TW200918669A - Method for producing polyhydroxyalkanonate (PHAs) by microorganisms utilizing different carbon sources - Google Patents

Abstract

A method for producing polyhydroxyalkanonate (PHAs) by microorganisms utilizing different carbon sources, which performed by utilizing a gene transfer technology to transfer a PhbCAB operon gene of Ralstonia eutropha capable of synthesizing poly 3 - hydroxybutyrate (PHB) by carbohydrate metabolism, into a microorganism belonging to genus Aeromonas, having amylase and capable of growing by utilizing starch, in which the PhbCAB operon gene has a sequence of SEQ ID NO.1;the genetically recombined Aeromonas can use hydrocarbons and carbohydrates as carbon sources simultaneously when appropriate culture medium and carbon sources are provided so as to synthesis and cumulate Polyhydroxyalkanoates (PHAs).

Description

200918669 IX. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for synthesizing polyhydroxyalkanoic acid (PHAs) by utilizing different carbon sources by microorganisms, particularly a technique utilizing gene transfer technology, which will be available Carbohydrate metabolism to synthesize poly-3-&-butyric acid (PHB); ?M) C45operon gene, which is transferred to microorganisms containing Aeromonas genus containing amylase-producing starch and induces the microorganism A method for synthesizing polybasic acid (PHAs) from different carbon sources. [Prior Art] In recent years, due to the rise of environmental awareness, the use of non-decomposable petrochemical plastic products, environmental damage and waste of resources has become an issue of concern and ambition; therefore, seeking low-cost, decomposable Alternative materials are currently the goal of all parties. In order to adapt to the changing environment of nature, microorganisms usually survive and produce specific energy substances in the body under adverse conditions, in which polyhydroxyalkanoates (PHAs) Because of its biocompatibility, biodepatibility, and plasticity, its physical properties are similar to those of plastic products (such as polyethylene p〇Iyeihylene), so it can be used as an alternative to petrochemical plastics. Bio-plastics 'to solve the environmental pollution problems caused by the decomposition of Long Petrochemical plastics, and can be further developed into high-value biomedical materials' for bio-defense and biomedical applications. Polyesters (PHAs) are polyesters that are used by certain bacteria to store energy. When these bacteria are in an environment where carbon sources are excessive and lack certain nutrients (eg, N, p, s, sputum, etc.) At that time, it will be used as a carbon source in the city (4), and as a carbon source, and stored in the amount of 200918669. It has been found that there are about 150 kinds of bacteria which can produce polyhydroxyalkanoic acid (PHAs), and these bacteria are not limited to the same genus (Genera), and include Gram-positive bacteria and Gram-negative bacteria. The structure of the polybasic acid (PHAs) is as shown in the formula (I), and the side chain R may be a different carbon number of the alkyl group to form different polyhydroxyalkanoic acids (PHAs); when the side chain carbon number is between When 3_5 carbons, it is a short-chain-length (scl-PHAs); when the side chain carbon number is 6-10 carbons, it is a medium-long carbon chain. Medium-chain-length (mcl-PHAs). R Ο

I II _ Ο - 0 Η - (CHA 0 – 00-3000 (I) n = 1, R = hydrogen, PHAs = poly(3-hydroxypropionate), P(3HP) R = methyl, PHAs = poly(3- Hydroxybutyrate), P(3HB) R = ethyl, PHAs = poly(3-hydroxyvalerate), P(3HV) R = propyl, PHAs = poly(3-hydroxycaproate), P(3HC) R = butyl, PHAs = poly(3 -hydroxyheptanoate), P(3HH) R = pentyl, PHAs = poly(3-hydroxyocatanoate), P(3H0) R = hexyl, PHAs = poly(3-hydroxynonanoate), P(3HN) R = heptyl, PHAs = poly( 3-hydroxydecanoate), P(3HD) R = octyl, PHAs = poly(3-hydroxyundecanoate), P(3HUD) R = nonyl, PHAs = poly(3-hydroxydodecanoate), P(3HDD) n = 2, R = hydrogen , PHAs = poly(4-hydroxybutyrate), P(4HB) n = 3, R = hydrogen, PHAs = poly(5-hydroxyvalerate), P(5HV) When using a medium with different carbon sources, the bacteria will be different The metabolic pathway to synthesize polyhydroxyalkanoic acids of different side chain lengths; among them, the more common structure is poly-3-hydroxybutyric acid 200918669 [Poly(3-hydroxybutyrate), P(3HB) or PHB], which is 3 - Polymerization of monomers via 3-hydroxybutyrate (3HB) And polybutyric acid vinegar-based valeric acid S-copolymer [卩〇1; 7(117(1]'〇乂>^1^ plus 6-117(11*〇乂}^16 yang 16), ?116\/'], is a copolymer formed by 3-mercaptoic acid (3HB) and 3-hydrovalerate (3HV) monomers. As shown in Figure 1, in the microbial organism, the metabolic pathways for the synthesis of polyhydroxyalkanoic acid (PHAs) have three 'pathway 1: glycolysis; pathway 2: fatty acid degradation (Fatty acids degradation); and pathway 3 : Fatty acids biosynthesis. In Route 1, the microorganisms use sugar as a carbon source, and the polysaccharides are subjected to glycolytic hydrolysis to form Acetyl-CoA, which utilizes β-ketothiolase (PhaA). The effect is to form Acetoacetyl CoA, and then Acetoacetyl CoA reductase (PhaB) is used to reduce Acetoacetyl CoA to (7?)-3. - Said ketidine coenzyme A [(/?)-3-Hydroxybutyryl-CoA], and finally, polymerized into a short carbon chain poly-3-hydroxybutyrate via PHB polymerase (PHB p〇lymerase, PhaC) PHB) 〇 For most species, the synthesis of PHB is mainly composed of β-ketothiolase, Acetoacetyl-CoA reductase and ΡΗΒpolymerase. The three enzymes act as 'these three enzymes are encoded by the μ, Canta, and 6C genes respectively. 'PHB synthetic gene operon (ie, household; ^CAB 〇per〇n) as shown in Figure 2, gene order For the household secret, Kun, the driver (pr〇m〇ter, labeled "p" in Figure 2) is located in the JL swim of the phbC gene. As shown in Figure 1, in the path from 2, microorganisms [eg: Pseudomonas Huili 200918669

Using fatty acids as a carbon source, fatty acids are degraded into short-chain fatty acid monomers via β-oxidation, and each degradation is reduced by 2 carbons to form different intermediates: 醯CoA

• (acyl-CoA), enoyl-CoA, 〇S> 3-hydroxyliposide coenzyme A • [(6)-3-hydroxyacyl-CoA], 3-ketoacylase A (3-ketoacyl- CoA); intermediates lipid oxime coenzyme A (enoyl-CoA), (3)-3-lipid 醯 coenzyme A [(<S)-3-hydroxyacyl-CoA], and 3-ketooxime enzyme A (3-ketoacyl) -CoA) by different enzymes separately [enoyl-CoA hydratase, epimerase, ketone oxime A reductase

C (Moacyi-CoA reductase)], which is catalyzed to form (8)-3·hydroxylipid coenzyme A

[(8)-3-hydroxyacyl-CoA] Finally, a polyhydroxyalkanoic acid (mcl_pHAs) which forms a medium-long carbon chain is catalyzed by a PHA polymerase (PHApolymerase, PhaC) synthesized by a gene. As shown in Figure 1, in pathway 3, microorganisms use carbohydrates as a carbon source to produce an intermediate product, A3-hydroxylipid-ACP (/?-3-hydrmyacyl_A〇V, via the fatty acid biosynthesis pathway; The encoded synthetic coenzyme a thiolase (c〇A transacylase) catalyzes the formation of a medium-long carbon chain poly-base acid (mcl_PHAs) in a mixed form. G 峨 魅 县 县 县 县 县 县 县 县A large number of subjects, the choice of medium carbon source will be a cost factor - the big factor; in the process of fermentation, carbohydrate is a relatively inexpensive substrate often used, the towel 'Lin class is more dependent on many farmers The cheap supplements obtained in the production of waste. The brain is currently mixed with hundreds of non-microorganisms to accumulate polypsylic acid (PHAs), but the choice of the medium is a carbon source, and most of them cannot be effectively utilized. As a carbon source for the production of polyhydroxyalkanoic acid (PHAs), there are many microorganisms including genetic recombination, such as recombinant Escherichia coli, etc. There are also many studies focusing on the use of inexpensive 200918669 carbon sources, such as agricultural waste. Photosynthesis Carbon dioxide is used to synthesize polyhydroxyalkanoic acid (PHAs), but since it is limited by the physiological characteristics of the microorganism itself, it cannot obtain good yields. Therefore, it is impossible to produce poly-based sulphuric acid by microorganisms at this stage in China. _Technology, directly applied to the scale of industrial production. Microorganisms that can grow with starch using amylase, such as: Bacillus and many species of Aeromonas, are known to produce extracellular amylase Microorganisms, when these microorganisms use starch, they will be hydrolyzed into glucose for further use; and the two genus have _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Such as 0 /? Μ α), etc. can also accumulate polyhydroxyalkanoic acid (PHAs) in vivo; however, wild strains of these strains use starch as a carbon source to produce polyhydroxyalkanoic acid (pHAs) However, it is far below the extent that it can be used in industrial production. According to the literature, Jer〇WiWay is synthesized and accumulated by using hydrocarbons or fatty acids (such as lauric acid) as carbon sources. Medium and long carbon chain Polyhydroxyalkanoic acid (mcl-PHAs); since detomonobacteria (deTOmonos) contain extracellular amylase, it can grow with starch as a carbon source, and then hydrolyze starch to glucose in the extracellular region, then use glucose to increase Its cell mass; however, when carbohydrates (including starch) are used as a carbon source, the number of polyhydroxyalkanoic acids (PHAs) accumulated by the genus is extremely small and can even be ignored. Aeromonas has The highly active amylase can effectively metabolize starch, but it cannot use starch to synthesize polyhydroxyalkanoic acid (PHAs), which is a big drawback in industrial applications. Microorganisms such as ew(10) corpse/ζα H16 can utilize glucose. Cumulative poly(3-hydroxybutyrate) (PHB) for carbon sources. However, the growth of H16 is slower, its PHB yield is low, and it is not possible to use the cheap carbon source of starch in 200918669; Wang and Lee reported in 1997 (Wang F. and Lee S Υ.

Production of poly ( 3-hydroxybutyrate ) by fed-batch culture of . filamentation-suppressed recombinant Escherichia coli. Appl. Em. Microbiol 1997· 63 :4765·9·) ' Correlation of e Ό/7/ζα HI6 synthetic PHB The gene operon is transferred to Escherichia coli (the coli after co-transplantation can be used to synthesize PHB by glucose. However, the transgenic Escherichia coli cannot use starch as a carbon source to produce ρΗβ. Thus, the above-mentioned microorganisms can be used. There are still many defects in the production of polyhydroxyalkanoic acid (PHAs), which is not a good designer and needs to be improved. The inventors of the present invention have derived from the above-mentioned methods for the production of polyhydroxyalkanoic acid (PHAs) by microorganisms. All the shortcomings are improved and innovated by Sui Si, and after years of painstaking research, I finally succeeded in researching and developing this method, which can make microorganisms use different carbon sources to synthesize polyhydroxyalkanoic acid (PHAs). The purpose is to provide a way for microorganisms to use different carbon sources to synthesize polyhydroxyalkanoic acid (PHAs). Carbohydrates as a carbon source for the synthesis of polyhydroxyalkanoic acid (PHAs) microorganisms, using gene transfer methods, provide another metabolic pathway for the synthesis of polyhydroxyalkanoic acid (pHAs), and become available as a carbon source to produce polyhydroxyl groups. Alkanoic acid (pHAs) 2 microorganisms. The second object of the present invention is to provide a method for synthesizing polybasic acid (PHAs) by using microorganisms using different carbon sources, in addition to utilizing gene transfer methods to transfer microorganisms. In addition to the synthesis of poly(3-hydroxybutyrate) (pHB) by using carbohydrate as a carbon source, it is also possible to use hydrocarbon as the carbon source to synthesize medium-long carbon chain poly-molecular acid (mcl_PHAs). The extracellular amylase of Aeromonas sp., such as Qing (4), when using starch, must first hydrolyze the secret into glucose. Therefore, the inventor of the present invention will touch 16 bacteria. The sugar can be metabolized to synthesize poly 3- The gene of butyl acetonate (ρηβ); the operon ϋ, the soil sequence (SEQH) NO: 1 jin) is transferred to the genus Aeromonas (4), which contains the extracellular phosphatase, which can be used as carbon. In the source of growth, the gene is recombined with P The erPer〇n gene of the genus Aeromonm (Zeromomw) bacteria, which can be used as a carbon source by using carbohydrates (eg, temple powder), synthesizes and accumulates a large amount of poly-I-peracetic acid vinegar and produces gas. The bacterium belonging to the genus Aspergillus itself can utilize hydrocarbons or fatty acids, and the ability to synthesize polyhydroxyalkanoic acid (mcl-PHAs) including medium-long carbon chains such as polyhydroxyhexanoate (PHHx) still exists, and thus the present invention The microbes provided, in the synthesis of S4PHAs), not only have the advantage of matrix selection, but also have the ability to form different groups of polybasic acid (PHAs). A method for synthesizing polyhydroxyalkanoic acid (PHAs) by using microorganisms using different carbon sources, comprising: (1) providing a genus Aeromonas (10) which can be used for starch growth. (2) providing a DNA construct having a gene; (3) transferring the DNA construct into the microorganism of the Aeromonas aeruginosa to obtain a polycarbonic acid (PHAs) which can be synthesized using different carbon sources. (4) providing the microorganism according to (3) a suitable medium and a carbon source to synthesize polyhydroxyalkanoic acid (PHAs) 〇200918669, wherein the gene is a gene related to the synthesis of poly-3-hydroxybutyrate (PHB) The gene is selected from the sequence of Sin / 〇 « / aewir 〇 p / zaH16, having the sequence of SEQ K) NO: 1. Wherein the carbon source is a carbohydrate and the carbohydrate is starch or glucose. Wherein the carbon source is a hydrocarbon and the hydrocarbon is lauric acid. The medium is M9 basal medium, LB medium, YT medium, 2xYT medium or 1/2 ΥΤ medium. [Examples] Example 1 Preparation of test strain and operan gene fragment The test strain was a strain of Aeromonas sp. Jeromo Cong/BCRC13018 (i.e., ATCC7966), which was purchased from a food factory. The operon gene was cloned from the H16 strain and its sequence is shown in SEQ ID NO: 1 'The gene is located in the pBHR68 plastid. In order to transfer the operon gene into the strain, the fragment (about 52 kb) carrying the operon gene sequence on the pBHR68 plastid was excised by the restriction enzyme also wHI and DM, and cloned into PRK415 with a broad host range ( TV) vector to obtain pRHBj plastid; since the ramo Na/^ strain itself has resistance to tetracydine (Tc) (Tc>, therefore, the restriction enzyme B_HUfpUC4K is carried on the plastid The anti-drug gene fragment (Km1) of the prime (ka_ydn, Km) was excised and cloned into the ρΚΗΒ]f body to obtain the pRHB-2 plastid with the operon gene sequence and the antibiotic gene (Km1). , pRHB2 plastid map is shown in Figure 3. 12 200918669 Example 2 to transfer the operan gene into Aeromonas aeruginosa dmwwwias • In this example, with Jerowowos·BCRC13018 strain (hereinafter referred to as Aeromonas hydrophila bacterium i As a target for gene transfer, the Aeromonas hydrophila strain was purchased from the Food Institute. I. Aeromonas ΛγώνφΜα Strain Competent Cell Preparation 3 ml of Jeramowos /7_y<ira/?Ma strain was inoculated with the bacteria solution. 150 ml LBB culture f ί based 500 ml cone Medium (LBB medium is formulated as: 1〇g/L trypsin (Tiyptone), 5 g/L yeast extract (Yeast extract), 5 g/L sodium chloride (NaCl), pH 7.35~7.4) 3〇t under 'shock culture at 200 rpm until the 〇D_ value is 0.4~0·6, the bath is ice bathed for 30 minutes. Then 'with 4,000 卬 111 at 4. (: centrifuge for 15 minutes, remove the supernatant After the solution, add 5〇1111 of 300 μΜ sucrose solution to resuspend the cells, and then repeat at 4° 〇〇〇 rpm at 4 (: 15 minutes under centrifugation). Repeat the above steps twice; then, add 15 ml. 10% glycerol, centrifuged at 6000 rpm for 15 minutes at 4 °C, remove the supernatant, and finally resuspend the cells by adding 1 μl of 1% giyCer〇1 solution, mix well, and dispense in 100 μM per tube. Into a microcentrifuge tube, store at -80 °C for use. II. Electroporation In this example, 'gene transfer by electroporation', the plastid obtained in Example 1 was transferred to the strain. The electroporation experiment requires operation in a sterile environment. First, the prepared strain competent cells (C〇mpetentceu) are dissolved in an ice water bath. Add a proper amount of pRHB_2 plastids to each of the micro-centrifuge tubes of 13 200918669 Competent cell bacterium (loo μι), and then transfer all the liquid to the colorimetric trough (_tte) placed on ice, and Avoid the generation of air bubbles, dry the moisture on the outer wall of the cuvette, and then electrolyze it. Add electricity to the 丨miLB liquid.

The base was uniformly mixed, and all the liquid was transferred to a new micro-ion tube, and the (iv) solution was added to LB solid medium (LB + Km4〇) containing μΕ/ml kanamycin, and cultured overnight at 3 〇t. III. Purification of plastid DNA In order to confirm whether the pRHB-2 plastid has been successfully transferred into the bacterium, first, the surviving strain was picked from the above lb + Km40 solid medium and named as 〇w〇_施(R) £), and overnight culture in a 2 ml LB liquid medium for plastid DNA extraction and examination of whether the plastid DNA is a pRHB-2 plastid.

On the next day, take 1_5 ml of the bacterial solution into a microcentrifuge tube and centrifuge at 13,000 rpm for 1 minute. After removing the supernatant, add 1 μμΐ Alkaline lysis solution I (50 mM glucose, 25 mM 1^-〇邛118. 〇'1〇11^£〇丁八,?118.〇) to resuspend the bacteria, then add 2〇〇0 just prepared Alkaline lysis solution II (0.2 NNaOH, 1% SDS), shake slightly, ice Bath 5~l〇 minutes' and add i5〇μ〗 Alkaline lysis solution III (3M sodium acetate, pH 4·8) 'The microcentrifuge tube is inverted upside down to complete the reaction. After 10~15 minutes in ice bath, at 13,000 rpm Centrifuge for 1 minute, transfer the supernatant to a new microcentrifuge tube, add 2 to 2.5 volumes of 95% iced alcohol, and place at -20 °C for 20 to 30 minutes to precipitate DNA, then 13,000. After centrifugation at rpm for 10 minutes, after removing the supernatant, the precipitate was washed twice with 1 ml of 70% alcohol, centrifuged at 13,000 rpm for 10 minutes, the alcohol was drained, air-dried, and the precipitate was dissolved in sterile water for use. Since the plastids of the derawowas strain are not easily extracted in large quantities, the plastids extracted from the above-mentioned plastids are transformed into E. coli XLl-Blue in order to confirm whether the 200918669 deramowos·(RE) is a successful transgenic person. 'Using E. coli's strong self-replication ability, copying the plastid DNA that is sent in large amount, and then purifying the plastid from E. coli xL^Biue's body and electrophoresing it with agarose gel Analysis confirmed that hydrophih (RE) was indeed a successful gene transfer and was a strain of Aeromonas hydrophila containing pmm-2 plastids. Example 3: Extraction of polyhydroxyalkanoic acid (PHAs) in Α/ό 《 "like" (RE) strain and analysis of strains containing strains of jrawonos such as i/rap/zf/or (RE) containing pRHB-2 plastids Based on the formula of M9 base medium (Minimal salt medium, 5x): 33.9 g/L NaHP04 '15 g/L KH2P〇4, 2.5 g/L NaC Bu 5 g/L NH4C1), and Different single carbon sources were added: glucose (gluc〇se), starch (starch) and lauric acid (Laurfc aC1d), each carbon source concentration was 4% 'put into a constant temperature shaking incubator at 2 rpm at 3 〇<>c under the lamp corpus callosum for 48 hours to induce the synthesis of __Shi (R £) strains

Ssc (PHAs) and the negative control group (negative control) of the brain strain without the gene transfer, such as the measurement of the growth curve of the bacterium I. Ten measurements of growth under different carbon source environments ▲ wide tear (10) seemingly add the grade ^ () growth curve 'measured by the spectrophotometer when the wavelength is _ (10) absorption value (〇 D_) ' _ Pei | ) ~1 () hourly 'every hour' test - times, then every * hour test, measuring the growth period of the bacteria and the stage of the ten-band period. 15 200918669 II. Analysis of the components in the bacteria by gas chromatography layer analyzer (GC) for 48 hours (RE) strain as a test sample, the sample of the bacteria sample is: weigh about 0.02 g of dry bacteria, will Put in a 50 ml threaded test tube, the tube is wound up in a spiral with a water stop, add 2 ml of 5% acid sterol and 2 ml of air, and hydrolyze at 105 〇c for more than 4 hours. After the reaction is completed, wait for the temperature to drop. To room temperature, add the internal standard (diphenol ester 85μ1), then add 1ml of IN NaCl' to stand for more than 30 minutes, wait for stratification, remove the lower layer of 500 μΐ into the sample bottle, and take the injection needle 2 Μΐ is in the gas chromatographic layer (''Gas Chromatography (GC)), the moving phase is air, nitrogen (ν2) and hydrogen (Η2), and the detector used is a flame ion detector (flame Ionization deteetm; FID). Standardized measurements of poly-3-hydroxybutyrate (PHB) standards using poly-3-hydroxybutyrate (PHB) standards and 3.9% PHB-co-HHX standards as controls. The line is shown in Figure 4, and the sample poly(3-hydroxybutyrate) (PHB) was quantified by the standard calibration curve. Extraction of polyhydroxyalkanoic acid (pHAs) with SDS-sub-gas sulphate U. Referring to the method published by Dong and Sun et al. in 2000, 100 ml of a different single carbon source was added to a 500 ml Erlenmeyer flask. 9 Basic medium, inoculate the pre-cultured bacteria solution of 3 Qiu, put it into a constant temperature shaking incubator, and carry it at 3 rpm at 3 rpm. After cultivating for 48 hours under the armpit, weigh the volume of each bacterial solution separately, and Pour into a large round tube at the bottom of the circle and centrifuge at 8 〇〇〇 rpm for 2 , minutes. After removing the supernatant, resuspend the cells in 5 ml of sterile water and pour into a dry empty plastic tube. Dry and weigh in the middle, scrape the dry bacteria as much as possible, and mix the dried bacteria with steaming water. 200918669 Reuse the nature of cell thermal expansion and contraction to destroy the cell wall by sudden heating and cooling. And the cell membrane' method is as follows: low temperature treatment at -80 ° C for 20 minutes, and treatment with l 〇 (TC high temperature for 15 minutes, about 2 to 3 times; reuse of ultrasonic cell pulverizer, with 12 W shock 1 Minutes. - Next, add 10 ml of 30% sodium hypogasate (NaOCl, polyhydroxyalkanoic acid) to each centrifuge tube. The extracting agent, which acts to remove the protein and other substances of the cells, is uniformly mixed, and then distilled water is added to the end of eight minutes, and centrifuged at 4, rpm for 4 minutes, and the supernatant is removed. Repeat the above steps until the precipitated white matter. Finally, dilute each sample with distilled water ('After homogenization, centrifuge again, remove the supernatant, mix with distilled water, and place 1〇5. (: Drying) The white powder after drying is a sample of polyhydroxyalkanoic acid (PHAs). IV. Analysis of the structure of PHA by nuclear magnetic resonance spectrometry (NMR) The polyhydroxyalkanoic acid (PHAS) sample extracted by the above method. A nuclear magnetic resonance spectrometer Pudeai·Magnetic KesGnance Spectroscopy (NMR) analysis was carried out, and the sample was taken to be about 6 g, and 1 ml of D-gas (D_chU)r〇f〇rm) was added thereto at 5 Torr. The sample is dissolved under the armpit, and the solution is centrifuged to the upper layer for clarification. Finally, the D_chl〇r〇f_ solution is placed in the analysis tube, and the sample is analyzed. v. The results are shown in the table. For the strains that have not been genetically transformed, the polyhydroxyalkane yield (59 escapes) when lauric acid is used as the single-carbon source is higher than the use of glucose or other carbohydrates. As a single-carbon source trainer, the yield of heterogeneous base ships (PHAs) ((4) sugar: 18 78%; temple powder: 8% milk) is much higher. , instrumentation and juice calculation of its poly 3-butyric acid ester (PHB) content, 17 200918669

Mro/strain also had the highest content of ρΗβ in lauric acid alone (glucose: 5.16%; temple powder: 1.63%; lauric acid: 68.28%); compared to glucose or starch as a single source When cultured, the S OD_ value of the starch cultivator was the most embarrassing, but the synthesis of polypyridyl acid (PHAs) product was not much, and the results showed that the jerowoww was like a strain in the synthesis of polyhydroxylated The pathway for acids (PHAs) is prone to the metabolism of fatty acids. According to the analysis results of the GC instrument, it is shown that when the ramo strain is cultured with lauric acid as a single carbon source, the type of polyhydroxyalkanoic acid (PHAs) product synthesized is phb-co-j^x, and poly-3-hydroxybutyrate The content of the acid ester (phb) is the highest (as shown in Figure 7_2). Table 1 Results of cultivation of Aeromonas aeruginosa strains with different carbon sources. Cultured cells before culture. Dry cell weight PHB PHB PHAs Medium 〇D6 ODODeo value (g/L) Concentration Cg/L) Content (%) Rate (%) M9+glucose 0.27 3.08 1.09 0.06 5.16 18.78 Aeromonas M9 + starch 0.32 4.45 1.74 0.03 1.63 30.58 hydrophila M9+ lauric acid - 1.32 0.90 68.28 59.32 Aeromonas M9+glucose 0.37 4.82 1.33 0.49 36.45 45.20 hydrophila M9 + starch 0.33 7.25 2.18 0.60 27.70 33.17 (RE) M9 + lauric acid - - 1.55 0.92 59.17 38.62 According to the analysis of the GC instrument, the derawoms (RE) strain is incubated with a carbohydrate such as glucose or starch as a single carbon source, and its peak ratio at 5 · Η minutes The strains were surprisingly many, showing that the amount of poly-3-hydroxybutyrate (PHB) synthesized by the gene-transferred (RE) strain was higher than that of the strain (Figures 5-2, 5-3' and Figures 6-2, 6). -3)). 200918669 The concentration of poly(3) hydroxybutyrate (ρΗβ) in the strains of Glucosamine as a single-carbon source and the coronation chamber (4), Μα (RE) strain were 5. Secret and 36.45%, respectively (see Table- and Figure 8). As shown in Table 1 and Figure 9, when the powder is cultured as a single carbon source, the content of poly 3_butyric acid vinegar (PHB) of the above two is 1.63% and 27.7%, respectively. When the laurel bribe is single-hybrid culture, the content of τ_(ρΗΒ) is 68.28% and 59.17%, respectively (as shown in Table 1 and Figure 1). It can be seen that the strain of Aeromonas aeruginosa with pRHB-2 plastids, such as M, can be used to transport carbon via carbohydrate metabolism under the culture of glucose or house powder as a single-carbon source. Source glycolytic, and will use the additional 0peron gene encoding to produce p-ketothiolase (beta-ketothiolase), acetamidine coenzyme a reductase (Acet〇acetyl c〇A and PHB polymerase (PHB P〇lymeraseg) An enzyme that synthesizes poly-3-butyrate (PHB) in bacteria and produces more poly-3-hydroxybutyrate (PHB) than the original strain (10) (see Table 1 and Figure). 8 and 9). The Aeromonas hydrophila strain and the ^ieromonas hydrophila (RE) strain were used to culture the PHB yield of the Jeramoms (RE) gene with pRHB-2 plastid when culturing with lauric acid as a single carbon source. Compared with the original strain Jeromomy, the PHB yield is slightly less (as shown in Table 1 and Figure 10). The genetically modified strain Jmwwwai (RE) is produced by culturing the polyhydroxyalkanoic acid (PHAs) produced by using starch as a single carbon source. The structure was analyzed by a resonance spectrometer (NMR), and the results are shown in Fig. 11 and Fig. 12, and the structure thereof was identified as poly-3-hydroxybutyrate (PHB). According to the examples provided by the present invention, the strain of Aeromonas aeruginosa 19 200918669 mainly utilizes a metabolic pathway of fatty acids to synthesize polyhydroxyalkanoic acid (PHAs), and the gene-transformed strain ^*0 is 0_(New) It is indeed possible to use the metabolic pathway of sugars to synthesize polyhydroxyalkanoic acid (PHAs), and the content of synthetic polyhydroxyalkanoic acid (pHAs) accounts for more than 27% of the dry weight of the cells; the cultivation of carbohydrates such as glucose or starch as a single carbon source The poly-based sulphuric acid (PHAs) produced by the time-based poly-hydroxybutyrate (PHB) with short carbon chain, showing that the mwoqing (RE) strain can take the metabolic pathway of sugars to synthesize short carbon. Chain of poly-3-hydroxybutyrate (PHB). The present invention provides a method for allowing microorganisms to utilize different carbon sources to synthesize polyhydroxyalkanoic acid (PHAs) when compared with other conventional techniques, and has the following Advantages: 1. The genetic recombination of Aeromonas aeruginosa obtained by the method provided by the present invention is still carried out, and (RE) can be cultured in different carbon sources (for example, lauric acid, starch or glucose). In the medium, and can convert the supplied carbon source into accumulated bacteria The lipid particles in the body, that is, different polyhydroxyalkanoic acids, such as PHBHHX (lauric acid) or PHB (starch or glucose), can be used as raw materials for biodegradable plastics after separation and purification. The genetically modified recombinant Aeromonas aeruginosa obtained by the method of the present invention can be used as a relatively inexpensive carbon source of starch, and a large amount of polyhydroxyalkanoic acid (PHAs) can be synthesized and analyzed by GC and NMR. It is identified as a short-chain poly-3-hydroxybutyrate (phb), and its content accounts for 27.7% of the dry weight of the cell, which is highly advantageous for industrial applications. 3. Recombinant Aeromonas aeruginosa (10) obtained by the method of the present invention still /^rap/2z7a (RE) can synthesize different polyhydroxyalkanoic acids (PHAs) by using different kinds of substrates, when using hydrocarbons It can synthesize the PHA of the medium-long carbon chain, and when using the carbohydrate, it is the synthetic PHB of 200918669. 4. This hair is said to be a secret, and the money is transferred to the __ surface. It is a kind of bacteria that contains the ubiquitous and private enzymes, so that it can produce polysilicic acid (review) as a carbon source. A large number of biodegradable bioplastic materials are produced to solve the problem of insufficient industrial strains for producing pHA. The detailed description above is a detailed description of one of the possible embodiments of the present invention, but the embodiment is not intended to be used in the specific embodiment of the present invention. It should be included in the patent scope of this case. The above-mentioned 'this case*' is a method in the method, and the microorganisms produced can enhance the above-mentioned multiple functions compared with the conventional microorganisms, and should have fully complied with the new and progressive legal patents of the inventions. I urge you to approve the application for this invention patent, so that you can invent the invention and feel it. [Simple illustration of the diagram] Figure 1 shows the metabolic pathway of microbial synthesis of polyhydroxyalkanoic acid (PHAS);

Figure 2 is a schematic diagram showing the structure of the PHB synthetic gene operon (also known as phbCAB operon). Figure 3 is a plastid map of pRHB-2; Figure 4 is a standard calibration curve for poly-3-hydroxybutyrate (PHB); 5-1 is a poly-3-hydroxybutyrate (PHB) standard GC profile 'PHB's retention front in 5 ii minutes' internal standard (Diphenol ester) retention front at 9.94 minutes; Figure 5-2 is Jeromems The GC spectrum of the strain was cultured with glucose as a single carbon source. The retention front of PHB was 5·12 minutes, and the retention front of the internal standard (Diphenol ester) was 9% 21 200918669 minutes; Figure 5-3 shows the derowowos (RE) strain. GC map with glucose as a single carbon source, the retention front of PHB is 5-11 minutes, and the retention front of Diphenol ester is at 9.95 minutes; Figure 6-1 shows poly-3-hydroxybutyrate (Fig. 6-1) PHB of the standard PHB, the retention front of PHB is 511 minutes, and the retention front of the internal standard (Diphenol ester) is 9.94 minutes; Figure 6-2 shows the Gc map of the strain cultured with starch as a single carbon source. The retention front of PHB is at 5.11 minutes, and the retention front of the internal standard (Diphenol ester) is at 9.95 minutes; Figure 6_3 is also a strain of ramoms (RE) Starch is the % of the single carbon source culture. The retention front of the PHB in the 5.11 minutes' internal standard (Diphen〇1 ester) is at 9.95 minutes; Figure 7-1 shows the 3.9% PHB-co-HHX standard. GC spectrum, PHB has a retention front of 5 ^ minutes, HHX has a retention front of 6.04 minutes, and internal standard (Diphen〇1 has a retention front of 9.95 minutes; Figure 7-2 shows a (10) os-run strain with lauric acid as a single carbon. The Gc map of the source culture, PHB Qiliufeng is 5. 丨 2 minutes 'HHX's (four) front is _ minutes, and the internal standard (Diphenol ester) is at 9.94 minutes; Figure 7-3 is a bribe / The ^_·/β(ΚΕ) strain was cultured with lauric acid as the single carbon source. The retention front of PHB was 5.12 minutes' internal standard (Diphen〇les (four)'s retention front was at 9.94 minutes; 22 200918669 Figure 8 is Jerawcwos · PHZ content (%) of strains with less strains and dermofos· /^drcipMa (RE) with glucose as a single carbon source; Figure 9 shows Jeramoms· /^t/rapMa strain and derawowos /^i/rapMa (RE) The PHB content (%) of the strain cultured with starch as a single carbon source; The picture shows the Aeromonas hydrophila strain anti-Aeromonas hydrophih (RE) strain PHB content (%) of lauric acid cultured as a single carbon source; Figure 11 is a hydrogen spectrum of polyhydroxyalkanoic acid (PHAs) synthesized by NMR analysis (RE) strain using a temple powder as a single carbon source C; 12 is a carbon spectrum of polyhydroxyalkanoic acid (PHAs) synthesized by NMR analysis of J eramo muscle y /2_>^/rapM<3 (RE) strain with starch as a single carbon source. [Main component symbol description] Benefit #»>> 23

Claims (1)

200918669 X. Patent application scope: 1. A method for synthesizing polypyridyl acid (PHAs) by using microorganisms with different carbon sources, including: (1) Providing a microorganism capable of using starch to grow Aeromonas (2) providing a DNA construct having a gene; () transferring the DNA construct to the microorganism of the genus Aeromonas to obtain a microorganism capable of synthesizing polyhydroxyalkanoic acid (pHAs) using different carbon sources; (4) providing the microorganism according to (3) a suitable medium and a carbon source for synthesizing polyhydroxyalkanoic acid [, (PHAs) 〇 2_ as described in claim 1 of the patent scope, enabling different microbial sources of carbon sources In the method of synthesizing polypyridyl acid (PHAs), wherein the Na(10) gene is a gene related to the synthesis of poly(3)-butyric acid vinegar (ρΗβ), having the sequence of SEQK) NO: 1. 3. If the application of the above-mentioned items in the first item can make the use of microorganisms differently to synthesize polypyridyl & l (PHAs), the δ hai family secret gene is selected from the group of Such as H16 strain. 4. A method according to claim 1, wherein the microorganism is capable of utilizing different carbon sources to synthesize polyhydroxyalkanoic acid (PHAs), wherein the carbon source is a carbohydrate. 5. A method of synthesizing polypyrrolic acid (PHAs) by using a different carbon source as described in the patent application, item 4, wherein the carbohydrate is starch. 6_ As claimed in the patent | & 4, which can be used to synthesize polypyrrolic acid (PHAs) by the microorganisms, wherein the carbohydrate is glucose. 7. A method of synthesizing polycarbamic acid (PHAs) by using a microbial paste to different carbon sources as described in claim 1, wherein the carbon source is a hydrocarbon. 24 200918669 8. A method of synthesizing polyhydroxyalkanoic acid (PHAs) by using a different carbon source, as described in claim 7, wherein the hydrocarbon is a fatty acid. 9. A method of allowing a microorganism to utilize a different carbon source to synthesize a polyphenol (PHAs) thereof as described in claim 8 wherein the fatty acid is lauric acid. 10. The method of claim 1, wherein the medium is a M9 basal medium, an LB medium, a 2xYT medium or a 1/2YT medium. C. 25 200918669 SEQUENCE LISTING <110>Yuanzhi University<120>>>210>><210><210><210> DNA < 213 > Ralstonia eutropha H16 < 400 > 1 CCCGGGCAAG TACCTTGCCG ACATCTATGC GCTGGCGCGC ACGCGCCTGG CGCGCGCCGG CTGTACCGAG GTCTACGGCG GCGACGCCTG CACCGTGGCC GACGCCGGTC GCTTCTACTC CTATCGGCGC GATGGCGTGA CCGGCCGCAT GGCCAGCCTG GTCTGGCTGG CGGACTGAGC CCGCCGCTGC CTCACTCGTC CTTGCCCCTG GCCGCCTGCG CGCGCTCGGC TTCAGCCTTG CGTCGGCGGC GGCCGGGCGT GCCCATGATG TAGAGCACCA CGCCACCGGC GCCATGCCAT ACATCAGGAA GGTGGCAACG CCTGCCACCA CGTTGTGCTC GGTGATCGCC ATCATCAGCG CCACGTAGAG CCAGCCAATG GCCACGATGT ACATCAAAAA TTCATCCTTC TCGCCTATGC TCTGGGGCCT CGGCAGATGC GAGCGCTGCA TACCGTCCGG TAGGTCGGGA AGCGTGCAGT GCCGAGGCGG ATTCCCGCAT TGACAGCGCG TGCGTTGCAA GGCAACAATG GACTCAAATG TCTCGGAATC GCTGACGATT CCCAGGTTTC TCCGGCAAGC ATAGCGCATG GCGTCTCCAT GCGAGAATGT CGCGCTTGCC GGATAAAAGG GGAGCCGCTA TCGGAATGGA CGCAAGCCAC GGCCGCAGCA GGTGCGGTCG AGGGCTTCCA GCCAGTTC CA GGGCAGATGT GCCGGCAGAC CCTCCCGCTT TGGGGGAGGC GCAAGCCGGG TCCATTCGGA TAGCATCTCC CCATGCAAAG TGCCGGCCAG GGCAATGCCC GGAGCCGGTT CGAATAGTGA CGGCAGAGAG ACAATCAAAT CATGGCGACC GGCAAAGGCG CGGCAGCTTC CACGCAGGAA GGCAAGTCCC AACCATTCAA GGTCACGCCG GGGCCATTCG ATCCAGCCAC ATGGCTGGAA TGGTCCCGCC AGTGGCAGGG CACTGAAGGC AACGGCCACG CGGCCGCGTC CGGCATTCCG GGCCTGGATG CGCTGGCAGG CGTCAAGATC GCGCCGGCGC AGCTGGGTGA TATCCAGCAG CGCTACATGA AGGACTTCTC AGCGCTGTGG CAGGCCATGG CCGAGGGCAA GGCCGAGGCC ACCGGTCCGC TGCACGACCG GCGCTTCGCC GGCGACGCAT GGCGCACCAA CCTCCCATAT CGCTTCGCTG CCGCGTTCTA CCTGCTCAAT GCGCGCGCCT TGACCGAGCT GGCCGATGCC GTCGAGGCCG ATGCCAAGAC CCGCCAGCGC ATCCGCTTCG CGATCTCGCA ATGGGTCGAT GCGATGTCGC CCGCCAACTT CCTTGCCACC AATCCCGAGG CGCAGCGCCT GCTGATCGAG TCGGGCGGCG AATCGCTGCG TGCCGGCGTG CGCAACATGA TGGAAGACCT GACACGCGGC AAGATCTCGC AGACCGACGA GAGCGCGTTT GAGGTCGGCC GCAATGTCGC GGTGACCGAA GGCGCCGTGG TCTTCGAGAA CGAGTACTTC CAGCTGTTGC AGTACAAGCC GCTGACCGAC AAGGTGCACG CGCGCCCGCT GCTGATGGTG CCGCCGTGCA TCAACAAGTA CTACATCCTG GACCTGC AGC CGGAGAGCTC GCTGGTGCGC CATGTGGTGG AGCAGGGACA TACGGTGTTT CTGGTGTCGT GGCGCAATCC GGACGCCAGC ATGGCCGGCA GCACCTGGGA CGACTACATC GAGCACGCGG CCATCCGCGC 60 120 180 240 360 360 420 480 900 960 10 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 200918669 CATCGAAGTC GCGCGCGACA TCAGCGGCCA GGACAAGATC AACGTGCTCG GCTTCTGCGT GGGCGGCACC ATTGTCTCGA CCGCGCTGGC GGTGCTGGCC GCGCGCGGCG AGCACCCGGC CGCCAGCGTC ACGCTGCTGA CCACGCTGCT GGACTTTGCC GACACGGGCA TCCTCGACGT CTTTGTCGAC GAGGGCCATG TGCAGTTGCG CGAGGCCACG CTGGGCGGCG GCGCCGGCGC GCCGTGCGCG CTGCTGCGCG GCCTTGAGCT GGCCAATACC TTCTCGTTCT TGCGCCCGAA CGACCTGGTG TGGAACTACG TGGTCGACAA CTACCTGAAG GGCAACACGC CGGTGCCGTT CGACCTGCTG TTCTGGAACG GCGACGCCAC CAACCTGCCG GGGCCGTGGT ACTGCTGGTA CCTGCGCCAC ACCTACCTGC AGAACGAGCT CAAGGTACCG GGCAAGCTGA CCGTGTGCGG CGTGCCGGTG GACCTGGCCA GCATCGACGT GCCGACCTAT ATCTACGGCT CGCGCGAAGA CCATATCGTG CCGTGGACCG CGGCCTATGC CTCGACCGCG CTGCTGGCGA ACAAGCTGCG CTTCGTGCTG GGTGCGTCGG GCCATATCGC CGGTGTGATC AACCCGCCGG CCAAGAACAA GCGCAGCCAC TGGACTAACG ATGCGCTGCC GGAGTCGCCG CAGCAATGGC TGGCCGGCGC CATCGAGCAT CACGGCAGCT GGTGGCCGGA CTGGACCGCA TGGCTGGCCG GGCAGGCCGG CGCGAAACGC GCCGCGCCCG CCAACTATGG CAATGCGCGC TATCGCGCAA TCGAACCCGC GCCTGGGCGA TACGTCAAAG CCAAGGCATG ACGCTTGCAT GAGTGCCGGC GTGCGTCATG CACGGCGCCG GCAGGCCTGC AGGTTCCCTC CCGTTTCCAT TGAAAGGACT ACACAATGAC TGACGTTGTC ATCGTATCCG CCGCCCGCAC CGCGGTCGGC AAGTTTGGCG GCTCGCTGGC CAAGATCCCG GCACCGGAAC TGGGTGCCGT GGTCATCAAG GCCGCGCTGG AGCGCGCCGG CGTCAAGCCG GAGCAGGTGA GCGAAGTCAT CATGGGCCAG GTGCTGACCG CCGGTTCGGG CCAGAACCCC GCACGCCAGG CCGCGATCAA GGCCGGCCTG CCGGCGATGG TGCCGGCCAT GACCATCAAC AAGGTGTGCG GCTCGGGCCT GAAGGCCGTG ATGCTGGCCG CCAACGCGAT CATGGCGGGC GACGCCGAGA TCGTGGTGGC CGGCGGCCAG GAAAACATGA GCGCCGCCCC GCACGTGCTG CCGGGCTCGC GCGATGGTTT CCGCATGGGC GATGCCAAGC TGGTCGACAC CATGATCGTC GACGGCCTGT GGGACGTGTA CAACCAGTAC CACATGGGCA TCACCGCCGA GAACGTGGCC AAGG7VATACG GCATCACACG CGAGGCGCAG GATGAGTTCG CCGTCGGCTC GCAGAACAAG GCCG7VAGCCG CGCAGAAGGC CGGCAAGTTT GACGAAGAGA TCGTCCCGGT GCTGATCCCG CAGCGCAAGG GCGACCCGGT GGCCTTCAAG ACCGACGAGT TCGTGCGCCA GGGCGCCACG CTGGACAGCA TGTCCGGCCT CAAGCCCGCC TTCGACAAGG CCGGCACGGT GACCGCGGCC AACGCCTCGG GCCTGAACGA CGGCGCCGCC GCGGTGGTGG TGATGTCGGC GGCCAAGGCC AAGGAACTGG GCCTGACCCC GCTGGCCACG ATCAAGAGCT ATGCCAACGC CGGTGTC GAT CCCAAGGTGA TGGGCATGGG CCCGGTGCCG GCCTCCAAGC GCGCCCTGTC GCGCGCCGAG TGGACCCCGC AAGACCTGGA CCTGATGGAG ATCAACGAGG CCTTTGCCGC GCAGGCGCTG GCGGTGCACC AGCAGATGGG CTGGGACACC TCCAAGGTCA ATGTGAACGG CGGCGCCATC GCCATCGGCC ACCCGATCGG CGCGTCGGGC TGCCGTATCC TGGTGACGCT GCTGCACGAG ATGAAGCGCC GTGACGCGAA GAAGGGCCTG GCCTCGCTGT GCATCGGCGG CGGCATGGGC GTGGCGCTGG CAGTCGAGCG CAAATAAGGA AGGGGTTTTC CGGGGCCGCG CGCGGTTGGC GCGGACCCGG CGACGATAAC GAAGCCAATC AAGGAGTGGA CATGACTCAG CGCATTGCGT ATGTGACCGG CGGCATGGGT GGTATCGGAA CCGCCATTTG CCAGCGGCTG GCCAAGGATG GCTTTCGTGT GGTGGCCGGT TGCGGCCCCA ACTCGCCGCG CCGCGAAAAG TGGCTGGAGC AGCAGAAGGC CCTGGGCTTC GATTTCATTG CCTCGGAAGG CAATGTGGCT GACTGGGACT CGACCAAGAC CGCATTCGAC AAGGTCAAGT CCGAGGTCGG CGAGGTTGAT GTGCTGATCA ACAACGCCGG TATCACCCGC GACGTGGTGT TCCGCAAGAT GACCCGCGCC GACTGGGATG CGGTGATCGA CACCAACCTG ACCTCGCTGT TCAACGTCAC CAAGCAGGTG 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 354 0 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 200918669 ATCGACGGCA TGGCCGACCG TGGCTGGGGC CGCATCGTCA ACATCTCGTC GGTGAACGGG CAGAAGGGCC AGTTCGGCCA GACCAACTAC TCCACCGCCA AGGCCGGCCT GCATGGCTTC ACCATGGCAC TGGCGCAGGA AGTGGCGACC AAGGGCGTGA CCGTCAACAC GGTCTCTCCG GGCTATATCG CCACCGACAT GGTCAAGGCG ATCCGCCAGG ACGTGCTCGA CAAGATCGTC GCGACGATCC CGGTCAAGCG CCTGGGCCTG CCGGAAGAGA TCGCCTCGAT CTGCGCCTGG TTGTCGTCGG AGGAGTCCGG TTTCTCGACC GGCGCCGACT TCTCGCTCAA CGGCGGCCTG CATATGGGCT GACCTGCCGG CCTGGTTCAA CCAGTCGGCA GCCGGCGCTG GCGCCCGCGT ATTGCGGTGC AGCCAGCGCG GCGCACAAGG CGGCGGGCGT TTCGTTTCGC CGCCCGTTTC GCGGGCCGTC AAGGCCCGCG AATCGTTTCT GCCCGCGCGG CATTCCTCGC TTTTTGCGCC AATTCACCGG GTTTTCCTTA AGCCCCGTCG CTTTTCTTAG TGCCTTGTTG GGCATAGAAT CAGGGCAGCG GCGCAGCCAG CACCATGTTC GTGCAGCGCG GCCCTCGCGG GGGCGAGGCT GCAGGCGCAC AAGGCGGCGG GCGTTTCGTT TCGCCGCCCG TTTCGCGGGC CGTCAAGGCC CGCGAATCGT TTCTGCCCGC GCGGCATTCC TCGCTTTTTG CGCCAATTCA CCGGGTTTTC CTTAAGCCCC GTCGCTTTTC TTAGTGCCTT GTTGGGCATA GAATCAGGGC AGCGGCGC AG CCAGCACCAT GTTCGTGCAG CGCGGCCCTC GCGGGGGCGA ATTC 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5204