TWI395815B - The application of pha synthetase gene phab and its enzyme from extreme halophilic archaea in bioplastic production - Google Patents

Description

Polyhydroxyalkanoate biosynthesis gene of extremely high-salt archaea phaB and its enzymes for bioplastic production

The present invention relates to a gene, protein, recombinant microorganism for producing a polyhydroxyalkyl ester. The invention also relates to a process for the production of polyhydroxyalkyl esters. The invention also relates to a method for screening polyhydroxyalkanoate biosynthesis-related genes of extreme halophilic archaea.

When bacteria and archaea grow under nutrient imbalance, the extra carbon source in the cell will accumulate in the cell in the form of polyhydroxyalkanoate (PHA); the accumulated PHA will be in the absence of carbon in the organism. It is degraded to Acetyl-CoA and then provided as a carbon source and energy source for cell growth via metabolic pathways. PHA characteristics are similar to the common plastic material polypropylene (PP), which can replace traditional petrochemical plastic products. Biodegradable plastics such as PHA have been mass-produced by microorganisms since the 1970s, but their cost is more expensive than petrochemical synthetic plastics, which is relatively uncommon; caused by plastics that cannot be degraded in recent years. Earth's environmental disasters make the importance of biodegradable plastics produced by microorganisms more prominent. Therefore, how to reduce the cost of production and purification to manufacture a variety of plastic materials is very important (Gross, RA and Kalra, B., Science , (2002), 297: 803-807; Madison, LL and Huisman, GW, Microbiol. Mol. Biol. Rev., (1999), 63: 21-53; Taguchi. S., and Doi, Y., Macromol. Biosci. , (2004), 4: 146-156). In addition, due to its good biocompatibility, PHA has attracted considerable attention in tissue engineering, tissue regeneration and tissue repair in recent years (Chen, GQ and Wu, Q., Biomaterials , (2005), 26:6565 -6578).

The production and research of PHA is mainly based on Ralstonia eutropha , Pseudomonas aeruginosa , Allochromatium vinosum , Bacillus megaterium , etc. in bacteria. Lord, in recent years, there have been many studies attempting to transfer the PHA biosythesis-related genes of bacteria to E. coli for performance and production, but for archaea (Archaea) which is a single-cell prokaryote with bacteria (or The related discussion of Taikoo) is quite limited. Extremely high-salt archaea in archaea needs to grow in a high-salt environment of at least 1.5 M NaCl (such as the Dead Sea, Yantian or Salt Lake), and these high-salt ecological environments usually have strong sunlight and evaporate, and the temperature can be as high as 50 ° C to 60 ° °C. Moreover, the molecular penetration adaptation mechanism of extremely high-salt archaea adapted to extracellular high concentration of sodium chloride (NaCl) is to accumulate a large amount of potassium ions in the cells, thereby balancing the intracellular and extracellular osmotic pressure and protecting the normal function of the protein, so high salt Biological proteins are resistant to high temperatures and high salts, and are still active even in organic chemical solvents, meeting the needs of industrial production (Mevarch, M. et al ., Biophys. Chem ., (2000), 86: 155-164). Compared with other archaea, the extremely high-salt archaea is easy to culture, grows fast and is not easily contaminated by other microorganisms. Extreme high-salt archaea is the earliest in the development of molecular biotechnology, and its transformation, genetic mutation and fermentation are all perfect (Allers, T. and Mevarch, M., Nat. Rev. Genet ., ( 2005), 6:58-73), it has considerable advantages in the development of PHA biosynthesis.

Feranandez-Castillo et al. first reported in 1986 that the extremely high-salt archaea, Haloferax mediterranei , uses PHA particles to store carbon sources (Feranandez-Castillo, R. et al ., Appl. Environ. Microbio. (1986), 51: 214-216). Previous studies by the inventors have shown that the H13 strain ( Haloterrigena sp. H13) accumulates PHA particles (Lai Meijin, (1995), Report of the National Institute of the National Academy of Sciences), and further analyzes the characteristics of this polyester. And its application in biomedical materials (Lai Meijin, (1999), report of the special research project of the National Academy of Sciences of the Executive Yuan), the results show that Haloterrigena sp. H13 can accumulate a large number of extremely high-salt archaea under the induction of low phosphorus and high glucose. Type polyester particles (hPHA), which are identified by chemical structure analysis, have a monomer composition of -[-O-CH(C≡CH)-CH(C ₂ H ₅ )-CHO-] _n -, and this The novel hPHA has a special unsaturated ethynyl group (C≡C), which is different from the published bacterial type PHA. This new type of polyester has also obtained the invention patent of the Republic of China Patent Certificate No. 198121. Fibroblast toxicity tests show that hPHA is not toxic to cells and can be applied to biomedical materials. Moreover, the cell attachment is much better than the polyhydroxybutyl ester (PHB) of the currently produced mass-produced oxyhydrogen bacteria, and the cell attachment amount of the polylactic acid polyester (PLGA and PLLA) which has been applied in tissue engineering is the same. It is non-toxic, has high compatibility, and has strong cell attachment. It shows that hPHA is a high-potential biomedical material. This part of the results has also been granted a patent (the invention patent of the Republic of China Patent Certificate No. I270376).

In summary, although the prior art has used the extremely high-salt archaea to produce PHA by fermentation technology, due to the high cost of the high-salt and high-temperature fermentation system, the production of PHA by the extremely high-salt archaea itself is still manufactured. The disadvantages are high cost and difficult to control operating conditions.

Summary of invention

Based on the above, the inventors have in view of the disadvantages of the prior art in the production of polyhydroxyalkyl esters (PHA), the high cost of the fermentation system and the difficulty in controlling the operating conditions, and the research and development can reduce the manufacturing cost of the polyhydroxyalkyl ester, and the stability is large and Efficient production of polyester technology.

In a first aspect, the invention provides an isolated nucleic acid molecule comprising a sequence selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO: 7; (ii) having a nucleotide sequence having a nucleotide sequence similarity greater than 90% of SEQ ID NO: 7; (iii) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13; and (iiii) a coding and SEQ ID NO: A nucleotide sequence having a similar amino acid sequence sequence of greater than 90%.

The present invention also provides a polypeptide having the amino acid sequence encoded by the nucleic acid molecule of the above first aspect and having the activity of PhaB _H13 .

In a second aspect, the present invention provides a vector comprising the control sequence of the nucleic acid molecule of the first aspect and the homologous control sequence of a non-salt genus H13 strain ( Haloterrigena sp. H13), wherein A nucleic acid molecule is operably linked to the control sequence.

In a third aspect, the invention provides a recombinant microorganism comprising a carrier as described above.

In a fourth aspect, the present invention provides a nucleic acid molecule according to the first aspect of the invention for use in the production of a polyhydroxyalkyl ester, the present invention provides a method for producing a polyester, comprising: providing a recombinant microorganism as described above; And growing the recombinant microorganism in a suitable environment to produce a polyhydroxyalkyl ester.

The inventors have confirmed through detailed experimental studies that the PHA biosynthesis-related genes of the extremely high-salt ancient strain H13 screened by the present invention contain phaB , and further analysis results confirm that they have extremely high potential and advantages in the production of polyester. .

Detailed description of the invention

In order to make the present invention more comprehensible, the terminology of the present invention is further described, and unless otherwise indicated, it is intended to be understood by those of ordinary skill in the art.

Plastics are known in the technical field to refer to the use of high molecular polymers as the main component, adding appropriate additives, such as plasticizers, stabilizers, flame retardants, lubricants, colorants, etc., plasticity (flexibility) after processing ) a material, or a rigid material formed by curing crosslinks. Bioplastic refers to a material obtained by treating as described above with a polymer component derived from a living body as a main component.

In addition to the specific sequences disclosed herein, a nucleic acid molecule also encompasses complementary sequences of the particular sequence, as well as conservative analogs, related naturally occurring structural variants or synthetic non-sequences. Naturally occurring analogs, such as: homologous sequences of degenerative codon substitutions. In particular, one nucleotide residue of a particular sequence as disclosed herein may be substituted with another nucleotide residue without affecting the activity of the polypeptide encoded by the particular sequence or itself as a promoter.

In accordance with the present invention, the term "Similarity" is defined herein as the degree of association between two sequences, which may be determined by the same sequence and/or conservative ratios between sequences.

According to the invention, the phrase "substantially identical" means that among different species, variations in the amino acid sequence do not necessarily affect the activity of the protein they constitute, so that as long as the amino acid sequence has a certain degree of similarity Does not affect the activity of the protein. The above degree of similarity is preferably 70% or more; more preferably 80% or more; and still more preferably 90% or more.

According to the present invention, the extreme halophilic archaea means an archaea which can be grown in a high salt environment, which has a high temperature, high salt protein and is active even in an organic solvent.

A method for screening a polyhydroxyalkyl ester biosynthesis-related gene of the extreme halophilic archaea of the present invention, comprising the steps of: providing a primer pair, wherein one of the primers has SEQ ID NO: 1 a sequence having another sequence having the sequence of SEQ ID NO: 2; providing a sample containing a polyhydroxyalkyl ester synthase gene and a sample containing halophilic archaea DNA; using the primer pair to amplify the polyhydroxyalkyl ester a synthetase gene to obtain a polyhydroxyalkyl ester synthase nucleic acid fragment; and detecting a genomic fragment of the halophilic archaeal DNA sample using the polyhydroxyalkyl ester synthase nucleic acid fragment as a probe, wherein The gene body fragment has at least one polyhydroxyalkyl ester biosynthesis-related gene.

The extremely high-salt archaea is an archaea belonging to a genus selected from the group consisting of Haladaptatus , Halalkalicoccus , Haloarcula , Halobacterium , Halobaculum , Halobiforma , Halococcus , Haloferax , Halogeometricum , Halomicrobium , Halopiger , Haloplanus, Haloquadratum, Halorhabdus, Halorubrum, Halosimplex, Halostagnicola, Haloterrigena, Halovivax, Natrialba, Natrinema, Natronobacterium, Natronococcus, Natronolimnobius, Natronomonas and Natronorubrum.

According to the invention, the term "polyhydroxyalkanoate synthase" as used herein, means an enzyme which converts hydroxyalkyl-CoA to a polyhydroxyalkyl ester and coenzyme A, possibly by one or several homo- or It consists of hetero units. PHA synthase (PHA synthase) is a key enzyme in the biosynthesis of polyester, which catalyzes the conversion of (R)-hydroxyacyl-CoA thioester to polyester and releases CoA.

The diversity of PHA synthase is divided into four types according to monomer composition and substrate specificity: (1) Type I PHA synthase: represented by Ralstonia eutropha The strain consists of PhaC protein with a molecular weight of 60 to 73 kDa. The β-HB-CoA of C3~C5 is used as a substrate to synthesize a short carbon chain type PHA. (2) Type II PHA synthase: a strain represented by Pseudomonas sp., consisting of PhaC1 and PhaC2 proteins with a molecular weight of 60 to 65 kDa, using C5~C16 β-HB-CoA is a acceptor and synthesizes a long carbon chain type PHA. (3) Type III PHA synthase: a representative strain of Allochromatium vinosum D, composed of PhaC and PhaE proteins, with a molecular weight of 40 kDa, with the first type and the first The similarity of the type 2 PHA synthetase amino acid sequence is about 21% to 28%. PhaE has a molecular weight of 40 kDa, and is not similar to any PHA synthase sequence. The third type PHA synthetase utilizes C3~C5 β-HB-CoA as a substrate to synthesize a short carbon chain type PHA. (4) Type IV PHA synthase: a representative strain of Bacillus , consisting of PhaC and PhaR proteins, PhaC has a molecular weight of 40 kDa, and PhaR has a molecular weight of 20 kDa, and is not associated with any PHA synthase sequence. Similarly, the use of C3-C5 β-HB-CoA as a substrate for the synthesis of short carbon chain PHA (Rehm, Biochem. J. , 376: 15-33, 2003; Rehm, Biotechnol. Lett. , 28: 207-213 , 2006; Rehm, Curr. Issues. Mol. Biol. , 9:41-62, 2007). The extremely high-salt archaea (太古生物) provided by the present invention is generally considered to belong to the third type, but the inventors' research results indicate that it should be an independent special high-salt archaea type.

According to the present invention, the aforementioned sample containing the polyhydroxyalkyl ester synthase gene may be any nucleic acid sample derived from a microorganism having the ability to produce a polyhydroxyalkyl ester, and therefore, is used in the present invention for screening extremely high-salt archaea (extreme The method of the polyhydroxyalkyl ester biosynthesis-related gene of halophilic archaea may contain samples of the polyhydroxyalkyl ester synthase gene from the following microorganisms including, but not limited to, Haloarcula sp. strains, such as Haloarcula sp. Strain HLR2; Haloferax mediterranei , such as the halophilic Dead Sea archaea strain deposited under the number ATCC43049; Haloterrigena sp.; ( Ralstonia eutropha ); Pseudomonas aeruginosa ; Allochromatium vinosum ; and Bacillus megaterium .

In a specific embodiment of the method for screening a polyhydroxyalkanoate biosynthesis-related gene of the extreme halophilic archaea of the present invention, the extremely high-salt archaea belongs to the salt terrestrial strain H13 ( Haloterrigena sp. H13).

According to the present invention, the aforementioned sample containing extremely high-salt archaeal DNA (DNA) is a DNA fragment derived from an extremely high-salt archaea, and the DNA fragment of the gene may be cleaved by a restriction enzyme or other organism. According to the technical method, specifically, the sample containing extremely high-salt archaeal DNA (DNA) is an extremely high-salt archaeal gene fragment cleaved by NotI.

According to the present invention, the term "polyhydroxyalkyl ester biosynthesis-related gene" as used herein, refers to a gene involved in the polyhydroxyalkyl ester biosynthesis pathway, including but not limited to: an enzyme gene, a transcriptional regulatory protein ( PHA biosynthesis transcriptional regulating protein) and a regulatory element.

According to the present invention, the enzyme gene may be phaC _H13, phaD _H13 of polyhydroxyalkanoates synthase (polyhydroxylalkanate sythetase) such as maoC _H13 alkenyl acyl coenzyme A hydrolase (enoyl-CoA hydratase), or such phaB _H13 of Nicotine indoleamine dinucleotide phosphate-dependent acetaminophen A reductase (NADP-depentent acetoacetyl-CoA reductase); the aforementioned transcription factor gene may be a polyhydroxyalkanoate synthesis transcriptional regulatory protein (PHA biosynthesis transcriptional) Regulating protein), such as phaR _H13 and phaT _H13 ; and the regulatory unit may be a promoter, an enhancer, a polyadenylation signal, a terminator of the aforementioned gene. ) and the like.

The method for screening polyhydroxyalkanoate biosynthesis-related genes for extremely high-salt archaea according to the present invention may be in a detection step of the method by a common method in the field of biotechnology (such as polymerase chain Lock reaction, northern ink stain method, southern ink stain method or instant polymerase chain reaction, etc.).

A vector according to the present invention comprising a nucleic acid molecule as described above and a control sequence for a homologous control sequence of a non-salt genus H13 strain ( Haloterrigena sp. H13), wherein the nucleic acid molecule is operably linked to the control sequence Connected.

The term "homology" as used herein, refers to the same gene or allelic genes that have a genetic loci that is typically configured in the same order.

The phrase "homology control sequence of the non-salt genus H13 strain ( Haloterrigena sp. H13)" as used herein , means a control sequence other than the homology control sequence of the H. serrata H13 strain ( Haloterrigena sp. H13). For example, a control sequence such as the T7 promoter of pET21b (T7 promoter) of a large number of heterologous genes in Escherichia coli BL21 (DE3).

The term "control sequence" as used herein, refers to a sequence that enables a nucleic acid sequence of a gene to be initiated or shut down, thereby affecting the expression of the nucleic acid sequence of the gene; for example, the control sequence may be a promoter, enhancer, poly A polyadenylation signal, terminator, and the like, which allow a protein or polypeptide coding sequence to be expressed in a host cell. The term "vector" refers to any recombinant vector which can be a recombinant expression system which can be constitutively or in any host cell either in vitro or in vivo . A selected nucleic acid sequence is inducibly expressed. The expression vector can be a linear or circular form, an episomal expression system, or a expression system incorporated into the genome of the host cell. The expression system may or may not have the ability to self-replicate, and may only initiate transient expressions in a host cell.

The term "operably linked" as used herein, means that a first sequence is disposed in close proximity to a second sequence such that the first sequence can affect or be in the second sequence. under. For example, a promoter sequence can be operably linked to a gene sequence, and is typically at the 5' end of the gene sequence such that expression of the gene sequence is under the control of the promoter sequence. In addition, a regulatory sequence can be operably linked to a promoter sequence to enhance the ability of the promoter sequence to initiate transcription. In this case, the regulatory sequence is typically located at the 5' end of the promoter sequence.

The vector according to the present invention may comprise a nucleic acid molecule according to the present invention and a target gene other than the homologous gene of the salt terrestrial H13 strain, wherein the nucleic acid molecule is operably linked to the target gene. In a specific embodiment of the invention, the aforementioned target gene encodes a PHA biosynthesis-related gene.

A recombinant microorganism according to the present invention, which is capable of heterologously expressing a PHA biosynthesis-related gene, the recombinant microorganism comprising any of the foregoing vectors, wherein the microorganism to be applied is all bacteria or archaea; more specifically, the foregoing The bacteria may be Escherichia coli, Ralstonia eutropha , Pseudomonas aeruginosa , Allochromatium vinosum or Bacillus megaterium . The aforementioned archaea may be an archaea belonging to the following genus: Haladaptatus , Halalkalicoccus , Haloarcula , Halobacterium , Halobaculum , Halobiforma , Halococcus , Haloferax , Halogeometricum , Halomicrobium , Halopiger , Haloplanus , Haloquadratum , Halorhabdus , Halorubrum , Halosimplex , Halostagnicola , Haloterrigena , Halovivax, Natrialba, Natrinema, Natronobacterium , Natronococcus, Natronolimnobius, Natronomonas and Natronorubrum.

A method for producing a polyester according to the present invention, wherein a suitable environment for the recombinant microorganism is an environment known in the art to allow the recombinant microorganism to properly proliferate and express and produce a polyester, such as a molecular biotechnology field, a microorganism Techniques well known in the field of fermentation; such as the method described in Lai Meijin's 1999 National Institute of Science and Technology Special Projects, the Republic of China Patent No. 00579390, and the Republic of China Patent No. I270376.

Due to the high cost of using high-salt archaea in high-salt and high-temperature fermentation systems, the inventors noted that if PHA biosynthetic-related genes are transferred to E. coli, they can be used not only to understand the function of these enzymes. It is active and can be used to transfer to industrially produced PHA strains such as Ralstonia eutropha to obtain a better polyester yield, or to produce such advantages of the novel polyester.

In summary, the present invention provides an application of extremely high-salt archaea comprising phaB in the production of polyester, and the features of the present invention are further described in detail below:

(1) Since the present invention provides the use of genes and proteins using the patent strain extremely high-salt archaea strain Haloterrigena sp. H13 (patent deposited strain BCRC 910151 of the Center for the Development of the Republic of China Food Industry Development Institute). Haloterrigena sp. H13 belongs to the Archaeea (archaea), is not pathogenic and is a high salt extreme organism. The high-altase archaea's extreme enzymes are salt tolerant, solvent resistant, antioxidant and high temperature stable, making them suitable for industrial and extracellular ( in vitro ) production applications.

(2) The present invention can be applied to the development of various biodegradable plastic PHA products, which helps to accelerate the global acceleration of biodegradable plastics to replace petrochemical plastics that are not friendly to the earth.

(3) The present invention provides a gene and sequence related to PHA synthesis of Haloterrigena sp. H13, which comprises a biosynthetic-related phaB , and a phaB gene of Haloterrigena sp. H13 is expressed in Escherichia coli BL21 (DE3) using the expression vector pET21b or pET28a. A technique that produces a large amount of protein, and PhaB _H13 expressed in E. coli, can be present as a soluble protein.

The inventor's laboratory uses a whole-genome sequence to complete the halophilic Dead Sea archaea (Haloarcula marismortui One of the ATCC43049 has been namedphaC The ORF sequence of the PHA synthetase (polyhydroxyalkanoate sythetase) gene is designed as a primer pair (SEQ ID NO: 1 and SEQ ID NO: 2) (Lin Ziyi, Master of Science in Life Science, National Chung Hsing University, 2006), inventor laboratory Purified from Yanshan, Tongluo Salt Field, Miaoli CountyHaloarcula Sp Strain HLR2 chromosomal DNA undergoes polymerase chain reaction to amplifyphaC _HLR2 (SEQ ID NO: 3) (You Yuting, Master's thesis, Department of Life Sciences, National Chung Hsing University, 2006).phaC _HLR2 Dead sea archaea with halophilicHaloarcula marismortui The PHA synthase amino acid sequence has a similarity of 97.1%, and its sequence has a catalytic ternary composed of (Cys-162)-(Asp-317)-(His-346) associated with PHA polymerization. Catalytic triad residues, therefore, should be assigned to the α/β-hydroxylase superfamily as well as the eubacterial PHA synthase.phaC _HLR2 It is indeed the PHA synthase gene (Lin Ziyi, same as above, 2006).

The inventors screened a NotI-cleaved fragment of the genus H13 ( Hloterrigena sp. H13) by Southern blotting using phaC _HLR2 as a probe, which has a full length of 4599 bp and has 5 complete ORFs. The sequence is a gene fragment of maoC, phaR, phaT, phaD, phaC, and phaB (as shown in the first figure). The complete sequence of the phaB gene is then complemented by the polymerase chain reaction (PCR) method and the DNA sequencing method. It was confirmed by analysis that the PHA synthetase gene cluster of Haloterrigena sp. H13 comprises phaC (SEQ ID NO: 4), phaD (SEQ ID NO: 5), maoC (SEQ ID NO: 6). ), phaB (SEQ ID NO: 7), phaR (SEQ ID NO: 8), and phaT (SEQ ID NO: 9), the functions and nomenclatures of which are based on similar proteins after comparison with the NCBI gene pool. The amino acid sequence was evaluated and analyzed. Among them, PhaC and PhaD are co-transcribed PHA synthase; PhaB _H13 is NADP-dependent acetoacetyl-CoA reductase; and MaoC is enoquinol acetoin A hydratase (enoyl) -CoA hydratase), PhaR is a transcriptional regulatory protein with a DNA-binding region that regulates phasins protein expression and controls PHA particle size; PhaT should be a transcriptional regulatory protein.

The gene length, G+C%, estimated isoelectric point, translated amino acid, and predicted molecular weight of the six PHA biosynthesis-related genes are shown in Table 1. Analysis of the amino acid composition of these proteins, as shown in Table 1, can be found that the isoelectric point is between about 3 and 4, the ratio of acidic amino acids is high, and the proportion of hydrophobic amino acids is also high, shown from Haloterrigena. The PHA biosynthesis protein of sp. H13 has high salt-tolerant protein properties and is negatively charged on the surface, which is resistant to high-ionic concentrations and low-water activity environments such as organic solvents.

The PhaC _H13 of the H13 strain of the genus L. is a PHA synthase. The sequence similarity results, the similarity of the amino acid sequence of PHA polymerase PhaC _H13 and the known bacterial first type or second type PhaC is about 20%, and The similarity of the third or fourth PHA synthetase of bacteria is about 40%; the similarity of PhaC to other extremely high-salt Pacific creatures is also less than 60%. Explicitly this PhaC _H13 and known PhaC have Great difference.

The upstream of phaC _H13 is phaD _H13 , which has a 4 base pair (bp) overlap with phaC _H13 , and only the region presumed to be a promoter is found upstream of phaD _H13 . The third- or fourth-type PHA synthase needs to be composed of an enzyme encoded by the phaEC or phaRC genes to form a hetero-subunits PHA synthetase, so it is preliminarily judged that PhaD _H13 is Play the role of PhaE or PhaR. Analysis of the PhaC _H13 amino acid sequence revealed an activation center composed of an amino acid residue (Cys-151)-(Asp-306)-(His-334) which is required for the polymerization of PHA synthase. Analysis by Northern blotting showed that phaC and phaD are co-transcribed.

According to sequence analysis and proteomic analysis, the PHA biosynthesis gene group of the H. serrata H13 strain and the general bacterial PHA biosynthesis gene group are different from maoC . The function of MaoC is enoyl-CoA hydratase, which should be the gene product of the upstream pathway of PHA biosynthesis. It is the main protein source of PHA biosynthesis, mainly β-oxidation of fatty acids. The intermediate product of the Fatty acid β-oxidation, trans-2-enoyl CoA, is converted to (R)-3-hydroxyindole Kiev A[(R)-3-hydroxyacyl- CoA] is then converted to PHA via PHA synthase. Different MaoCs and their mutant proteins can change their specificity and produce different kinds of monomers with different carbon chain lengths. Therefore, the genes and proteins of MaoC _H13 of the H13 strain are produced in various novel biodegradable polyesters. The potential.

PhaB functions as a nicotine indole diamine nucleotide-reductive acetoacetyl-CoA reductase, and is also a gene product of the upstream pathway of PHA biosynthesis, which can The acetaminophen A is converted to (R)-3-(R)-3-hydroxyindole Kiev enzyme A and then reacted to PHA via PHA synthase. PhaR _H13 is an AbrB-like protein, which is a transcriptional regulatory protein with a DNA binding region, regulates the expression of Phasins protein, and controls the size of PHA particles. PhaT should be a transcriptional regulatory protein. PhaT _H13 overlaps with phaR _H13 , so there is a correlation in gene transcription. At present, the true function of PhaT _H13 is unknown, but from the molecular weight and hydrophobic distribution, PhaT _H13 may be a combination of Phasins and PHA.

The present invention further utilizes the PHA biosynthesis-related proteins obtained by the present invention to further utilize the heterologous expression of Escherichia coli, which mainly uses a primer to carry out a polymerase chain reaction (PCR) to introduce a specific restriction enzyme enzyme cleavage into both ends of the sequence, and performs After the selection, the same restriction enzymes were used to clone the gene fragment into the same cleavage expression vector pET21b or pET28a. Thereafter, it was transformed into E. coli BL21 (DE3) for expression, and induced with different concentrations of Isopropyl-β-D-Thiogalactoside (IPTG) and different times. As an analysis of the heterologous protein expression of these genes, PhaC _H13 , PhaD _H13 , PhaB _H13 , MaoC _H13, PhaR _H13 and PhaT _H13 can all be expressed in E. coli under IPTG induction (as shown in Figures 2 and 3). ). These recombinant proteins are all presented as soluble proteins. Results confirmed by heterologous performance of E. coli can be a lot to show PhaC _H13 and PhaD _H13, were tested for protein activity that a lot of performance, and to determine the function PhaC _H13 and PhaD _H13 protein. In terms of activity testing, it mainly uses (R,S)-3-hydroxybutyridinase A[(R,S)-3-Hydroxybutyryl-CoA,3HB-CoA] to react with an enzyme. If it has the activity of PHA synthase, It will polymerize (R)-3HB-CoA into PHB and release Coenzyme A. Further, it is decomposed into thionitrobenzoate (TNB) by coupling with CoA with 5,5'-Dithiobis (2-nitrobenzoic acid, DTNB); yellow is detected by OD _412. The amount of TNB is used to estimate the release of CoA, and the activity of PHA polymerase is pushed back by the release of CoA (M h, U., et al., Biochem . 38: 826-837, 1999; Valentin, HE, and Steinb Chel. A., Appl. Microbiol. Biotechnol ., 40: 699-709, 1994).

The present invention utilizes a heterologous expression in E. coli and the resulting purified protein PhaD _H13 and PhaC _H13 (second diagram), to 3-hydroxybutyrate acyl coenzyme A is receiving quality, for PhaDC _H13 synthase characterization, as in the fourth diagram shows only PhaD _H13 and PhaC _H13 exist (expressed in PhaDC _H13) have activity, PhaDC _H13 specific activity of about 5 U / mg, wherein the enzyme activity unit (unit, U) to be converted 1 μmol per minute by mass (1 mol /min)(M H et al., supra, 1999). According to the results of different temperature tests, PhaDC _H13 was the optimum reaction temperature at 45 °C. According to the results of heat stability test, PhaDC _H13 was found to have 50% enzyme activity after being treated at 75 ° C for 5 minutes (as shown in the fifth figure). Tested at different ion concentrations, PhaDC _H13 was found to be active in the presence of potassium or sodium ions from 1 M to 4 M, with the highest enzyme activity at 4 M potassium ion concentration (as shown in Figure 6). The results of a long-term (three-week) preservation test in a high salt environment showed that PhaDC _H13 still retained 90% activity (as shown in Figure 7).

The above results show that the PHA biosynthesis enzyme from Haloterrigena sp. H13 has the characteristics of high salt tolerance, can adapt to low water activity environment such as high ion concentration, and has good stability to heat, suitable for industrial and extracellular production applications. Therefore, it has a good applicability in the future production and development of PHA.

PHA synthase is the key enzyme for PHA biosynthesis. The activity of PHA synthase and the specificity of the receptor determine the PHA yield and monomer diversity. Therefore, the invention patents currently in PHA are heavily focused on PHA synthetases, such as U.S. Patent Nos. 5,968,805, 6,475,734 B1, 6,903,220 B2 and 6,972,192 B2. However, different PHA monomer metabolism pathways, combined with PHA synthase, are also the key to PHA producing different monomer compositions. Therefore, in recent years, some invention patents have also targeted proteins of the PHA metabolic pathway, such as U.S. Patent No. 7183987 B2. Therefore, in the present invention, in addition to the gene sequence of the PHA synthase in the Haloterrigena sp. H13 gene, the gene upstream of the PHA biosynthesis pathway is also found, and in the future, the heterologous expression can be used together with the PHA synthase, which can be in a test tube. Or in a heterologous host, PHA can be synthesized more efficiently, and then PHA can be tailored to different needs depending on the needs of different uses.

In summary, the present invention provides a molecular method for screening PHA biosynthesis genes, which can be used for screening and recombination of PHA biosynthesis proteins, and to prepare a PFA biosynthesis enzyme with high specificity and high efficiency; the present invention also provides extreme The gene sequence of the polyhydroxyalkanoate (PHA) biosynthesis gene group of the high-salt archaea Haloterrigena sp. H13 is used as the basis for the research and development of PHA biosynthesis and regulation. More importantly, the present invention provides a technique for expressing a large number of genes such as maoC, phaD, phaC, phaB and phaR of Haloterrigena sp. H13 in a heterologous expression vector pET21b or pET28a in Escherichia coli BL21 (DE3), and expressing in Escherichia coli. PhaC _H13 , PhaD _H13 , PhaB _H13 , MaoC _H13 and PhaR _H13 are all present as soluble proteins. Different MaoC and its mutant proteins can change their substrate specificity and produce different kinds of monomers with different carbon chain lengths. Therefore, the gene and protein of MaoC _H13 of Haloterrigena sp. H13 provide a good molecular material for research and development.

The heterologously expressed PhaDC _H13 protein has PHA synthetase activity when PhaD _H13 and PhaC _{H13 are} simultaneously present. Moreover, the PHA synthetase can withstand high temperature, high potassium, sodium ion concentration and long-term (three weeks) preservation, and still retain 90% activity. The development of the PHA synthase gene of Archean organisms not only enhances the phaC gene pool for the construction of more diverse PhaCs and more novel PHAs, but also develops extreme enzymes that utilize high-salt Pacific organisms. Expression in E. coli can be a large number of active and archaeal enzymes PhaD _H13 PHA biosynthesis genes and PhaC _H13 carrier, PhaD _H13 and purified recombinant protein PhaC _H13 stable salt, and low resistance to high-temperature water activity PHA is synthesized and can be used to produce bioplastics (polyester, polyhydroxyalkyl ester, PHA) either intracellularly or extracellularly.

The invention is further illustrated by the following examples which are not intended to limit the invention. A person skilled in the art can make some modifications and modifications without departing from the scope of the invention.

Example

Example 1: Extremely high salt archaea Haloterrigena Cultivation of sp. H13

The extremely high-salt archaea Haloterrigena sp. H13 (the patent-registered strain CCRC 91015 of the Center for the Development of the Research Institute of Food Industry Development of the Republic of China) was isolated from a single colony, and a single colony was inoculated to 5 mL of 2% NaCl-containing NHA liquid medium ( Each liter of water contains 250 g NaCl, 10 g MgSO ₄ .7H ₂ O, 5 g KCl, 0.2 g CaCl ₂ .2H ₂ O, 1 g KNO ₃ and 5 g casein amino acid (Casamino acid), and 1 N NaOH was adjusted to pH 7.22. The cells were shaken at 80 rpm for 2 to 3 days with a 45 ° C constant temperature shaking bath (TKS Shaking Bath, SB302). Using a sterilized micro-dispenser, 1 mL of the bacterial solution was taken up to a mid-log phase (OD ₆₀₀ value of 0.5 to 0.6), and inoculated to a 25% NaCl-containing NHB liquid medium at a ratio of 1:20. The 45 ° C constant temperature shaking water bath (TKS Shaking Bath, SB302) was shaken at 80 rpm for 2 to 3 days.

Example 2: Extraction of chromosomal DNA from extremely high-salt archaea

The bacterial culture to the mid-log phase was incubated at a high speed centrifuge (Sorvall RC5C, DuPont Co.) at 6761 xg (8000 rpm) for 20 minutes at 4 °C. After removing the supernatant, use an appropriate amount of basal salt medium (suspend the cells with the above NHA liquid medium and casein amino acid to make the final suspension 1 mL. Dissolve 9 mL. The lysis solution (formulated by 8 mL of TE buffer (TE buffer) and 1 mL of 10% SDS) was added to the cell suspension, gently shaken and mixed, and 100 μL of proteinase K was added. Gg/mL) and treated at 55 ° C for 3 hours or overnight. Add 1 mL of 5 M NaCl (to a final concentration of 0.5 M), mix well and place on ice for 1 hour on ice. Take shake every 15 minutes. Evenly, the temperature can be evenly lowered and the SDS and protein can be precipitated. After the addition, there will be a white precipitate and the protein precipitation caused by the instantaneous change of salinity. Centrifuge (Sigma, 2K15) at 20,000 xg for 20 minutes at 4 °C. The supernatant was recovered, an equal volume of isopropanol was added and mixed to precipitate the DNA. The chromosomal DNA was picked up using a sterilized glass rod, air-dried for 5 minutes, and immersed in 70% alcohol for 5 minutes. Air dry for 5 minutes, repeat 3 times to ensure complete Remove salt and isopropanol or other substances. Add 100 μL of TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0) to dissolve the DNA back into the solution. Add 1 μL. ribonuclease a (RNase a). stored at 4 ℃. Press the DNA is completely dissolved, to a spectrophotometer (Ultropspec 1000, Pharmacia Biotech) Determination of the ratio of OD ₂₆₀ / OD ₂₈₀ after dilution, DNA and RNA, the OD ₂₆₀ / OD The ratio of ₂₈₀ should be 1.8 and 2.0. If the ratio is too low, it indicates the presence of protein impurities, which must be repurified and the DNA content calculated. When the ratio of OD ₂₆₀ is 1, the double-stranded DNA concentration is 50 μg/μL. Electrophoresis was carried out using a 0.8% agarose gel at 50 V for 1 hour and labeled with λ/ Hin d III Digest DNA Marker (Protec Biotech).

Example 3: Preparation of DNA probes

The ORF sequence designation primer gene pair (SEQ ID NO. 1 and SEQ ID NO) using the known ultra-high salt archaea Haloarcula marismortui ATCC43049, which is known to be fully genetically sequenced, is designated as the PHA synthase ( phaC ) gene (Polyhydroxyalkanoate synthase, phaC ). 2) (Lin Ziyi, Master's thesis, Department of Life Sciences, National Chung Hsing University, 2006), Haloarcula sp . strain HLR2 purified from Yanshan, Tongluo Salt Field, Miaoli County (published in the Food Industry Research Institute for bio-storage) And the research center, the chromosomal DNA with the accession number BCRC AR10013) was used as a template for polymerase chain reaction to obtain the product of phaC _HLR2 (SEQ ID NO. 3) as a probe (You Yuting, Master of Science in Life Science, National Chung Hsing University, 2006). Labeling was then carried out using a DIG system (Roche, DIG Labeling Kit), and this labeled DNA was used as a nucleic acid probe in the following examples.

Example 4: Southern blotting method to detect extremely high salt archaea PHA synthase

The Halocerrigena sp H13 chromosomal nucleic acid was subjected to nucleic acid electrophoresis analysis after restriction enzyme enzyme NotI, and then the gel was added to 0.25 N HCl buffer for 20 minutes, the liquid was removed, and a denature buffer (0.5 N NaOH; After 1.5 minutes of 1.5 M NaCl), the liquid was removed, and a neutralization buffer [1.5 M NaCl; 1 M Tris] was added for 20 minutes and finally stored in a neutralization buffer. A HybondTM-N+Nylon membrane (Amersham, Co.) was prepared and subjected to vacuum evacuation using a vacuum bucker (Gucuum Blotter, GHE 5560). After blotting of ultraviolet fixing device ^{(XL-1000 UV Crosslinker Linker TM} ) cross-linked to an automatic mode (auto cross-link) (1200 X 100 μJ / cm 2) treated twice, the nucleic acids immobilized on the membrane. Hybridization and immunodetection (Roche) were performed using the nucleic acid probe prepared as described above. 300 ng of template DNA ( phaC _HLR2 ) was taken from Roche's DIG Labeling Kit and quantified to 15 μL with sterilized secondary deionized water. After heating for 10 minutes in a 100 ° C water bath, it was placed on ice for 5 minutes. Then, 2 μL of Hexanucleotide Mix 10X (vial 5), 2 μL of dNTP Labeling Mix (vial 6), and 1 μL of Klenow enzyme labeling grade (vial 7) were added, mixed uniformly, and placed in a 37 ° C water bath for 20 hours. Thereafter, the mixture was heated in a 65 ° C water bath for 10 minutes, and then placed on ice for 5 minutes to terminate the reaction, and stored at -20 ° C. After probe preparation, probe calibration efficiency determination and DNA hybridization were performed according to Roche's DIG Labeling Kit instructions. The signaled nucleic acid fragments are then recovered and stored in a vector that has been subjected to the same restriction enzyme enzyme to establish a desired gene pool.

Example 5: Gene bank search and nucleic acid sequencing

From the gene pool established above, an individual selected strain is selected for amplification, and the plastid nucleic acid is extracted, and then heated to be a single-stranded nucleic acid, and spotted on a HybondTM-N ⁺ Nylon (Amersham, Co.) membrane. Above, it is treated twice with an ultraviolet fixture to fix the nucleic acid on the membrane. Hybridization and immunodetection (Roche) were performed using the nucleic acid probe prepared as described above. The plastid nucleic acid with the signal is then sequenced with an ABI 3700 sequencer to obtain the complete sequence. The resulting sequences were analyzed and annotated using BLAST (NCBI).

Example 6: Heterologous expression of proteins

After the primers designed with NdeI and HindIII cleavage, the PCR reaction of specific plastids can be used to increase the product of primers containing NdeI and HindIII cleavage at both ends. -Easy vector (Promega Co.). Then, the NdeI and HindIII cleavage sites were used to select the fragment and bind to the expression vector pET-21b or pET-28a by the same enzyme cleavage activity for 16 hours, and then transformed into E. coli JM101 for screening, and screening by drug resistance genes. Thereafter, the extracted plastids were confirmed by electrophoresis analysis using NdeI and HindIII. The confirmed plastid was transformed into the expression strain Escherichia coli BL21 (DE3), induced by different concentrations of Isopropyl-β-D-Thiogalactoside (IPTG), and utilized. Protein electrophoresis analysis. The heterogeneous manifestation of protein.

The method of protein purification is described in the manual of "A handbook for high-level expression and purification of 6xHis-tagged proteins" by Amersham Biosciences. The detailed steps are as follows. Show: Select a single colony of E. coli BL21 (DE3) containing a specific gene expression vector to 5 mL of LB liquid medium containing the corresponding antibiotic [containing 50 to 100 μg/mL penicillin or connamycin ( Kanamycin) was incubated overnight at 37 ° C with shaking at 200 rpm. Take 5 mL to 500 mL of LB liquid medium (containing 50 to 100 μg/mL penicillin or kanamycin) containing the corresponding antibiotics at 37 ° C, and shake culture at 200 rpm for about 2 hours. When the OD ₆₀₀ is 0.5, IPTG is added to a final concentration of 50 μM, followed by induction at 200 rpm at 37 ° C for 5 to 6 hours, and placed on ice for 10 minutes, centrifuged at 4 ° C. The supernatant was removed, suspended in 50 mM Tris buffer (containing 25% NaCl, 0.2% KCl, 0.5% MgSO ₄ ‧7H ₂ 0) and disrupted with a ultrasonic oscillator (Sonics & Materials, Inc. VCX-750 Watt) The bacteria [40% intensity, pulse on for 1 second, pulse off for 2 seconds)], centrifuged at 12599 xg for 20 minutes at 4 ° C, and the supernatant was taken. Was added 2 mL of ^{Ni Sepharose TM 6 Fast Flow resin (} Amersham Co.), placed on ice, with gentle agitation for 20 minutes. Open the column to collect the flow through (FT), then add 10 mL [5 column volume] 50 mM Tris buffer (containing 25% NaCl, 0.2% KCl, 0.5% MgSO _{4 ).} ‧7H ₂ 0 and 0 mM imidazole), the supernatant was collected. Thereafter, elution was carried out using concentrations of 10, 20, 50, 100, 200, 300, and 500 mM imidazole, and collected in test tubes, and different target proteins were transcribed according to the concentration of imidazole. The collected tube protein samples were analyzed by 12.5% SDS-PAGE, and the samples with the highest purity and high yield were selected for the following activity analysis.

Example 7: Activity test of PHA synthase

PHA polymerization enzyme activity analysis, mainly to detect the PHA polymerization reaction, will release CoA, using DTNB will bond with CoA, and decompose into TNB, while TNB is yellow, with obvious absorption peak at OD ₄₁₂ nm, Therefore, the amount of TNB can be measured by a spectrophotometer (Ultropspec 1000, Pharmacia Biotech), and then the content of CoA can be estimated, and the activity of PHA polymerase can be pushed back by the release amount of CoA (Valentin and Steinbuchel, 1994). Activity tests are divided into continuous and discontinuous. The buffer for the continuous reaction was 50 mM Tris buffer, 25% NaCl, 0.2% KCl, 0.5% MgSO ₄ . 7H ₂ 0, 1 mM DTNB, 1 mM HBCoA and PHA polymerase were added and reacted at room temperature. The amount of change in OD ₄₁₂ mm. The discontinuous reaction consisted of 50 mM Tris buffer, 25% NaCl, 0.2% KCl, 0.5% MgSO ₄ . 7H ₂ 0, ₁ mM HBCoA, and PHA polymerase at 45 ° C in a total volume of 320 μL of reaction. The reaction was carried out, and 40 μL of the supernatant was taken at a specific time, and 100 μL of 10% TCA was added to terminate the reaction. The supernatant was centrifuged, mixed with 800 μL of 1 mM DTNB, and the amount of change of OD ₄₁₂ mm was detected to estimate the activity of PHA polymerase (M). H et al. , supra, 1999). CoA is ε412 = 13600 cm ^-1 M ^-1 (Ellamn, 1959) at OD ₄₁₂ mm. Enzyme activity unit can convert 1 μmol of mass per minute (1 μmol/min) (M H et al. , 1999). This study adopted discontinuous reaction proceeds, PhaC _H13 and PhaD _H13 ratio was added with purified to give a 1: 1 mixture by hydroxybutyrate acyl coenzyme A (3-Hydroxybutyryl-CoA) as by mass of the total reaction volume of 320 μL, in 45 The reaction was carried out at °C.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention.

The first panel shows the arrangement of the PHA biosynthetic gene group of Haloterrigena strain H13.

The second panel shows a protein electrophoresis pattern of a large amount of the product of the homologous synthesis and regulation of PHA synthesis in Haloterrigena strain H13 by E. coli BL21 (DE3).

The third panel shows a large number of electrophoresis patterns of Hatoerrigena strain H13 and PhaT protein expressed in Escherichia coli BL21 (DE3), wherein Lane 1 (Lane 1) is a crude extractable protein from cells of Escherichia coli (DE3), diameters 2 and 3. (Lane 2 and 3) are crude extraction of soluble proteins from cells obtained after induction with E. coli having pET28a-PhaT _H13 at 50 μM and 500 μM IPTG, respectively.

The fourth graph shows PhaD _H13 and purified by PhaC _H13 PHA biosynthesis release test measurement CoA, CoA to release results obtained from the plotted time pair.

The fifth panel shows the PHA biosynthesis activity versus temperature for PhaDC _H13 .

Figure 6 shows a plot of PHA biosynthesis activity versus ionic strength for PhaDC _H13 .

Figure 7 shows a plot of PHA biosynthesis activity versus storage time for PhaDC _H13 .

<110> National Chung Hsing University

<120> Polyhydroxyalkanoate biosynthesis gene group of extreme high-salt archaea and application of enzyme in bioplastic production

<130>

<160> 15

<170> PatentIn version 3.4

<210> 1

<211> 20

<212> DNA

<213> Artificial

<220>

<223> syntheic DNA

<400> 1

<210> 2

<211> 18

<212> DNA

<213> Artificial

<220>

<223> Synthetic DNA

<400> 2

<210> 3

<211> 1434

<212> DNA

<213> Haloarcula sp. HLR2

<400> 3

<210> 4

<211> 1617

<212> DNA

<213> Haloterrigena sp. H13

<400> 4

<210> 5

<211> 549

<212> DNA

<213> Haloterrigena sp. H13

<400> 5

<210> 6

<211> 642

<212> DNA

<213> Haloterrigena sp. H13

<400> 6

<210> 7

<211> 744

<212> DNA

<213> Haloterrigena sp. H13

<400> 7

<210> 8

<211> 321

<212> DNA

<213> Haloterrigena sp. H13

<400> 8

<210> 9

<211> 486

<212> DNA

<213> Haloterrigena sp. H13

<400> 9

<210> 10

<211> 538

<212> PRT

<213> Haloterrigena sp. H13

<400> 10

<210> 11

<211> 182

<212> PRT

<213> Haloterrigena sp. H13

<400> 11

<210> 12

<211> 213

<212> PRT

<213> Haloterrigena sp. H13

<400> 12

<210> 13

<211> 247

<212> PRT

<213> Haloterrigena sp. H13

<400> 13

<210> 14

<211> 106

<212> PRT

<213> Haloterrigena sp. H13

<400> 14

<210> 15

<211> 161

<212> PRT

<213> Haloterrigena sp. H13

<400> 15

Claims (7)

An isolated nucleic acid molecule selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO: 7; (ii) having the nucleotide of SEQ ID NO: a nucleotide sequence having a sequence similarity greater than 90%; (iii) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 13; and (iiii) encoding and amino acid sequence similarity of SEQ ID NO: 13. More than 90% of the nucleotide sequence. An isolated polypeptide having the amino acid sequence encoded by the nucleic acid molecule of claim 1 and having PhaB _H13 activity. A vector comprising a control sequence of a nucleic acid molecule according to claim 1 and a homologous control sequence of a non-salt genus H13 strain ( Haloterrigena sp. H13), wherein the nucleic acid molecule is operably linked The control sequences are connected. A recombinant microorganism comprising at least one carrier as described in claim 3 of the patent application. The recombinant microorganism of claim 4, wherein the microorganism is a bacterium or an archaea (Archaea). The recombinant microorganism according to claim 4, wherein the microorganism is selected from the group consisting of Escherichia coli, Haloterrigena sp., Ralstonia eutropha ), Pseudomonas aeruginosa , Allochromatium vinosum , Bacillus megaterium , and Haloferax mediterranei . A method for producing a polyester, comprising: providing a recombinant microorganism according to any one of claims 4 to 6; and growing the recombinant microorganism in a suitable environment to produce a polyhydroxy group Alkyl ester.