Application of transcription factor GAL4 in improving yield of ganoderic acid Y in saccharomyces cerevisiae and method for preparing ganoderic acid Y

Abstract

The application belongs to the field of synthetic biology and microbial metabolic engineering, and particularly relates to application of a transcription factor GAL4 in improving yield of ganoderic acid Y of Saccharomyces cerevisiae and a method for preparing ganoderic acid Y. The application takes Saccharomyces cerevisiae as a host bacterium, overexpresses a transcription factor GAL4 gene, and improves yield of ganoderic acid Y of Saccharomyces cerevisiae. In a specific experiment, the yield of ganoderic acid Y of Saccharomyces cerevisiae overexpressing the GAL4 gene is increased from 12.91 mg / L of the starting bacterium to 19.81 mg / L, about 53.4%. Therefore, the application has great application value.

Description

Technical Field

[0001] This invention relates to the fields of synthetic biology and microbial metabolic engineering, specifically to the application of transcription factor GAL4 in increasing the yield of ganoderic acid Y in Saccharomyces cerevisiae and a method for preparing ganoderic acid Y. Background Technology

[0002] Ganoderma triterpenoids are a class of natural products with important medicinal value, among which ganoderic acid Y (GA-Y) is one of the key representative compounds and has potential medicinal value. However, the content of this type of compound in natural sources is low, and traditional extraction methods have problems such as long cycle, high cost and unstable yield, which make it difficult to meet the needs of industrial production.

[0003] In recent years, metabolic engineering strategies based on *Saccharomyces cerevisiae* have been widely used for the heterologous synthesis of Ganoderma lucidum triterpenes. By introducing key synthetic enzymes, the target product can be synthesized in *Saccharomyces cerevisiae*. However, existing studies have mostly focused on modifications at the structural gene level, with relatively little systematic optimization at the transcriptional regulatory level. This has resulted in insufficient overall coordination of metabolic pathway expression, limiting further enhancement of the target product.

[0004] Therefore, developing a method to improve the efficiency of GA-Y synthesis at the transcriptional regulation level is of great significance for enhancing the biosynthesis level of Ganoderma lucidum triterpenes. Summary of the Invention

[0005] To address the needs of existing technologies, previous research by the inventors has focused on improving substrate utilization efficiency by constructing xylose utilization pathways to promote triterpenoid synthesis from the carbon source supply level, while also optimizing the expression of key structural genes to increase yield. However, this approach essentially focuses on the regulation of single genes or local pathways, with limited coordinating effects on the entire metabolic network. Furthermore, considering that the synthesis of ganoderic acid Y involves multiple steps, especially the complex modification process involving P450, there may be issues of uneven flux distribution and expression imbalance. Therefore, this invention aims to address this issue from the transcriptional regulation level, attempting to achieve overall regulation of multiple genes through transcription factors, seeking new regulatory targets, and ultimately realizing this invention through research and exploration.

[0006] Therefore, the purpose of this invention is to provide a method for increasing the yield of ganoderic acid Y in Saccharomyces cerevisiae by expressing the transcription factor GAL4, so as to solve the problem of low yield of ganoderic acid Y in the prior art.

[0007] To achieve the above objectives, the present invention adopts the following technical solution.

[0008] This invention provides the application of transcription factor GAL4 in increasing the yield of ganoderic acid Y, the amino acid sequence of which is shown in SEQ ID NO:6, and the application is for increasing the yield of ganoderic acid Y in Saccharomyces cerevisiae.

[0009] Specifically, this is achieved by using Saccharomyces cerevisiae as the host microorganism and overexpressing the transcription factor GAL4 gene.

[0010] Preferably, the brewing yeast used as the starting strain has the ability to produce ganoderic acid Y.

[0011] In a specific embodiment, the cytochrome P450 enzyme genes CYP5150L8 and CYP512W2 are overexpressed in the starting strain, Saccharomyces cerevisiae. The amino acid sequences of the cytochrome P450 enzyme genes CYP5150L8 and CYP512W2 are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively.

[0012] The present invention also provides a recombinant Saccharomyces cerevisiae that produces ganoderic acid Y, which overexpresses the cytochrome P450 enzyme genes CYP5150L8 and CYP512W2 and the transcription factor GAL4 in Saccharomyces cerevisiae. The amino acid sequences of the cytochrome P450 enzyme genes CYP5150L8 and CYP512W2 are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, and the amino acid sequence of the transcription factor GAL4 is shown in SEQ ID NO:6.

[0013] The present invention also provides a method for preparing ganoderic acid Y, which includes the step of fermenting and culturing an engineered strain of Saccharomyces cerevisiae that overexpresses the transcription factor GAL4 gene to produce ganoderic acid Y, wherein the amino acid sequence of the transcription factor GAL4 is shown in SEQ ID NO:6.

[0014] Preferably, the engineered Saccharomyces cerevisiae also simultaneously overexpresses the cytochrome P450 enzyme genes CYP5150L8 and CYP512W2, the amino acid sequences of which are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively.

[0015] Specifically, the fermentation culture medium is YPD24 medium, which includes 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose and 40 g / L glycerol.

[0016] More specifically, the fermentation temperature is 25-35℃, and the fermentation time is 60 h-180 h; it further includes the steps of collecting the fermentation products and separating ganoderic acid Y.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] First, the yield of the target product was significantly increased. The yield of ganoderic acid Y in *Saccharomyces cerevisiae* overexpressing the GAL4 gene increased from 12.91 mg / L in the original strain to 19.81 mg / L, an increase of approximately 53.4%. Simultaneously, it also promoted an overall increase in metabolic flux along the pathway; specifically, the yield of GA-HLDOA in *Saccharomyces cerevisiae* overexpressing the GAL4 gene increased from 81.97 mg / L in the original strain to 101.07 mg / L, indicating that this method can enhance the metabolic efficiency of the entire triterpenoid synthesis pathway. Furthermore, the regulatory mechanism demonstrates universality; this invention achieves yield enhancement through transcriptional regulation rather than single enzyme optimization, providing a novel regulatory strategy for the metabolic engineering of complex natural products. Detailed Implementation

[0019] The present invention will be further described below with reference to the embodiments, but the present invention is not limited to the following embodiments.

[0020] Example 1: Construction of engineered strains

[0021] Using the engineered Saccharomyces cerevisiae strain YL-T3 (Wang, WF, H. Xiao, and JJ Zhong, Biosynthesis of a ganoderic acid in Saccharomyces cerevisiae by expressing acytochrome P450 gene from Ganoderma lucidum[J]. Biotechnol Bioeng, 2018. 115(7): p. 1842-1854.) as the chassis strain, the cytochrome P450 enzyme genes CYP5150L8 and CYP512W2 from Ganoderma lucidum were integrated into the pRS426 vector to construct the engineered strain YL-T3 / L8-W2, which was used for the biosynthesis of ganoderic acid Y.

[0022] The nucleotide sequence of the CYP5150L8 gene is shown in SEQ ID NO:1, and the amino acid sequence it encodes is shown in SEQ ID NO:2; the nucleotide sequence of the CYP512W2 gene is shown in SEQ ID NO:3, and the amino acid sequence it encodes is shown in SEQ ID NO:4.

[0023] Using the pRS314 vector as a backbone, the GAL4 gene derived from *Saccharomyces cerevisiae* was inserted into its multiple cloning site to construct the GAL4 expression plasmid. The GAL4 gene is derived from *Saccharomyces cerevisiae*, and its nucleotide sequence is shown in SEQ ID NO:5, while the encoded amino acid sequence is shown in SEQ ID NO:6.

[0024] Meanwhile, the pRS314 vector without the GAL4 gene inserted was used as an empty plasmid control.

[0025] Subsequently, the GAL4 expression plasmid and the empty plasmid control were transformed into the YL-T3 / L8-W2 strain, respectively. Transformation was performed using the lithium acetate / PEG method. The transformed strains were plated on the corresponding selection medium and cultured at 30°C for 2-3 days to obtain the GAL4 engineered strain 5F8 and the empty plasmid control strain.

[0026] Example 2: Fermentation Culture

[0027] Empty plasmid control bacteria and GAL4 engineered strain 5F8 were inoculated into YPD24 medium and fermented at 30℃ for 120 h.

[0028] The composition of the YPD24 culture medium is as follows:

[0029] Yeast extract: 10 g / L

[0030] Peptone: 20 g / L

[0031] Glucose: 20 g / L

[0032] Glycerin: 40 g / L.

[0033] Example 3 Product Detection

[0034] After fermentation, the target product in the fermentation broth was quantitatively analyzed.

[0035] The fermentation broth was processed as follows: 3 mL of fermentation broth was taken, centrifuged to remove the supernatant, and an equal volume of methanol was added to the cell precipitate. After mixing, the mixture was extracted by shaking at 30°C. After centrifugation, the supernatant was collected, and the precipitate was extracted twice more. The extracts were combined, concentrated, redissolved in methanol, and filtered through a 0.22 μm organic phase filter membrane before being used for detection.

[0036] The product was detected by high-performance liquid chromatography (HPLC) using a liquid chromatography system equipped with a diode array detector at a detection wavelength of 214 nm. Chromatographic separation was performed using a Kinetex Biphenyl column (150 mm × 4.6 mm), with water as mobile phase A and methanol / acetic acid as mobile phase B, at a flow rate of 0.5 mL / min using a gradient elution program.

[0037] The method uses standards to perform quantitative analysis of the target product, and it has good repeatability and reliability.

[0038] The results are shown in Table 1.

[0039] Table 1

[0040] ;

[0041] The results in the table show that, compared with the control strain, the yield of ganoderic acid Y in the GAL4 engineered strain increased by approximately 53.4%. Simultaneously, the yield of GA-HLDOA increased by approximately 23.3%, indicating that GAL4 may enhance the overall flux of metabolic pathways by regulating the expression of genes related to triterpenoid synthesis, thereby promoting the accumulation of the target product. Therefore, the results demonstrate that overexpression of the GAL4 gene significantly increases the yield of ganoderic acid Y, while simultaneously increasing GA-HLDOA.

Claims

1. Application of transcription factor GAL4 in increasing the yield of ganoderic acid Y, wherein the amino acid sequence of transcription factor GAL4 is shown in SEQ ID NO:6, and the application is to increase the yield of ganoderic acid Y in Saccharomyces cerevisiae that overexpresses the cytochrome P450 enzyme genes CYP5150L8 and CYP512W2. The application described herein is achieved by overexpressing the transcription factor GAL4 gene using Saccharomyces cerevisiae as the host microorganism; the amino acid sequences of the cytochrome P450 enzyme genes CYP5150L8 and CYP512W2 are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively.

2. A recombinant brewing yeast that produces ganoderic acid Y, characterized in that, Overexpression of cytochrome P450 enzyme genes CYP5150L8 and CYP512W2 and transcription factor GAL4 in Saccharomyces cerevisiae, wherein the amino acid sequences of cytochrome P450 enzyme genes CYP5150L8 and CYP512W2 are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively, and the amino acid sequence of transcription factor GAL4 is shown in SEQ ID NO:

6.

3. A method for preparing ganoderic acid Y, characterized in that, It includes the step of fermenting and culturing engineered Saccharomyces cerevisiae overexpressing the transcription factor GAL4 gene to produce ganoderic acid Y, wherein the amino acid sequence of the transcription factor GAL4 is shown in SEQ ID NO:6; The engineered Saccharomyces cerevisiae also simultaneously overexpresses the cytochrome P450 enzyme genes CYP5150L8 and CYP512W2, the amino acid sequences of which are shown in SEQ ID NO:2 and SEQ ID NO:4, respectively.

4. The method as described in claim 3, characterized in that, The fermentation culture medium is YPD24 medium, which includes 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose and 40 g / L glycerol.

5. The method as described in claim 3, characterized in that, The fermentation temperature is 25-35℃, and the fermentation time is 60 h-180 h; the fermentation product is further collected and ganoderic acid Y is separated.