Application of lupinol synthase gene in construction of engineering bacteria with high yield of lupinol

Abstract

The present application is suitable for the technical field of synthetic biology and metabolic engineering, and provides application of a lupinol synthase gene in construction of an engineered bacterium with high yield of lupinol, wherein the lupinol synthase gene is at least one of LUS1 and LUS2, and the amino acid sequence of the encoded protein of LUS1 and LUS2 is shown in SEQ ID NO. 1-2. Based on the heterologous expression of the lupinol synthase gene LUS1 and LUS2 and in combination with a metabolic flow optimization strategy, efficient, stable and green production of lupinol is achieved.

Description

Technical Field

[0001] This invention belongs to the fields of synthetic biology and metabolic engineering technology, specifically relating to the application of the lupin alcohol synthase gene in the construction of engineered bacteria that produce high levels of lupin alcohol. Background Technology

[0002] Lupeol (LUP) is a pentacyclic triterpenoid compound widely found in higher plants and is an important precursor to many medicinally active substances (such as betulinic acid and betulinol). Studies have shown that lupeol and its derivatives possess significant anti-inflammatory, antitumor, antioxidant, lipid-lowering, and hepatoprotective activities, and have broad application prospects in the fields of pharmaceuticals, health products, and cosmetics.

[0003] Currently, lupeol is mainly derived from natural plant extracts, such as birch, soybeans, and aloe vera. However, these plants contain extremely low levels of lupeol, and the extraction process is limited by season, environment, and raw material sources, resulting in high production costs and low efficiency, making large-scale industrialization impossible. While chemical synthesis is feasible, its reaction routes are long and complex, requiring the use of environmentally unfriendly reagents such as strong acids or bases, which makes it difficult to meet the requirements of green manufacturing and the development of the biopharmaceutical industry.

[0004] In recent years, with the development of synthetic biology technology, the production of natural products using microbial cell factories has become a research hotspot. Saccharomyces cerevisiae, due to its clear genetic background, mature metabolic network, ease of genetic modification, and the availability of an endogenous MVA pathway to provide precursors for triterpenoid synthesis, has been widely used as a production platform for triterpenoid compounds. The synthesis of lupeol uses squalene (SQ) as a precursor, which is catalyzed by squalene epoxidase (SE) to produce 2,3-squalene oxide. In Saccharomyces cerevisiae, this process is driven by ScERG1, followed by cyclization of 2,3-squalene oxide by lupeol synthase (LUS) to produce lupeol (e.g., squalene oxidase). Figure 1 (As shown). However, existing research still faces the following major problems: low lupin alcohol synthase activity and limited precursor supply. Therefore, applying a highly active lupin alcohol synthase gene and optimizing the synergistic expression ratio of squalene epoxidase and lupin alcohol synthase to significantly increase lupin alcohol production while ensuring stable strain growth is a key technical problem that urgently needs to be solved in the field of synthetic biology. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the lupin alcohol synthase gene in the construction of engineered bacteria that produce high levels of lupin alcohol, thereby addressing the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] On the one hand, the present invention provides the application of the lupin alcohol synthase gene in the construction of engineered bacteria that produce high levels of lupin alcohol, wherein the lupin alcohol synthase gene is at least one of LUS1 and LUS2, and the amino acid sequences of the proteins encoded by LUS1 and LUS2 are shown in SEQ ID NO.1-2.

[0008] On the other hand, the present invention provides an engineered strain that produces high levels of lupeol, which is obtained by constructing a strain of Saccharomyces cerevisiae J01 as the substrate by expressing one or more copies of the genes LUS2 and ScERG1.

[0009] On the other hand, the present invention provides an application of the above-mentioned engineered bacteria that produce high levels of lupeol in the fermentation of lupeol.

[0010] Compared with the prior art, the specific beneficial effects of the present invention are as follows:

[0011] This invention achieves efficient lupeol production by heterologous expression of lupeol synthase genes LUS1 and LUS2, combined with a metabolic flux optimization strategy. The lupeol yield is 323.78 mg / L in shake-flask fermentation and 2.11 g / L in a 5 L bioreactor, realizing efficient, stable and green production of lupeol. Attached Figure Description

[0012] Figure 1 This invention provides a pathway for the synthesis of lupeol in specific embodiments.

[0013] Figure 2 The standard curve for lupeol detection provided in this embodiment of the invention;

[0014] Figure 3 The yield and growth of lupeol of the strain provided in the embodiments of the present invention are shown in Figure A, where A represents the yield and B represents the growth.

[0015] Figure 4 The yield and growth of strain LUP3 provided in this embodiment of the invention were obtained by fermentation in a 5 L bioreactor. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0017] 1. Culture medium used in the embodiments of the present invention:

[0018] SC medium: Adenine hemisulfate 18 mg / L, L-alanine 76 mg / L, L-arginine 76 mg / L, L-aspartic acid 76 mg / L, L-glutamate 76 mg / L, L-glutamine 76 mg / L, glycine 76 mg / L, L-isoleucine 76 mg / L, L-phenylalanine 76 mg / L, L-proline 76 mg / L, L-serine 76 mg / L, L-threonine 76 mg / L, L-tryptophan 76 mg / L, L-tyrosine 76 mg / L, L-valine 76 mg / L, L-methionine 76 mg / L, L-lysine 76 mg / L, L-uracil 76 mg / L, L-leucine 380 mg / L, L-histidine hydrochloride 76 mg / L, amino acid-free yeast nitrogen source 1.7 g / L, ammonium sulfate 5 g / L, glucose 20 g / L. For amino acid-deficient culture media, the amount of amino acids should be reduced, while for solid culture media, 20 g / L agar should be added.

[0019] YPD medium: peptone 20 g / L, yeast extract 10 g / L, glucose 20 g / L;

[0020] 2×YPG medium: peptone 40 g / L, yeast extract 20 g / L, galactose 40 g / L;

[0021] All culture media must be autoclaved at 121°C for 15 minutes before use.

[0022] 2. Method for constructing the strain in the embodiments of the present invention:

[0023] All strains were constructed using CRISPR-Cas9 technology. Chromosome sequences were downloaded from the *Saccharomyces cerevisiae* genome dataset. Homologous arm sequences within 1000 bp upstream and downstream of the locus were retrieved based on the gRNA sequence of the locus. Primers for the upstream and downstream arms, target gene, promoter, and terminator were manually designed using snapgene software. Homologous linkers were added to the 5' ends of the corresponding primers according to the requirements of subsequent fusion PCR. Primers for strain identification and PCR were designed using NCBI primers. Novozymes high-fidelity enzyme P515 was used to perform PCR on these small fragments of the upstream and downstream arms, target gene, promoter, and terminator. These small fragments were then subjected to fusion PCR using TOYOBO KOD FX enzyme to obtain a long fragment for subsequent integration. *Saccharomyces cerevisiae* strains were streaked on SC plates and incubated at 30°C for 72 h to prepare competent cells. Transformation was performed using PEG3350-LiAc-mediated chemical transformation. The cells were plated on corresponding auxotrophic plates, and after single colonies grew, a portion was picked and placed in 10 μL of 20 mM... In a PCR tube containing NaOH, alkaline lysis was performed at 99℃ for 20 min and 4℃ for 5 min. The lysis products were used as templates for colony PCR to screen for positive clones. The PCR products were sequenced for further verification. The correctly sequenced clones were streaked on SC agar medium and cultured at 30℃ for 72 h. The pCUT plasmid was removed, and the newly grown single colonies were picked and cultured in YPD liquid medium at 30℃ and 220 rpm for 24 h. The strains were then placed in cryovials containing 20% ​​glycerol and stored at -80℃.

[0024] 3. The shake-flask fermentation process in this embodiment of the invention:

[0025] Activate the glycerol bacteria by streaking them onto YPD plates and incubate at 30°C and 220 rpm for 72 h. Pick three single colonies from each plate and transfer them to a 50 mL centrifuge tube containing 10 mL of YPD liquid medium. Ensure that the amount of bacteria picked is consistent each time. Incubate at 30°C and 220 rpm for 24 h. Re-inoculate the seed culture into a 100 mL Erlenmeyer flask with a baffle containing 20 mL of 2×YPG liquid medium. Ensure that the inoculum has a uniform OD600 of 0.2 and incubate at 30°C and 220 rpm for 120 h.

[0026] 4. The fermentation process using a 5 L bioreactor in this embodiment of the invention is as follows:

[0027] Glycerin bacteria were activated by streaking on YPD plates and cultured at 30℃ and 220 rpm for 72 h. Single colonies were then inoculated into 10 mL centrifuge tubes containing 1 mL of YPD liquid medium and cultured for 24 h to obtain the primary seed culture. The primary seed culture was inoculated at 1% into 200 mL of YPD liquid medium and cultured for 24 h to obtain the secondary seed culture. In a 5 L bioreactor, 2.5 L of 2×YPG medium was added, and the secondary seed culture was inoculated at an inoculation rate of 8%. The temperature was maintained at 30℃, with an aeration rate of 1.5–3 mL / min and a stirrer speed of 220–800 rpm. The dissolved oxygen concentration was maintained at 30%. Feed pump 1 was connected to a solution containing 50 g / L yeast extract and 100 g / L peptone, and supplemented at a rate of 2.5 mL / h for 40 h after inoculation. Feed pump 2 was connected to 3M NaOH solution to maintain the pH at 5.5. Feed pump 3 was connected to 10% antifoaming solution, and feed pump 4 was connected to a solution containing 500 g / L yeast extract and 100 g / L peptone. Inoculate with g / L galactose solution, and feed at a rate of 2.5~4 mL / h after 24 h; culture for a total of 120 h.

[0028] 5. Detection of strain growth and products in embodiments of the present invention:

[0029] Mix the fermented bacterial culture thoroughly, transfer 50 μL to a cuvette, dilute with 950 μL of YPG medium, mix well, and detect growth using a handheld OD analyzer. Transfer the mixed bacterial culture to a 2 mL lysate tube, add 200 μL of 2 mm diameter glass beads, and then add ethyl acetate. Homogenize at 6500 rpm for 1 min using a high-speed homogenizer, repeating 10 times with 15 s intervals between each homogenization. Centrifuge at 13000 rpm for 10 min, and carefully transfer the top ethyl acetate layer to an EP tube. Concentrate using a vacuum concentrator in V-AL mode at 30℃ for 20 min. Add 200 μL of the derivatization reagent N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) to the dried EP tube. Incubate in a metal bath at 80℃ for 40 min. Filter the derivatized product using a 0.22 μm nylon filter.

[0030] GC-MS analysis was performed using an Agilent 8890B gas chromatograph equipped with an HP-5MS capillary column and a 5977B mass spectrometer detector. Helium was used as the carrier gas at a flow rate of [missing value], the temperature was 250 °C, and the injection volume was 2 μL. The initial temperature was 80 °C, and the temperature was increased to 300 °C at a rate of 20 °C / min, held for 15 min. Mass spectrometry used an EI ion source with an electron energy of 70 eV and an ion source temperature of 230 °C. In full scan mode, the m / z range was 50–1000. In ion detection mode, the detectable ions for lupeol were m / z = 188.8 and 73.0.

[0031] The standard curve for the detection of lupeol is as follows: Figure 2 As shown.

[0032] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0033] Example 1: Construction of strains expressing different LUS and detection of products:

[0034] First, a screening based on the birch transcriptome database was performed, and the conserved structure of all genes was predicted. The birch lupin synthase gene LUS1 (encoding protein sequence as shown in SEQ ID NO.1) with a complete conserved structure was obtained. LUS2 (encoding protein sequence as shown in SEQ ID NO.2) with a similarity of 70.07% was also found. This round of construction was based on CRISPR-Cas9 technology, using Saccharomyces cerevisiae strain J01 as the chassis. The LUS1 or LUS2 gene was integrated at the 308a site, resulting in engineered strains LUP1 and LUP2. The engineered strains were fermented using 2×YPG medium. After fermentation, there was no significant difference in growth among the strains. The fermentation product analysis showed that the lupin alcohol yields of strains LUP1 and LUP2 were 133.5 mg / L and 167.35 mg / L, respectively (e.g., SEQ ID NO.1). Figure 3 (As shown).

[0035] Example 2: Construction of LUP pathway enhanced strains and product detection:

[0036] To further optimize pathway expression, the squalene epoxidase genes ScERG1 (SGD: S000003407) and LUS2 of *Saccharomyces cerevisiae* were overexpressed in strain LUP2 to obtain strain LUP3. Fermentation and product analysis of LUP3 showed that the LUP yield of LUP3 was significantly increased, reaching 323.78 mg / L (e.g., ...). Figure 3 As shown in the figure, it is 1.93 times higher than LUP2, which is the highest yield achieved by shake flask fermentation to date.

[0037] Example 4: Production of lupeol by engineered strain LUP3C1 in a 5 L bioreactor:

[0038] Fermentation was performed in a 5 L bioreactor using LUP3. The culture was activated by streaking on YPD plates and incubated at 30°C and 220 rpm for 72 h. Single colonies were then inoculated into 10 mL centrifuge tubes containing 1 mL of YPD liquid medium and incubated for 24 h to obtain the primary seed culture. The primary seed culture was then inoculated at 1% into 200 mL of YPD liquid medium and incubated for 24 h to obtain the secondary seed culture. 2.5 L of 2×YPG medium was added to the 5 L bioreactor. The secondary seed culture was inoculated at an 8% v / v inoculation rate. The temperature was maintained at 30°C. Aeration was controlled at 1.5–3 mL / min. The stirrer speed was 220–800 rpm. The dissolved oxygen concentration was controlled at 30%. Feed pump 1 was connected to a solution containing 50 g / L yeast extract and 100 g / L peptone, and supplemented at a rate of 2.5 mL / h for 40 h after inoculation. Feed pump 2 was connected to a 3M... NaOH solution was used to maintain the pH at 5.5; feed pump 3 was connected to a 10% antifoaming agent solution; feed pump 4 was connected to a 500 g / L galactose solution. Feeding was carried out at a rate of 2.5–4 mL / h after 24 h of inoculation; the mixture was co-cultured for 120 h. The highest yield was observed at 96 h of fermentation, reaching 2.11 g / L, with an OD of 103.5 (e.g., ...). Figure 4 (As shown).

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of the lupin alcohol synthase gene in constructing engineered bacteria that produce high levels of lupin alcohol, characterized in that, The lupin alcohol synthase gene is at least one of LUS1 and LUS2, and the amino acid sequences of the proteins encoded by LUS1 and LUS2 are shown in SEQ ID NO. 1-2.

2. An engineered strain that produces high levels of lupeol, characterized in that, The engineered bacteria were constructed using Saccharomyces cerevisiae strain J01 as the substrate bacteria by expressing one or more copies of the genes LUS2 and ScERG1.

3. The engineered strain for high lupeol production as described in claim 2, characterized in that, Both genes LUS2 and ScERG1 are expressed by galactose-inducible strong promoters GAL1 or GAL7.

4. The application of an engineered strain that produces high levels of lupeol as described in claim 2 or 3 in the fermentation production of lupeol.

5. The application as described in claim 4, characterized in that, Includes the following steps: The engineered bacteria that produce high levels of lupeol as described in claim 2 or 3 are inoculated into a fermentation medium, and shake-flask fermentation is performed. After fermentation, a bacterial solution containing lupeol is obtained.

6. The application according to claim 5, characterized in that, The fermentation medium comprises 40 g / L peptone, 20 g / L yeast extract, and 40 g / L galactose.

7. The application according to claim 5, characterized in that, The specific operation of the shake flask fermentation is as follows: temperature is 30℃, pH is 5.5, aeration rate is 1.5-3 mL / min, rotation speed is 200-800 r / min, and dissolved oxygen is maintained at 30%; 24 h after inoculation, a solution containing 50 g / L yeast extract and 100 g / L peptone is added at a rate of 2.5 mL / h, and a solution containing 500 g / L galactose is added at a rate of 2.5-4 mL / h; the culture is continued for 120 h.

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