Modified polypeptide having homoserine acetyltransferase activity and microorganism expressing it

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology, the modified polypeptide having homoserine-O-acetyltransferase activity having the amino acid sequence of SEQ ID NO:17 or at least 95% homologous thereto, in which the amino acid at position 111 from the start amino acid methionine, of the sequence is substituted with glutamic acid. The invention also relates to a method for producing O-acetyl homoserine, comprising culturing the microorganism belonging to the genus Escherichia, transformed with polynucleotide, encoding the said polypeptide.

EFFECT: high production yield of O-acetyl homoserine.

18 cl, 5 dwg, 3 tbl, 5 ex

 

The prior art prior to the invention

1. The technical field to which the invention relates

The present invention relates to a polypeptide which is modified so that it has homeserverconsoletab activity, codereuse it to the polynucleotide, a recombinant vector containing the polynucleotide, a microorganism which is transformed with a recombinant vector, and a method for producing O-acetylserine using a microorganism.

2. Description related field

Methionine is one of essential amino acids in the body, and is widely used as animal feed and food additives, and medical component aqueous solutions and other starting substances for medicines. Methionine acts as a precursor of choline (lecithin) and creatinine, and it is also used as starting substances for the synthesis of cysteine and taurine. Additionally, it functions as a sulfur donor.

S-adenosyl methionine derived from L-methionine serves as a methyl donor in the body and it is involved in the synthesis of various neurotransmitters in the brain. It was also revealed that the methionine and/or S-adenosyl-L-methionine (SAM) prevents the accumulation of lipids in the liver and arteries and is effective in the treatment of depression, inflamed�I, liver disease and muscle pain.

Methionine can be synthesized chemically or biologically for use in animal feed, food and pharmaceuticals.

During the chemical synthesis of L-methionine is mainly obtained by hydrolysis of 5-(β-methylmercaptide)hydantoin. However, chemically synthesized methionine has the disadvantage that it is obtained only in a mixed form of L-type and D-type.

In the biological synthesis of L-methionine in patent publication U.S. No. US2005/0054060A1 described method of synthesis of homocysteine or methionine directly using H2S or CH3SH, without the use of cysteine, by modifying cystathionine synthase for obtaining microorganisms. In this method, the modified cystathionine synthesis directly injected into the cells for the synthesis of methionine by the method of intracellular synthesis of methionine. However, there are particular problems in that, this method yields only a small amount of methionine due to the inhibitory activities of the synthesized methionine, due to the use of intracellular metabolic pathways of methionine, and H2S or CH3SH also cause cytotoxicity.

To solve these problems, the authors present invention has developed a two-stage method of conversion of the precursor L-methionine� in L-methionine by enzymatic reaction (PCT/KR2007/003650). This two-stage method can solve the problems described above, the cytotoxicity of H2S or CH3SH and inhibition of metabolic process resulting L-methionine. In addition, this method is characterized in that it is very effective for selectively receiving only L-methionine, and not mixed form of D-methionine and L-methionine.

In this two-stage method O-suktinilholin and O-acetylcoumarin can be used as a precursor of methionine. During the reaction the conversion of methionine O-acetylserotonin is preferred O-succinylamino in respect of yield ratio of precursor to methionine. Specifically you can get of 0.91 mole of methionine from 1 mol O-acetylserine, whereas 1 mol of O-succinylamino you can only get to 0.67 mol of methionine. Thus, the cost of obtaining the end product of methionine can be reduced through the use of O-acetylserine as a precursor of methionine, and high product yield O-acetylserine is a determining factor for mass production of methionine.

The use of O-acetylserine or O-succinylcholine as a precursor of methionine depends on the type of microorganisms. In detail, microorganisms belonging to the genus Escherichia, Enterobacteria, Salmonella and Bacillu, produce O-suktinilholin from homoserine and succinyl-COA via L-homoserine O-succinylsuccinate (Biochemistry, 1999 Oct. 26, 38(43): 14416-23), and microorganisms belonging to the genus Corynebacterium, Leptospira, Deinococcus, Pseudomonas and Mycobacterium, produce O-acetylserotonin from homoserine and acetyl-COA via L-homoserine O-acetyltransferase (Journal of Bacteriology, Mar. 2002, p. 1277-1286).

Thus, expression of O-acetylgalactosaminyltransferase, mediated by the introduction of metX, alien gene required for the biosynthesis of O-acetylserine using microorganisms of the genus Escherichia, which is used to produce recombinant proteins for experimental and industrial purposes. However, there are problems associated with the negative attitude of consumers to the introduction of foreign genes into microorganisms used to produce food, and the proof of security of the introduction of foreign genes.

Thus, the authors of the present invention have made efforts to obtain a strain of the genus Escherichia which produces O-acetylcoumarin, preferable in respect of output of a product, without the introduction of foreign genes. As a result, the authors present invention found that gomoserinlaktonazy activity can be converted into homeserverconsoletab activity using a modified polyp�of ptid, obtained by substitution of glutamic acid for the amino acid at position 111 O-succinylsuccinate, which is derived from E. coli, thus performing the present invention.

Summary of the invention

The aim of the present invention is the provision of a modified polypeptide in which the polypeptide having homoserine-O-activitiesfree activity, is converted to have homeserverconsoletab activity.

Another objective of the present invention is the provision of a polynucleotide encoding the above modified polypeptide.

Another objective of the present invention is the provision of a recombinant vector containing the polynucleotide sequences that are functionally associated with the above-mentioned polynucleotide.

Another objective of the present invention is the provision of a microorganism containing the above polynucleotide.

Another objective of the present invention is to provide a microorganism which is transformed into a recombinant vector, functionally associated with the above-mentioned polynucleotide.

Another objective of the present invention is the provision of a method for producing O-acetylserine with the use of a microorganism that expresses modificirovannaya, having homeserverconsoletab activity.

Brief description of the drawings

Fig. 1 is a diagram showing a recombinant vector which is functionally linked to a polynucleotide that encodes a modified polypeptide of the present invention.

Fig. 2 shows a homology comparison of the primary amino acid sequence of homoserine-O-succinylsuccinate between variants of E. coli.

Fig. 3 and 4 shows a homology comparison of the primary amino acid sequences of the mutant homoserine-O-succinylsuccinate resistant to regulation with feedback methionine, in which for comparison, the primary amino acid sequence of homoserine-O-succinylsuccinate wild-type, resistant to regulation with feedback the mutant homoserine-O-succinylsuccinate met10A and met11A described in PCT publication no WO 2008/127240, and resistant to regulation with feedback the mutant homoserine-O-succinylsuccinate described in PCT publication no WO 2005/108561, and

Fig. 5 is a diagram showing the receiving FRT-odnostadiinoi deletion cassette by PCR by the method of overlapping the splice extensions to replace the promoter pro on the acs promoter in the chromosome.

Detailed description of preferred variants of implementation�Oia

In one of the aspects to achieve the above objectives, the present invention relates to a modified polypeptide having homoserine-O-acetyltransferase activity containing the amino acid sequence of SEQ OD No:17 or at least 95% homologous to it, in which the amino acid at position 111 from the starting amino acid methionine, the sequence is substituted with glutamic acid.

In the framework of the invention, the polypeptide having homoserine-O-activitiesfree activity means the polypeptide having the activity to synthesize O-suktinilholin from homoserine and succinyl-COA presented in the pathways of biosynthesis of methionine, as shown by the following reaction scheme.

homoserine + succinyl-COA -> O-suktinilholin

The polypeptide having homoserine-O-activitiesfree activity, may be a recombinant polypeptide, which is derived from a microorganism of the genus Enterobacteria, Salmonella, Pseudomonas, Bacillus or Escherichia, preferably a recombinant polypeptide having gomoserinlaktonazy activity, which is derived from a microorganism of the genus Escherichia, and more preferably a recombinant polypeptide having homoserine-O-activitiesfree activity, which derives from E. coli.

In the present invention a polypeptide possessing�th homoserine-O-activitiesfree activity can include a polypeptide having gomoserinlaktonazy activity, which consists of the amino acid sequence SEQ ID NO:17 or at least 95% homologous to her, provided that he has activity specified in the above reaction scheme.

In the examples of the present invention compared the homology of amino acid sequences of homoserine-O-succinylsuccinate between different types of E. coli. As a result there were less than 5% variation in the polypeptides of homoserine-O-succinylsuccinate between different types of E. coli (thus, they have at least 95% homology), but did not exist significant differences in homoserine-O-activitiesfree activity (Fig. 2). These results indicate that the polypeptide having 95% or more homology to the polypeptide of SEQ ID NO:17 of the present invention, also have the identical homoserine-O-activitiesfree activity, which is understandable to experts in this field, and which the authors of the present invention was demonstrated.

In the framework of the invention, the term "modified polypeptide" means a polypeptide having homoserine-O-acetyltransferase activity, obtained by replacement of part of the amino acid sequence of the polypeptide having homoserine-O-succinates�different activity, different from wild type. Thus, the modified polypeptide of the present invention means a modified polypeptide having similar activity, as in the following reaction scheme, which has substrate specificity for acetyl-COA unlike succinyl-COA derived by replacement of a part of the amino acid sequence of the polypeptide having homoserine-O-activitiesfree activity.

homoserine + acetyl-COA -> O-acetylcoumarin

In the present invention, the above modified polypeptide may be a modified polypeptide in which the amino acid at position 111 of the polypeptide having the amino acid sequence of SEQ ID NO:17, or a polypeptide having 95% or more homology sequences, replacing glutamic acid(SEQ ID NO:18), and the amino acid at position 112 of the polypeptide optionally is replaced by a threonine (SEQ ID NO:19) or histidine (SEQ ID NO:20).

Revealed that additional substitution of threonine or histidine in the amino acid leucine at position 112 amplifies homeserverconsoletab activity (tables 2 and 3).

According to one preferred embodiment of the above, the modified polypeptide may be a polypeptide containing any of the amino acid sequences of SEQ I NO:18-20.

In the examples of the present invention receive a plasmid capable of expressing a polypeptide, where the amino acid glycine at position 111 gomoserinlaktonazy, coded metA genome of E. coli consisting of the nucleotide sequences provided by SEQ ID NO:39, replaced by glutamic acid, and a plasmid capable of expressing a polypeptide, where the amino acid at position 112 in addition to the above substitution replaced by threonine or histidine (example 2).

In addition, experimental examples of the present invention demonstrated that only O-suktinilholin was producyrovtsa CJM2 pCL_Pcj1_metA(wt) and CJM3 pCL_Pcj1_metA(wt) transformed with plasmid containing the gene for wild-type metA (SEQ ID NO:39). In contrast, only O-acetylcoumarin accumulated a strain transformed by a plasmid containing the gene encoding the modified polypeptide of the present invention (experimental example 2, tables 2 and 3).

Thus, the microorganism expressing the modified polypeptide of the present invention, it is preferred that it is capable of producing O-acetylserotonin as a precursor of methionine, with high yield without the introduction of foreign genes homeserverconsoletab activity.

In the present invention specified above� the modified polypeptide may be resistant to regulation with feedback methionine due to the replacement of part of amino acids of the polypeptide, having gomoserinlaktonazy activity. Thus, the most active gomoserinlaktonazy is regulated by the inhibition of the feedback a small amount of methionine in the medium, and, thus, the modified polypeptide of the present invention may be resistant to regulation with feedback methionine for mass production of O-acetylserine.

In the present invention can carry out the replacement of amino acids to eliminate regulation with feedback methionine by the method described in PCT publication no WO 2008/127240. In greater detail, the regulation feedback methionine can be eliminated by substitution of Proline for leucine at position 29, a substitution of glycine to the amino acid position 114, the substitution of serine for the amino acid at position 140 of the polypeptide having gomoserinlaktonazy activity, or one or more combinations of the three substitutions of amino acids. Preferably it is possible to replace two or more and most preferably three of amino acid.

According to one preferred embodiment of the modified polypeptide that is resistant to regulation with feedback methionine, may be a modified polypeptide containing any amino acid sequence selected from the amino acid pic�of egovernance SEQ ID NO:21-23.

In the examples of the present invention the amino acid at position 29, 114 and 140 recombinant polypeptide having gomoserinlaktonazy activity that encodes metA gene of E. coli, was replaced by Proline, glycine and serine, respectively, so as to eliminate the regulation with feedback methionine. In addition, designed a plasmid containing polynucleotides that encode modified polypeptides having homeserverconsoletab activity, which represent [pCL_Pcj1_metA#11(EL)], obtained by substitution of glutamic acid for the amino acid at position 111, [pCL_Pcj1_metA#11(ET)], obtained by substitution of glutamic acid and threonine for the amino acid at position 111 and 112, and [pCL_Pcj1_metA#11(EH)], obtained by substitution of glutamic acid and histidine for the amino acid at position 111 and 112 (example 3).

In addition, experimental examples of the present invention demonstrated that among the strains expressing the modified polypeptides that are resistant to regulation with feedback methionine, for strains CJM2 pCL_Pcj1_metA(#11)EH and CJM3 pCL_Pcj1_metA(#11)EH, obtained by substitution of glutamic acid and histidine for the amino acid at position 111 and 112, demonstrated high production O-acetylserine of 11.1 g/l and 24.8 g/l, respectively, and the accumulation of O-acetylserine are similar such as policemilitary foreign gene homeserverconsoletab (experimental example 2, tables 2 and 3).

In another aspect, the present invention relates to polynucleotide, codereuse modified polypeptide or a recombinant vector containing the polynucleotide sequences that are functionally associated with the polynucleotide.

In the present invention, the above polynucleotide is a nucleotide polymer consisting of nucleotide monomers covalently linked in a chain, and its examples are a chain of DNA or RNA with a predefined or more long, and it represents a polynucleotide encoding the above modified polypeptide.

In the present invention, the above polynucleotide may be a polynucleotide that contains any of the nucleotide sequences SEQ ID NO:24-29.

In the framework of the invention specified above, the term "recombinant vector" is a means for expression of the modified polypeptide by introducing DNA into a host cell to obtain a microorganism expressing a modified polypeptide of the present invention, it is possible to use a known expression vectors such as plasmid vector, Kemeny vector and the vector based on the bacteriophage. Specialists in this field can easily get a vector by any known method using the technology R�combinant DNA.

In the present invention the recombinant vector may be a vector pACYC177, pACYC184, pCL1920, pECCG117, pUC19, pBR322 or pMW118 and preferably the vector pCL1920.

The term "functionally linked" means that the regulatory sequence of the expression is bound in such a way that regulates the transcription and translation of the polynucleotide sequence that encodes a modified polypeptide, and includes the maintenance of a specific broadcast frame such that the modified polypeptide encoded polynucleotide sequence that is produced when the polynucleotide sequence is expressed under the control of regulatory sequences (including promoter).

In another aspect, the present invention relates to a microorganism containing the polynucleotide encoding the above modified polypeptide, and the microorganism which is transformed with recombinant vector and functionally linked to the polynucleotide that encodes the above modified polypeptide.

In the framework of the invention, the term "transformation" refers to the way in which the gene is introduced into a host cell for expressively in the host cell. The transformed gene, if it is in the condition expression in the host cell, can be integrated into the chromosome of the cell�of ozaina or can exist independently of the chromosome.

In addition, the gene contains DNA and RNA as a polynucleotide capable of encoding a polypeptide. Gene can introduce in any form provided that it can be inserted into a host cell, and it can be expressed in it. For example, the gene can be introduced into the host cell in the form of expressing cassette that represents the polynucleotide construct containing all the elements for expression of the gene itself. Typically, expression cassette contains the promoter, the signal termination of transcription, the binding site of the ribosome and the signal translation termination, which are functionally associated with the gene. Expressing cassette can be in the form of an expression vector that is able to self-replicate. The above gene can also be inserted into a host cell either by itself or in the form of polynucleotide constructs so that he was functionally associated with sequence, required for expression in the host cell.

The above microorganism is a prokaryotic or eukaryotic microorganism that is capable of expressing the modified polypeptide by the content of the polynucleotide encoding the modified polypeptide, or by transformation of the recombinant vector and functionally linked to the polynucleotide�m, encoding a modified polypeptide, and, for example, it may be a microorganism belonging to the genus Escherichia, Bacillus, Aerobacter, Serratia, Providencia, Erwinia, Schizosaccharomyces, Enterobacteria, Zygosaccharomyces, Leptospira, Deinococcus, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Salmonella, Streptomyces, Pseudomonas, Brevibacterium or Corynebacterium.

In the present invention, the microorganism expresses a polypeptide having homoserine-O-activitiesfree activity. For example, it may be a microorganism belonging to the genus Bacillus, Escherichia, or Salmonella Enterobacteria, preferably a microorganism belonging to the genus Escherichia and more preferably E. coli.

In the examples of the present invention have the strains CJM2 pCL_Pcj1_metAEL, CJM2 pCL_Pcj1_metAET and CJM2 pCL_Pcj1_metAEH E. coli transformed with a recombinant vector containing the polynucleotide encoding the modified polypeptide of the present invention (example 2 and experimental example 2), and strains CJM2 pCL_Pcj1_metA(#11)EL, CJM2 pCL_Pcj1_metA(#11)ET, and CJM2 pCL_Pcj1_metA(#11)EH E. coli transformed with a recombinant vector containing the polynucleotide encoding the modified polypeptide, resistant to regulation with feedback methionine and having homoserine-O-acetyltransferase activity of the present invention (example 3 and experimental example 2). Among the above strains strains CJM2 pCL_Pcj1_metA(#11)EL, CJM2 pCL_Pcj1_metA(#11)ET � CJM2 pCL_Pcj1_metA(#11)EH meant as CA05-0546, CA05-0547 and CA05-0548, respectively, and deposited with the Korean center of cultures of microorganisms as of 14 December 2010, and they are assigned access numbers KCCM11145P, KCCM11146P and KCCM11147P respectively.

The present invention relates to a modified polypeptide having homoserine-O-acetyltransferase activity, which replaced part of the amino acid sequence of the polypeptide having homoserine-O-activitiesfree activity. Thus, it is preferable that when the modified polypeptide of the present invention is expressed in a microorganism expressing a polypeptide having only homoserine-O-activitiesfree activity, a polypeptide having homoserine-O-acetyltransferase activity, can be expressed without introducing a foreign gene, such as metX encoding homoserine-O-acetyltransferase.

In the present invention, the above microorganism may be a microorganism which is additionally modified so that it had increased acetyl-COA synthetase activity, or additionally modified so that it had pantothenticus activity resistant to the regulation with feedback from the accumulation of COA, to produce large quantities of O-acetylserine.

In this image�hetenyi acetyl-COA synthetase and pantothenticus, that occur from a variety of microorganisms, and genes encoding proteins with such activity, generally referred to as acs and coaA, respectively.

In the present invention the increase of acetyl-COA synthetase activity can be obtained by increasing the gene expression by modifying the nucleotide sequences of the promoter region and region 5'-UTR of the gene acs, encodes an acetyl-COA-synthetase, and you can increase the activity of a protein by introduction of mutations in the ORF region of the corresponding gene, and you can increase the level of expression of a protein by introduction of additional copies of the corresponding gene in the chromosome or by introduction of the corresponding gene with its own promoter or reinforced by another promoter in the strain.

More specifically, the acetyl-COA sintetizou activity can be increased by replacing the promoter with increased activity, the introduction of mutations in the promoter for increasing the activity or increasing the number of copies of the gene, and thus, the present invention relates to a method of improving the product O-acetylserine and obtained by the method of E. coli. To replace the promoter with increased activity can be used pTac, pTrc, pPro, pR and pL, for which it is known that they have increased activity.

According to one preferred embodiment of the present invention relates to a Pro�userwise O-acetylcoumarin strain in which acs gene involved in the biosynthesis of acetyl-COA is expressed by replacing the constitutional promoter expression, promoter pro, on its promoter. Promoter pro may constitute part or all of SEQ ID NO:30.

The present invention further relates to a microorganism, which is administered in a modified pantothenticus resistant to the regulation of the inhibition of the feedback the accumulation of COA in the pathways of biosynthesis of COA. More specifically, the amino acid arginine at position 106 in the amino acid sequence pantothenticus replaced with alanine (SEQ ID NO:40), in such a way that it becomes resistant to the regulation of the inhibition of the feedback accumulation of COA, leading to the improvement of products O-acetylserine.

In the present invention, the above microorganism may be a microorganism, which increased the number of copies of one or more genes selected from the group consisting of a gene encoding fosfoenolpiruvatcarboksilaza (ppc), the gene encoding aspartataminotransferaza (aspC), and the gene encoding aspartylphenylalanine (asd), or a gene promoter replacing the promoter with increased activity or introduce a mutation to possess high activity.

In the present invention a series of enzymes have a asset�awn for the synthesis of O-acetylserine from fosfoenolpiruvata, as shown in the following schemes of reactions. Thus, it is possible to induce the accumulation of O-acetylserine in the cell by increasing the expression of genes with such activities.

Fosfoenolpiruvatcarboksilaza (ppc)

Fosfoenolpiruvat + H2O + CO2<-> oxaloacetate + phosphate

Aspartate aminotransferase (aspC)

Oxaloacetate + glutamine <-> aspartate + α-Ketoglutarate

Aspartoacylase (thrA)

Aspartate + ATP <-> aspartyl-4-phosphate + ADP

(asd)

Aspartyl-4-phosphate + NADPH <-> aspartoacylase + phosphate + NADP+

Homoerythromycin (thrA)

Aspartoacylase + NADPH <-> homoserine

In the schemes of reactions thrA gene, encoding the bifunctional enzyme aspartoacylase/gomoserinlaktonazy, pre-amplify by weakening the regulation of the inhibition of the feedback in the strain CJM2 in experimental example 2, and the remaining three of the enzyme can be enhanced by increasing the number of copies of the gene, replacement of the promoter of the above gene to the promoter with increased activity or the introduction of mutations in the promoter to increase the activity.

In the framework of the invention the term "increased number of copies" means the additional introduction of the desired gene into the chromosome or by introduction of a plasmid containing the encoding�schy the corresponding enzyme gene.

In the examples of the present invention the strain CJM2-AP received by deletions of the promoter acs metA and strain CJM2 with the deletion of the metB and replacing its promoter pro, and then transformed to have resistance to regulation with feedback coaA thus, to obtain the strain CJM2-AP/CO, which has an increased pool of acetyl-COA, followed by obtaining strain CJM3 containing two copies of the three genes ppc, aspC and asd. In the future, designed strains CJM3 entered pCL_Pcj1_metA#11(EL), pCL_Pcj1_metA#11(EH) and pCL_Pcj1_metA#11(ET) in the form CA05-0578, CA05-0579 and CA05-0580 respectively, and deposited at the Korean center of cultures of microorganisms as of December 12, 2011, and assigned a number of access KCCM11228P, KCCM11229P and KCCM11230P respectively (experimental example 2).

In another aspect, the present invention relates to a method for producing O-acetylserine, comprising the steps of culturing a microorganism containing the polynucleotide encoding the modified polypeptide or a microorganism, which is transformed by the recombinant vector and functionally linked to a polynucleotide that encodes a modified polypeptide, and obtaining O-acetylserine, which is produced during the above cultivation of the microorganism.

In the present invention the receiving O-acetylserine with the use of a microorganism expressing the modification�qualified polypeptide, can be performed in a suitable environment and conditions, known in this field. Specialists in this field is well understood that the method of cultivation can be easily adjusted in accordance with the selected strain.

Examples of a method of cultivation include, but are not limited to, periodic, continuous and fed by culture. Used in the cultivation environment must meet the specific culture conditions of the strain.

Used in the present invention, the medium can contain any carbon source is sucrose, glucose, glycerol and acetic acid, or combination thereof, and an example of a source of nitrogen that must be used are organic nitrogen sources such as peptone, yeast extract, meat extract, malt extract, liquid corn steep liquor and soybean meal, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, or combinations thereof.

As a source of phosphate Wednesday may contain a primary acidic potassium phosphate, secondary acid potassium phosphate and containing the corresponding sodium salt. The medium may also contain a metal salt such as magnesium sulfate or iron sulfate. In addition, you can also add amino acids, vitamins, and relevant previous�tennice. Environment or the precursors can be added to the culture batch-type or continuous type. Under cultivation it is possible to adjust the pH of the culture by the addition of appropriate compounds, such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid, and under cultivation it is possible to suppress the formation of foam using protivovospalitel, such as an ester of polyglycol and fatty acids.

To maintain aerobic culture conditions can introduce oxygen or an oxygen-containing gas into the culture. To maintain anaerobic conditions and microaerobic conditions may not enter a gas or can introduce nitrogen, hydrogen or carbon dioxide. The culture temperature can be between 27°C to 37°C, and preferably from 30°C to 35°C. the cultivation Period can be prolonged under the condition that produced the desired substance, and preferably is from 10 to 100 hours.

Further in the present description the present invention is described in more detail with respect to examples and experimental examples. However, these examples are given solely for illustrative purposes and do not imply that the invention is limited to these examples.

Example 1: Construction of plasmids containing homoserine-O-succinylsuccinate and homoserine-O-acetyltransferase

For gene amplification etA, encoding homoserine-O-succinylsuccinate, PCR was performed using the chromosome of the strain E. coli W3110 (access number ATCC9637), purchased from the American type culture collection, as template and primers SEQ ID NO:1 and SEQ ID NO:2.

Used in PCR primers were obtained on the basis of the sequence of the chromosome of E. coli NC_000913 registered in the gene Bank of the National Institute of health (NIH), and the primers SEQ ID NO:1 and SEQ ID NO:2 contained the restriction sites EcoRV and HindIII, respectively.

SEQ ID NO:1

5' AATTGATATCATGCCGATTCGTGTGCCGG 3'

SEQ ID NO:2

5' AATTAAGCTTTTAATCCAGCGTTGGATTCATGTG 3'

For amplification of metX gene encoding homoserine-O-acetyltransferase (SEQ ID NO:44) was performed by PCR using as matrix of Deinococcus radiodurans chromosome and primers SEQ ID NO:3 and SEQ ID NO:4. Primers SEQ ID NO:3 and SEQ ID NO:4 contained the restriction sites EcoRV and HindIII, respectively.

SEQ ID NO:3

5' AATTGATATCATGACCGCCGTGCTCGC 3'

SEQ ID NO:4

5' AATTAAGCTTTCAACTCCTGAGAAACGCCCC 3'

PCR was performed under the following conditions: denaturation at 94°C for 3 minutes, 25 cycles consisting of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds and polymerization at 72°C for 5 min, and polymerization at 72°C for 7 minutes.

The resulting PCR products are cloned into the plasmid pCL1920 containing the promoter cj1 (KR 2006-0068505), after treatment with restriction enzymes EcoRV and HindIII, respectively. DH5α E. coli transformation�Ali cloned plasmids and selected transformed E. coli DH5α in LB medium for Petri dishes, containing 50 µg/ml spectinomycin, thus, to obtain the plasmid. The resulting plasmid was designated as a pCL_Pcj1_metA and pCL_Pcj1_metXdr respectively.

Example 2: Construction of a modified polypeptide having homoserine-O-acetyltransferase activity

The amino acid glycine (Gly) at position 111 O-succinylsuccinate replaced by glutamic acid (Glu) using the plasmid pCL_Pcj1_metA obtained in example 1 as template and the kit for site-directed mutagenesis (Stratagene, USA) (G111E). Sequences of the used primers represent are as stated below:

SEQ ID NO:5

5' ttgtaactggtgcgccgctggaactggtggggtttaatgatgtc 3'

SEQ ID NO:6

5' gacatcattaaaccccaccagttccagcggcgcaccagttacaa 3'

The constructed plasmid containing the mutant gene G111E metA, denoted as pCL_Pcj1_metA(EL).

In addition, the amino acid glycine (Gly) at position 111 O-succinylsuccinate replaced by glutamic acid (Glu) and the amino acid leucine at position 112 O-succinylsuccinate replaced by threonine (L112T) or histidine (L112H). This time the sequences of the used primers represent are as stated below:

Substitution of threonine for leucine

SEQ ID NO:7

5' tgtaactggtgcgccgctggaaaccgtggggtttaatgatgtcg 3'

SEQ ID NO:8

5' cgacatcattaaaccccacggtttccagcggcgcaccagttaca 3'

Substitution of histidine for leucine

SEQ ID NO:9

5' tgtaactggtgcgccgctggaacatgtggggtttaatgatgtcg 3'

SEQ ID NO:10

5' cgacatcattaaaccccacatgttccagcggcgcaccagttaca 3'

Among the constructed plasmid containing the gene metA plasmid, in which the amino acid glycine at position 111 replaced by glutamic acid and the amino acid leucine at position 112 is replaced by threonine, denoted as pCL_Pcj1_metA(ET). As well as the plasmid containing the gene metA in which the amino acid glycine at position 111 replaced by glutamic acid and the amino acid leucine at position 112 is replaced with histidine, was designated as a pCL_Pcj1_metA(EH).

Example 3: Construction resistant to regulation with feedback of the modified polypeptide with homoserine-O-acetyltransferase activity

Gene metA with resistance to regulation with feedback methionine (metA#11) was constructed using plasmid pCL_Pcj1_metA obtained in example 1 as a matrix, in the same manner as in example 2. Specifically, the method described in PCT publication no WO 2008/127240, was replaced by serine, glutamic acid and phenylalanine at position 29, 114, and 140 O-succinylsuccinate by Proline (S29P), glycine (E114G) and serine (F140S), respectively. Sequences of primers used are those as described below.

Substitution of Proline for serine

SEQ ID NO:11

5' ATGACAACTTCTCGTGCGCCTGGTCAGGAAATTCG 3

SEQ ID NO:12

5' CGAATTTCCTGACCAGGCGCACGAGAAGTTGTCAT 3'

Substitution of glycine for glutamic acid

SEQ ID NO:13

5' CGCCGCTGGGCCTGGTGGGGTTTAATGATGTCGCT 3'

SEQ ID NO:14

5' AGCGACATCATTAAACCCCACCAGGCCCAGCGGCG 3'

Replacement CE�ins phenylalanine

SEQ ID NO:15

5' CACGTCACCTCGACGCTGAGTGTCTGCTGGGCGGT 3'

SEQ ID NO:16

5' ACCGCCCAGCAGACACTCAGCGTCGAGGTGACGTG 3'

Each of the mutations were sequentially introduced into the design of the plasmid containing the gene metA(#11) with three mutations, which were identified as pCL_Pcj1_metA#11.

Then designed plasmids for expression of polypeptides containing mutations identical mutations, modified polypeptides having homoserine-O-acetyltransferase activity in example 2, using as the matrix of the resulting plasmids pCL_Pcj1_metA#11.

Among the constructed plasmid containing the gene metA#11 plasmid in which the amino acid glycine at position 111 replaced by glutamic acid, was designated as a pCL_Pcj1_metA#11(EL) containing the gene metA#11 plasmid in which the amino acid glycine at position 111 replaced by glutamic acid and the amino acid leucine at position 112 is replaced by threonine, denoted as pCL_Pcj1_metA#11(ET), and containing the gene metA#11 plasmid in which the amino acid glycine at position 111 replaced by glutamic acid and the amino acid leucine at position 112 is replaced by histidine, marked as pCL_Pcj1_metA#11(EH).

Experimental example 1: Comparison of the homology between gomoserinlaktonazy E. coli and resistant to regulation with feedback gomoserinlaktonazy E. coli

Compared the primary amino acid sequence [SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43 �on order] options homoserine-O-succinylsuccinate 09:H4 E. coli (strain HS), 0139:H28 E. coli (strain E24377A) and 0157:H7 E. coli (strain ATCC8739) using the program CLC Main Workbench (CLC bio, Denmark).

As shown in Fig. 2, observed less than 5% variation in the primary amino acid sequences of homoserine-O-succinylsuccinate variants of E. coli (Fig. 2).

Also compared the primary amino acid sequence of the mutant homoserine-O-succinylsuccinate resistant to regulation with feedback methionine, using the abovementioned program. For comparison, the primary amino acid sequence of homoserine-O-succinylsuccinate wild-type, resistant to regulation with feedback the mutant homoserine-O-succinylsuccinate met10A and met11A described in PCT publication no WO 2008/127240, and resistant to regulation with feedback the mutant homoserine-O-succinylsuccinate described in PCT publication no WO 2005/108561.

As shown in Fig. 3 and 4, observed less than 5% variation in the primary amino acid sequences of the mutant homoserine-O-succinylsuccinate resistant to regulation with feedback methionine (Fig. 3 and 4).

These results indicate that the polypeptides of homoserine-O-succinylsuccinate contained in E. coli, had a 95% or higher homology between them, and there was no great difference gomoserinlaktonazy� activity even when the difference of sequences less than 5%.

Experimental example 2: Comparison of substrate specificity and activity modified polypeptides having homeserverconsoletab activity

2-1: Getting tested strains

2-1-1. Deletion of the genes metA and metB

For comparison of activity-modified polypeptides, producing an excess amount of O-acetylserine, received strain is for storing homoserine and containing a deletion of utilization of O-acetylserine. Strain with a deletion of the genes metA and metB are obtained using the methods of examples 1-1 to 1-4, described in patent publication EP2108693A2, on the basis of the threonine producing strain FTR2533 (KCCM 10541), described in PCT/KR2005/00344. The strain was designated as CJM2. CJM2 is a strain that accumulates a large number of homoserine and produces O-acetylserotonin or O-suktinilholin depending on the introduced gene.

2-1-2. Replacement of the promoter acs

To obtain an excess amount of O-acetylserine necessary to ensure production of homoserine and acetyl-COA. First, to ensure the provision of acetyl-COA, the promoter of the gene acs (acetyl-COA synthetase) was replaced by a constitutive promoter pro SEQ ID NO:30, therefore, to induce the constitutive sverkhekspressiya the desired gene. To replace the promoter conducted a modified FRT-one-step PCR (PNAS (2000) vol.97: 6640-6645). To obtain the Cass�you as shown in Fig. 5, obtained from pKD3 (PNAS (2000) vol.97: 6640-6645) resistant to chloramphenicol FRT cassette was subjected to PCR using SEQ ID NO:31 and SEQ ID NO:33, and the region of the promoter pro was subjected to PCR using SEQ ID NO:32 and SEQ ID NO:34. Two of the PCR product was subjected to PCR by the method of overlapping the splice extensions to obtain a separate cassette (the cassette with the remote acs promoter replaced by the promoter pro) (Nucleic Acids Res. 1988 August 11, 16(15): 7351-7367). PCR was performed under the following conditions: 30 cycles consisting of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds and polymerization at 72°C for 1 minute.

SEQ ID NO:31

5' AGGGGCTTCATCCGAATTGCGCCATTGTTGCAATGGCGGTGCTGGAGCTGCTTCGAAGTTC 3'

SEQ ID NO:32

5' GATATTCATATGGACCATGGCTCGAGCATAGCATTTTTATCC 3'

SEQ ID NO:33

5' GGATAAAAATGCTATGCTCGAGCCATGGTCCATATGAATATC 3'

SEQ ID NO:34

5' CGATGTTGGCAGGAATGGTGTGTTTGTGAATTTGGCTCATATGTACCTTTCTCCTCTTTA 3'

The resulting PCR product was subjected to electrophoresis on a 1.0% agarose gel, and then purified the DNA from the band of about 1.2 T. D. the Resulting DNA fragment was electroporative in strain CJM2 pre-transformed with pKD46 vector (PNAS (2000) vol.97: 6640-6645). Before electroporation strain CJM2 transformed with pKD46 was cultivated at 30°C in LB medium containing 100 μg/l of ampicillin and 5 mm L-arabinose to until OD600 reached about 0.6. Then the cultivated strain is repeatedly washed with sterilized distilled water and twice 10% glyc�Rene. Electroporation was performed at 2500 V. the Resulting strain sow a stroke on Wednesday LB Petri dish containing 25 µg/l of chloramphenicol, followed by cultivation at 37°C over night. Then, the thus selected strain exhibiting resistance to chloramphenicol.

PCR was performed using a selected strain as template and the same primers under the same conditions. The deletion of acs promoter and promoter replacement pro installed confirmation of the gene size of 1.2 T. D. on a 1.0% agarose gel. Then strain transformed with pCP20 vector (PNAS (2000) vol.97: 6640-6645), and was cultured in LB medium. Designed ultimate strain with the remote acs promoter and replaced by the promoter pro, in which the size of the gene was reduced to 150 BP by 1.0% agarose gel by PCR in the same experimental conditions, and confirmed that the marker gene of chloramphenicol was removed. Designed strain was designated as CJM2-AP.

2-1-3. Replacement resistant to regulation with feedback coaA

To obtain a strain CJM2-AP with resistance to regulation with feedback coaA PCR was performed using gdnc w3110 as template and primers SEQ ID NO:35 and SEQ ID NO:36, containing the restriction site EcoRI to obtain the coaA gene encoding pantothenticus. As the polymerase used DNA polymerase PfuUltra high precision™ (Stratagene) and PCR was carried out in �conditions 30 cycles, consisting of denaturation denaturation at 96°C for 30 seconds, annealing at 50°C for 30 seconds and polymerization at 72°C for 2 minutes.

After processing the received coaA gene and the plasmid pSG76C (Journal of Bacteriology, July 1997, 4426-4428) restriction enzyme EcoRI, was ligated with each other. DH5α E. coli transformed designed plasmid, and then selected transformed E. coli DH5α in LB medium for Petri dishes containing 25 μg/ml of chloramphenicol, to get the pSG-76C-coaA.

SEQ ID NO:35

5' ATGAGTATAAAAGAGCAAAC 3'

SEQ ID NO:36

5' TTATTTGCGTAGTCTGACC 3'

pSG-76C-coaA (R106A) designed using the resulting pSG-76C-coaA and primers SEQ ID NO:37 and SEQ ID NO:38 site-directed mutagenesis (Stratagene, USA).

SEQ ID NO:37

5' GGAAAAGTACAACCGCCgccGTATTGCAGGCGctatt 3'

SEQ ID NO:38

5' AATAGCGCCTGCAATACggcGGCGGTTGTACTtttcc 3'

Strain CJM2-AP transformed with plasmid pSG76C-coaA (R106A) and were cultured in medium LB-Cm (10 g/l yeast extract, 5 g/l NaCl, 10 g/l of tryptone, 25 mg/l chloramphenicol) for selection of colonies resistant to chloramphenicol. Select the transformants was a strain in which pSG76c-coaA (R106A) mostly built in an area coaA genome.

Strain with a built-genome coaA (R106A) transformed vector pASceP (Journal of Bacteriology, July 1997, 4426-4428) expressing the restriction enzyme I-SceI, which breaks down the plot of the I-SceI contained in pSG76c, followed by selection of strains on the environment, LB-Ap (10 g/l yeast extra�the one 5 g/l NaCl, 10 g/l of tryptone, 100 mg/l ampicillin). CoaA gene was amplified from selected strains by using primers SEQ ID NO:35 and SEQ ID NO:36 and replacement coaA (R106) in amplification gene was confirmed by sequencing in the company Macrogen (Korea) (Nucleic Acids Research, 1999, Vol.27, No. 22 4409-4415). The resulting strain was designated as CJM2-AP/CO. Strain CJM2-AP/CO is a strain having an increased pool of homoserine and acetyl-COA.

2-1-4. The increase in the number of copies of key genes of the biosynthetic pathways of homoserine

Despite the fact that the strain CJM2 or CJM2-AP/CO is a strain that produces excessive amounts of homoserine, increased the number of copies of three genes ppc, aspC and asd, in order still further to improve the production of homoserine. Plasmids pSG7 6c-2ppc, pSG76c-2aspC and pSG76c-2asd designed by the methods described in the examples from <1-1> to <1-3> publication of patent No. KR2011-0023703, and injected the plasmid into a strain CJM2-AP/CO to obtain a strain containing two copies of the three genes, by way of example, <1-5>. The resulting strain was designated as CJM3. CJM3 is a strain that accumulates a large number of homoserine compared to the strain CJM2 and produces O-acetylserotonin or O-suktinilholin depending on the injected plasmid.

2-2: Experimental methods and experimental results

Received two strains CJM2 and CJM3 obtained as competent cells awodele 9 plasmids pCL_Pcj1_metX, pCL_Pcj1_metA, pCL_Pcj1_metA(EL), pCL_Pcj1_metA(EH), pCL_Pcj1_metA(ET), pCL_Pcj1_metA#11, pCL_Pcj1_metA#11(EL), pCL_Pcj1_metA#11(EH) and pCL_Pcj1_metA#11(ET) to competent cells by electroporation, respectively.

Among them, strains CJM2 which inserted pCL_Pcj1_metA#11(EL), pCL_Pcj1_metA#11(EH) and pCL_Pcj1_metA#11(ET), denoted as CA05-0546, CA05-0547 and CA05-0548 respectively. They were deposited in the Korean center of cultures of microorganisms as of December 14, 2010 and assigned access numbers KCCM11145P, KCCM11146P and KCCM11147P respectively.

In addition, strains CJM3 which inserted pCL_Pcj1_metA#11(EL), pCL_Pcj1_metA#11(EH) and pCL_Pcj1_metA#11(ET), denoted as CA05-0578, CA05-0579 and CA05-0580 respectively. They were deposited in the Korean center of cultures of microorganisms as of December 12, 2011 and assigned access numbers KCCM11228P, KCCM11229P and KCCM11230P respectively.

In a further test conducted in a flask for comparison of types and products of the methionine precursor, which was producirovanie each strain, which were introduced in 9 types of plasmids. In the test in the flask after the stroke wiring each strain in LB medium for Petri dishes and culturing them in the incubator at 31°C for 16 hours, individual colonies were inoculable in 3 ml of LB medium, and then cultured in the incubator at 200 rpm/31°C for 16 hours.

25 ml of medium for the production of a precursor of methionine from table 1 were placed in a 250 ml flask and added to it every 500 ál of culture BU�ionov. Then flasks were incubated in incubator at 200 rpm/31°C for 40 hours and compared HPLC type and predecessor products of methionine produced by each of the strains, which were introduced in the plasmid. The results are presented in table 2 (results of type strains CJM2) and table 3 (results of type strains CJM3).

Table 1
CompositionConcentration (per liter)
Glucose70 g
Ammonium sulfate25 g
KH2PO41 g
MgSO4·7H2O0.5 g
FeSO4·7H2O5 mg
MnSO4·8H2O5 mg
ZnSO45 mg
Calcium carbonate30 g
Yeast extract2 g
Methionine0.3 g
Threonine1.5 g

Table 2
StrainsODThe consumption of sugar (g/l)The product (g/l)The number of products (g/l)
CJM2 pCL_Pcj1_metX35,663,8O-acetylcoumarin12,3
CJM2 pCL_Pcj1_metA (wild type)Of 31.349,1O-suktinilholin2,7
CJM2 pCL_Pcj1_metA ELOf 32.648,3O-acetylcoumarin2,5
CJM2 pCL_Pcj1_metA ET33,650,2O-acetylcoumarin2,0
CJM2 pCL_Pcj1_metA EH31,947,5O-acetylcoumarin3,1
CJM2 pCL_Pcj1_metA(#11) 29,556,2O-suktinilholin11,3
CJM2 pCL_Pcj1_metA(#11)EL32,749,0O-acetylcoumarin7,8
CJM2 pCL_Pcj1_metA(#11)ET3853,7O-acetylcoumarin6
CJM2 pCL_Pcj1_metA(#11)EH34,559,1O-acetylcoumarin11,1

Table 3
StrainsODThe consumption of sugar (g/l)The product (g/l)The number of products (g/l)
CJM3 pCL_Pcj1_metX17,267,0O-acetylcoumarin23,7
CJM3 pCL_Pcj1_metA (wild type)18,860,5O-suktinilholin 1,2
CJM3 pCL_Pcj1_metA EL18,560,5O-acetylcoumarin2,1
CJM3 pCL_Pcj1_metA ET18,061,0O-acetylcoumarin2,2
CJM3 pCL Pcj1_metA EHOf 17.862,2O-acetylcoumarin3,2
CJM3 pCL_Pcj1_metA(#11)14,667,0O-suktinilholin16,1
CJM3 pCL_Pcj1_metA(#11)EL17,163,2O-acetylcoumarin12,5
CJM3 pCL_Pcj1_metA(#11)ET18,265,1O-acetylcoumarin16,7
CJM3 pCL_Pcj1_metA(#11)EH19,067,8O-acetylcoumarin24,8

As shown in tables 2 and 3, only O-su�similkameen was producyrovtsa pCL_Pcj1_metA (wild-type), containing the gene for wild-type metA, but only O-acetylcoumarin accumulated strains containing three mutant metA gene of the present invention. Thus, ironically, homeserverconsoletab activity of the polypeptide is modified in homeserverconsoletab activity and substitution of amino acids.

In addition, among the three mutant strains type CJM3 strain (EL) obtained by substitution of glutamic acid for the amino acid at position 111, had produced 2.1 g/l O-acetylserine, whereas strain (EH), get an additional substitution of histidine for leucine at position 112, had produced 3.2 g/l O-acetylserine, which is the highest product yield O-acetylserine.

For strains expressing the modified polypeptides having homeserverconsoletab activity resistant to the regulation feedback methionine, also demonstrated similar results. Specifically, the strain introduced with the gene metA#11(EH), which possessed resistance to regulation with feedback methionine and included the replacement of glutamic acid and histidine for the amino acid at position 111 and 112, had produced the largest number of O-acetylserine (24.8 g/l), indicating that it accumulates O-acetylserotonin on the similar level with the introduction of a foreign gene homeserverconsoletab (CM3 pCL_Pcj1_metX, Of 23.7 g/l).

The effect of the invention

The present invention O-acetylcoumarin can be obtained from homoserine without the introduction of a foreign gene in a microorganism that expresses an enzyme that converts homoserine in O-suktinilholin, and the aforementioned O-acetylcoumarin can be used as a precursor for obtaining methionine. Thus, when applying the present invention to obtain methionine for use in foods, it is preferable that you can solve the problems of anxiety and negative attitudes of consumers in regards to the introduction of foreign genes and provide evidence of the safety of introduction of alien genes.

1. The modified polypeptide c homoserine-O-acetyltransferase activity with an amino acid sequence of SEQ ID NO:17 or at least 95% homology with it, in which the amino acid at position 111 from the starting amino acid methionine in the sequence is replaced by glutamic acid.

2. The modified polypeptide of claim 1, wherein the amino acid at position 112 of the polypeptide optionally is replaced by threonine or histidine.

3. The modified polypeptide according to claim 1 or 2, where the modified polypeptide has any of the amino acid sequences of SEQ ID NO:18-20.

4. The modified polypeptide according to p 1, where the modified polypeptide exhibits resistance to regulation with feedback methionine at amino acid changes.

5. The modified polypeptide according to claim 4, wherein the amino acid at position 29 is replaced with Proline, at position 114 is replaced by a glycine, at position 140 is replaced by a serine, or one or more combinations thereof.

6. The modified polypeptide according to claim 5, where the amino acid at position 112 of the polypeptide optionally is replaced by threonine or histidine.

7. The modified polypeptide according to claim 5 or 6, where the modified polypeptide has any of the amino acid sequences of SEQ ID nos:21-23.

8. The polynucleotide encoding the modified polypeptide of claim 1.

9. The polynucleotide according to claim 8, wherein the polynucleotide is any one of the nucleotide sequences SEQ ID NO:24-29.

10. A recombinant expression vector containing polynucleotide sequences that are functionally associated with the polynucleotide of claim 8.

11. The microorganism of the genus Escherichia for products on-acetyllactosamine containing the polynucleotide according to claim 8.

12. The microorganism according to claim 11, where the microorganism is additionally modified to possess increased acetyl-COA synthetase activity compared to the endogenous acetyl-COA synthetase activity, or modify, that he had pantothenticus activity, resistant to re�ulali inhibiting the feedback accumulation of COA.

13. The microorganism according to claim 11, where increasing the number of copies of one or more genes selected from the group consisting of a gene encoding fosfoenolpiruvatcarboksilaza (ppc), the gene encoding aspartataminotransferaza (aspC), and the gene encoding (asd), or a gene promoter replacing the promoter with increased activity, it is administered or mutation, that he had increased activity.

14. The microorganism of the genus Escherichia for products on-acetylserine transformed with a recombinant vector according to claim 10.

15. The microorganism according to claim 14, where the microorganism belongs to the genus Escherichia.

16. The microorganism according to claim 15, where the microorganism is an E. coli.

17. The microorganism according to claim 16, where the microorganism deposited under the access number KCCM11145P, KCCM11146P, KCCM11147P, KCCM11228P, KCCM11229P or KCCM11230P.

18. A method of producing O-acetylserine, comprising culturing a microorganism according to any one of claims. 11-17, and getting O-acetylserine, which is produced during cultivation of the microorganism.



 

Same patents:

FIELD: biotechnology.

SUBSTANCE: recombinant strains Rhodococcus erythropolis 37 p16-Ami RNCIM Ac-1937 and Rhodococcus erythropolis HX7 p16-Ami RNCIM Ac-1938 are constructed, constitutively producing the enzyme acylamidase with acylating activity. Also, the method of synthesis of N-substituted acrylamides is created, in particular N-isopropylacrylamide, and N-dimethylaminopropylacrylamide, using these strains as a biocatalyst.

EFFECT: invention enables to improve the efficiency of preparing N-substituted acrylamides.

3 cl, 1 tbl, 1 dwg, 9 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes a method for obtaining immobilised biocatalyst for synthesis of water solutions of amides, including acrylamide and nicotinamide from nitriles of carboxylic acids. Chitosan microgranules with diameter of 1-2 mm are obtained as a result of forcing-through of its 2-4% solution in 2% acetic acid by means of an extrusion machine to 1M of potassium hydroxide solution. Then, chitosan is activated with 0.01-0.5% solution of benzoquinone in the ratio of 1:1. Then, ferment preparation of nitrile hydrase is immobilised at 20 minutes of incubation at the temperature of 22-25°C.

EFFECT: immobilised nitrile hydrase at conversion of nitriles keeps its activity at least in fifty cycles of reactions.

5 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to biotechnology, in particular, to obtaining water solutions of acrylamide. Method is realised by hydration of acrylonitrile under action of water suspension of biocatalyst - strain of Rhodococcus rhodochrous M-8, possessing nitrile hydratase activity. Water suspension of biocatalyst contains stabilising additive, polyacrylic acid in amount 0.0015-0.015 wt % and disodium salt of ethylenediaminetetraacetic acid in amount 0.0005-0.003 wt % being used as such.

EFFECT: invention makes it possible to increase efficiency of biocatalyst action in obtaining target product, corresponding in quality to laid technical demands and to simplify the process.

2 tbl, 2 ex

FIELD: food industry.

SUBSTANCE: method for production of at least one organic compound having at least 3 carbon atoms or at least 2 carbon atoms and at least one nitrogen atom by way of fermentation involves the following stages: a1) milling cereal crop grains representing a starch source. The produced milled material contains at least 20 wt % of starch-free solid components of the starch source. At a2) stage one performs suspending the milled material in a water liquid and partial fermentative hydrolysis of starch in the milled material and in case of necessity - subsequent saccharification. One produces a liquid (1) containing monosaccharides or oligosaccharides; at b) stage one performs addition of the liquid (1) at a concentration of at least 50 wt % to a fermentative medium containing a microorganism producing the organic compound under fermentative conditions.

EFFECT: target product yield enhancement.

17 cl, 7 tbl

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology. The Rhodococcus rhodochrous NCIMB 41164 strain produces nitrile hydrase. The strain is cultured in a culture medium containing urea or a urea derivative. Urea or derivative there of is added to the culture medium at least 6 hours after the microorganism begins to grow. Biocatalytic activity of the Rhodococcus rhodochrous NCIMB 41164 strain increases and is equal to 250-300000 mcmol/min/g of the dry biomass of cells. Nitrile hydrase obtained from said strain can convert (meth)acrylonitrile to (meth)acrylamide. The invention also discloses methods of producing amide from the corresponding nitrile. The methods involve reaction of nitrile hydrase in an aqueous medium in the presence of a biocatalyst which is a Rhodococcus rhodochrous NCIMB 41164 strain and the amide is (meth)acrylamide. The invention also discloses a suspension for storing biocatalyst, containing inactively growing cells of this strain, water and optionally residual components of the fermentation broth. The biocatalyst is stored at temperature higher than freezing point, preferably higher than 0°C and specifically from 4 to 30°C.

EFFECT: higher biocatalytic activity of the Rhodococcus rhodochrous NCIMB 41164 strain.

23 cl, 5 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: method provides the use of said strain as a biocatalyst for N-substituted aliphatic acrylamide synthesis of acrylamide and primary aliphatic amines. The invention allows producing high-yield N-substituted aliphatic acrylamides. The yield of N-iaopropylacrylamide is 11 g/l, of N-dimethylaminopropiylacrylamide - 5 g/l.

EFFECT: Rhodococcus erythropolis bacteria strain, 37 RNCIM No As-1793 exhibit acylating activity and is capable to synthesise N-substituted aliphatic acrylamides.

2 cl, 5 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to a method of obtaining ethylene unsaturated amide or ethylene unsaturated carboxylic acid or its salt from the corresponding ethylene unsaturated nitrile, in which the nitrile is hydrated or hydrolysed in an aqueous medium in the presence of a biocatalyst, where the nitrile contains more than 2 parts by weight per million acrolein and amide or carboxylic acid or its salt contains 2 parts by weight per million acrolein. The method can be used to obtain acrylamide or acrylic acid (salt) of high purity from low-quality acrylonitrile containing a lot of acrolein. Suitable biocatalysts include Rhodococcus bacteria.

EFFECT: invention increases efficiency of producing ethylene unsaturated amide or ethylene unsaturated carboxylic acid or its salt from the corresponding ethylene unsaturated nitrile and provides a method of producing a polymer of ethylene unsaturated monomer.

26 cl, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to bioengineering and provides a cyanide-resistant nitrile hydratase, produced by a Pseudomonas family microorganism, which has high resistance to cyanides. The invention also relates to use of such a nitrile hydratase to obtain amides from nitriles in the presence of cyanides.

EFFECT: invention enables obtaining highly effective amides.

18 cl, 7 dwg, 11 ex

FIELD: chemistry; biotechnologies.

SUBSTANCE: method of producing immobilised biocatalyst includes trapping of Rhodococcus type microorganism cells of nitrile-hydrase activity on carbon carriers by delivering cellular suspension through the column, filled with the carrier, or by joint centrifugation of cells and carriers. Herewith carbon carriers are active carbons made of vegetative and polymer raw materials with porous structure parameters, including total pore space 0.4-1.8 cm3/g and micropore volume concentration 45-92%. Produced biocatalyst is used in method to produce aqueous amide solutions by carboxylic acid nitrile hydration.

EFFECT: high amide yield and increased time for amide synthesis biocatalyst application.

4 cl, 2 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: proposed aqueous solution of acrylamide is obtained through hydration of acrylonitrile in an aqueous solution in the presence of a biocatalyst. To carry out the reaction, a device is used, comprising a reactor and a centrifuge attached to it. To separate the biocatalyst from the aqueous solution of acrylamide, the centrifuge operates in a semi-continuous mode, at least. The centrifuge has optical means of controlling parameters of the clarified liquid medium tapped from it. The control results are used for controlling operation of the centrifuge. The method has the highest possible environmental friendliness and allows for minimising quantity of by-products.

EFFECT: design of an efficient method of making an aqueous solution of acrylamide.

16 cl, 1 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions concerns recombinant DNA pA3, recombinant plasmid DNA pQE 30-pA3, strain E.coli M 15-A3, recombinant polypeptide A3, test systems for the qualitative detection and quantitative determination of microalbuminuria. The characterised recombinant DNA pA3 is produced by a polymerase chain reaction with the use of chromosomal DNA of the strain DG 13 of group B streptococci and structured primers. An amplification fragment has been cloned by a system of expression vectors pQE-pQE 30 in E.coli M 15 to produce recombinant DNA pQE 30-pA3, which codes an amino acid sequence of the recombinant polypeptide A3 having an ability to bind human serum albumin (HSA) selectively.

EFFECT: presented inventions can be used in the medical practice and industry for producing the test systems for the detection and quantitative determination of microalbuminuria - one of the earliest signs of renal irritation in the patients suffering from diabetes mellitus and essential arterial hypertension.

6 cl, 9 dwg, 1 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to biotechnology and can be used for recombinant production of human tissue factor (hTF). Constructed is a plasmid pHYP-10ETFCS6 having a length of 5,912 b.p. with a physical map presented on Fig. 2, for expression in a bacterium of the genus Escherichia, which is a precursor of the mutant [C245S] hTF containing an inseparable N-terminal leader peptide containing a deca-histidine cluster and an enterokinase identification sequence fused in a frame with a sequence coding the above mutein fused in the frame with the sequence coding the additional inseparable C-terminal peptide containing the deca-histidine cluster. A method for producing the precursor of the mutein[C245S]hTF contains culturing the producing bacterium in a nutrient medium, recovering inclusion bodies, solubilising the precursor protein, performing a metal chelator chromatography in the denaturation environment, re-folding and diafiltration of the protein solution. A method for producing the mature mutein[C245S] hTF involves detecting the N-terminal leader peptide from the above mutein precursor with using enterokinase and recovering the target protein.

EFFECT: invention enables increasing the level of biosynthesis and yield of pro-coagulation active hTF.

9 cl, 5 dwg, 1 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: claimed inventions deal with an isolated polynucleotide, coding a polypeptide, involved in biosynthesis of pyripyropene A, a vector and a host cell, including such a polypeptide, and methods of obtaining pyripyropene A precursors, including the host cell cultivation. The claimed polynucleotide codes the polypeptide, possessing any one or more of the activities - polyketide synthase, prenyltransferase, hydroxylase, acetyltransferase or adenylate synthase.

EFFECT: claimed inventions make it possible to synthesise pyripyropene A, which is an insecticidal agent, and can be used in the formation of plant resistance to pest insects.

16 cl, 11 dwg, 1 tbl, 11 ex

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, in particular to obtaining recombinant enzyme-labelled antigen G2 of hantavirus Dobrava. Essence of invention consists in the following: claimed method of obtaining antigen G2 of hantavirus Dobrava consists in expression of antigen in cells of E.coli in form of enzyme-labelled antigen of G2 hantaviruses based on HT protein antigen. Beta-galactosidase, which is highly active stable enzyme, serves enzyme label for protein antigen. Invention can be used to increase specificity and reproducibility of immunosorbent assay in HFRS diagnostics.

EFFECT: presence of commercially available chromogenic substrate of beta-galactosidase (X-gal) makes it possible to quickly estimate result of immunosorbent reaction visually or by change of optic density of solution in visible area.

6 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry and represents a new bifunctional PSH protein containing human peroxiredoxin Prx6 and manganese superoxide dismutase MnSOD possessing the antioxidant activity of superoxide dismutase and peroxidase. What is also described is a chimeric nucleic acid coding the presented protein. A method for preparing the presented protein by culturing cells of the strain E.coli BL21(PSH) transformed by constructed recombinant expression vector based on pET22b(+) plasmid is disclosed.

EFFECT: invention enables producing high-yield protein PSH possessing the high antioxidant activity.

4 cl, 6 dwg, 3 tbl, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology, namely to recombinant obtaining G-CSF, and can be used for production of G-CSF in cells of E.coli. For effective production of protein in cells of E.coli G-CSF-coding DNA sequence is optimised. On the basis of obtained optimised DNA sequence plasmid pAS017, also including NdeI/BamHI-fragment of DNA of pETM-50 vector and having physical map, presented on the drawing, is constructed.

EFFECT: invention provides effective production of protein in cells of Ecoli.

2 cl, 1 dwg, 1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly to genetically engineered production of human proteins, and may be used for preparing human epidermal growth factor (hEGF) in bacterial cells in the form of glutathione-3-transferase fusion protein. What is constructed is the recombinant DNA coding GST-hEGF fusion protein which consists of an amino acid sequence of glutathione-S-transferase and an amino acid sequence of human epidermal growth factor divided by a cleavage site by enterokinase, and characterised by the nucleotide sequence SEQ ID NO:1. The KpnI/XhoI fragment of the vector pET41 and the above recombinant DNA are used to create the recombinant plasmid pAS007 for expression of GST-hEGF fusion protein in E.coli cells.

EFFECT: invention enables reaching high GST-hEGF expression levels in Ecoli cells.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, gene and protein engineering and specifically to recombinant plasmid DNA pG1-Rm7, which facilitates synthesis of hybrid protein G1-Rm7 in Escherichia coli cells, which is capable of biding the tumour necrosis factor and has bioluminescence of luciferase Renilla muelleri, where said plasmid DNA includes the nucleotide sequence SEQ ID NO: 1 and can be in medicine. The invention also relates to the protein pG1-Rm7 having molecular weight of 65.4 kDa, consisting of a single-strand anti tumour necrosis factor antibody, a GGSGGS peptide and modified luciferase Renalla muelleri and characterised by SEQ ID NO: 2.

EFFECT: invention enables to obtain a highly sensitive reporter for detecting a tumour necrosis factor via bioluminescent analysis.

2 cl, 4 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology and concerns preparing a genetic construct providing a synthesis of p35d recombinant protein in Escherichia coli cells. There are presented: recombinant plasmid DNA pQE-p35d providing the synthesis of p35d recombinant protein of cowpox virus and containing in accordance with physical and genetic map presented on Fig. 2: pQE30 plasmid vector, a fragment coding MRGSHHHHHHG oligopeptice and a fragment of 17 base pairs, coding a fragment of p35 protein of cowpox virus within 1 to 239 amino acid residues (Fig.1a); Escherichia coli XL1Blue/pQE-p35d B-1252 bacterial strain that is a producer of p35d recombinant protein of cowpox virus, containing recombinant plasmid DNA pQE-p35d deposited in the Collection of Bacteria, Bacteriophages and Fungi of FBUN GNTs VB Vector, registration No. B-1252, and p35d recombinant protein of cowpox virus.

EFFECT: solutions may be used to engineer the test systems and to prepare orthopoxvirus split vaccines.

3 cl, 7 dwg, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of obtaining a recombinant antigen G2 of hantaviruses. The disclosed method is characterised by that the DNA structure pGHF, which encodes a fused protein of three parts, where N-terminal position is occupied by a green fluorescent protein GFP, central position is occupied by a peptide of 73 amino acid residues with the amino acid sequence SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEW KDPDGMLKDHLNILVTKDIDFDT, and the C-terminal position is occupied by a mini-domain Foldon of coliphage fibritin JS98C3 (dwg 2), is introduced into E. coli cells; cells transformed by this structure are cultured, the biomass is lysed, the insoluble fraction of the lysate is separated by centrifuging, the product of expression in the form of inclusion bodies is solubilised with methylated spirit, chromatography is carried out and the obtained product is used to detect specific antibodies in serum of patients with hemorrhagic fever with renal syndrome.

EFFECT: disclosed solution improves repeatability and sensitivity of immunoenzymatic assay when diagnosing hemorrhagic fever with renal syndrome.

7 dwg

FIELD: chemistry.

SUBSTANCE: group of inventions relates to biotechnology, particularly to mutant O-phosphoserine sulfhydrylase (OPSS) from Mycobacterium smegmatis with an amino acid sequence corresponding to SEQ ID NO: 1 which is devoid of three to seven C-terminal amino acid residues. The inventions also relate to a nucleic acid molecule encoding the mutant OPSS, an expression vector carrying the nucleic acid molecule, and a transformant transformed by the expression vector. In addition, a method is provided for producing cysteine in which O-phospho-L-serine (OPS) is reacted with a sulphide in the presence of the mutant OPSS.

EFFECT: muntant OPSS has improved enzymatic activity and can be used for environmentally friendly production of L-cysteine in conditions through a simple enzymatic conversion reaction; the group of inventions provides high output of L-cysteine.

16 cl, 7 dwg, 20 tbl, 19 ex

Up!