CN108239630B

CN108239630B - Method for modifying terpene synthase

Info

Publication number: CN108239630B
Application number: CN201710297632.2A
Authority: CN
Inventors: 刘天罡; 卞光凯; 韩以超; 侯安伟; 苑玉杰; 刘然
Original assignee: J1 Biotech Co ltd
Current assignee: Wuhan Hesheng Technology Co ltd
Priority date: 2016-12-27
Filing date: 2017-04-28
Publication date: 2021-07-16
Anticipated expiration: 2037-04-28
Also published as: CN108239630A

Abstract

The invention provides a terpene synthase, a nucleic acid molecule, a construction body, a recombinant cell, an application and a method for obtaining the terpene synthase. The terpene synthase has a mutation, as compared to a wild-type terpene synthase, of at least one of: aromatic amino acids in hydrophobic or neutral amino acids in the active region are mutated to non-aromatic amino acids; and a non-aromatic amino acid among hydrophobic or neutral amino acids in the active region is mutated to an aromatic amino acid. Under the catalytic action of the terpene synthase, the abundance of a main product in a catalytic product can be changed, so that the original by-product is changed into the main product or a new terpenoid is obtained.

Description

Method for modifying terpene synthase

Technical Field

The present invention relates to the field of biology. In particular, the invention relates to a method of engineering terpene synthases.

Background

Terpenoids are a generic term for compounds containing isoprene units. To date, approximately 76000 terpenoids have been found in animals, plants, and microorganisms. Is widely applied to perfume production industry, health care product industry, agricultural production field and medical industry.

However, terpene synthases are still under development.

Disclosure of Invention

The present invention aims to solve at least to some extent at least one of the technical problems of the prior art. To this end, the invention proposes a terpene synthase, a nucleic acid molecule, a construct, a recombinant cell, a use and a method for obtaining a terpene synthase.

It should be noted that the present invention has been completed based on the following findings of the inventors:

in order to obtain better performing terpene synthases, the industry sometimes needs to make certain modifications to wild-type terpene synthases. Traditional methods often modify the active cavity of terpene synthases by means of saturation mutagenesis or scan different positions by means of mutation to alanine. The method usually needs to establish a large library, the whole process consumes large manpower and material resources, and finally, the property change of a plurality of mutated proteins is not obvious.

In view of the above, the inventors have unexpectedly found that, by exchanging aromatic amino acids and non-aromatic amino acids for sites related to the functional specificity of wild-type terpene synthase, on one hand, the steric hindrance effect of the whole enzyme catalysis is changed, and on the other hand, the interaction between pi bonds and carbenium ions in an active cavity is influenced, which finally has a large influence on the whole reaction. Therefore, under the catalytic action of the converted new terpenoid synthase, the abundance of the main product in the catalytic product can be changed, the original by-product can be changed into the main product, and the new terpenoid can be obtained.

To this end, in one aspect of the invention, the invention features a terpene synthase. According to embodiments of the invention, the terpene synthase has a mutation, as compared to the wild-type terpene synthase, of at least one of: aromatic amino acids in hydrophobic or neutral amino acids in the active region are mutated to non-aromatic amino acids; and a non-aromatic amino acid among hydrophobic or neutral amino acids in the active region is mutated to an aromatic amino acid.

The inventors surprisingly found that, by exchanging hydrophobic amino acids or aromatic amino acids and non-aromatic amino acids in neutral amino acids in the active region of the wild-type terpene synthase, on the one hand, the steric hindrance effect of the whole enzyme catalysis is changed, and on the other hand, the interaction between pi bonds and carbenium ions in the active cavity is influenced, which finally has a large influence on the whole reaction. Thus, under the catalytic action of the terpene synthase according to the embodiment of the invention, the abundance of the main product in the catalytic product can be changed, the original side product can be changed into the main product, or a new terpenoid can be obtained.

According to an embodiment of the present invention, the above terpene synthase may also have the following additional technical features:

according to an embodiment of the present invention, the mutation site is 65 th, 69 th, 85 th, 88 th, 89 th, 159 th, 186 th, 191 th, 219 th, 222 th, 307 th, 311 th or 314 th site. Therefore, under the catalytic action of the terpene synthase according to the embodiment of the invention, the abundance of the main product in the catalytic product can be further changed, the original by-product is changed into the main product, or a new terpenoid is obtained.

According to an embodiment of the invention, the aromatic amino acid is selected from tryptophan, phenylalanine or tyrosine and the non-aromatic amino acid is selected from alanine, cysteine, glycine, glutamine, leucine, methionine, asparagine, serine, threonine, isoleucine or valine. Therefore, under the catalytic action of the terpene synthase according to the embodiment of the invention, the abundance of the main product in the catalytic product can be further changed, the original by-product is changed into the main product, or a new terpenoid is obtained.

According to an embodiment of the invention, the mutation comprises a substitution or a modification.

According to embodiments of the invention, the wild-type terpene synthase has the amino acid sequence of SEQ ID NO: 1 to 5 or a pharmaceutically acceptable salt thereof.

In another aspect of the invention, the invention features a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the terpene synthase described above. Thus, under the catalytic action of the terpene synthase encoded by the nucleic acid molecule according to the embodiment of the invention, the abundance of the main product in the catalytic product can be changed, the original by-product can be changed into the main product, or a new terpenoid can be obtained.

In yet another aspect of the invention, the invention features a construct. According to an embodiment of the invention, the construct comprises a nucleic acid molecule as described above. Therefore, under the catalytic action of terpene synthases expressed by the constructs according to the embodiments of the present invention, the abundance of the main product in the catalytic product can be changed, the original by-products can be changed into the main product, or new terpenoids can be obtained.

In yet another aspect of the invention, the invention features a recombinant cell. According to an embodiment of the invention, the recombinant cell contains a nucleic acid molecule as described above. Therefore, the obtained terpene synthase can change the abundance of the main product in the catalytic product, change the original by-product into the main product or obtain a new terpenoid under the catalytic action of the recombinant cell cultured.

In a further aspect of the invention, the invention provides the use of a terpene synthase or nucleic acid molecule or construct or recombinant cell as described above for the synthesis of terpenoids. Therefore, under the catalytic action of the mutated terpene synthase, the abundance of the main product in the catalytic product can be changed, the original by-product is changed into the main product, or a new terpenoid is obtained.

In yet another aspect of the invention, the invention provides a method of obtaining the terpene synthase described above. According to an embodiment of the invention, the method comprises: culturing the recombinant cell described above under conditions suitable for expression of the terpene synthase so as to obtain the terpene synthase. Therefore, under the catalytic action of the obtained terpene synthase, the abundance of the main product in the catalytic product can be changed, the original by-product is changed into the main product, or a new terpenoid is obtained.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 shows a chromatogram according to one embodiment of the invention.

Detailed Description

The following describes embodiments of the present invention in detail. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.

The invention provides a terpene synthase, a nucleic acid molecule, a construction body, a recombinant cell, an application and a method for obtaining the terpene synthase. Which will be described in detail below, respectively.

Terpene synthases

In one aspect of the invention, the invention features a terpene synthase. According to embodiments of the invention, the terpene synthase has a mutation, as compared to the wild-type terpene synthase, of at least one of: aromatic amino acids in hydrophobic or neutral amino acids in the active region are mutated to non-aromatic amino acids; and a non-aromatic amino acid among hydrophobic or neutral amino acids in the active region is mutated to an aromatic amino acid.

The inventors surprisingly found that, by exchanging hydrophobic amino acids or aromatic amino acids and non-aromatic amino acids in neutral amino acids in the active region of the wild-type terpene synthase, on the one hand, the steric hindrance effect of the whole enzyme catalysis is changed, and on the other hand, the interaction between pi bonds and carbenium ions in the active cavity is influenced, which finally has a large influence on the whole reaction. Specifically, the terpene synthase catalytic substrate obtained through mutation can change the abundance of a main product in a catalytic product, so that the original by-product is changed into the main product or a new terpenoid can be obtained.

According to an embodiment of the present invention, the mutation site is 65 th, 69 th, 85 th, 88 th, 89 th, 159 th, 186 th, 191 th, 219 th, 222 th, 307 th, 311 th or 314 th site.

The inventor finds that compared with other amino acid sites for coding terpene synthases, the functional specificity (activity) of the amino acid sites is stronger, hydrophobic amino acids or aromatic amino acids and non-aromatic amino acids in neutral amino acids are exchanged, and the obtained terpene synthase catalytic substrate can further change the abundance of a main product in a catalytic product, so that the original by-product is changed into the main product or a new terpene compound is obtained.

According to an embodiment of the invention, the aromatic amino acid is selected from tryptophan (W), phenylalanine (F) or tyrosine (Y), and the non-aromatic amino acid is selected from alanine (a), cysteine (C), glycine (G), glutamine (Q), leucine (L), methionine (M), asparagine (N), serine (S), threonine (T), isoleucine (I) or valine (V). The inventor finds that the terpene synthase catalytic substrate obtained by interchanging the aromatic amino acid and the non-aromatic amino acid can further change the abundance of the main product in the catalytic product, change the original by-product into the main product or obtain a new terpene compound.

According to an embodiment of the invention, the mutation comprises a substitution or a modification. According to particular embodiments of the present invention, terpene synthases are obtained by site-directed mutagenesis of a gene encoding a wild-type terpene synthase.

According to embodiments of the invention, the wild-type terpene synthase has the amino acid sequence of SEQ ID NO: 1 to 5 or a pharmaceutically acceptable salt thereof. According to a particular embodiment of the invention, the wild-type terpene synthase is isolated from two fungi, Fusarium graminearum and Alternaria alternata.

Wild-type terpene synthase 1 (abbreviated as FgMS) has the amino acid sequence of SEQ ID NO: 1, specifically the following amino acid sequence:

MDFTYRYSFEPTDYDTDGLCDGVPVRMHKGADLDEVAIFKAQYDWEKHVGPKLPFRGALGPRHNFICLTLPECLPERLEIVSYANEFAFLHDDITDVESAETVAAENDEFLDALQQGVREGDIQSRESGKRHLQAWIFKSMVAIDRDRAVAAMNAWATFINTGAGCAHDTNFKSLDEYLHYRATDVGYMFWHALIIFGCAITIPEHEIELCHQLALPAIMSVTLTNDIWSYGKEAEAAEKSGKPGDFVNALVVLMREHNCSIEEAERLCRARNKIEVAKCLQVTKETRERKDVSQDLKDYLYHMLFGVSGNAIWSTQCRRYDMTAPYNERQQARLKQTKGELTSTYDPVQAAKEAMMESTRPEIHRLPTPDSPRKESFAVRPLVNGSGQYNGNNHINGVSNEVDVRPSIERHASTKRATSADDIDWTAHKKVDSGADHKKTLSDIMLQELPPMEDDVVMEPYRYLCSLPSKGVRNKTIDALNFWLKVPIENANTIKAITESLHGSSLMLDDIEDHSQLRRGKPSAHAVFGEAQTINSATFQYIQSVSLISQLRSPKALNIFVDEIRQLFIGQAYELQWTSNMICPPLEEYLRMVDGKTGGLFRLLTRLMAAESTTEVDVDFSRLCQLFGRYFQIRDDYANLKLADYTEQKGFCEDLDEGKFSLPLIIAFNENNKAPKAVAQLRGLMMQRCVNGGLTFEQKVLALNLIEEAGGISGTEKVLHSLYGEMEAELERLAGVFGAENHQLELILEMLRID

wild-type terpene synthase 2 (abbreviated D510A) has the amino acid sequence of SEQ ID NO: 2, specifically the amino acid sequence shown in the specification as follows:

MDFTYRYSFEPTDYDTDGLCDGVPVRMHKGADLDEVAIFKAQYDWEKHVGPKLPFRGALGPRHNFICLTLPECLPERLEIVSYANEFAFLHDDITDVESAETVAAENDEFLDALQQGVREGDIQSRESGKRHLQAWIFKSMVAIDRDRAVAAMNAWATFINTGAGCAHDTNFKSLDEYLHYRATDVGYMFWHALIIFGCAITIPEHEIELCHQLALPAIMSVTLTNDIWSYGKEAEAAEKSGKPGDFVNALVVLMREHNCSIEEAERLCRARNKIEVAKCLQVTKETRERKDVSQDLKDYLYHMLFGVSGNAIWSTQCRRYDMTAPYNERQQARLKQTKGELTSTYDPVQAAKEAMMESTRPEIHRLPTPDSPRKESFAVRPLVNGSGQYNGNNHINGVSNEVDVRPSIERHASTKRATSADDIDWTAHKKVDSGADHKKTLSDIMLQELPPMEDDVVMEPYRYLCSLPSKGVRNKTIDALNFWLKVPIENANTIKAITESLHGSSLMLADIEDHSQLRRGKPSAHAVFGEAQTINSATFQYIQSVSLISQLRSPKALNIFVDEIRQLFIGQAYELQWTSNMICPPLEEYLRMVDGKTGGLFRLLTRLMAAESTTEVDVDFSRLCQLFGRYFQIRDDYANLKLADYTEQKGFCEDLDEGKFSLPLIIAFNENNKAPKAVAQLRGLMMQRCVNGGLTFEQKVLALNLIEEAGGISGTEKVLHSLYGEMEAELERLAGVFGAENHQLELILEMLRID

wild-type terpene synthase 3 (abbreviated as FgGS) has the sequence of SEQ ID NO: 3, specifically the following amino acid sequence:

MDPYSETSDLVDISRFDTHGLGANYKLRRHKFEHLADTGCHKARSDWVKYIGPLTEFGGCNHINGNFSAVVLPLCRPDRLELIAYVLEFAFLHDSVLESENTSPESEVQAEAGLRLLYERCISRLLQTDEVCAKKIAKTWKDAINTTTKDKNVDFQSIEDYLEFRMIDTGAPFVEALMLFGLGMSLSPQEDDALGHVIRPCFAALALTNDYFSFDREIEEVDTSTLINSVAIVMRIQSLDIPTAKTIINETIQKYEREFLRRIDEYKQHKGPISNKIEQYMEAMTYQISGNLVWSLNCPRYNPDYRYGLEACQHEG

wild-type terpene synthase 4 (abbreviated as GGPPS-Aa) has the amino acid sequence of SEQ ID NO: 4, specifically the following amino acid sequence:

MRYQYSERVESHRYRDDGLANNIHLRIHKDSYKEVIGTLRAQNDWSRLVSSMTKYHGGLGDLFSFISVTIPECLPERLEVVAYANEYAFLYDDQMERLDLKDFREGRDDMLDIFGIHGGASNLEDRRPEKTLQLQIFDELMAIDQDRAIVTMQAWAKFIDLASRTRVEPFNTLAAYLPSRTIDAGELFWFGMLTFAMALTIPAHELDVCMRLARPGYEAISLINDIYSWPKERAEAEKAGQDYVFNAVWVVMKERKCDEQKATEFCKNLARQSIQDFSTSVNTPQVTELSCDSRTYLGAVRLSYVGNLVWSIYCPRYNIAVPVYHSKL

wild-type terpene synthase 5 (AaTS for short) has the sequence of SEQ ID NO: 5, specifically the following amino acid sequence:

the catalytic substrate for the mutated terpene synthase is not strictly limited, and an existing substrate may be selected according to actual needs. According to a specific embodiment of the invention, the catalytic substrate may be geranyl pyrophosphate, isopentenyl pyrophosphate, allyl pyrophosphate, farnesyl pyrophosphate, geranylgeranyl pyrophosphate or geranylfarnesyl pyrophosphate.

Nucleic acid molecules

In another aspect of the invention, the invention features a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the terpene synthase described above. Thus, the nucleic acid molecules according to embodiments of the invention encode terpene synthase catalytic substrates that are capable of altering the abundance of the main product in the catalytic product, changing the original byproduct to the main product, or obtaining new terpenoids.

According to an embodiment of the invention, the nucleotide sequence encoding the wild-type terpene synthase is as follows:

nucleic acid molecule 1 has the sequence of SEQ ID NO: 6, and can code FgMS, and the specific nucleotide sequence is as follows:

ATGGATTTCACCTACCGTTATAGCTTTGAACCGACCGACTACGATACCGACGGTCTGTGCGACGGTGTGCCGGTTCGTATGCACAAGGGTGCGGATCTGGACGAAGTGGCGATCTTCAAAGCGCAGTATGACTGGGAGAAGCACGTTGGCCCGAAACTGCCGTTCCGTGGTGCGCTGGGTCCGCGTCACAACTTTATTTGCCTGACCCTGCCGGAATGCCTGCCGGAACGTCTGGAGATCGTGAGCTACGCGAACGAGTTCGCGTTTCTGCACGACGATATTACCGATGTGGAAAGCGCGGAGACCGTTGCGGCGGAAAACGATGAGTTCCTGGACGCGCTGCAGCAAGGTGTTCGTGAAGGCGACATCCAAAGCCGTGAGAGCGGCAAGCGTCACCTGCAGGCGTGGATTTTTAAAAGCATGGTGGCGATCGATCGTGACCGTGCGGTTGCGGCGATGAACGCGTGGGCGACCTTCATTAACACCGGTGCGGGCTGCGCGCACGATACCAACTTTAAGAGCCTGGACGAGTACCTGCACTATCGTGCGACCGACGTGGGTTACATGTTCTGGCACGCGCTGATCATTTTTGGCTGCGCGATCACCATTCCGGAGCACGAAATCGAGCTGTGCCACCAGCTGGCGCTGCCGGCGATTATGAGCGTGACCCTGACCAACGACATCTGGAGCTATGGTAAAGAAGCGGAGGCGGCGGAAAAGAGCGGTAAACCGGGCGACTTCGTTAACGCGCTGGTTGTGCTGATGCGTGAACACAACTGCAGCATTGAGGAAGCGGAGCGTCTGTGCCGTGCGCGTAACAAGATCGAGGTGGCGAAATGCCTGCAAGTTACCAAGGAAACCCGTGAGCGTAAAGATGTGAGCCAGGATCTGAAGGACTACCTGTATCACATGCTGTTTGGTGTTAGCGGCAACGCGATCTGGAGCACCCAGTGCCGTCGTTACGACATGACCGCGCCGTATAACGAACGTCAGCAAGCGCGTCTGAAGCAAACCAAAGGCGAGCTGACCAGCACCTACGATCCGGTTCAGGCGGCGAAGGAAGCGATGATGGAGAGCACCCGTCCGGAAATTCACCGTCTGCCGACCCCGGACAGCCCGCGTAAAGAGAGCTTCGCGGTGCGTCCGCTGGTTAACGGTAGCGGCCAATATAACGGTAACAACCACATTAACGGCGTGAGCAACGAAGTGGACGTTCGTCCGAGCATCGAGCGTCACGCGAGCACCAAACGTGCGACCAGCGCGGACGACATCGATTGGACCGCGCACAAGAAAGTTGATAGCGGTGCGGACCACAAGAAAACCCTGAGCGACATTATGCTGCAGGAACTGCCGCCGATGGAGGACGATGTGGTTATGGAACCGTACCGTTATCTGTGCAGCCTGCCGAGCAAGGGTGTGCGTAACAAAACCATTGATGCGCTGAACTTTTGGCTGAAGGTTCCGATCGAAAACGCGAACACCATCAAAGCGATTACCGAGAGCCTGCACGGCAGCAGCCTGATGCTGGACGACATCGAAGACCACAGCCAACTGCGTCGTGGCAAGCCGAGCGCGCACGCGGTGTTCGGCGAGGCGCAGACCATTAACAGCGCGACCTTTCAGTACATTCAAAGCGTGAGCCTGATCAGCCAACTGCGTAGCCCGAAAGCGCTGAACATCTTCGTTGATGAAATTCGTCAGCTGTTTATCGGTCAAGCGTACGAGCTGCAGTGGACCAGCAACATGATCTGCCCGCCGCTGGAGGAATATCTGCGTATGGTTGACGGCAAGACCGGTGGCCTGTTCCGTCTGCTGACCCGTCTGATGGCGGCGGAAAGCACCACCGAGGTGGATGTTGACTTTAGCCGTCTGTGCCAACTGTTCGGTCGTTACTTTCAGATCCGTGACGATTATGCGAACCTGAAGCTGGCGGATTACACCGAACAGAAAGGTTTCTGCGAGGACCTGGACGAGGGCAAATTCAGCCTGCCGCTGATCATTGCGTTTAACGAGAACAACAAGGCGCCGAAAGCGGTGGCGCAACTGCGTGGCCTGATGATGCAGCGTTGCGTGAACGGTGGCCTGACCTTCGAACAAAAGGTTCTGGCGCTGAACCTGATTGAGGAAGCGGGTGGCATCAGCGGTACCGAGAAAGTGCTGCACAGCCTGTATGGCGAAATGGAGGCGGAACTGGAGCGTCTGGCGGGTGTTTTTGGCGCGGAGAACCACCAGCTGGAACTGATTCTGGAGATGCTGCGTATCGACTAA

specifically, SEQ ID NO: 6 from TTT to CTG corresponding to the nucleotide sequence shown in SEQ ID NO: 1 to leucine from a phenylalanine at position 65 of the amino acid sequence shown in 1; or converting SEQ ID NO: 6 from ACC to TTC, corresponding to the nucleotide sequence shown in SEQ ID NO: 1 to phenylalanine from threonine 69 of the amino acid sequence shown in the sequence table; or converting SEQ ID NO: 6 from AAC to TTC, corresponding to the nucleotide sequence shown in SEQ ID NO: 1 to phenylalanine from asparagine at position 85 of the amino acid sequence shown in figure 1; or converting SEQ ID NO: 6 from GCG to TTC corresponding to the nucleotide sequence shown in SEQ ID NO: 1 to phenylalanine from alanine at position 88 of the amino acid sequence shown in figure 1; or converting SEQ ID NO: 6 from TTT to CTG corresponding to the nucleotide sequence shown in SEQ ID NO: 1 to leucine from the 89 th site of the amino acid sequence shown in the specification; or converting SEQ ID NO: 6 from TTC to GGC, corresponding to the nucleotide sequence shown in SEQ ID NO: 1 to glycine at position 159 of the amino acid sequence shown in figure 1; or converting SEQ ID NO: 6 from GTG to TTC, corresponding to the nucleotide sequence shown in SEQ ID NO: 1 to phenylalanine from a valine mutation at position 186 of the amino acid sequence shown in 1; or converting SEQ ID NO: 6 from TGG to GTG corresponding to the nucleotide sequence shown in SEQ ID NO: 1, wherein the 191 th tryptophan of the amino acid sequence shown in the specification is mutated into valine; or converting SEQ ID NO: 6 from ATT to TTC, corresponding to the nucleotide sequence shown in SEQ ID NO: 1 to phenylalanine from isoleucine at position 219 of the amino acid sequence shown in figure 1; or converting SEQ ID NO: 6 from GTG to TTC, corresponding to the nucleotide sequence shown in SEQ ID NO: 1 to phenylalanine from a valine mutation at position 222 of the amino acid sequence shown in 1; or converting SEQ ID NO: 6 is mutated from GGT to TTC at the 919-th 921 th nucleotide of the nucleotide sequence shown in SEQ ID NO: 1 to phenylalanine from glycine at position 307 of the amino acid sequence shown in the figure; or converting SEQ ID NO: 6 from AAC to TTC, corresponding to the nucleotide sequence shown in SEQ ID NO: 1 to phenylalanine from the 311 th glutamine mutation of the amino acid sequence shown in the figure; or converting SEQ ID NO: 6 from TGG to GGT corresponding to the sequence shown in SEQ ID NO: 1 to glycine at position 314 of the amino acid sequence shown in figure 1. Thus, a mutated nucleic acid molecule was obtained. Expressing the mutated nucleic acid molecule to obtain terpene synthase.

Nucleic acid molecule 2 has the sequence of SEQ ID NO: 7, encoding D510A, wherein the specific nucleotide sequence is as follows:

ATGGATTTCACCTACCGTTATAGCTTTGAACCGACCGACTACGATACCGACGGTCTGTGCGACGGTGTGCCGGTTCGTATGCACAAGGGTGCGGATCTGGACGAAGTGGCGATCTTCAAAGCGCAGTATGACTGGGAGAAGCACGTTGGCCCGAAACTGCCGTTCCGTGGTGCGCTGGGTCCGCGTCACAACTTTATTTGCCTGACCCTGCCGGAATGCCTGCCGGAACGTCTGGAGATCGTGAGCTACGCGAACGAGTTCGCGTTTCTGCACGACGATATTACCGATGTGGAAAGCGCGGAGACCGTTGCGGCGGAAAACGATGAGTTCCTGGACGCGCTGCAGCAAGGTGTTCGTGAAGGCGACATCCAAAGCCGTGAGAGCGGCAAGCGTCACCTGCAGGCGTGGATTTTTAAAAGCATGGTGGCGATCGATCGTGACCGTGCGGTTGCGGCGATGAACGCGTGGGCGACCTTCATTAACACCGGTGCGGGCTGCGCGCACGATACCAACTTTAAGAGCCTGGACGAGTACCTGCACTATCGTGCGACCGACGTGGGTTACATGTTCTGGCACGCGCTGATCATTTTTGGCTGCGCGATCACCATTCCGGAGCACGAAATCGAGCTGTGCCACCAGCTGGCGCTGCCGGCGATTATGAGCGTGACCCTGACCAACGACATCTGGAGCTATGGTAAAGAAGCGGAGGCGGCGGAAAAGAGCGGTAAACCGGGCGACTTCGTTAACGCGCTGGTTGTGCTGATGCGTGAACACAACTGCAGCATTGAGGAAGCGGAGCGTCTGTGCCGTGCGCGTAACAAGATCGAGGTGGCGAAATGCCTGCAAGTTACCAAGGAAACCCGTGAGCGTAAAGATGTGAGCCAGGATCTGAAGGACTACCTGTATCACATGCTGTTTGGTGTTAGCGGCAACGCGATCTGGAGCACCCAGTGCCGTCGTTACGACATGACCGCGCCGTATAACGAACGTCAGCAAGCGCGTCTGAAGCAAACCAAAGGCGAGCTGACCAGCACCTACGATCCGGTTCAGGCGGCGAAGGAAGCGATGATGGAGAGCACCCGTCCGGAAATTCACCGTCTGCCGACCCCGGACAGCCCGCGTAAAGAGAGCTTCGCGGTGCGTCCGCTGGTTAACGGTAGCGGCCAATATAACGGTAACAACCACATTAACGGCGTGAGCAACGAAGTGGACGTTCGTCCGAGCATCGAGCGTCACGCGAGCACCAAACGTGCGACCAGCGCGGACGACATCGATTGGACCGCGCACAAGAAAGTTGATAGCGGTGCGGACCACAAGAAAACCCTGAGCGACATTATGCTGCAGGAACTGCCGCCGATGGAGGACGATGTGGTTATGGAACCGTACCGTTATCTGTGCAGCCTGCCGAGCAAGGGTGTGCGTAACAAAACCATTGATGCGCTGAACTTTTGGCTGAAGGTTCCGATCGAAAACGCGAACACCATCAAAGCGATTACCGAGAGCCTGCACGGCAGCAGCCTGATGCTGGCCGACATCGAAGACCACAGCCAACTGCGTCGTGGCAAGCCGAGCGCGCACGCGGTGTTCGGCGAGGCGCAGACCATTAACAGCGCGACCTTTCAGTACATTCAAAGCGTGAGCCTGATCAGCCAACTGCGTAGCCCGAAAGCGCTGAACATCTTCGTTGATGAAATTCGTCAGCTGTTTATCGGTCAAGCGTACGAGCTGCAGTGGACCAGCAACATGATCTGCCCGCCGCTGGAGGAATATCTGCGTATGGTTGACGGCAAGACCGGTGGCCTGTTCCGTCTGCTGACCCGTCTGATGGCGGCGGAAAGCACCACCGAGGTGGATGTTGACTTTAGCCGTCTGTGCCAACTGTTCGGTCGTTACTTTCAGATCCGTGACGATTATGCGAACCTGAAGCTGGCGGATTACACCGAACAGAAAGGTTTCTGCGAGGACCTGGACGAGGGCAAATTCAGCCTGCCGCTGATCATTGCGTTTAACGAGAACAACAAGGCGCCGAAAGCGGTGGCGCAACTGCGTGGCCTGATGATGCAGCGTTGCGTGAACGGTGGCCTGACCTTCGAACAAAAGGTTCTGGCGCTGAACCTGATTGAGGAAGCGGGTGGCATCAGCGGTACCGAGAAAGTGCTGCACAGCCTGTATGGCGAAATGGAGGCGGAACTGGAGCGTCTGGCGGGTGTTTTTGGCGCGGAGAACCACCAGCTGGAACTGATTCTGGAGATGCTGCGTATCGACTAA

nucleic acid molecule 3 has the sequence of SEQ ID NO: 8 and encoding FgGS, wherein the specific nucleotide sequence is as follows:

ATGGATCCCTACAGTGAAACATCAGATCTTGTTGACATTTCTCGCTTCGACACCCACGGCCTTGGAGCTAATTACAAACTACGACGACATAAGTTCGAACACCTAGCTGACACTGGATGTCACAAAGCAAGGTCAGATTGGGTAAAATACATTGGCCCTCTTACTGAATTCGGAGGCTGCAATCACATCAACGGGAATTTCTCTGCTGTAGTGTTGCCATTGTGCAGACCTGACCGCCTGGAGCTTATAGCATATGTACTCGAATTCGCATTTCTTCATGATTCCGTTCTCGAGTCAGAAAACACGTCTCCGGAATCCGAAGTGCAAGCCGAGGCTGGTCTACGCCTCTTATATGAACGATGCATAAGTCGACTCTTGCAGACAGACGAAGTATGCGCCAAAAAGATTGCAAAGACGTGGAAAGACGCGATCAACACAACTACAAAGGATAAGAACGTGGACTTCCAATCTATAGAAGACTACTTGGAGTTTCGCATGATTGATACTGGTGCACCGTTCGTCGAGGCCCTCATGCTTTTTGGATTGGGCATGTCGCTTTCACCGCAAGAAGATGATGCTCTTGGTCACGTTATTCGGCCATGTTTCGCCGCTTTGGCGTTGACGAACGACTACTTTTCGTTTGATCGAGAGATAGAAGAAGTCGATACTTCTACTCTTATCAACTCGGTTGCCATAGTAATGCGAATTCAGAGTCTGGACATTCCCACCGCCAAGACAATTATCAATGAGACTATACAGAAGTACGAGCGAGAGTTCCTCCGACGCATTGATGAGTACAAACAGCACAAAGGACCAATCTCTAACAAGATTGAACAATACATGGAAGCTATGACTTATCAGATCAGTGGGAATTTAGTATGGAGTCTGAATTGTCCTAGATATAATCCTGACTATCGGTACGGACTGGAGGCTTGTCAGCACGAGGGTTGA

nucleic acid molecule 4 has the sequence of SEQ ID NO: 9, encoding GGPPS-Aa, and the specific nucleotide sequence is as follows:

ATGTCTACTGAAACGCATCCTTTCGCCTCGCCGAACGCCATACCACCTCGAACCAGCTCTACTGGCCAAGTCACGAACGGCTATCCTATAAATCCGCGGCACAGCGTCTTGCGCCCGCTCTCAGAAATTGACTGGATGAGCCAAAGTAAAAAGAGCAAGACCTCACACGTTTCCACCGAACCACTCAACAGCACACAACCACACACACGCACGCTGTCGCAACCACAGTCGCAGCCCGACCCTATGAACCTCGAAGAAGTCAGCACAAACTACCCCACCCCGCTCTCCCCGCCGAGTGACACCAAGAACCTGGGCGAAGACCTCATATACGGCAACGGCGCAGCATGGACAGAAGAGAAGGAGCGCATACTGCTGGGGCCTTATGATTACCTTTGGGGTCACCCGGGCAAGGACATAAGGTCACAATGCATAGCAGCGTTCAACCTGTGGCTGAAAGTACCACCAGAGCGGCTTGAGGTCATAACGCGCGCGGTGGGCATGCTACACACAGCATCTCTTTTGGTCGACGATGTCGAAGACAGCTCAATATTACGGCGAGGCATTCCTGTCGCGAATAGCATATTCGGCGTTGCGCAGACGATCAACTCGGCGAACTACGTATACTTCAAGGCGTTGCAGGAGCTGATGCACATGGGCAATCCCAAGCTCATCGAGATCTTCACAGAAGAGCTGTTGAACCTGCACAGAGGCCAGGGAATGGATCTGTACTGGCGGGACAGTTTGACATGTCCTAGCGAAGCAGATTACCTAGAGATGGTAGGCAACAAGACCGGTGGCCTGTTCAGGCTAGCGATCAAGCTCATGCAGGCCGAAAGCGCAGTACAAGTCGACTGCGCACCCCTCGTCTCCACAATCGGCCTCCTCTTCCAGATCCTCGACGATCACCTCAATCTCTCCCCCACGTCGGGCTACTCCTCGCTCAAAGGCCTCTGCGAAGACCTCACCGAAGGCAAATTCTCCTTCCCCGTCATCCACGCTATCCGCGCCGACCCGTCGAACCAGATCCTCATCAACATCCTCAAGCAGAAAACTACAGATGAGGAGGTCAAGCGCTATGCGCTCAAGTACATGGAGAGTAAGGGTAGCTTTGAATATTCCAAGAGGGTTATTGATGACTTGAGGGGGAAGACGGAGGGGCTTGTCAGTGGGATTGAGAAGGGGTTGGGCGAGGAGGGGACGCAGGGGGCGGAGGCGTTGAGGAAAATGTTAGGGAGGTTGGTGTTGAGGTAG

nucleic acid molecule 5 has the sequence of SEQ ID NO: 10, encoding AaTS, and the specific nucleotide sequence is as follows:

ATGCGTTACCAGTATAGCGAGCGTGTGGAAAGCCACCGTTATCGTGACGATGGTCTGGCGAACAACATTCACCTGCGTATCCACAAGGATAGCTACAAAGAAGTGATTGGCACCCTGCGTGCGCAAAACGACTGGAGCCGTCTGGTTAGCAGCATGACCAAGTATCACGGTGGCCTGGGCGACCTGTTCAGCTTTATTAGCGTTACCATCCCGGAATGCCTGCCGGAGCGTCTGGAAGTGGTTGCGTACGCGAACGAGTATGCGTTCCTGTACGACGATCAGATGGAACGTCTGGACCTGAAAGATTTCCGTGAGGGTCGTGACGATATGCTGGACATCTTTGGCATTCACGGTGGCGCGAGCAACCTGGAGGATCGTCGTCCGGAAAAGACCCTGCAGCTGCAAATTTTTGACGAGCTGATGGCGATTGACCAGGATCGTGCGATCGTGACCATGCAAGCGTGGGCGAAATTCATCGATCTGGCGAGCCGTACCCGTGTTGAACCGTTTAACACCCTGGCGGCGTATCTGCCGAGCCGTACCATTGACGCGGGCGAGCTGTTCTGGTTTGGCATGCTGACCTTCGCGATGGCGCTGACCATCCCGGCGCACGAACTGGATGTGTGCATGCGTCTGGCGCGTCCGGGTTATGAGGCGATCAGCCTGATTAACGACATCTACAGCTGGCCGAAGGAACGTGCGGAGGCGGAAAAAGCGGGCCAGGATTACGTGTTTAACGCGGTTTGGGTGGTTATGAAGGAGCGTAAATGCGACGAACAAAAGGCGACCGAGTTCTGCAAAAACCTGGCGCGTCAGAGCATCCAAGATTTTAGCACCAGCGTGAACACCCCGCAAGTTACCGAGCTGAGCTGCGACAGCCGTACCTATCTGGGTGCGGTTCGTCTGAGCTACGTGGGCAACCTGGTTTGGAGCATTTATTGCCCGCGTTACAACATCGCGGTGCCGGTTTACCACAGCAAGCTGTA

construct

In another aspect of the invention, the invention features a nucleic acid molecule. According to an embodiment of the invention, the construct comprises a nucleic acid molecule as described above. The construct is introduced into a host cell to express a terpene synthase. Therefore, the obtained terpene synthase catalytic substrate can change the abundance of the main product in the catalytic product, change the original by-product into the main product or obtain new terpenoid.

Recombinant cell

In yet another aspect of the invention, the invention features a recombinant cell. According to an embodiment of the invention, the recombinant cell contains a nucleic acid molecule as described above. Therefore, the terpene synthase catalytic substrate obtained by culturing the recombinant cell can change the abundance of the main product in the catalytic product, change the original by-product into the main product or obtain a new terpenoid.

Use of

In a further aspect of the invention, the invention provides the use of a terpene synthase or nucleic acid molecule or construct or recombinant cell as described above for the synthesis of terpenoids. Therefore, the terpene synthase catalytic substrate after mutation can change the abundance of the main product in the catalytic product, change the original by-product into the main product or obtain a new terpenoid.

Method for obtaining terpene synthase

In yet another aspect of the invention, a method of obtaining the foregoing terpene synthases is provided. According to an embodiment of the invention, the method comprises: culturing the recombinant cell described above under conditions suitable for expression of the terpene synthase so as to obtain the terpene synthase. Therefore, the obtained terpene synthase catalytic substrate can change the abundance of the main product in the catalytic product, change the original by-product into the main product or obtain new terpenoid.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

The sequences of 51 fungal terpene synthases that were well-validated for function in literature reports before 8 months of 2016 were subjected to multiple sequence alignments. The substrate binding regions of these proteins were then analyzed in conjunction with representative crystal structure data to exclude the very conserved catalytic related sites and obtain amino acid sites that determine functional specificity (see Table 1). The 65 th, 69 th, 85 th, 88 th, 89 th, 159 th, 186 th, 191 th, 219 th, 222 th, 307 th, 311 th, 314 th sites of the corresponding terpene synthases FgMS have 13 sites in total. Wherein the grey portion is an aromatic amino acid.

TABLE 1 amino acid positions of terpene synthases

Example 2 modification of phenylalanine at position 65 of wild-type terpene synthase FgMS to leucine

In this example, wild-type FgMs terpene synthases were mutated such that phenylalanine at position 65 was altered to leucine as compared to the wild-type terpene synthase, as follows:

converting SEQ ID NO: 6, the 193 rd-position 195 th nucleotide of the nucleotide sequence shown in the specification is mutated from TTT to CTG to obtain the mutant gene F65L. The gene is obtained and then cloned to an expression vector of pet28a, and transformed into an expression host E.coli BL21(DE 3). After transformation, single clones were picked up in LB medium containing the corresponding antibiotic and cultured overnight at 37 ℃ and 220 rpm. Transferred to 1L of fresh LB medium containing the corresponding antibiotic at a temperature of 37 ℃ and at 220rpm to OD₆₀₀About 0.6-0.8, cooling to 16 deg.C, adding IPTG with final concentration of 0.1mM, culturing at 16 deg.C and 220rpm for 16-18 h. The cells were harvested by centrifugation at 8000rpm for 5min, after which the cells were thoroughly resuspended in 30-40mL of protein purification Buffer A (Buffer A: 50mM Tris-HCl, 300mM NaCl, 4 mM. beta. -mercaptoethanol, pH7.6) and disrupted by sonication (pulse 5s, pause 8s, sonication 5 min). Centrifugation was carried out at 12,000g for 30min or more at 4 ℃ and 12,000g, the supernatant was collected and centrifuged at 20,000rpm for 1 hour at 4 ℃ and the supernatant was collected, filtered through a 0.45 μm filter, and 6% Buffer B (Buffer B: 500mM imidazole added to Buffer A) was added to make imidazole about 30mM and mixed well for use.

Histidine-tagged proteins were purified using the Biologic DuoFlow Chromatography System from Bio-Rad. The protein separation column was loaded onto the FPLC for control, the flow rate of the FPLC was always 1.5mL/min, and the flow rate of the sample for autosampling was 2 mL/min. The resulting supernatant sample was purified in a first step by Biorad using a 5mL Hitrap HP Ni-NTA column equilibrated with 30mL (6 column volumes) of Buffer A (Buffer A: 50mM Tris-HCl, 300mM NaCl, 4 mM. beta. -mercaptoethanol, pH7.6), then the prepared 30mL of supernatant was loaded onto the column by an autosampler, the column was washed with 20mL of Buffer A (4 column volumes), at which time a linear gradient of Buffer B (50mM Tris-HCl, 150mM NaCl, 250mM Imidazole pH7.6) was initiated, Buffer B increased from 0% to 100% in a flow rate of 100mL (20 column volumes), and then the column was washed with 20mL (4 column volumes) of 100% Buffer B. The histidine-tagged protein of interest was collected by UV absorption and detected by SDS-PAGE. The relatively pure fractions were selected, collected, concentrated to 2.5mL by centrifugation through Amicon Centricon-10 (molecular weight below 10,000 is filtered off), which is a centrifugation and concentration tube from Millipore, desalted through PD-10 column from Pharmacia and exchanged into buffer C (20mM Tris-HCl, 10mM NaCl, pH 7.6).

The protein volume coming out of the PD-10 column was diluted to 3.5mL and the sample was loaded onto an ion exchange column Hitrap16/10Q/FF and purified with FPLC. After the ion exchange column is loaded with the sample, the column is washed by 20mL (1 column volume) of buffer C, and then gradient elution is carried out by using buffer D (20mM Tris-HCl, 1M NaCl, pH7.6), wherein the buffer D is increased from 0% to 30% in a flow rate of 20 mL; after a further flow of 40mL (2 column volumes), buffer D increased from 30% to 50%; after a further 20mL flow (1 column volume), buffer D increased from 50% to 100%; finally, the column was washed with 100% of 20mL buffer D. The eluted target protein was collected by UV absorption and detected by SDS-PAGE.

The resulting protein was concentrated to 2mL by centrifugation, and then loaded onto a gel filtration Superdex 200 column (column), which was equilibrated with buffer E (50mM phosphate buffer containing 10% glycerol, pH7.6) on FPLC for 240mL (2 column volumes) before loading, and the eluted target protein was recovered by UV absorption. Concentrating the sample to 2mL by using a protein centrifugal concentration column, subpackaging, quickly freezing by using liquid nitrogen, and storing in a refrigerator at the temperature of-80 ℃.

To test the activity of the mutant protein and the original protein, 10. mu.M of the purified protein was reacted with 100. mu.M of a substrate (one of geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP) or geranylfarnesyl pyrophosphate (GFPP)) and 2mM of magnesium ions as a metal catalyst under 200. mu.L of 50mM PB buffer (pH 7.6) and 10% concentration of glycerol at 30 ℃ overnight. The product was extracted with n-hexane to give a final sample and analyzed by GC-MS. The results are shown in FIG. 1(1), and the function of terpene synthase is changed as follows: 1. to generate new sesterterpene products 53, 55 and 56; 2. the

original sesterterpene products

1 and 2 disappeared.

Example 3 modification of phenylalanine at position 89 of wild-type terpene synthase FgMS to leucine

The method of example 2 was followed to convert SEQ ID NO: 6 from TTT to CTG, so that the phenylalanine at the 89 th position is changed into leucine compared with the wild terpene synthase.

Verification was performed as in example 2, and the results are shown in fig. 1(2), with the following changes to the product: 1. the content ratio of the sesterterpene main product 1 is increased from 92.82 percent to 100 percent; 2. the content ratio of the original diterpene by-product 35 is increased from 1.90% to 49.50%, and the product becomes a main product.

Example 4 modification of phenylalanine at position 159 of FgMS, a wild-type terpene synthase, to Glycine

The method of example 2 was followed to convert SEQ ID NO: 6 from TTC to GGC, so that the phenylalanine at the 159 th position of the obtained terpene synthase is changed into glycine compared with the wild terpene synthase.

The validation was carried out as in example 2, and the results are shown in FIGS. 1(3), with the following changes in the product: 1. the content ratio of the generated new product 49 of sesterterpene is 100 percent; 2. the

original sesterterpene products

1 and 2 disappeared; 3. the content ratio of the original diterpene by-product 35 is increased from 1.90% to 66.60%, and the product becomes a main product; 4. the content ratio of the original sesquiterpene by-product 10 is increased from 2.80% to 29.10%.

Example 5 Tryptophan at position 191 of wild-type terpene synthase FgMS was modified to valine

The method of example 2 was followed to convert SEQ ID NO: 6 from TGG to GTG, so that compared with the wild type terpene synthase, tryptophan at position 191 is changed into valine.

Verification was performed as in example 2, and the results are shown in fig. 1(4), with the following changes to the product: 1. the selection range of the substrate is reduced, and GFPP can not be converted any more; 2. the content ratio of the original diterpene by-product 35 is increased from 1.90% to 100%, and the original diterpene by-product becomes a unique main product; 3. the content ratio of the original sesquiterpene by-product 9 is increased from 28.90% to 49.80%.

Example 6 Tryptophan at position 314 of wild-type terpene synthase FgMS was modified to Glycine

The method of example 2 was followed to convert SEQ ID NO: 6 from TGG to GGT, so that the tryptophan at position 314 is changed into glycine compared with the wild-type terpene synthase.

The validation was carried out as in example 2, and the results are shown in FIGS. 1(5), with the following changes in the product: 1. the selection range of the substrate is reduced, and GFPP can not be converted any more; 2. the content ratio of the original diterpene by-product 35 is increased from 1.90% to 100%, and the original diterpene by-product becomes a unique main product; 3. the content ratio of the original sesquiterpene by-product 9 is increased from 28.90% to 47.40%.

Example 7 threonine at position 69 of wild-type terpene synthase FgMS engineered phenylalanine

The method of example 2 was followed to convert SEQ ID NO: 6 from ACC to TTC, so that the threonine at position 69 is transformed into phenylalanine compared with the wild terpene synthase.

Verification was performed as in example 2, and the results are shown in fig. 1(6), with the following changes to the product: 1. the selection range of the substrate is reduced, and GFPP can not be converted any more; 2. the content ratio of the original sesquiterpene by-product 23 is increased from 9.10% to 27.60%.

Example 8 modification of asparagine at position 85 of wild-type terpene synthase FgMS to phenylalanine

The method of example 2 was followed to convert SEQ ID NO: 6 is mutated from AAC to TTC at the 253-255 th nucleotide of the nucleotide sequence shown in the figure 6, so that compared with the wild-type terpene synthase, the asparagine at the 85 th position is transformed into phenylalanine.

Verification was performed as in example 2, and the results are shown in fig. 1(7), with the following changes to the product: 1. the selection range of the substrate is reduced, and GFPP can not be converted any more; 2. the content ratio of the original diterpene by-product 35 is increased from 1.90% to 98.60%; 3. the content ratio of the original sesquiterpene by-product 10 is increased from 2.80% to 35.40%.

Example 9 alanine modification of wild-type terpene synthase FgMS at position 88 to phenylalanine

The method of example 2 was followed to convert SEQ ID NO: 6 from GCG to TTC, so that the alanine at the 88 th position of the obtained terpene synthase is changed into phenylalanine compared with the wild terpene synthase.

Verification was performed as in example 2, and the results are shown in fig. 1(8), with the following changes to the product: 1. the content ratio of the sesterterpene byproducts is increased from 1.66 percent to 19.50 percent; 2. the content ratio of the original diterpene by-product 35 is improved from 1.90% to 99.00%.

Example 10 modification of valine at position 186 of wild-type terpene synthase FgMS to phenylalanine

The method of example 2 was followed to convert SEQ ID NO: 6 from the 556-558 th nucleotide of the nucleotide sequence shown in the specification, mutation is carried out from GTG to TTC, so that compared with the wild-type terpene synthase, valine at the 186 th position is changed into phenylalanine.

Verification was performed as in example 2, and the results are shown in fig. 1(9), with the following changes to the product: 1. the selection range of the substrate is reduced, and GFPP can not be converted any more; 2. the content ratio of the original diterpene by-product 35 is increased from 1.90 percent to 98.70 percent; 3. the content ratio of the original sesquiterpene by-product 10 is increased from 2.80% to 23.10%.

Example 11 isoleucine at position 219 of wild-type terpene synthase FgMS was engineered to phenylalanine

The method of example 2 was followed to convert SEQ ID NO: 6 from ATT to TTC, so that the isoleucine at position 219 is changed into phenylalanine compared with the wild terpene synthase.

Verification was performed as in example 2, and the results are shown in fig. 1(10), with the following changes in the product: the substrate selection range is reduced, and GFPP and GGPP can not be converted any more.

Example 12 modification of valine at position 222 of wild-type terpene synthase FgMS to phenylalanine

The method of example 2 was followed to convert SEQ ID NO: 6 from GTG to TTC, so that the valine at the 222 th position of the obtained terpene synthase is changed into phenylalanine compared with the wild terpene synthase.

Verification was performed as in example 2, and the results are shown in fig. 1(11), with the following changes to the product: 1. the selection range of the substrate is reduced, and GFPP can not be converted any more; 2. the content ratio of the original diterpene by-product 35 is increased from 1.90% to 63.00%.

Example 13 Glycine modification of wild-type terpene synthase FgMS at position 307 to phenylalanine

The method of example 2 was followed to convert SEQ ID NO: 6, the nucleotide at the 919-921 th site is mutated from GGT to TTC, so that the glycine at the 307 th site is transformed into phenylalanine compared with the wild type terpene synthase.

Verification was performed as in example 2, and the results are shown in fig. 1(12), with the following changes to the product: 1. the selection range of the substrate is reduced, and GFPP can not be converted any more; 2. the content ratio of the original diterpene by-product 35 is increased from 1.90 percent to 96.70 percent; 3. the content ratio of the original sesquiterpene by-product 10 is increased from 2.80% to 22.30%.

Example 14 transformation of Glutamine at position 311 of wild-type terpene synthase FgMS to phenylalanine

The method of example 2 was followed to convert SEQ ID NO: 6, the nucleotide at the 931 and 933 site is mutated from AAC to TTC, so that compared with the wild type terpene synthase, the glutamine at the 311 site is transformed into phenylalanine.

Verification was performed as in example 2, and the results are shown in fig. 1(13), with the following changes to the product: the substrate selection range is reduced, and GFPP and GGPP can not be converted any more.

Example 15

According to the results of examples 1-14, D510A, FgGS, GGPPS-Aa, AaTS and NCBI database seq id nos XP _018034954.1, AHY23929.1, XP _002846409.1, XP _003025181.1, XP _003236661.1, CEF73922.1, XP _009262810.1, OBS27829.1, XP _011317573.1, EYB24413.1, XP _011317623.1, OBS27869.1, XP _009262762.1, XP _003343918.1 and kfa74407.1, which have higher homology with terpene synthase FgMS, can be modified in the same way, including exchanging aromatic amino acids with non-aromatic amino acids in hydrophobic or neutral amino acids in the active region, to achieve the effects of changing substrate diversity, changing terpene product abundance and synthesizing new compounds.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

SEQUENCE LISTING

<110> Wuhan Zhen Zhi Biotechnology GmbH

<120> a method for engineering terpene synthase

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<170> PatentIn version 3.5

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Met Asp Phe Thr Tyr Arg Tyr Ser Phe Glu Pro Thr Asp Tyr Asp Thr

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Asp Gly Leu Cys Asp Gly Val Pro Val Arg Met His Lys Gly Ala Asp

20 25 30

Leu Asp Glu Val Ala Ile Phe Lys Ala Gln Tyr Asp Trp Glu Lys His

35 40 45

Val Gly Pro Lys Leu Pro Phe Arg Gly Ala Leu Gly Pro Arg His Asn

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Phe Ile Cys Leu Thr Leu Pro Glu Cys Leu Pro Glu Arg Leu Glu Ile

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Val Ser Tyr Ala Asn Glu Phe Ala Phe Leu His Asp Asp Ile Thr Asp

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Val Glu Ser Ala Glu Thr Val Ala Ala Glu Asn Asp Glu Phe Leu Asp

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Ala Leu Gln Gln Gly Val Arg Glu Gly Asp Ile Gln Ser Arg Glu Ser

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Gly Lys Arg His Leu Gln Ala Trp Ile Phe Lys Ser Met Val Ala Ile

130 135 140

Asp Arg Asp Arg Ala Val Ala Ala Met Asn Ala Trp Ala Thr Phe Ile

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Asn Thr Gly Ala Gly Cys Ala His Asp Thr Asn Phe Lys Ser Leu Asp

165 170 175

Glu Tyr Leu His Tyr Arg Ala Thr Asp Val Gly Tyr Met Phe Trp His

180 185 190

Ala Leu Ile Ile Phe Gly Cys Ala Ile Thr Ile Pro Glu His Glu Ile

195 200 205

Glu Leu Cys His Gln Leu Ala Leu Pro Ala Ile Met Ser Val Thr Leu

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Thr Asn Asp Ile Trp Ser Tyr Gly Lys Glu Ala Glu Ala Ala Glu Lys

225 230 235 240

Ser Gly Lys Pro Gly Asp Phe Val Asn Ala Leu Val Val Leu Met Arg

245 250 255

Glu His Asn Cys Ser Ile Glu Glu Ala Glu Arg Leu Cys Arg Ala Arg

260 265 270

Asn Lys Ile Glu Val Ala Lys Cys Leu Gln Val Thr Lys Glu Thr Arg

275 280 285

Glu Arg Lys Asp Val Ser Gln Asp Leu Lys Asp Tyr Leu Tyr His Met

290 295 300

Leu Phe Gly Val Ser Gly Asn Ala Ile Trp Ser Thr Gln Cys Arg Arg

305 310 315 320

Tyr Asp Met Thr Ala Pro Tyr Asn Glu Arg Gln Gln Ala Arg Leu Lys

325 330 335

Gln Thr Lys Gly Glu Leu Thr Ser Thr Tyr Asp Pro Val Gln Ala Ala

340 345 350

Lys Glu Ala Met Met Glu Ser Thr Arg Pro Glu Ile His Arg Leu Pro

355 360 365

Thr Pro Asp Ser Pro Arg Lys Glu Ser Phe Ala Val Arg Pro Leu Val

370 375 380

Asn Gly Ser Gly Gln Tyr Asn Gly Asn Asn His Ile Asn Gly Val Ser

385 390 395 400

Asn Glu Val Asp Val Arg Pro Ser Ile Glu Arg His Ala Ser Thr Lys

405 410 415

Arg Ala Thr Ser Ala Asp Asp Ile Asp Trp Thr Ala His Lys Lys Val

420 425 430

Asp Ser Gly Ala Asp His Lys Lys Thr Leu Ser Asp Ile Met Leu Gln

435 440 445

Glu Leu Pro Pro Met Glu Asp Asp Val Val Met Glu Pro Tyr Arg Tyr

450 455 460

Leu Cys Ser Leu Pro Ser Lys Gly Val Arg Asn Lys Thr Ile Asp Ala

465 470 475 480

Leu Asn Phe Trp Leu Lys Val Pro Ile Glu Asn Ala Asn Thr Ile Lys

485 490 495

Ala Ile Thr Glu Ser Leu His Gly Ser Ser Leu Met Leu Asp Asp Ile

500 505 510

Glu Asp His Ser Gln Leu Arg Arg Gly Lys Pro Ser Ala His Ala Val

515 520 525

Phe Gly Glu Ala Gln Thr Ile Asn Ser Ala Thr Phe Gln Tyr Ile Gln

530 535 540

Ser Val Ser Leu Ile Ser Gln Leu Arg Ser Pro Lys Ala Leu Asn Ile

545 550 555 560

Phe Val Asp Glu Ile Arg Gln Leu Phe Ile Gly Gln Ala Tyr Glu Leu

565 570 575

Gln Trp Thr Ser Asn Met Ile Cys Pro Pro Leu Glu Glu Tyr Leu Arg

580 585 590

Met Val Asp Gly Lys Thr Gly Gly Leu Phe Arg Leu Leu Thr Arg Leu

595 600 605

Met Ala Ala Glu Ser Thr Thr Glu Val Asp Val Asp Phe Ser Arg Leu

610 615 620

Cys Gln Leu Phe Gly Arg Tyr Phe Gln Ile Arg Asp Asp Tyr Ala Asn

625 630 635 640

Leu Lys Leu Ala Asp Tyr Thr Glu Gln Lys Gly Phe Cys Glu Asp Leu

645 650 655

Asp Glu Gly Lys Phe Ser Leu Pro Leu Ile Ile Ala Phe Asn Glu Asn

660 665 670

Asn Lys Ala Pro Lys Ala Val Ala Gln Leu Arg Gly Leu Met Met Gln

675 680 685

Arg Cys Val Asn Gly Gly Leu Thr Phe Glu Gln Lys Val Leu Ala Leu

690 695 700

Asn Leu Ile Glu Glu Ala Gly Gly Ile Ser Gly Thr Glu Lys Val Leu

705 710 715 720

His Ser Leu Tyr Gly Glu Met Glu Ala Glu Leu Glu Arg Leu Ala Gly

725 730 735

Val Phe Gly Ala Glu Asn His Gln Leu Glu Leu Ile Leu Glu Met Leu

740 745 750

Arg Ile Asp

755

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Met Asp Phe Thr Tyr Arg Tyr Ser Phe Glu Pro Thr Asp Tyr Asp Thr

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Asp Gly Leu Cys Asp Gly Val Pro Val Arg Met His Lys Gly Ala Asp

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Leu Asp Glu Val Ala Ile Phe Lys Ala Gln Tyr Asp Trp Glu Lys His

35 40 45

Val Gly Pro Lys Leu Pro Phe Arg Gly Ala Leu Gly Pro Arg His Asn

50 55 60

Phe Ile Cys Leu Thr Leu Pro Glu Cys Leu Pro Glu Arg Leu Glu Ile

65 70 75 80

Val Ser Tyr Ala Asn Glu Phe Ala Phe Leu His Asp Asp Ile Thr Asp

85 90 95

Val Glu Ser Ala Glu Thr Val Ala Ala Glu Asn Asp Glu Phe Leu Asp

100 105 110

Ala Leu Gln Gln Gly Val Arg Glu Gly Asp Ile Gln Ser Arg Glu Ser

115 120 125

Gly Lys Arg His Leu Gln Ala Trp Ile Phe Lys Ser Met Val Ala Ile

130 135 140

Asp Arg Asp Arg Ala Val Ala Ala Met Asn Ala Trp Ala Thr Phe Ile

145 150 155 160

Asn Thr Gly Ala Gly Cys Ala His Asp Thr Asn Phe Lys Ser Leu Asp

165 170 175

Glu Tyr Leu His Tyr Arg Ala Thr Asp Val Gly Tyr Met Phe Trp His

180 185 190

Ala Leu Ile Ile Phe Gly Cys Ala Ile Thr Ile Pro Glu His Glu Ile

195 200 205

Glu Leu Cys His Gln Leu Ala Leu Pro Ala Ile Met Ser Val Thr Leu

210 215 220

Thr Asn Asp Ile Trp Ser Tyr Gly Lys Glu Ala Glu Ala Ala Glu Lys

225 230 235 240

Ser Gly Lys Pro Gly Asp Phe Val Asn Ala Leu Val Val Leu Met Arg

245 250 255

Glu His Asn Cys Ser Ile Glu Glu Ala Glu Arg Leu Cys Arg Ala Arg

260 265 270

Asn Lys Ile Glu Val Ala Lys Cys Leu Gln Val Thr Lys Glu Thr Arg

275 280 285

Glu Arg Lys Asp Val Ser Gln Asp Leu Lys Asp Tyr Leu Tyr His Met

290 295 300

Leu Phe Gly Val Ser Gly Asn Ala Ile Trp Ser Thr Gln Cys Arg Arg

305 310 315 320

Tyr Asp Met Thr Ala Pro Tyr Asn Glu Arg Gln Gln Ala Arg Leu Lys

325 330 335

Gln Thr Lys Gly Glu Leu Thr Ser Thr Tyr Asp Pro Val Gln Ala Ala

340 345 350

Lys Glu Ala Met Met Glu Ser Thr Arg Pro Glu Ile His Arg Leu Pro

355 360 365

Thr Pro Asp Ser Pro Arg Lys Glu Ser Phe Ala Val Arg Pro Leu Val

370 375 380

Asn Gly Ser Gly Gln Tyr Asn Gly Asn Asn His Ile Asn Gly Val Ser

385 390 395 400

Asn Glu Val Asp Val Arg Pro Ser Ile Glu Arg His Ala Ser Thr Lys

405 410 415

Arg Ala Thr Ser Ala Asp Asp Ile Asp Trp Thr Ala His Lys Lys Val

420 425 430

Asp Ser Gly Ala Asp His Lys Lys Thr Leu Ser Asp Ile Met Leu Gln

435 440 445

Glu Leu Pro Pro Met Glu Asp Asp Val Val Met Glu Pro Tyr Arg Tyr

450 455 460

Leu Cys Ser Leu Pro Ser Lys Gly Val Arg Asn Lys Thr Ile Asp Ala

465 470 475 480

Leu Asn Phe Trp Leu Lys Val Pro Ile Glu Asn Ala Asn Thr Ile Lys

485 490 495

Ala Ile Thr Glu Ser Leu His Gly Ser Ser Leu Met Leu Ala Asp Ile

500 505 510

Glu Asp His Ser Gln Leu Arg Arg Gly Lys Pro Ser Ala His Ala Val

515 520 525

Phe Gly Glu Ala Gln Thr Ile Asn Ser Ala Thr Phe Gln Tyr Ile Gln

530 535 540

Ser Val Ser Leu Ile Ser Gln Leu Arg Ser Pro Lys Ala Leu Asn Ile

545 550 555 560

Phe Val Asp Glu Ile Arg Gln Leu Phe Ile Gly Gln Ala Tyr Glu Leu

565 570 575

Gln Trp Thr Ser Asn Met Ile Cys Pro Pro Leu Glu Glu Tyr Leu Arg

580 585 590

Met Val Asp Gly Lys Thr Gly Gly Leu Phe Arg Leu Leu Thr Arg Leu

595 600 605

Met Ala Ala Glu Ser Thr Thr Glu Val Asp Val Asp Phe Ser Arg Leu

610 615 620

Cys Gln Leu Phe Gly Arg Tyr Phe Gln Ile Arg Asp Asp Tyr Ala Asn

625 630 635 640

Leu Lys Leu Ala Asp Tyr Thr Glu Gln Lys Gly Phe Cys Glu Asp Leu

645 650 655

Asp Glu Gly Lys Phe Ser Leu Pro Leu Ile Ile Ala Phe Asn Glu Asn

660 665 670

Asn Lys Ala Pro Lys Ala Val Ala Gln Leu Arg Gly Leu Met Met Gln

675 680 685

Arg Cys Val Asn Gly Gly Leu Thr Phe Glu Gln Lys Val Leu Ala Leu

690 695 700

Asn Leu Ile Glu Glu Ala Gly Gly Ile Ser Gly Thr Glu Lys Val Leu

705 710 715 720

His Ser Leu Tyr Gly Glu Met Glu Ala Glu Leu Glu Arg Leu Ala Gly

725 730 735

Val Phe Gly Ala Glu Asn His Gln Leu Glu Leu Ile Leu Glu Met Leu

740 745 750

Arg Ile Asp

755

<210> 3

<211> 316

<212> PRT

<213> Artificial Sequence

<220>

<223> SEQ ID NO：3

<400> 3

Met Asp Pro Tyr Ser Glu Thr Ser Asp Leu Val Asp Ile Ser Arg Phe

1 5 10 15

Asp Thr His Gly Leu Gly Ala Asn Tyr Lys Leu Arg Arg His Lys Phe

20 25 30

Glu His Leu Ala Asp Thr Gly Cys His Lys Ala Arg Ser Asp Trp Val

35 40 45

Lys Tyr Ile Gly Pro Leu Thr Glu Phe Gly Gly Cys Asn His Ile Asn

50 55 60

Gly Asn Phe Ser Ala Val Val Leu Pro Leu Cys Arg Pro Asp Arg Leu

65 70 75 80

Glu Leu Ile Ala Tyr Val Leu Glu Phe Ala Phe Leu His Asp Ser Val

85 90 95

Leu Glu Ser Glu Asn Thr Ser Pro Glu Ser Glu Val Gln Ala Glu Ala

100 105 110

Gly Leu Arg Leu Leu Tyr Glu Arg Cys Ile Ser Arg Leu Leu Gln Thr

115 120 125

Asp Glu Val Cys Ala Lys Lys Ile Ala Lys Thr Trp Lys Asp Ala Ile

130 135 140

Asn Thr Thr Thr Lys Asp Lys Asn Val Asp Phe Gln Ser Ile Glu Asp

145 150 155 160

Tyr Leu Glu Phe Arg Met Ile Asp Thr Gly Ala Pro Phe Val Glu Ala

165 170 175

Leu Met Leu Phe Gly Leu Gly Met Ser Leu Ser Pro Gln Glu Asp Asp

180 185 190

Ala Leu Gly His Val Ile Arg Pro Cys Phe Ala Ala Leu Ala Leu Thr

195 200 205

Asn Asp Tyr Phe Ser Phe Asp Arg Glu Ile Glu Glu Val Asp Thr Ser

210 215 220

Thr Leu Ile Asn Ser Val Ala Ile Val Met Arg Ile Gln Ser Leu Asp

225 230 235 240

Ile Pro Thr Ala Lys Thr Ile Ile Asn Glu Thr Ile Gln Lys Tyr Glu

245 250 255

Arg Glu Phe Leu Arg Arg Ile Asp Glu Tyr Lys Gln His Lys Gly Pro

260 265 270

Ile Ser Asn Lys Ile Glu Gln Tyr Met Glu Ala Met Thr Tyr Gln Ile

275 280 285

Ser Gly Asn Leu Val Trp Ser Leu Asn Cys Pro Arg Tyr Asn Pro Asp

290 295 300

Tyr Arg Tyr Gly Leu Glu Ala Cys Gln His Glu Gly

305 310 315

<210> 4

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> SEQ ID NO：4

<400> 4

Met Arg Tyr Gln Tyr Ser Glu Arg Val Glu Ser His Arg Tyr Arg Asp

1 5 10 15

Asp Gly Leu Ala Asn Asn Ile His Leu Arg Ile His Lys Asp Ser Tyr

20 25 30

Lys Glu Val Ile Gly Thr Leu Arg Ala Gln Asn Asp Trp Ser Arg Leu

35 40 45

Val Ser Ser Met Thr Lys Tyr His Gly Gly Leu Gly Asp Leu Phe Ser

50 55 60

Phe Ile Ser Val Thr Ile Pro Glu Cys Leu Pro Glu Arg Leu Glu Val

65 70 75 80

Val Ala Tyr Ala Asn Glu Tyr Ala Phe Leu Tyr Asp Asp Gln Met Glu

85 90 95

Arg Leu Asp Leu Lys Asp Phe Arg Glu Gly Arg Asp Asp Met Leu Asp

100 105 110

Ile Phe Gly Ile His Gly Gly Ala Ser Asn Leu Glu Asp Arg Arg Pro

115 120 125

Glu Lys Thr Leu Gln Leu Gln Ile Phe Asp Glu Leu Met Ala Ile Asp

130 135 140

Gln Asp Arg Ala Ile Val Thr Met Gln Ala Trp Ala Lys Phe Ile Asp

145 150 155 160

Leu Ala Ser Arg Thr Arg Val Glu Pro Phe Asn Thr Leu Ala Ala Tyr

165 170 175

Leu Pro Ser Arg Thr Ile Asp Ala Gly Glu Leu Phe Trp Phe Gly Met

180 185 190

Leu Thr Phe Ala Met Ala Leu Thr Ile Pro Ala His Glu Leu Asp Val

195 200 205

Cys Met Arg Leu Ala Arg Pro Gly Tyr Glu Ala Ile Ser Leu Ile Asn

210 215 220

Asp Ile Tyr Ser Trp Pro Lys Glu Arg Ala Glu Ala Glu Lys Ala Gly

225 230 235 240

Gln Asp Tyr Val Phe Asn Ala Val Trp Val Val Met Lys Glu Arg Lys

245 250 255

Cys Asp Glu Gln Lys Ala Thr Glu Phe Cys Lys Asn Leu Ala Arg Gln

260 265 270

Ser Ile Gln Asp Phe Ser Thr Ser Val Asn Thr Pro Gln Val Thr Glu

275 280 285

Leu Ser Cys Asp Ser Arg Thr Tyr Leu Gly Ala Val Arg Leu Ser Tyr

290 295 300

Val Gly Asn Leu Val Trp Ser Ile Tyr Cys Pro Arg Tyr Asn Ile Ala

305 310 315 320

Val Pro Val Tyr His Ser Lys Leu

325

<210> 5

<211> 328

<212> PRT

<213> Artificial Sequence

<220>

<223> SEQ ID NO：5

<400> 5

Met Arg Tyr Gln Tyr Ser Glu Arg Val Glu Ser His Arg Tyr Arg Asp

1 5 10 15

Asp Gly Leu Ala Asn Asn Ile His Leu Arg Ile His Lys Asp Ser Tyr

20 25 30

Lys Glu Val Ile Gly Thr Leu Arg Ala Gln Asn Asp Trp Ser Arg Leu

35 40 45

Val Ser Ser Met Thr Lys Tyr His Gly Gly Leu Gly Asp Leu Phe Ser

50 55 60

Phe Ile Ser Val Thr Ile Pro Glu Cys Leu Pro Glu Arg Leu Glu Val

65 70 75 80

Val Ala Tyr Ala Asn Glu Tyr Ala Phe Leu Tyr Asp Asp Gln Met Glu

85 90 95

Arg Leu Asp Leu Lys Asp Phe Arg Glu Gly Arg Asp Asp Met Leu Asp

100 105 110

Ile Phe Gly Ile His Gly Gly Ala Ser Asn Leu Glu Asp Arg Arg Pro

115 120 125

Glu Lys Thr Leu Gln Leu Gln Ile Phe Asp Glu Leu Met Ala Ile Asp

130 135 140

Gln Asp Arg Ala Ile Val Thr Met Gln Ala Trp Ala Lys Phe Ile Asp

145 150 155 160

Leu Ala Ser Arg Thr Arg Val Glu Pro Phe Asn Thr Leu Ala Ala Tyr

165 170 175

Leu Pro Ser Arg Thr Ile Asp Ala Gly Glu Leu Phe Trp Phe Gly Met

180 185 190

Leu Thr Phe Ala Met Ala Leu Thr Ile Pro Ala His Glu Leu Asp Val

195 200 205

Cys Met Arg Leu Ala Arg Pro Gly Tyr Glu Ala Ile Ser Leu Ile Asn

210 215 220

Asp Ile Tyr Ser Trp Pro Lys Glu Arg Ala Glu Ala Glu Lys Ala Gly

225 230 235 240

Gln Asp Tyr Val Phe Asn Ala Val Trp Val Val Met Lys Glu Arg Lys

245 250 255

Cys Asp Glu Gln Lys Ala Thr Glu Phe Cys Lys Asn Leu Ala Arg Gln

260 265 270

Ser Ile Gln Asp Phe Ser Thr Ser Val Asn Thr Pro Gln Val Thr Glu

275 280 285

Leu Ser Cys Asp Ser Arg Thr Tyr Leu Gly Ala Val Arg Leu Ser Tyr

290 295 300

Val Gly Asn Leu Val Trp Ser Ile Tyr Cys Pro Arg Tyr Asn Ile Ala

305 310 315 320

Val Pro Val Tyr His Ser Lys Leu

325

<210> 6

<211> 2268

<212> DNA

<213> Artificial Sequence

<220>

<223> SEQ ID NO：6

<400> 6

atggatttca cctaccgtta tagctttgaa ccgaccgact acgataccga cggtctgtgc 60

gacggtgtgc cggttcgtat gcacaagggt gcggatctgg acgaagtggc gatcttcaaa 120

gcgcagtatg actgggagaa gcacgttggc ccgaaactgc cgttccgtgg tgcgctgggt 180

ccgcgtcaca actttatttg cctgaccctg ccggaatgcc tgccggaacg tctggagatc 240

gtgagctacg cgaacgagtt cgcgtttctg cacgacgata ttaccgatgt ggaaagcgcg 300

gagaccgttg cggcggaaaa cgatgagttc ctggacgcgc tgcagcaagg tgttcgtgaa 360

ggcgacatcc aaagccgtga gagcggcaag cgtcacctgc aggcgtggat ttttaaaagc 420

atggtggcga tcgatcgtga ccgtgcggtt gcggcgatga acgcgtgggc gaccttcatt 480

aacaccggtg cgggctgcgc gcacgatacc aactttaaga gcctggacga gtacctgcac 540

tatcgtgcga ccgacgtggg ttacatgttc tggcacgcgc tgatcatttt tggctgcgcg 600

atcaccattc cggagcacga aatcgagctg tgccaccagc tggcgctgcc ggcgattatg 660

agcgtgaccc tgaccaacga catctggagc tatggtaaag aagcggaggc ggcggaaaag 720

agcggtaaac cgggcgactt cgttaacgcg ctggttgtgc tgatgcgtga acacaactgc 780

agcattgagg aagcggagcg tctgtgccgt gcgcgtaaca agatcgaggt ggcgaaatgc 840

ctgcaagtta ccaaggaaac ccgtgagcgt aaagatgtga gccaggatct gaaggactac 900

ctgtatcaca tgctgtttgg tgttagcggc aacgcgatct ggagcaccca gtgccgtcgt 960

tacgacatga ccgcgccgta taacgaacgt cagcaagcgc gtctgaagca aaccaaaggc 1020

gagctgacca gcacctacga tccggttcag gcggcgaagg aagcgatgat ggagagcacc 1080

cgtccggaaa ttcaccgtct gccgaccccg gacagcccgc gtaaagagag cttcgcggtg 1140

cgtccgctgg ttaacggtag cggccaatat aacggtaaca accacattaa cggcgtgagc 1200

aacgaagtgg acgttcgtcc gagcatcgag cgtcacgcga gcaccaaacg tgcgaccagc 1260

gcggacgaca tcgattggac cgcgcacaag aaagttgata gcggtgcgga ccacaagaaa 1320

accctgagcg acattatgct gcaggaactg ccgccgatgg aggacgatgt ggttatggaa 1380

ccgtaccgtt atctgtgcag cctgccgagc aagggtgtgc gtaacaaaac cattgatgcg 1440

ctgaactttt ggctgaaggt tccgatcgaa aacgcgaaca ccatcaaagc gattaccgag 1500

agcctgcacg gcagcagcct gatgctggac gacatcgaag accacagcca actgcgtcgt 1560

ggcaagccga gcgcgcacgc ggtgttcggc gaggcgcaga ccattaacag cgcgaccttt 1620

cagtacattc aaagcgtgag cctgatcagc caactgcgta gcccgaaagc gctgaacatc 1680

ttcgttgatg aaattcgtca gctgtttatc ggtcaagcgt acgagctgca gtggaccagc 1740

aacatgatct gcccgccgct ggaggaatat ctgcgtatgg ttgacggcaa gaccggtggc 1800

ctgttccgtc tgctgacccg tctgatggcg gcggaaagca ccaccgaggt ggatgttgac 1860

tttagccgtc tgtgccaact gttcggtcgt tactttcaga tccgtgacga ttatgcgaac 1920

ctgaagctgg cggattacac cgaacagaaa ggtttctgcg aggacctgga cgagggcaaa 1980

ttcagcctgc cgctgatcat tgcgtttaac gagaacaaca aggcgccgaa agcggtggcg 2040

caactgcgtg gcctgatgat gcagcgttgc gtgaacggtg gcctgacctt cgaacaaaag 2100

gttctggcgc tgaacctgat tgaggaagcg ggtggcatca gcggtaccga gaaagtgctg 2160

cacagcctgt atggcgaaat ggaggcggaa ctggagcgtc tggcgggtgt ttttggcgcg 2220

gagaaccacc agctggaact gattctggag atgctgcgta tcgactaa 2268

<210> 7

<211> 2268

<212> DNA

<213> Artificial Sequence

<220>

<223> SEQ ID NO：7

<400> 7

atggatttca cctaccgtta tagctttgaa ccgaccgact acgataccga cggtctgtgc 60

gacggtgtgc cggttcgtat gcacaagggt gcggatctgg acgaagtggc gatcttcaaa 120

gcgcagtatg actgggagaa gcacgttggc ccgaaactgc cgttccgtgg tgcgctgggt 180

ccgcgtcaca actttatttg cctgaccctg ccggaatgcc tgccggaacg tctggagatc 240

gtgagctacg cgaacgagtt cgcgtttctg cacgacgata ttaccgatgt ggaaagcgcg 300

gagaccgttg cggcggaaaa cgatgagttc ctggacgcgc tgcagcaagg tgttcgtgaa 360

ggcgacatcc aaagccgtga gagcggcaag cgtcacctgc aggcgtggat ttttaaaagc 420

atggtggcga tcgatcgtga ccgtgcggtt gcggcgatga acgcgtgggc gaccttcatt 480

aacaccggtg cgggctgcgc gcacgatacc aactttaaga gcctggacga gtacctgcac 540

tatcgtgcga ccgacgtggg ttacatgttc tggcacgcgc tgatcatttt tggctgcgcg 600

atcaccattc cggagcacga aatcgagctg tgccaccagc tggcgctgcc ggcgattatg 660

agcgtgaccc tgaccaacga catctggagc tatggtaaag aagcggaggc ggcggaaaag 720

agcggtaaac cgggcgactt cgttaacgcg ctggttgtgc tgatgcgtga acacaactgc 780

agcattgagg aagcggagcg tctgtgccgt gcgcgtaaca agatcgaggt ggcgaaatgc 840

ctgcaagtta ccaaggaaac ccgtgagcgt aaagatgtga gccaggatct gaaggactac 900

ctgtatcaca tgctgtttgg tgttagcggc aacgcgatct ggagcaccca gtgccgtcgt 960

tacgacatga ccgcgccgta taacgaacgt cagcaagcgc gtctgaagca aaccaaaggc 1020

gagctgacca gcacctacga tccggttcag gcggcgaagg aagcgatgat ggagagcacc 1080

cgtccggaaa ttcaccgtct gccgaccccg gacagcccgc gtaaagagag cttcgcggtg 1140

cgtccgctgg ttaacggtag cggccaatat aacggtaaca accacattaa cggcgtgagc 1200

aacgaagtgg acgttcgtcc gagcatcgag cgtcacgcga gcaccaaacg tgcgaccagc 1260

gcggacgaca tcgattggac cgcgcacaag aaagttgata gcggtgcgga ccacaagaaa 1320

accctgagcg acattatgct gcaggaactg ccgccgatgg aggacgatgt ggttatggaa 1380

ccgtaccgtt atctgtgcag cctgccgagc aagggtgtgc gtaacaaaac cattgatgcg 1440

ctgaactttt ggctgaaggt tccgatcgaa aacgcgaaca ccatcaaagc gattaccgag 1500

agcctgcacg gcagcagcct gatgctggcc gacatcgaag accacagcca actgcgtcgt 1560

ggcaagccga gcgcgcacgc ggtgttcggc gaggcgcaga ccattaacag cgcgaccttt 1620

cagtacattc aaagcgtgag cctgatcagc caactgcgta gcccgaaagc gctgaacatc 1680

ttcgttgatg aaattcgtca gctgtttatc ggtcaagcgt acgagctgca gtggaccagc 1740

aacatgatct gcccgccgct ggaggaatat ctgcgtatgg ttgacggcaa gaccggtggc 1800

ctgttccgtc tgctgacccg tctgatggcg gcggaaagca ccaccgaggt ggatgttgac 1860

tttagccgtc tgtgccaact gttcggtcgt tactttcaga tccgtgacga ttatgcgaac 1920

ctgaagctgg cggattacac cgaacagaaa ggtttctgcg aggacctgga cgagggcaaa 1980

ttcagcctgc cgctgatcat tgcgtttaac gagaacaaca aggcgccgaa agcggtggcg 2040

caactgcgtg gcctgatgat gcagcgttgc gtgaacggtg gcctgacctt cgaacaaaag 2100

gttctggcgc tgaacctgat tgaggaagcg ggtggcatca gcggtaccga gaaagtgctg 2160

cacagcctgt atggcgaaat ggaggcggaa ctggagcgtc tggcgggtgt ttttggcgcg 2220

gagaaccacc agctggaact gattctggag atgctgcgta tcgactaa 2268

<210> 8

<211> 951

<212> DNA

<213> Artificial Sequence

<220>

<223> SEQ ID NO：8

<400> 8

atggatccct acagtgaaac atcagatctt gttgacattt ctcgcttcga cacccacggc 60

cttggagcta attacaaact acgacgacat aagttcgaac acctagctga cactggatgt 120

cacaaagcaa ggtcagattg ggtaaaatac attggccctc ttactgaatt cggaggctgc 180

aatcacatca acgggaattt ctctgctgta gtgttgccat tgtgcagacc tgaccgcctg 240

gagcttatag catatgtact cgaattcgca tttcttcatg attccgttct cgagtcagaa 300

aacacgtctc cggaatccga agtgcaagcc gaggctggtc tacgcctctt atatgaacga 360

tgcataagtc gactcttgca gacagacgaa gtatgcgcca aaaagattgc aaagacgtgg 420

aaagacgcga tcaacacaac tacaaaggat aagaacgtgg acttccaatc tatagaagac 480

tacttggagt ttcgcatgat tgatactggt gcaccgttcg tcgaggccct catgcttttt 540

ggattgggca tgtcgctttc accgcaagaa gatgatgctc ttggtcacgt tattcggcca 600

tgtttcgccg ctttggcgtt gacgaacgac tacttttcgt ttgatcgaga gatagaagaa 660

gtcgatactt ctactcttat caactcggtt gccatagtaa tgcgaattca gagtctggac 720

attcccaccg ccaagacaat tatcaatgag actatacaga agtacgagcg agagttcctc 780

cgacgcattg atgagtacaa acagcacaaa ggaccaatct ctaacaagat tgaacaatac 840

atggaagcta tgacttatca gatcagtggg aatttagtat ggagtctgaa ttgtcctaga 900

tataatcctg actatcggta cggactggag gcttgtcagc acgagggttg a 951

<210> 9

<211> 1251

<212> DNA

<213> Artificial Sequence

<220>

<223> SEQ ID NO：9

<400> 9

atgtctactg aaacgcatcc tttcgcctcg ccgaacgcca taccacctcg aaccagctct 60

actggccaag tcacgaacgg ctatcctata aatccgcggc acagcgtctt gcgcccgctc 120

tcagaaattg actggatgag ccaaagtaaa aagagcaaga cctcacacgt ttccaccgaa 180

ccactcaaca gcacacaacc acacacacgc acgctgtcgc aaccacagtc gcagcccgac 240

cctatgaacc tcgaagaagt cagcacaaac taccccaccc cgctctcccc gccgagtgac 300

accaagaacc tgggcgaaga cctcatatac ggcaacggcg cagcatggac agaagagaag 360

gagcgcatac tgctggggcc ttatgattac ctttggggtc acccgggcaa ggacataagg 420

tcacaatgca tagcagcgtt caacctgtgg ctgaaagtac caccagagcg gcttgaggtc 480

ataacgcgcg cggtgggcat gctacacaca gcatctcttt tggtcgacga tgtcgaagac 540

agctcaatat tacggcgagg cattcctgtc gcgaatagca tattcggcgt tgcgcagacg 600

atcaactcgg cgaactacgt atacttcaag gcgttgcagg agctgatgca catgggcaat 660

cccaagctca tcgagatctt cacagaagag ctgttgaacc tgcacagagg ccagggaatg 720

gatctgtact ggcgggacag tttgacatgt cctagcgaag cagattacct agagatggta 780

ggcaacaaga ccggtggcct gttcaggcta gcgatcaagc tcatgcaggc cgaaagcgca 840

gtacaagtcg actgcgcacc cctcgtctcc acaatcggcc tcctcttcca gatcctcgac 900

gatcacctca atctctcccc cacgtcgggc tactcctcgc tcaaaggcct ctgcgaagac 960

ctcaccgaag gcaaattctc cttccccgtc atccacgcta tccgcgccga cccgtcgaac 1020

cagatcctca tcaacatcct caagcagaaa actacagatg aggaggtcaa gcgctatgcg 1080

ctcaagtaca tggagagtaa gggtagcttt gaatattcca agagggttat tgatgacttg 1140

agggggaaga cggaggggct tgtcagtggg attgagaagg ggttgggcga ggaggggacg 1200

cagggggcgg aggcgttgag gaaaatgtta gggaggttgg tgttgaggta g 1251

<210> 10

<211> 986

<212> DNA

<213> Artificial Sequence

<220>

<223> SEQ ID NO：10

<400> 10

atgcgttacc agtatagcga gcgtgtggaa agccaccgtt atcgtgacga tggtctggcg 60

aacaacattc acctgcgtat ccacaaggat agctacaaag aagtgattgg caccctgcgt 120

gcgcaaaacg actggagccg tctggttagc agcatgacca agtatcacgg tggcctgggc 180

gacctgttca gctttattag cgttaccatc ccggaatgcc tgccggagcg tctggaagtg 240

gttgcgtacg cgaacgagta tgcgttcctg tacgacgatc agatggaacg tctggacctg 300

aaagatttcc gtgagggtcg tgacgatatg ctggacatct ttggcattca cggtggcgcg 360

agcaacctgg aggatcgtcg tccggaaaag accctgcagc tgcaaatttt tgacgagctg 420

atggcgattg accaggatcg tgcgatcgtg accatgcaag cgtgggcgaa attcatcgat 480

ctggcgagcc gtacccgtgt tgaaccgttt aacaccctgg cggcgtatct gccgagccgt 540

accattgacg cgggcgagct gttctggttt ggcatgctga ccttcgcgat ggcgctgacc 600

atcccggcgc acgaactgga tgtgtgcatg cgtctggcgc gtccgggtta tgaggcgatc 660

agcctgatta acgacatcta cagctggccg aaggaacgtg cggaggcgga aaaagcgggc 720

caggattacg tgtttaacgc ggtttgggtg gttatgaagg agcgtaaatg cgacgaacaa 780

aaggcgaccg agttctgcaa aaacctggcg cgtcagagca tccaagattt tagcaccagc 840

gtgaacaccc cgcaagttac cgagctgagc tgcgacagcc gtacctatct gggtgcggtt 900

cgtctgagct acgtgggcaa cctggtttgg agcatttatt gcccgcgtta caacatcgcg 960

gtgccggttt accacagcaa gctgta 986

Claims

1. A terpene synthase comprising a mutation in one of the following mutations in a wild-type terpene synthase:

the threonine at the 69 th site is mutated into phenylalanine, the asparagine at the 85 th site is mutated into phenylalanine, the alanine at the 88 th site is mutated into phenylalanine, the phenylalanine at the 89 th site is mutated into leucine, the phenylalanine at the 159 th site is mutated into glycine, the valine at the 186 th site is mutated into phenylalanine, the tryptophan at the 191 th site is mutated into valine, the valine at the 222 th site is mutated into phenylalanine, the glycine at the 307 th site is mutated into phenylalanine or the tryptophan at the 314 th site is mutated into glycine;

the amino acid sequence of the wild type terpene synthase is shown as SEQ ID NO: 1 is shown.

2. A nucleic acid molecule encoding the terpene synthase of claim 1.

3. A construct comprising the nucleic acid molecule of claim 2.

4. A recombinant cell comprising the nucleic acid molecule of claim 2.

5. Use of the terpene synthase of claim 1 or the nucleic acid molecule of claim 2 or the construct of claim 3 or the recombinant cell of claim 4 for the synthesis of terpenoids.

6. A method of obtaining the terpene synthase of claim 1, comprising:

culturing the recombinant cell of claim 4 so as to obtain the terpene synthase.