TWI276684B - Engineering of metabolic control - Google Patents

Abstract

The invention features a method of producing heterologous molecules in cells under the regulatory control of a metabolite and metabolic flux. The method can enhance the synthesis of heterologous polypeptides and metabolites.

Description

1276684

V. INSTRUCTIONS INSTRUCTIONS (1) GOVERNMENT RIGHTS This invention is based on the rights of the US Department of Energy (number: DE-FGG3-98Em298) and BES-981 4097) Piedding ~ > Supported by Yuan Cheng. The government has a particular background in the invention ... the use of technology has allowed host cells to engineer the desired compounds (e.g., multi-peptides and secondary metabolites). The large-scale production of multi-peptides in the cells of the industrial process provides the production of protein for medical use and enzymes for industrial use. Secondary metabolites are natural products that have long been known for their biological and medicinal importance. Because of the structural complexity of these molecules and oysters, traditional chemical synthesis; the effort of 3 匕 1 is required, and expensive precursors and cofactors (c 〇fact 〇r ) can be used to prepare such Compound. In recent years, the performance of heterologous proteins in cells has prompted the heterogeneous biosynthetic pathway to be easier and feasible to produce metabolites from inexpensive starting materials in microbes. In this way, many compounds have been produced, including polyacetamide and its derivatives (polyketides), /3-actam antibiotics, monoterpenes, steroids, and aromatic compounds. SUMMARY OF THE INVENTION The present invention is based in part on the discovery that heterologous polypeptones and metabolites are produced using a promoter that is subject to secondary metabolism.

105T-3479-?F*ptd Page 4 1276684 V. INSTRUCTIONS (2) The concentration of a substance (for example, acetylphosphonium phosphate) is enhanced by the regulatory expression of a multi-peptide such as a biosynthetic enzyme. The term "heterologous," (θ refers to a multi-peptide or metabolite introduced by humans. Heterologous multi-peptide = thank-you can be the same as the endogenous nature of natural existence. The term "metabolite" is Refers to an organic compound that is the product of one or more biochemical reactions. The metabolite itself may be a precursor to other reactions. The secondary g-glycogen is a metabolite derived from other metabolites. 疋 Thus, in one form of the invention A nucleic acid sequence comprising an acid sequence comprising a nucleic acid sequence encoding a heterologous polypeptide. Examples of bacterial host cells Escherichia coli, Bacillus subtilis , Salmonella typhimurium, Agrobacterium tumefaciens, Thermus thermophilus, and Rhizobium leguminosarum cells. The nucleic acid sequence is operably linked to a promoter that is regulated by a response Controlled by proteins. In other words, 'nucleic acid sequences are designed to allow nucleotide sequences to be expressed in vitro and in vivo. Type, is connected to a promoter sequence. "Promoter ,, ^ guiding means any DNA fragment transcribed material. The promoter is controlled by an anti-regulatory protein (eg, ntrC, phoB, ph〇P, ompR, cheY, creB or torR) of 9 strains of E. coli or homologs from other species. In addition, the regulatory protein can also be a cluster in situ group (c 1 u s t e r

1057-3479-PF.ptd 1 1276684 V. Description of invention (3) orthologous group ) (COG) Another member of COG0745, as defined by the URL http : "www·ncbi.nlm.nih· gov/COG / Tatusov et al., Nucleic Acids Res. 2000, 28:33-36). In a specific embodiment, the starting soil is a gold-clad knot. The term "ntrC" refers to the E. coli ntrC protein (SWISSPROT: P0671 3, http://www.expasy.ch/) and homologs in other suitable species. As used herein, "bonding" refers to the physical association of an equilibrium binding constant (KD) of less than 丨〇〇nM, preferably less than 1 nM, which is Escherichia coli g InAP1_filament, for example, disclosed The sequence of the gates of a 61^81^ registration number 042 1 is located between approximately 93 and approximately 323 (Reitzer & Magasanik (1 985),

Proc· Natl· Acad· Sci· USA, 82:1 979-1 983 ). This region includes the untranslated sequences of the g 1 nA gene. In addition, the translated fusion can be constructed between a sequence encoding g 1 nA and a sequence encoding a heterologous polypeptide. The host cells are modified by genetic engineering techniques so that the promoter can be controlled in the gas 3#ft T (i n t s ence of nitrogen starvation). For example, the gene of the host red dragonfly - 1 尧 — - ^ ^ 皇 屋 ^ rabbit ϋ, for deletion or mutation repair, decoration, this gene is a member of ifLi? COG0642, like ^ http: / / w ww · ncbi · nlm .nih· gov/COG/ For example, CglnL, Miaocheng, Tu Q, cr and C or en Qiaoyi (Tatusov et al.). In another embodiment, the histidine protein kinase is specific for the regulatory regulatory protein that controls the promoter. Histamine protein kinase can be encoded by glnL, for example, E. coli glnL (swisspr〇t:

1276684 V. Description of invention (4) P0671 2, http: "www .Λ w· exPasy· ch/ ) 〇 鋥 纟 纟 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + Shengyuetai or metabolite I is regulated by the use of acid for heterologous* proliferation, for example, the main cell can be in any desired condition, nitrogen depleted rnw hung (n 〇gen starvation), + Nitr〇gen poor conditions or conditions rich in nitrogen (mtrogen rich). The biosynthesis of the heterologous protein encoded by the nucleic acid sequence may be a metabolite, octapeptide, which may be a mammalian protein, for example, a secreted growth factor, a single sputum. In the example of the 浐鲈101, the stromal or extracellular matrix component. In the original, for example,; r white primordial, yum peptide may be the anti-face protein required for vaccines. A set of bacteria, fungi, or single-cell biological pathogens:: = a form characterized by a - nucleic acid sequence. This set is more visible as needed. 2f is the protein-controlled starter host, so that ^μ # I -Τ γ y匕3, genetically engineered bacteria modified by two: if: nitrogen is not present, subject to Acetate == This kit can also provide instructions for its use. The nucleic acid sequence may comprise a sequence which is ligated into the polylinker, and may be ligated to the promoter of the reaction-regulated egg mussel & In the case of the E. coli glnAP2 promoter in the kit, and the bacterial host, the mutant or the deleted E. coli cell is trapped... the cell contains a "gene in another aspect of the invention, which is characterized by A first performance 1272684 V. Description of the invention (5) Host cells of the card E (cassette). The first performance cassette comprises a promoter (e.g., any of the promoters described above) and a nucleic acid sequence encoding an enzyme required for the synthesis of a heterologous metabolite. As used herein, "enzyme," refers to a multi-peptide that can catalyze a chemical reaction or multiple reactions. The nucleic acid sequence I is operably linked to a promoter, which can be subjected to nitrogen in the absence of nitrogen. Regulation of B-phosphoric acid. Host cells also contain additional nucleic acid sequences for the expression of other enzymes required for the synthesis of metabolites. Such additional sequences may be endogenous sequences that represent endogenous enzymes, or may behave differently. The sequence of the introduction of the source enzyme.

In one embodiment, the heterologous metabolite is isoprenoid (isoprenoid), polypyridyl acid salt (p〇iyhydroxyalkanoate), ♦ B 1 and its bamboo organism, /3 - endoprost antibiotic, Or a precursor thereof (eg, an upstream metabolite) or a derivative thereof (eg, a downstream metabolite). For example, isoprenoids can be carotenoids, sterols, paclitaxel, biguanides, gibberellin, and quinone. Examples of specific isoprenoids include isoamyl diphosphate, dimercaptopropyl diphosphate, geranyl diphosphate, farnesyl diphosphate, diphosphonium geranyl Yak-based vinegar and phytoene phytoene °

Examples of specific carotenoids include ·5-carotene, carotene, astaxanthin, zeaxanthin, zeaxanthin-/5-glycan, hexahydrofuran (phytofluene) ), neurosporine, iutein and torulene. When the desired heterologous metabolite is isodecadiene, the heterologous enzyme may be isopentenyl isomerate,

1057-3479-PF.ptd Page 8 1276684 V. INSTRUCTIONS (6) Dicalcium yttrium geranyl geranyl vinegar synthase or budeoxy glucoside 5 _ acid synthase. When the desired heterologous metabolite is a polyhydroxyalkanoate, the heterologous enzyme may be a 3-ketoximino reductase or a poly-3-hydroxyalkanoate polymerase. The host cell can be a bacterial fine &'', e.g., an E. coli cell. The host cell can be genetically engineered as needed by deleting or mutating the above gene (e.g., a gene encoding a histidine protein kinase). In a specific embodiment, the host cell further comprises a second expression card comprising a nucleic acid sequence encoding a phosphoenolpyruvate (pEp) synthetase operably linked to a promoter (eg, glnAp2) It is controlled by the concentration of cyanoic acid. In another aspect of the invention, a method of producing a heterologous isoprenoid in a host cell is provided. This method involves the extensive expression of phosphoenolpyruvate synthetase and the biosynthetic enzymes required to synthesize heterologous isoprenoids. In a specific embodiment, the gene encoding the pyruvate kinase or the discolinyl pyruvate carboxylase in the cell is deleted or rendered incapable by genetic engineering. In another embodiment, a gene encoding a phytic acid enolpyruvate carboxykinase is expressed in large amounts in a host cell. Still another aspect of the invention is a method of producing lycopene in a host cell. The method comprises the following heterologous enzymes: budeoxy-D-xylulose 5-phosphate synthase, geranyl geranyl diester synthase, phytoene synthase, and octahydro tomato Erythrase saturation enzyme. In a specific embodiment of the method, for example, a g 1 nAp2 promoter is used to express a large amount of prenyl ester isomerase. In another concrete

1057-3479-PF-ptd Page 9 1276684

A large amount of phosphoenol C is used in the glnAp2 promoter, for example, a citrate synthase. Another aspect of the invention features a nucleic acid sequence comprising a right promoter and an enzyme-producing enzyme sequence encoding a first metabolite. The promoter is operably linked to this sequence and is regulated by a 2's metabolite, 'the concentration of this S metabolite is representative of the utilization of the precursor prior to biosynthesis for the first generation & In a specific embodiment, the second thank-on is ^^6* /Wrt λ* produced from the precursor for biosynthesis of the first metabolite. In one embodiment, the first metabolite is a polyhydroxyl group. An alkanoate (eg, polyhydroxybutyrate), and a nucleic acid sequence encoding a synthetic enzyme, (eg, [3-, 3-ketoxime A (coA) reductase or poly-3-3 hydroxyoctyl coenzyme A polymerase ). In another example, the first metabolite is polyethyl hydrazine and its derivatives, a dot-indoleamine antibiotic or an aromatic compound. In still another example, the first metabolite is isoprenoid, for example, isoprenoid as referred to herein. The nucleic acid sequence encodes a biosynthetic enzyme for the production of isoprenoids, for example, isopentenyl isomerate diphosphate, geranyl geranyl diacetate synthase, 1-deoxyated wood Sugar-regulating 5_acid-synthesizing enzyme, acid-enolpyruvate synthase, di-n-farnesyl farefin synthase, di-loaded geranyl-based geranyl ester synthase, phytoene synthase , phytoene desaturase or lycopene cyclase. An isoprene-like precursor can be pyruvic acid. The concentration of pyruvic acid is related to the concentration of acetic acid and acetic acid. Therefore, in this example, the second metabolite is acetylphosphonium

acid. Promoter-responsive protein

1057-3479-PF-ptd Page 10 1272684 V. INSTRUCTIONS (8) (For example, ntrC can be combined only in a specific embodiment, the promoter is a CAP I-binding mammal, a mover, and its junction The promoter Gisl, Msn2 regulatory signal can be used to regulate the transcription of the protein-regulated nucleic acid sequence in the eukaryotic organism. The host-dependent cell production depends on the desired product protein or metabolite activity Microbiol. The reaction regulates the protein) and is controlled. Such a promoter determines the amino acid in the host cell to be acid-reacted. The promoter in response to a particular 'concentration of acetamidine phosphate is with E. coli eg E. coli g 1 n A p 2 promoter. It can be regulated by cyclic adenosine monophosphate (cAMP). The promoter can be: a bacterial promoter that catabolizes the activated protein). The promoter may be a protein containing CRAMP, CREM or ATF-1. The yeast cell may be a promoter regulated by cAMP or a promoter that binds to protein or Msn4. Another possible shell of the promoter

Take fructose-1 - tartaric acid or fructose beta - 粦 粦 acid. Promote such a promoter. Tian Weicai's trapped FruR column can be contained in a plastid. This sequence can also contain, from, and filterable markers. The sequence may further comprise yeast ^ ^ 囷 replication origin and appropriate screenable markers, , 5 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , One of the standard techniques is to make cells, and the king's mouth is added to an agent (for example, genes required for induction and synthesis). It has been extensively induced to induce a growth retardation, resulting in growth retardation. I Dong, and (Kurland and Dong, 1 996, Mol. 氐 21:1-4). The implementation of the external supply inducer is based on

I 1276684 can't monitor the 'naturally savvy, the more succulent ‘the synthesis of the characteristics of the production of the physiological surface should be provided to provide engineering and mobilization with the best synthesis. The central component of the source of the multi-peptide is utilized in this technique operably the promoter makes regulation. It can be included in the gene therapy for the growth and recovery of specific metabolic factors, and the method of gene therapy will be transferred to a genetic engineering project. The state of the high state reaction controls and reduces the current regulation method. </ br> will be described in the following five, invention description (9), and rate. The opposite order. In the present cells and heterologous control loop peptides or metabolites provide enhancer formation. It is beneficial to other inventions. Other aspects of the invention and the detailed description of the invention utilize the method of producing or metabolizing the present invention and encoding the nucleic acid sequence of the nucleic acid sequence of the selected state. It is controlled by a combination of specific promoter peptides for cell changes. The facts, reduced use of the state of the reaction, for example, or a variety of specific purposes and advantages are easy to see. The metabolically controlled protein is a phenotype (the toxic byproduct of the use of the plastid source in the process of the use of each plastid source to refine and change the heterologous multi-win recombinant pathway) There are detailed instructions and application specifics, which are used in a specific method for the use of protein, which includes the current standard method, to construct a mover (for example, a promoter that is subjected to cell physiology or species). For example, the bacterial plastid, the genomic promoter is in the nucleic acid sequence, so that the coding to the promoter regulates the cellular metabolism of the peptides. Some applications 1276864 V. Inventive Note (10) and have the replication of bacteria from calling the standard techniques in this technique. The marker ° performance card E can be inserted into the semi-genome of the fine-moon pack. If it is used for the heterologous multi-site peptide gI. during the growth period or the stationary phase, the promoter is selected. For example, the pair can be selected. </ br> = this is selected) Reactive age narrative, ten signals (for example, the accumulation of the second letter period period. The first logarithmic growth period or static drive, intermediate products or waste before production can be glycolytic of Interproduct = sub-signal, + decomposed waste product (for example, the signal of the nutritional status of acetic acid or B.), the 疋度疋一 (body 丨 ΐ ΐ E E: promoter can be based on large The results of the scale performance analysis were experimentally selected from the results of the experiment, and the results were analyzed by the hybridization of the acetylated phosphate to the microarray prepared by the cells cultured in acetic acid, and the signal and A reference signal (for example, in (9) prepared by cells in the early logarithmic growth phase, the comparison is: identification. The experiment can be performed simultaneously on prokaryotic and eukaryotic cells; than bacteria, yeast, plants, and mammalian cells.) For examples of such experiments in biology, see TaUat et al., (2〇〇〇), Nat.

Bi〇techn〇l. 1 8:679-82 and Liao (2000), Bi’otechh〇1; Prog·1 6:278-86. Once the gene that is expressed under the desired conditions is identified, its promoter can be applied to the performance card. In addition, experiments can also be done by externally adding a desired molecule (for example, in a metabolic pathway 1057-3479-PF· ptd第13页

1276684 V. The precursor of the invention (11), or M + Fen If. The number of stages or in the synthesis of 'sutake: Dan: 4's stay (for example, after growing to iP it ^ ^ aa ^ , It is carried out while growing. The promoter can be identified based on the gene being induced. Nothing to move in. In an example, what? The production of engineering controlled by the object. It can be used in the fine cells to metabolize the movers, while the Z = ' of the second metabolite is regulated by a second metabolite. For example, if the first metabolite is from the precursor = : to be acetaminophen. In a nutrient-rich environment, the cells are stored in a' with the parental age, which is the product of propionic acid. Excess acetamidine 7 enzyme A" in / Α τρ and acetic acid, in the form of waste products, the concentration of 驮 驮 increases with increasing cell density. The concentration of acetic acid, B = and acetamidine is Interrelated by the following biochemical reactions:

(1) Acetyl Coenzyme A + Pi &lt;-&gt; Ethyl Phosphate + Coenzyme A (2) Ethyl Phosphate + ADP &lt;-&gt; Acetic Acid + ατρ

Therefore, the acid-breaking concentration of 'Southern Acetate' represents the excess of Ethyl Coenzyme A and the excess of C-I acid. For example, a host cell modified by deletion or mutation of §丨nL will turn the function of the generation into an acetaminophosphate-dependent (to call et al. (1 992), J. Bacteriol· 1 74: 606 6 0 70 ). With this

In this way, a promoter regulated by ntirC, for example, the glnAp2 promoter, can be used to control gene expression in response to acetaminophen acid. The g i nAp2 promoter can be obtained using standard techniques well known in the art. For example, primers that adhere to the ginAp2 promoter can be designed and synthesized. Polymerase chain reaction (pCR)

1276684 V. Inventive Note (12)) can be used to amplify a nucleic acid fragment containing a glnAp2 promoter. This fragment is now available for further construction. Similarly, E. coli strains containing a histidine kinase gene (e.g., g 1 nL ) deletion can also be readily prepared. See, Link et al., (1997), J. Bacteriol.

1 7 9 ( 2 0 ) : 6 2 2 8 - 6 2 3 7, detailing a possible method. The sequence encoding the desired heterologous peptide is optionally ligated to the downstream of the g 1 ηAp2 promoter such that the sequence is operably linked to the promoter. Host cells with an inactivated g UL gene can then be transformed with these sequences. The transformed strain was cultured and the production of multi-peptide was monitored during the culture. At high cell densities, strong protein expression can be observed, like Farmer aZ

Liao (20 00), Nat. Biotechnol. 1 8: 533-537, the entire contents of which is incorporated herein by reference. 70 Mammalian cells can be used as host cells for the production of polypeptides. For example, a promoter that responds to cAMP can be selected. The promoter may comprise a cAMP reactive element (CRE), the

I C two CREM or ATW. The coding sequence required by standard techniques well known in the art can be placed in the promoter of the milk animal cells. In some examples, the construct can be inserted into a virus; 7 for example, a virus that is not activated. Such specific production can be produced in the regulation of gene therapy or in the production of enzymes. Plant cells can also be used as a host to produce a suitable promoter, for example, for plants. Alternatively, a promoter of the reaction can be selected for use in the preparation, production, metabolite or precursor identification by microarray experiments. In an appropriate two: things. The promoter can be used in the vector of the field, the desired promoter

1276684 V'Inventive Note (13) 'After fusion to the desired coding sequence, this construct is electrotransformed into Agrobacterium tumefaciens and then transformed into plant cells using standard techniques well known in the art. In another embodiment, the yeast cells are manipulated to exhibit a heterologous polypeptide or metabolite under metabolic control. For example, Saccharomyces cerevisiae Ί, the promoter may be a CAMP-regulated promoter, for example, a promoter that binds to the protein Gisl, Msn2 or Msn4. The regulation of all yeast genes that respond to various metabolic conditions is increasing. More research. For example,

DeRi Si et al., (1 997), Science 278·680-686, describe the almost complete S. cerevisiae gene, a post-transcriptional test under various metabolic conditions. Promoters that are regulated by the desired metabolite can be selected based on such data. The production of yeast plastids and the transformation of yeast are well known to those skilled in the art. Various metabolic pathways can be reconstructed using the above-described performance techniques. For example, 'the pathway for the production of arbutin can be introduced into Escherichia coli by constructing the expression vector of the following gene: dxs derived from Escherichia coli (encoding 1-deoxy-D-xylulose 5-phosphate synthase), Gp s of Archaeoglobus fulgidus (coded geranyl dicarboxylate (GGPP) synthase) and Erwinia

105T-34T9-PF-ptd Page 16 CrtBI (encoding phytoene synthase and saturase, respectively). These genes can reside on single or multiple plastids&apos; or can be incorporated into the chromosome of E. coli. Further, the acid-saturated pyruvate synthase can be expressed in a large amount by any method, for example, by fusion to the ginAp2 promoter by 1276684 V, invention (14). The isopentenyl diphosphate isomerase may also be expressed in large quantities by any method, for example, by fused to glnAP2 in another embodiment to produce a polyhydroxyalkanoate (PHA) (eg, polyhydroxybutyrate) The route of the acid salt can be carried out in E. coli. Pha is a family of linear esters of hydroxy acids with a variety of thermoplastic and commercial uses. A Pseudomonas aemginosa gene encoding a 3-ketooxime A reductase and a poly-3-hydroxysodium sulphate polymerase can be placed under the control of a desired promoter (eg, glnAp2) because The concentration of acetaminophen coenzyme a can represent the utilization of the precursor prior to PHA synthesis. Without further elaboration, it is believed that the above description has been adapted to the invention. Therefore, the following examples are intended to be illustrative only and not to limit the scope of the invention. All publications cited herein, the entire contents of which are incorporated herein by reference. : Method:

Culture conditions · All E. coli strains were cultured in a conical flask containing the specified culture medium in a water bath shaker (Model G 7 6 ; Newman BrUnswick Scientific, Edis〇n, NJ). The culture is cultured at a minimum nutrient requirement consisting of a salt 34 defined by the foot containing 〇·5 % (w/v) glucose or a salt defined by ye containing eclipse, %, (gravity/volume) glucose. Culture in liquid. The salt defined by YE is 14 grams per liter Κ 2 ΗΡ 04 1 6 grams KH2P04, 5 grams (NH4) 2S04, 1 gram MgS04 and 1 mg thiamine

1276684 V. Description of invention (15) Composition of the prime minister. The turbidity of the cells was monitored by a spectrophotometer at a wavelength of 550 nm.

Metabolite measurements: acetic acid, pyruvic acid, and other organic acids are analyzed using high performance chromatography (HPLC; Cons tame trie 3500 solvent delivery system and spectroscopic monitor 3 1 〇 〇 variable wavelength detector; LDC

Analytical, Riviera Beach, FL), maintained at 65 ° C organic acid column (Aminex HPX-87H, Bi〇-Rad Lab·, Hercules,

CA). The mobile phase consisted of 〇·〇1 N sulfuric acid and the flow rate was maintained at 0.6 mL/min. The peaks off the column are detected at a wavelength of 21 〇 nm. Glucose was measured using the Sigma kit number 31 5 -1 〇(). To quantify lycopene, 1 ml of the bacterial culture was extracted with acetone, centrifuged, and the absorbance of the supernatant was measured at a wavelength of 474 nm. The concentration of lycopene is calculated by comparing the absorbance value with a standard curve. X Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-pAGE) and Enzyme Analysis: The procedure for SDS-PAGE is as described in Lae_u (197〇) “饨” 227:680-685. The measurement of point-galactosidase activity, = lle "1 992" was carried out by the method described in "Short-term course of bacterial genetics" (Lengyigang Shangbei to publication, Cold Spring Harbor, New York).

As a result, the two sexes were integrated into the index of lacZ glucose change. 'And the use of acetamidine phosphorus in the desired metabolic pathway in E. coli, the use of glnAp2 promoter heterologous increase in the concentration of acetamidine phosphate can be excessive glucose. The invention is characterized by host cells, nucleic acid sequences in Western language as a signal to regulate Rate-Controlled Enzyme 1276684 V. INSTRUCTIONS (16) A method of performance in melon to completely utilize excess carbon and change its orientation 'so that it does not produce a toxic product (acetic acid).

To test the potential of g 1 n A p 2 as a dynamic controller for product expression, the nucleic acid sequence containing the heterologous 1 a c Z gene was constructed and operably linked to the iglnAp2 promoter. The g 1 nAp2 promoter region comprising the promoter and the ntrC-binding site is readily available by standard methods well known to those skilled in the art. The glnAp2 promoter is derived from E. coli genomic DNA using a forward primer 5, -CAGCTGCAAAGGTCATTGCACCAAC (containing an engineered Pvul Ϊ position) and a reverse primer.

5'-GGTACCAGTACGTGTTCAGCGGACATAC (containing an engineered ΚρηΙ position) was obtained by PCR amplification reaction. These two primers increase the area between positions 93 and 343 in the public 0^ sequence 16 (661^31^ registration number %1 0421 ).

The PCR fragment of glnAp2 was also cloned into the EcoRI site of pRS551, thus producing p2GFPuv, which contains glnAp2 before the promoterless lacZ gene. The glnAp2-lacZ region was transferred to ARS45 via homologous recombination (simons j et al. (1987), Gene 53:85-96), and the phage Ap2GFPuv 〇JCL1595 and JCL1596 were generated by avoiding 11^?2- The 13〇2 fusion was inserted into BW13177 (lacX74) by infection with 1?26??11 phage (Silfavy et al. (1984) Gene Fusion Experiment, Cold Spring Harbor Laboratory Publishing, Cold Spring Harbor, New York) And in the chromosome of BW1 8302 (lacXgln200 1 ; Feng et al., supra). This strain contains the gln20 0 1 dual gene, which consists of an internal deletion between the crypto 2 3 and 1 8 2 of the GlnL coding sequence, and is speculated to be

1057-3479-PF-ptd Page 19 1276684 V. INSTRUCTIONS (17) Ineffective mutations (Feng et al., supra).

The temporal relationship of the proced-galactosidase (calli-Ga 1 ) activity was measured in wild type and glnL mutant strains. The activity of glnAp2-stone-Gal increased in a time-dependent manner, similar to the concentration of acetic acid secreted from the glnL host (JCL1 596), but not for the genotyped wild-type control group j (JCL1595). Induction of promoter activity was found. Table 1: J0·galactoside-activity/3·galactosidase activity of glnAp2-lacZ (nmd/min·mg protein) Net nAp2_lacZ &lt; 100 〜100 in wild type (TCL1595) at 6 hours and 11 hours In the gtaL (TCL1590) glnApiMacZ ~ 700 ~ 1500 in the (VJS632) PUc-lacZ -500 -550

j Therefore, the lack of ginL, glnAp2, can be reflected by the secretion of acetic acid

I show excessive carbon changes. As the cells approach the post-exponential phase, the biosynthetic requirements decrease and the cells begin to show excess carbon, as evidenced by increased acetic acid production. At this point in time, approximately 6 hours, unexpectedly, the activity of glnAp2- /3 -Gal began to increase to approximately 7 〇〇 nm 〇 1 / mM protein (see Table 1), while in the wild type strain (JCL1595) )middle

1057-3479-PF.ptd Page 20 1276684 V. Description of invention (18)

Rate, approximately 100 nmol/min - mg protein (see Table 1). After 1 hour, the glnAp2-wan-Gai activity without glnL is non-significant, about 1 500 nm〇l/min-mg protein (see Table 1. glnAp2 is also in the opposite lac promoter) Induced in (piac). The chromosomal he activity in the VJS632 (lac+) strain is also rapidly increased after induction of wipTG (heterologous 'IL galactose ridges) and reaches a fixed degree in the cells, approximately 550 Nm〇l / min - mg protein (see Table 1), and is not subject to the culture period. / / In E. coli, using the glnAP2 promoter heterologous fusion to and aroG will be the performance of two different metabolic enzymes, phosphoric acid Enolpyruvate synthase (PPS) and 3-deoxy-])-arabinose heptasaccharide-sodium citrate (3) human liver) synthase (aroG) were placed under the control of the glnAp2 promoter. As a control group, the same two proteins were also expressed in large amounts from the tac promoter. Under the same genetic background and environmental conditions, the tac promoter showed static control. The standard method of expressing pps leads to growth retardation (Patnaik et al., (1 992), J. Bacterioi. 174:7527-7532). The plastid PAR0G was constructed by selecting a PCR fragment containing ar〇G pRW5tkt into the EcoRI-BamHI position of PJF118EH. The plastid pPS706 was first described in the above mentioned document by Patnaik et al. Both plastids express individual genes under the control of the Ptac promoter. The PCR #段' containing the glnAp2 promoter was selected to the plastid pAROG and the EcoRV-EcoRI position of pPS7 06

1057-3479-PF-ptd Page 21 1276684 V. INSTRUCTION DESCRIPTION (19) The plastids P2AR0G3 and pPSG706 containing individual genes are controlled under the control of the g 1 ηΑρ2 promoter, respectively. j The host strain BW1 8302 ( lacXgln2〇〇l ) was transformed into all four plastids. The strain having the individual plastids was cultured in a culture solution of M 〇 salt-glucose. The growth was compared after 5 hours.

Table 2: Growth of a large number of strains of growth without plastids after 5 hours Ptac-aroG ~0·5 glnAp2-aroG ~0_5 pu, PPs 〜0 · 12 glnAp2-pps __ ^0-4 as before As shown, a large amount of pps is expressed using Ptac-pps, resulting in significant growth retardation. However, unexpectedly, the use of glnAp2 resulted in near normal growth (Table 2). After 15 hours, proteins were separated from each strain and analyzed on a H) % SDS-PAGE gel. Compared to ^. When the control of the ppS gene was transferred to the glnAP2 promoter, at least more than |% of the PPS protein was expressed. Another surprising finding is that protein, its traditional large-scale performance is not significantly unfavorable, compared to the scorpion promoter, in the cell extracts expressed by the glnAp2 promoter,

1276684 V. Description of invention (20) Less than 300% performance. In Escherichia coli, the production of lycopene by a large amount of idi | We use dxs derived from Escherichia coli (encoding 1-deoxy-D-xylone | sugar 5-phosphate synthase), from / dg /d's gps (coded two-loaded yak-geranyl geranyl-based vinegar (GGPP) synthase) and Erwinia (price wima-derived crtBI (respectively encoding phytoene synthase and a gene that expresses genes such as a saturating enzyme, and reconstitutes lycopene in E. coli. Insert these genes into

PCL1 920 (a low copy number of plastids) to form pcw9 and can be expressed in large quantities at the same time. We used the g 1 ηAp2 promoter to control the performance of i d i (isopentenyl diphosphate acetyl isomerase). The construct containing the 丨d i gene is derived from the promoterless vector PJF118. The glnAp2 promoter was inserted to form a P2IDI. As

In the control group, the ptac promoter was inserted to form pTacIIH. These plasmids were separately introduced into a glnL strain (Bwl83〇2) containing pCW9. At 26 hours, the P2IDI-containing strain (ginAp2-idi) produced 丨00 mg/liter of lycopene in a defined culture medium containing glucose. The other side = two under the same conditions, the strain containing ptac - idi produced only a small amount of τ erythropoietin (less than 5 gram / liter). In addition, the p2IDI strain produced less than almost three times more acetic acid than pTacm, indicating that the carbon flowing to the acetic acid had been diverted to phytoene.

1276684 V. INSTRUCTIONS (21) Table 3: In the batch culture of E. coli, the carbon yield of tomato red turbidity is the bankruptcy rate of tomato red erythropoietin (mole end/mole) only host (BW183 〇2) 0.0000 +pTacIDI (PUc-idi) 0.0003 +pTacIDI (PtAC-idi)/pPS184 (Ptac-pps) 0.0012 +p2IDI (glnAp2-idi) 0.014 ~ 4p2IDI(glnAp2-idi)/pPSG184 (glnAp2-pps) 0.022 Using PPS to enhance lycopene yield from another compatible plastid PPSG18, pps are expressed in large quantities from ginAp2' while the remaining phytoerythrin pathway (dxs, gps, cr t BI ) gives two pCLl 920 expressions . The common performance of ppS and sputum in the % erythromycin pathway increased the final lycopene potency by 5% and increased the yield by a factor of three, from 〇·05 mg/ml/hr to 〇·16 mg. /ml/hr (Table 3). This is the opposite of containing pTacIDI and pPS184 (p^_idi +

A concomitant strain of Ptac-pps) in which no significant yield improvement was observed and substantial growth inhibition occurred. Other host cells used to make phytoerythrin introduce the pykF::cat and pykA::kan dual genes into the wild-type strain' to generate two single mutations (JCL1610 (pykF) and JCL1612 (pykA)), and a double mutation. (JCL1613 (pykF pykA) strain (P〇n^e et al., (i995), J. Bacteriol 177: 5719-5722). The double canine strain can reach approximately 14 mg of lycopene per gram of stem cells.

Page 24 1276684 V. INSTRUCTIONS (22) The final lycopene potency, and each single mutant strain produces a lycopene titer of 2.5 mg of erythromycin per gram of stem cells. A single p y k mutation produces lycopene at a level similar to that of the wild type strain, approximately 4 mg lycopene per gram of stem cells. In addition, the large performance of P c k (phosphonium citrate pyruvate kinase) increased the final lycopene titer by a factor of about three. The large performance of ppc (Acetyl enol propionate carboxylase) reduced the production of lycopene by about 30%. Microbial deposit

The recombinant plastids P2IDI (in E&gt; coli DMa), PPSG184 (in co· coli DH5 α ), and pcW9/p2IDi (in Ε· coli JCL1613) used in this case have been in accordance with the provisions of Article 26 of the Patent Law. Registered at the Center for Fungi of the Food Industry Development Institute on October 4, 1989, the registration numbers are CCRC94031 9, CCRC 940320 and CCRC 940321. Other specific embodiments of the invention have been described in the following. However, it is to be understood that the invention may be variously modified without departing from the spirit and scope of the invention. Accordingly, other specific embodiments are also within the scope of the present invention. For example, all of the above-described multi-peptides and homologs of genes are also within the scope of the present invention.

1057-3479-PF-ptd第25页

Claims (1)

1276684 __Case No. 89121439 VI. Scope of Patent Application _?β^3ι a μ _ Charge Amendment Announcement j j · An E. coli host cell comprising a nucleic acid sequence comprising a promoter and a coded two-filler Acid isoamyl ester isomerase, dibromide geranyl geranyl synthase, I-deoxyxylulose 5-phosphate synthase, phytoene synthase or october red a nucleic acid sequence of a desaturase; the nucleic acid sequence is operably linked to the promoter, the promoter is under the control of ntrC, the host cell line is deleted or mutated by genetic engineering techniques, glnL, such that the promoter is In the presence of nitrogen, it is the regulation of acetylphosphoric acid. Thousands of times to the host cell 2 · The large intestine rod as described in claim 1 of the scope of the patent, wherein the promoter is. 3. The E. coli host according to claim 1, wherein the isoprene is a carotenoid. 4. The Escherichia coli host according to claim 1, wherein the isoprene is one of lycopene, /5-carotene, and a precursor thereof. ', 瑕月素 or 5 · E. coli as described in the scope of the patent application, which further comprises a cell encoding phosphoenolpyruvate synthetase, 6 · the large intestine rod according to claim 1 The host cell further comprises a nucleic acid cassette, which comprises a nucleic acid sequence of the main cell, pyruvate synthase, and operably encodes a phosphoenol. The promoter is regulated by the concentration of acetyl phosphate. To a promoter, a method for producing a variant in a bacterial host cell comprising the method of providing a host fine-olefin as described in claim i, wherein the promoter is activated by acetamidine phosphate, Cell, and 〃 Bu, cultivate the host fine 1276684 Amendment No. 891214卯 VI. Patent application area 0 where the culture is cultured, where the culture is the host 8. The scope of claim 7 includes nitrogen-rich conditions. d. 9. If the conditions of item 7 of the scope of patent application include growth to the logarithmic growth period. 4 million 沄 10·If the towel please patent scope? The conditions of the item include growth to the stationary phase. The method of righteousness 11. As described in the method of claim 7, the cell is produced by the isoprenoid-comb-dye #青素.戌 疋 疋 lycopene, /3 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ And at least 5 mg of L-1 tomato red is produced. The method in which p and the soil H are heavy in the host cell as described in the first paragraph of the patent application, including the heterologous 1-deoxy-D-xylulose%:-forming enzyme, The source of diphosphoric acid geranyl geranyl ester synthesis &amp; mother: axillary phytoene synthase and heterologous phytoene desaturase. 1 4 · A kit comprising: ^) a nuclear sequence. The nucleic acid sequence comprises a promoter controlled by ntr C. a prenyl isomerase encoding a dipentyl diphosphate, a geranyl diphosphate synthase , i-deoxy-xylo-5-phosphate synthase, octopus: sequence 1 combination: octahydro-denyl desaturase or cycline ring: the enzyme allows the promoter to be in a defined host cell, Received B 1057-3479-PF3.ptc Page 27 1276864 --- 89121439_年月日日_Amendment___ Six, the scope of application for the regulation of indigo acid; (2) and the host cell of this definition is carried out by genetic engineering techniques Modification of deletion or mutation of g 1 n L. 1 5 · a nucleic acid sequence comprising a promoter and a succinyl isocyanate isomerase, a geranyl geranyl diphosphate synthase, a budeoxyxylulose 5-phosphate a synthetase, phytoene synthase or octahydroparaffin desaturase sequence operably linked to the promoter '5H promoter is controlled by ntr C' such that the promoter is in the group In the absence of amino acid protein kinase, it is regulated by the acid filling. The nucleic acid sequence of claim 15, wherein the isoprene is a carotenoid. The nucleic acid sequence of claim 15, wherein the isoprene is one of lycopene, cold-carotene, astaxanthin or a precursor thereof. The nucleic acid sequence of claim 5, wherein the promoter is glnAp2. An E. coli host cell comprising: (1) an inactivated histidine protein kinase gene; (2) a nucleic acid sequence comprising a nucleic acid sequence encoding a phosphoenolpyruvate synthetase operably linked Up to a g 1 n Ap2 promoter, which is under the control of ntrC and ethylene phosphate; and (3) a nucleic acid sequence encoding a yttrium geranyl geranyl ester to synthesize a scorpion serotonin synthase And a octahydroquinone saturase. 2 0 · E. coli host as described in claim 19 l〇57-3479-PF3.ptc Page 28 1276684 _ Case No. 89121439_Year of the month _«_ Six, the scope of the patent application, including a nucleic acid encoding isovaleryl isomerase isomerase. The E. coli host cell of claim 20, wherein the nucleic acid encoding the prenyl isocyanate isomerase is operably linked to a glnAp2 promoter. 2. The E. coli host cell according to claim 19, wherein the E. coli host cell exhibits a lycopene. 2 3. The E. coli host cell of claim 19, wherein the lycopene is present in an amount greater than 5 mg/L. End of section ❿ 1057-3479-PF3.ptc Page 29