31 results about "Aminolevulinic Acid Synthetase" patented technology

The invention discloses a method to prepare engineering bacteria of 5-amino levulinic acid, which comprises the following steps: 1) extracting total genom DNA from bacterial liquid of red ball bacterium; 2)augmenting 5- glycyl ethylformic acid synthase gene with polyose chain reaction; 3) connecting with colonic carrier pMD-18T simple; proceeding DNA testing sequence; 4) connecting the goal gene segment with expressing carrier pET28a; constructing recon pET28a-R. S. hemA; 5) transforming the recon pET28a-R. S. hemA to host bacteria; getting the product. This invention possesses higher enzyme activity, which output of external cell ALA can reach 6. 6g / L.

The present invention relates to a method for cultivating engineering bacterium capable of high yielding 5-amino acetylpropionic acid. The engineering bacterium is colibacillus containing 5-amino acetylpropionic acid synthetase gene of Rhodoblastus acidophilus PSB-1 strain in photosynthetic bacteria. The engineering bacterium is firstly cultured in the culture medium containing peptone, yeast extract and inorganic salt as well as glycine, succinic acid and levulic acid for 2-4 hr and then further cultured in the culture medium with inducing agent added for additional 5-14 hr to reach the maximum output of 5-amino acetylpropionic acid. The present invention has not only high 5-amino acetylpropionic acid output, but also simple technological process, environment friendship and capacity of meeting industrial production requirement.

The invention provides a 5-aminolevulinic acid (ALA) synthetase. The amino acid sequence of the ALA synthetase has mutation at the amino acid residues of the 40th site, the 365th site, the 75th site,the 29th site and the 44th site corresponding to the amino acid sequence as shown in SEQ ID NO:1. The ALA synthetase provided by the invention obviously improves the activity and also partially relieves the feedback inhibition of hemachrome. Furthermore, the invention also provides methods for preparing and modifying the ALA synthetase, an expression vector containing the ALA synthetase, host cells containing the ALA synthetase, and application of the ALA synthetase in ALA production.

The invention discloses a method to prepare engineering bacteria of 5-glycyl propionic acid and methods for using them, which comprises the following steps: incorporating radial agrobacteriocin; setting the preserved number at CGMCC No. 1938; activating engineering bacteria with LB medium flat; getting monoclonal of the engineering bacteria; seeding to shake; culturing; getting first-grade seed; seeding the first-grade in shake; culturing; getting second-grade seed; seeding the second-grade seed in fermenter; culturing; cooling and evoking with isopropyl-beta-D-sulfo-galactose-glycoside; culturing continually; proceeding supplementing material culture. This engineering bacteria possesses higher ALA synthase expression, simple craft, low cost and good industrial prospect.

The present invention relates to one kind of engineering bacterium capable of producing 5-amino acetylpropionic acid. The engineering bacterium is colibacillus containing 5-amino acetylpropionic acid synthetase gene of Rhodoblastus acidophilus PSB-1 strain in photosynthetic bacteria. The engineering bacterium is first cultured in the culture medium containing peptone, yeast extract and inorganic salt as well as glycine, succinic acid and levulic acid for 2-4 hr and then further cultured in the culture medium with inducing agent added for additional 5-14 hr to reach the maximum output of 5-amino acetylpropionic acid. The present invention has not only high 5-amino acetylpropionic acid output, but also simple technological process, environment friendship and capacity of meeting industrial production requirement.

An engineered bacterium able to generate 5-aminolevulic acid is disclosed, which contains the 5-aminolevulic acid, synthetase gene of radial Agrobacterium. Its configuring process includes such steps as extracting genom DNA from Agrobacterium liquid, PCR amplification of 5-aminoevulic acid synthetase gene, linking it with carrier pGEM-T, sequencing linking sequenced target gene fragment with expression carrier pET28 a to configue recombination pET28a-A.R-hemA, and transfering it in host.

Microorganisms producing 5-aminolevulinic acid which show a 5-aminolevulinic acid synthetase activity of 2 to 7 (nmol / min / mg protein) under aerobic culture conditions with a dissolved oxygen concentration of 0.70 to 6.60 ppm; a process for producing 5-aminolevulinic acid characterized by culturing one or more microorganisms and harvesting 5-aminolevulinic acid from the obtained culture; a process for producing 5-aminolevulinic acid characterized by culturing 5-aminolevulinic acid-producing microorganisms in a medium containing 5 to 500 .mu.M of iron components; and a method for culturing 5-aminolevulinic acid-producing microorganisms.

A method of continuously producing 5-aminolevulinic acid employs the photosynthetic bacteria-derived photosynthetic membrane vesicle, succinyl-CoA synthetase, and 5-aminolevulinic acid synthase. The enzymatic synthesis of 5-aminolevulinic acid directly from succinic acid and glycine may be simple, but the synthesis is not inexpensive due to the supply of ATP and CoA, which are relatively expensive reactants. The photosynthetic membrane vesicle is used together with succinyl-CoA synthetase and 5-aminolevulinic acid synthase, thereby enabling the re-use of adenosine diphosphate or CoA in reaction. Accordingly, relatively expensive 5-aminolevulinic acid can be efficiently produced at low manufacturing costs from succinic acid and glycine.

The invention discloses a recombinant escherichia coli strain for producing 5-aminolevulinic acid (5-ALA). The invention also discloses a way for efficiently synthesizing the 5-aminolevulinic acid, wherein the 5-aminolevulinic acid synthetase (HemL and HemA) of the escherichia coli is enhanced, and the strain has the capability of synthesizing the 5-aminolevulinic acid preliminarily; the expression of the 5-aminolevulinic acid efflux protein eamA gene is enhanced, and the 5-aminolevulinic acid efflux capability of the strain is improved; an exogenous 5-aminolevulinic acid synthetase hemA gene is introduced, so that the 5-aminolevulinic acid synthesis capability of the strain is enhanced; galR, glk and ppc genes of a glucose utilization way are modified, and the utilization efficiency of glucose is improved; and genes (hemF, poxB and aceB) of metabolic bypasses are knocked out. The recombinant Escherichia coli strain constructed by the invention has the capability of efficiently synthesizing 5-aminolevulinic acid by using glucose and glycine, so that the recombinant Escherichia coli strain has the application of industrially producing 5-aminolevulinic acid.

The invention discloses a method for increasing the yield of 5-aminolevulinic acid synthesized by corynebacterium glutamicum, and belongs to the technical field of bioengineering. According to the method, the construction of the recombinant corynebacterium glutamicum for knocking out alpha-ketoglutaric acid inhibitory protein and succinate dehydrogenase and overexpressing 5-aminolevulinic acid synthetase is successfully realized. A 5-L fermentation tank batch fermentation strategy is adopted, fermentation conditions are optimized, finally, recombinant corynebacterium glutamicum (C. glutamicum) 13032 / delta odhI delta sdhA / pXMJ19-hemA is fermented for 84 h, 25.05 g / L of 5-ALA is synthesized within 48 h under the condition that glycine with the final concentration of 4 g / L is added, the productivity is 0.52 g / L / h, and at the moment, the sugar-acid conversion rate reaches 16.79%. The high yield of the 5-aminolevulinic acid is realized, and the highest yield of one-step fermentation starting from glucose as a carbon source to the current is obtained.

An engineered bacterium able to generate 5-aminolevulic acid is disclosed, which contains the 5-aminolevulic acid, synthetase gene of radial Agrobacterium. Its configuring process includes such steps as extracting genom DNA from Agrobacterium liquid, PCR amplification of 5-aminoevulic acid synthetase gene, linking it with carrier pGEM-T, sequencing linking sequenced target gene fragment with expression carrier pET28 a to configue recombination pET28a-A.R-hemA, and transfering it in host.

The invention provides a recombinant methanol utilization type yeast and especially provides a recombinant bacterium provided with an exogenous ALA(5-aminolevulinic acid) synthetase expression vector, a method for producing ALA by utilizing the recombinant bacterium and a method for establishing the expression vector and obtaining a recombinant engineering strain. The recombinant bacterium having the characteristics is fermented in a culture medium containing glycerin, a proper amount of glycine, succinic acid and a certain carbon source, and high-level ALA accumulation can be obtained. The novel methanol utilization type pichia pastoris can achieve green and pollution-free production of 5-aminolevulinic acid and can be widely applied to the field of agriculture and feeds.

The invention discloses a rhodopseudomonas palustris 5-aminolevulinic acid synthetase mutant and application thereof. The amino acid sequence of the rhodopseudomonas palustris 5-aminolevulinic acid synthetase mutant is as shown in SEQ ID NO.1. The invention further discloses a preparation method of the rhodopseudomonas palustris 5-aminolevulinic acid synthetase mutant. Compared with non-mutated 5-aminolevulinic acid synthetase, the rhodopseudomonas palustris 5-aminolevulinic acid synthetase mutant disclosed by the invention has the advantages that the enzyme activity is improved, and the capability of demodulating feedback inhibition of high-concentration heme is also improved, so that the capability of producing 5-ALA by a host cell disclosed by the invention is remarkably improved and is improved by about 40%.

Nucleic acid sequences mediating chemical compound induced 5-aminolevulinate synthase gene (ALAS1) expression are disclosed. Said sequences comprise at least a DR-4 binding site. Furthermore, in vitro methods for testing chemical compounds for modulation of heme and / or P 450 cytochromes synthesis are described.

The invention provides a rhodobacter capsulatus 5-aminolevulinic acid synthetase mutant and application thereof. The amino acid sequence of the rhodobacter capsulatus 5-aminolevulinic acid synthetase mutant is as shown in SEQ ID NO. 1. Compared with wild type rhodobacter capsulatus 5-aminolevulinic acid synthetase, the rhodobacter capsulatus 5-aminolevulinic acid synthetase mutant has the advantages that the yield of 5-aminolevulinic acid in host cells is increased by about 22%; in the presence of 20 [mu] M heme, the mutant 5-aminolevulinic acid synthetase C201A can maintain relatively high relative enzyme activity.

The invention discloses a recombinant Escherichia coli strain for producing 5-aminolevulinic acid (5-ALA); the invention also discloses a high-efficiency approach for synthesizing 5-aminolevulinic acid: strengthening the 5-aminolevulinic acid of Escherichia coli itself Aminolevulinic acid synthase (HemL and HemA), the strain initially has the ability to synthesize 5-aminolevulinic acid; strengthen the expression of the 5-aminolevulinic acid efflux protein eamA gene, and increase the 5-aminolevulinic acid efflux of the strain Removal ability; introduce exogenous 5-aminolevulinic acid synthase hemA gene to enhance the 5-aminolevulinic acid synthesis ability of the strain; transform galR, glk, ppc genes in the glucose utilization pathway to improve glucose utilization efficiency; knock out metabolism Bypassed genes (hemF, poxB, and aceB). The recombinant Escherichia coli strain constructed by the invention has the ability to efficiently utilize glucose and glycine to synthesize 5-aminolevulinic acid, making it useful for industrial production of 5-aminolevulinic acid.

The invention discloses a method for improving insect resistance of a plant. The method is characterized in that a 5-aminolevulinic acid synthetase gene is introduced and expressed into the plant to obtain the insect resistance of a transgenic plant. The invention also provides a method for preparing the transgenic plant with improved insect resistance, wherein the method comprises the following steps of transforming the plant by using the plant expression vector containing the 5-aminolevulinic acid synthetase gene, and carrying out screening to obtain the transgenic plant with improved insectresistance. Experimental results show that each strain of the 5-ALAS transgenic rice shows enhanced resistance response to the rice planthopper, so that the method has relatively high popularizationand application values.

The present invention provides a 5-aminolevulinic acid synthetase (ALA synthetase), the amino acid sequence of the ALA synthetase is mutated in part of the amino acid residues corresponding to the amino acid sequence shown in SEQ ID NO:1. The ALA synthetase of the present invention significantly improves the activity and the production of ALA. At the same time, the invention also provides a method for preparing and transforming the enzyme, an expression vector containing the enzyme, a host cell and the application of the enzyme in ALA production.

The present invention discloses a mutant of Rhodopseudomonas palustris 5-aminolevulinic acid synthase and its application. The amino acid sequence of the mutant of Rhodopseudomonas palustris 5-aminolevulinic acid synthase is shown in SEQ ID NO.1 shown. The Rhodopseudomonas palustris 5-aminolevulinic acid synthetase mutant of the present invention not only improves the enzyme activity compared with the unmutated 5-aminolevulinic acid synthase, but also improves the feedback of a higher concentration of heme. The ability to inhibit, which makes the ability of the host cell of the present invention to produce 5‑ALA significantly improved, about 40%.

The invention discloses a method for improving the low-temperature stress resistance of rice and a plant expression vector used in the method. The method is characterized in that a 5-aminolevulinic acid synthetase gene is introduced and expressed in a plant, so that the low-temperature stress resistance of the transgenic plant is improved. The invention also provides a method for preparing transgenic rice with the improved low-temperature stress resistance, which comprises the following steps of transforming the plant by using the plant expression vector containing the 5-aminolevulinic acid synthetase gene, and performing screening to obtain the transgenic plant with the improved low-temperature stress resistance. Experimental results show that the low-temperature stress resistance of eachstrain of the 5-ALAS transgenic rice is greatly enhanced, so that the method has the popularization and application value in the cultivation of low-temperature stress-resistant varieties such as cold-resistant varieties.

The invention discloses a method for improving insect resistance of plants, which is characterized in that the insect resistance of transgenic plants is obtained by introducing and expressing a 5-aminolevulinic acid synthase gene in plants. The present invention also provides a method for preparing transgenic plants with improved insect resistance, which comprises transforming plants with a plant expression vector containing 5-aminolevulinic acid synthase gene, and screening to obtain transgenic plants with improved insect resistance. The experimental results of the present invention show that each strain of the transgenic 5-ALAS rice shows enhanced resistance to planthoppers, and therefore has a strong value for popularization and application.

The present invention provides a rhodobacter capsulated 5-aminolevulinic acid synthase mutant and its application. The amino acid sequence of the capsulated rhodobacter 5-aminolevulinic acid synthase mutant is shown in SEQ ID NO.1. Compared with the wild-type Rhodobacter capsula 5-aminolevulinic acid synthase mutant of the present invention, the production of 5-aminolevulinic acid in host cells is increased by about 22%; in the presence of 20 μM heme, the mutant 5‑aminolevulinic acid synthase C201A can maintain a relatively high relative enzyme activity.

The invention provides a 5-aminolevulinic acid synthetase (ALA synthetase). Amino acid residues of the ALA synthetase at the 11 position of the amino acid sequence shown in SEQ ID NO:1 are Ile, Ser, Met, Cys and Val. The invention also provides a method for preparing the ALA synthetase, an expression vector and host cell which contain the ALA synthetase, the application of the ALA synthetase in ALA production and a method for modifying the ALA synthetase to improve the activity of the ALA synthetase.

The invention provides a 5-aminolevulinic acid producing strain, and a construction method and application thereof. According to the invention, a ribosome binding site (RBS) sequence of 5-aminolevulinic acid synthetase and phosphoenolpyruvate carboxylase in a starting strain is optimized, so that capacity of producing 5-aminolevulinic acid by the strain is greatly improved, and the strain is suitable for large-scale production.

Systems and methods for increasing protoporphyrin IX accumulation in a target cell population using aminolevulinate synthase variants. Aminolevulinic acid-mediated photodynamic therapy is a promising approach to treating dysplasic disorders such as cancer and atherosclerosis, but is limited by the lack of a means to deliver optimal quantities of aminolevulinic acid selectively to the target cells, and thereby ensure the best therapeutic response. The disclosed invention provides a means for enhancing the natural production of aminolevulinic acid selectively within target cells to levels predetermined to give an optimal therapeutic response, and is expected to lead to increased efficacy of treatment, possibly broadening the scope of diseases treatable by photodynamic therapy considerably. The disclosed invention is also amenable to patient specific therapy, meaning that a patient's target cells could be used to screen for the aminolevulinic acid delivery system most appropriate for the patient's needs.

The invention provides a 5-aminolevulinic acid synthetase (ALA synthetase). The C-terminal tail amino acid residue of the ALA synthetase is deleted or is non-alanine; and / or, the amino acid residue atthe 403rd position corresponding to SEQ ID NO:1 of the amino acid sequence of the ALA synthetase is deleted or is non-alanine; and / or, 1-N arbitrary amino acid residues are added to the tail positionof the C terminal of the ALA synthetase, and have the ALA synthetase activity. The invention also provides a method for preparing the enzyme, an expression vector containing the enzyme, a host cell,application of the enzyme in ALA production and a method for modifying the ALA synthetase to improve the activity of the ALA synthetase.

The invention discloses a pig 5-aminolevulinic acid synthetase 1 gene as a genetic marker of pig litter size traits and an application thereof. The nucleotide sequence of the genetic marker is shown as SEQ ID NO.1, and R at a 26-site base in the sequence is T or C. The mutation causes polymorphism of a 5'flanking region of the porcine 5-aminolevulinic acid synthase 1 gene. According to the invention, the SNP locus of the 5'flanking region of the pig ALAS1 gene is discovered for the first time; the relationship between the SNP locus and the pig litter size traits is analyzed; the molecular marker related to the pig litter size is found; and a rapid detection method is established.