Mutated glycerol-3-phosphate acyltransferase and its encoding gene and use

Abstract

The application relates to a mutant enzyme of 3-phosphoglyceroyltransferase, a coding gene and application thereof, and belongs to the fields of biochemistry, molecular biology and metabolism. 58 GPAT9 mutant genes are constructed by using a site-directed mutagenesis technique, and the influence of single and multiple amino acid site changes on GPAT9 enzyme activity is analyzed by combining a GPAT-specific yeast genetic complementation method. It is found that the changes of six amino acid residues (85, 114, 119, 230, 237 and 322) located outside the acyltransferase conserved domain in AtGPAT9 can significantly affect the enzyme activity. There is interaction among the amino acids, for example, the simultaneous mutation of Y85W / N119H / S237N of the three sites can greatly increase the activity of AtGPAT9, accelerate the growth of yeast and promote the synthesis of triacylglycerol, and the triacylglycerol content in the yeast cells expressing the mutant enzyme is increased by 45.7% compared with that of the yeast cells expressing the wild type BnGPAT9.

Description

Technical Field

[0001] This invention belongs to the fields of biochemistry, molecular biology and metabolic technology, and relates to the modification of the nucleotide sequence of the 3-phosphoglyceryl acyltransferase gene and the amino acid sequence of the corresponding enzyme protein, as well as the uses of these mutant genes and enzyme proteins. Background Technology

[0002] The de novo glycerolipid biosynthesis pathway is one of the most fundamental metabolic processes in cells. Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial step in the de novo synthesis of glycerolipids, generating lysophosphatidic acid (LPA), which is then converted to phosphatidic acid (PA) by LPA acyltransferase (LPAAT). PA is an important signaling molecule regulating various cellular processes in eukaryotes and is also a precursor in the biosynthesis of polar glycerophospholipids and neutral triacylglycerols (TAGs). PA is converted to diacylglycerols under the catalysis of phosphatases, and the latter can be converted to TAGs by acyl-CoA-dependent diacylglycerol acyltransferase (DGAT) and / or phospholipid-dependent diacylglycerol acyltransferase (PDAT). TAG biosynthesis is a key trait in oilseed crops and is closely related to diseases such as obesity in humans. Therefore, manipulating TAG biosynthesis using genetic or chemogenetic methods to increase the oil content of oilseed crops while reducing obesity-related human diseases has significant practical implications.

[0003] Numerous biochemical studies have demonstrated the crucial role of acyltransferases in TAG biosynthesis. However, the intrinsic relationship between the structure and function of these enzymes remains poorly understood, including GPAT, which participates in the first step of acylation; only a few reports exist regarding its structure-function relationship. Acyltransferases such as GPAT, LPAAT, and dihydroxyacetone-phosphate acyltransferase (DHAPAT) are known to contain four highly conserved domains. Histidine (H) and aspartic acid (D) in domain I, glycine (G) in domain III, and proline (P) in domain IV are essential for GPAT catalysis; while arginine (R) in domain II and glutamate (E) in domain III play a role in binding the substrate 3-glycerophosphate. To date, our understanding of the roles of other amino acid residues outside the conserved domains in regulating acyltransferase activity and their potential interactions with amino acid residues within the conserved domains is extremely limited.

[0004] It is known that GPAT9, located in the endoplasmic reticulum of plant cells, participates in the biosynthesis of membrane lipids and TAGs; its functional loss leads to abnormal seed development and weakened lipid synthesis. Consistent with this, our previous yeast genetic complementation studies showed that heterologous expression of BnGPAT9 from rapeseed could restore the growth defects caused by the lack of GPAT enzyme activity in the yeast conditionally lethal double knockout mutant (ZAFU1). However, unexpectedly, Arabidopsis thaliana AtGPAT9 did not possess this complementation ability, despite the close evolutionary relationship between AtGPAT9 and BnGPAT9, with a 94.1% amino acid sequence identity, and complete identical amino acid residues in the four conserved acyltransferase domains. Therefore, we hypothesize that certain amino acid residues outside the conserved domains play an important regulatory role in GPAT9 activity. Summary of the Invention

[0005] To address the aforementioned issues, this study fully utilizes the different properties exhibited by AtGPAT9 and BnGPAT9 in yeast heterologous systems. By combining site-directed mutagenesis and yeast genetic complementation techniques, it analyzes the effects of changes in single and multiple amino acid residues on GPAT9 enzyme activity, identifies new key active sites, and deepens the understanding of the intrinsic relationship between the structure and function of acyltransferases. This provides a theoretical basis for the molecular modification and structural optimization of acyltransferases, the improvement of TAG synthesis pathways in eukaryotes, and the construction of novel de novo TAG synthesis pathways.

[0006] To achieve the above objectives, the specific solution adopted by the present invention is as follows:

[0007] Firstly, the mutant enzyme of 3-phosphoglyceryl acyltransferase is a site-directed mutation of a single or multiple amino acid residues at the key active site of 3-phosphoglyceryl acyltransferase GPAT9, which alters the enzyme activity of GPAT9. The key active sites include positions 85, 114, 119, 230, 237, and 322 of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4.

[0008] Furthermore, the protein encoded by the amino acid containing the site-directed mutation has stronger enzymatic activity than wild-type 3-phosphoglyceryl acyltransferase. The site-directed mutation of the single amino acid residue is as follows (I) or (II): (I) Compared with the wild-type BnGPAT9 with an amino acid sequence such as SEQ ID NO: 4, the 114th amino acid residue is changed from threonine to leucine, or the 230th amino acid residue is changed from asparagine to aspartic acid. (II) Compared with the wild-type AtGPAT9 with an amino acid sequence such as SEQ ID NO: 2, the amino acid residue at position 119 is changed from asparagine to histidine; The site-directed mutations of the plurality of amino acid residues include any one of the following (1) to (11): (1) Compared with the wild-type AtGPAT9 with an amino acid sequence such as SEQ ID NO: 2, the amino acid residues at positions 85 and 119 are changed from tyrosine and asparagine to tryptophan and histidine, respectively. (2) Compared with the wild-type AtGPAT9 with an amino acid sequence such as SEQ ID NO: 2, the amino acid residues at positions 85, 114, and 119 are changed from tyrosine, leucine, and asparagine to tryptophan, threonine, and histidine, respectively. (3) Compared with the wild-type AtGPAT9 with an amino acid sequence such as SEQ ID NO: 2, the amino acid residues at positions 85, 119, and 237 are changed from tyrosine, asparagine, and serine to tryptophan, histidine, and asparagine, respectively. (4) Compared with the wild-type AtGPAT9 with an amino acid sequence such as SEQ ID NO: 2, the amino acid residues at positions 85, 114, 119, and 237 are changed from tyrosine, leucine, asparagine, and serine to tryptophan, threonine, histidine, and asparagine, respectively. (5) Compared with the wild-type AtGPAT9 with an amino acid sequence such as SEQ ID NO: 2, the amino acid residues at positions 85, 119, and 230 are changed from tyrosine, asparagine, and aspartic acid to tryptophan, histidine, and asparagine, respectively. (6) Compared with the wild-type AtGPAT9 with an amino acid sequence such as SEQ ID NO: 2, the amino acid residues at positions 85, 119, and 235 are changed from tyrosine, asparagine, and alanine to tryptophan, histidine, and threonine, respectively. (7) Compared with the wild-type AtGPAT9 with an amino acid sequence such as SEQ ID NO: 2, the amino acid residues at positions 85 and 237 are changed from tyrosine and serine to tryptophan and asparagine, respectively. (8) Compared with the wild-type AtGPAT9 with an amino acid sequence such as SEQ ID NO: 2, the amino acid residues at positions 40, 85, and 237 are changed from serine, tyrosine, and serine to arginine, tryptophan, and asparagine, respectively. (9) Compared with the wild-type AtGPAT9 with an amino acid sequence such as SEQ ID NO: 2, the amino acid residues at positions 119 and 237 are changed from asparagine and serine to histidine and asparagine, respectively. (10) Compared with the wild-type AtGPAT9 with an amino acid sequence such as SEQ ID NO: 2, the amino acid residues at positions 119, 230, and 237 are changed from asparagine, aspartic acid, and serine to histidine, asparagine, and asparagine, respectively. (11) Compared with the wild-type AtGPAT9 with an amino acid sequence such as SEQ ID NO:2, the amino acid residues at positions 119, 235, and 237 are changed from asparagine, alanine, and serine to histidine, threonine, and asparagine, respectively.

[0009] Secondly, the gene encoding the aforementioned 3-phosphoglyceryl acyltransferase mutant enzyme.

[0010] Thirdly, the application of the aforementioned mutant enzymes or genes in the regulation of lipid synthesis.

[0011] Beneficial Effects: This invention constructed 58 GPAT9 mutant genes using site-directed mutagenesis technology. Combined with GPAT-specific yeast genetic complementation, the effects of single and multiple amino acid site alterations on GPAT9 enzyme activity were analyzed. Analysis of 19 amino acid residues in AtGPAT9 and BnGPAT9 revealed that individual mutations at the six N-terminal phosphorylation sites of AtGPAT9 (T10A, S11A, S13A, S28A, S30A, S31A) did not enhance the activity of AtGPAT9 during heterologous expression in yeast. Conversely, alterations to the other six amino acid residues located outside the conserved acyltransferase domain (85, 114, 119, 230, 237, 322) significantly affected GPAT9 enzyme activity. Interactions among these amino acids were discovered. For example, simultaneous mutations at all three sites (Y85W / N119H / S237N) significantly increased AtGPAT9 activity, accelerated yeast growth, and promoted TAG synthesis. The TAG content in yeast expressing this mutant enzyme was 45.7% higher than that expressing wild-type BnGPAT9. More notably, the simultaneous presence of phosphorylated amino acid residues at positions 114 and 237 was detrimental to acyltransferase activity, suggesting that plant GPAT9 activity may be regulated by phosphorylation and non-phosphorylation mechanisms.

[0012] This study identified six previously unreported key regulatory sites for GPAT enzyme activity. Among them, W85 and H119 are essential for the normal function of GPAT9, while L114, D230, N237, and A322 are beneficial for maintaining GAPT9 activity. These findings broaden our understanding of the intrinsic relationship between the structure and function of acyltransferases and provide a theoretical basis for the molecular modification and structural optimization of different acyltransferases. Attached Figure Description

[0013] Figure 1 This is an amino acid sequence alignment diagram of Arabidopsis thaliana AtGPAT9 and rapeseed BnGPAT9. In the diagram, identical amino acid residues are indicated by black shading, similar amino acid residues by gray shading, and different amino acid residues by white shading. The conserved acyltransferase domains are labeled AT1, AT2, AT3, and AT4. The predicted transmembrane regions are labeled TM1, TM2, and TM3. "*" indicates amino acid residues essential for GPAT catalysis, and "^" indicates amino acid residues essential for binding the substrate 3-glycerophosphate.

[0014] Figure 2 This is a predicted three-dimensional structure diagram of AtGPAT9 and BnGPAT9; in the diagram, amino acid residues are represented by abbreviations and sites, such as S237 representing serine at position 237.

[0015] Figure 3The effects of different single point mutations on the enzyme activities of AtGPAT9 and BnGPAT9 are shown in the figure. In the figure, A represents the effect of a single point mutation on the enzyme activity of BnGPAT9, and B represents the effect of a single point mutation on the enzyme activity of AtGPAT9. To evaluate the effect of amino acid residue substitution on GPAT9 enzyme activity, AtGPAT9 or BnGPAT9 carrying different mutation sites were expressed in the conditionally lethal double mutant yeast ZAFU1. The activity of the expressed product was generally positively correlated with the growth rate of the yeast. The related yeast cell concentration gradient dilution culture experiments were repeated at least 4 times at different time points, and the trend of the results was consistent each time. In addition to using the empty vector pYES2 as a negative control, wild-type Arabidopsis thaliana AtGPAT9 and rapeseed BnGPAT9 were used as negative and positive controls, respectively.

[0016] Figure 4 The effect of leucine substitution at position 114 (T114L) on BnGPAT9 activity; the figure shows that the transformed yeast was photographed after 48 h of growth on solid medium to compare the effects of wild-type BnGPAT and mutant genes on the growth rate of ZAFU1 mutant yeast cells; after about 3 days of growth, yeast colonies expressing wild-type BnGPAT9 became visible, while the T114L mutation advanced the appearance of visible colonies by about 1 day; BnGPAT9 (T114L): BnGPAT9 carrying T114L.

[0017] Figure 5 This study investigated the effects of multiple site mutations on AtGPAT9 enzyme activity. In the figure, A represents the effects of mutations at sites 85, 119, and 237 on AtGPAT9 enzyme activity; B represents the effects of combinations of mutations at site 114 and other sites on AtGPAT9 enzyme activity; C represents the effects of combinations of mutations at site 85 and other sites on AtGPAT9 enzyme activity; and D represents the effects of combinations of mutations at site 119 and other sites on AtGPAT9 enzyme activity. To assess the effects of amino acid residue substitutions on GPAT9 enzyme activity, AtGPAT9 carrying different mutation sites was expressed in the conditionally lethal double mutant yeast ZAFU1. The activity of the expressed product was generally positively correlated with the yeast growth rate. Related yeast cell concentration gradient dilution experiments were repeated at least four times at different time points, with consistent trends in each result. In addition to using the empty vector pYES2 as a negative control, wild-type Arabidopsis thaliana AtGPAT9 and rapeseed BnGPAT9 served as negative and positive controls, respectively.

[0018] Figure 6The expression of AtGPAT9 with different mutation sites had an effect on the cell density of ZAFU1 yeast strains at different growth stages. Results are expressed as mean ± standard deviation (n=3). AtN119H, AtY85W / N119H, and AtY85W / N119H / S237N represent Arabidopsis AtGPAT9 strains carrying the N119H, Y85W / N119H, and 85W / N119H / S237N mutation sites, respectively. Brassica rapa BnGPAT9 was used as a positive control, and the empty vector pYES2 was used as a negative control.

[0019] Figure 7 The expression of AtGPAT9 with different mutation sites affected the TAG content of ZAFU1 yeast strains during the platform growth period. Results are presented as mean ± standard deviation (n=3), and statistical analysis was performed using one-way ANOVA. * indicates a significant difference compared to ZAFU1 cells expressing BnGPAT9 (*p<0.05). AtN119H, AtY85W / N119H, and AtY85W / N119H / S237N represent Arabidopsis AtGPAT9 cells carrying the N119H, Y85W / N119H, and 85W / N119H / S237N mutation sites, respectively; BnGPAT9 from rapeseed served as a positive control. Detailed Implementation

[0020] The nucleotide sequence of AtGPAT9 described in this application is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2.

[0021] The nucleotide sequence of BnGPAT9 described in this application is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.

[0022] This application focuses on protecting the following sequences:

[0023] SEQ ID NO: 5, the nucleotide sequence AtGPAT9 (A355C / A356A / T357C), differs from the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1) in that it changes from AAT to CAC at positions 355, 356, and 357. Proteins encoded by nucleotide sequences containing these mutation sites exhibit stronger enzymatic activity than proteins encoded by the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1).

[0024] SEQ ID NO: 6, amino acid sequence AtGPAT9(N119H), compared with wild-type amino acid sequence AtGPAT9(SEQ ID NO: 2), the amino acid residue at position 119 is changed from asparagine to histidine. The protein encoded by the amino acid sequence containing the above mutation site has stronger enzyme activity than the protein encoded by wild-type amino acid sequence AtGPAT9(SEQ ID NO: 2).

[0025] SEQ ID NO: 7, the nucleotide sequence AtGPAT9 (T253T / A254G / C255G / A355C / A356A / T357C), compared with the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1), has the following changes: at positions 253, 254, and 255, TAC is replaced by TGG; and at positions 355, 356, and 357, AAT is replaced by CAC. The protein encoded by the nucleotide sequence containing these mutation sites exhibits stronger enzymatic activity than the protein encoded by the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1).

[0026] SEQ ID NO: 8, amino acid sequence AtGPAT9 (Y85W / N119H), compared with wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2), has amino acid residues at positions 85 and 119 changed from tyrosine and asparagine to tryptophan and histidine, respectively. The protein encoded by the amino acid sequence containing the above mutation sites has stronger enzyme activity than the protein encoded by wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2).

[0027] SEQ ID NO: 9, the nucleotide sequence AtGPAT9 (T253T / A254G / C255G / T340A / T341C / G342G / A355C / A356A / T357C), compared with the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1), has the following changes: at positions 253, 254, and 255, TAC is changed to TGG; at positions 340, 341, and 342, TTG is changed to ACG; and at positions 355, 356, and 357, AAT is changed to CAC. The protein encoded by the nucleotide sequence containing these mutation sites has stronger enzymatic activity than the protein encoded by the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1).

[0028] SEQ ID NO: 10, amino acid sequence AtGPAT9 (Y85W / L114T / N119H), compared with wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2), has amino acid residues at positions 85, 114, and 119 changed from tyrosine, leucine, and asparagine to tryptophan, threonine, and histidine, respectively. The protein encoded by the amino acid sequence containing the above mutation sites has stronger enzymatic activity than the protein encoded by wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2).

[0029] SEQ ID NO: 11, the nucleotide sequence AtGPAT9 (T253T / A254G / C255G / A355C / A356A / T357C / A709A / G710A / T711T), compared with the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1), has the following changes: at positions 253, 254, and 255, TAC is changed to TGG; at positions 355, 356, and 357, AAT is changed to CAC; and at positions 709, 710, and 711, AGT is changed to AAT. The protein encoded by the nucleotide sequence containing these mutation sites has stronger enzymatic activity than the protein encoded by the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1).

[0030] SEQ ID NO: 12, amino acid sequence AtGPAT9 (Y85W / N119H / S237N), compared with wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2), has amino acid residues at positions 85, 119, and 237 changed from tyrosine, asparagine, and serine to tryptophan, histidine, and asparagine, respectively. The protein encoded by the amino acid sequence containing the above mutation sites has stronger enzymatic activity than the protein encoded by wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2).

[0031] SEQ ID NO: 13, the nucleotide sequence AtGPAT9 (T253T / A254G / C255G / T340A / T341C / G342G / A355C / A356A / T357C / A709A / G710A / T711T), compared with the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1), has the following changes: at positions 253, 254, and 255, TAC is changed to TGG; at positions 340, 341, and 342, TTG is changed to ACG; at positions 355, 356, and 357, AAT is changed to CAC; and at positions 709, 710, and 711, AGT is changed to AAT. The protein encoded by the nucleotide sequence containing these mutation sites exhibits stronger enzymatic activity than the protein encoded by the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1).

[0032] SEQ ID NO: 14, amino acid sequence AtGPAT9 (Y85W / L114T / N119H / S237N), compared with wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2), has amino acid residues at positions 85, 114, 119, and 237 changed from tyrosine, leucine, asparagine, and serine to tryptophan, threonine, histidine, and asparagine, respectively. The protein encoded by the amino acid sequence containing the above mutation sites has stronger enzymatic activity than the protein encoded by wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2).

[0033] SEQ ID NO: 15, the nucleotide sequence AtGPAT9 (T253T / A254G / C255G / A355C / A356A / T357C / G688A / A689A / C690C), compared with the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1), has the following changes: at positions 253, 254, and 255, TAC is changed to TGG; at positions 355, 356, and 357, AAT is changed to CAC; and at positions 688, 689, and 690, GAC is changed to AAC. The protein encoded by the nucleotide sequence containing these mutation sites exhibits stronger enzymatic activity than the protein encoded by the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1).

[0034] SEQ ID NO: 16, amino acid sequence AtGPAT9 (Y85W / N119H / D230N), compared with wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2), has amino acid residues at positions 85, 119, and 230 changed from tyrosine, asparagine, and aspartic acid to tryptophan, histidine, and asparagine, respectively. The protein encoded by the amino acid sequence containing the above mutation sites has stronger enzymatic activity than the protein encoded by wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2).

[0035] SEQ ID NO: 17, the nucleotide sequence AtGPAT9 (T253T / A254G / C255G / A355C / A356A / T357C / G703A / C704C / T705T), compared with the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1), has the following changes: at positions 253, 254, and 255, TAC is changed to TGG; at positions 355, 356, and 357, AAT is changed to CAC; and at positions 703, 704, and 705, GCT is changed to ACT. The protein encoded by the nucleotide sequence containing these mutation sites exhibits stronger enzymatic activity than the protein encoded by the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1).

[0036] SEQ ID NO: 18, amino acid sequence AtGPAT9 (Y85W / N119H / A235T), compared with wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2), has amino acid residues at positions 85, 119, and 235 changed from tyrosine, asparagine, and alanine to tryptophan, histidine, and threonine, respectively. The protein encoded by the amino acid sequence containing the above mutation sites has stronger enzymatic activity than the protein encoded by wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2).

[0037] SEQ ID NO: 19, the nucleotide sequence AtGPAT9 (T253T / A254G / C255G / A709A / G710A / T711T), compared with the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1), has the following changes: at positions 253, 254, and 255, TAC is replaced with TGG; and at positions 709, 710, and 711, AGT is replaced with AAT. The protein encoded by the nucleotide sequence containing these mutation sites exhibits stronger enzymatic activity than the protein encoded by the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1).

[0038] SEQ ID NO: 20, amino acid sequence AtGPAT9 (Y85W / S237N), compared with wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2), has amino acid residues at positions 85 and 237 changed from tyrosine and serine to tryptophan and asparagine, respectively. The protein encoded by the amino acid sequence containing the above mutation sites has stronger enzymatic activity than the protein encoded by wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2).

[0039] SEQ ID NO: 21, the nucleotide sequence AtGPAT9 (A118C / G119G / C120C / T253T / A254G / C255G / A709A / G710A / T711T), compared with the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1), has the following changes: AGC is changed to CGC at positions 118, 119, and 120; TAC is changed to TGG at positions 253, 254, and 255; and AGT is changed to AAT at positions 709, 710, and 711. The protein encoded by the nucleotide sequence containing these mutation sites has stronger enzymatic activity than the protein encoded by the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1).

[0040] SEQ ID NO: 22, amino acid sequence AtGPAT9 (S40R / Y85W / S237N), compared with wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2), has amino acid residues at positions 40, 85, and 237 changed from serine, tyrosine, and serine to arginine, tryptophan, and asparagine, respectively. The protein encoded by the amino acid sequence containing the above mutation sites has stronger enzymatic activity than the protein encoded by wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2).

[0041] SEQ ID NO: 23, the nucleotide sequence AtGPAT9 (A355C / A356A / T357C / A709A / G710A / T711T), compared with the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1), has the following changes: at positions 355, 356, and 357, AAT is replaced with CAC; at positions 709, 710, and 711, AGT is replaced with AAT. The protein encoded by the nucleotide sequence containing these mutation sites exhibits stronger enzymatic activity than the protein encoded by the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1).

[0042] SEQ ID NO: 24, amino acid sequence AtGPAT9 (N119H / S237N), compared with wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2), has amino acid residues at positions 119 and 237 changed from asparagine and serine to histidine and asparagine, respectively. The protein encoded by the amino acid sequence containing the above mutation sites has stronger enzymatic activity than the protein encoded by wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2).

[0043] SEQ ID NO: 25, the nucleotide sequence AtGPAT9 (A355C / A356A / T357C / G688A / A689A / C690C / A709A / G710A / T711T), compared with the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1), has the following changes: AAT is replaced with CAC at positions 355, 356, and 357; GAC is replaced with AAC at positions 688, 689, and 690; and AGT is replaced with AAT at positions 709, 710, and 711. The protein encoded by the nucleotide sequence containing these mutation sites has stronger enzymatic activity than the protein encoded by the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1).

[0044] SEQ ID NO: 26, amino acid sequence AtGPAT9 (N119H / D230N / S237N), compared with wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2), has amino acid residues at positions 119, 230, and 237 changed from asparagine, aspartic acid, and serine to histidine, asparagine, and asparagine, respectively. The protein encoded by the amino acid sequence containing the above mutation sites has stronger enzymatic activity than the protein encoded by wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2).

[0045] SEQ ID NO: 27, the nucleotide sequence AtGPAT9 (A355C / A356A / T357C / G703A / C704C / T705T / A709A / G710A / T711T), compared with the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1), has the following changes: at positions 355, 356, and 357, AAT is replaced by CAC; at positions 703, 704, and 705, GCT is replaced by ACT; and at positions 709, 710, and 711, AGT is replaced by AAT. The protein encoded by the nucleotide sequence containing these mutation sites exhibits stronger enzymatic activity than the protein encoded by the wild-type nucleotide sequence AtGPAT9 (SEQ ID NO: 1).

[0046] SEQ ID NO: 28, amino acid sequence AtGPAT9 (N119H / A235T / S237N), compared with wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2), has amino acid residues at positions 119, 235, and 237 changed from asparagine, alanine, and serine to histidine, threonine, and asparagine, respectively. The protein encoded by the amino acid sequence containing the above mutation sites has stronger enzymatic activity than the protein encoded by wild-type amino acid sequence AtGPAT9 (SEQ ID NO: 2).

[0047] SEQ ID NO: 29, nucleotide sequence BnGPAT9 (A340T / C341T / G342G), compared with wild-type nucleotide sequence BnGPAT9 (SEQ ID NO: 3), has its nucleotide sequence changed from ACG to TTG at positions 340, 341, and 342. The protein encoded by the nucleotide sequence containing these mutation sites has stronger enzymatic activity than the protein encoded by the wild-type nucleotide sequence BnGPAT9 (SEQ ID NO: 3).

[0048] SEQ ID NO: 30, amino acid sequence BnGPAT9(T114L), compared with wild-type amino acid sequence BnGPAT9(SEQ ID NO: 4), has a threonine residue at position 114 changed to leucine. The protein encoded by the amino acid sequence containing the above mutation site has stronger enzymatic activity than the protein encoded by wild-type amino acid sequence BnGPAT9(SEQ ID NO: 4).

[0049] SEQ ID NO: 31, the nucleotide sequence BnGPAT9 (A688G / A689A / C690C), differs from the wild-type nucleotide sequence BnGPAT9 (SEQ ID NO: 3) in that it changes from AAC to GAC at positions 688, 689, and 690. Proteins encoded by nucleotide sequences containing these mutation sites exhibit stronger enzymatic activity than proteins encoded by the wild-type nucleotide sequence BnGPAT9 (SEQ ID NO: 3).

[0050] SEQ ID NO: 32, amino acid sequence BnGPAT9(N230D), compared with wild-type amino acid sequence BnGPAT9(SEQ ID NO: 4), has amino acid residue 230 changed from asparagine to aspartic acid. The protein encoded by the amino acid sequence containing the above mutation site has stronger enzyme activity than the protein encoded by wild-type amino acid sequence BnGPAT9(SEQ ID NO: 4).

[0051] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.

[0052] 1. Materials and Methods

[0053] 1.1 Sequence Analysis

[0054] The amino acid sequences of AtGPAT9 (GENEBAKN accession number: ACT32031.1) and BnGPAT9 (GENEBAKN accession number: ANV28166.1) were aligned using Vector NTI 11.5.4 software. Transmembrane domains and protein topological isomorphism models were predicted using TMHMM 2.0 and Protter. Three-dimensional structures were predicted using I-TASSER.

[0055] 1.2 Site-directed gene mutation

[0056] The coding sequences of AtGPAT9 and BnGPAT9 were cloned into the pMD19-T vector using BamHI / XhoⅠ and BamHI / EcoRI double restriction sites, respectively, to obtain new plasmids pMD19-T-AtGPAT9 and pMD19-T-BnGPAT9. Using these as templates, site-directed mutagenesis was performed on AtGPAT9 and BnGPAT9. The specific methods are as follows: the entire plasmid was amplified by PCR using primers containing the mutation sites (Table S1); the plasmid DNA was digested and purified with Dpn I, then transformed into *E. coli* and sequenced; plasmids with correct sequences were double-digested with BamHI and XhoⅠ and ligated into the pYES2-yADH1-Kan V2 yeast expression vector, which had undergone the same restriction enzyme digestion treatment. The resulting recombinant plasmids were then subjected to DNA sequencing analysis again to ensure the correctness of the mutation sites. It should be noted that the pMD19-T plasmid was chosen as an intermediate plasmid in the site-directed mutagenesis process, rather than directly performing site-directed mutagenesis of the GPAT9 gene on the pYES2-yADH1-Kan V2 plasmid, because the latter's DNA length (6998bp) is 2.6 times that of the former (2660bp). This strategy can reduce the frequency of potential erroneous bases caused by prolonged PCR amplification time.

[0057] 1.3 Yeast genetic transformation

[0058] The conditionally lethal yeast double mutant ZAFU1 (BY4742, gat1Δgat2Δ+[pGAL1::AtGPAT1Leu2]) can grow on galactose-based media but loses its ability to grow on glucose-based media. A yeast genetic complementation method based on the ZAFU1 strain exhibits high specificity for GPAT identification. Therefore, this study used it to identify the effects of different amino acid residue mutations on GPAT9 activity.

[0059] The recombinant yeast expression plasmid was introduced into ZAFU1 competent yeast cells using a standard lithium acetate-based method. After 4 hours of recovery, the transformation solution was plated onto SC-Ura-His-Leu medium (using glucose (Glu) or galactose (Gal) as the carbon source, and cultured at 30°C for 3–5 days. To accurately compare the effects of different amino acid residue mutations on enzyme activity, we simultaneously performed a serial dilution experiment on different single yeast colonies growing on galactose medium: well-grown single colonies were randomly selected from galactose medium and transferred to SC-Ura-His-Leu+Gal liquid medium, and cultured at 30°C with shaking for 1–2 days until OD. 600 The concentration of the bacterial culture should be 2.0–3.0, diluted to an OD value of [missing value]. 600 The effective values ​​were 1.0, 0.2, 0.04, 0.008, and 0.00016. 5 μL of each value was inoculated onto SC-Ura-His-Leu+Glu and SC-Ura-His-Leu+Gal solid medium and incubated at 30°C for 3–5 days.

[0060] 1.4 Yeast growth curve determination and lipid analysis

[0061] ZAFU1 cells expressing different GPAT9 mutant genes were cultured in SC-Ura-His-Leu+Gal liquid medium at 30℃ until OD. 600 The concentration was 3.0–4.0, diluted and inoculated into SC-Ura-His-Leu+Glu liquid medium to OD. 600 The concentration was set to 0.1, and the culture was carried out with shaking and OD values ​​recorded periodically. 600 value.

[0062] Cells harvested during the platform's growth period were dried in a vacuum freeze dryer, and total yeast lipids were extracted and spotted onto silica gel plates. The total lipids were then separated by thin-layer chromatography. A 0.05% primrose yellow chromogenic agent was sprayed onto the plates, and the lipids were observed under ultraviolet light. TAG was extracted from the silica gel, and its content was quantitatively analyzed by gas chromatography.

[0063] 2 Results and Analysis

[0064] 2.1 Structural differences between AtGPAT9 and BnGPAT9

[0065] Early studies found that Arabidopsis thaliana AtGPAT9 and rapeseed BnGPAT9 exhibited different activities when heterologously expressed in yeast. To identify potential key regulatory sites, sequence alignment of AtGPAT9 and BnGPAT9 was first performed. Both AtGPAT9 and BnGPAT9 consist of 376 amino acids, with a sequence identity of 94.1%. The four conserved acyltransferase domains and the C-terminal hydrophobic pentapeptide domain (–ILARL–) essential for endoplasmic reticulum localization are completely identical. Both also contain multiple potential phosphorylation sites at the N-terminus, mainly composed of serine and threonine residues. Figure 1 They differ in a total of 22 amino acid residues, of which 11 have similar properties. Transmembrane domain predictions based on TMHMM and Protter show that the transmembrane regions have high structural similarity, but 7 amino acid residues differ across the three transmembrane regions. Figure 1 Furthermore, based on I-TASSER-based three-dimensional structure prediction, differences in amino acid residues at positions 40, 109, 114, 119, 230, 235, 237, and 322 between AtGPAT9 and BnGPAT9 may cause differences in their three-dimensional spatial structures. Therefore, these sites became the focus of analysis in this study. Figure 2 ).

[0066] 2.2 Site-directed mutagenesis of AtGPAT9 and BnGPAT9

[0067] To clarify which amino acid sites play a crucial regulatory role in GPATase activity, this study employed site-directed mutagenesis to replace corresponding amino acid residues in AtGPAT9 and BnGPAT9, based on the differences in amino acid residues between them. Specifically, this involved simultaneously replacing one or more amino acid residues in AtGPAT9 with the exact same amino acid residues at the corresponding positions in BnGPAT9, and vice versa. In this study, a total of 58 different GPAT9 mutant genes were constructed (Table 1) for subsequent structure-function studies.

[0068] Table 1: Site-directed mutations of single and multiple amino acid residues in AtGPAT9 and BnGPAT9.

[0069] In Table 1, each mutation is represented by the first letter abbreviation of the species and the abbreviation of the amino acid residues before and after the mutation. For example, AtS40R / S237N represents the change of serine (S) to arginine (R) at position 40 of AtGPAT9 and the change of serine (S) to asparagine (N) at position 237. * indicates that heterologous expression of the gene can restore the growth defects of the yeast double mutant ZAFU1; the number of * indicates the magnitude of the restoration ability. For example, wild-type AtGPAT9 lacks complementation ability, while wild-type BnGPAT9 has a complementation ability of two (**).

[0070] The primer sequences used for site-directed mutagenesis of Arabidopsis thaliana AtGPAT9 and rapeseed BnGPAT9 are shown in Table 2.

[0071] Table 2: Primer sequences used for site-directed mutagenesis of Arabidopsis thaliana AtGPAT9 and rapeseed BnGPAT9.

[0072] 2.3 Effect of single amino acid residue mutation on GPAT9 activity

[0073] Different GPAT9 mutant genes were cloned into plasmids containing the glucose-inducible promoter (ADH1). Empty vectors and plasmids containing wild-type AtGPAT9 were used as negative controls, and plasmids containing wild-type BnGPAT9 were used as positive controls. These recombinant plasmids were introduced into the ZAFU1 strain, and the ability of different GPAT9 mutant genes to restore the growth defects of ZAFU1 on glucose medium was determined. The effect of different mutation sites on GPAT9 enzyme activity could be assessed relatively directly based on the growth rate of the transformed yeast cells.

[0074] The results showed that when the ZAFU1 mutant was cultured on glucose medium, single-site substitution of W85Y or H119N resulted in BnGPAT9 losing its ability to recover from the mutant's growth defects. Figure 3 A) indicates that W85 and H119 are essential for the normal function of BnGPAT9. Furthermore, compared to wild-type BnGPAT9, BnGPAT9 containing the N237S or A322G mutation sites showed a decreased promoting effect on the growth of ZAFU1 strain. Figure 3 A) indicates that these two sites are also involved in the regulation of BnGPAT9 activity. Conversely, the other five site substitutions (R40S, C87F, I102F, F109I, T235A) had no significant effect on BnGPAT9 activity. Figure 3 A). Of particular note is that the unit-point substitution of N230D and T114L enhanced BnGPAT9 activity, with this upregulation being especially pronounced for BnT114L. For example... Figure 4As shown, compared with wild-type BnGPAT9, expression of BnGPAT9 containing the T114L mutation site in yeast mutants can significantly increase cell growth rate. After 2 days of culture, the number of colonies expressing BnGPAT9 (T114L) on glucose medium is about four times that of wild-type BnGPAT9, and each single colony has a larger surface area.

[0075] Similarly, yeast genetic complementation was performed on 19 different AtGPAT9 mutant genes. Six of these proteins exhibited substitutions at the N-terminal potential phosphorylation sites: T10A, S11A, S13A, S28A, S30A, and S31A. The remaining 13 showed substitutions at S40R, Y85W, F87C, F102I, I109F, L114T, N119H, D230N, A235T, S237N, G322A, L335H, and P355S. The results showed that replacing the six N-terminal potential phosphorylation sites with neutral amino acid residues did not improve AtGPAT9 activity. Figure 3 B). Except for N119H, other single-point mutations did not have a noticeable effect on the activity of AtGPAT9 in yeast heterologous systems (Table 1). In yeast concentration gradient dilution culture experiments, N119H substitution restored the growth defect of AtGPAT9 in the ZAFU1 mutant on glucose, but this effect was relatively weak (Table 1). Figure 5 D).

[0076] 2.4 Interaction of multiple amino acid residue mutations on GPAT9 activity

[0077] Based on the hypothesis that both adjacent and non-adjacent amino acid residues may have specific interactions affecting enzyme activity, we further constructed 28 AtGPAT9 mutant enzymes with substitutions of 2–4 amino acid residues, taking advantage of the amino acid differences between AtGPAT9 and BnGPAT9. Consistent with the important regulatory roles of W85, H119, and N237 sites on BnGPAT9 activity, simultaneous substitution of two or three corresponding amino acid residues (Y85W, N119H, S237N) on AtGPAT9 significantly improved AtGPAT9 enzyme activity. Specifically, AtGPAT9 enzymes carrying the Y85W / N119H, Y85W / S237N, N119H / S237N, and Y85W / N119H / S237N mutations could complement the growth defects of the ZAFU1 strain. Figure 5A); however, the effect of single point mutations on AtGPAT9 activity was very limited (Table 1). Further investigation revealed that AtGPAT9 carrying the Y85W / N119H or Y85W / N119H / S237N mutation combination promoted the growth of ZAFU1 strain more than wild-type BnGPAT9, and had a similar effect to BnGPAT9 carrying the T114L mutation. Figure 3 A, Figure 5 A).

[0078] To better understand the regulatory effect of the 114th amino acid on GAPT9 activity and its interaction with other amino acids, we replaced the leucine (L114) at position 114 of the two-site mutant AtGPAT9 (Y85W / N119H) and the three-site mutant AtGPAT9 (Y85W / N119H / S237N) with the threonine (T) present at the corresponding position in BnGPAT9. The results showed that the activity of the generated three-site mutant AtGPAT9 (Y85W / L114T / N119H) was significantly weaker than that of AtGPAT9 (Y85W / N119H), but the activity of the four-site mutant AtGPAT9 (Y85W / L114T / N119H / S237N) was still comparable to that of AtGPAT9 (Y85W / N119H / S237N). Figure 5 B). One possible explanation for this interesting phenomenon is that when two potential phosphorylation sites, T114 and S237, are simultaneously present in AtGPAT9(Y85W / L114T / N119H) (containing S237) or BnGPAT9(N237S) (containing T114), the phosphorylation level of GPAT9 may be intensified, thereby inhibiting enzyme activity. Therefore, we hypothesize that plant GPAT9 activity may be regulated by phosphorylation and non-phosphorylation mechanisms, and the two potential phosphorylation sites T114 and S237 present in wild-type GPAT9 may have a negative effect on acyltransferase activity.

[0079] Further experiments revealed that amino acid residue 230 interacts with amino acid residues 85 and 119, thus affecting GPAT activity. Although D230N itself or its combination mutations with A235T and S237N did not significantly affect AtGPAT9 enzyme activity (Table 1). When bound to N119H, Y85W / N119H, or Y85W / S237N, the D230N mutation inhibits AtGPAT9 activity. This is because, compared to strains expressing enzymes containing the N119H, Y85W / N119H, or Y85W / S237N mutation sites, ZAFU1 strains expressing AtGPAT9(N119H / D230N), AtGPAT9(Y85W / N119H / D230N), or AtGPAT9(Y85W / D230N / S237N) showed significantly reduced growth rates or no growth at all on glucose medium, suggesting that the substitution of D230N is detrimental to AtGPAT9 activity (Table 1). Figure 5 B, C, D). This hypothesis is supported by the following results: N230D substitution can improve BnGPAT9 activity (B, C, D). Figure 3 A). However, it should be noted that the activities of AtGPAT9(N119H / D230N / S237N) and AtGPAT9(N119H / S237N) are comparable, indicating that the negative effect of D230N depends on the interactions of other amino acids (Table 1). Figure 5 D).

[0080] Other interesting phenomena are that the triple mutant combinations of N119H / D230N / S237N and N119H / A235T / S237N can enhance AtGPAT9 activity, enabling it to rescue ZAFU1 growth defects. However, the quadruple mutant combination of N119H / D230N / A235T / S237N, obtained by adding A235T and D230N to the former and the latter respectively, causes the corresponding protein to lose GPAT activity, just like wild-type AtGPAT9, and it cannot restore the growth defects of ZAFU1 (Table 1). Figure 5 (D) Furthermore, neither single nor double substitutions of S237N and G322A enhanced the activity of AtGPAT9 expressed in yeast (Table 1), which is inconsistent with the aforementioned phenomenon that substitutions of N237S or A322G led to a decrease in BnGPAT9 activity. Therefore, although retaining a non-phosphorylated amino acid (asparagine, N) at position 237 is beneficial to BnGPAT9 activity, its effect depends on the properties of the amino acids at other positions.

[0081] In summary, the enzyme activity of GPAT9 is influenced by the properties of amino acid residues at positions 85, 114, 119, 230, 237, and 322, and there are interactions between them. When these amino acid residues are tryptophan (W), leucine (L), histidine (H), aspartic acid (D), asparagine (N), and alanine (A), respectively, the acyltransferase activity is higher. The fact that these multi-site mutations enhance AtGPT9 enzyme activity demonstrates that molecular design can effectively modify and optimize the structure of acyltransferases, giving them new properties. This is highly beneficial for the future artificial manipulation of de novo synthesis of glycerides.

[0082] 2.5 Effects of Heterologous Expression of Mutant Enzymes on Yeast Lipid Synthesis

[0083] To further investigate the impact of amino acid site mutations on GPAT9 function, this study selected three AtGPAT9 mutant enzymes with different activities, containing mutations of N119H, Y85W / N119H, and Y85W / N119H / S237N, respectively, and expressed them heterologously. The effect of their expression on TAG synthesis in yeast cells was then measured. Wild-type BnGPAT9 was used as the positive control in this experiment. When the corresponding yeast cells entered the plateau growth phase, total lipids were extracted, separated by thin-layer chromatography, and the TAG content was determined by gas chromatography.

[0084] It should be noted that due to differences in the activity of different mutant enzymes, the culture time required for the corresponding yeast cells to reach the plateau growth phase varies, and the cell density during the plateau growth phase is also not entirely the same. For example, the cell density (expressed as OD) of yeast cells expressing BnGPAT9, AtGPAT9(Y85W / N119H), and AtGPAT9(Y85W / N119H / S237N) during the plateau growth phase is different. 600 The values ​​are 3.72, 4.40, and 5.32 respectively. Figure 6 Generally speaking, the greater the enzyme activity, the faster the cell growth and the higher the cell density. Figure 6 ).

[0085] Lipid analysis showed that expression of AtGPAT9 (Y85W / N119H / S237N) increased the TAG content in ZAFU1 mutant yeast cells to 0.51%, while expression of BnGPAT9 and AtGPAT9 (Y85W / N119H) resulted in oil contents of 0.35% and 0.39%, respectively. The former increased the oil content by 45.7% and 30.8%, respectively, compared to the latter two. Figure 7 Consistent with the relatively low activity of AtGPAT9(N119H), yeast cells expressing this enzyme had a TAG content of only 0.22%, significantly lower than yeast cells expressing BnGPAT9 (0.35%). Figure 7The above results indicate that the ability of yeast cells to synthesize TAG is regulated by GPAT activity, and the magnitude of GPAT activity is closely related to the properties of amino acids at specific positions. Therefore, optimizing the amino acid composition of GPAT9 through molecular design will help modify the ability of cells to synthesize TAG.

[0086] 3 Discussion

[0087] Enhancing lipid synthesis in oilseed crops is crucial for ensuring a balance between edible oil supply and demand. Conversely, increased lipid synthesis in human cells is not beneficial to health, as it can induce obesity and cardiovascular diseases. Currently, attempts are being made to manipulate TAG biosynthesis using genetic or chemogenetic methods, but several factors influence the effectiveness of these efforts. One such factor is the limited understanding of the intrinsic relationship between enzyme structure and function in the TAG synthesis pathway. This hinders enzyme structure optimization and limits the development of technologies that regulate enzyme activity based on post-translational modification mechanisms. This study utilizes GPAT-specific yeast genetic complementation to identify key amino acid sites controlling the activity of plant GPAT9 enzyme and the interactions between different sites. This deepens our understanding of the intrinsic relationship between acyltransferase structure and function, providing a foundation for future effective manipulation of TAG synthesis in eukaryotes using synthetic biology and other methods.

[0088] Plant GPAT9 shares structural similarities with mammalian GPAT3, and both are involved in the biosynthesis of polar membrane lipids and neutral triglycerides. Previous studies have shown that although AtGPAT9 and BnGPAT9 share more than 90% sequence similarity, their activities during heterologous expression in yeast differ significantly. This characteristic has helped us effectively identify candidate sites for regulating enzyme activity. Building on this, this study is the first to experimentally clarify the important regulatory role of six amino acid sites outside the conserved domain of acyltransferases in plant GPAT enzyme activity.

[0089] 3.1 The roles of W85 and H119 may be related to their influence on the membrane binding properties of GPAT9.

[0090] Both the N-terminus and C-terminus of AtGPAT9 are exposed to the cytoplasm, implying that the protein should have an even number of transmembrane domains. However, this contradicts bioinformatics predictions that AtGPAT9 contains three potential transmembrane domains. One possible explanation for this phenomenon is that several proline residues at the N-terminus may form a hinge-like structure, preventing hydrophobic domain I from crossing the membrane and instead allowing it to attach to the surface of the endoplasmic reticulum. Based on the fact that amino acid residues at positions 85 and 119 are located in the predicted first and second hydrophobic domains, respectively, it is speculated that replacing the hydrophobic tryptophan (W) at position 85 with a hydrophilic tyrosine (Y) or replacing the positively charged histidine (H) at position 119 with neutral asparagine (N) might alter the membrane assembly of GPAT9.

[33] This may be one reason why the enzyme activity of AtGPAT9 is lower than that of BnGPAT9.

[0091] 3.2 The activity of GPAT9 may be regulated by phosphorylation mechanisms.

[0092] Our results indicate that while individual mutations at the six N-terminal phosphorylation sites of AtGPAT9 (T10A, S11A, S13A, S28A, S30A, S31A) do not enhance AtGPAT9 activity during heterologous expression in yeast, the T114L substitution enhances BnGPAT9 enzyme activity, while the N237S substitution decreases it. When amino acid residues at positions 114 and 237 are non-phosphorylated amino acids, namely leucine (L) and asparagine (N), instead of the potential phosphorylation sites threonine (T) and serine (S), both AtGPAT9 and BnGPAT9 mutant enzymes maintain high activity. Therefore, it is reasonable to speculate that GPAT9 activity is regulated by phosphorylation, and increased phosphorylation at positions 114 and 237 is detrimental to maintaining acyltransferase activity. This hypothesis is supported to some extent by previous studies. Protein phosphorylation and non-phosphorylation are important ways to regulate enzyme activity. Studies have reported that mammalian mitochondrial GPAT (mtGPAT1) regulates its activity through phosphorylation of its C-terminal S632 and S639 residues. Phosphorylation of the C-terminal amino acid residues of yeast GPAT (Gat1p, Gat2p) can also downregulate enzyme activity.

[0093] 3.3 Interactions at multiple amino acid sites affect acyltransferase activity

[0094] When multiple amino acid residues are mutated simultaneously, their physical interactions can lead to epistatic effects within the protein molecule. Certain combinations of amino acid residue mutations can produce synergistic effects, i.e., positive epistatic effects; for example, any combination of mutations involving Y85W, N119H, and S237N can enhance AtGPAT9 activity. Figure 5 A). Conversely, other mutant combinations may create antagonistic effects, leading to negative upsigma effects, downregulating enzyme activity, or completely impairing protein function. For example, a certain antagonistic relationship between four amino acid residues in the AtGPAT9 (N119H / D230N / A235T / S237N) mutant enzyme prevents it from functioning in yeast ZAFU1. Figure 5 D). The positions of amino acid residues at positions 230, 235, and 237 are close together. Figure 2 It is located between arginine (R215) in conserved domain II of acyltransferase and glutamate (E245) in domain III; given that these two amino acid residues, R215 and E245, are crucial for substrate binding of GPAT.

[18] It is speculated that the complex interaction between amino acid residues at positions 230, 235, and 237 and other sites may affect enzyme activity by interfering with the three-dimensional structure of the glycerol 3-phosphate substrate binding region.

[0095] Interestingly, although both N237S and A322G single point mutations can reduce the activity of BnGPAT9 ( Figure 3 A), but neither single nor double substitution of S237N and G322A can enhance the activity of AtGPAT9, which indicates that the effects of amino acids at positions 237 and 322 are greatly influenced by the physicochemical properties of other amino acids. However, it should be noted that their mechanisms of action may differ. As mentioned earlier, the phosphorylation state of amino acid 237 may have a regulatory effect on enzyme activity, while the substitution of alanine (A) at position 322 with glycine (G) may affect the stability of the protein conformation. This is because the glycine side chain is small, with only one hydrogen atom, which is unfavorable for the stability of the α-helix structure. Consistent with this, three-dimensional structure prediction shows that A322 in BnGPAT9 is different from G322 in AtGPAT9 ( Figure 2 The former is spatially closer to amino acid residues 114, 119, 230, 235, and 237 (which may be related to the formation of the enzyme's active site), which may have a positive effect on GPAT9 activity. Given the important roles of amino acids 237 and 322, it is necessary to further investigate which substitutions at these two amino acid sites are beneficial for enhancing acyltransferase activity.

[0096] In summary, this paper reports for the first time six GPAT9 enzyme activity regulatory sites located outside the conserved regions of acyltransferases and their complex interaction effects, thus deepening our understanding of the intrinsic relationship between acyltransferase structure and function and providing a theoretical basis for the molecular modification and structural optimization of acyltransferases. Furthermore, the constructed GPAT9 variant gene can be used to explore the biosynthetic mechanism of TAG in plants, particularly the regulatory role of phosphorylation-nonphosphorylation mechanisms on GPAT enzyme activity. However, the regulatory mechanisms of the interaction effects of the aforementioned six key active sites remain unclear and require further investigation.

[0097] It should be noted that the above-described embodiments should be understood as illustrative, not as limiting the scope of protection of this invention. The scope of protection of this invention is defined by the claims. For those skilled in the art, some non-essential improvements and adjustments made to this invention without departing from the essence and scope of this invention still fall within the scope of protection of this invention.

Claims

A mutant enzyme of 1,3-phosphoglyceryl acyltransferase, characterized in that: it is Site-directed mutations of a single or multiple amino acid residues occur at key active sites of the wild-type 3-phosphoglyceryl acyltransferase AtGPAT9. The site-directed mutation of the single amino acid residue is as follows: The amino acid residue at position 119 of wild-type AtGPAT9, as shown in SEQ ID NO: 2, is changed from asparagine to histidine. The site-directed mutation of the plurality of amino acid residues can be any one of the following: (1) The amino acid residues at positions 85 and 119 of the wild-type AtGPAT9 in the amino acid sequence SEQ ID NO:2 were changed from tyrosine and asparagine to tryptophan and histidine, respectively. (2) The amino acid residues at positions 85, 114 and 119 of the wild-type AtGPAT9 amino acid sequence such as SEQ ID NO: 2 are changed from tyrosine, leucine and asparagine to tryptophan, threonine and histidine, respectively. (3) The amino acid residues at positions 85, 119 and 237 of the wild-type AtGPAT9 amino acid sequence such as SEQ ID NO: 2 are changed from tyrosine, asparagine and serine to tryptophan, histidine and asparagine, respectively. (4) The amino acid residues at positions 85, 114, 119 and 237 of the wild-type AtGPAT9 amino acid sequence such as SEQ ID NO: 2 are changed from tyrosine, leucine, asparagine and serine to tryptophan, threonine, histidine and asparagine, respectively. (5) The amino acid residues at positions 85, 119 and 230 of the wild-type AtGPAT9 amino acid sequence such as SEQ ID NO: 2 are changed from tyrosine, asparagine and aspartic acid to tryptophan, histidine and asparagine, respectively. (6) The amino acid residues at positions 85, 119 and 235 of the wild-type AtGPAT9 amino acid sequence such as SEQ ID NO: 2 are changed from tyrosine, asparagine and alanine to tryptophan, histidine and threonine, respectively. (7) The amino acid residues at positions 119 and 237 of the wild-type AtGPAT9 in the amino acid sequence SEQ ID NO: 2 are changed from asparagine and serine to histidine and asparagine, respectively; (8) The amino acid residues at positions 119, 230 and 237 of the wild-type AtGPAT9 amino acid sequence such as SEQ ID NO: 2 are changed from asparagine, aspartic acid and serine to histidine, asparagine and asparagine, respectively. (9) The amino acid residues at positions 119, 235 and 237 of the wild-type AtGPAT9 amino acid sequence such as SEQ ID NO:2 are changed from asparagine, alanine and serine to histidine, threonine and asparagine, respectively.

2. A gene encoding the mutant enzyme of the 3-phosphoglyceryl acyltransferase of claim 1.

3. The use of heterologous expression of the mutant enzyme (1) or (3) of the 3-phosphoglyceryl acyltransferase described in claim 1 in improving triacylglycerol synthesis in yeast cells lacking 3-phosphoglyceryl acyltransferase activity.

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