Linoleate 13-Hydratase Engineering for 13-HOD Productivity
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Solution Overview
Problem
Current methods for producing 13-hydroxy-9(Z)-octadecenoic acid and δ-decalactone have limitations in productivity, with existing enzymes like linoleate 13-hydratase from Lactobacillus acidophilus exhibiting suboptimal activity.
Innovation Solution
A method involving transformed microorganisms that produce proteins with specific amino acid sequences or modifications, such as those from Lactobacillus gallinarum, Lactobacillus gasseri, and Streptococcus mutans, which have enhanced linoleate 13-hydratase activity, is used to produce 13-hydroxy-9(Z)-octadecenoic acid, followed by conversion to δ-decalactone using microorganisms with reduced aldehyde oxidase activity like Yarrowia lipolytica.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If linoleate 13-hydratase from Lactobacillus acidophilus is used to produce 13-HOD, then the production method is established, but the productivity is limited due to suboptimal enzyme activity
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the linoleate 13-hydratase enzyme. Specifically, it introduces substitutions at positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 to enhance enzyme activity and productivity while maintaining the catalytic function for converting linoleic acid to 13-HOD
Solution Approach 2:
The patent creates composite enzyme structures by combining multiple amino acid sequence variants and modifications. It uses chimeric proteins composed of fragments from different sources (e.g., L. acidophilus, L. plantarum, L. casei) to generate enzymes with superior activity, stability, and productivity compared to the native enzyme
2Productivity
If traditional biochemical conversion methods are used, then the process is simple, but the overall efficiency of production is low
Solution Approach 1:
The patent merges multiple functions into a single transformed microorganism system. It combines the enhanced linoleate 13-hydratase enzyme production, linoleic acid conversion capability, and 13-HOD synthesis function within engineered bacterial hosts (e.g., E. coli, Lactococcus lactis), creating an integrated production platform that enhances overall efficiency while managing system complexity
Solution Approach 2:
The transformed microorganisms are engineered to autonomously perform the biochemical conversion process. The engineered bacteria self-regulate enzyme production, substrate uptake, and product synthesis through integrated metabolic pathways, reducing the need for external intervention and manual processing steps
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
This approach significantly enhances the productivity of 13-hydroxy-9(Z)-octadecenoic acid and δ-decalactone production, surpassing the activity of traditional enzymes and improving the overall efficiency of the biochemical conversion process.
Implementation Method 1
a protein having a linoleate 13-hydratase activity
Implementation Method 2
microorganisms with reduced aldehyde oxidase activity like Yarrowia lipolytica
Data Source
AI summary
The present invention provides a method of producing 13-hydroxy-9(Z)-octadecenoic acid, productivity of which has been enhanced. Specifically, the present invention provides a method of producing 13-hydroxy-9(Z)-octadecenoic acid, by producing 13-hydroxy-9(Z)-octadecenoic acid from linoleic acid in the presence of a transformed microorganism that produces a protein such as the following: (A) a protein having an amino acid sequence of SEQ ID NOs: 4, 5, 8 to 10, 13, or 14; (B) a protein having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence of SEQ ID NOs: 4, 5, 8 to 10, 13, or 14, and having a linoleate 13-hydratase activity; and (C) a protein having an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NOs: 4, 5, 8 to 10, 13 or 14, and having a linoleate 13-hydratase activity.