Modified UGT Polypeptides for Steviol Glycoside Fermentation
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Solution Overview
Problem
Current methods for producing steviol glycosides from Stevia rebaudiana are limited by variable yields, high production costs, and the need for substantial land and labor, with existing microorganisms used in fermentative processes not efficiently producing high amounts of desired steviol glycosides or achieving desired ratios.
Innovation Solution
Identification and use of new UDP-glycosyltransferase (UGT) polypeptides with modified amino acid sequences, specifically with substitutions at positions 35, 189, 280, 284, 285, 334, or 373, to enhance the production of steviol glycosides in recombinant cells, such as Yarrowia lipolytica, leading to higher yields and reduced production of undesirable by-products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional extraction from Stevia plant is used, then steviol glycosides can be obtained, but yields are variable and affected by agriculture and environmental conditions
Solution Approach 1:
The patent replaces the mechanical/agricultural extraction system with a biochemical fermentation system using recombinant microorganisms. The microorganisms express plant enzymes (UGT1, UGT2, UGT3) that catalyze the conversion of steviol to steviol glycosides through controlled fermentation, eliminating dependence on agricultural variables and environmental conditions while providing consistent, scalable production.
2Productivity
If Stevia cultivation is expanded to increase production, then more steviol glycosides can be obtained, but substantial land area and intensive labour are required
Solution Approach 1:
The patent replaces extensive agricultural cultivation with controlled microbial fermentation. Recombinant microorganisms are cultured in bioreactors using simple carbon sources, enabling high-volume production of steviol glycosides without requiring additional land area or intensive agricultural labor, thus decoupling production volume from land use.
3Quantity of substance
If existing microorganisms are used for fermentative production, then some steviol glycosides can be produced, but high amounts of desired steviol glycosides or desired ratios are not achieved
Solution Approach 1:
The patent divides the glycosylation pathway into distinct enzymatic steps, each catalyzed by a specific UGT enzyme (UGT1 for C-13 glucosylation, UGT2 for C-19 glucosylation, UGT3 for further glucosylation). By independently expressing and optimizing these segmented enzymatic functions in recombinant microorganisms, precise control over the production of specific steviol glycosides and their ratios is achieved.
Solution Approach 2:
The patent optimizes multiple parameters including the selection of specific UGT enzyme variants with enhanced activity, cultivation conditions, carbon source composition, and aeration rates. These parameter changes enable the microorganisms to produce high amounts of desired steviol glycosides with controlled ratios, overcoming the limitations of existing microorganisms.
4Productivity
If fermentation process is used to avoid land and labor requirements, then production can be scaled, but yields of desired steviol glycosides are still limited
Solution Approach 1:
The patent employs systematic optimization of fermentation parameters including aeration rates (e.g., 0.5-2.0 v/v/min), carbon source concentrations, pH levels, and temperature. These parameter changes enhance the metabolic activity of recombinant microorganisms, significantly increasing the yield of steviol glycosides while maintaining the scalability advantages of fermentation processes.
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 modified UGT polypeptides increase the production of steviol glycosides in recombinant cells, making recovery easier and achieving higher yields with improved substrate specificity and stability, thus overcoming the limitations of traditional methods.
Implementation Method 1
UDP-glycosyltransferases (UGTs) are enzymes that catalyse the transfer of a glycosyl group from an activated nucleotide sugar to an acceptor molecule
Data Source
AI summary
The present disclosure relates a polypeptide having UGT activity, which polypeptide comprises an amino acid sequence which, when aligned with a polypeptide having UGT activity comprising the sequence set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid corresponding to any of amino acids at positions35, 189, 280, 284, 285, 334 or 373,said positions being defined with reference to SEQ ID NO: 2 and wherein the polypeptide has one or more modified properties as compared with a reference polypeptide having UGT activity. A polypeptide according to the disclosure may be used in a recombinant cell for the production of steviol or a steviol glycoside.