A View to a Cell
Breast milk is the best nutrition for new-born infants. In cases where breast milk is not or insufficiently available, FrieslandCampina Domo wants to offer the highest quality infant formula fulfilling the nutritional needs of the infant. Oligosaccharides are a vitally important component of human milk, that are hardly present in cow’s milk. Therefore, infant formula based on cow’s milk is currently lacking human milk oligosaccharides. FrieslandCampina Domo is dedicated to developing human milk oligosaccharides for application in infant formula. Currently, over 200 human milk oligosaccharides (HMOs) have been identified in human milk. 2′-Fucosyllactose (2’-FL) is the most abundant oligosaccharide in human milk, making up 20 to 30% of all oligosaccharides in human milk. With Aequival® 2′-FL, FrieslandCampina Domo offers the first human milk oligosaccharide (not derived from human milk) for application in next generation infant formula. 2’-FL is produced by fermentation, using E. coli as microbial cell factory.
The goal of this project was to gain more insight in the mechanisms of the E. coli fermentation process yielding 2’-FL and to identify and validate potential improvement routes that lead to higher 2’-FL yields. Several work packages and tasks were performed with the following objectives
• Identify the metabolic bottlenecks that prevent more substrate going into the 2’-FL production pathway.
• Improving productivity and yield of 2’-FL
• Determine the stability of expression of the 2’-FL pathway genes and – if necessary – improve it.
• Identify how acetate is produced and suggest genetic interventions to reduce acetate production
The efficient production of 2’-FL contributes to the aim of MMIP D2 to produce a healthy and sustainable food supply
This project entails making a product (Aequival®) and its optimized production process available to supply a healthy food component.
WP1. Metabolic model
Task 1.1. Conceptual metabolic model
A conceptual model linking substrate consumption to product formation by redox cofactors and energy generation.
Task 1.2. Genome-scale metabolic model (GEM)
A genome-scale metabolic model of E. coli with the reactions necessary for 2’-FL production.
Task 1.3. Identification of bottlenecks
Bottlenecks preventing higher 2’-FL production in the current production strain
Task 1.4. Model verification
A verified model using existing and new fermentation data from WP2 to WP4.
Task 1.5. Genetic modifications
The selected genetic modifications from task 1.3., realized by Glycosyn.
Task 1.6. Strain evaluation
The genetically modified strain evaluated by FrieslandCampina.
WP2. Physiological studies
Task 2.1. Medium optimization
A medium composition resulting in improved 2’-FL-production.
Task 2.2. Transcriptome analysis
Genes identified to be involved in the production of 2’-FL
Task 2.3. Limited metabolomics
Knowledge about availability of selected intermediates of 2’FL-production
Task 2.4. Genetic modifications
Proposed genetic modifications, to be realized by Glycosyn.
Task 2.5. Alternative cultivation protocols
Alternative cultivation protocols to improve 2’-FL production. To be assessed by Friesland Campina.
WP3. Stability of expression
Task 3.1. Determining the 2’-FL production capacity during fermentation process
The capacity of the production strain to produce 2’-FL during a fed-batch fermentation.
Task 3.2 and 3.3. Stability of expression of the 2’-FL pathway
Stability of expression of the genes of the 2’-FL pathway during the fermentation
Task 3.4. Options for genetic modifications
Options for genetic modifications to improve expression of the genes involved in the 2’-FL pathway.
Task 3.5. Strain evaluation
Effect of genetic modifications (realized by Glycosyn) on 2’-FL production is evaluated (by FrieslandCampina).
WP4. Acetate formation
Knowledge about acetate production by E. coli and a proposal for genetic engineering of the 2’-FL production strain to reduce acetate production.