Abstract:
The furfural-resistant strain containing the furfural-resistant gene according to the present disclosure may be effectively grown in a furfural-containing medium. Accordingly, the problem that microorganism fermentation was difficult because toxic by-products such as furfural are contained in a hydrolysate derived from inedible lignocellulosic biomass may be solved. Further, according to the method for producing a strain of the present disclosure, the resistant gene may be selected from relatively small number of target genes. Thus, time, cost and the like for developing the resistant strain may be saved. Further, this method for identifying genes may be broadly applied to methods for identifying various unknown functional genes in addition to the furfural-resistant gene.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims priority to Korean Patent Application No. 10-2014-0036970, filed on Mar. 28, 2014, and all the benefits accruing therefrom under 35 U.S.C. §119, the contents of which in its entirety are herein incorporated by reference. 
       BACKGROUND 
       [0002]    1. Field 
         [0003]    Disclosed herein is a gene having resistance to furfural, which is a by-product of a hydrolysate derived from lignocellulosic biomass, and a strain comprising the same. 
         [0004]    Description about National Support Research and Development 
         [0005]    This study is conducted by the support of Korea Ministry of Science, ICT and Future Planning under the supervision of Korea Institute of Science and Technology, and research title is development of technique for manufacturing next generation fuel/material by integrated utilization of lignocellulosic biomass (2N36860) (Research management agency: Korea Research Council of Fundamental Science and, Grant Number: CAP-11-1: Creative Allied Program[CAP]). 
         [0006]    2. Description of the Related Art 
         [0007]    As concerns about global warming and oil resource depletion rise, world bio research fields pay more attention to development of alternative energy sources and alternative materials of petrochemicals. For this, studies into production of bio-fuels and platform chemicals from lignocellulosic biomass by fermentation of microorganism strain are actively going on. The lignocellulosic biomass mainly consists of cellulose, hemicellulose, lignin and the like, and can be converted to renewable fuels, plastics and other various chemicals by fermentation of microorganism. 
         [0008]    In order to use the lignocellulosic biomass to microorganism fermentation, a pre-treatment process for hydrolyzing cellulose to sugar is needed. However, various by-products, which are made during the pre-treatment process, as well as sugars such as glucose and xylose, which can be used by microorganisms, are contained in a hydrolysate, which is made during the pre-treatment for sugar hydrolysis using acid at high temperature and pressure. Examples of the by-products made during the pre-treatment process for sugar hydrolysis are furfural, hydroxymethylfurfural (HMF), acetic acid and the like. Because these have toxicity to microorganisms, there is a problem of decreased productivity on the fermentation process using the hydrolysate. Accordingly, development of a strain having resistance to a toxic material in the hydrolysate is necessary. 
         [0009]    Furfural made from xylose, among the by-product existing in the hydrolysate, is one of important materials inhibiting microorganism fermentation in the hydrolysate, and the toxicity of the hydrolysate has considerable relevance to the concentration of the furfural. The furfural is converted to furfuryl alcohol, which is relatively less toxic than the furfural in cells. In the process, it uses NADH or NADPH, and intracellular amount of the NAD(P)H becomes unbalanced, thereby inhibiting cell growth and fermentation. In addition, it is known that its toxicity becomes severe because it causes genetic mutation, makes cell membrane weak, and interacts with other materials in the hydrolysate such as hydroxymethylfurfural and acetic acid. 
       REFERENCES OF THE RELATED ART 
     Patent Document 
       [0000]    
       
         International Patent Publication No. WO2012-135420A2 (2012 Oct. 4) 
         Korean Patent Publication No. 10-2003-0040605 (2003 May 23) 
       
     
       Non-Patent Document 
       [0000]    
       
         Wang, X., E. Miller, et al., 2011, “Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in  Escherichia coli  strains engineered for the production of ethanol and lactate.” Applied and environmental microbiology 77(15): 5132-5140 
         Sasano, Y., D. Watanabe, et al., 2012, “Overexpression of the yeast transcription activator Msn2 confers furfural resistance and increases the initial fermentation rate in ethanol production.” Journal of bioscience and bioengineering 113(4): 451-455 
       
     
       SUMMARY 
       [0014]    The present disclosure is directed to providing a gene having resistance to furfural, which is a toxic by-product contained in a hydrolysate derived from lignocellulosic biomass, and a strain containing the same. 
         [0015]    In one aspect, there is provided a furfural-resistant gene containing at least one genetic sequence selected from the group consisting of the cg1661 (SEQ ID No.: 1) and cg1310 (SEQ ID No.: 2) gene, and a furfural-resistant strain containing the same. 
         [0016]    In another aspect, there is provided a screening method of the furfural-resistant gene, which contains: selecting a target gene for identifying the furfural-resistant gene; and after inserting the target gene into a wild type and overexpressing thereof, selecting a gene showing furfural resistance as the furfural-resistant gene. 
         [0017]    The furfural-resistant strain containing the furfural-resistant gene according to the present disclosure shows faster growth speed than the wild type (wild type strain) in the furfural-containing medium. Thus, it may increase production rate of the material, which is produced by the wild type. Accordingly, the problem that microorganism fermentation was difficult because toxic by-products such as furfural are contained in a hydrolysate derived from inedible lignocellulosic biomass may be solved. Thus, it may effectively produce food additives, feed additives and the like such as amino acids from inedible biomass as well as the existing starch-based biomass without growth inhibition or production rate reduction by the toxic materials in a hydrolysate. 
         [0018]    Further, according to the method for producing a strain of the present disclosure, the resistant gene may be selected from relatively small number of target genes. Thus, time, cost and the like for developing the resistant strain may be saved. Further, this method for identifying genes may be broadly applied to methods for identifying various unknown functional genes in addition to the furfural-resistant gene. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0019]    The above and other aspects, features and advantages of the disclosed exemplary embodiments will be more apparent from the following detailed description taken in conjunction with the accompanying drawings in which: 
           [0020]      FIG. 1  is a graph showing growth curve of  Corynebacterium glutamicum  wild type strain (wild type) in a medium containing furfural of various concentrations (0, 6.5, 13 and 20 mM); 
           [0021]      FIGS. 2   a  to  2   c  are graphs showing the result of analyzing the concentrations of furfural and furfuryl alcohol in the furfural-containing medium according to the growth of  Corynebacterium glutamicum , wherein  FIG. 2   a  is the case of the medium containing 6.5 mM furfural,  FIG. 2   b  is the case of the medium containing 13 Mm furfural, and  FIG. 2   c  is the case of the medium containing 20 mM furfural; 
           [0022]      FIG. 3  is an image schematically showing the process of homology search of FucO in  Corynebacterium glutamicum;    
           [0023]      FIG. 4  is a graph comparing relative growth of target gene-transformed strains (pAN6-cg0310, pAN6-cg1310, pAN6-cg1661 and pAN6-cg3374) and a positive control group (pAN6-b2799) in the medium containing 6.5 mM furfural, with growth rate of a negative control group (pAN6) in the medium not containing the furfural, respectively; and 
           [0024]      FIG. 5  is a graph comparing relative growth of target gene-transformed strains (pAN6-cg0310, pAN6-cg1310, pAN6-cg1661 and pAN6-cg3374) and a positive control group (pAN6-b2799) in the medium containing 13 mM furfural, with growth rate of a negative control group (pAN6) in the medium not containing the furfural, respectively. 
       
    
    
     DETAILED DESCRIPTION 
       [0025]    Exemplary embodiments now will be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments are shown. 
         [0026]    Embodiments of the present disclosure provide a furfural-resistant gene containing at least one genetic sequence selected from the group consisting of cg1661 (SEQ ID No.: 1) and cg1310 (SEQ ID No.: 2) gene. 
         [0027]    The furfural is an organic compound made from by-products during sugar hydrolysis process of various biomass, and has a chemical formula of OC 4 H 3 CHO and a chemical structure of the following formula (I). The furfural is toxic, and therefore, it deteriorates production speed of microorganism and production rate of fermentation product when fermenting microorganism in a hydrolysate of biomass. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0028]    Accordingly, embodiment of the present disclosure provides a furfural-resistant strain, which contains the furfural-resistant gene, thereby having excellent growth capability under furfural-containing environment. Specifically, the furfural-resistant strain according to embodiments of the present disclosure is a strain, wherein the furfural-resistant gene containing the cg1661 (SEQ ID No.: 1) and cg1310 (SEQ ID No.: 2) gene is inserted in a wild type. In one embodiment, the wild type is  Corynebacterium glutamicum , specifically  Corynebacterium glutamicum  ATCC 13032 (ACCESSION NC — 006958, VERSION NC — 006958.1 GI:62388892). The insertion of the gene is conducted by inserting the furfural-resistant gene into the wild type using a vector and then overexpressing thereof, and the vector may be, for example, a plasmid such as pAN6. In one embodiment, the furfural-resistant strain is  Corynebacterium glutamicum  ATCC 13032/pAN6-cg1661 (KCTC 12565BP). 
         [0029]    The furfural-resistant strain according to embodiments of the present disclosure may be grown in a furfural-containing medium. Further, growth of the furfural-resistant strain according to one embodiment in the furfural-containing medium is increased about 1.4 folds or more, more specifically about 1.8 folds or more, compared to a wild type. When the wild type is  Corynebacterium glutamicum , the  Corynebacterium glutamicum  wild type (wild type strain) is hard to be grown in the hydrolysate derived from lignocellulosic biomass due to toxicity of the furfural contained in the hydrolysate. However, the furfural-resistant strain according to embodiments of the present disclosure has furfural resistance by containing the furfural-resistant gene of the present disclosure. Thus, it may be effectively grown in the hydrolysate derived from lignocellulosic biomass, thereby having further increased amino acid productivity, compared to the wild type. 
         [0030]    Accordingly, embodiments of the present disclosure may provide a method for producing amino acids, which contains inoculating the furfural-resistant strain into the hydrolysate derived from lignocellulosic biomass. 
         [0031]    Further, another embodiment of the present disclosure may provide a method for screening the furfural-resistant gene, which contains: 
         [0032]    selecting a target gene for identifying the furfural-resistant gene; and 
         [0033]    after inserting the target gene into the wild type and overexpressing thereof, selecting a gene showing furfural resistance as the furfural-resistant gene. 
         [0034]    In order to select the target gene for identifying the furfural-resistant gene, embodiments of the present disclosure may contains at least one of: 
         [0035]    searching genes, which are expected to have furfural-resistance through literature search, and selecting a gene, which is similar with the gene searched through the literature, among genes of the wild type, as the target gene; and 
         [0036]    analyzing gene expression pattern depending on furfural stress using microarray, and selecting a gene showing high expression level as the target gene. 
         [0037]    In one embodiment, selecting the target gene through literature search may contain searching furfural-resistant genes known in literature, and selecting genes showing high similarity in the wild type by homology search as the target gene. For example, from [Wang, X., E. Miller, et al., 2011, “Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in  Escherichia coli  strains engineered for the production of ethanol and lactate.” Applied and environmental microbiology 77(15): 5132-5140], it may be found that the NADH-dependent oxidoreductase (fucO) converts the furfural to furfuryl alcohol (see the following chemical formula (II)). Then, the genes in the wild type, which are similar with the searched FucO, may be selected as the target gene. At this time, the contents of the literature in its entirety are herein incorporated by reference. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0038]    In one embodiment, when the parent strain is  Corynebacterium glutamicum , cg1310 (rolM, maleylacetate reductase, SEQ ID No.: 2), which is similar with the genes searched by the literature in  Corynebacterium glutamicum  genes, may be selected as the target gene. 
         [0039]    In another embodiment, specifically, selecting the target gene using microarray may contain: treating the furfural to the wild type at different concentrations, conducting microarray to mRNA of the wild type, and then selecting genes, whose gene expression change depending on furfural stress, namely, whose expression pattern increased by the furfural, is judged to have relevance to furfural resistance, as the target gene. For example, when using  Corynebacterium glutamicum  as the wild type, cg0310 (SEQ ID No.: 3), cg3374 (SEQ ID No.: 4), cg1661 (SEQ ID No.: 1) and cg3399 (SEQ ID No.: 5) may be selected as the target gene. 
         [0040]    In embodiments of the present disclosure, a gene having high resistance to the furfural may be finally selected as the furfural-resistant gene, by inserting the selected target gene into the wild types and overexpressing thereof. Specifically, the selected target genes are cloned and inserted into the wild types to obtain target gene-overexpressed strains, and then these strains are cultured in the furfural-containing medium. Then, their growth speed is compared with growth speed of strains with an expression vector (e.g.: pAN6), in which the target gene is not inserted, and the target genes, which are inserted in the strains showing increased growth speed, may be selected as the furfural-resistant genes. For example, when using  Corynebacterium glutamicum  as the wild type, the target genes cg1310, cg0310, cg3374, cg1661 and cg3399 are genetically recombined and inserted into the wild type, respectively, and overexpressed. Then, a recombinant strain, which substantially shows higher cell growth rate than the wild type, is selected, and the target gene inserted in the recombinant strain is finally selected as the furfural-resistant genes. In one embodiment, the final furfural-resistant gene selected in the above step may contain at least one gene of membrane protein (efflux permease)-type cg1661 and reductase-type cg1310. 
         [0041]    The examples (and experiments) will now be described. The following examples (and experiments) are for illustrative purposes only and not intended to limit the scope of the present disclosure. 
       Example 1 
     Confirm of Growth Inhibitory Effect of Furfural in Wild type 
       [0042]    In order to develop a furfural-resistant strain, first of all,  Corynebacterium glutamicum  is selected as a wild type, and then, growth inhibitory effect of furfural at  Corynebacterium glutamicum  wild type strain (wild type), which is  Corynebacterium glutamicum  ATCC 13032, depending on concentration, and concentrations of furfural and furfuryl alcohol in a culture solution depending on cell growth are confirmed. 
         [0043]    Specifically, as the culture solution, 2% glucose as a carbon source is added to a minimal medium, CGXII medium, and furfural of 6.5 mM, 13 mM and 20 mM are added thereto, respectively, to compare growth speed with the case not adding the furfural. At this time, the composition of the CGXII medium is 20 g/L (NH 4 ) 2 50 4 , 5 g/L Urea, 1 g/L KH 2 PO 4 , 1 g/L K 2 HPO 4 , 0.25 g/L MgSO 4 .7H 2 O, 42 g/L MOPS, 10 mg/L CaCl 2 , 0.2 mg/L Biotin, 0.03 g/L Protecatechuic acid, Trace metal (10 mg/L FeSO 4 .7H 2 O, 10 mg/L MnSO 4 .H 2 O, 1 mg/L ZnSO 4 .7H 2 O, 0.2 mg/L CuSO 4 .7H 2 O, 0.02 mg/L NiCl 2 .6H 2 O). Culture is conducted at 30° C. after inoculating the cells (initial OD 600 =1) into the culture solution 50 mL in a 250 mL Erlenmeyer flask. As a result of the culture, as shown in  FIG. 1 , it is confirmed that the growth speed of the strain becomes slow as the furfural concentration in the medium is increased, and the growth is reduced at about 50% or more even at the lowest concentration of 6.5 mM, compared with the medium not containing the furfural. 
         [0044]    Further, in order to check the concentration changes of the furfural and furfuryl alcohol in the medium according to the growth of  Corynebacterium glutamicum  in the medium containing 6.5 mM, 13 mM and 20 mM furfural by the culture time, respectively, the culture solution is analyzed using gas chromatography (GC). At this time, the culture solutions obtained by each collection time are centrifuged at 12,000 rpm for 5 min, and the supernatants are filtered and used for the analysis. The results of the culture solution analysis are shown in  FIG. 2   a ,  FIG. 2   b  and  FIG. 2   c . As shown in  FIGS. 2   a, b and c , it is confirmed that entire furfural added to the medium is introduced into the cells regardless of the concentration and converted to furfuryl alcohol. 
       Example 2 
     Selection of Target Gene for identifying Furfural-Resistant Gene 
       [0045]    In order to select a target gene for identifying the furfural-resistant gene, two methods, i.e., a method of searching literatures and a method using microarray are used. 
         [0046]    Method of Searching Literatures 
         [0047]    Because it is confirmed that the furfural added in the medium is converted to the furfuryl alcohol in the cells in Example 1, literature search is conducted to find a furfural reductase. As a result, FucO gene as a furfural reductase using NADH in  E. coli  is found in [Wang, X., E. Miller, et al., 2011, “Increased furfural tolerance due to overexpression of NADH-dependent oxidoreductase FucO in  Escherichia coli  strains engineered for the production of ethanol and lactate.” Applied and environmental microbiology 77(15): 5132-5140]. And, in order to find similar genes with the gene in  Corynebacterium glutamicum , homology search is conducted using Blastx (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The searching process is schematically shown in  FIG. 3 . 
         [0048]    As a result, among the genes, which are expected to have similarity, cg1310 (SEQ ID No.: 2) showing increased expression at microarray is selected as the target gene. And, information about the selected target gene, and expression level of the target gene depending on the furfural concentration in the medium, which is obtained by analyzing gene expression pattern thereof depending on the furfural concentration using microarray, are shown in the following Table 1. 
         [0000]    
       
         
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 1 
               
             
             
               
                   
                   
               
               
                   
                 Gene Expression Level 
               
             
          
           
               
                   
                   
                   
                 Furfural 
                 Furfural 
                 Furfural 
               
               
                   
                   
                   
                 Conc. in 
                 Conc. in 
                 Conc. in 
               
               
                 Gene 
                 Gene 
                   
                 Medium: 
                 Medium: 
                 Medium: 
               
               
                 No. 
                 Name 
                 Annotated Protein 
                 6.5 mM 
                 13 mM 
                 20 mM 
               
               
                   
               
               
                 cg1310 
                 rolM 
                 Maleylacetate 
                 1.476 
                 2.872 
                 2.183 
               
               
                   
                   
                 reductase 
               
               
                   
               
             
          
         
       
     
         [0049]    The gene expression level of the above Table 1 shows the relative mRNA expression level of the target gene cg1310 of the wild type strain,  Corynebacterium glutamicum  ATCC 13032, grown in the medium containing the furfural, against the wild type strain grown without stress in the medium not containing the furfural. As a result of analysis, compared with the medium not containing the furfural, the mRNA expression level of the target gene is increased in the medium containing the furfural, and the mRNA expression level of the target gene is further increased when the furfural concentration is increased from 6.5 mM to 13 mM. 
         [0050]    Method Using Gene Expression Analysis 
         [0051]    DNA Microarray is used as a method for analyzing gene expression depending on the furfural stress. 
         [0052]    Specifically, among genes corresponding to the module classified at http://www.coryneregnet.de/ of entire genes, and genes, whose corresponding protein names contain at least one of transport, permease and pump, genes showing increased expression level at all furfural concentrations are selected, and then, genes showing about 10 folds or more increased expression level at least one concentration are selected. And, among the selected genes, existing genes, which are known to response to stress, are excluded by literature search, and cg0310 (SEQ ID No.: 3), cg1661 (SEQ ID No.: 1), cg3374 (SEQ ID No.: 4) and cg3399 (SEQ ID No.: 5) are selected as the target gene. These are catalase, efflux pump in the cell membrane, oxidoreductase and permease-type genes. Then, information of the target genes selected by microarray, and expression levels of the target genes depending on the furfural concentration in the medium, which are obtained by analyzing gene expression pattern thereof using microarray, are shown in the following Table 2. 
         [0000]    
       
         
               
               
             
               
               
               
               
               
               
             
           
               
                   
                 TABLE 2 
               
             
             
               
                   
                   
               
               
                   
                 Gene Expression Level 
               
             
          
           
               
                   
                   
                   
                 Furfural 
                 Furfural 
                 Furfural 
               
               
                   
                   
                   
                 Conc. in 
                 Conc. in 
                 Conc. in 
               
               
                 Gene 
                 Gene 
                   
                 Medium: 
                 Medium: 
                 Medium: 
               
               
                 No. 
                 Name 
                 Annotated Protein 
                 6.5 mM 
                 13 mM 
                 20 mM 
               
               
                   
               
               
                 cg0310 
                 katA 
                 catalase 
                 3.219 
                 3.443 
                 10.956 
               
               
                 cg1661 
                 — 
                 Arsenite efflux 
                 7.240 
                 3.071 
                 13.186 
               
               
                   
                   
                 pump ACR3 or 
               
               
                   
                   
                 related permease 
               
               
                 cg3374 
                 cye1 
                 Putative NADH- 
                 1.461 
                 3.219 
                 29.016 
               
               
                   
                   
                 dependent flavin 
               
               
                   
                   
                 oxidoreductase 
               
               
                 cg3399 
                 — 
                 Permease of the 
                 4.485 
                 6.529 
                 15.694 
               
               
                   
                   
                 major facilitator 
               
               
                   
                   
                 superfamily 
               
               
                   
               
             
          
         
       
     
         [0053]    The gene expression levels of the above Table 2 show the relative mRNA expression levels of the target genes of the wild type strain grown in the medium containing the furfural, against the wild type strain grown without stress in the medium not containing the furfural. As a result of analysis, compared with the medium not containing the furfural, the mRNA expression levels of each target gene are increased in the medium containing the furfural, and the mRNA expression levels of the target genes are further increased as the furfural concentration is increased. 
       Example 3 
     Confirm of Resistant Effect of Target Gens in Furfural-Containing Medium 
       [0054]    A test for confirming resistant effect of the target genes selected in Example 2 is conducted. At this time, among the target genes of Example 2, cg3399 is excluded from this test because it is not grown after culturing in a medium containing 20 mM furfural. 
         [0055]    Specifically, as the target genes of the present disclosure, cg0310, cg1310, cg1661 and cg3374, and b2799 (ACCESSION NP — 417279, VERSION NP — 417279.2 GI:345452723), which is FucO gene of  E. coli  K-12 MG1655 (L-1,2-propanediol oxidoreductase), as a positive control group are transformed into a wild type strain,  Corynebacterium glutamicum  ATCC 13032, using a  Corynebacterium glutamicum  expression vector, pAN6, under the conditions of 25 μF, 200Ω and 2,500 V by electroporation, respectively. As a negative control group, pAN6, which is not inserted with any other genes, is also transformed into a wild type strain with the same method. The pAN6 (Kan′) is a high copy plasmid, and a shuttle vector derived from pEKEx2 for gene expression regulation of  Corynebacterium glutamicum/E. coli  (P tac , lacI q , pBL1 oriV C.glutamicum , pUC18 oriV E.coli ). This may be confirmed in [Frunzke, J., Engels, V., Hasenbein, S., Gätgens, C., Bott, M., 2008. Co-ordinated regulation of gluconate catabolism and glucose uptake in  Corynebacterium glutamicum  by two functionally equivalent transcriptional regulators, GntR1 and GntR2. Molecular microbiology. 67, 305-322], and the contents of the literature in its entirety are herein incorporated by reference. After the transformation, cells are selected on BHIS (37 g/L brain heart infusion (Difco), 91 g/L sorbitol) agar plate containing 25 μg/ml Kanamycin. As a result, recombinant strains pAN6-cg0310, pAN6-cg1310, pAN6-cg1661, pAN6-cg3374 and pAN6-b2799 are manufactured, respectively. 
         [0056]    The above pAN6 contains lacI gene. Accordingly, gene expression may be controlled by IPTG induction. Thus, in order to confirm the furfural resistance of the recombinant strains, IPTG is added to the culture at the concentration of 0.5 mM at the time of inoculating the strains for induction, thereby overexpressing the genes. Then, the recombinant strains are cultured in CGXII medium supplemented with 6.5 mM and 13 mM furfural, respectively. And, OD 600  value by the time is compared with OD 600  value of the negative control group in the medium not containing the furfural, and the results are shown in  FIG. 4  and  FIG. 5 . 
         [0057]    As a result, it is found that the pAN6-cg1310 and pAN6-cg1661 have the highest furfural resistance because they show about 1.5 folds and about 1.8 folds higher cell growth than the negative control group (pAN6) in the medium not containing the furfural. Accordingly, these are decided as the final furfural-resistant genes. 
         [0058]    [Accession No.]
       Depositary Authority: Korean Collection for Type Culture (Republic of Korea)   Accession No.: KCTC 12565BP   Date of Deposit: 2014 Mar. 5.       
 
         [0062]    While the exemplary embodiments have been shown and described, it will be understood by those skilled in the art that various changes in form and details may be made thereto without departing from the spirit and scope of the present disclosure as defined by the appended claims.