التفاصيل البيبلوغرافية
| العنوان: |
Intermediates and enzymes of the non-mevalonate isoprenoid pathway |
| Document Number: |
20080318227 |
| تاريخ النشر: |
December 25, 2008 |
| Appl. No: |
11/897349 |
| Application Filed: |
August 30, 2007 |
| مستخلص: |
The invention provides a protein in a form that is functional for the enzymatic conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-butenyl 4-diphosphate notably in its (E)-form of the non-mevalonate biosynthetic pathway to isoprenoids. The invention also provides a protein in a form that is functional for the enzymatic conversion of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, notably in its (E)-form, to isopentenyl diphosphate and/or dimethylallyl diphosphate. Further, screening methods for inhibitors of these proteins are provided. Further, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate is provided and chemical and enzymatic methods of its preparation. |
| Inventors: |
Bacher, Adelbert (Garching, DE); Rohdich, Felix (Zolling, DE); Adam, Petra (Kramsach, AT); Amslinger, Sabine (Weiden, DE); Eisenreich, Wolfgang (Freising, DE); Hecht, Stefan (Bad Aibling, DE) |
| Claim: |
1-103. (canceled) |
| Claim: |
104. A protein in a form that is functional for the enzymatic conversion of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, in its (E)-form, to isopentenyl diphosphate and/or dimethylallyl diphosphate. |
| Claim: |
105. The protein according to claim 104, wherein it is in a form functional for said conversion in the presence of FAD and NAD(P)H. |
| Claim: |
106. The protein according to claim 105, wherein it is in a form functional for said conversion in the presence of a metal ion selected from the group of manganese, iron, cobalt, or nickel ion. |
| Claim: |
107. The protein according to claim 104, wherein it has a sequence encoded by the ispH (formerly lytB) gene of E. coli or a function-conservative homologue of said sequence. |
| Claim: |
108. A purified isolated nucleic acid encoding a protein in a form that is functional for the enzymatic conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-butenyl 4-diphosphate in its (E)-form and/or a protein in a form that is functional for the enzymatic conversion of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, in its (E)-form, to isopentenyl diphosphate and/or dimethylallyl diphosphate, with or without introns. |
| Claim: |
109. A DNA expression vector containing the sequence of the nucleic acid according to claim 108. |
| Claim: |
110. Cells, cell cultures, organisms or parts thereof recombinantly endowed with the sequence of the nucleic acid according to claim 108, wherein said cell is selected from the group consisting of bacterial, protozoal, fungal, plant, insect and mammalian cells. |
| Claim: |
111. Cells, cell cultures, organisms or parts thereof according to claim 110, wherein it is recombinantly endowed with a vector containing a nucleic acid sequence encoding a protein in a form that is functional for the enzymatic conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-butenyl 4-diphosphate in its (E)-form and/or a protein in a form that is functional for the enzymatic conversion of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, in its (E)-form, to isopentenyl diphosphate and/or dimethylallyl diphosphate, and wherein said cell is optionally further endowed with at least one gene selected from the group consisting of dxs, dxr, ispD (formerly ygbP); ispE (formerly ychB); ispF (formerly ygbB) of E. coli, a function-conservative homologue thereof, and a function-conservative fusion, deletion or insertion variant of any of the above genes. |
| Claim: |
112. Cells, cell cultures, or organisms or parts thereof transformed or transfected for an increased rate of formation of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, in its (E)-form, compared to cells, cell cultures, or organisms or parts thereof absent said transformation or transfection. |
| Claim: |
113. Cells, cell cultures, or organisms or parts thereof transformed or transfected for an increased rate of conversion of (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate to isopentenyl diphosphate and/or dimethylallyl diphosphate compared to cells, cell cultures, or organisms or parts thereof absent said transformation or transfection. |
| Claim: |
114. Cells, cell cultures, or organisms or parts thereof according to claim 110 transformed or transfected for an increased expression level of a protein in a form that is functional for the enzymatic conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-butenyl 4-diphosphate in its (E)-form and/or a protein in a form that is functional for the enzymatic conversion of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, in its (E)-form, to isopentenyl diphosphate and/or dimethylallyl diphosphate compared to cells, cell cultures, or organisms or parts thereof absent said transformation or transfection. |
| Claim: |
115. Cells, cell cultures or organisms or parts thereof according to claim 110, characterized by the recombinant endowment with sets of genes selected from the following group: ispC (formerly dxr), ispD, ispE, ispF, ispG (formerly gcpE); or ispC, ispD, ispE, ispF, ispG, ispH (formerly lytB); or dxs, ispC, ispD, ispE, ispF, ispG; or dxs, ispC, ispD, ispE, ispF, ispG, ispH; or dxs, ispC, ispG, or dxs, ispC, ispG, ispH of E. coli or a function-conservative homologue thereof and/or a function-conservative fusion, deletion or insertion variant of any of the above genes. |
| Claim: |
116. Cells, cell cultures or organisms or parts thereof according to claim 115, characterized by further recombinant endowment(s) with gene(s) being functional for biosynthetic steps downstream from the C5 isoprenoids. |
| Claim: |
117. Cells, cell cultures or organisms or parts thereof according to claim 110, wherein at least one gene of said recombinant endowments is equipped with artificial ribosomal binding site(s) for expression of the corresponding gene product(s) at a rate enhanced compared to the rate in the absence of the artificial ribosomal binding site(s). |
| Claim: |
118. Cells, cell cultures or organisms or parts thereof according to claim 110, wherein at least one of said recombinant endowments is due to a high copy replication vector. |
| Claim: |
119. Cells, cell cultures or organisms or parts thereof according to claim 110, wherein they are of bacterial, protozoal, fungal, plant or animal origin. |
| Claim: |
120. Use of the cells, cell cultures or organisms, or parts thereof according to claim 110 or disruption products thereof for the enhanced rate of in vivo formation or for the efficient in vitro production of a biosynthetic intermediate or product of the non-mevalonate isoprenoid biosynthetic pathway. |
| Claim: |
121. Use according to claim 120, wherein said intermediate or product is a C5-isoprenoid intermediate compound; or a >C5-isoprenoid compound; or a terpenoid compound. |
| Claim: |
122. Use according to claim 120, wherein the rate of formation or production is enhanced by providing a source for CTP. |
| Claim: |
123. Use according to claim 122, wherein the source for CTP is at least one member selected from the group consisting of cytidine, uridine, cytosine, uracil, ribose, ribose 5-phosphate and any biosynthetic precursor of CTP. |
| Claim: |
124. Use according to claim 120, wherein the rate of formation or production is enhanced by providing a source for phosphorylation enhancement. |
| Claim: |
125. Use according to claim 124, wherein the source for phosphorylation enhancement is glycerol 3-phosphate, phosphoenolpyruvate, inorganic phosphate, inorganic pyrophosphate or any organic phosphate or pyrophosphate. |
| Claim: |
126. Use according to claim 120, wherein the rate of formation or production is enhanced by providing a source for reduction equivalents. |
| Claim: |
127. Use according to claim 126, wherein the source for reduction equivalents is succinate, lipids, glucose, glycerol or lactate. |
| Claim: |
128. Use of the cells, cell cultures or organisms or parts thereof according to claim 110 for the production of a protein in an enzymatically competent form for the conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate. |
| Claim: |
129. Use of the cells, cell cultures or organisms or parts thereof according to claim 110 for the production of a protein in an enzymatically competent form for the conversion of (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate into isopentenyl diphosphate and/or dimethylallyl diphosphate. |
| Claim: |
130. Use of the cells, cell cultures or organisms or parts thereof according to claim 110 for the production of proteins in an enzymatically competent form for the conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate into isopentenyl diphosphate and/or dimethylallyl diphosphate. |
| Claim: |
131. A method of altering the expression level of the gene product(s) of ispG and/or ispH in cells comprising: (a) transforming host cells with the ispG and/or ispH gene; and (b) growing the transformed host cells of step (a) under conditions that are suitable for the efficient expression of ispG and/or ispH, resulting in production of altered levels of the ispG and/or ispH gene product(s) in the transformed cells relative to expression levels of untransformed cells. |
| Claim: |
132. A process for the efficient in vivo synthesis of (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate; or isopentenyl diphosphate or dimethylallyl diphosphate; in salt form or in protonated form, by the following steps: (a) culturing cells, recombinantly endowed according to claim 110 for said synthesis for a predetermined period of time at a predetermined temperature; (b) optionally adding glucose to a predetermined final concentration and further culturing for a predetermined period of time; (c) harvesting the cells; (d) preparing a crude extract from the harvested cells; (e) separating and purifying optionally isotope-labelled (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate; or isopentenyl diphosphate; or dimethylallyl diphosphate; in salt form or in protonated form. |
| Claim: |
133. Cells, cell cultures or organisms or parts thereof for the efficient formation of a biosynthetic product or intermediate of the non-mevalonate pathway to isoprenoids or terpenoids, characterized by (a) first recombinant endowment with a gene functional for the biosynthesis of 1-deoxy-D-xylulose 5-phosphate from 1-deoxy-D-xylulose; (b) capability for the uptake of 1-deoxy-D-xylulose; and (c) recombinant endowment(s) with gene(s) being functional for the conversion of 1-deoxy-D-xylulose 5-phosphate into desired downstream C5-intermediate(s) of said pathway. |
| Claim: |
134. Cells, cell cultures or organisms or parts thereof according to claim 133, wherein said gene(s) of said second recombinant endowment(s) code(s) for enzyme(s) for the formation of at least one of the following C5-intermediates of the non-mevalonate isoprenoid pathway: (a) 2C-methyl-D-erythritol 4-phosphate; (b) 4-diphosphocytidyl-2C-methyl-D-erythritol; (c) 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate; (d) 2C-methyl-D-erythritol 2,4-cyclodiphosphate; (e) 1-hydroxy-2-methyl-2-butenyl 4-diphosphate; (f) isopentenyl diphosphate; (g) dimethylallyl diphosphate. |
| Claim: |
135. Cells, cell cultures or organisms or parts thereof according to claim 133, characterized by the recombinant endowment with sets of genes as follows: (a) xylB, dxr; or (b) xylB, dxr, ispD (formerly ygbP); or (c) xylB, dxr; ispD, ispE (formerly ychB), or (d) xylB, dxr, ispD, ispE, ispF (formerly ygbB); or (e) xylB, dxr, ispD, ispE, ispF, ispG (formerly gcpE); or (f) xylB, dxr, ispD, ispE, ispF, ispG, ispH (formerly lytB) of E. coli or a function-conservative homologue thereof and/or a function-conservative fusion, deletion or insertion variant of any of the above genes. |
| Claim: |
136. Cells, cell cultures or organisms or parts thereof according to claim 133, characterized by the recombinant endowment with xylB and ispG (formerly gcpE) and optionally at least one gene selected from the following group: dxr; ispD (formerly ygbP); ispE (formerly ychB); ispF (formerly ygbB) of E. coli or a function-conservative homologue thereof, or a function-conservative fusion, deletion or insertion variant of any of the above genes. |
| Claim: |
137. Cells, cell cultures or organisms or parts thereof according to claim 133, characterized by the recombinant endowment with xylB and ispH (formerly lytB) and optionally at least one gene selected from the following group: dxr; ispD (formerly ygbP); ispE (formerly ychB); ispF (formerly ygbB); ispG (formerly gcpE) of E. coli or a function-conservative homologue thereof, or a function-conservative fusion, deletion or insertion variant of any of the above genes. |
| Claim: |
138. Cells, cell cultures or organisms or parts thereof according to claim 133, characterized by the recombinant endowment with xyl, ispG, (formerly gcpE) and ispH (formerly lytB) and optionally at least one gene selected from the following group: dxr, ispD (formerly ygbP); ispE (formerly ychB); ispF (formerly ygbB); of E. coli or a function-conservative homologue thereof, or a function-conservative fusion, deletion or insertion variant of any of the above genes. |
| Claim: |
139. A process for the efficient in vivo synthesis of 2C-methyl-D-erythritol 4-phosphate; or 4-diphosphocytidyl-2C-methyl-D-erythritol; or 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate; or 2C-methyl-D-erythritol 2,4-cyclodiphosphate; or (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate; or isopentenyl diphosphate or dimethylallyl diphosphate; in salt form or in protonated form, by the following steps: (a) culturing cells, preferably bacterial cells, recombinantly endowed according to claim 133 for said synthesis for a predetermined period of time at a predetermined temperature; (b) adding 1-deoxy-D-xylulose to a predetermined final concentration and further culturing for a predetermined period of time; (c) harvesting the cells; (d) preparing a crude extract from the harvested cells; (e) separating and purifying optionally isotope-labelled 2C-methyl-D-erythritol 4-phosphate; or 4-diphosphocytidyl-2C-methyl-D-erythritol; or 4-diphosphocytidyl-2C-methyl-D-erythritol 2-phosphate; or 2C-methyl-D-erythritol 2,4-cyclodiphosphate; or 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, notably (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate; or isopentenyl diphosphate; or dimethylallyl diphosphate; in salt form or in protonated form, by preparative chromatography. |
| Claim: |
140. The process according to claim 132, wherein a source for CTP, is added in step (a). |
| Claim: |
141. The process according to claim 132, wherein a source of phosphorylation activity is added in step (a). |
| Claim: |
142. The process according to claim 132, wherein a source of reduction equivalents, is added in step (a). |
| Claim: |
143. A vector comprising a sequence coding for one of the recombinant endowments as defined according to claim 133. |
| Claim: |
144. The protein according to claim 104, wherein it is a plant protein, notably from Arabidopsis thaliana. |
| Claim: |
145. The protein according to claim 104, wherein it is a bacterial protein, notably from E. coli. |
| Claim: |
146. The protein according claim 104, wherein it is a protozoal protein, notably from Plasmodium falciparum. |
| Claim: |
147. Cells, cell cultures or organisms or parts thereof according to claim 111, characterized by the recombinant endowment with sets of genes selected from the following group: ispC (formerly dxr), ispD, ispE, ispF, ispG (formerly gcpE); or ispC, ispD, ispE, ispF, ispG, ispH (formerly lytB); or dxs, ispC, ispD, ispE, ispF, ispG; or dxs, ispC, ispD, ispE, ispF, ispG, ispH; or dxs, ispC, ispG; or dxs, ispC, ispG, ispH of E. coli or a function-conservative homologue thereof and/or a function-conservative fusion, deletion or insertion variant of any of the above genes. |
| Claim: |
148. A process for the efficient in vivo synthesis of (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate; or isopentenyl diphosphate or dimethylallyl diphosphate, in salt form or in protonated form, comprising the following steps: (a) culturing cells, recombinantly endowed according to claim 112 for said synthesis for a predetermined period of time at a predetermined temperature; (b) optionally adding glucose to a predetermined final concentration and further culturing for a predetermined period of time; (c) harvesting the cells; (d) preparing a crude extract from the harvested cells; and (e) separating and purifying optionally isotope-labelled (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate; or isopentenyl diphosphate; or dimethylallyl diphosphate, in salt form or in protonated form. |
| Claim: |
149. A process for the efficient in vivo synthesis of (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate; or isopentenyl diphosphate or dimethylallyl diphosphate, in salt form or in protonated form, comprising the following steps: (a) culturing cells, recombinantly endowed according to claim 113 for said synthesis for a predetermined period of time at a predetermined temperature; (b) optionally adding glucose to a predetermined final concentration and further culturing for a predetermined period of time; (c) harvesting the cells; (d) preparing a crude extract from the harvested cells; and (e) separating and purifying optionally isotope-labelled (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate; or isopentenyl diphosphate; or dimethylallyl diphosphate, in salt form or in protonated form. |
| Claim: |
150. The process according to claim 139, wherein a source for CTP is added in step (a). |
| Claim: |
151. The process according to claim 139, wherein a source of phosphorylation activity is added in step (a). |
| Claim: |
152. The process according to claim 139, wherein a source of reduction equivalents is added in step (a). |
| Current U.S. Class: |
435/6 |
| Current International Class: |
12; 12; 40; 40; 12; 12; 12; 12; 01; 01; 07; 61; 07; 12; 01; 12; 12; 12 |
| رقم الانضمام: |
edspap.20080318227 |
| قاعدة البيانات: |
USPTO Patent Applications |