Direct cloning method and application of large biosynthetic gene cluster

Abstract

The invention relates to direct cloning of a biosynthetic gene cluster, in particular to a direct cloning method and application of an ultra-large biosynthetic gene cluster, a microbial genome is extracted from a low-melting-point agarose gel block through combination, and the ultra-large gene cluster can be rapidly cloned in a targeted manner by utilizing the specific cutting characteristic of CRISPR / Cas and a saccharomyces cerevisiae TAR technology. According to the method, mechanical shearing can be avoided by extracting and treating the genome in the thallus embedding block, and the operation is simple, so that the integrity of an ultra-large target gene cluster can be maintained to the maximum extent, and particularly, the probability that rearrangement is extremely easy to occur in a yeast body due to excessive overlapped fragments can be reduced for (ultra)-large PKS / NRPS gene clusters. CRISPR / Cas mediated cleavage can occur at any DNA site, and a target gene cluster can be released in a most accurate and fastest manner, so that targeted cloning is not limited by restriction enzyme cleavage sites any more. The high efficiency of the TAR technology enables the target cloning of (ultra) large gene clusters to be simple and feasible.

Description

Technical Field

[0001] This invention relates to the direct cloning of biosynthetic gene clusters, and particularly to methods and applications for the direct cloning of large biosynthetic gene clusters. Background Technology

[0002] The rapid development of modern DNA sequencing technology has led to an exponential increase in the number of potential natural product (NP) biosynthetic gene clusters (BGCs) in microbial genomes in databases. The vast majority of these BGCs have not yet been functionally identified and are thus considered silent / orphan BGCs. Their encoded products represent a vast treasure trove of small molecule compounds, injecting new vitality into the development of small molecule drugs. However, due to the lack of universal strategies or technologies for high-throughput activation of silent BGCs, prioritizing their activation remains a challenge for researchers. To date, for most researchers, amplifying or completely de novo synthesizing a large BGC (e.g., >30kb) via PCR remains difficult. Therefore, direct cloning and heterologous expression of BGCs are considered a commonly used strategy for activating silent BGCs. Over the past decade, various methods for directly cloning target BGCs have been reported.

[0003] Currently, there are three main strategies for obtaining target BGCs: 1. Library construction; 2. PCR-based bottom-up assembly; 3. Direct cloning. Among them, the first strategy belongs to non-targeted cloning, while the latter two belong to targeted cloning.

[0004] 1. Library Construction: This strategy requires extensive screening (1000–2000 clones), which is time-consuming and labor-intensive. Due to its non-targeting nature, this strategy is not suitable for cloning and activating specific BGCs from a large number of uncharacterized BGCs. However, this strategy currently has the highest upper limit for the size of BGCs that can be cloned; for example, Hashimoto et al. successfully cloned the polyketide compound quinolidomicin BGC (>215kb) using the BAC vector. Furthermore, the construction of such ultra-large gene cluster libraries requires highly specialized operational techniques, making it difficult for most researchers to implement.

[0005] 2. PCR-based bottom-up assembly: This strategy often needs to be used in conjunction with other assembly methods (e.g., IIS-type restriction endonucleases, Gibson assembly), but the assembly efficiency is limited by the length of the target BGCs, the number of repetitive sequences, and the GC content. Furthermore, random mutations are easily introduced during multiple PCR amplifications and assembly processes.

[0006] 3. Direct Cloning: To date, the largest BGC cloned using direct cloning is a 145kb candicidin BGC (75% GC content) from Streptomycesalbus J1074, which was directly cloned using the CAT-FISHING method. Therefore, although the reported direct cloning methods can solve the problem of direct cloning of the vast majority of BGCs, many ultra-large BGCs (e.g., >150kb) have not yet been obtained by direct cloning. All direct cloning strategies need to address the following three issues: (1) how to prepare high-quality genomic DNA; (2) how to cut the bilateral boundaries of the target BGC; and (3) how to assemble the target BGC with a linearized capture plasmid into a circular plasmid. Summary of the Invention

[0007] The purpose of this invention is to overcome the aforementioned problems of existing large target BGCs. It innovatively combines the extraction of microbial genomes from low-melting-point agarose gel blocks, the specific cutting characteristics of CRISPR / Cas, and the TAR technology of *Saccharomyces cerevisiae* to achieve rapid and targeted cloning of ultra-large BGCs. Extracting and processing the genome in the cell embedding block avoids mechanical shearing, simplifying the operation and maximizing the preservation of the integrity of ultra-large target gene clusters. This is especially true for large PKS / NRPS gene clusters, reducing the probability of rearrangements in yeast due to excessive overlapping fragments. CRISPR / Cas-mediated cutting can occur at relatively arbitrary DNA sites, releasing the target BGC in the most precise and rapid manner, freeing targeted cloning from the limitations of restriction enzyme sites. The high efficiency of TAR technology makes the targeted cloning of (ultra)large BGCs simple and feasible.

[0008] The present invention adopts the following technical solution:

[0009] A first aspect of the present invention provides a method for direct cloning of large biosynthetic gene clusters, comprising the following steps:

[0010] S1. Embed the bacterial cells in agarose to obtain an embedded block containing the genomic DNA of the target strain;

[0011] S2. Based on the CRISPR / Cas system, specific cutting is performed on both sides of the target BGC in the embedded block;

[0012] S3. Construct the target BGC capture plasmid;

[0013] S4. Directly clone the target BGC using yeast TAR technology.

[0014] Preferably, the CRISPR / Cas system is CRISPR / Cas9 or CRISPR / Cas12a.

[0015] Preferably, the target BGC is longer than 30kb, especially longer than 150kb.

[0016] Preferably, in step S3, the BAC carrier is used as the starting carrier.

[0017] Preferably, step S1 includes the following steps:

[0018] S1-1. Collect bacterial cells;

[0019] S1-2. Suspended bacterial cells form a bacterial solution;

[0020] S1-3. Mix the agarose solution with the bacterial culture, cool and solidify to obtain the pre-embedded block;

[0021] S1-4. The pre-embedded blocks are sequentially treated with lysozyme, proteinase K, and PMSF to obtain embedded blocks containing the genomic DNA of the target strain.

[0022] Preferably, step S2 includes the following steps:

[0023] S2-1. Based on the two sides of the target BGC in the embedding block, design crRNA template primers to obtain UP-crRNA at the upstream boundary and DN-crRNA at the downstream boundary of the target BGC.

[0024] S2-2. Specifically cut the genomic DNA in the embedded block to obtain the target BGC.

[0025] S2-3. Digest the embedded block with agarose enzyme to obtain a DNA mixture containing the target BGC.

[0026] In a second aspect, the present invention provides an application of the above-described method for direct cloning of large biosynthetic gene clusters, for directly cloning large biosynthetic gene clusters in microbial genomes.

[0027] A third aspect of the present invention provides a method for directly cloning rifamycin BGC from the A. mediterranei genome, using the above-described direct cloning method for large biosynthetic gene clusters.

[0028] In a fourth aspect, the present invention provides a method for increasing the yield of rifamycin in *A. mediterranei*, wherein rifamycin BGC is cloned using the above-mentioned direct cloning method for large biosynthetic gene clusters, and then the rifamycin BGC construct vector is transferred into *A. mediterranei* to overexpress rifamycin BGC.

[0029] In a fifth aspect, the present invention provides a method for directly cloning a super-large polyketide synthase (PKS) gene cluster from A. pretoriensis, using the above-described method for direct cloning of large biosynthetic gene clusters.

[0030] By implementing the above technical solution, the present invention has the following advantages:

[0031] This invention innovatively combines the extraction of microbial genomes from low-melting-point agarose gel blocks, the specific cleavage characteristics of CRISPR / Cas, and the TAR technology of Saccharomyces cerevisiae to achieve rapid and targeted cloning of ultra-large BGCs.

[0032] Embedding bacterial cells in low-melting-point agarose followed by subsequent genome extraction (including lysozyme and proteinase K treatment) significantly reduces mechanical shearing of the target genome, ensuring its integrity and greatly increasing capture efficiency. This DNA preparation technique is also relatively simple and reproducible. Furthermore, considering the high homologous recombination efficiency in yeast, for very large gene clusters (e.g., greater than 150 kb), processing in gel blocks reduces the probability of rearrangements / deletions occurring in yeast due to excessive overlapping fragments.

[0033] The CRISPR / Cas system was used to specifically cut the two sides of the target BGC in the embedding gel block. After treatment with agarose enzyme, a genomic mixture containing the complete target BGC fragment was obtained. Theoretically, the genomic DNA other than the target BGC fragment is also relatively complete, which greatly reduces the recombination of interfering sequences and linearized capture plasmids. Attached Figure Description

[0034] Figure 1 This is a flowchart of the direct cloning method for large gene clusters disclosed in this invention;

[0035] Figure 2 This is a schematic diagram of constructing a pCLBT01 carrier based on pCL01 in an embodiment of the present invention;

[0036] Figure 3 Electrophoresis diagram for PCR verification of the resistance gene and upstream and downstream homologous arm interfaces in pCLBT01-Vrif vector;

[0037] Figure 4 This is an electrophoresis image of the PCR verification of the resistance gene and the upstream and downstream homologous arm interfaces in pCLBT01-V157.

[0038] Figure 5 KpnI restriction map of pCLBT01-rif plasmid (clones 3, 5 and 8);

[0039] Figure 6The SalI and EcoRI double digestion map of pCLBT01-157k plasmid (clones 12, 46 and 85);

[0040] Figure 7 Growth curves of A. mediterranei U32 and its derivative strains;

[0041] Figure 8 The graph shows the rifamycin yield curves for A. mediterranei U32 and its derivative strains. Detailed Implementation

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] The effectiveness and practicality of the direct cloning method in this invention are tested below by directly cloning rifamycin BGC (rif, PKS, 93kb) from A. mediterranei U32 and by directly cloning a super-large PKS gene cluster (abbreviated as 157k) of approximately 157kb from the genome of A. pretoriensis NRRL B-24133.

[0044] Example 1

[0045] Preparation of high-quality genomic DNA from the target strain

[0046] Fresh seeds of *Amylopectinobacterium* were obtained on Bennet solid medium. The bacterial cells were then transferred to Bennet liquid medium using a bamboo stick and cultured at 30°C and 220 rpm for 24-26 hours on a shaker. Fresh bacterial cells were collected for the preparation of high-quality genomic DNA. The specific steps are as follows:

[0047] (1) Collect bacterial cells: Collect 4-5 mL of bacterial solution, centrifuge at 10000 rpm for 5 min, discard the supernatant, and weigh;

[0048] (2) Clean the bacterial cells: Add 1.5 mL of TE25S buffer to suspend the cells, centrifuge at 10000 rpm for 5 min, discard the supernatant, and repeat 1-2 times;

[0049] (3) Based on the wet weight of the bacterial cells, suspend them in TE25S buffer to approximately 2 g / mL;

[0050] (4) Prepare 1.5% low melting point agarose using TE25S and mix it with the bacterial solution in step (3) in equal volume;

[0051] (5) Preparation of embedding blocks: Inject the mixed liquid from step (4) into the mold, about 80 μL per hole, at 4℃.

[0052] Cooling and solidification;

[0053] (6) Lysozyme treatment: Place the embedding block in TE buffer, add lysozyme to make the final concentration 2 mg / mL, and incubate at 37°C for about 2 hours.

[0054] (7) Proteinase K treatment: Replace the solution in step (6) with NDS solution, add NDS solution, the working concentration of proteinase K is 2 mg / mL, and incubate at 50℃ for 4-6 hours until the embedded block is completely transparent.

[0055] (8) PMSF treatment: Replace the solution in step (7), add TE buffer, and then add PMSF to make the working concentration 0.1mM. Let it stand at room temperature for 1h. The genomic DNA embedding block has now been prepared.

[0056] Example 2

[0057] Cas12a specific cuts on both sides of the target BGC

[0058] First, primers for the synthesis of crRNA were designed and synthesized. The crRNA primers required for rif BGC capture are as follows:

[0059] T7p-DR-F(5'-3'):

[0060]

[0061] UP-rif-R(5'-3'):

[0062]

[0063] DN-rif-R(5'-3'):

[0064]

[0065] The crRNA primers required for 157k BGC capture are as follows:

[0066] T7p-DR-F(5'-3'):

[0067]

[0068] UP-157k-R(5'-3'):

[0069]

[0070] DN-157k-R(5'-3'):

[0071]

[0072] Next, double-stranded template DNA that can be transcribed into crRNA was obtained directly through primer annealing and extension. Subsequently, UP-crRNA upstream of the target BGC / DN-crRNA downstream of the target BGC was obtained through a commercially available T7 RNA polymerase in vitro transcription system. The T7 RNA polymerase in vitro transcription system is shown in Table 1 below.

[0073] Table 1. In vitro transcription system of T7 RNA polymerase

[0074] System components Volume (40 μL) 5×Buffer 8μL NTPs (10mM) 8μL T7 RNA polymerase 4μL RRI 2μL DNA template 3μL DNase / RNase, Free Water 15μL

[0075] React at 37℃ for 12-16 hours.

[0076] Next, after purifying the crRNA, Cas12a can specifically cleave the genomic DNA in the embedded block under the guidance of UP-crRNA / DN-crRNA. The reaction is carried out at 37°C for 2 hours to release the target BGC. The system for CRISPR / Cas12a to cleave the genome in the embedded block is shown in Table 2.

[0077] Table 2. Genomic structure in CRISPR / Cas12a cleavage embedding blocks.

[0078]

[0079]

[0080] Finally, the solution in the above reaction system was replaced with sterile ddH2O, and the gel block was digested with agarose enzyme and treated at 42°C for 30 min to obtain a DNA mixture containing the target BGC.

[0081] Example 3

[0082] Selection of capture vector and construction of target BGC capture plasmid

[0083] Given that we will later need to introduce rifamycin BGC(rif) into *A. mediterranei* to test whether adding a copy of rif can increase rifamycin yield, we need to modify the *Saccharomyces cerevisiae* / *Escherichia coli* / *Streptomyces* triple shuttle vector pCL01, which will be... The int-attP element was cloned into the pCL01 vector to construct pCLBT01. The construction of the pCLBT01 vector was carried out in two steps: (1) using plasmid pRT803 as a template and primers pRT124F and pRT28R for amplification. (2) pCL01 was linearized using restriction endonucleases EcoRI and PmeI, containing the int-attP element sequence; The fragment of the int-attP element was seamlessly spliced ​​with the linearized pCL01 to obtain pCLBT01, and the interface was designed with AscI and NsiI restriction sites, which facilitates the subsequent construction of plasmids containing homologous arms (see [link to documentation]). Figure 2 ).

[0084] pRT124F(5'-3'):

[0085]

[0086] pRT28R(5'-3'):

[0087]

[0088] Among them, ATGCAT is the NsiI recognition site, and GGCGCGCC is the AscI recognition site.

[0089] Next, using pCLBT01 as the starting vector, a capture plasmid containing homologous arms at the boundary of a rif (~93kb) was constructed. First, sequences approximately 4.2kb in length were selected at each end of the rif boundary as homologous arms. Using primers UP-4kF1 / R1 and DN-4kF2 / R2, the upstream homologous arm UP-rif (4167bp) and the downstream homologous arm DN-rif (4276bp) were amplified by PCR and recovered by agarose gel electrophoresis. Simultaneously, the pCLBT01 vector was linearized using the restriction enzyme NsiI and recovered by agarose gel electrophoresis. The recovered PCR product and the linearized vector pCLBT01 were seamlessly spliced ​​together under the mediation of Ezmax recombinase to obtain the capture plasmid pCLBT01-Vrif (verification results are available in [link to documentation]). Figure 3 At the same time, the AsiSI restriction site (GCGATCGC) is introduced to facilitate the subsequent linearization of the plasmid.

[0090] UP-4kF1(5'-3'):

[0091] AGCGGGCGCGCCATCATGCAGGCGTTCTGGTTCAAGTCCG.

[0092] UP-4KR1(5'-3'):GCGGTTCGCGATCGCACGTGCCCATCCGGGTAAAC.

[0093] DN-4KF2(5'-3'):CACGTGCGATCGCGAACCGCGTCGAGAGCATGAG.

[0094] DN-4KR2(5'-3'):

[0095] GTTTATTTTTCTAAATACAGGGAAACGACCGCTGAAACC.

[0096] The PCR primers for pCLBT01-Vrif validation are as follows:

[0097] aprF(5'-3'):CATGTGCAGTCCATCAG.

[0098] aprR(5'-3'):GATCCGCTCCACGTGTTG.

[0099] Vrif-J1F(5'-3'):CGCCACCCTGAGACGAAG.

[0100] Vrif-J1R(5'-3'):CCACTTGTCGCCCTGGAG.

[0101] Vrif-J2F(5'-3'):CCGGGCGTCTACTACTCC.

[0102] Vrif-J2R(5'-3'):GGCGATCCCCCTAGAGTC.

[0103] To test the direct cloning capability of the method of this invention, we also cloned a PKS gene cluster of approximately 157 kb with unknown function from the genome of *A. pretoriensis* NRRL B-24133 as test material. The capture plasmid pCLBT01-V157 was constructed using pCLBT01 as the starting vector (validation results are shown in [link to validation results]). Figure 4 The sequences of its upstream homologous primer UP-157F1 / R1 and downstream homologous primer DN-157F2 / R2 are as follows.

[0104] UP-157F1(5'-3'):

[0105] GCGGGCGCGCCATCATGCAGGTCAACGAGGAGGGGGA.

[0106] UP-157R1(5'-3'):CGCGCAAGGAAATGCATGCGACGGAATCCCGCAAG.

[0107] DN-157F2(5'-3'):GCATGCATTTCCTTGCGCGCCTTGGT.

[0108] DN-157R2(5'-3'):

[0109] CCAGGTGGCACGTTTATGCAGTTGGGGAGCGGTGTGCC.

[0110] Example 4

[0111] Direct cloning of target BGC using yeast TAR technology

[0112] For the capture plasmid pCLBT01-Vrif, we linearized it using AsiSI and recovered it by electrophoresis, while for pCLBT01-V157, we linearized it using NsiI and recovered it. Next, the linearized capture plasmid, along with a Cas12a-digested DNA mixture containing the target BGC, was transformed into *Saccharomyces cerevisiae* VL6-48 protoplasts. After mixing with yeast tryptophan-deficient medium (Trp-) containing 1.5% low-melting-point agarose, the mixture was plated on Trp- plates and incubated at 30°C for 3–5 days. Clones were then picked and validated.

[0113] Example 5

[0114] Extraction and validation of target BGC plasmid

[0115] In the direct cloning of RIF experiments, a total of 86 transformants that could grow on tryptophan-deficient solid medium were obtained from 6 transformation plates. All transformants were then transferred to tryptophan-deficient solid medium for expansion culture, and 11 transformants containing RIF were obtained by colony PCR screening.

[0116] Next, we extracted plasmids from the positive yeast clones and transformed them into E. coli EPI300. Under culture conditions with added inducers, we extracted large quantities of the pCLBT01-rif plasmid containing rif. Finally, restriction mapping analysis of clones 3, 5, and 8 revealed that the banding patterns of these three clones were consistent (KpnI restriction mapping analysis results). Figure 5 The presence of these features suggests that no major DNA rearrangements or deletions occurred in the rif. Therefore, the above results indicate that we have successfully cloned rif BGC.

[0117] Similarly, for the 157k gene cluster, we obtained four positive clones, pCLBT01-157k, from 105 transformant clones after multiple rounds of PCR screening. Subsequently, restriction enzyme digestion was performed on the plasmids of three of these positive clones (12, 46, and 85) for verification. SalI and EcoRI double digestion results showed that the banding patterns of the three independent positive clones were consistent. Figure 6 This suggests the absence of significant large-fragment rearrangements or deletions. Finally, whole-sequence sequencing confirmed that the sequence of clone 12 was identical to the genome sequence.

[0118] In summary, using two (super)large BGCs, rif and 157k, as examples, we have demonstrated the high efficiency of the direct cloning method in this invention, which greatly enriches the synthetic biology toolbox and provides a very important optional tool for the study of gene (cluster) function in the post-genomic era.

[0119] Example 6

[0120] Effect of rifamycin BGC overexpression on yield

[0121] After successfully cloning the rif, we wanted to know if introducing another copy of the rif into *A. mediterranei* would increase rifamycin production. Subsequently, pCLBT01-rif (and the empty vector pCLBT01) was introduced into *A. mediterranei* U32 competent cells via electroporation and cultured on Bennet solid medium containing apramycin (Apr) for 5–7 days. Next, transformants grown on Apr-resistant plates were validated by PCR, and positive clones showing integration of pCLBT01-rif (and the empty vector pCLBT01) onto the *A. mediterranei* U32 chromosome were selected.

[0122] Next, using wild-type *A. mediterranei* U32 and U32-pCLBT01 strains transformed with the empty pCLBT01 vector (U32-p1 and U32-p3) as controls, the growth curves and rifamycin synthesis capabilities of the rif overexpression strains U32-rif (U32-rif-1A, U32-rif-1B, and U32-rif-2A) were analyzed. The culture conditions were Bennet liquid medium with 80 mM KNO3 added. The results showed that the overall growth trend of wild-type U32 was consistent with that of the two U32-p1 and U32-p3 strains, while the overall growth trend of the three U32-rif strains was consistent. Furthermore, the growth of the U32-rif strains was slightly lower than that of U32, U32-p1, and U32-p3 strains, and they reached their peak growth later. Figure 7 Wild-type U32 produced rifamycin at levels comparable to the two strains, U32-p1 and U32-p3, reaching peak rifamycin production around 120 hours of fermentation, with yields ranging from 680 to 750 μg / mL. In contrast, the three U32-rif strains reached peak rifamycin production around 132 hours of fermentation, with yields ranging from 887 to 1325 μg / mL. This means that the highest rifamycin yield was approximately 40% higher than that of U32 and the two U32-p1 and U32-p3 strains. Figure 8 ).

Claims

1. A method for direct cloning of large biosynthetic gene clusters, characterized in that, Includes the following steps: S1. Embed the bacterial cells in agarose to obtain an embedded block containing the genomic DNA of the target strain; S2. Based on the CRISPR / Cas system, specific cutting is performed on both sides of the target gene cluster in the embedding block; S3. Construct a target gene cluster capture plasmid; S4. Directly clone the target gene cluster using yeast TAR technology.

2. The method for direct cloning of ultra-large biosynthetic gene clusters according to claim 1, characterized in that, The CRISPR / Cas system is either CRISPR / Cas9 or CRISPR / Cas12a.

3. The method for direct cloning of ultra-large biosynthetic gene clusters according to claim 1, characterized in that, The target gene cluster is longer than 150kb.

4. The method for direct cloning of large biosynthetic gene clusters according to claim 1, characterized in that, In step S3, the BAC carrier is used as the starting carrier.

5. The method for direct cloning of large biosynthetic gene clusters according to claim 1, characterized in that, Step S1 includes the following steps: S1-1. Collect bacterial cells; S1-2. Suspended bacterial cells form a bacterial solution; S1-3. Mix the agarose solution with the bacterial culture, cool and solidify to obtain the pre-embedded block; S1-4. The pre-embedded blocks are sequentially treated with lysozyme, proteinase K, and PMSF to obtain embedded blocks containing the genomic DNA of the target strain.

6. The method for direct cloning of large biosynthetic gene clusters according to claim 1, characterized in that, Step S2 includes the following steps: S2-1. Based on the two sides of the target gene cluster in the embedding block, design crRNA template primers to obtain UP-crRNA upstream of the target BGC and DN-crRNA downstream of the target BGC. S2-2. Specifically cut the genomic DNA in the embedded block to obtain the target BGC. S2-3. Digest the embedded block with agarose enzyme to obtain a DNA mixture containing the target BGC.

7. The application of the direct cloning method for large biosynthetic gene clusters as described in any one of claims 1-6, characterized in that, Large biosynthetic gene clusters used for direct cloning of microbial genomes.

8. A method for directly cloning rifamycin BGC from the genome of Amycolatopsis mediterranei, characterized in that, The method for direct cloning of large biosynthetic gene clusters as described in any one of claims 1-6 is employed.

9. A method for increasing the yield of rifamycin from *A. mediterranei*, characterized in that, Rifamycin BGC was cloned using the direct cloning method for large biosynthetic gene clusters as described in any one of claims 1-6, and then the rifamycin BGC construct vector was transformed into A. mediterranei to overexpress rifamycin BGC.

10. A method for directly cloning polyketide synthase (PKS) gene clusters from Amycolatopsis pretoriensis, characterized in that, The method for direct cloning of large biosynthetic gene clusters as described in any one of claims 1-6 is employed.

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