Engineered Yeast HMO Production to Limit Unwanted Byproducts
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Solution Overview
Problem
The challenge in the production of human milk oligosaccharides (HMOs) lies in the generation of unwanted byproducts during the heterologous production of compounds like 2′-fucosyllactose (2′-FL) in yeast, which hinders efficient and high-purity synthesis.
Innovation Solution
Genetically modified host cells, such as yeast cells, are engineered to express specific enzymes like fucosyltransferase, GDP-mannose dehydratase, and lactose permease, which enhance the biosynthetic pathway for HMOs, ensuring high selectivity and catalytic efficiency, thereby producing HMOs with high purity and yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If heterologous production of 2'-FL is performed in yeast using four non-native enzymes, then HMO production capability is achieved, but unwanted byproducts are generated
Solution Approach 1:
The patent removes the problematic GDP-fucose transporter enzyme from the biosynthetic pathway, keeping only the essential four enzymes (fucosyltransferase, lactose permease, GDP-mannose 4,6-dehydratase, and fucose synthase). This extraction eliminates the source of unwanted byproducts while preserving HMO production capability.
Solution Approach 2:
The patent optimizes the expression levels and activity of the four selected enzymes to achieve high catalytic efficiency and selectivity. By carefully controlling enzyme parameters, the pathway produces desired HMOs with high purity (20-70% w/w yield) while minimizing byproduct formation.
2Productivity
If traditional HMO production methods are used, then production capability is achieved, but purity is reduced due to unwanted byproducts
Solution Approach 1:
The patent converts the potential harm of byproduct formation into a benefit by carefully selecting and optimizing a minimal enzyme set that channels metabolic flux exclusively toward desired HMO products. The streamlined pathway acts as a beneficial constraint that enhances purity while maintaining productivity.
Solution Approach 2:
The patent applies local quality optimization by ensuring each of the four enzymes operates with high selectivity and catalytic efficiency at its specific position in the pathway. This localized optimization of enzyme performance ensures high overall pathway purity.
3Adaptability or versatility
If multiple non-native enzymes are introduced for HMO synthesis, then biosynthetic capability is achieved, but process complexity increases
Solution Approach 1:
The patent segments the HMO biosynthetic pathway into four distinct, well-defined enzymatic steps, each catalyzed by a specific non-native enzyme. This segmentation provides clarity and control, reducing process complexity while maintaining biosynthetic capability.
Solution Approach 2:
The patent uses a host yeast cell that provides universal metabolic support functions (substrate provision, energy generation, cellular machinery) for the four specialized non-native enzymes. This multi-functionality reduces overall system complexity by leveraging the host's existing capabilities.
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 engineered host cells achieve high-purity and high-titer production of HMOs, such as 2′-FL, with yields ranging from 20% to 70% (w/w) and productivities up to 5 g/L/hr, addressing the issue of unwanted byproducts in traditional methods.
Implementation Method 1
fucosyltransferase having an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOS: 1-41
Implementation Method 2
GMD having an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOS: 42-64
Implementation Method 3
fucose synthase having an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOS: 100-103
Implementation Method 4
lactose permease having an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOS: 65-99
Data Source
AI summary
Provided herein are host cells capable of producing a human milk oligosaccharide (HMO), such as yeast cells that include one or more heterologous nucleic acids encoding one or more enzymes of the HMO biosynthetic pathway, such as a fucosyltransferase, GDP-mannose dehydratase, lactose permease, and/or fucose synthase. Also provided are fermentation compositions including the disclosed host cells, as well as related methods of producing and recovering HMOs generated by the host cells.