CN112522219B - Key amino acid site for controlling lipopeptide chain length change and mutant and application thereof - Google Patents

Abstract

The invention discloses a key amino acid site for controlling lipopeptide chain length change, a mutant and an application thereof, which are characterized in that the key site of an initial condensation structural domain (Cs) in rhizopus amide and rhizopus peptide is subjected to point mutation or combined mutation, and various novel lipopeptide derivatives with different fatty acid chain lengths can be obtained. For rhizopus amide, the Q36, Y138, R148 sites are decisive, Q136, M143 is ancillary, mutants R148A, R148G, Q36G + R148G, Y138A + R148G, Q136A + Y138A + R148G, Y138A + M143A + R148G, Q136A + Y138A + M143A + R148G all tend to obtain long chain acyl lipopeptide products, and mutants R148V, R148L and R148M tend to produce short chain acyl lipopeptide derivatives. The invention can effectively guide the reformation of lipopeptide which has medicinal prospect even has been prepared, and has wide application value.

Description

Key amino acid site for controlling lipopeptide fat chain length change and mutant and application thereof

Technical Field

The invention relates to a lipopeptide compound synthesis related amino acid, a mutant and an application thereof, in particular to a key amino acid site for controlling lipopeptide chain length change, a mutant and an 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 the module, it transfers the downstream substrate-adenylated amino acid activated by the adenylation domain to the condensation domain and subsequently condenses with upstream substrates, including intermediates synthesized from the last module or starting substrates synthesized from related genes (starting condensation domains), such as fatty acids and 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 means of modification of the lipopeptide lipid chain are mainly 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, which is mainly anteisoundecanoyl, A21978C2, which is isododecanoyl, and A21978C3, which is anteisotridecanoyl. The research shows that when the length of the lipid chain at the N end of the product exceeds C11, the product has greater 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, it would be beneficial to explore a combinatorial biosynthesis method for directed modification of lipopeptide chain length based on elucidating the key amino acids for specific control of the fatty acyl substrate in the Cs domain of non-ribosomal lipopeptides, or to provide great benefits for modification of potential lipopeptides and even drug-already-formed lipopeptides, such as providing competitive advantages for the fatty acyl substrate during daptomycin 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 crystal structure-based key amino acid sites and mutants and applications for controlling the change in the lipid chain length of lipopeptides, 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 invention aims to provide a key amino acid site for controlling the change of the lipopeptide fat chain length, a mutant and application thereof.

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

The invention relates to a key amino acid site for controlling the change of the lipopeptide lipid chain length, wherein the key amino acid site is positioned in an initial condensation domain, the initial condensation domain is named as a Cs domain, the Cs domain refers to a domain which is responsible for catalyzing the condensation of an initial lipoyl substrate and a first aminoacyl substrate in the lipopeptide biosynthesis process, and exists on lipopeptide synthesis gene clusters of various bacteria and fungi, and the Cs domain has specificity to the lipopeptide lipid chain and mediates the initiation of lipopeptide biosynthesis; the Cs structural domain has crystal structure data, and the key amino acid sites for controlling the change of the lipid chain length of the lipopeptide are determined to be three key sites and two auxiliary sites for determining the lipid chain length in the Cs structural domain according to the crystal structure data;

the method is characterized in that:

the Cs domain mediates the initial synthesis of rhizopus amide by initial condensation of the C2 fatty acyl substrate with a leucyl substrate, wherein the C2 fatty acyl substrate is acetyl-CoA (C2-CoA), the leucyl substrate is L-type leucyl-peptide carrier protein, abbreviated as L-Leu-PCP, and the leucyl substrate, when reacted in vitro, is the substrate analog L-type leucyl-N-acetylcysteamine, abbreviated as: L-Leu-SNAC; the rhizopus amide is abbreviated as rzmA, and the biosynthesis gene cluster of the rhizopus amide is derived from a bacterium Parabrukholderia rhizoxinica HKI 454; three key amino acid sites for controlling the change of the lipopeptide fat chain length start from the initial amino acid methionine, namely glutamine-Q36 at the 36 th position, tyrosine-Y138 at the 138 th position and arginine-R148 at the 148 th position, two sites playing an auxiliary role are glutamine-Q136 at the 136 th position and methionine-M143 at the 143 th position, and related amino acid sequences are shown as SEQ NO. 1.

The mutant obtained by performing point mutation or combined mutation on the key amino acid site for controlling the change of the lipopeptide lipid chain length is characterized in that: the mutants which are obtained by carrying out point mutation or combined mutation on the Cs domain of rzmA and generate changes on the specificity of a lipid chain comprise 5 single mutants which are respectively named as R148G, R148A, R148M, R148L and R148V in sequence and/or 5 combined mutants which are respectively named as Q36G + R148G, Y138A + R148G, Q136A + Y138A + R148G, Y138A + M143A + R148G, Q136A + Y138A + M143A + R148G in sequence.

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

Wherein: single mutants R148G, R148A, R148M, R148L or R148V catalyse the synthesis of C2 to C10 lipid chain length rzmA derivatives in the heterologous host schlegellella brevitalea DSM7029, or/and the single mutants catalyse the condensation of C2 to C10-CoA with L-Leu-SNAC under ex vivo conditions; combination mutants Q36G + R148G, Y138A + R148G, Q136A + Y138A + R148G, Y138A + M143A + R148G or Q136A + Y138A + M143A + R148G catalyze the synthesis of C10 to C16 lipid chain length rzmA derivatives in the heterologous host Schlegellella brevitalea DSM7029 or the combination mutants catalyze the condensation of C10 to C16-CoA with L-Leu-SNAC under ex vivo conditions.

The key amino acid site for controlling the change of the lipopeptide lipid chain length is positioned in an initial condensation domain, the initial condensation domain is named as a Cs domain, the Cs domain refers to a domain which is responsible for catalyzing the condensation of an initial fatty acyl substrate and a first aminoacyl substrate in the lipopeptide biosynthesis process of lipopeptide and exists on lipopeptide synthesis gene clusters of various bacteria and fungi, and the Cs domain has specificity to the lipopeptide lipid chain and mediates the initiation of lipopeptide biosynthesis; the Cs structure domain is subjected to homologous modeling by using crystal structure data of a rhizopus amide (rzmA) Cs structure domain, and key amino acid sites for controlling the change of the lipid chain length of the lipopeptide are determined according to the structure, namely three key sites and two auxiliary sites for determining the lipid chain length in the Cs structure domain;

the method is characterized in that:

the Cs domain mediates initial synthesis of Rhizopus peptide by initial condensation of C8 fatty acyl substrate with valyl substrate, wherein the C8 fatty acyl substrate is C8-CoA, the valyl substrate is L-type valyl-peptide carrier protein, abbreviated as L-Val-PCP, the Rhizopus peptide is abbreviated as holoA, and its biosynthetic gene cluster is derived from bacterium Parabrukholderia rhizoxinica HKI 454; three key amino acid sites for controlling the change of the lipopeptide lipid chain length start from the initial amino acid methionine, namely glutamine-Q37 at the 37 th site, tyrosine-Y139 at the 139 th site and alanine-A149 at the 149 th site, two sites playing auxiliary roles are glutamine-Q137 at the 137 th site and methionine-M144 at the 144 th site, and related amino acid sequences are shown as SEQ NO. 2.

The mutant obtained by performing point mutation or combined mutation on the key amino acid site for controlling the change of the lipopeptide lipid chain length is characterized in that: the mutants which are obtained by carrying out point mutation or combined mutation on the Cs domain of holoA and generate changes on the specificity of a lipid chain comprise 5 single mutants which are respectively named as A149G, A149V, A149M, A149L and A149R in turn and/or 5 combined mutants which are respectively named as Q37G + A149G, Y139A + A149G, Q137A + Y139A + A149G, Y139A + M144A + A149G, Q137A + Y139A + M144A + A149G in turn.

Use of the above mutants for the preparation of lipopeptide derivatives with different lipid chain lengths, wherein: single mutants A149G, A149V, A149M, A149L or A149R catalyse the synthesis of C2 to C10 lipid chain length holA derivatives in a heterologous host Schlegellella brevitalea DSM 7029.

The key amino acid site for controlling the change of the lipopeptide lipid chain length is positioned in an initial condensation domain, the initial condensation domain is named as a Cs domain, the Cs domain refers to a domain which is responsible for catalyzing the condensation of an initial fatty acyl substrate and a first aminoacyl substrate in the lipopeptide biosynthesis process of lipopeptide and exists on lipopeptide synthesis gene clusters of various bacteria and fungi, and the Cs domain has specificity to the lipopeptide lipid chain and mediates the initiation of lipopeptide biosynthesis; the Cs structural domain is subjected to homologous modeling by using crystal structure data of a rhizopus amide (rzmA) Cs structural domain, and key amino acid sites for controlling the change of the lipid chain length of the lipopeptide are determined according to the structure, namely three key sites and two auxiliary sites for determining the lipid chain length in the Cs structural domain;

the method is characterized in that:

the Cs domain mediates initial synthesis of daptomycin by initial condensation of a C10 fatty acyl substrate with a tryptophanyl substrate, wherein the C10 fatty acyl substrate is C10-ACP and the tryptophanyl substrate is an L-form tryptophanyl-peptide carrier protein, abbreviated as L-Trp-PCP; daptomycin is abbreviated as dap, and a biosynthesis gene cluster of daptomycin is derived from streptomyces roseosporus NRRL 11379; the three key amino acid sites for controlling the change of the lipopeptide fat chain length are alanine-A34 at the 34 th site, tyrosine-L137 at the 137 th site and alanine-A147 at the 147 th site respectively from the initial amino acid methionine, and the two sites playing an auxiliary role are phenylalanine-F135 at the 135 th site and leucine-L142 at the 142 th site, and related amino acid sequences are shown as SEQ NO. 3.

The mutant obtained by performing point mutation or combined mutation on the key amino acid site for controlling the change of the lipopeptide lipid chain length is characterized in that: there are 5 single mutants, respectively designated as a147G, a147V, a147M, a147L, a147R, and/or 5 combined mutants, respectively designated as a34G + a147G, L137A + a147G, F135A + L137A + a147G, L137A + L142A + a147G, F135A + L137A + L142A + a147G, which are obtained by point mutation or combined mutation of the Cs domain of dap to produce changes in lipid chain specificity.

The use of the above mutants in the preparation of lipopeptide derivatives with different lipid chain lengths.

The method provides key amino acid sites for controlling the change of the lipopeptide chain length in the Cs domain, realizes the directional modification of the lipopeptide chain length through point mutation and combined mutation, and provides technical support for modifying 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, the C2 acyl of the lipopeptide rzmA is successfully prolonged to the length of C12-C16 acyl by using key site mutation and combined mutation, and the antitumor activity of the obtained C14-rzmA novel derivative is improved by at least 17 times compared with that of an original product.

The invention has the following beneficial effects and remarkable advantages: 1) the invention utilizes key site mutation or combined mutation for directly modifying Cs, is not limited by species, has extremely low invasion to Cs and has small influence on the yield of final products. 2) The invention provides a basis for the modification of a non-ribosomal lipopeptide lipid chain, and provides a reference and a technical method for the further optimization of a compound for subsequent patent medicine, such as daptomycin, and the further development and modification of potential medicines.

Drawings

FIG. 1a is a schematic diagram of the co-crystallization of the Cs domain mutant R148A of rzmA with its most preferred fatty acyl substrate C8-CoA (PDB No. 7C 1L). b is the binding pocket of the amino acid and fatty acid chain that determines the specificity of the fatty acyl substrate after amplification. Including Q36, Y138, a148 (originally R), Q136, M143, and active center H140.

FIG. 2: a is rhizopus amide (Rhizommide) all products, including original products and derivative products generated by mutation, wherein 1a (C2-rzmA) is the original product generated by rzmA of the original gene cluster, and the rest are products generated by mutants; b is the product of rhizopus peptide (holrhizoxin), including original product and derivative product generated by mutation, wherein 2C (C6-holA) and 2d (C8-holA) are the original product generated by the original gene cluster holA, and the rest is the product generated by the mutant; c and d are the in vivo experimental results (in vivo) of point mutations at the mutant R148 (for rzmA gene cluster) and A149 (for holoA gene cluster), respectively;

FIG. 3: a is rzm-Cs in vitro reaction process and product schematic, L-Leu-SNAC substrate is the analogue of its original substrate L-Leu-PCP, Cn-CoA is its natural substrate; b is the in vitro LC-MS experimental result (in vitro) of rzmA-Cs Wild Type (WT) and the mutant at R148 site; and c and d are results of in vitro and in vivo mutation LC-MS of other key sites including Q36 and Y138 and auxiliary sites Q136 and M143 in Cs of the rzmA gene cluster respectively, and all the mutations are subjected to further mutation on the basis of R148G.

Detailed Description

The present invention will be described in detail below with reference to specific 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 molecular manipulations, such as direct cloning, mutation, 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, for example, reference 1.Wangh, Liz, JianR, et al, ExoCET, exonuclease in a video assembly combining with RecET combining for high efficiency direct DNA cloning from complex genes [ J ]. Nucleic Acids Research2018 Mar 16; 46(5), e28.doi:10.1093/nar/gkx1249. and document 2.Wangh, BianX, Xiao AL. improved laboratory mutagenesis by recombination using ccdB for counterselection [ J ]. Nucleic Acids Research2014 Mar; 42(5), e37.doi:10.1093/nar/gkt1339. The construction of GB2005-dir, GB2005-red-gyrA4622, GB2005, and vector p15A-cm-hyg-ccdB, R6K-amp-ccdB, R6k-tnpA-km involved in recombination in the examples is as described above. The pET28a vector is purchased from Addgene, and the Escherichia coli BL21(DE3) is purchased from China general microbiological culture Collection center (CGMCC). The antibiotics used and the concentrations were also performed according to the above-mentioned documents, and the abbreviations of the antibiotics used are kanamycin: km, chloramphenicol: cm, apramycin: and (3) apra.

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

Primer synthesis: the primers used in the examples of the present invention were all prepared by Biotechnology engineering (Shanghai) Ltd. The Universal DNA purification recovery kit was purchased from tiangen biochemical technology (beijing) ltd.

For the cultivation of E.coli, the example used a low-salt Luria-Bertani medium (LB medium) with the formulation: 10g of tryptone (tryptone), 5g of yeast extract (yeast extract), 1g of sodium chloride (NaCl), 12% agar powder added into a solid culture medium, 1000mL of double distilled water added into the solid culture medium, and sterilizing the solid culture medium 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 MgCl 2.2H2O, 10mL/l glycerol, 12% agar powder added to the solid medium, double distilled water added to 1000mL, and sterilization at 115 ℃ for 30 min. Corresponding media components were purchased from OXIOD.

Example 1:

the first key site of rzmA and holoA, namely R148/A149 site, is subjected to point mutation, and is positioned at the entrance position of a binding pocket (as shown in figure 1), mutants R148G, R148A, R148V, R148L, R148M, A149G, A149V, A149L, A149M and R148R are obtained, the catalytic activity of the Cs domain is verified from the in vitro and in vivo level, and the mutation effect of the corresponding mutant is verified from the in vitro and in vivo level for rzmA. For holoA, the Cs domains based on rzmA and holoA are the same enzyme, the catalytic mechanism is similar, and therefore, the mutation effect of the holoA corresponding mutant is verified from the in vivo experimental level on the basis of the rzmA experiment.

The in vivo level is based on the ability of the full-length rzmA/holoA gene cluster and its mutants to catalyze the formation of the final product rzmA/holoA and its derivatives (structure is shown in FIG. 2, a), and the quantification adopts relative quantification, namely the amount of the final product/derivative in the mutant is relative to the amount of the wild-type final product.

The in vitro reaction is based on the ability of the protein ex vivo to catalyze the condensation of a fatty acyl substrate (here, fatty acyl-coa) with an aminoacyl substrate (here, the analog aminoacyl-SNAC, structure shown in figure 3, a), using relative quantitation, i.e., the amount of product obtained in the mutant relative to the amount of product of the wild-type protein.

The specific implementation steps are as follows:

(1) according to anti mash: (https://antismash.secondarymetabolites.org/#！/start) The predicted Cs domain region of rzmA has the amino acid sequence shown in SEQ ID NO: 1 is shown. And (2) carrying out sequence alignment with Cs of holoA, wherein the Cs amino acid sequence of the holoA is shown as SEQ ID NO: 2, performing homologous modeling on the Cs domain of the holA (Swiss model: https:// swissmodel. expasy.org/interactive) by using the crystal structure (PDB: 7C1H) of the previously obtained rzmA Cs domain, combining the Cs sequence difference, searching a key site for controlling the specificity of the fatty acyl substrate, and searching an amino acid near the reaction activity center (H140 for rzmA) according to the principle

In rzmA Cs, the final product is C2 acyl, so the amino acid side chain is larger, and the accommodated substrate is smaller, such as arginine (R). In Cs of holoA, the key amino acid is smaller than that in rzmA, such as alanine (A), because the final product is C8 acyl, and the smaller amino acid is located at the entrance position of the binding pocket to accommodateThe larger substrate enters the binding pocket. Accordingly, R148 in rzmA Cs was initially located, with a149 in the corresponding position in holoa Cs.

(2) The procedures were as described in 1 (1.Wangh, LiZ, JianR, et al. Exocet: exonuclease in visual analysis combined with RecET recombination for highly affecting direct DNA cloning from complex genes [ J ]. Nucleic Acids Research2018 Mar 16; 46(5) e28.doi:10.1093/nar/gkx1249) after PCR to obtain a p15A-cm-hyg-ccdB vector with homologous arm as template, the primers were p1-p2 for rzmA p3-p4 for holA (as shown in SEQ ID NO: 4-7) directly cloned with the digested DSM aScI/rmA, the full-length genomic sequence of rzmA of rZR gene 7370, the full-length genomic sequence of rzRS # rBmS + RG + rBmS + rzrBmS + rBmS + rzrS + rS + rzrS + rS + rzrS + rBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrBrB And (4) holoA. Then, the Red/ET Loop recombination technique was used, and the specific procedure was as described in document 2, (2.WangH, BianX, XiaL, ET al. improved amplified Nucleic acid mutagenesis by side recombination using ccdB for correlation selection [ J ]. Nucleic Acids Research2014 Mar; 42(5): e37.doi:10.1093/nar/gkt1339.), the tnPA-IR-km gene cassette obtained by PCR was inserted into the p15A-cm-rzmA/holA plasmid, the primer was p5-p6 (as shown in SEQ ID NO: 8-9), and the template was R6K-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 p9, p10 (shown as SEQ ID NO: 10-13) with homology arms for PCR amplification of amp-ccdB gene cassettes, inserting amp-ccdB gene cassettes at positions R148 or A149 of p15A-tnPA-km-rzm and p15A-tnPA-km-hol respectively, and performing Red/ET loop recombination in Escherichia coli GB2005-Red-gyrA462 to construct plasmids p15A-tnPA-km-rzm-amp-ccdB for R148 and p15A-tnPA-km-hol-amp-ccdB for A149. After obtaining the plasmid, synthesized oligonucleotides with mutation sites are used for carrying out traceless repair on the plasmid with the amp-ccdB gene cassette, and for rzmA, the constructed mutations are R148A, R148G, R148V, R148L and R148M respectively, and the traceless repair oligonucleotides are O1-O5 respectively (shown as SEQ ID NO: 14-18). For holoA, the constructed mutations are A149R, A149G, A149V, A149L and A149M respectively, and the traceless repair oligonucleotides are O6-O10 respectively (as shown in SEQ ID NOS: 19-23). The above mutants were electrotransformed in DSM7029WT for heterologous expression at a voltage of 1250V, the procedure was carried out according to the instructions of an Eppendorf Eporator electrotransformer, and the amount of DNA added was 5. mu.g. The constructed strains are 7029-R148A, 7029-R148G, 7029-R148V, 7029-R148L, 7029-R148M, 7029-A149R, 7029-A149G, 7029-A149V, 7029-A149L and 7029-A149M respectively. The substrate specificity control of R148/A149 can be verified from the in vivo level (in vivo) after fermentation expression, product identification and quantification of these mutants. 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 1 and FIGS. 2a, b, c, d, and the bolded group is the main product or the derivative with the best yield.

Table 1: the first key site R148/A149 and the corresponding mutant in vivo experimental level generated derivatives. For rzmA, R148G, R148V, R148L, R148M, R148 are wild type controls. The corresponding holoA is A149G, A149V, A149L and A149M, and the wild type is A149.

And (4) conclusion: for rzmA, R148G produced derivatives of C2 to C10 lipid chain length, with optimal production of C8-rzmA relative to the wild type product C2-rzmA of R148. R148A produced derivatives of C2 to C8 lipid chain length, with optimal production of C8-rzmA lipid chain derivatives. R148V produced derivatives of C2 to C6 lipid chain length, with optimal production of C6-rzmA lipid chain derivatives. R148L produced derivatives of C2 to C6 lipid chain length, with optimal production of C4-rzmA lipid chain derivatives. R148M produced derivatives of C2 to C4 lipid chain length, with optimal production of C4-rzmA lipid chain derivatives. For holoa, a149G can produce derivatives of C6 to C10 lipid chain length, with optimal production of C8-holoa lipid chain derivatives, relative to the wild-type a149 product C6 to C8-holoa. A149V produced derivatives of C4 to C8 lipid chain length, with the best yield of C6-holA lipid chain derivatives. A149L produced derivatives of C4 to C8 lipid chain length, with the best yield of C6-holA lipid chain derivatives. A149M produced derivatives of C2 to C6 lipid chain length, with the best yield of C6-holA lipid chain derivatives. A149R produced derivatives of C2 to C4 lipid chain length, with the best yield of C2-holA lipid chain derivatives. From the above results, it is clear that at the in vivo experimental level, the R148/A149 site plays a crucial role in the control of the specificity of the lipopeptide lipid chain, and by manipulating this site, derivatives with extended or shortened lipid chains can be obtained.

(3) At the in vitro protein level, the control effect of the R148 position in rzmA on the fatty acyl substrate was verified. Selecting a Cs region of rzmA (amino acids M1-D431, see SEQ ID NO: 1), carrying out Red/ET linear recombination with a pET28a vector digested with NdeI/Xhol after PCR amplification by using primers with homologous arms as p11 and p12 (see SEQ ID NO: 24-25), constructing pET 28-rzmA-Cs, then inserting an amp-ccdB gene box and further carrying out traceless repair by using oligonucleotides with mutation sites, wherein the primers with homologous arms, p7 and p8 as well as p9 and p10 (see SEQ ID NO: 10-13) are used for PCR amplification of the amp-ccdB gene box, and the oligonucleotides with mutation sites are respectively O1-O5 (see SEQ ID NO: 14-18), so as to obtain mutants of pET28a-rzmA-Cs, which correspond to R148A, R G, R148 and R39148 and R3985. The mutated and original Cs-bearing pET28a plasmid was subsequently electroporated into E.coli BL21(DE3) at an electrotransformation voltage of 1250V, in accordance with the instructions of the Eppendorf electrotransformer, with an amount of DNA of 500ng being added. The obtained strains with mutant plasmids are BL21-R148, BL21-R148A, BL21-R148G, BL21-R148V, BL21-R148L and BL 21-R148M.

The method comprises the following steps of fermenting the strain and then extracting Cs protein: the cells were scraped from the plate and cultured overnight at 37 ℃ at 200rpm in 50mL of a kanamycin-resistant LB liquid (15. mu.g/mL). The next day, the cells were inoculated in 2 liters of LB liquid with the same resistance at an inoculum size of 1% for 2.5 hours, tempered to 18 ℃ and precooled for about 3 hours, and then 0.1mM isoproyl-1-thio-. beta. -D-galactopyranoside (IPTG) was added to induce protein expression. After culturing for 12 to 18 hours, the cells were collected by centrifugation at 4200rpm for 20 minutes using a low-temperature centrifuge at 4 ℃. The supernatant was discarded, and 80ml of a resuspension solution (50mM Tris-HCl,300mM NaCl and 50mM imidazole, pH 7.5) was added to resuspend the cells. Ultrasonic crushing, and the procedure is as follows: 20% power, 1 second for 2 seconds, 20 minutes, when broken, the container is placed on ice. The nickel column matrix was centrifuged at 24000rpm for 30min at 4 ℃ during which three column volumes were first washed with eluent (50mM Tris-HCl,300mM NaCl and 300mM imidazole, pH 7.5) followed by equilibration of two column volumes with resuspension liquid. Placing into a chromatography cabinet for precooling. Transferring the centrifuged crushing liquid supernatant into a clean glass container, pouring the crushed liquid supernatant into a column in several times, and hanging 80ml of supernatant within 2-4 h. After hanging the column, three column volumes were washed with resuspension to remove contaminating proteins. Two column volumes were equilibrated with assay solution (50mM Tris-HCl,300mM NaCl, pH 7.5) to remove imidazole. Subsequently, 100U of Thrombin enzyme (purchased from Beijing Solebao technologies Co., Ltd.) dissolved in the assay solution was added thereto, and the enzyme was cleaved overnight at 4 ℃. The next day, the enzyme digestion solution was collected, 2. mu.L of Nanodrop was used for concentration determination, and 5-10. mu.L of SDS-PAGE gel (purchased from Osry Biotechnology Ltd.) was used for verification, indicating that the band size was 48 KD. The remaining protein was concentrated to 20-30mg/mL using a protein concentration tube (Millipore), and then dispensed into 1.5mL EP tubes and stored at-80 ℃.

The in vitro experiment of the enzyme proves that the aminoacyl substrate is a substrate analogue L-Leu-SNAC, synthesized according to reference 3 (3.Ehmann, D.E., Trauger, J.W., Stachelhaus, T. & Walsh, C.T.Aminoacyl-SNACs as small-molecule substrates for the condensation domains of non-ribosomal peptide synthesis, chemistry & biology7, 765. 772, doi:10.1016/s1074-5521(00)00022-3 (2000)), fatty acyl substrates were purchased from sigma (Cn-CoA) including C2-CoA, C4-CoA, C6-CoA, C8-CoA, C10-CoA, in vitro, protein 20. mu.M, L-Leu-SNAC 8mM, 80. mu.M each from C2-CoA to C10-CoA, 20. mu.l reaction system, and fatty acyl substrate from C2 to C10 at the same concentration were added to simulate the in vivo environment, and the substrate competition effect was evaluated. After reaction at 30 ℃ for 100min, the reaction was terminated with the same volume of methanol, and after removal of protein by centrifugation at 15000rpm, the product obtained by the protein reaction was detected and quantified by LC-MS. The key role of position R148 was examined at the in vitro level and the results are shown in table 2 and fig. 3a, b, with the bold group as the major product.

Table 2: in vitro experiments prove that the Cs domain and mutant of rzmA have the capability of catalyzing different fatty acyl substrates (fatty acyl coenzyme A) and acyl substrates (L-Leu-SNAC), and relative quantification of enzyme catalysis products is carried out by taking wild type R148 as one hundred percent

And (4) conclusion: from the in vitro experimental level, the key site R148 in the rzmA Cs is verified, and compared with the C2-Leu-SNAC product catalyzed by the wild type R148, the R148G mutant can catalyze the C8 to C10-Leu-SNAC, and the yield of the C8-Leu-SNAC is optimal. The R148A mutant can catalyze C6-C10-Leu-SNAC, and the yield of C8-Leu-SNAC is optimal. The R148V mutant can catalyze C6-C10-Leu-SNAC, and the yield of C6-Leu-SNAC is optimal. The R148L mutant can catalyze C4 to C8-Leu-SNAC, and the yield of C6-Leu-SNAC is optimal. The R148M mutant can catalyze C2-C6-Leu-SNAC, and the yield of C4-Leu-SNAC is optimal. It is verified from the in vitro experimental level that the R148 site plays a decisive role in the specific control of the lipopeptide lipid chain, and by manipulating this site, derivatives of the extended or shortened lipopeptide chain can be obtained, and the Cs domains of the same type can be manipulated accordingly.

(4) Finally, the crucial role of the R148/A149 site in the binding process of the acyl substrates of the lipids is demonstrated by crystallographic data and point mutations to rzmA and holoA gene clusters or Cs proteins, which are located at the entrance position of the pocket and can be manipulated to realize the directional modification of lipopeptide.

Example 2:

further co-crystallization studies were carried out on the Cs domain of rzmA, i.e., the R148A mutant was co-crystallized with its optimal fatty acyl substrate C8-CoA to successfully obtain the crystal structure (PDB No. 7C1L), and then the decisive amino acids inside the pocket were further analyzed, the analysis strategy was close to the fatty acyl base end of C8-CoA (C8-CoA: (C1L))

Internal) as shown in FIG. 1. The four amino acids of the alternative amino acids Q36, Q136, Y138 and M143 are obtained by analysis. Because the aforementioned key site at position R148 is at the entrance position of the pocket, in order to obtain a lipopeptide derivative with a longer lipid chain, the inventors further extended the interior of the pocket, i.e., single mutation or combined mutation for alternative amino acids Q36, Q136, Y138, M143, on the basis of opening the entrance of the binding pocket (R148G mutant), to obtain a lipopeptide derivative with a long chain.

Experiments are verified at in vivo and in vitro experimental levels, the in vivo level is based on the capability of catalyzing the full-length rzmA gene cluster and the mutant thereof to form the final product rzmA and the derivative thereof (the structure is shown in figure 2 and a), the quantification adopts relative quantification, namely the quantity of the final product/derivative in the mutant is relative to the quantity of the wild type final product, and a quantification instrument is LC-MS. The in vitro reaction is based on the ability of the in vitro protein to catalyze the condensation of a fatty acyl substrate (here, fatty acyl-coa) with an acyl substrate (here, the analog L-Leu-SNAC, structure shown in fig. 3, a), and is relatively quantitative, i.e., the amount of product obtained in the mutant is relative to the amount of product of the wild-type protein, and the quantitative instrument is liquid mass spectrometry (LC-MS).

The specific implementation steps are as follows:

(1) at the in vivo experimental level, further mutations were performed using the aforementioned mutant with R148G on the p15A vector, using the Red/ET-based ccdB reverse screening technique, the procedure was performed as in reference 2, (2.WangH, BianX, XiaoL, ET al. improved seamless mutagenesis by recombinant binding using ccdB for systematic selection [ J ]. Nucleic Acids Research2014 Mar; 42(5) e37.doi:10.1093/nar/gkt 1339.). After obtaining the amp-ccdB gene cassette by utilizing a primer PCR with a homologous arm, further performing traceless repair by using oligonucleotide of a mutation site, and constructing the mutation as follows: Q36G, Q136A, Y138A, M143A, Q136A + Y138A, Y138A + M143A, Q136A + Y138A + M143A, wherein the primer used when the Q36A mutation is added to the amp-ccdB gene cassette is p A-p A (shown in SEQ ID NO: 26-27), the primer used when the Q A-amp-ccdB gene cassette is obtained, the primer used when the Q136A mutation is added to the amp-ccdB gene cassette is p A-p A (shown in SEQ ID NO: 28-29), the plasmid Q136-amp-ccdB is obtained, the oligonucleotide used for traceless repair is O A-O A (shown in SEQ ID NO: 30-36), and the mutant plasmids Q36A, Q136A, Y138A, M143A, Q143 36136 + Y138A + M143 + M A + Y A + M A + Y143 + M A can be obtained respectively. For the Q36G mutation, the plasmid for traceless repair is Q36-amp-ccdB, and for Q136A, Y138A, M143A, Q136A + Y138A, Y138A + M143A, Q136A + Y138A + M143A, the plasmid for traceless repair is Q136-amp-ccdB. The above mutants were electrotransformed in DSM7029WT for heterologous expression at a voltage of 1250V, the procedure was carried out according to the instructions of an Eppendorf Eporator electrotransformer, and the amount of DNA added was 5. mu.g. The constructed bacterial strains are 7029-R148G-Q36G, 7029-R148G-Q136A, 7029-R148G-Y138A, 7029-R148G-M143A, 7029-R148G-Q136A-Y138A, 7029-R148G-Y138A-M143A and 7029-R148G-Q136A-Y138A-M143A respectively. After fermentation expression, product identification and quantification of the mutant strains, the fatty acyl substrate specificity control of the sites Q36, Q136, Y138 and M143 can be verified from the in vivo level (in vivo). 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 3 and FIG. 3c, with the bolded group being the main product or the best derivative.

(2) At the in vitro experimental level, the aforementioned R148G mutant on pET28a vector was used, using the Red/ET based ccdB reverse screening technique, the procedure was performed according to literature 2, (2.Wangh, BianX, Xiao, ET al, improved release mutagenesis by recombinant binding using ccdB for the correlation [ J ]. Nucleic Acids Research2014 Mar; 42(5) e37.doi:10.1093/nar/gkt 1339.). After obtaining the amp-ccdB gene cassette by utilizing a primer PCR with a homologous arm, further performing traceless repair by using oligonucleotide of a mutation site, and constructing the mutation as follows: Q36G, Q136A, Y138A, M143A, Q136A + Y138A, Y138A + M143A, Q136A + Y138A + M143A, wherein the primer used when the Q36A mutation is added to the amp-ccdB gene cassette is p A-p A (shown as SEQ ID NO: 26-27), the plasmid Q A-amp-ccdB is obtained, the primer used when the Q136A mutation is added to the amp-ccdB gene cassette is p A-p A (shown as SEQ ID NO: 28-29), the plasmid Q136-amp-ccdB is obtained, the oligonucleotide used for traceless repair is O A-O A (shown as SEQ ID NO: 30-36), and the mutant plasmids Q36, Q136A, Y138, M143A, Q136 + Y36138, Y A + M36143 + Y A + M A + Y36143 + A + M A + 36143 + A can be respectively obtained. For the Q36G mutation, the plasmid for traceless repair is Q36-amp-ccdB, and for Q136A, Y138A, M143A, Q136A + Y138A, Y138A + M143A, Q136A + Y138A + M143A, the plasmid for traceless repair is Q136-amp-ccdB. The mutant plasmid and pET28a plasmid with original Cs were then electroporated into E.coli BL21(DE3) at an electrotransformation voltage of 1250V, in accordance with the instructions for an Eppendorf electrotransformer, with an amount of 500ng DNA added. The obtained strain with the mutant plasmid is BL21-R148G-Q36G, BL21-R148G-Q136A, BL21-R148G-Y138A, BL21-R148G-M143A, BL21-R148G-Q136A-Y138A, BL21-R148G-Y138A-M143A, BL 21-R148G-Q136A-Y138A-M143A.

The method comprises the following steps of fermenting the strain and then extracting Cs protein: the cells were scraped from the plate and cultured overnight at 37 ℃ at 200rpm in 50mL of a kanamycin-resistant LB liquid (15. mu.g/mL). The next day, the cells were inoculated in 2 liters of LB liquid with the same resistance at an inoculum size of 1% for 2.5 hours, tempered to 18 ℃, pre-cooled for about 3 hours, and then added with 0.1mM isoproyl-1-thio-. beta. -D-galactopyranoside (IPTG) to induce protein expression. After culturing for 12 to 18 hours, the cells were collected by centrifugation at 4200rpm for 20 minutes using a low-temperature centrifuge at 4 ℃. The supernatant was discarded, and 80ml of a resuspension solution (50mM Tris-HCl,300mM NaCl and 50mM imidazole, pH 7.5) was added to resuspend the cells. Ultrasonic crushing, and the procedure is as follows: 20% power, 1 second for 2 seconds, 20 minutes, when broken, the container is placed on ice. The nickel column matrix was centrifuged at 24000rpm for 30min at 4 ℃, during which three column volumes were first washed with eluent (50mM Tris-HCl,300mM NaCl and 300mM imidazole, pH 7.5) and subsequently two column volumes were equilibrated with resuspension liquid. Placing into a chromatography cabinet for precooling. Transferring the centrifuged supernatant of the crushed liquid into a clean glass container, pouring the supernatant into a column in several times, and hanging 80ml of supernatant within 2-4 h. After hanging the column, three column volumes were washed with resuspension to remove contaminating proteins. Two column volumes were equilibrated with assay solution (50mM Tris-HCl,300mM NaCl, pH 7.5) to remove imidazole. Subsequently, 100U of Thrombin enzyme (purchased from Beijing Solebao technologies Co., Ltd.) dissolved in the assay solution was added thereto, and the enzyme was cleaved overnight at 4 ℃. The next day, the enzyme digestion solution was collected, 2. mu.L of Nanodrop was used for concentration determination, and 5-10. mu.L of SDS-PAGE gel (purchased from Osry Biotechnology Ltd.) was used for verification, indicating that the band size was 48 KD. The remaining protein was concentrated to 20-30mg/mL using a protein concentration tube (Millipore), and then dispensed into 1.5mL EP tubes and stored at-80 ℃.

The in vitro experiment of the enzyme proves that the aminoacyl substrate is a substrate analogue L-Leu-SNAC, synthesized according to reference 3 (3.Ehmann, D.E., Trauger, J.W., Stachelhaus, T. & Walsh, C.T.Aminoacyl-SNACs as small-molecule substrates for the condensation domains of non-ribosomal peptide synthesis, chemistry & biology7, 765. 772, doi:10.1016/s1074-5521(00)00022-3 (2000)), fatty acyl substrates were purchased from sigma (Cn-CoA) including C2-CoA, C4-CoA, C6-CoA, C8-CoA, C10-CoA, in vitro, protein 20. mu.M, L-Leu-SNAC 8mM, 80. mu.M each from C2-CoA to C10-CoA, 20. mu.l reaction system, and fatty acyl substrate from C2 to C10 at the same concentration were added to simulate the in vivo environment, and the substrate competition effect was evaluated. After reaction at 30 ℃ for 100min, the reaction was terminated with the same volume of methanol, and after removal of protein by centrifugation at 15000rpm, the product obtained by the protein reaction was detected and quantified by LC-MS. The key role of the Q36, Q136, Y138, M143 sites was examined at the in vitro level and the results are shown in table 3 and fig. 3 d.

(3) The cytotoxicity of the original product C2-rzmA and the modified derivative products C8-rzmA and C14-rzmA are measured, and the cell strain is a human breast cancer cell measured by using a microplate reader (VERSA max microplate reader, MD, USA): MCF-7 and human colon cancer cells: HCT-116. Experimental reagent: the compounds were prepared as 10mM stock solutions and dispensed and stored at-20 ℃ until use. Positive control: doxorubicin was dissolved in cellular DMSO as a 10mM stock solution and stored at-20 ℃ until use. Other reagents: MTT (M2128, Sigma, USA), DMSO (cell culture grade CAS-NO:67-68-5, Applichem, Germany), DMSO (analytical grade, batch number: 20151102, national drug group, China); fetal bovine serum (Gibco, South America); medium (Hyclone, USA); doxorubicin (Doxorubicin, Sigma, usa).

The experimental method comprises the following steps: inoculating cells in 96-well plate at cell density of 3-4 × 10 ³ A hole. Placing in 5% CO ₂ Culturing in a 37 ℃ cell culture box, adding a sample to be detected with specified concentration after the cells adhere to the wall, wherein the negative control group is DMSO with the same concentration, and three parallel holes are arranged on the medicament with the same concentration. After 48h of the drug-added incubation, 20. mu.L of MTT (5mg/mL) was added to each well, the incubation was continued for 4h, after the supernatant was aspirated by a pump, 150. mu.L of DMSO was added, the OD value of each well was measured at 570nm with a microplate reader, and the IC50 value was calculated using IC50 software (Prism 5.0). The experiment was repeated three times.

Statistics were performed on IC50 for 3 independent calculations, expressed as mean ± standard deviation (s.d). Wherein the activities of the C2-rzmA to MCF-7 and HCT-116 are both more than 100 mu M, the activities of the C8-rzmA to MCF-7 and HCT-116 are respectively 6.95 +/-1.46 mu M and 70.53 +/-9.03 mu M, while the activities of the C14-rzmA to MCF-7 and HCT-116 are increased to 5.261 +/-0.609 mu M and 5.832 +/-0.396 mu M, and the activity of the derivative is improved by at least 17 times.

Table 3: verifying the Cs domain of rzmA at in vitro and in vivo levels, the critical sites within the pocket include critical sites Q36 and Y138 and helper sites Q136, M143. All mutations were subjected to further combinatorial mutations based on the R148G mutant. The final product or the enzyme-catalyzed product was relatively quantified with respect to the best derivative catalyzed by the R148G mutant, i.e., the derivative C8-rzmA in the R148G mutant was 100% for in vivo experiments, and the enzyme-catalyzed product C8-Leu-SNAC in the R148G mutant was 100% for in vitro experiments.

And (4) conclusion: from the in vivo experimental level it was verified that the key sites Q36, Q136, Y138 and M143 in the Cs domain of rzmA, in addition to the original products i.e. C2 to C6-rzmA, mutant R148G + Q36G could produce derivatives of C G to C G-rzmA, where C G-rzmA is the best yield derivative, R148G + Y138G could produce derivatives of C G to C G-rzmA, where C G-rzmA is the best yield derivative, R148G + Y138 + M143G could produce derivatives of C G to C G-rzmA, where C G-rzmA is the best yield derivative, R148Y G + Y138 + M143G could produce derivatives of C G to C G-rzmA, where C G-rzmA is the best yield derivative, R G + Q136 + Y138M 143M G + z 72M G-rzmA is the best yield derivative, where C G-rzmA is the best yield derivative. From in vitro experimental level, it was verified that the key sites in the Cs domain of rzmA, Q36, Q136, Y138 and M143, mutant R148G + Q36G, could produce products from C10 to C12-Leu-SNAC, with C10-Leu-SNAC being the best-yielding product, R148G + Y138A could produce products from C10 to C14-Leu-SNAC, with C14-Leu-SNAC being the best-yielding product, R148 14 + Q136 14 + Y138 14 could produce products from C14 to C14-Leu-SNAC, with C14-Leu-SNAC being the best-yielding product, R148 14 + Y138 14 + M143 14 could produce products from C14 to C14-Leu-SNAC, with C14-Leu-SNAC being the best-yielding product, R148 + Q136, Y138 + M143 14 + M14-SNAC could produce the best-C14-Leu-SNAC, with C14-Leu-C14 being the best-C-Leu-yielding product.

(4) Finally, in vitro and in vivo experiments and crystallography data can prove that in the four key amino acids Q36, Q136, Y138 and M143 in the binding pocket, Q36 and Y138 play a decisive role, Q136 and M143 play an auxiliary role, and the directional modification of lipopeptide can be realized by carrying out point mutation or combined mutation on the four sites, and the activity of the lipopeptide can be influenced significantly.

Example 3:

the key sites of the Cs domain of daptomycin are verified from in vitro and in vivo experimental levels, and comprise three key amino acid sites. The procedure was carried out analogously to examples 1 and 2.

Wherein said Cs domain mediates initial synthesis of daptomycin by initial condensation of a C10 fatty acyl substrate with a tryptophanyl substrate, wherein said C10 fatty acyl substrate is C10-ACP and said tryptophanyl substrate is an L-form tryptophanyl-peptide carrier protein, abbreviated L-Trp-PCP; daptomycin is abbreviated as dap, and a biosynthesis gene cluster of daptomycin is derived from streptomyces roseosporus NRRL 11379; the three key amino acid sites for controlling the change of the lipopeptide fat chain length are alanine-A34 at the 34 th site, tyrosine-L137 at the 137 th site and alanine-A147 at the 147 th site respectively from the initial amino acid methionine, and the two sites playing an auxiliary role are phenylalanine-F135 at the 135 th site and leucine-L142 at the 142 th site, and related amino acid sequences are shown as SEQ NO. 3.

There are 5 single mutants, respectively designated as a147G, a147V, a147M, a147L, a147R, and/or 5 combined mutants, respectively designated as a34G + a147G, L137A + a147G, F135A + L137A + a147G, L137A + L142A + a147G, F135A + L137A + L142A + a147G, which are obtained by point mutation or combined mutation of the Cs domain of dap to produce changes in lipid chain specificity.

The constructed mutant is expressed in Streptomyces roseosporus NRRL11379, and finally, an engineering strain with dap derivatives with different lipid chain lengths or directed high-yield daptomycin (C10 lipid chain length) is obtained.

Sequence listing

<110> Shandong university

<120> key amino acid sites for controlling lipopeptide lipid chain length change, mutants and applications thereof

<141>2020-12-07

<160>36

<210>1

<211>431

<212>PRT

<213>DSM 19002

<221> amino acid sequence of Cs domain of rzmA

<222>（1）…（431）

<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 D 431

<210> 2

<211> 431

<212>PRT

<213> DSM 19002

<221> amino acid sequence of holA Cs

<222>（1）…（431）

<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 D 431

<210> 3

<211> 463

<212> PRT

<213> Streptomyces roseosporus NRRL11379

<221> amino acid sequence of Cs of dap

<222>（1）…（463）

<400> 3

MDMQSQRLGV TAAQQSVWLA GQLADDHRLY HCAAYLSLTG SIDPRTLGTA VRRTLDETEA 60

LRTRFVPQDG ELLQILEPGA GQLLLEADFS GDPDPERAAH DWMHAALAAP VRLDRAGTAT 120

HALLTLGPSR HLLYFGYHHI ALDGYGALLH LRRLAHVYTA LSNGDDPGPC PFGPLAGVLT 180

EEAAYRDSDN HRRDGEFWTR SLAGADEAPG LSEREAGALA VPLRRTVELS GERTEKLAAS 240

AAATGARWSS LLVAATAAFV RRHAAADDTV IGLPVTARLT GPALRTPCML ANDVPLRLDA 300

RLDAPFAALL ADTTRAVGTL ARHQRFRGEE LHRNLGGVGR TAGLARVTVN VLAYVDNIRF 360

GDCRAVVHEL SSGPVRDFHI NSYGTPGTPD GVQLVFSGNP ALYTATDLAD HQERFLRFLD 420

AVTADPDLPT GRHRLLSPGT RARLLDDSRG TERPVPRATL PEL 463

<210> 4

<211> 93

<212> DNA

<213> Artificial sequence

<221> p1

<222>（1）…（93）

<400> 4

TGATGCTGTG ATTGGCTTGC GTGAATTTAT CCGGCGTCTG GAGAGGGATT TAATCCCATA 60

CGCGCTAACC gatcttaagg atctccaggc a 93

<210> 5

<211> 91

<212> DNA

<213> Artificial sequence

<221> p2

<222>（1）…（91）

<400> 5

ACAATCGGTC AAGCAAGACA GTTAAAAAGA ACAGGGCAAG CCAGTACGTC GCACAGGCGC 60

GGCGCCATTG taagacgtcg atatctggcg a 91

<210> 6

<211> 91

<212> DNA

<213> Artificial sequence

<221> p3

<222>（1）…（91）

<400> 6

CAAAGTGGAA AATGCACGCA TTTCTCAGCA GCCGGGAAAA CCCACAAATG AATTGCACGT 60

CAGCAGTTGG taagacgtcg atatctggcg a 91

<210> 7

<211> 91

<212> DNA

<213> Artificial sequence

<221> p4

<222>（1）…（91）

<400> 7

GGCAGCATCT TCATCGGCGG CGCTGTGGTG CAATGGCTGC GCGACGGGCT GGGAATCATC 60

AAACAGGCGG gatcttaagg atctccaggc a 91

<210> 8

<211> 70

<212> DNA

<213> Artificial sequence

<221> p5

<222>（1）…（70）

<400> 8

CTGAGGTCAT TACTGGATCT ATCAACAGGA GTCCAAGCGA GCTCGATATC tgcatccgat 60

gcaagtgtgt 70

<210> 9

<211> 89

<212> DNA

<213> Artificial sequence

<221> p6

<222>（1）…（89）

<400> 9

GTTTGGGCAG CGGATAATGC ATACGTAGTG GACATGACGC TAGCGTCCAT aatctgtacc 60

tccttaagtc agaagaactc gtcaagaag 89

<210> 10

<211> 91

<212> DNA

<213> Artificial sequence

<221> p7

<222>（1）…（91）

<400> 10

GTTAAAAGTG GCGCCCGCGC AATGGATGTG GTATCAGCGC TATCACCATA TCATGATGGA 60

TGGATATGGt tttgtttatt tttctaaata c 91

<210> 11

<211> 93

<212> DNA

<213> Artificial sequence

<221> p8

<222>（1）…（93）

<400> 11

CACATTCCGC CGGCGTCGTT CCCTCGCATA GCGCGCTATA CACGTATGCA ACGCGTTGCG 60

CGATCAGGTA tttgttcaaa aaaaagcccgc tc 93

<210> 12

<211> 91

<212> DNA

<213> Artificial sequence

<221> p9

<222>（1）…（91）

<400> 12

GCTGAAAGTG GCGCCCAAGC AGTGGATTTG GTATCAGCGC TATCACCATA TCATGATGGA 60

TGGCTATGGT tttgtttatt tttctaaata c 91

<210> 13

<211> 78

<212> DNA

<213> Artificial sequence

<221> p10

<222>（1）…（78）

<400> 13

CACGCTCCGC GCACAGCGCG CTGTACACTT GCGCAACACG CTGCGCGATG AGCACtttgt 60

tcaaaaaaaa gcccgctc 78

<210> 14

<211> 130

<212> DNA

<213> Artificial sequence

<221>O1

<222>（1）…（130）

<400> 14

AAGTGGCGCC CGCGCAATGG ATGTGGTATC AGCGCTATCA CCATATCATG ATGGATGGAT 60

ATGGTgctTA CCTGATCGCG CAACGCGTTG CATACGTGTA TAGCGCGCTA TGCGAGGGAA 120

CGACGCCGGC 130

<210> 15

<211> 130

<212> DNA

<213> Artificial sequence

<221>O2

<222>（1）…（130）

<400> 15

AAGTGGCGCC CGCGCAATGG ATGTGGTATC AGCGCTATCA CCATATCATG ATGGATGGAT 60

ATGGTggaTA CCTGATCGCG CAACGCGTTG CATACGTGTA TAGCGCGCTA TGCGAGGGAA 120

CGACGCCGGC 130

<210> 16

<211> 130

<212> DNA

<213> Artificial sequence

<221>O3

<222>（1）…（130）

<400> 16

AAGTGGCGCC CGCGCAATGG ATGTGGTATC AGCGCTATCA CCATATCATG ATGGATGGAT 60

ATGGTgtaTA CCTGATCGCG CAACGCGTTG CATACGTGTA TAGCGCGCTA TGCGAGGGAA 120

CGACGCCGGC 130

<210> 17

<211> 130

<212> DNA

<213> Artificial sequence

<221>O4

<222>（1）…（130）

<400> 17

AAGTGGCGCC CGCGCAATGG ATGTGGTATC AGCGCTATCA CCATATCATG ATGGATGGAT 60

ATGGTttaTA CCTGATCGCG CAACGCGTTG CATACGTGTA TAGCGCGCTA TGCGAGGGAA 120

CGACGCCGGC 130

<210> 18

<211> 130

<212> DNA

<213> Artificial sequence

<221>O5

<222>（1）…（130）

<400> 18

AAGTGGCGCC CGCGCAATGG ATGTGGTATC AGCGCTATCA CCATATCATG ATGGATGGAT 60

ATGGTatgTA CCTGATCGCG CAACGCGTTG CATACGTGTA TAGCGCGCTA TGCGAGGGAA 120

CGACGCCGGC 130

<210> 19

<211> 128

<212> DNA

<213> Artificial sequence

<221>O6

<222>（1）…（128）

<400> 19

GCTGAAAGTG GCGCCCAAGC AGTGGATTTG GTATCAGCGC TATCACCATA TCATGATGGA 60

TGGCTATGGT cggGTGCTCA TCGCGCAGCG TGTTGCGCAA GTGTACAGCG CGCTGTGCGC 120

GGAGCGTG 128

<210> 20

<211> 128

<212> DNA

<213> Artificial sequence

<221>O7

<222>（1）…（128）

<400> 20

GCTGAAAGTG GCGCCCAAGC AGTGGATTTG GTATCAGCGC TATCACCATA TCATGATGGA 60

TGGCTATGGT ggaGTGCTCA TCGCGCAGCG TGTTGCGCAA GTGTACAGCG CGCTGTGCGC 120

GGAGCGTG 128

<210> 21

<211> 128

<212> DNA

<213> Artificial sequence

<221>O8

<222>（1）…（128）

<400> 21

GCTGAAAGTG GCGCCCAAGC AGTGGATTTG GTATCAGCGC TATCACCATA TCATGATGGA 60

TGGCTATGGT gtaGTGCTCA TCGCGCAGCG TGTTGCGCAA GTGTACAGCG CGCTGTGCGC 120

GGAGCGTG 128

<210> 22

<211> 128

<212> DNA

<213> Artificial sequence

<221>O9

<222>（1）…（128）

<400> 22

GCTGAAAGTG GCGCCCAAGC AGTGGATTTG GTATCAGCGC TATCACCATA TCATGATGGA 60

TGGCTATGGT ttaGTGCTCA TCGCGCAGCG TGTTGCGCAA GTGTACAGCG CGCTGTGCGC 120

GGAGCGTG 128

<210> 23

<211> 128

<212> DNA

<213> Artificial sequence

<221>O10

<222>（1）…（128）

<400> 23

GCTGAAAGTG GCGCCCAAGC AGTGGATTTG GTATCAGCGC TATCACCATA TCATGATGGA 60

TGGCTATGGT atgGTGCTCA TCGCGCAGCG TGTTGCGCAA GTGTACAGCG CGCTGTGCGC 120

GGAGCGTG 128

<210> 24

<211> 60

<212> DNA

<213> Artificial sequence

<221> p11

<222>（1）…（60）

<400> 24

TCATCACAGC AGCGGCCTGG TGCCGCGCGG CAGCCATATG gacgctagcg tcatgtccac 60

<210> 25

<211> 60

<212> DNA

<213> Artificial sequence

<221> p12

<222>（1）…（60）

<400> 25

TTGTTAGCAG CCGGATCTCA GTGGTGGTGG TGGTGGTGtt agtcgagcag gtcgatgctg 60

<210> 26

<211> 99

<212> DNA

<213> Artificial sequence

<221> p13

<222>（1）…（99）

<400> 26

ATCCGCTGCC CAAACGGAGA TTTGGCTAGC GCAGCAACTG TATCCAGACA GCCCCGTCTA 60

CAACATTGCG ttaattaatt tgtttatttt tctaaatac 99

<210> 27

<211> 101

<212> DNA

<213> Artificial sequence

<221> p14

<222>（1）…（101）

<400> 27

AGACGGCTGG CTCAATCACG CCTTCAATCA CCGTGTACTG CGCAATGTTG TAGACGGGGC 60

TGTCTGGATA ttaattaatt tgttcaaaaa aaagcccgct c 101

<210> 28

<211> 99

<212> DNA

<213> Artificial sequence

<221> p15

<222>（1）…（99）

<400> 28

CAATCTGACG CAGGGACCGC TTTTTTGCTA TGCGTTGTTA AAAGTGGCGC CCGCGCAATG 60

GATGTGGTAT ttaattaatt tgtttatttt tctaaatac 99

<210> 29

<211> 101

<212> DNA

<213> Artificial sequence

<221> p16

<222>（1）…（101）

<400> 29

ATCCACCATA TCCATCCATC ATGATATGGT GATAGCGCTG ATACCACATC CATTGCGCGG 60

GCGCCACTTT ttaattaatt tgttcaaaaa aaagcccgct c 101

<210> 30

<211> 123

<212> DNA

<213> Artificial sequence

<221> O17

<222>（1）…（123）

<400> 30

CAAACGGAGA TTTGGCTAGC GCAGCAACTG TATCCAGACA GCCCCGTCTA CAACATTGCG 60

gggTACACGG TGATTGAAGG CGTGATTGAG CCAGCCGTCT TTGAAGCGGC ATTGCGTCAA 120

GTC 123

<210> 31

<211> 123

<212> DNA

<213> Artificial sequence

<221> O18

<222>（1）…（123）

<400> 31

CAGGGACCGC TTTTTTGCTA TGCGTTGTTA AAAGTGGCGC CCGCGCAATG GATGTGGTAT 60

gcgCGCTATC ACCATATCAT GATGGATGGA TATGGTGGAT ACCTGATCGC GCAACGCGTT 120

GCA 123

<210> 32

<211> 123

<212> DNA

<213> Artificial sequence

<221> O19

<222>（1）…（123）

<400> 32

ACCGCTTTTT TGCTATGCGT TGTTAAAAGT GGCGCCCGCG CAATGGATGT GGTATcagcg 60

cgctCACCAT ATCATGATGG ATGGATATGG TGGATACCTG ATCGCGCAAC GCGTTGCATA 120

CGTG 123

<210> 33

<211> 130

<212> DNA

<213> Artificial sequence

<221> O20

<222>（1）…（130）

<400> 33

CGCTTTTTTG CTATGCGTTG TTAAAAGTGG CGCCCGCGCA ATGGATGTGG TATcagcgct 60

atcaccatat catggcgGAT GGATATGGTG GATACCTGAT CGCGCAACGC GTTGCATACG 120

TGTATAGCGC 130

<210> 34

<211> 124

<212> DNA

<213> Artificial sequence

<221> O21

<222>（1）…（124）

<400> 34

ACCGCTTTTT TGCTATGCGT TGTTAAAAGT GGCGCCCGCG CAATGGATGT GGTATgcgcg 60

cgctCACCAT ATCATGATGG ATGGATATGG TGGATACCTG ATCGCGCAAC GCGTTGCATA 120

CGTG 124

<210> 35

<211> 130

<212> DNA

<213> Artificial sequence

<221> O22

<222>（1）…（130）

<400> 35

CGCTTTTTTG CTATGCGTTG TTAAAAGTGG CGCCCGCGCA ATGGATGTGG TATcagcgcg 60

ctcaccatat catggcgGAT GGATATGGTG GATACCTGAT CGCGCAACGC GTTGCATACG 120

TGTATAGCGC 130

<210> 36

<211> 130

<212> DNA

<213> Artificial sequence

<221> O23

<222>（1）…（130）

<400> 36

CGCTTTTTTG CTATGCGTTG TTAAAAGTGG CGCCCGCGCA ATGGATGTGG TATgcgcgcg 60

ctcaccatat catggcgGAT GGATATGGTG GATACCTGAT CGCGCAACGC GTTGCATACG 120

TGTATAGCGC 130

Claims (4)

1. A single mutant or a combined mutant which is obtained by carrying out point mutation or combined mutation on a Cs domain of rhizopus amide and has changed specificity on a lipid chain, and is characterized in that: the Cs domain refers to the domain of the initial condensation of the C2 fatty acyl substrate with the leucyl substrate during lipopeptide biosynthesis, where the C2 fatty acyl substrate refers to C2-CoA, the leucyl substrate refers to the L-type leucyl-peptide carrier protein, abbreviated as L-Leu-PCP, and the leucyl substrate, when reacted in vitro, refers to the substrate analog L-type leucyl-N-acetylcysteamine, abbreviated as: L-Leu-SNAC; the rhizopus amide is abbreviated as rzmA, and the biosynthesis gene cluster of the rhizopus amide is derived from a bacterium Paraburkholderirhizoxidiica HKI 454; the single mutants are R148G, R148A, R148M, R148L and R148V respectively; the combined mutants are respectively Q36G + R148G, Y138A + R148G, Q136A + Y138A + R148G, Y138A + M143A + R148G, Q136A + Y138A + M143A + R148G; the amino acid sequence is obtained by point mutation or combined mutation of glutamine-Q36 at the 36 th position, tyrosine-Y138 at the 138 th position, arginine-R148 at the 148 th position, glutamine-Q136 at the 136 th position and methionine-M143 at the 143 th position of the amino acid sequence shown in SEQ NO. 1.

2. Use of the mutant of claim 1 for the preparation of lipopeptide derivatives with different lipid chain lengths, wherein: in an in vivo experiment in the heterologous host schlegellella brevitalea DSM7029, R148G can produce derivatives of C2 to C10 lipid chain length, R148A can produce derivatives of C2 to C8 lipid chain length, R148V can produce derivatives of C2 to C6 lipid chain length, R148L can produce derivatives of C2 to C6 lipid chain length, R148M can produce derivatives of C2 to C4 lipid chain length; the combination mutant R148G + Q36G can produce derivatives of C8 to C14-rzmA, R148G + Y138A can produce derivatives of C8 to C14-rzmA, R148G + Q136A + Y138A can produce derivatives of C10 to C16-rzmA, R148G + Y138A + M143A can produce derivatives of C8 to C16-rzmA, R148G + Q136A + Y138A + M143A can produce derivatives of C10 to C16-rzmA; in vitro experiments, the mutant R148G can catalyze to obtain C8-C10-Leu-SNAC, the mutant R148A can catalyze to obtain C6-C10-Leu-SNAC, the mutant R148V can catalyze to obtain C6-C10-Leu-SNAC, the mutant R148L can catalyze to obtain C4-C8-Leu-SNAC, and the mutant R148M can catalyze to obtain C2-C6-Leu-SNAC; the combination mutant R148G + Q36G may produce a product from C10 to C12-Leu-SNAC, R148G + Y138A may produce a product from C10 to C14-Leu-SNAC, R148G + Q136A + Y138A may produce a product from C10 to C14-Leu-SNAC, R148G + Y138A + M143A may produce a product from C10 to C14-Leu-SNAC, R148G + Q136A + Y138A + M143A may produce a product from C12 to C16-Leu-SNAC.

3. A single mutant or a combinatorial mutant with altered lipid chain specificity obtained by point mutation or combinatorial mutation of the Cs domain of rhizopus peptide, characterized by: the Cs domain refers to the domain of the initial condensation of the C8 fatty acyl substrate with a valyl substrate during lipopeptide biosynthesis, wherein the C8 fatty acyl substrate refers to C8-CoA, the valyl substrate refers to L-type valyl-peptide carrier protein, abbreviated as L-Val-PCP, the Rhizopus peptide, abbreviated as holoA, and its biosynthetic gene cluster is derived from the bacterium Paraburkholderia rhizoxidica HKI 454; the single mutants are A149G, A149V, A149M, A149L and A149R respectively; the combined mutants are respectively Q37G + A149G, Y139A + A149G, Q137A + Y139A + A149G, Y139A + M144A + A149G, Q137A + Y139A + M144A + A149G; the peptide is obtained by point mutation or combined mutation of glutamine-Q37 at the 37 th position, tyrosine-Y139 at the 139 th position, alanine-A149 at the 149 th position, glutamine-Q137 at the 137 th position and methionine-M144 at the 144 th position of an amino acid sequence shown in SEQ NO. 2.

4. Use of the mutant of claim 3 for the preparation of lipopeptide derivatives with different lipid chain lengths, wherein: in vivo experiments in the heterologous host schlegellella brevitalea DSM7029, a149G can produce derivatives of C6 to C10 lipid chain length, a149V can produce derivatives of C4 to C8 lipid chain length, a149L can produce derivatives of C4 to C8 lipid chain length, a149M can produce derivatives of C2 to C6 lipid chain length, and a149R can produce derivatives of C2 to C4 lipid chain length.

Images