阅读说明：本技术 温度优化的l-阿拉伯糖异构酶突变体 (Temperature optimized L-arabinose isomerase mutants ) 是由 A·法尔维克 J·克拉伦 H·迪茨 H·约亨斯 M·默特尔 于 2020-03-16 设计创作，主要内容包括：本发明涉及在包含高浓度二价金属离子的原料内具有高催化活性的温度优化的L-阿拉伯糖异构酶,编码本发明所述的L-阿拉伯糖异构酶的核酸序列,包含编码本发明所述的L-阿拉伯糖异构酶的所述核酸序列的载体,包含本发明所述的L-阿拉伯糖异构酶的组合物,包含本发明所述的L-阿拉伯糖异构酶的酵母细胞,本发明所述的L-阿拉伯糖异构酶、所述组合物或所述酵母细胞在具有高含量二价金属离子的原料的发酵中的用途。(The present invention relates to a temperature optimized L-arabinose isomerase with high catalytic activity in a feedstock comprising a high concentration of divalent metal ions, a nucleic acid sequence encoding the L-arabinose isomerase according to the present invention, a vector comprising the nucleic acid sequence encoding the L-arabinose isomerase according to the present invention, a composition comprising the L-arabinose isomerase according to the present invention, a yeast cell comprising the L-arabinose isomerase according to the present invention, the use of the L-arabinose isomerase according to the present invention, the composition or the yeast cell for the fermentation of a feedstock with a high content of divalent metal ions.)

1. A temperature optimized L-arabinose isomerase with high catalytic activity in a feedstock comprising a high concentration of divalent metal ions, which has a sequence identity of at least 97.0% to SEQ ID No. 2.

2. The temperature-optimized L-arabinose isomerase of claim 1 comprising the amino acid sequence of SEQ ID NO 4: deletions and mutations at positions 40, 41, 42, 43, 239, 240, 241, 242, 243, 244, 246, 247, 248, 249, 250 and 251.

3. The temperature-optimized L-arabinose isomerase of claim 2, wherein the at least one mutation is a substitution selected from: permutations at positions 40, 41, 43, 239, 246, 247, 248, 249, 250 and 251.

4. The temperature-optimized L-arabinose isomerase of claim 2 or claim 3 wherein the at least one mutation is a substitution selected from: a40K, S41K, K43D, M239I, H246P, D247K, K248A, Y249S, V250M, and H251D.

5. The temperature-optimized L-arabinose isomerase of claim 2, wherein the at least one mutation is a deletion selected from: deletions at positions 42, 240, 241, 242, 243 and 244.

6. The temperature-optimized L-arabinose isomerase of claim 5, wherein the at least one mutation is a deletion selected from: g42, V240, E241, G242, D243 and N244.

7. The temperature-optimized L-arabinose isomerase of claim 2 comprising SEQ ID NO 4 with substitutions at positions 40, 41 and 43 and/or at positions 239, 246, 247, 248, 249, 250 and 251.

8. The temperature-optimized L-arabinose isomerase of claim 2 or claim 7 comprising SEQ ID NO 4 with deletions at position 42 and/or at positions 240, 241, 242, 243 and 244.

9. The temperature-optimized L-arabinose isomerase of any of the preceding claims, which has a sequence identity of at least 98.0% with SEQ ID NO 2.

10. The temperature-optimized L-arabinose isomerase of any of the preceding claims, which has a sequence identity of at least 99.0% with SEQ ID NO 2.

11. The temperature-optimized L-arabinose isomerase according to any one of the preceding claims, which is selected from the group consisting of: 6, 11, 13, 15 and 17.

12. The temperature-optimized L-arabinose isomerase according to any of the preceding claims, which, when incubated at 30 ℃ for 20 minutes at pH 7, has an L-arabinose content of 400mM and MgCl₂Having a content of at least 5mM in the starting material28.5kat/mol of catalytic activity of the enzyme.

13. A nucleic acid sequence encoding the temperature-optimized L-arabinose isomerase of any one of claims 1 to 12.

14. Use of the temperature optimized L-arabinose isomerase according to any one of claims 1 to 12 in a fermentation process of a lignocellulosic hydrolysate.

15. A composition comprising the temperature-optimized L-arabinose isomerase of any one of claims 1 to 12.

16. Use of a composition according to claim 15 in a fermentation process of a lignocellulosic hydrolysate.

17. A vector comprising the nucleic acid sequence of claim 13.

18. A yeast cell comprising the nucleic acid sequence of claim 13.

19. Use of a yeast cell according to any of the claims 18 in a fermentation process of a feedstock with a high content of divalent metal ions.

Brief Description of Drawings

FIG. 1 shows a composition comprising 1 or 5mM MnCl₂、MgCl₂Or CaCl₂And a comparison of the enzymatic activities at 30 ℃ of the substrates for L-arabinose of 400mM SEQ ID NO:8 (prior art L-arabinose isomerase) and SEQ ID NO:6 (L-arabinose isomerase according to the invention).

FIG. 2 shows a MgCl composition containing 1 or 5mM MgCl₂Or CaCl₂And a comparison of the enzymatic activities at 30 ℃ and 50 ℃ of the substrates of L-arabinose of 400mM SEQ ID NO:8 (prior art L-arabinose isomerase) and SEQ ID NO:6 (L-arabinose isomerase according to the invention).

Example 1

Cloning of vectors for L-arabinose isomerase Gene expression in E.coli

Methods of manipulation of nucleic acid molecules are well known to those skilled in the art and are incorporated herein by reference (1.Molecular cloning: a laboratory Manual, Michael R.Green and Joseph Sambrook, 4 th edition; 2.Current Protocols in Molecular Biology, ISSN: 1934-.

SEQ ID NOs 1 and 3, encoding SEQ ID NOs 2 and 4, respectively, were synthesized by Geneart (Regensburg, Germany) and then used as templates in PCR reactions for cloning. In two sequential PCRs, the sequence encoding the N-terminal 6XHis tag followed by the TEV protease site (ENLYFQS) was added to the ORF. The resulting open reading frames are SEQ ID NO 5 and SEQ ID NO 7, respectively. The corresponding amino acid sequences are SEQ ID NO 6 and SEQ ID NO 8. For the first PCR reaction, the primer pairs were designed to match the 5 'and 3' ends of SEQ ID NOs 1 and 3, respectively. The forward primers (bound to the 5 'end of the respective ORFs) had a 5' extension comprising the TEV protease site sequence and a portion of the 6xHis tag sequence (12 bp). The reverse primer has a 5' extension suitable for use in Gibson cloning into the target vector. The forward primer of the second reaction is designed to bind to the 5 'extension of the first forward primer and has a 5' extension comprising the remaining 6xHis tag sequence and sequences that allow for Gibson cloning into the target vector. The reverse primers used for the first and second reactions were identical. Following the supplier's recommendations for dNTP, primer and buffer concentrations, two PCR reactions were set up using Q5 high fidelity DNA polymerase (HF buffer system). Amplification of the PCR products was accomplished in an Eppendorf Thermocycler using the standard procedure of Phusion polymerase (98 ℃, 30 seconds initial denaturation followed by 98 ℃, 20 seconds-60 ℃, 20 seconds-72 ℃, 30 cycles of 30 seconds, and a final extension phase of 5 minutes at 72 ℃). PCR products of the expected size were purified by preparative, ethidium bromide-stained TAE-agarose gel electrophoresis and recovered from the gel using Promega Wizard SV-PCR and gel purification kits.

Plasmid pET-22b was linearized by digestion with the restriction endonucleases HindIII and NdeI, and the digested fragments were separated by agarose gel electrophoresis. The linearized vector backbone was recovered from the gel following the instructions of the Promega Wizard SV-PCR and gel purification kit. The amplified PCR product was cloned into the digested vector backbone using the Gibson cloning method (NEBuilder) according to the manufacturer's instructions (vector insertion ratio 1:3, 0.1pmol total DNA, 20. mu.l reaction). Transformation was carried out into competent E.coli Mach1 cells according to the supplier's protocol. Transformants were grown on LB-ampicillin plates overnight and the correct plasmids were tested by plasmid miniprep and control digestion and DNA sequencing. Using Promega PureYield^TMPlasmid midi prep System larger amounts of Plasmid DNA were prepared from the confirmed clones.

Expression of L-arabinose isomerase and purification of L-arabinose isomerase in E.coli

Coli Rosetta under IPTG (isopropyl. beta. -D-1-thiogalactopyranoside) inducible promoter by using standard molecular biology techniques^TMThe L-arabinose isomerase is expressed in the cell. A preculture of 200mL Lysogene broth containing 34. mu.g/mL chloramphenicol and 100. mu.g/mL ampicillin was inoculated directly with the transformed E.coli cells and allowed to grow overnight at 37 ℃ with shaking at 250 rpm. 250ml Terrific broth of 8 main cultures containing 100. mu.g/ml ampicillin in 2L shake flasks were inoculated to an OD of 0.1₆₀₀And incubated overnight at 37 ℃ with shaking at 250 rpm. When the culture reached 1,0OD₆₀₀When the temperature was lowered to 20 ℃ and the expression of AI was induced by the addition of 1mM IPTG. After 21.5 hours, cells were harvested by centrifugation (10 min, 10,000x g). Pellets were washed once with brine (0.9% NaCl) and combined into one tube. The pellets were then stored at-20 ℃ until cell lysis.

For cell lysis, the cell pellet was thawed, resuspended in 70ml binding buffer (20mM Trizma base, 500mM NaCl, 20mM imidazole, pH 7.4), and protease inhibitor (cOmplexate) added^TMEDTA-free protease inhibitor cocktail), lysozyme and Benzonase nuclease (Sigma-Aldrich, 62970 and E1014). The cell suspension was incubated on ice for 30 minutes. Then, while the cells were kept on ice, the cells were lysed by sonication at the following settings: 4x 1 min, amplitude 70%, cycle 0.6, 1 min apart. Cells were lysed by centrifugation at 20,000x g for 60 minutes at 4 ℃. The clear supernatant was filtered (0.2 μm) and used to purify the corresponding AI.

Use ofExplorer 900 and 5mL HisTrap HP columns (GE life sciences). The column is equilibrated with binding buffer and then the sample is loaded onto the column. The column was washed with binding buffer until the absorbance at 280nm had been normalized. Elution buffer (20mM Trizma base, 500mM NaCl, 500mM imidazole, pH 7.4) was used to exceed 10 column volumes (50mL, flow)Elution was performed with a gradient of 3ml/min, 17 min). Fractions of 2ml were collected and then analyzed by SDS-PAGE (Biorad Criterion XT 4-12% Bis-Tris) according to the manufacturer's instructions. Fractions containing the respective AI proteins were pooled and dialyzed against 2X 2L dialysis buffer (50mM HEPES, pH 7.0, 10mM EDTA) for 96h at 4 ℃ with one buffer change in order to remove divalent cations from the proteins. AI concentration was determined photometrically by absorbance at 280 nm.

Proteins with SEQ ID NO 6 and SEQ ID NO 8 were obtained at concentrations of 15.4 and 12.3g/L, respectively.

Measurement of L-arabinose isomerase Activity

The purified protein was assayed in an endpoint measurement to determine the catalytic activity of the AI protein. The amount of the AI active product L-ribulose with L-arabinose as substrate was quantified by HPLC (Dionex Ulimate 3000 with RI detector Shodex RI-101) using a Biorad Aminex HPX-87P column (with corresponding pre-column). The settings are as follows:

eluent: MilliQ water

Flow rate: 0.6ml/min

Column oven: 80 deg.C

Operating time: 40 minutes

Sample introduction volume: 20 μ l

And (3) detection: RI, 50 ℃, polarity (+), data acquisition rate 10Hz

The correction ranges for L-arabinose and L-ribulose are 0.25-10 mg/ml and 0.05-2 mg/ml, respectively.

Tests were performed beforehand to determine the reaction conditions, in particular the amount of protein and the reaction time, to ensure that the measurement was performed in the linear range. The final components of the reaction are shown in table 1.

Components	Final concentration
		HEPES pH 7	100mM
MnCl2/MgCl2/CaCl2	1/5mM
		L-arabinose	400mM
Purified AI proteins	0.375μM

Mu.l protein solution (purified AI diluted to 0, 75. mu.M in 100mM HEPES, pH 7) was added to 20. mu.l MnCl₂/MgCl₂/CaCl₂In solution (10 or 50mM in 100mM HEPES, pH 7). Mu.l of concentrated L-arabinose solution (1M in 100mM HEPES, pH 7) was added, and the reaction was mixed and centrifuged. The reaction was carried out in a thermal cycler at 30 ℃ or 50 ℃ for 20 minutes. The reaction was stopped by heat treatment at 95 ℃ for 10 minutes, then diluted 1:5 with water, filtered and analyzed by HPLC. Each reaction was performed in triplicate. The enzyme activity was calculated from the amount (in moles) of L-ribofuranose produced after 1200 seconds for 75 picomoles of enzyme. The unit is kat/mole_EnzymeWherein kat is mole_Ribulose/s。

As can be seen in FIG. 1, the enzyme according to SEQ ID NO:6 has a higher catalytic activity than the enzyme according to SEQ ID NO:8 at low and high concentrations of all divalent metal cations at 30 ℃. In Mn²⁺Has a catalytic activity at least 15% higher in the presence of Mg²⁺And Ca²⁺In the presence of (a), the catalytic activity is at least 30% higher. Furthermore, FIG. 2 shows the enzyme according to SEQ ID NO 6 on divalent cations Mg at elevated temperatures of 30 ℃ and 50 ℃²⁺And Ca²⁺Is superior to the enzyme according to SEQ ID NO 8. In summary, this tableThe L-arabinose isomerase according to SEQ ID No. 6 showed significantly increased temperature tolerance in the presence of high concentration of divalent metal cation compared to the enzyme according to SEQ ID No. 8, which has high catalytic activity.

Description of sequence listing:

1, SEQ ID NO: modified DNA sequence of L-arabinose isomerase, codon optimized

2, SEQ ID NO: amino acid sequence of modified L-arabinose isomerase

3, SEQ ID NO: DNA sequence of wild-type L-arabinose isomerase, codon-optimized

4, SEQ ID NO: amino acid sequence of the wild-type L-arabinose isomerase from Lactobacillus (Lactobacillus atri) DSM 16041

5, SEQ ID NO: modified DNA sequence of L-arabinose isomerase, N-terminal 6xhis tag, codon optimized

6 of SEQ ID NO: modified amino acid sequence of L-arabinose isomerase, N-terminal 6xhis tag

7, SEQ ID NO: DNA sequence of wild-type L-arabinose isomerase, N-terminal 6xhis tag, codon optimized

8, SEQ ID NO: amino acid sequence of wild-type L-arabinose isomerase, N-terminal 6xhis tag

9 of SEQ ID NO: modified DNA sequence of L-arabinose isomerase, codon optimized

10, SEQ ID NO: modified DNA sequence of L-arabinose isomerase, codon optimized

11, SEQ ID NO: amino acid sequence of modified L-arabinose isomerase

12, SEQ ID NO: modified DNA sequence of L-arabinose isomerase, codon optimized

13 in SEQ ID NO: amino acid sequence of modified L-arabinose isomerase

14, SEQ ID NO: modified DNA sequence of L-arabinose isomerase, N-terminal 6xhis tag, codon optimized

15, SEQ ID NO: modified amino acid sequence of L-arabinose isomerase, N-terminal 6xhis tag

16 in SEQ ID NO: modified DNA sequence of L-arabinose isomerase, N-terminal 6xhis tag, codon optimized

17 in SEQ ID NO: amino acid sequence of modified L-arabinose isomerase, N-terminal 6xhis tag.

Examples and figures

The invention is described below by way of example and figures. The examples and figures are for illustrative purposes only and do not limit the scope of the invention and the claims in any way.