DGAT1 Enzyme Expression in Recombinant Yeast for Triacylglycerol Synthesis
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Solution Overview
Problem
Current methods for producing polyunsaturated fatty acids (PUFAs) from microorganisms face challenges due to the limited ability of certain diacylglycerol acyltransferases (DGATs) to promote lipid accumulation, particularly DGAT1 from Mortierella alpina, which has not been extensively studied, hindering the large-scale production of PUFAs.
Innovation Solution
A recombinant Saccharomyces cerevisiae expressing the diacylglycerol acyltransferase 1 (DGAT1) from Mortierella alpina, using the pYES2-Madgat1b vector, is developed to enhance triacylglycerol synthesis and PUFAs production by increasing the enzyme's activity and fatty acid content.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DGAT1 from Mortierella alpina is used to promote lipid accumulation, then triacylglycerol synthesis is enhanced, but the enzyme's ability to promote lipid accumulation is limited
Solution Approach 1:
The patent applies parameter changes by modifying the expression system and culture conditions of DGAT1 from Mortierella alpina in Saccharomyces cerevisiae. By optimizing the host organism, expression vector, and cultivation parameters, the enzyme's activity and lipid accumulation capability are significantly enhanced, transforming a limited enzyme into a highly effective lipid production system.
2Quantity of substance
If polyunsaturated fatty acids are obtained from aquatic phytoplankton and deep-sea fish, then PUFAs are produced, but the growth cycle is long and cultivation cost is high
Solution Approach 1:
The patent uses copying by expressing the DGAT1 gene from Mortierella alpina in the model organism Saccharomyces cerevisiae. This creates a recombinant yeast system that copies the lipid synthesis capability of the original organism, enabling rapid production of PUFAs without the long growth cycles associated with aquatic phytoplankton and deep-sea fish.
Solution Approach 2:
The invention changes the production system from natural aquatic sources to a recombinant microbial system. This parameter change transforms the production methodology, achieving the same PUFAs output with dramatically reduced growth cycles and lower cultivation costs through controlled fermentation processes.
3Quantity of substance
If polyunsaturated fatty acids are obtained from aquatic phytoplankton and deep-sea fish, then PUFAs are produced, but the cultivation cost is high
Solution Approach 1:
The patent creates a copy of the DGAT1 enzyme function in a cost-effective host organism (Saccharomyces cerevisiae). This recombinant system replaces expensive aquatic source cultivation with affordable microbial fermentation, significantly reducing production costs while maintaining high PUFAs yield.
Solution Approach 2:
The invention employs a cheap and rapidly growing microbial system (recombinant yeast) instead of expensive long-lived aquatic organisms. The yeast can be cultured in inexpensive media and harvested quickly, embodying the principle of using economical, short-cycle production systems to replace costly, time-consuming traditional methods.
4Productivity
If DGAT1 from Mortierella alpina is expressed in Saccharomyces cerevisiae, then enzyme activity is increased, but the expression system complexity increases
Solution Approach 1:
The patent uses a well-established expression system (pYES2 vector in Saccharomyces cerevisiae) to copy and express the DGAT1 gene. This approach leverages the simplicity and reliability of the yeast expression system while achieving high enzyme activity, avoiding the need for complex synthetic biology approaches.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The recombinant Saccharomyces cerevisiae shows a 1.94-fold increase in total fatty acid content and a 12.09-fold increase in triacylglycerol content, with significant increases in monounsaturated fatty acids, effectively improving PUFAs production and paving the way for large-scale industrial production.
Implementation Method 1
Acyl coenzyme A:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) is the key rate-limiting enzyme in the Kennedy pathway that can catalyze the process of adding a fatty acid acyl to the position sn-3 of diacylglycerol
Implementation Method 2
The recombinant Saccharomyces cerevisiae shows a 1.94-fold increase in total fatty acid content and a 12.09-fold increase in triacylglycerol content
Data Source
AI summary
Disclosed is a diacylglycerol acyltransferase 1, a recombinant Saccharomyces cerevisiae containing the diacylglycerol acyltransferase 1, and application thereof in production of triacylglycerol. The diacylglycerol acyltransferase 1 of the invention has a function of catalyzing synthesis of triacylglycerol. After the recombinant Saccharomyces cerevisiae containing the diacylglycerol acyltransferase 1 of the invention is subjected to induction culture for 48 h, the content of total fatty acid and triacylglycerol in the recombinant Saccharomyces cerevisiae containing the diacylglycerol acyltransferase 1 can be respectively increased by 1.94 folds and 12.09 folds as compared with those of Saccharomyces cerevisiae without the recombinant diacylglycerol acyltransferase 1. The instant invention provides a method for improving the ability of microorganisms to produce polyunsaturated fatty acids (PUFAs) by means of genetic engineering.