Variant YOR1 Transporter Engineering for Higher HMO Export in Yeast
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for producing human milk oligosaccharides (HMOs) in yeast strains are limited by low yield and purity, as conventional ABC transporters like Sc.YOR1 are inefficient in exporting these compounds across cell membranes.
Innovation Solution
Genetically modify yeast cells to express variant ABC transporter polypeptides, such as YOR1_A446L, YOR1_T220Q, and others, which enhance the export of HMOs, thereby increasing yield and purity compared to strains expressing the parental Sc.YOR1 polypeptide.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional ABC transporter Sc.YOR1 is used in yeast strains, then the yeast can produce HMOs, but the yield and purity of HMOs are limited due to inefficient export across cell membranes
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at defined positions in the YOR1 protein sequence (e.g., positions 64, 1127, 1175, 220, 226, 256, 373, 446, 795, 1014, 1167). These sequence parameter modifications create variant YOR1 proteins with enhanced HMO export functionality, directly resolving the contradiction between yield and purity by improving the transporter's efficiency and specificity.
Solution Approach 2:
The patent uses copying by creating multiple variant versions of the YOR1 protein (YOR1-64H, YOR1-1127A, YOR1-1175Y, etc.) that replicate the parental Sc.YOR1 structure but with modified amino acid sequences. These copies inherit the basic transporter function while incorporating improved export characteristics through the introduced substitutions, enabling simultaneous improvement of yield and purity.
2Productivity
If the parental Sc.YOR1 polypeptide is expressed in yeast, then the yeast strain is stable and easy to construct, but the HMO export efficiency is low resulting in limited production
Solution Approach 1:
The patent applies local quality by making targeted modifications at specific locations within the YOR1 protein sequence rather than completely redesigning the transporter. Each variant introduces substitutions at particular positions (e.g., position 64, 1127, 1175) to locally improve export efficiency while maintaining the overall protein structure and yeast strain stability, thus improving productivity without excessive complexity.
Solution Approach 2:
The patent segments the improvement strategy into multiple independent YOR1 variants, each with specific amino acid substitutions at different positions. This segmentation allows systematic evaluation of individual mutation effects and enables selection of the optimal variant combination, managing the complexity of protein engineering through modular approach.
3Productivity
If variant YOR1 polypeptides with multiple amino acid substitutions are introduced, then HMO export efficiency and production yield increase, but the genetic modification complexity increases
Solution Approach 1:
The patent applies partial action by introducing a specific number of amino acid substitutions (e.g., 1-6 substitutions per variant) rather than complete redesign. Each variant represents a partial modification that provides sufficient improvement in HMO export efficiency while maintaining manageable genetic modification complexity. The patent identifies optimal substitution counts that balance productivity enhancement with construction feasibility.
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 yeast strains exhibit enhanced production and improved purity of HMOs, with yields potentially increasing by at least 5% to over 100% and maintaining high HMO: lactose ratios.
Implementation Method 1
particular variants of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter polypeptide YOR1 exhibit the ability to export human milk oligosaccharides (HMOs) across cell membranes more effectively
Data Source
AI summary
Provided herein are genetically modified yeast cells capable of producing one or more human milk oligosaccharides (HMOs) and methods of making such cells. The yeast cells are engineered to comprise a heterologous nucleic acid encoding a variant ABC transporter protein YOR1 from Saccharomyces cerevisiae with improved 2′-fucosyllactose export activity and one or more heterologous nucleic acids that encode enzymes of a HMO biosynthetic pathway. Also provided are fermentation compositions including the disclosed genetically modified yeast cells, and related methods of producing and recovering HMOs generated by the yeast cells.