Z, Jia R, et al, ExoCET, exoenzyme in vitro assembly with RecET assembly for high throughput efficient direct DNA cloning from complex genes [ J ]. Nucleic Acids Research 2018Mar 16; 46(5), e28.doi:10.1093/nar/gkx1249. and document 2.Wang H, Bian X, Xia L, et al. improved laboratory mutagenesis by recombination using ccdB for counterselection [ J ]. Nucleic Acids Research 2014 Mar; 42(5), e37.doi:10.1093/nar/gkt1339. In the examples, the recombinant GB2005-dir, GB2005-red-gyrA4622, GB2005, and the vector p15A-cm-hyg-ccdB, pR6K-amp-ccdB, pR6k-tnpA-km, pR6K-apra-phiC31 were constructed as described above. The antibiotics used and the concentrations were also carried out according to the above-mentioned documents, and the abbreviations used for the antibiotics are respectively kanamycin: km, chloramphenicol: cm, apramycin: apra, gentamicin: genta.

All restriction enzymes referred to in the examples were purchased from New England Biolabs (NEB). Polymerase Chain Reaction (PCR) the polymerases involved were purchased from TAKARA. The strains Schlegellella brevitalea DSM7029 and Paraburkholderia rhizoxidica HKI 454(DSM19002) were purchased from the German Collection of strains DSMZ.

Such as those involving Red/ET homologous recombination in Schlegellella brevialea DSM7029, the procedure was carried out according to the reference 3, Wang X, Zhou H, Chen H, ET al, discovery of recombinant enzymes minor minning of crystalline biochemical genes in Burkholderials species [ J ]. Proceedings of the National Academy of Sciences,2018,115(18):201720941. the construction involving the strain Red α β DSM7029 is described above. Primer synthesis: the primers used in the examples of the present invention were prepared by Biotechnology engineering (Shanghai) Co., Ltd. The Universal DNA purification recovery kit was purchased from tiangen biochemical technology (beijing) ltd.

In the examples, for the cultivation of E.coli, a low-salt Luria-Bertani medium (LB medium) was used, and the formulation was: tryptone (10 g), yeast extract (yeast ext)ract)5g, sodium chloride (NaCl)1g, 12% agar powder in solid medium, double distilled water 1000mL, and sterilization at 121 ℃ for 20 min. For the cultivation of Schlegellella brevitalea DSM7029 and Paraburkholderia rhizoxinica HKI 454(DSM19002) CYMG medium was used with the formula: 8g/l Casein peptone (Casein peptone),4g/l yeast extract, 4.06g/l MgCl₂·2H₂O,10mL/l of glycerol, 12% agar powder added in the solid culture medium, double distilled water added to 1000mL, and sterilization at 115 ℃ for 30 min. Corresponding media components were purchased from OXIOD.

Example 1:

exchanging rzmA and Cs-AL regions of holoA to obtain mutant rzmACsrzmA and holoA, and verifying the catalytic activity of the mutant from in vitro and in vivo levels. The method is characterized in that the capacity of catalyzing a full-length rzmA/holA gene cluster and corresponding mutants to form final products rzmA/holA and derivatives thereof is taken as a standard (the structure is shown in figure 1, a and b), absolute quantification is adopted, the derivatives and wild lipopeptide products are separated and purified, the mutants are quantified, and a quantification instrument is a liquid chromatography-mass spectrometry (LC-MS).

The specific implementation steps are as follows:

(1) according to anti mash (https://antismash.secondarymetabolites.org/#！/start) Predicting the Cs-AL regions of rzmA and holoA, and determining the amino acid sequences of the Cs-AL regions as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

(2) The procedures were as described in article 1 (1.Wang H, Li Z, Jia R, et al. Exocet: exonuclease in vitro assembly combined with RecET combining for high efficiency direct DNA cloning from complex genes [ J ]. Nucleic Acids Research 2018Mar 16; 46(5): e28.doi:10.1093/nar/gkx1249) using p15A-cm-hyg-ccdB as template PCR to obtain p15A-cm vector with homologous arm, primers p1-p2 for rzp 3-p4 for A (as shown in SEQ ID NO: 5-8), direct cloning with cut DSM19002, (EcaScI/rI gene cutting RH), genome sequence of rzP 3-p4 for A (as shown in SEQ ID NO: 5-8), full-length RBrRS sequence of rRS # 12370, RBrRS # of RBsJR-cluster, RBsJrRS # 3600, plasmids p15A-cm-rzmA and p 15A-cm-holoA with rzmA and holoA full-length gene clusters are obtained. Then, the Red/ET loop recombination technology is used, and the specific steps are as per document 2, (2.Wang H, Bian X, Xia L, ET al. improved Nucleic acid mutagenesis by recombination using ccdB for counting selection [ J ]. Nucleic Acids Research 2014 Mar; 42(5): e37.doi:10.1093/nar/gkt1339.), the tnPA-IR-km gene cassette obtained by PCR is inserted into the p15A-cm-rzmA/holA plasmid, the primer is p5-p6 (as shown in SEQ ID NO: 9-10), and the template is pR6 np 6K-tA-km. The gene cassette can express transposase and is used for inserting plasmids with gene clusters into the genome of host bacteria so as to facilitate the stable expression of the gene clusters. Synthesizing primers p7, p8 and p7, p9 (shown as SEQ ID NO: 11-13) with homologous arms for PCR amplification of the Cs-AL regions of rzmA and holoA respectively, wherein the templates are p15A-tnpA-km-rzm and p15A-tnpA-km-hol respectively, the step can obtain the Cs-AL regions of rzmA and holoA with homologous arms and km resistance respectively, wherein the homologous arms of the rzmA-Cs-AL region can be used for loop recombination with the plasmid p 15A-cm-holoA with the holoA gene cluster, the homologous arms of the holoA-Cs-AL region can be used for loop recombination with the plasmid p15A-cm-rzmA with the rzmA gene cluster, the host bacteria for Red ET/recombination is Escherichia coli GB2005-Red-gyrA462, and the constructed mutants are respectively: mutant plasmid p 15A-tnpA-km-rzmaCholA of the Cs-AL region of chimeric holA and mutant plasmid p15A-tnpA-km-holacsrzmA of the Cs-AL region of chimeric rzmA.

The above mutant and wild type plasmids were separately transformed into DSM7029WT for heterologous expression at 1250V according to the instructions of an Eppendorf Eporator electrotransformer, and the amount of DNA added was 5. mu.g. The constructed strains are respectively mutant 7029-rzmaCholA, 7029-holACsrzmA, wild type 7029-rzmA and wild type 7029-holA.

The effect of the Cs-AL region exchange can be verified from the in vivo level (in vivo) after the mutant and the wild type are subjected to fermentation expression, and the product is identified and quantified. The strain is fermented by inoculating 500 μ l of overnight strain into 50ml of CYMG liquid culture medium, fermenting for 72 hr, adding adsorption resin XAD-16 (2%) into the fermentation liquid, adsorbing for one day, centrifuging to collect the strain and resin, pouring off the supernatant, eluting with 40ml of methanol for 2 hr, evaporating methanol with rotary evaporator, adding 1ml of imported methanol, and detecting the product with liquid chromatography-mass spectrometry (LC-MS). The fermentation and quantitative results are shown in Table 1 and FIGS. 1a, b, and c.

Table 1: absolute quantitation of mutant rzmaCholA, holoacsrzmA and wild-type rzmA after heterologous expression in DSM7029

And (4) conclusion: for rzmA, when the Cs-AL region is replaced by holA, the derivative of C8-rzmA can be directionally obtained, and the yield of the rzmA is reduced by a small extent compared with the wild type. In the case of holoA, however, the C2-holoA derivative can be obtained in an oriented manner when the Cs-AL region is replaced by rzmA, and the yield is comparable to that of the wild type. From the above results, it can be seen that, at the in vivo experimental level, the replacement of the Cs-AL region can realize the directional manipulation of the lipopeptide lipid chain, and the derivatives with the extended or shortened lipopeptide chain can be obtained.

Example 2:

further, the glbA gene cluster derived from DSM7029 was manipulated to replace the Cs-AL region of glbA with Cs-AL from holoA of the hetero-bacterium DS19002 and Cs-AL from glpA of the homo-bacterium DSM 7029. The obtained mutants are glbACsholA and glbACsglpA respectively, and the catalytic activity of the mutants is verified from in vitro and in vivo levels. The method is characterized in that the full-length glbA gene cluster and the capability of the corresponding mutant for catalyzing and forming the final product glbA and the derivative thereof are taken as the standard (the structures are shown in figure 2, a and b), absolute quantification is adopted for quantification, the derivative and the wild-type lipopeptide product are separated and purified, and then the mutant is quantified by taking the purified derivative and the wild-type lipopeptide product as the standard, and a liquid chromatography-mass spectrometry (LC-MS) is taken as a quantification instrument.

The Red/ET recombination system and operation in DSM7029 referred to in example 2 was performed as described in reference 3(Wang X, Zhou H, Chen H, ET al, discovery of recombinant enzymes administration of crystalline biochemical genes in Burkholderia species [ J ]. Proceedings of the National Academy of Sciences,2018,115(18):201720941.) using the strain DSM7029 with Red α β (Red α β 7029).

The specific implementation steps are as follows:

(1) the replacement of the Cs of the slipsin by the Cs of the rhizopus peptide and the Cs of the slipsin is directly carried out in Red alpha beta 7029, and because apra resistance is required to be used, the aforementioned plasmid p15A-tnPA-km-holA is firstly subjected to loop recombination in Escherichia coli GB2005-Red, and the apra resistance is inserted to obtain a plasmid p15A-tnPA-km-holA-apra, the primer pair is p10 and p11 (shown as SEQ ID NO: 14-15), and the template is pR6K-apra-phiC31 plasmid. Subsequently, the homopolar primer pair p12, p13 (shown in SEQ ID NO: 16-17) was used to PCR amplify the apra-resistant holoA-Cs-AL fragment using p 15A-tnPA-km-holoA-apra as a template.

The acquisition of the apra-resistant glpA-Cs-AL fragment was performed by overlap PCR using the previously described plasmid p15A-p15A-tnpA-km-holA-apra, primers p14 and p15 (SEQ ID NOS: 18-19), the glpA-Cs fragment PCR from the DSM7029 genome, primers p16 and p17 (SEQ ID NOS: 20-21), and after obtaining the two fragments, the overlap PCR amplification was performed using p15, p16 and the two fragments as primers and template, and the amplification method was performed according to the Primerstax instructions of TAKARA.

The acquisition of the glbA-Cs-apra fragment was similar to that of the glpA-Cs-apra fragment, the amplification primer of the apra fragment was p18-19 (shown in SEQ ID NOS: 22-23), the amplification primer of the glbA-Cs fragment was p20-p21 (shown in SEQ ID NOS: 24-25), and then overlapping PCR amplification was performed using p19, p20 and the two fragments as primers and template, and the amplification method was performed according to the PrimerstamMax instruction of TAKARA.

The three fragments with apra resistance comprise a HolA-Cs-AL, a glpA-Cs-AL and a glbA-Cs-AL, wherein the Cs is placed in a km-resistant constitutive promoter P_Tn5-kmUnder control of (c). Because the original glpA-Cs can interfere recombination when the glpA-Cs-apra is inserted, a Red alpha beta DSM7029 strain with deleted glpA-Cs needs to be constructed, and is named as the Red alpha beta DSM7029 delta glpACs, and the construction process is as follows: PCR amplification of a gene-resistant fragment with homologous arms, with primer pairs p22, p23 (as shown in SEQ ID NOS: 26-27), according to reference 3(Wang X, Zhou H, Chen H, et alnome mining of cryptic biosynthetic gene clusters in Burkholderiales species[J]Proceedings of the National Academy of Sciences,2018,115(18):201720941.) recombination resulted in the knock-out strain Red α β DSM7029 Δ glpACs using primer pairs p24, p25 (as shown in SEQ ID NO: 28-29) was subjected to colony PCR, and the PCR protocol was performed in accordance with the instruction of Primerstart Max of TAKARA, whereby the fragment length of the successfully knocked-out group amplified fragment was 1.3 kb. Then the aforementioned Cs-AL fragments including holoA-Cs-AL, glpA-Cs-AL and glbA-Cs-AL were electroporated into the strain Red α β DSM7029 Δ glpACs, and recombination was carried out as described in literature 3 to obtain a recombinant strain 7029-glbACsholA in which Cs-AL of glbA was replaced by Cs-AL of holoA, respectively; (ii) strain 7029-glbACsglpA in which Cs-AL of glbA is replaced by Cs-AL of glpA; wild-type glbA Cs-AL is with apra resistance and P_Tn5-kmThe Cs-AL replacement of the glbA promoter strain 7029-glbACs, which serves as a control for mutant quantification, is convenient for comparison.

The role of the key exchange domain can be verified at the in vivo level (in vivo) after the fermentation expression, product identification and quantification of these mutant strains. The strain is fermented by inoculating 500 μ l of overnight strain into 50ml of CYMG liquid culture medium, fermenting for 72 hr, adding adsorption resin XAD-16 (2%) into the fermentation liquid, adsorbing for one day, centrifuging to collect the strain and resin, pouring off the supernatant, eluting with 40ml of methanol for 2 hr, evaporating methanol with rotary evaporator, adding 1ml of imported methanol, and detecting the product with liquid chromatography-mass spectrometry (LC-MS). The fermentation results are shown in Table 2 and FIGS. 2, a-c.

Table 2: validation of the role of the Key exchange Domain in glbA from in vitro and in vivo levels

And (4) conclusion: for glbA, when the Cs-AL region is replaced by holoA, the derivative of C8-glbA can be obtained in an oriented mode, and the yield of the derivative is greatly improved (increased by about 16 times) relative to the wild type; when the Cs-AL region is replaced by glpA, the C10-glbA derivative can be obtained in an oriented manner, and the yield is also greatly improved (about 8 times).

According to the results, on the level of in vivo experiments, the replacement of the Cs-AL region can realize the directional control of the lipopeptide lipid chain, and the yield of the derivatives is excellent, thereby being beneficial to the construction of engineering bacteria.

Sequence listing

<110> Shandong university

<120> key exchange structural domain for controlling lipopeptide chain length change and mutant and application thereof

<141>2020-12-07

<160>27

<210>1

<211>479

<212>PRT

<213> Paraburkholderia rhizoxinica HKI 454

<221> amino acid sequence of Cs-AL region of rzmA

<222>（1）…（479）

<400> 1

MDASVMSTTY ALSAAQTEIW LAQQLYPDSP VYNIAQYTVI EGVIEPAVFE AALRQVIDEA 60

DTLRLQFIDS DDGLRQRIGT PAWSMPVLDL TAQADPQAAA QAWMRADYQQ PVNLTQGPLF 120

CYALLKVAPA QWMWYQRYHH IMMDGYGRYL IAQRVAYVYS ALCEGTTPAE CDFGSILQLL 180

ESDAQYQISA QRAQDEAYWL KHCANWSEPA TLASRSAPVL QQRLRQTTYL AIQALGDTAP 240

DARRLAQFMT AAMAAYLYRF TGEQDVVLGL PVKVRFGADR HIPGMKSNTL PLRLTMRPGM 300

NLSSLMQQAA QEMQSGLRHQ RYPSEALRRQ LGMPSGQRLF GTTVNVMPFD LDLSFGGYSA 360

TNHNLLNGPA EDLMLGVYWT PGSHQLRIDF DANPACYTPE GLGAHQRRFI RFMQVLAADA 420

TQPIDSIDLL DANERHRLLV EWNATQRDYP AHLCVHQLFE AQVERTPKAT ALVYEDQTL 479

<210> 2

<211> 479

<212>PRT

<213> Paraburkholderia rhizoxinica HKI 454

<221> amino acid sequence of Cs-AL region of holA

<222>（1）…（479）

<400> 2

MSAGAISLTT YPLSSAQTEI WLAQQLHSGS PVYNIAQYTV IEGAIDPTVF EAALRQVIDE 60

ADSLRLQFVE SEAGLRQRIG SPAWSMPVLN LTAEAESQAV AQAWMRTDYE EPVDLMQGPL 120

FQYALLKVAP KQWIWYQRYH HIMMDGYGAV LIAQRVAQVY SALCAEREPA PCTFGSVLKL 180

LESDAQYRAS AQREKDEAYW LKHCAHWPVP ATLAGRAAPA LQHRLRQTAY LATQALGDAA 240

SDVGRLAQFL TAALATYLHR MTRAQDVALG LPVTARLGAN RHIPGVVSNT VPLRFTFEAK 300

MTLASLLQQA TQIQRGFRYQ RYPSEALRRK LELMPGQALF GATVNVMPFD YDLSFDGYPS 360

SNHNLLNGPV EDLMLAVYWT PDNPQLRIDF DVNPACYTAE ELEAHRCRFV RFMQALAADV 420

TQPIGEIDLL DTEERHRLLI EWNATQQDYP AHQCIHQLFE AQVEHTPEAM ALVYEEQTL 479

<210> 3

<211> 489

<212> PRT

<213> DSM7029

<221> amino acid sequence of Cs-AL region of glbA

<222>（1）…（489）

<400> 3

MQANHNTPSY PLSPAQQEIW IAEQLNPGTG VYNTAGYADI RGPVDVARFG AALRQTMAEA 60

DCLRAVFHDH GEGPRQTPND TMAWPFPLID VSGEPDPQAA AEAWMQADLA QPPDFAHGPL 120

FRMALFRAAP DRFFWYLCGH HLVADAFGLT LLMHRLAEIY SALMLQQAPP PAWFGKLEDM 180

IAADRDYHTS DALEHDRTYW ISRLAGCPEA VSLSTGTAAA AAAPPGKFHR QWAELPAATG 240

DALRAIARDN GAGVPPLLIA LVAAYLYRMT GQEDLVLGLP VTGRPGRELR RIPGMLANVL 300

PLRFEMTPNL GFGALFQQTA REVRQALRHQ RYRGETMLRD LQQAGAVARL YAHNVNVMAF 360

DSEVWFAGHA GRTCNLSNGP VDDLSLTIYD DGEGKGLRIA FDAPAALYTE DELSGHRERF 420

IRLANTLVAD LGAPIAAVDL MAAEERQRLL TDWNVAEPSP RRTTLASLFE QQAGRAPDAV 480

ALVASEERL 489

<210> 4

<211> 507

<212> PRT

<213> DSM7029

<221> amino acid sequence of Cs-AL region of glpA

<222>（1）…（507）

<400> 4

MFPSRQDPTA SSPPRAAAAD APTLHELASV QQVVWLDQVL DPGVPSYNLG AMWQIEGDLD 60

PVLFESVLNE LAAAHDALRL VLQLEAGVAR QRVLPELEVK LPVIDLSMHD DARDRAWQHA 120

QTSFATPFQL YGEPLWQTQL LRVGPSSFYW LHRMHHVIGD GATVSLTYLA AWDLYKRRLA 180

GDTTPLEPGP SYLEFLDDDS AYLRSPRYQR DEQFWKQRYA QLPAPLLPVR EGVEQRQALP 240

SGQTSLYIER PRLRRFKALA ARFGCTLPHA MLALLAAYFA RAHQAPEVVI GVPVHNRGTA 300

RQKRTWGMFS SIIPVAIDVD PAQSFAALMQ GVAAELKRCY RHQRFPIADI NRALRLGQHG 360

RKQLFDVTLA LEEYPMDIYL GDRKLRVTKM YSGFEQTPLA VCLCDYHEDE PVAVRFNYNT 420

GAFEHEAAED LPRRIGVLLD AVLDAGDDRP LDDLRWFDDA EREQVLHTWN ATAQRYAEHD 480

RCLHEMFEAQ VQRTPDAVAV VAGEQRV 507

<210> 5

<211> 93

<212> DNA

<213> Artificial sequence

<221> p1

<222>（1）…（93）

<400> 5

TGATGCTGTG ATTGGCTTGC GTGAATTTAT CCGGCGTCTG GAGAGGGATT TAATCCCATA 60

CGCGCTAACC gatcttaagg atctccaggc a 93

<210> 6

<211> 91

<212> DNA

<213> Artificial sequence

<221> p2

<222>（1）…（91）

<400> 6

ACAATCGGTC AAGCAAGACA GTTAAAAAGA ACAGGGCAAG CCAGTACGTC GCACAGGCGC 60

GGCGCCATTG taagacgtcg atatctggcg a 91

<210> 7

<211> 91

<212> DNA

<213> Artificial sequence

<221> p3

<222>（1）…（91）

<400> 7

CAAAGTGGAA AATGCACGCA TTTCTCAGCA GCCGGGAAAA CCCACAAATG AATTGCACGT 60

CAGCAGTTGG taagacgtcg atatctggcg a 91

<210> 8

<211> 91

<212> DNA

<213> Artificial sequence

<221> p4

<222>（1）…（91）

<400> 8

GGCAGCATCT TCATCGGCGG CGCTGTGGTG CAATGGCTGC GCGACGGGCT GGGAATCATC 60

AAACAGGCGG gatcttaagg atctccaggc a 91

<210> 9

<211> 70

<212> DNA

<213> Artificial sequence

<221> p5

<222>（1）…（70）

<400> 9

CTGAGGTCAT TACTGGATCT ATCAACAGGA GTCCAAGCGA GCTCGATATC tgcatccgat 60

gcaagtgtgt 70

<210> 10

<211> 89

<212> DNA

<213> Artificial sequence

<221> p6

<222>（1）…（89）

<400>10

GTTTGGGCAG CGGATAATGC ATACGTAGTG GACATGACGC TAGCGTCCAT aatctgtacc 60

tccttaagtc agaagaactc gtcaagaag 89

<210> 11

<211> 25

<212> DNA

<213> Artificial sequence

<221> p7

<222>（1）…（25）

<400> 11

ctgaggtcat tactggatct atcaa 25

<210> 12

<211> 70

<212> DNA

<213> Artificial sequence

<221> p8

<222>（1）…（70）

<400> 12

ATGAGCTGGT GTGCCAAGCG GTTGGCCTGC GCATTCAGTT GCGTATAGCT gagcgtttgc 60

tcctcgtaga 70

<210> 13

<211> 75

<212> DNA

<213> Artificial sequence

<221> p9

<222>（1）…（75）

<400> 13

ATCAGCTGAT GCGCCAGACG GTTGGCCCGC GCATTCAGCT CAGCGTAACT cagtgtttga 60

tcttcataga ccaac 75

<210> 14

<211> 75

<212> DNA

<213> Artificial sequence

<221> p10

<222>（1）…（75）

<400> 14

ATCTCTTCAA ATGTAGCACC TGAAGTCAGC CCCATACGAT ATAAGTTGTT tgatcatatg 60

cggattagaa aaaca 75

<210> 15

<211> 74

<212> DNA

<213> Artificial sequence

<221> p11

<222>（1）…（74）

<400> 15

TTCCGCTTCC TTTAGCAGCC CTTGCGCCCT GAGTGCTTGC GGCAGCGTGA tagtgcttgg 60

attctcacca ataa 74

<210> 16

<211> 95

<212> DNA

<213> Artificial sequence

<221> p12

<222>（1）…（95）

<400> 16

CCGGGCCGAC CCCCAGGCCG ATCAGGTGGT GCGCCAGGCG GTTGGCGCGC TCGTTGAGCG 60

CGGCATAGGT gagcgtttgc tcctcgtaga ccaac 95

<210> 17

<211> 93

<212> DNA

<213> Artificial sequence

<221> p13

<222>（1）…（93）

<400> 17

GCGCACTGGA CCGACCTGCG CCCACGCAGC GTGGCCCGCC GCGCACACGC CCAACCGGAG 60

GCCTGAGGAC tcatctcgtt ctccgctcat gag 93

<210> 18

<211> 34

<212> DNA

<213> Artificial sequence

<221> p14

<222>（1）…（34）

<400> 18

GAACACAATC TGTACCTCCt taagtcagaa gaac 34

<210> 19

<211> 96

<212> DNA

<213> Artificial sequence

<221> p15

<222>（1）…（96）

<400> 19

GCGCACTGGA CCGACCTGCG CCCACGCAGC GTGGCCCGCC GCGCACACGC CCAACCGGAG 60

GCCTGAGGAC tcatctcgtt ctccgctcatg agctc 96

<210> 20

<211> 90

<212> DNA

<213> Artificial sequence

<221> p16

<222>（1）…（90）

<400> 20

CCGGGCCGAC CCCCAGGCCG ATCAGGTGGT GCGCCAGGCG GTTGGCGCGC TCGTTGAGCG 60

CGGCATAGGT cacgcgctgc tcgccggcca 90

<210> 21

<211> 41

<212> DNA

<213> Artificial sequence

<221> p17

<222>（1）…（41）

<400>21

AGGAGGTACA GATTgtgttc ccttcccgac aagatcccac c 41

<210> 22

<211> 43

<212> DNA

<213> Artificial sequence

<221> p18

<222>（1）…（43）

<400> 22

GTGATTCGCT TGCATaatct gtacctcctt aagtcagaag aac 43

<210> 23

<211> 93

<212> DNA

<213> Artificial sequence

<221> p19

<222>（1）…（93）

<400> 23

GCGCACTGGA CCGACCTGCG CCCACGCAGC GTGGCCCGCC GCGCACACGC CCAACCGGAG 60

GCCTGAGGAC atgcaagcga atcacaacac ccc 93

<210> 24

<211> 90

<212> DNA

<213> Artificial sequence

<221> p20

<222>（1）…（90）

<400> 24

CCGGGCCGAC CCCCAGGCCG ATCAGGTGGT GCGCCAGGCG GTTGGCGCGC TCGTTGAGCG 60

CGGCATAGGT gaggcgctct tctgacgcga 90

<210> 25

<211> 37

<212> DNA

<213> Artificial sequence

<221> p21

<222>（1）…（37）

<400> 25

AGGAGGTACA GATTatgcaa gcgaatcaca acacccc 37

<210> 26

<211> 90

<212> DNA

<213> Artificial sequence

<221>p22

<222>（1）…（90）

<400> 27

GCACCGGCGC GCCGGCCTGC CGCGGTCCGC GTTCCAGCGA CGTCCCTGCC AGGAGTGTTT 60

CGTGGTTGCT gaattacatt cccaaccgcg 90

<210> 27

<211> 89

<212> DNA

<213> Artificial sequence

<221>p23

<222>（1）…（89）

<400> 27

TCGGCCCCAC CCCGCGGCGG CGCAGTTCTC GCGCCAGCTG GTTGGCCCGC GCGTTCAGCT 60

CGGCATAGCT cgaaacgacc tgcaactta 89

Claims

1. A key exchange domain that controls lipopeptide lipid chain length changes, said key exchange domain being an initial condensation domain and its interface with an adjacent adenylation domain, the initial condensation domain being named the Cs domain and the adenylation domain being named the a domain; the boundary name of the Cs structural domain and the A structural domain is a Cs-A linker region; wherein the Cs domain is a domain responsible for catalyzing the condensation of an initiating fatty acyl substrate with a first acyl substrate during lipopeptide biosynthesis, the Cs domain being specific for a lipopeptide's lipid chain and mediating initiation of lipopeptide biosynthesis; the A domain refers to an aminoacyl substrate that catalyzes the formation of an upstream Cs domain from a free amino acid, the aminoacyl substrate being an aminoacyl-peptide carrier protein; the exchange structure domain is a Cs structure domain and a Cs-A linker region and is named as a Cs-AL structure domain;

the method is characterized in that:

2. A mutant obtained by exchanging Cs-AL domains using the key exchange domain for controlling lipopeptide lipid chain length change according to claim 1, wherein: the rhizopus amide biosynthesis gene cluster mutant with the rhizopus peptide Cs-AL structural domain obtained after the Cs-AL structural domain of the rhizopus amide and the Cs-AL structural domain of the rhizopus peptide are exchanged is named rzmaCholA; the mutant of the rhizopus peptide biosynthetic gene cluster with the Cs-AL domain of the rhizopus amide is obtained after the Cs-AL domain of the rhizopus peptide is replaced by the Cs-AL domain of the rhizopus amide and is named as holoacsrzmA; the mutant of the slipperine biosynthetic gene cluster with the rhizopus peptide Cs-AL structural domain, which is obtained after the Cs-AL structural domain of the slipperine is replaced by the Cs-AL structural domain of the rhizopus peptide, is named as glbACsholA; the mutant of the biotransformation gene cluster of the sliding rod rhzomorph with the Cs-AL structural domain of the sliding rod rhzomorph is obtained after the Cs-AL structural domain of the sliding rod rhzomorph is replaced by the Cs-AL structural domain of the sliding rod rhzomorph and is named as glbACsglpa; the amino acid sequence of the rhizopus amide Cs-AL structural domain is shown as SEQ NO.1, the amino acid sequence of the rhizopus peptide Cs-AL structural domain is shown as SEQ NO.2, the amino acid sequence of the Cs-AL structural domain of the slipperine is shown as SEQ NO.3, and the amino acid sequence of the Cs-AL structural domain of the gramicidin is shown as SEQ NO. 4.

3. Use of the mutant of claim 2 for the preparation of lipopeptide derivatives with different lipid chain lengths.

4. Use according to claim 3, characterized in that: mutant rzmaCsyla catalyzes the synthesis of rzmA derivatives of C8 lipid chain length in a heterologous host Schlegellella brevitaea DSM 7029; the mutant holoacsrzma catalyzes the synthesis of C2 lipid chain length holoa derivatives in a heterologous host schlegellella brevitalea DSM 7029; the mutant glbACsholA catalyzes the synthesis of a C8 lipid chain length glbA derivative in the homologous host Schlegellella brevitalea DSM 7029; the mutant glbACsglpa catalyzes the synthesis of the C10 lipid chain length glbA derivative in the homologous host Schlegellella brevitalea DSM 7029.