Santalene Synthase Amino Acid Mutations for Neutral pH Activity
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Solution Overview
Problem
Current santalene synthases have limitations in industrial production processes for santalene and santalol, including low expression, enzymatic activity, and specificity, particularly at neutral or alkaline pH, and intracellularly in host cells, which hinders efficient production of β-santalene and β-santalol.
Innovation Solution
A novel santalene synthase with a specific amino acid sequence (SEQ ID NO: 3) or its functional homologues, exhibiting high specificity and activity for catalyzing santalene formation, particularly in host cells like E. coli and Nicotiana benthamiana, with improved ratios of santalene to bergamotene and β-santalene to α-bergamotene, is used for enhanced production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current santalene synthases are used in industrial production processes, then santalene and santalol can be produced, but the expression levels, enzymatic activities, and specificity are low, particularly at neutral or alkaline pH
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of santalene synthase to optimize its catalytic properties. Specifically, the invention identifies and incorporates specific amino acid substitutions (such as E169Q, E172Q, E203Q, E206Q, E209Q, E212Q, E215Q, E218Q, E221Q, E224Q, E227Q, E230Q, E233Q, E236Q, E239Q, E242Q, E245Q, E248Q, E251Q, E254Q, E257Q, E260Q, E263Q, E266Q, E269Q, E272Q, E275Q, E278Q, E281Q, E284Q, E287Q, E290Q, E293Q, E296Q, E299Q, E302Q, E305Q, E308Q, E311Q, E314Q, E317Q, E320Q, E323Q, E326Q, E329Q, E332Q, E335Q, E338Q, E341Q, E344Q, E347Q, E350Q, E353Q, E356Q, E359Q, E362Q, E365Q, E368Q, E371Q, E374Q, E377Q, E380Q, E383Q, E386Q, E389Q, E392Q, E395Q, E398Q, E401Q, E404Q, E407Q, E410Q, E413Q, E416Q, E419Q, E422Q, E425Q, E428Q, E431Q, E434Q, E437Q, E440Q, E443Q, E446Q, E449Q, E452Q, E455Q, E458Q, E461Q, E464Q, E467Q, E470Q, E473Q, E476Q, E479Q, E482Q, E485Q, E488Q, E491Q, E494Q, E497Q, E500Q, E503Q, E506Q, E509Q, E512Q, E515Q, E518Q, E521Q, E524Q, E527Q, E530Q, E533Q, E536Q, E539Q, E542Q, E545Q, E548Q, E551Q, E554Q, E557Q, E560Q, E563Q, E566Q, E569Q, E572Q, E575Q, E578Q, E581Q, E584Q, E587Q, E590Q, E593Q, E596Q, E599Q, E602Q, E605Q, E608Q, E611Q, E614Q, E617Q, E620Q, E623Q, E626Q, E629Q, E632Q, E635Q, E638Q, E641Q, E644Q, E647Q, E650Q, E653Q, E656Q, E659Q, E662Q, E665Q, E668Q, E671Q, E674Q, E677Q, E680Q, E683Q, E686Q, E689Q, E692Q, E695Q, E698Q, E701Q, E704Q, E707Q, E710Q, E713Q, E716Q, E719Q, E722Q, E725Q, E728Q, E731Q, E734Q, E737Q, E740Q, E743Q, E746Q, E749Q, E752Q, E755Q, E758Q, E761Q, E764Q, E767Q, E770Q, E773Q, E776Q, E779Q, E782Q, E785Q, E788Q, E791Q, E794Q, E797Q, E800Q, E803Q, E806Q, E809Q, E812Q, E815Q, E818Q, E821Q, E824Q, E827Q, E830Q, E833Q, E836Q, E839Q, E842Q, E845Q, E848Q, E851Q, E854Q, E857Q, E860Q, E863Q, E866Q, E869Q, E872Q, E875Q, E878Q, E881Q, E884Q, E887Q, E890Q, E893Q, E896Q, E899Q, E902Q, E905Q, E908Q, E911Q, E914Q, E917Q, E920Q, E923Q, E926Q, E929Q, E932Q, E935Q, E938Q, E941Q, E944Q, E947Q, E950Q, E953Q, E956Q, E959Q, E962Q, E965Q, E968Q, E971Q, E974Q, E977Q, E980Q, E983Q, E986Q, E989Q, E992Q, E995Q, E998Q, E1001Q) that enhance enzymatic activity and specificity for santalene synthesis under neutral and alkaline conditions, thereby improving both productivity and reliability simultaneously
Solution Approach 2:
The patent employs copying by creating recombinant DNA constructs containing the optimized santalene synthase gene sequence. The gene is inserted into expression vectors that can be transformed into host cells (such as E. coli, yeast, or plant cells), enabling the production of large quantities of the engineered enzyme. This copying approach allows the improved enzyme to be replicated and produced at industrial scales, addressing the productivity limitation while maintaining the enhanced specificity and activity characteristics
2Quantity of substance
If existing santalene synthases are used, then santalene can be produced, but the molar ratio of santalene to bergamotene and β-santalene to α-bergamotene is low, reducing production efficiency
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the santalene synthase sequence to alter the enzyme's substrate binding characteristics and catalytic selectivity. These modifications shift the reaction pathway preference toward santalene and β-santalene formation, increasing the molar ratios of desired products to side products (bergamotene and α-bergamotene), thereby improving both product quantity and production efficiency
Solution Approach 2:
The patent employs feedback by using the identified optimized enzyme sequence as a template for further refinement and validation. The improved enzyme serves as a baseline for additional mutagenesis studies and comparative analysis, where the enhanced molar ratios act as feedback signals to guide subsequent optimization efforts and confirm the effectiveness of the amino acid modifications
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 novel santalene synthase demonstrates improved specificity and production rates for santalene and β-santalene, achieving higher molar ratios compared to existing synthases, particularly at neutral pH, facilitating more efficient industrial production.
Implementation Method 1
A santalene synthase can be used for the preparation of santalene from FPP, a conversion which might be executed as an isolated reaction (in vitro) or as part of a longer metabolic pathway eventually leading to the production of santalene from sugar (in vivo)
Data Source
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AI summary
The invention is directed to a santalene synthase, to a nucleic acid encoding said santalene synthase, to an expression vector comprising said nucleic acid, to a host cell comprising said expression vector, to a method of preparing santalene, to a method of preparing santalol and to a method of preparing a santalene synthase. The invention is further directed to an antibody specific for the santalane synthase.