Variant Ester Synthases for Higher FAME Yield and Lower Byproducts
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Solution Overview
Problem
Existing ester synthases, such as those from Marinobacter hydrocarbonoclasticus, produce low yields of fatty esters and significant amounts of beta hydroxy esters, limiting the production of biofuels and chemicals like fatty acid methyl esters (FAME) in recombinant host cells.
Innovation Solution
Engineering variant ester synthase and thioesterase enzymes with specific mutations to enhance their activity, allowing them to produce higher yields of fatty esters, particularly FAME, while reducing beta hydroxy ester production, and enabling the use of short-chain alcohols without requiring additional enzyme co-expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild type ester synthase from Marinobacter hydrocarbonoclasticus is used, then the enzyme can produce fatty esters, but the yield is low and significant amounts of beta hydroxy esters are produced
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations at positions 4, 5, 7, 15, 24, 30, 33, 39, 40, 41, 43, 44, 58, 73, 76, 77, 78, 80, 98, 99, 101, 102, 111, 122, 131, 146, 147, 149, 150, 155, 157, 162, 164, 166, 170, 171, 172, 173, 177, 179, 182, 184, 185, 186, 187, 188, 190, 192, 193, 195, 197, 201, 202, 203, 206, 207, 212, 216, 219, 234, 239, 242, 243, 244, 246, 255, 257, 258, 259, 260, 262, 263, 264, 265, 266, 267, 285, 288, 289, 293, 294, 301, 302, 303, 304, 306, 307, 309, 310, 311, 313, 314, 315, 316, 317, 319, 320, 323, 328, 334, 348, 349, 351, 352, 353, 356, 357, 360, 366, 375, 381, 393, 394, 395, 409, 411, 413, 420, 424, 442, 443, 447, 454, 455, 457, 458, 461, 466, 468, and 472. These mutations change the enzyme's catalytic parameters to improve fatty ester yield and reduce beta hydroxy ester byproduct formation.
Solution Approach 2:
The patent applies local quality by making specific localized changes at particular amino acid positions within the ester synthase enzyme. Each mutation at a specific position (e.g., position 4, 5, 7, etc.) modifies the local properties of the enzyme's active site or regulatory regions, thereby improving catalytic efficiency for fatty ester production while reducing unwanted side reactions that produce beta hydroxy esters.
2Productivity
If additional enzyme co-expression is used to improve fatty ester production, then yield increases, but system complexity increases
Solution Approach 1:
The patent applies universality by designing a variant ester synthase that performs multiple functions within a single enzyme molecule. The mutated enzyme not only catalyzes the primary reaction to produce fatty esters with high efficiency but also inherently reduces beta hydroxy ester byproduct formation, eliminating the need for additional co-expressed enzymes to achieve optimal production.
Solution Approach 2:
The patent applies merging by combining the functions of multiple enzymes into a single variant ester synthase. Instead of requiring co-expression of multiple enzymes to achieve high fatty ester yield and low byproduct formation, the patent merges these functions into one genetically modified enzyme that accomplishes both objectives simultaneously, thereby simplifying the expression system.
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 variant enzymes increase the titer of fatty esters by at least 5% compared to wild-type enzymes, improving the efficiency and yield of fatty ester production in recombinant host cells, including the production of fatty acid methyl esters and other esters using a simplified one-gene-system.
Implementation Method 1
The disclosure provides variant ester synthase and thioesterase enzymes with improved ester synthase properties
Data Source
AI summary
The disclosure relates to enzyme variants with improved ester synthase properties for the production of fatty acid esters. Further contemplated are recombinant host cells that express such variants, cell cultures comprising the recombinant host cells and fatty acid ester compositions produced by such recombinant host cells.