Determination and optimization of key amino acid sites to control sesquiterpene synthesis

Abstract

The invention discloses judgment and optimization of key amino acid sites for controlling sesquiterpene synthesis, and determines amino acid residues lysine or arginine or glutamine related to sesquiterpene synthesis for point mutation and combined mutation of various plant sesquiterpene synthase. Lysine or arginine or glutamine is located in a cavity among 5-10 amino acids in front of the first long alpha helix at the N end, alpha helixes (also the longest alpha helix in the protein) communicating the N end and the C end of the protein and the last 1-7 amino acids at the C end at the site. The method for determining the key amino acid can be used for efficiently screening the sesquiterpene synthase with activity, and the optimization of the amino acid of the key residue can be used for guiding the transformation of the high-yield sesquiterpene synthase for industrial production. The invention has important application value and economic benefit in the fields of biological synthase identification of sesquiterpenoids, biosynthesis of drugs, fermentation production of spices, synthesis of industrial raw materials and the like.

Description

Identification and optimization of key amino acid sites controlling sesquiterpene synthesis Technical Field

[0001] This invention relates to the field of biology, specifically to a method for identifying and optimizing key amino acid sites that control sesquiterpene synthesis. Background Technology

[0002] Sesquiterpene synthases are core enzymes in the biosynthesis of terpenoids in organisms. They catalyze the cyclization and rearrangement of farnesyl pyrophosphate precursors (FPP) to generate various sesquiterpene skeletons. Engineered strains with these characteristics are widely used in the pharmaceutical, fragrance, and food industries. Many known natural terpenoid drugs are synthesized through skeleton modification via this pathway, such as artemisinin and bisabolol.

[0003] Previous studies have demonstrated that magnesium ion-enriched cavities in sesquiterpene synthases anchor pyrophosphate groups in FPP substrates via metal and hydrogen bonds, leading to their removal. Subsequently, the unstable, positively charged isopentenyl group undergoes self-cyclization to generate diverse parent nuclei. This invention identifies a key amino acid site controlling sesquiterpene synthesis and proposes methods for its identification and optimization. Summary of the Invention

[0004] To address the limitations of existing research, this invention proposes a novel method for identifying and optimizing a key amino acid site controlling sesquiterpene synthesis. This invention selected various plant sesquiterpene synthases and conducted research on key residues at this site, as shown in SEQ NO. 1-3. Through site-directed mutagenesis, the key residues were determined to be arginine, lysine, or glutamine.

[0005] One aspect of the present invention provides a method for determining key amino acid sites controlling sesquiterpene synthesis: performing three-dimensional modeling of sesquiterpene synthase with known amino acid sequences to determine whether there is a cavity between the "1-10 amino acids before the first long α-helix at the N-terminus, the α-helix at the N-terminus and C-terminus of the connecting protein (which is also the longest α-helix in this type of protein), and the last 1-7 amino acids at the C-terminus," and whether there is lysine, arginine, or glutamine exposed in the cavity on the "α-helix at the N-terminus and C-terminus of the connecting protein."

[0006] In another aspect of the present invention, a method for optimizing key amino acids for controlling sesquiterpene synthesis is provided: for the three-dimensional modeling results, the amino acids exposed in the cavity of the "α-helix of the N-terminus and C-terminus of the connecting protein" are mutated at a single point or by combination to lysine, arginine or glutamine.

[0007] The beneficial effects of this invention are as follows: 1. This invention can rapidly determine the activity of sesquiterpene synthase by detecting the presence of key amino acids in the three-dimensional modeling results of the amino acid sequence. 2. This invention can improve enzyme activity and yield by modifying key residues of sesquiterpene synthase. Attached Figure Description

[0008] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0009] Figure 1 shows the three-dimensional structural model and key amino acid sites of SEQ NO.1. Red represents the 5-10 amino acids before the first long α-helix at the N-terminus; yellow represents the α-helix connecting the N-terminus and C-terminus of the protein; and green represents the last 1-7 amino acids at the C-terminus.

[0010] Figure 2 shows the total ion chromatogram of the yeast fermentation product of SEQ NO.2 as detected by GC-MS. Black represents the blank control, and red represents the fermentation of SEQ NO.2.

[0011] Figure 3 is a comparison of the changes in yeast fermentation yield of the SEQ NO.1 mutant.

[0012] Figure 4 is a comparison of the changes in yeast fermentation yield of the SEQ NO.2 mutant.

[0013] Figure 5 is a comparison of the changes in yeast fermentation yield of the SEQ NO.3 mutant.

[0014] Figure 6 is a comparison of changes in yeast fermentation yield after optimization of amino acids according to SEQ NO.2. Detailed Implementation

[0015] The present invention will now be described in detail with reference to specific accompanying drawings and embodiments. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

[0016] The identification and optimization of key amino acid sites controlling sesquiterpene synthesis, and the specific implementation process are as follows:

[0017] Example 1: A rapid preliminary assessment of sesquiterpene synthase activity was conducted by performing three-dimensional modeling of the amino acid sequence to identify a cavity located "5-10 amino acids before the first long α-helix at the N-terminus, the α-helices at the N-terminus and C-terminus of the connecting protein (also the longest α-helix in this type of protein), and the last 1-7 amino acids at the C-terminus" (Figure 1). For example, in SEQ NO.1, this cavity is located between amino acids 26-34, 228-242, and 538-546. The α-helices at the N-terminus and C-terminus of this cavity contain one of the following: lysine, arginine, or glutamine (Figure 1). For example, in SEQ NO.1, this is arginine at position 239 (R239). The identified amino acid coding gene was cloned or synthesized, cloned into the pESC-URA vector, and transformed into engineered Saccharomyces cerevisiae for fermentation. The fermentation broth was extracted with dodecane and the product was detected by GC-MS. For SEQ NO.2, which had not been previously reported, a new sesquiterpene product was successfully detected (Figure 2).

[0018] Example 2: Determination of Key Amino Acids in Sesquiterpene Synthases. In the three-dimensional modeling of protein amino acids, lysine, arginine, and glutamine in the cavity of the "α-helix connecting the N-terminus and C-terminus of the protein" were mutated to alanine. Amino acid side-chain deletion studies were conducted to minimize the impact on the overall three-dimensional structural stability. The gene encoding the mutated amino acids was synthesized, cloned into the vector pESC-URA, and transformed into engineered Saccharomyces cerevisiae for fermentation. The fermentation broth was extracted with dodecane and the products were analyzed by GC-MS. For SEQ NO.1, after the mutation of arginine-R239 at amino acid position 239 to alanine-R239A, the characteristic sesquiterpene product almost completely disappeared (Figure 3). For SEQ NO.2, after the mutation of lysine-K238 at amino acid position 238 to alanine-K238A, the characteristic sesquiterpene product almost completely disappeared (Figure 4). For SEQ NO.3, after the arginine-R246 at position 25 of the amino acid sequence is mutated to alanine-R246A, the characteristic sesquiterpene product almost completely disappears (as shown in Figure 5).

[0019] Example 3: Optimization of Key Amino Acids for Sesquiterpene Synthase. In the three-dimensional modeling of protein amino acids, the amino acid at the "α-helix connecting the N-terminus and C-terminus of the protein" was mutated to one of lysine, arginine, or glutamine, or a combination thereof. The gene encoding the determined amino acid mutation was synthesized, cloned into the pESC-URA vector, and transformed into engineered Saccharomyces cerevisiae for fermentation. The fermentation broth was extracted with dodecane and the products were analyzed by GC-MS. For SEQ NO.2, the yield of the characteristic sesquiterpene product increased after the lysine-K238 mutation at position 238 of the amino acid sequence was changed to arginine-K238R (Figure 6); the yield of the characteristic sesquiterpene product increased after the leucine-L232 mutation at position 232 of the amino acid sequence was changed to glutamine-L232Q (Figure 6); the yield of the characteristic sesquiterpene product increased further than that of the single mutation after the leucine-L232 mutation at position 232 of the amino acid sequence was changed to glutamine and the lysine mutation at position 238 was changed to arginine-L232Q+K238R (Figure 6).

[0020] For the recombinant cell acquisition and fermentation protocol in Examples 1, 2, or 3, we adopted the following steps: After synthesis, the target gene was cloned into the pESC-URA vector, transformed into yeast PPB25 competent cells, and transformants were screened on SD-URA medium. Positive transformants were cultured in SD-URA liquid medium at 37 °C with shaking at 200 rpm until A600 = 0.4 ~ 0.6. They were then inoculated at a ratio of 1:100 into 20 ml of SD-URA liquid medium and cultured at 37 °C with shaking at 200 rpm for 12 h. 2 ml of n-dodecane was added to the top layer of the culture medium, and the culture was continued with shaking for 72 h. The upper organic liquid was then aspirated, ultracentrifuged, and transferred to an end-capped tube. The product was detected by gas chromatography-mass spectrometry (GC-MS).

[0021] For the GC-MS detection in Examples 1, 2, or 3, we used the following protocol. The GC-MS instrument was an Agilent 7890B-5977B, and the column was a 19091S-433. The GC furnace was initially set at 85°C and held for 5 min, then increased to 185°C at a rate of 3°C / min and held at 185°C for 10 min. The temperature was then increased to 250°C at a rate of 5°C / min and held for another 5 min. The split ratio was 1:10, and the injection volume was 1 μL. The interface temperature was maintained at 250°C. ◦ C. Mass spectra scanned in full scan mode, with a range of 33 ~ 350 amu; ion source temperature 230 °C, solvent delay time 15 min.

[0022] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

[0023] SEQ NO.*

[0024] SEQ NO.2MCPEQENDLRPTADFPPDIWGDQFLVYDEEEQDHGVEQVVEDLKLKVKKEILATLNVPAEHTNLLKLVDTIQRLGISYYFEEEINEALQHIYDEYGDNWTGGCPSVWFRLLRQQGFFVSCGIFNKDKDGAFKESLMNDVQGLLDLYEAAYMRVPGEVILDDALVFTRSRLDDISKDPLHGTNIDSTQIQEALTQPIVKRLPRLEALRYIPFYQRQDSHNEHLLKLAKLGFNLLQSLHKKEISQLSKWWKSYDVTNNFPYARDRLVECYFWIQGVYFEPKYFQSRILLTKNLAMASILDDTYDAYSTCEELELFTKAIDMWSITCYDVLPKYMKPLYQMVMELYKEMEETMADEGKSYLLNYLKESIKEFITGYMIEAKWRNEGYMPTLEEHVSVSLITSGYKYLITASFVGMGDVITEESFKWVSTNPPIVISSCVVGRFRDDVITHKEEQERNHVPSVIECYMKQFDVTKDHAYGLANKKVEDAWKEIIRESLTCKDVPMPLIMRVINFTRVIHVMYKDQDNYTHVGDEMKNHIRSLLIDTMST*

[0025] SEQ NO.3MAPSNIVVQSSSTPPVAGGDEEFAPSVWGDFFVTYATPVSQASEQRMSERAELLKAQVRQAFDAASMDVAGLITYVDTLERLGLDNHFRDLIGAALERIGAEELPEHGGGLHIVALRFRLLRQHGIWVSTDVFDAFREDAGGFCSSLCSDDPRGLLSLYNAAHMAVPGEVVLDDAIAFARGRLLDIISKGEVRSPVSEQITRALDIPLPRFTRRLETMHYIAEYEHEEAHDGLLLELARLNFVLVRALHLRELKDLSLWWRELYNTVKLPYARDRMVEIYFWTCGMLHEEEYSLARMFFAKTFGMVSLMDDTFDVHATLDECHKLKEAMQRWDESEVSILPEYLRLLYIKTLSNFKEFEEILEPNKKYRMAYTKEAYKLCSKNYLKEAIWSNQKYQPSFKEHEELSIMTSGLPMLTILTLMGFGDEATPEAFEWVSSVPEMVRAGSQVTRFLNDLSSYKLGKNKKDMPGSVETYMVENGLTGDEAAAAIAALLENRWRILNQTRMEIDHTLLPAAQVVLNMARANEIIYLHGRDAYTFGADLKDLVTTLFLKQVLPL*

Claims

1. A method for identifying and optimizing key amino acid sites controlling sesquiterpene synthesis, characterized in that: For sesquiterpene synthase proteins with known amino acid sequences, the key residues in the cavity between the three-dimensional model are lysine, arginine, or glutamine, which are located in the cavity between the first long α-helix at the N-terminus, the α-helix at the N-terminus and C-terminus of the protein (which is also the longest α-helix in this type of protein), and the last 1-7 amino acids at the C-terminus. For example, lysine, arginine, or glutamine in the cavity between amino acids 26-34, 228-242, and 538-546 of SEQ NO.1 is a key residue; it is composed of arginine-R239 at position 239 of the amino acid sequence shown in SEQ NO.1, lysine-K238 at position 238 of the amino acid sequence shown in SEQ NO.2, or arginine-R246 at position 246 of the amino acid sequence shown in SEQ NO.

3.

2. The method according to claim 1, characterized in that: When optimizing the amino acid at this site, the amino acid located on the "α-helix at the N-terminus and C-terminus of the connecting protein" is mutated to lysine, arginine, or glutamine, as well as combinations of these mutations. For example, the leucine at position 232 of the amino acid sequence shown in SEQ NO.2 is mutated to glutamine-L232Q, or the lysine at position 238 of the amino acid sequence shown in SEQ NO.2 is mutated to arginine-K238R, or a combination mutation is made where the leucine at position 232 of the amino acid sequence shown in SEQ NO.2 is mutated to glutamine and the lysine at position 238 is mutated to arginine-L232Q+K238R.

3. A recombinant vector comprising the encoding gene of claim 1, 2 or 3.

4. A recombinant strain comprising the encoding gene of claim 1, 2 or 3.

5. A host cell containing the encoding gene of claim 1, 2 or 3.

6. The application according to claim 3, characterized in that, The yield of sesquiterpene alcohols in the modified terpene compound products changed.

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