Lumazine and riboflavin synthase

Abstract

Through function complementation of E. coli auxotrophs, the ultimate and pentultimate enzymes of the spinach riboflavin biosynthetic pathway have been cloned, namely, lumazine synthase (LS) and riboflavin synthase (RS). This invention relates to the isolation of nucleic acid fragments from plants or fungi that encode LS protein. The invention also relates to the isolation of nucleic acid fragments from plants or fungi that encode RS protein. In addition, the invention also relates to the construction of chimeric genes encoding all of a portion of LS, in sense or antisense orientation, wherein the expression of the chimeric gene results in production of altered levels of plant LS in a transformed host cell. Furthermore, the invention also relates to the construction of chimeric genes encoding all of a portion of RS, in sense or antisense orientation, wherein the expression of the chimeric gene results in production of altered levels of plant or fungal RS in a transformed host cell. In vivo and in vitro methods to identify herbicide or fungicide candidates are included that evaluate the ability of a chemical compound to inhibit the activity of a plant or fungal LS enzyme or a plant or fungal RS enzyme.

What is claimed is:  
     
         1 . An isolated nucleic acid fragment encoding a plant LS enzyme, selected from the group consisting of: 
 (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6;    (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6;    (c) an isolated nucleic acid fragment encoding a polypeptide having at least 72% identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6; and    (d) an isolated nucleic acid fragment that is complementary to (a), (b) or (c).    
     
     
         2 . The isolated nucleic acid fragment of  claim 1  selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5.  
     
     
         3 . The isolated nucleic acid fragment of  claim 1  encoding a plant LS enzyme, wherein the plant is spinach, tobacco or arabidopsis.  
     
     
         4 . A polypeptide encoded by the isolated nucleic acid fragment of  claim 1 .  
     
     
         5 . The polypeptide of  claim 4  having at least 72% identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.  
     
     
         6 . The polypeptide of  claim 4  selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.  
     
     
         7 . A chimeric gene comprising the isolated nucleic acid fragment of  claim 1  operably linked to suitable regulatory sequences.  
     
     
         8 . A transformed host cell comprising a host cell and the chimeric gene of  claim 7 .  
     
     
         9 . The transformed host cell of  claim 8  wherein the host cell is a plant cell.  
     
     
         10 . The transformed host cell of  claim 8  wherein the host cell is  E. coli.    
     
     
         11 . A method of altering the level of expression of a plant LS enzyme in a host cell comprising: 
 (a) transforming a host cell with the chimeric gene of claim  7 ; and    (b) growing the transformed host cell of step (a) under conditions that are suitable for expression of the chimeric gene, resulting in production of altered levels of a plant LS enzyme in the transformed host cell relative to expression levels of an untransformed host cell.    
     
     
         12 . A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a plant LS enzyme comprising: 
 (a) probing a cDNA or genomic library with the nucleic acid fragment of  claim 1;     (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of  claim 1;  and    (c) sequencing the cDNA or genomic fragment that comprises the clone identified in step (b), wherein the sequenced cDNA or genomic fragment encodes all or substantially all of the amino acid sequence encoding a plant LS enzyme.    
     
     
         13 . A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a plant LS enzyme comprising: 
 (a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5; and    (b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector, wherein the amplified cDNA insert encodes a portion of an amino acid sequence encoding a plant LS enzyme.    
     
     
         14 . The product of the method of  claim 12  or 13.  
     
     
         15 . An in vivo method for identifying as a herbicide candidate a chemical compound that inhibits the activity of a plant LS enzyme encoded by the isolated nucleic acid fragment of  claim 1 , the method comprising the steps of: 
 (a) disrupting the endogenous LS gene of a suitable microbial host cell, rendering growth of the microbial host cell dependent on added riboflavin;    (b) transforming the altered microbial host cell of step (a) with a chimeric gene comprising the isolated nucleic acid fragment of  claim 1  encoding a plant LS enzyme and operably linked to at least one suitable regulatory sequence that allows its expression by the microbial host cell;    (c) growing the transformed microbial host cell of step (b) under conditions that allow expression of the chimeric plant LS gene;    (d) contacting the transformed microbial host cell of step (c) while it is growing exponentially with a chemical compound of interest and in both the presence and absence of added riboflavin;    (e) identifying as a herbicide candidate the chemical compound of interest that inhibits growth of the transformed microbial host cell only when grown in the absence of added riboflavin.    
     
     
         16 . The method of  claim 15  wherein the nucleic acid fragment is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 and wherein the LS enzyme is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.  
     
     
         17 . An in vitro method for identifying as an herbicide candidate a chemical compound that inhibits the activity of a plant LS enzyme encoded by the isolated nucleic acid fragment of  claim 1 , the method comprising the steps of: 
 (a) transforming a host cell with a chimeric gene comprising the isolated nucleic acid fragment of  claim 1  encoding a plant LS enzyme, the chimeric gene operably linked to at least one suitable regulatory sequence;    (b) growing the transformed host cell of step (a) under conditions suitable for expression of the chimeric gene resulting in production of the plant LS enzyme;    (c) purifying the plant LS enzyme expressed by the transformed host cell;    (d) contacting the plant LS enzyme with a chemical compound of interest; and    (e) identifying as an herbicide candidate the chemical compound of interest that reduces the activity of the plant LS enzyme relative to the activity of the plant LS enzyme in the absence of the chemical compound of interest.    
     
     
         18 . The method of  claim 17  wherein the nucleic acid fragment is selected from the group consisting of SEQ ID NO: l, SEQ ID NO: 3 and SEQ ID NO: 5 and the LS enzyme is selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6.  
     
     
         19 . An isolated plant nucleic acid fragment encoding a plant RS enzyme selected from the group consisting of: 
 (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10;    (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO:10;    (c) an isolated nucleic acid fragment encoding a polypeptide having at least 70% identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10; and    (d) an isolated nucleic acid fragment that is complementary to (a), (b) or (c).    
     
     
         20 . The isolated nucleic acid fragment of  claim 19  selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 9.  
     
     
         21 . The isolated nucleic acid fragment of  claim 19  encoding a plant RS enzyme, wherein the plant is spinach or arabidopsis.  
     
     
         22 . A polypeptide encoded by the isolated nucleic acid fragment of  claim 19 .  
     
     
         23 . The polypeptide of  claim 22  having at least 70% identity with the amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10.  
     
     
         24 . The polypeptide of  claim 22  selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10.  
     
     
         25 . A chimeric gene comprising the isolated nucleic acid fragment of  claim 19  operably linked to suitable regulatory sequences.  
     
     
         26 . A transformed host cell comprising a host cell and the chimeric gene of  claim 25 .  
     
     
         27 . The transformed host cell of  claim 26  wherein the host cell is a plant cell.  
     
     
         28 . The transformed host cell of  claim 26  wherein the host cell is  E. coli.    
     
     
         29 . A method of altering the level of expression of a plant RS enzyme in a host cell comprising: 
 (a) transforming a host cell with the chimeric gene of claim  25 ; and    (b) growing the transformed host cell of step (a) under conditions that are suitable for expression of the chimeric gene, resulting in production of altered levels of a plant RS enzyme in the transformed host cell relative to expression levels of an untransformed host cell.    
     
     
         30 . A method of obtaining a nucleic acid fragment encoding all or substantially all of the amino acid sequence encoding a plant RS enzyme comprising: 
 (a) probing a cDNA or genomic library with the nucleic acid fragment of claim  19 ;    (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of claim  19 ; and    (c) sequencing the cDNA or genomic fragment that comprises the clone identified in step (b) wherein the sequenced cDNA or genomic fragment encodes all or substantially all of the amino acid sequence encoding a plant RS enzyme.    
     
     
         31 . A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a plant RS enzyme comprising: 
 (a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence selecting from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 9; and    (b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector, wherein the amplified cDNA insert encodes a portion of an amino acid sequence encoding a plant RS enzyme.    
     
     
         32 . The product of the method of  claim 30  or  31 .  
     
     
         33 . An in vivo method for identifying as an herbicide candidate a chemical compound that inhibits the activity of a plant RS enzyme encoded by the isolated nucleic acid fragment of  claim 19 , the method comprising the steps of: 
 (a) disrupting the endogenous RS gene of a suitable microbial host, rendering growth of the microbial host dependent on added riboflavin;    (b) transforming the altered microbial host cell of step (a) with a chimeric gene comprising the nucleic acid fragment of  claim 19  encoding a plant RS enzyme and operably linked to at least one suitable regulatory sequence that allows its expression by the transformed microbial host cell;    (c) growing the transformed microbial host cell of step (b) under conditions that allow expression of the chimeric plant RS gene;    (d) contacting the transformed microbial host cell of step (c) while it is growing exponentially with a chemical compound of interest and in both the presence and absence of added riboflavin; and    (e) identifying as an herbicide candidate the chemical compound of interest that inhibits growth of the transformed microbial host cell only when grown in the absence of added riboflavin.    
     
     
         34 . The method of  claim 33  wherein the nucleic acid fragment is selected from the group consisting of SEQ ID NO: 7 and, SEQ ID NO: 9 and wherein the RS enzyme is selected from the group consisting of SEQ ID NO: 8, and SEQ ID NO: 10.  
     
     
         35 . An in vitro method for identifying as an herbicide candidate a chemical compound that inhibits the activity of a plant RS enzyme encoded by the nucleic acid fragment of  claim 19 , the method comprising the steps of: 
 (a) transforming a suitable microbial host cell with a chimeric gene comprising the nucleic acid fragment of  claim 19  encoding a plant RS enzyme, the chimeric gene operably linked to at least one suitable regulatory sequence;    (b) growing the transformed microbial host cell of step (a) under conditions suitable for expression of the chimeric gene resulting in the production of the plant RS enzyme;    (c) purifying the plant RS enzyme expressed by the transformed microbial host cell;    (d) contacting the plant RS enzyme with a chemical compound of interest; and    (e) identifying as an herbicide candidate the chemical compound of interest that reduces the activity of the plant RS enzyme relative to the activity of the plant RS enzyme in the absence of the chemical compound of interest.    
     
     
         36 . The method of  claim 35  wherein the nucleic acid fragment is selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 9 and wherein the RS enzyme is selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 10.  
     
     
         37 . An isolated nucleic acid fragment encoding a fungal RS enyme selected from the group consisting of: 
 (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO: 12;    (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or is homologous to at least a substantial portion of the amino acid sequence set forth in SEQ ID NO: 12;    (c) an isolated nucleic acid fragment that is complementary to (a) or (b).    
     
     
         38 . The isolated nucleic acid fragment of  claim 37  as set forth in SEQ ID NO: 11.  
     
     
         39 . The isolated nucleic acid fragment of  claim 37  encoding a fungal RS enzyme obtained from  Magnaporthe grisea.    
     
     
         40 . A polypeptide that is encoded by the isolated nucleic acid fragment of  claim 37 .  
     
     
         41 . The polypeptide of  claim 40  having the amino acid sequence as set forth in SEQ ID NO: 12.  
     
     
         42 . A chimeric gene comprising the isolated nucleic acid fragment of  claim 37  operably linked to suitable regulatory sequences.  
     
     
         43 . A transformed host cell comprising a host cell and the chimeric gene of  claim 42 .  
     
     
         44 . The transformed host cell of  claim 43  wherein the host cell is a plant cell.  
     
     
         45 . The transformed host cell of  claim 43  wherein the host cell is  E. coli.    
     
     
         46 . A method of altering the level of expression of a fungal RS in a host cell comprising: 
 (a) transforming a host cell with the chimeric gene of claim  42 ; and    (b) growing the transformed host cell of step (a) under conditions that are suitable for expression of the chimeric gene, resulting in production of altered levels of a  Magnaporthe grisea  RS enzyme relative to expression levels of an untransformed host cell.    
     
     
         47 . A method of obtaining a nucleic acid fragment encoding all or substantially all of the amino acid sequence encoding a fungal RS comprising 
 (a) probing a cDNA or genomic library with the isolated nucleic acid fragment of claim  37 ;    (b) identifying a DNA clone that hybridizes with the isolated nucleic acid fragment of claim  37 ; and    (c) sequencing the cDNA or genomic fragment that comprises the clone identified in step (b), wherein the sequenced cDNA or genomic fragment encodes all or substantially all of the amino acid sequence encoding a fungal RS.    
     
     
         48 . A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a fungal RS comprising 
 (a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence set forth in SEQ ID NO: 11; and    (b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector, wherein the amplified cDNA insert encodes a portion of an amino acid sequence encoding a  Magnaporthe grisea  RS enzyme.    
     
     
         49 . The product of the method of  claim 47  or  48 .  
     
     
         50 . An in vivo method for identifying as a fungicide candidate a chemical compound that inhibits the activity of a fungal RS enzyme encoded by the isolated nucleic acid fragment of  claim 37 , the method comprising the steps of: 
 (a) disrupting the endogenous RS gene of a suitable microbial host, rendering growth of the microbial host dependent on added riboflavin;    (b) transforming the altered microbial host cell of step (a) with a chimeric gene comprising a nucleic acid fragment of  claim 37  encoding a fungal RS enzyme and operably linked to at least one suitable regulatory sequence that allows its expression by the transformed microbial host cell;    (c) growing the transformed microbial host cell of step (b) under conditions that allow expression of the chimeric fungal RS gene;    (d) contacting the transformed microbial host cell of step (c) while it is growing exponentially with a chemical compound of interest and in both the presence and absence of added riboflavin; and    (e) identifying as a fungicide candidate the chemical compound of interest that inhibits growth of the transformed host cell only when grown in the absence of added riboflavin.    
     
     
         51 . The method of  claim 50  wherein the isolated nucleic acid fragment corresponds to the sequence in SEQ ID NO: 11 and wherein the RS enzyme fragment corresponds to the sequence in SEQ ID NO: 12.  
     
     
         52 . An in vitro method for identifying a chemical compound that inhibits the activity of a fungal RS enzyme encoded by the isolated nucleic acid fragment of  claim 37 , the method comprising the steps of 
 (a) transforming a host cell with a chimeric gene comprising an isolated nucleic acid fragment of  claim 37  encoding a fungal RS enzyme, the gene operably linked to at least one suitable regulatory sequence;    (b) growing the transformed host cell of step (a) under conditions suitable for expression of the chimeric gene resulting in the production of the fungal RS enzyme;    (c) purifying the fungal RS enzyme expressed by the transformed host cell;    (d) contacting the fungal RS enzyme with a chemical compound; and    (e) identifying as a fungicide candidate the chemical compound of interest that reduces the activity of the fungal RS enzyme relative to the activity of the fungal RS enzyme in the absence of the chemical compound of interest.    
     
     
         53 . The method of  claim 52  wherein the isolated nucleic acid fragment corresponds to the sequence in SEQ ID NO: 11 and wherein the fungal RS enzyme fragment corresponds to the sequence in SEQ ID NO: 12.  
     
     
         54 . An isolated nucleic acid fragment encoding a fungal LS enzyme selected from the group consisting of 
 (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence set forth in SEQ ID NO: 38;    (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or is homologous to at least a substantial portion of the amino acid sequence set forth in SEQ ID NO: 38; and    (c) an isolated nucleic acid fragment that is complementary to (a) or (b).    
     
     
         55 . The isolated nucleic acid fragment of  claim 54  as set forth in SEQ ID NO: 37.  
     
     
         56 . The isolated nucleic acid fragment of  claim 54  encoding a fungal LS enzyme obtained from  Magnaporthe grisea.    
     
     
         57 . A polypeptide that is encoded by the isolated nucleic acid fragment of  claim 54 .  
     
     
         58 . The polypeptide of  claim 60  having the amino acid sequence as set forth in SEQ ID NO: 38.  
     
     
         59 . A chimeric gene comprising the isolated nucleic acid fragment of  claim 54  operably linked to suitable regulatory sequences.  
     
     
         60 . A transformed host cell comprising a host cell and the chimeric gene of  claim 59 .  
     
     
         61 . The transformed host cell of  claim 60  wherein the host cell is a plant cell.  
     
     
         62 . The transformed host cell of  claim 60  wherein the host cell is  E. coli.    
     
     
         63 . A method of altering the level of expression of a fungal LS enzyme in a host cell comprising 
 (a) transforming a host cell with the chimeric gene of claim  59 ; and    (b) growing the transformed host cell of step (a) under conditions that are suitable for expression of the chimeric gene, resulting in production of altered levels of a fungal LS enzyme relative to the expression of an untransformed host cell.    
     
     
         64 . A method of obtaining a nucleic acid fragment encoding all or substantially all of the amino acid sequence encoding a fungal LS enzyme comprising 
 (a) probing a cDNA or genomic library with the nucleic acid fragment of claim  54 ;    (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of claim  54 ; and    (c) sequencing the CDNA or genomic fragment that comprises the clone identified in step (b), wherein the sequenced cDNA or genomic fragment encodes all or substantially all of the amino acid sequence encoding a fungal LS enzyme.    
     
     
         65 . A method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a fungal LS enzyme comprising 
 (a) synthesizing an oligonucleotide primer corresponding to a portion of the sequence set forth in SEQ ID NO: 37; and    (b) amplifying a cDNA insert present in a cloning vector using the oligonucleotide primer of step (a) and a primer representing sequences of the cloning vector wherein the amplified cDNA insert encodes a portion of an amino acid sequence encoding a fungal LS enzyme.    
     
     
         66 . The product of the method of  claim 64  or  65 .  
     
     
         67 . An in vivo method for identifying as a fungicide candidate a chemical compound that inhibis the activity of a fungal LS enzyme encoded by the isolated nucleic acid fragment of  claim 54 , the method comprising the steps of 
 (a) disrupting the endogenous LS gene of a suitable microbial host, rendering growth of the microbial host dependent on added riboflavin;    (b) transforming the altered microbial host cell of step (a) with a chimeric gene comprising a nucleic acid fragment of  claim 54  encoding a fungal LS enzyme and operably linked to at least one suitable regulatory sequence that allows its expression by the transformed microbial host cell;    (c) growing the transformed microbial host cell of step (b) under conditions suitable for expression of the chimeric fungal LS gene;    (d) contacting the transformed microbial host cell of step (b) while it is growing exponentially with a chemical compound of interest and in both the presence and absence of added riboflavin; and    (e) identifying as a fungicide candidate the chemical compound of interest that inhibitis growth of the transformed microbial host cell only when grown in the absence of added riboflavin.    
     
     
         68 . The method of  claim 67  wherein the isolated nucleic acid fragment corresponds to the sequence in SEQ ID NO: 37 and wherein the LS enzyme fragment corresponds to the sequence in SEQ ID NO: 38.  
     
     
         69 . An in vitro method for identifying as a fungicide candidate a chemical compound that inhibits the activity of a fungal LS enzyme encoded by the isolated nucleic acid fragment of  claim 54 , the method comprising the steps of 
 (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment of  claim 54  encoding a fungal LS enzyme, the gene operably linked to at least one suitable regulatory sequence;    (b) growing the transformed host cell of step (a) under conditions suitable for expression of the chimeric gene resulting in the production of the fungal LS enzyme;    (c) purifying the fungal LS enzyme expressed by the transformed host cell;    (d) contacting the fungal LS enzyme with a chemical compound of interest; and    (e) identifying as a fungicide candidate the chemical compound of interest that reduces the activity of the fungal LS enzyme relative to the activity of the fungal LS enzyme in the absence of the chemical compound of interest.    
     
     
         70 . The method of  claim 69  wherein the isolated nucleic acid fragment corresponds to the sequence in SEQ ID NO: 37 and wherein the fungal LS enzyme fragment corresponds to the sequence in SEQ ID NO: 38.  
     
     
         71 . An isolated nucleic acid fragment selected from the group consisting of: 
 (a) an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 35;    (b) an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding all or a substantial portion of the amino acid sequence selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 35; and    (c) an isolated nucleic acid fragment that is complementary to (a) or (b).    
     
     
         72 . The isolated nucleic acid fragment of claim  71  selected from the group consisting of SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: 36.  
     
     
         73 . A polypeptide encoded by the isolated nucleic acid fragment of claim  71 .  
     
     
         74 . The polypeptide of claim  73  selected from the group consisting of SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33 and SEQ ID NO: 35.