JP7565569B2 - Mutant isoprene synthase and screening method therefor - Google Patents

Description

The present invention relates to mutant isoprene synthases and screening methods for the same.

There are as many as 70,000 compounds made of isoprene units, including terpenes, terpenoids, and carotenoids, but the activity of the enzymes that synthesize these compounds is very low. The need for improvement is widely recognized.

However, in both terpene and isoprenoid synthesis methods, the products are colorless and volatile, and there was no easy way to observe the activity of terpene and isoprene synthases. There is an example of obtaining highly active mutants by examining a library of tens of thousands of mutants one by one using chromatography methods such as HPLC and GC-MS (Patent Document 1). In order to improve the biosynthetic enzymes of terpene and isoprenoids, a high-throughput activity detection technology is required (Non-Patent Document 1).

The present inventors previously developed methods to visualize "substrate consumption" (Patent Document 2, Patent Document 3, Patent Document 4, Patent Document 5). These methods involve "creating an E. coli strain with a carotenoid biosynthetic pathway and introducing a group of terpene synthase genes into the cells." The higher the activity of the terpene enzyme, the more it deprives the coexisting carotenoid biosynthetic pathway of substrates, and therefore the higher the substrate consumption, the less carotenoid biosynthesis will occur. As a result, by selecting clones with pale colony color, highly active terpene enzyme mutants were obtained.

The mevalonate pathway, an isoprenoid precursor pathway found in eukaryotes, also contains mevalonate, an intermediate that can be added to the medium. It was known that adding too much mevalonate to the medium would similarly result in excessive accumulation of DMAPP (dimethylallyl diphosphate), causing cell growth inhibition (Non-Patent Document 2). Recently, a case has been reported in which it was used to improve isoprene synthase (Non-Patent Document 3). In this report, a state of reduced growth was created by overexpressing HMG1, the rate-limiting enzyme of the mevalonate pathway found in yeast, and an active mutant of isoprene synthase (IspS) was obtained based on the recovery of growth. However, the cytotoxicity obtained by this method was very low, and the cell density difference that could be created overnight was only two-fold. It is difficult to put this method into practical use as a screening and selection technique.

Examples of improved activity of isoprene synthase are reported in Patent Document 1 and Non-Patent Document 3. This paper reports that three active mutations, F340L, I478V, and A570T, are found, and that among these, the W-mutants of F340L+A570T are considered to be the best.
However, the mutant isoprene synthase obtained in the present invention has a structure that is clearly different from that of the previously reported mutants.

U.S. Patent No. 9,273,298 JP 2019-170350 A JP 2018-198576 A JP 2014-223038 A JP 2011-125334 A

M. Tashiro and T. Umeno: Chemistry and Biology, 54, 562-567 (2016) VJJ Martin et al., Nat. Biotech., 21, 796 (2003) Wang, Metabolic Engineering, 39, 257 (2017)

The objective of the present invention is to provide a mutant isoprene synthase and a screening method for the same.

As a result of extensive research, the inventors of the present invention have developed a method for screening mutant isoprene synthases by introducing an isoprene synthase gene with random mutations into cells expressing a gene for an enzyme with dimethylallyl alcohol (DMAOH) phosphorylation activity, and using DMAOH sensitivity as an indicator. They have also confirmed that mutant isoprene synthases can be obtained by this method, thereby completing the present invention.

That is, the present invention is as follows.
1. A mutant isoprene synthase or a derivative thereof, characterized in that it has amino acid deletions at positions 1 to 52 in the amino acid sequence shown in SEQ ID NO:7 and has amino acid substitutions at one, two or three sites selected from the group consisting of positions 218, 587 and 593.
2. The mutant isoprene synthase or a derivative thereof according to the preceding item 1, wherein the mutant isoprene synthase synthesizes a higher amount of isoprene than a wild-type isoprene synthase.
3. The mutant isoprene synthase or derivative thereof according to the above item 1 or 2, wherein the amino acid substitution is at least one selected from the following group:
H218R, T587A and F593L.
4. The mutant isoprene synthase or derivative thereof according to any one of the above items 1 to 3, wherein the amino acid substitution is one selected from the following (1) or (2):
(1) H218R, T587A, and (2) F593L.
5. A mutant isoprene synthase or a derivative thereof, which has amino acid deletions at positions 1 to 52 in the amino acid sequence shown in SEQ ID NO:7 and has amino acid substitutions of H218R and T587A.
6. A mutant isoprene synthase or a derivative thereof, which has amino acid deletions at positions 1 to 52 in the amino acid sequence shown in SEQ ID NO:7 and has an amino acid substitution of F593L.
7. A mutant isoprene synthase gene comprising any one of the following genes (1) to (7):
(1) A gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 12,
(2) A gene encoding a polypeptide in which 1 to 20 amino acids have been substituted, deleted, inserted and/or added in the amino acid sequence set forth in SEQ ID NO: 11 or 12, and which has substantially the same activity as the activity of producing isoprene using dimethylallyl diphosphate as a substrate, which is possessed by a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 12.
(3) A gene encoding a polypeptide having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 11 or 12 and having substantially the same activity as the activity of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 12 to produce isoprene using dimethylallyl diphosphate as a substrate.
(4) A gene consisting of DNA having the base sequence set forth in SEQ ID NO: 13 or 14.
(5) A gene that hybridizes under stringent conditions with DNA having a base sequence complementary to DNA having the base sequence set forth in SEQ ID NO: 13 or 14, and encodes a polypeptide having an activity of producing isoprene using dimethylallyl diphosphate as a substrate that is substantially equivalent to the activity of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 or 12.
(6) A gene consisting of DNA in which 1 to 50 bases in the base sequence set forth in SEQ ID NO: 13 or 14 have been substituted, deleted, inserted and/or added; and
(7) A gene consisting of DNA having a homology of 90% or more to the DNA consisting of the base sequence set forth in SEQ ID NO: 13 or 14.
8. A mutant isoprene synthase having an amino acid sequence selected from any one of the following (1) to (3):
(1) an amino acid sequence set forth in SEQ ID NO: 11 or 12;
(2) an amino acid sequence comprising 1 to 20 amino acids substituted, deleted, inserted and/or added in the amino acid sequence set forth in SEQ ID NO: 11 or 12, and having an activity of producing isoprene using dimethylallyl diphosphate as a substrate, which is possessed by a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 12; and
(3) An amino acid sequence having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 11 or 12, and having activity of producing isoprene using dimethylallyl diphosphate as a substrate, which is possessed by a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 12.
9. A method for screening for mutant isoprene synthases, comprising introducing a library of mutated isoprene synthase genes into cells capable of synthesizing dimethylallyl diphosphate from dimethylallyl alcohol, and using an increase or decrease in the cell proliferation rate of the cells as an indicator.
10. The screening method according to the preceding item 9, wherein the increase in the cell proliferation rate of the cells is an increase in the amount of isoprene synthesized using dimethylallyl diphosphate as a substrate.
11. The screening method according to the above 9 or 10, wherein the cell proliferation rate of the cells is a cell proliferation rate in the presence of dimethylallyl alcohol.
12. The screening method according to any one of the preceding aspects 9 to 11, wherein the cell is Escherichia coli.
13. The screening method according to any one of items 9 to 12 above, wherein the cell is a cell transformed with a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:6.
14. A method for producing a cell containing dimethylallyl diphosphate, comprising the step of introducing or adding dimethylallyl alcohol to a cell capable of synthesizing dimethylallyl diphosphate from dimethylallyl alcohol,
The method for producing the cell is characterized in that the cell is a cell transformed with a mutant isopentenyl phosphate kinase.
15. The method according to item 14 above, wherein the cell contains more dimethylallyl diphosphate than a wild-type cell.

The present invention provides a mutant isoprene synthase that synthesizes a higher amount of isoprene than the wild-type isoprene synthase, as well as a method for screening mutants of the enzyme.

Plasmid MAP (pJ211-J23119-IPK _mut ). Colony counts of IPK _mut mutants. Expression of the IPK mutant conferred sensitivity to DMAOH in host cells. Sensitivity of host cells to DMAOH by IPK expression (liquid culture). Effect of DMAOH concentration in the medium on the viable cell count of IPK-expressing strains. Plasmid MAP (pT5/lacO-IspS). Plasmid MAP (PJ211-pJ23119-IPK _mut ). DMAOH/IPK _mut resistance of IspS mutants (second screening). The experiment was carried out twice, with an interval of two days. Amino acid mutation sites of IspS activity mutants. Plasmid MAP (pT5-IspS). Plasmid MAP (pMEV-idi). Comparison of isoprene synthesis ability between wild-type IspS and active mutants (upper layer of dodecane) (N = 5). Comparison of isoprene synthesis ability between the wild type and active mutants (N=2). DMAPP sensitivity of E. coli strains expressing mutant and wild-type IspS.

(Summary of the Invention)
The present invention relates to a "mutant isoprene synthase" and a "screening method for mutant isoprene synthase."

The present invention relates to methods for improving the activity of isoprene synthase as well as to mutant isoprene synthases (IspS).
In the present invention, (1) we established a selection system based on avoidance of toxicity by accumulation of DMAPP (dimethylallyl diphosphate), the direct substrate of IspS, rather than toxicity due to accumulation of isomeric IPP, (2) a precursor supply pathway realized by a single enzyme, and (3) a substrate excess state was not an endogenous pathway but could be changed by the amount of precursor (DMAOH) added to the medium, thus establishing a screening method that can efficiently obtain highly active mutant IspS.
In the present invention, DMAPP excess is created using an artificial enzyme with a low number of steps (2 steps) realized with one enzyme. As shown below, this method easily realizes a selection system with a selectivity of 1000 to 10,000 times. Furthermore, the selection pressure can be freely set by the DMAOH solution added to the medium.

The present invention also covers a "method for producing cells containing large amounts of DMAPP (a method for inducing abnormal accumulation of DMAPP by phosphorylating DMAOH in cells)" for use in the above screening method.

(Mutant isoprene synthase or its derivatives)
The mutant isoprene synthase or a derivative thereof of the present invention means an enzyme which is different in one or more amino acids or bases compared to the wild-type isoprene synthase.
Wild-type isoprene synthase (IspS) is produced in plants such as the Populus genus, and is known to synthesize isoprene from dimethylallyl diphosphate (DAMPP) as a substrate.
In particular, it is preferable that the mutant isoprene synthase or a derivative thereof of the present invention synthesizes a higher amount of isoprene (has a higher synthetic activity) than the wild-type isoprene synthase.
The derivative of the present invention also includes a protected derivative, a glycosylated derivative, an acylated derivative, or an acetylated derivative of the mutant isoprene synthase.
The protected derivatives, glycosylated derivatives, acylated derivatives, or acetylated derivatives can be obtained by methods known per se.
In addition, the mutant of the present invention is preferably a mutant that enhances the enzymatic activity, expression level, or stability of the expressed enzyme. Also preferred are mutants that enhance substrate specificity. In particular, mutants that enhance enzymatic activity are preferred.
In the screening method of the present invention, a library of mutants (a gene library in which substitutions, deletions, insertions and/or additions have been introduced into the wild type) is created, and from the library, mutants having high enzymatic activity, expression level or stability in the host can be obtained.
The genes may be of any origin, such as genes extracted from natural cells, genes in a constructed library, chemically synthesized genes, PCR amplified genes, etc. Since the screening method of the present invention uses the substrate consumption ability of the expressed enzyme as an index, it is also possible to screen unidentified genes.
The isoprene synthase gene (a gene library in which substitutions, deletions, insertions, and/or additions have been introduced into the wild type) can be introduced into cells capable of synthesizing dimethylallyl diphosphate from dimethylallyl alcohol by a standard method used in recombinant gene technology, for example, by inserting the gene to be screened into a vector and transforming the cells.

The mutant isoprene synthase or a derivative thereof of the present invention has the following mutations in its amino acid sequence compared to the isoprene synthase derived from Populus alba (SEQ ID NO: 7).
Deletions at positions 1 to 52 and substitutions at positions 218, 587 and/or 593.
Furthermore, the mutant isoprene synthase of the present invention has the following mutations in its amino acid sequence compared to the isoprene synthase derived from Populus alba (SEQ ID NO: 7).
M1-T52del, and H218R, T587A and/or F593L.

The mutant isoprene synthase of the present invention has any one of the following mutations in its amino acid sequence, as compared with the isoprene synthase derived from Populus alba (SEQ ID NO: 7):
(1) M1-T52del, H218R, T587A (SEQ ID NO: 11)
(2) M1-T52del, F593L (SEQ ID NO: 12)

The mutant isoprene synthase of the present invention has one or more amino acid mutations as shown below compared to the isoprene synthase derived from Populus alba (SEQ ID NO: 7), and it has been confirmed in the Examples below that the mutant isoprene synthase of the present invention synthesizes a higher amount of isoprene than the wild-type isoprene synthase derived from Populus alba.
(1) M1-T52del, H218R, T587A (SEQ ID NO: 11)
(2) M1-T52del, F593L (SEQ ID NO: 12)

A high isoprene synthesis amount (isoprene synthesis activity) is 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 2.0 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times or more, 2.5 times or more, 2.6 times or more, 2.7 times or more, 2.8 times or more, 2.9 times or more, 3.0 times or more, about 4.0 times (or more), about 5.0 times (or more), about 6.0 times (or more), about 7.0 times (or more), about 8.0 times (or more), about 9.0 times (or more), about 10 times (or more), about 11 times (or more) compared to the wild-type isoprene synthase derived from Populus alba (SEQ ID NO: 7). ), about 12 times (or more), about 13 times (or more), about 14 times (or more), about 15 times (or more), about 16 times (or more), about 17 times (or more), about 18 times (or more), about 19 times (or more), about 20 times (or more), about 21 times (or more), about 22 times (or more), about 23 times (or more), about 24 times (or more), about 25 times (or more), about 26 times (or more), about 27 times (or more), about 28 times (or more), about 29 times (or more), about 30 times (or more), about 31 times (or more), about 35 times (or more), and about 40 times (or more) of isoprene synthesis amount can be exemplified.

(Mutant isoprene synthase gene)
The mutant isoprene synthase gene of the present invention is: (1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 12; (2) a gene encoding a polypeptide in which 1 to 20 amino acids, preferably 1 to 15, more preferably 1 to 10, and most preferably 1 to 5 amino acids in the amino acid sequence set forth in SEQ ID NO: 11 or 12, have been substituted, deleted, inserted, and/or added, and which has substantially the same activity as the activity of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 12 to produce isoprene using dimethylallyl diphosphate as a substrate; or (3) a gene having 90% or more homology to the amino acid sequence set forth in SEQ ID NO: 11 or 12 and having the activity of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 12 to produce isoprene using dimethylallyl diphosphate as a substrate. (4) a gene encoding a polypeptide having substantially the same activity as the base sequence set forth in SEQ ID NO: 13 or 14; (5) a gene encoding a polypeptide that hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence set forth in SEQ ID NO: 13 or 14 and has substantially the same activity of producing isoprene using dimethylallyl diphosphate as a substrate as the polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 or 12; (6) a gene consisting of DNA in which 1 to 50 bases have been substituted, deleted, inserted and/or added in the base sequence set forth in SEQ ID NO: 13 or 14; and (7) a gene consisting of DNA having 90% or more homology to the base sequence set forth in SEQ ID NO: 13 or 14.
The above-mentioned gene (1) is preferably a gene encoding a mutant isoprene synthase obtained by the screening method of the present invention for a mutant isoprene synthase. The mutant isoprene synthase has an activity of producing isoprene using dimethylallyl diphosphate as a substrate.
The gene (2) above is a gene encoding a polypeptide into which a mutation has been introduced into the mutant isoprene synthase of the present invention to such an extent that the enzyme activity is not lost. Such mutations include not only mutations occurring in nature, but also artificial mutations. Examples of means for generating artificial mutations include site-directed mutagenesis (Nucleic Acids Res. 10, 6487-6500, 1982). The number of mutated amino acids is usually 20 or less, preferably 10 or less, more preferably 5 or less, and most preferably 3 amino acids. Whether or not the polypeptide into which the mutation has been introduced retains the enzyme activity can be determined, for example, by introducing a gene encoding the polypeptide into Escherichia coli or the like, expressing it, and examining whether or not the Escherichia coli or the like can produce isoprene using dimethylallyl diphosphate as a substrate. Since the enzyme activity is likely to be lost when a mutation is introduced into the position or site of these amino acids, it is preferable to introduce the mutation into an amino acid other than these amino acids.
The above (2), (3) and/or (5) "activity substantially equivalent to the activity of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 or 12 to produce isoprene using dimethylallyl diphosphate as a substrate" may mean that the level of activity is stronger or weaker than the activity of a polypeptide having the amino acid sequence set forth in SEQ ID NO: 11 or 12 to produce isoprene using dimethylallyl diphosphate as a substrate. For example, examples of activity include 0.1x, 0.2x, 0.3x, 0.4x, 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, 1.0x, 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, 2.0x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3.0x, 4.0x, 5.0x, 10x, 15x, 20x, 30x, 40x, and 50x.
Alternatively, homology can be calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, using default or initial setting parameters).
The gene (3) above includes a gene encoding a polypeptide having a homology of 90% or more, preferably 93% or more, more preferably 95% or more, even more preferably 98% or more, and most preferably 99% or more to the amino acid sequence set forth in SEQ ID NO: 11 or 12.
The gene (5) above is a gene obtained by utilizing hybridization between DNAs. The "stringent conditions" in this gene refer to conditions under which only specific hybridization occurs and non-specific hybridization does not occur. Such conditions are usually hybridization in a buffer containing 5×SSC and 1% SDS at 37° C. and washing treatment with a buffer containing 1×SSC and 0.1% SDS at 37° C., preferably hybridization in a buffer containing 5×SSC and 1% SDS at 42° C. and washing treatment with a buffer containing 0.5×SSC and 0.1% SDS at 42° C., and more preferably hybridization in a buffer containing 5×SSC and 1% SDS at 65° C. and washing treatment with a buffer containing 0.2×SSC and 0.1% SDS at 65° C. Whether or not the DNA obtained by hybridization encodes a polypeptide having activity can be determined, for example, by introducing the DNA into Escherichia coli or the like, expressing it, and examining whether or not the Escherichia coli or the like can produce isoprene using dimethylallyl diphosphate as a substrate. The DNA obtained by hybridization usually has high homology with the gene (SEQ ID NO: 13 or 14) in (4) above. High homology refers to a homology of 90% or more, preferably a homology of 95% or more, more preferably a homology of 98% or more, and most preferably a homology of 99% or more.
The gene (6) above is a gene consisting of DNA having the base sequence set forth in SEQ ID NO: 13 or 14, in which 1 to 50, preferably 1 to 30, more preferably 1 to 20, particularly preferably 1 to 10, and most preferably 1 to 5 base sequences have been substituted, deleted, inserted and/or added.
The gene (7) above includes a gene consisting of DNA having a homology of 90% or more, preferably 93% or more, more preferably 95% or more, even more preferably 98% or more, and most preferably 99% or more to the DNA consisting of the base sequence set forth in SEQ ID NO: 13 or 14.

(Mutant isoprene synthase)
The mutant isoprene synthase of the present invention comprises: (1) an amino acid sequence set forth in SEQ ID NO: 11 or 12; (2) an amino acid sequence in which 1 to 20, preferably 1 to 15, more preferably 1 to 10, and most preferably 1 to 5 amino acids have been substituted, deleted, inserted, and/or added in the amino acid sequence set forth in SEQ ID NO: 11 or 12, and which has substantially the same activity as the activity of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 12 to produce isoprene using dimethylallyl diphosphate as a substrate; or (3) an amino acid sequence which has 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 11 or 12, and which has substantially the same activity as the activity of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 12 to produce isoprene using dimethylallyl diphosphate as a substrate.
From the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptide when introducing mutations into the peptide, for example, mutual substitutions between homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) can be easily envisioned.

(Screening Method of the Present Invention)
The screening method for mutant isoprene synthase of the present invention involves introducing a mutant isoprene synthase gene (preferably an isoprene synthase gene library, in particular an isoprene synthase gene library into which random mutations have been introduced) into cells capable of synthesizing dimethylallyl diphosphate (DAMPP) from dimethylallyl alcohol (DMAOH) (preferably a cell into which isopentenyl phosphate kinase (IPK) has been introduced, more preferably Escherichia coli into which a mutant isopentenyl phosphate kinase has been introduced) in order to search for mutant isoprene synthases, and using an increase or decrease in the cell proliferation rate (preferably the possibility of cell proliferation) of the introduced cells in the presence of graded concentrations of dimethylallyl alcohol (any method is acceptable as long as DMAOH is present in the cells; for example, DMAOH may be added to the cells, or DMAOH may be added to the culture medium) as an indicator for screening for mutant isoprene synthases.
Furthermore, the present invention relates to a method for screening for a mutant isoprene synthase gene that is more active than the wild-type isoprene synthase, by comparing the cell growth rate of the introduced cells in the presence of graded concentrations of dimethylallyl alcohol with that of cells into which wild-type isoprene synthase has been introduced, and selecting cells that have a higher cell growth rate (or cells that require a higher concentration of dimethylallyl alcohol to stop cell growth).
Furthermore, the present invention relates to a screening method for comparing the cell growth rates of two or more clone-derived cells obtained by introducing an isoprene synthase gene library, preferably an isoprene synthase gene library into which random mutations have been introduced, in the presence of graded concentrations of dimethylallyl alcohol, and selecting cells having a higher cell growth rate than at least one other clone-derived cell (or cells having a higher dimethylallyl alcohol concentration required to stop cell growth than at least one other clone-derived cell), preferably cells having the 1st to 5th highest cell growth rates, the 1st to 10th highest cell growth rates, or the 1st to 50th highest cell growth rates (or cells having the 1st to 5th highest dimethylallyl alcohol concentration required to stop cell growth), more preferably any one or more cells among the cells having the highest cell growth rate (or cells having the highest dimethylallyl alcohol concentration required to stop cell growth). This screening method allows for obtaining mutant isoprene synthase genes with high isoprene synthase activity among cells into which an isoprene synthase gene library, preferably an isoprene synthase gene library into which random mutations have been introduced, has been introduced.

(Mutant isopentenyl phosphate kinase)
Wild-type isopentenyl phosphate kinase (IPK) is produced in archaea such as Thermoplasma acidophilum and catalyzes the synthesis of isopentenyl diphosphate (IPP) or dimethylallyl phosphate (DMAP), which are the basic building blocks of isopentenyl compounds, to produce isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DAMPP).
Similarly, it has been reported that a mutant isopentenyl phosphate kinase with three amino acid mutations, V73I, Y141V, and K204G, in IPK acquires monophosphorylation activity for dimethylallyl alcohol (DMAOH, also known as DMA), converting DMAOH to DMAP (LIU, Ying, et al. Scientific Reports, 2016, 6: 24117.).
The "mutant isopentenyl phosphate kinase" in the present invention is an enzyme (including a group of enzymes) involved in the consumption of dimethylallyl alcohol. In particular, the mutant isopentenyl phosphate kinase is not particularly limited as long as it has the action of directly or indirectly converting dimethylallyl alcohol (DMAOH) into dimethylallyl diphosphate (DAMPP).

(Cells capable of synthesizing dimethylallyl diphosphate from dimethylallyl alcohol)
In the present invention, the "cell capable of synthesizing dimethylallyl diphosphate from dimethylallyl alcohol" is not limited as long as it is a cell capable of biosynthesizing dimethylallyl diphosphate from dimethylallyl alcohol. It may be a natural cell or a cell transformed by genetic recombination. Examples of genetically modified cells include Escherichia coli, Bacillus subtilis, and yeast, which are cells that are easy to manipulate for genetic recombination.

(Increase or decrease in cell proliferation rate in the presence of dimethylallyl alcohol)
In the present invention, "an increase or decrease in the cell proliferation rate in the presence of dimethylallyl alcohol" refers to an increase or decrease in the cell proliferation rate in the presence of dimethylallyl alcohol due to an increase or decrease in the isoprene synthesis activity in the cells using dimethylallyl diphosphate (DAMPP) as a substrate.
That is, in the screening method of the present invention, a mutant isoprene synthase gene is obtained using as an indicator the cell growth rate in the presence of dimethylallyl alcohol of cells capable of synthesizing dimethylallyl diphosphate from dimethylallyl alcohol into which a mutant isoprene synthase gene has been introduced (or the cell growth rate in the presence of dimethylallyl alcohol of cells capable of synthesizing dimethylallyl diphosphate from dimethylallyl alcohol into which an isoprene synthase gene with random mutations has been introduced).
Furthermore, in the screening method of the present invention, the cell growth rate in the presence of dimethylallyl alcohol of cells capable of synthesizing dimethylallyl diphosphate from dimethylallyl alcohol into which a mutant isoprene synthase gene has been introduced is compared with the cell growth rate in the presence of dimethylallyl alcohol of cells capable of synthesizing dimethylallyl diphosphate from dimethylallyl alcohol into which a wild-type isoprene synthase gene has been introduced; cells with increased cell growth rate in the presence of dimethylallyl alcohol are selected; and the gene introduced into those cells is determined to be a mutant isoprene synthase gene with higher activity than the wild-type isoprene synthase.
Furthermore, in the screening method of the present invention, the cell growth rates in the presence of dimethylallyl alcohol of cells capable of synthesizing dimethylallyl diphosphate from methylallyl alcohol derived from two or more clones obtained by introducing an isoprene synthase gene library, preferably an isoprene synthase gene library into which random mutations have been introduced, are compared, cells with an increased (faster) cell growth rate in the presence of dimethylallyl alcohol are selected, and the gene introduced into the cells is determined to be a mutant isoprene synthase gene with high isoprene synthase activity.
The higher the isoprene synthesis activity of the mutant isoprene synthase using dimethylallyl diphosphate (DAMPP) as a substrate, the more dimethylallyl diphosphate it can consume, and therefore the more effectively it can counteract the cell growth inhibitory effect of dimethylallyl diphosphate.
Therefore, in cells that have been introduced with a mutant isoprene synthase gene and are capable of synthesizing dimethylallyl diphosphate from dimethylallyl alcohol (DMAOH), the higher the isoprene synthesis activity, the higher the cell growth rate in the presence of dimethylallyl alcohol, and the higher the cell growth rate in the presence of higher concentrations of dimethylallyl alcohol.

(How DMAOH phosphorylation leads to high DMAPP accumulation)
The present invention also relates to a method for accumulating high concentrations of DMAPP in cells capable of synthesizing dimethylallyl diphosphate from dimethylallyl alcohol (a method for producing cells containing a large amount of DMAPP).Specifically, by introducing the above-mentioned isopentenyl phosphate kinase gene (particularly, mutant isopentenyl phosphate kinase gene) into cells and then introducing dimethylallyl alcohol into the cells, high concentrations of DMAPP can be accumulated.The cells are preferably cells transformed with mutant isopentenyl phosphate kinase.
Furthermore, the cells are characterized by containing more DMAPP than wild-type cells. "Containing more DMAPP" means that the cells are more than 1.05 times, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, about 4.0 times (or more), about 5.0 times (or more), about 6.0 times (or more), about 7.0 times (or more), about 8.0 times (or more), about 9.0 times (or more), about 10 times (or more), about 11 times (or more) compared to the wild-type cells. Examples of DMAPP amounts in cells include about 12-fold (or more), about 13-fold (or more), about 14-fold (or more), about 15-fold (or more), about 16-fold (or more), about 17-fold (or more), about 18-fold (or more), about 19-fold (or more), about 20-fold (or more), about 21-fold (or more), about 22-fold (or more), about 23-fold (or more), about 24-fold (or more), about 25-fold (or more), about 26-fold (or more), about 27-fold (or more), about 28-fold (or more), about 29-fold (or more), about 30-fold (or more), about 31-fold (or more), about 35-fold (or more), and about 40-fold (or more).

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[DMAOH sensitivity of IPK _mut expressing strain]
1. Construction of expression system The gene for IPK derived from the scientific name (Thermoplasma acidophilum) was synthesized in a codon-optimized form for E. coli (total length 260 amino acids, 783-bases: SEQ ID NO: 6). In accordance with the report, the two amino acids "MM" at the N-terminus were removed from the natural sequence, and 17 amino acids "MRGSHHHHHHTDPALRA" were added (M1-M2delinsMRGSHHHHHHTDPALRA), and three amino acid mutations V73I, Y141V, and K204G were introduced. According to the report, the introduction of these mutations results in the acquisition of monophosphate activity of DMAOH. This was inserted downstream of the J23119-promoter using the restriction enzymes BamHI and XhoI to obtain pJ211-J23119-IPK _mut (RBS score set to 2048) (Reference: LIU, Ying, et al. Scientific Reports, 2016, 6: 24117.).
The plasmid MAP (pJ211-J23119-IPK _mut : sequence number 1) is shown in FIG.

2. DMAOH sensitivity of strains expressing IPK _mut mutants (agar medium)
E. coli XL1 Blue was transformed with pJ211-J23119-IPK _mut to form colonies. The colonies were picked and cultured overnight in LB medium, then diluted and 300 cells were plated on LB agar medium containing various concentrations of dimethylallyl alcohol (DMAOH). The colonies were left to stand at 37°C for 24 hours, and the number of colonies formed was counted (Figure 2).
E. coli expressing an IPK mutant (IPK _mut ) showed significantly higher sensitivity to DMAOH than strains that did not express IPK. The IPK _mut- expressing strain showed a decrease in cfu when DMAOH was added, and the survival rate was 0% at 10 mM or higher (blue line in Figure 2). DMAOH itself is not very toxic, and E. coli that did not express IPK (expressed GFP instead) did not show a decrease in colony number even when DMAOH was added up to 20 mM (green line in Figure 2).

3. DMAOH sensitivity of strains expressing IPK _mut mutants (liquid medium)
We investigated whether DMAOH sensitivity was also expressed in a liquid culture system (Figure 3).
Specifically, E. coli strains carrying PJ211-IPK _mut (XL1 blue: blue line in Fig. 3) and E. coli strains carrying PJ211-empty (yellow line in Fig. 3) were placed in LB medium with various DMAOH concentrations and cultured at 37°C for 12 hours with shaking. The cell density (OD) of each strain was measured.
In liquid culture, the sensitivity of IPK _mut -expressing strains to DMAPP was much more pronounced than on solid medium: at a DMAOH concentration of just 2 mM, cell density increase was virtually completely abolished, whereas the growth of the control strain not expressing IPK _mut was completely unaffected at this concentration.

4. DMAOH sensitivity of strains expressing _{IPK mut} mutants (solid medium)
To investigate how efficiently the excess DMAPP produced by this method could inhibit cell proliferation, the number of cells seeded on a plate was increased from 2 to 2 million, and the viable cell count was examined when DMAOH was added at various concentrations (0-20 mM) (Figure 4).
It was confirmed that the higher the DMAOH concentration, the lower the viable cell count. The viable cell count ratios at DMAOH concentrations of 5 mM, 10 mM, and 20 mM were 555/1,670/1, and 20,000/1, respectively, compared to the medium without DMAOH. This is clearly different from the weak bacteriostatic effect (bacterial density is halved) brought about by the enhancement of the mevalonate pathway, and by simple calculation, it is a concentration efficiency of 1,000 to 20,000 times.

From the above experiments, it was confirmed that when IPK _mut is expressed in the expression strain, the DMAPP concentration gradually increases depending on the concentration of DMAOH added to the medium. In other words, it was revealed that cell growth is stopped very efficiently by IPK _mut overaccumulating DMAPP through two-step phosphorylation, starting from DMAOH, which is less toxic. This "abnormal accumulation" of DMAPP can only be eliminated by a strong enzyme activity that consumes DMAPP. It has become possible to selectively grow only host cells that have a DMAPP-consuming enzyme with a higher enzyme activity.

The advantages of the method of the present invention are: 1) if DMAOH is not administered, abnormal accumulation of DMAPP does not occur (its cytotoxicity is not constantly expressed, but is "conditional" and is exerted only upon the addition of the prodrug DMAOH); and 2) it has an extremely high proliferation inhibitory effect (up to 20,000-fold).
The method of the present invention differs from conventional methods in that it can screen the consumption activity of DMAPP (the direct source of isoprene) rather than IPP (an additional unit in the formation of the isoprene skeleton), and furthermore, the addition of DMAOH was able to produce a three-order reduction in the viable cell count.

[Evolutionary engineering of IspS]
1. Construction of expression system
The gene (SEQ ID NO: 10) of IspS derived from Populus alba (573 amino acids (M1-T52del) with the N-terminal 52 amino acids of the full-length 595 amino acids (1815-bases) (SEQ ID NO: 7) truncated and a stop codon, totaling 1722-bases) was inserted downstream of PT5/LacO using the restriction enzymes BamHI and XhoI. The RBS score calculated by RBScalculator (https://salislab.net/software/predict_rbs_calculator) was 17,091.
The plasmid MAP (pT5/lacO-IspS: sequence number 2) is shown in FIG.

2. Library (first generation)
The IspS gene was amplified by epPCR.
The reaction conditions are as follows:
Template plamsid amount: 5 ng/reaction volume 50 μL
PCR Buffer NEB 10x Thermo Pol Reaction Buffer
dNTP concentration: 0.2 mM each, Mg concentration: 0.2 mM, Mn concentration: 10, 20, and 50 μM
NEB Taq DNA Polymerase, 5 units
Final yield: 5000 ng, amplification rate: 1000-fold Primer sequence 1: Fwd: 5-CTCGAAAATAATAAAGGGAAAATCAG-3 (SEQ ID NO: 8)
Primer sequence 2: Rev: 5- GTAGGGAACTGCCAGGCATCAAATA-3 (SEQ ID NO: 9)

The resulting DNA was gel extracted, further purified, and ligated into the vector under the following reaction conditions.
Vector: 100 ng, insert: 122 ng, reaction volume: 10 μL
Reaction time: 16 hours, reaction temperature: 16℃, NEB T4 DNA Ligase, 40 units

The obtained ligation solution was desalted and electroporated into E. coli strain BW25113. 1440 μL of SOC was added to 60 μL of competent cells and curation cultured at 37°C for 1 h (total volume 1.5 mL). 10 μL (1/150) was plated on an agar plate. The remaining 1490 μL was diluted with 40 mL of LB and cultured overnight (12 h) at 37°C with shaking. 2 mL of the obtained bacterial cell suspension was mini-prepped.
The next day, 196 colonies for Mn10, 512 colonies for Mn20, and 396 colonies for Mn50 were obtained on the agar plate, and the respective library sizes were determined to be 2.9×10 ⁴ for Mn10, 7.7×10 ⁴ for Mn20, and 5.9×10 ⁴ for Mn50.

3. Selection of highly active clones (primary screening)
The obtained IspS library was introduced into an IPK _mut expression strain (XL1-Blue strain with pJ211-IPK _mut ). Specifically, 5 μL of the IspS library plasmid was mixed with 100 μL of competent cells and left on ice for 15 minutes. 1150 μL of SOC was added and the cells were curated at 37°C for 1 hour (total volume 1250 μL).
The plasmid MAP (PJ211-pJ23119-IPK _mut : sequence number 3) is shown in Figure 6.
The obtained transformants were plated on LB-Agar plates (9 cm diameter) containing 0 mM, 12 mM, and 14 mM (0, 0.86 g/L, 1.01 g/L) DMAOH at approximately 500 cells/plate (5 plates were plated for each, so the screening size was approximately 2,500 for each). Total screening size: 2500 x 2 conditions x 3 libraries = 1.5 x ¹⁰⁴ .
The plates were incubated at 37°C for 24 hours to allow colony formation. As shown in Table 1, 12 colonies grew on the plate containing 12 mM DMAOH, and one colony grew on the plate containing 14 mM DMAOH (hit rate in primary screening: 13÷1.5× ¹⁰⁴ ×100＝0.087%).

Results of the primary screening. With this construct, it was confirmed that the viable cell rate of the wild-type IspS expression strain was 1/100 or less at 11 mM or more.

4. Secondary screening A total of 13 clones obtained above were isolated and miniprepared. They were treated with the restriction enzymes Hind III and EcoRI to selectively cleave the contaminating IPK _mut plasmid. The 13 isolated clones and the wild type were each reintroduced into a fresh IPK _mut expression strain (XL1-Blue strain with pJ211-IPK _mut introduced).
The transformants were plated on LB-Agar plates (9 cm diameter) containing 0, 11, 12, 13 or 14 mol/L DMAOH at 2000 cells/plate and allowed to stand at 37°C for 24 hours to form colonies. The viable cell count ratios on DMAOH (11, 12, 13, 14 mM)-containing media (relative to media without DMAOH) for each mutant are shown in Figure 7.
Nine of the 13 (shown in gray in the figure) did not show significant DMAOH resistance compared to the wild type, but strains expressing the four mutants showed significantly higher DMAOH resistance than the parent (wild type, wt) (Figure 7).

5. Sequencing The sequences of the four proteins determined to have increased activity above (Mn10-12A, Mn10-12B, Mn20-12E, and Mn50-12A) are shown in Table 2.

Genotypes of IspS mutants.

In the two mutants Mn10-12A and Mn50-12A, no mutations were found in the structural gene of ispS, but base substitutions were introduced into LacO. These are "expression mutants" in which the transcription level has increased due to a decrease in affinity for lacI. On the other hand, there were two mutants that were difficult to consider as expression mutants. Among the mutations found there, amino acid substitutions were found in Mn10-12B (H218R, T587A) and Mn20-12E (F593L). These were mapped to the position in Figure 8 on the structure (PDB 3N0G). In both cases, the residues were located on the surface rather than inside the reactive pocket.

6. Plasmid transfer (conversion of expression system)
To examine whether the Mn10-12B and Mn20-12E enzymes have properties that enhance E. coli activity rather than being expression mutants with high enzyme activity, the reading frames were excised with the restriction enzymes BamHI and XhoI and placed downstream of a constant promoter (PT5) that does not contain lacO.
The plasmid MAP (pT5-IspS: sequence number 4) is shown in FIG.

7. Comparison of product characteristics by GC analysis
pT5-IspS(wt), pT5-IspS(Mn10-12B), and pT5-IspS(Mn10-12E) were each introduced into the E. coli strain XL1 Blue, into which a precursor supply pathway (mevalonate pathway) had been introduced using pMEV-idi (SEQ ID NO:5).
The plasmid MAP (pMEV-idi) is shown in FIG.

7-1. Analysis of dodecane layer The isoprene production ability of the transformants thus obtained was measured. Specifically, the transformants (50 μL of OD2 pre-culture solution: equivalent to 5× ¹⁰⁷ cells) were inoculated into 5 mL of TB medium, and one-fifth of the medium volume of dodecane (1 mL) was layered on top of the medium, followed by shaking culture at 37°C for 24 hours. Dodecane (0.25 mL) was then collected, and 0.5 μL of it (1/500: 1/2000 of the total amount of dodecane layered on top of the medium) was injected into GC-FID.
GC analysis conditions: Rtx-5 (Shimadzu), oven temperature 250℃
The temperature ramp protocol was 35°C for 5 min, then ramped at 30°C/min to 150°C and 20°C/min to 200°C.
Both E. coli expressing Mn10-12B and Mn20-12E produced higher amounts of isoprene than E. coli expressing IspS(wt) (FIG. 11).

7-2. Headspace analysis The Mn10-12B mutant, which showed significant activity, was reevaluated using a different culture and measurement protocol than the above. Specifically, the transformant (100 μL of preculture solution at OD ~ 2: equivalent to 1 × 10 ⁸ cells) was inoculated into 10 mL TB medium in a 50 mL flask and cultured at 30 degrees for 48 hours with shaking. During the culture, the flask was tightly tied with aluminum foil and a rubber band to keep it airtight throughout. Then, 10 mL of air from the headspace of each sample was collected using a gas sampler (NeedlEx, Shinwa Kako). The components adsorbed to the solid-phase adsorbent from the collected gas were injected into GC-FID.
GC analysis conditions: Rtx-5 (Shimadzu), oven temperature 250℃
Heating protocol: 30°C for 5 min, then heated at 20°C/min up to 200°C.
The results are shown in Figure 12.
E. coli expressing Mn10-12B exhibited higher isoprene production than E. coli expressing IspS(wt) (FIG. 12).
These results suggest that a highly active mutant isoprene synthase was isolated by using the ability to rescue DMAPP from excessive accumulation through phosphorylation of DMAOH by IPK _mut as an indicator.

8. Evolution of DMAOH resistance The obtained mutant plasmids were introduced into E. coli XL1 Blue expressing IPK _mut , and their DMAOH sensitivity was examined (Fig. 13). The mutants showed clearly higher DMAOH resistance than the wild type. The DMAOH sensitivity of the IPK _mut expression strain was examined again. The cell viability rapidly decreased with the amount of DMAOH added, and in this expression system, the cells were completely killed by 5 mM DMAOH (Fig. 13, dark blue line). The strain expressing the wild type IspS has DMAPP removal activity, so its viability increases (Fig. 13, light blue line). The strain expressing the mutants obtained this time (Fig. 13, purple and red lines) clearly acquired DMAOH resistance, reflecting their high DMAOH consumption activity (isoprene synthesis activity: Fig. 13).
Even with the active mutant, if more DMAOH is added, cell growth stops (IC99-12 mM). By setting DMAOH=12 mM, a more active second generation mutant can be obtained using this mutant as the parent (starting point).

The present invention can provide a mutant isoprene synthase that synthesizes a higher amount of isoprene than the wild-type isoprene synthase, as well as a method for screening mutants of the enzyme.

Claims (8)

A method for screening mutant isoprene synthases, comprising introducing a library of mutated isoprene synthase genes into cells capable of synthesizing dimethylallyl diphosphate from dimethylallyl alcohol, and using an increase or decrease in a cell proliferation rate of the cells as an indicator, the method comprising the steps of:
A screening method, wherein the cell is a cell transformed with a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:6.
The method of claim 1 , wherein the increase in the cell proliferation rate of the cells is an increase in the amount of isoprene synthesized using dimethylallyl diphosphate as a substrate.
The method of any one of claims 1 to 2, wherein the cell proliferation rate of the cells is the cell proliferation rate in the presence of dimethylallyl alcohol.
The method of any one of claims 1 to 3, wherein the cell is Escherichia coli.
A mutant isoprene synthase having amino acid deletions at positions 1 to 52 in the amino acid sequence shown in SEQ ID NO:7 and having only amino acid substitutions of H218R and T587A.
The mutant isoprene synthase according to claim 5 , wherein the mutant isoprene synthase synthesizes a higher amount of isoprene than a wild-type isoprene synthase.
A mutant isoprene synthase having amino acid deletions at positions 1 to 52 in the amino acid sequence shown in SEQ ID NO:7 and having only the amino acid substitution F593L.
A mutant isoprene synthase gene comprising any one of the following genes (1) to (2):
(1) a gene encoding a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 11 or 12, and (2) a gene consisting of DNA consisting of the base sequence set forth in SEQ ID NO: 13 or 14.