Vitamin D3 (Cholecalciferol) Bioproduction Engineering Service

Vitamin D3 (Cholecalciferol) is an essential fat-soluble vitamin crucial for calcium homeostasis and bone health, widely used in pharmaceuticals and food fortification. Conventional production methods include extraction from lanolin (sheep wool grease), which yields low purity in lanolin extraction and is dependent on animal sources. Chemical synthesis involves complex, energy-intensive processes, especially the need for a chemical synthesis requires light steps (UV irradiation) to convert 7-dehydrocholesterol (7-DHC) to Vitamin D3, which is difficult to scale and control. Biosynthesis in engineered yeast offers a sustainable, high-purity alternative.

CD Biosynsis offers a synthetic biology service focused on highly efficient Vitamin D3 precursor production in Saccharomyces cerevisiae (baker's yeast). Our core strategy involves modification of yeast ergosterol synthesis pathway . We engineer the upstream mevalonate (MVA) pathway to maximize the flow of carbon flux, and critically, we delete key downstream genes (like Erg6 or Erg5) to block the conversion of 7-dehydrocholesterol (7-DHC) to ergosterol . This forces the accumulation of the direct Vitamin D3 precursor, 7-DHC. Crucially, this is coupled with the optimization of 7-dehydrocholesterol reductase (ERG4). We knock down or eliminate the native Erg4 reductase activity to ensure 7-DHC is not consumed by the cell's endogenous pathways, guaranteeing maximum yield of the final precursor. This high-purity 7-DHC can then be easily extracted and irradiated to yield Vitamin D3, or the full pathway can be integrated. This integrated approach aims to deliver a high-yield, pure, and non-animal-sourced Vitamin D3 precursor, simplifying the final conversion.

Pain Points

Developing a superior Vitamin D3 production route faces these key limitations:

• Low Purity in Lanolin Extraction: Lanolin (the primary source) contains various sterols and impurities, leading to low purity and high refinement costs for the extracted 7-DHC precursor. It also relies on a non-sustainable animal source.
• Chemical Synthesis Requires Light Steps: The conversion of 7-DHC to Vitamin D3 requires UV irradiation , which is a complex photochemical process to scale up, requiring specialized equipment and strict control to prevent the formation of toxic byproducts.
• Metabolic Bottlenecks: The upstream mevalonate (MVA) pathway in yeast is tightly regulated, leading to insufficient supply of sterol precursors needed for high 7-DHC production.
• Precursor Consumption: In wild-type yeast, the synthesized 7-DHC is immediately converted into the final membrane sterol, Ergosterol , by the Erg4 reductase, preventing accumulation.

A successful solution must reroute the yeast metabolic pathway to accumulate the key precursor, 7-DHC, at high concentration and purity.

Solutions

CD Biosynsis utilizes advanced metabolic engineering to optimize 7-DHC precursor production in S. cerevisiae:

Modification of Yeast Ergosterol Synthesis Pathway

           

We delete or attenuate key downstream enzymes (e.g., Erg4, Erg6) to halt the conversion of 7-DHC to the final product, forcing the precursor to accumulate.

Optimization of 7-dehydrocholesterol reductase

We use CRISPR/Cas9 to eliminate the native Erg4 (7-DHC reductase) activity to ensure minimal loss of 7-DHC precursor to ergosterol synthesis, maximizing purity.

MVA Pathway Flux Enhancement

We overexpress key rate-limiting enzymes (e.g., HMG-CoA reductase) in the mevalonate pathway to significantly increase the upstream supply of the base sterol precursors.

Host Tolerance and Stability Engineering

We modify cell membrane composition or introduce protective systems to ensure the highly modified yeast strain remains robust and stable under high-yield fermentation conditions.

This systematic approach is focused on rebuilding the yeast sterol pathway to efficiently accumulate the high-value 7-DHC precursor.

Advantages

Our Vitamin D3 precursor engineering service is dedicated to pursuing the following production goals:

High 7-DHC Purity and Titer

Targeted pathway modification eliminates side products, leading to ultra-high purity precursor ready for final conversion.

Sustainable, Non-Animal Source

Production is vegan/vegetarian-friendly , utilizing renewable sugar feedstock instead of animal-derived lanolin.

Simplified Purification

The high purity of the synthesized 7-DHC significantly reduces downstream separation complexity and cost . [Image of Cost Reduction Icon]

Bioproduction Flexibility

The yeast host is suitable for large-scale fermentation , ensuring flexibility in production capacity.

Consistent Batch Quality

Genetic control ensures stable yield and reproducible sterol profile , a critical requirement for pharmaceutical intermediates.

We provide a specialized platform aimed at maximizing the yield and minimizing the cost of the Vitamin D3 precursor, 7-DHC.

Process

Our Vitamin D3 strain engineering service follows a rigorous, multi-stage research workflow:

• Ergosterol Pathway Downregulation: Use CRISPR technology to knockout the Erg4 gene (7-DHC reductase) and/or other sterol pathway enzymes that compete for 7-DHC.
• MVA Pathway Optimization: Overexpress the key upstream genes (e.g., HMG-CoA reductase, tHMG1) to boost metabolic flow to the sterol synthesis pathway.
• Host Optimization for Tolerance: Modify membrane sterol composition or introduce heterologous proteins to increase host tolerance to the accumulated 7-DHC.
• Fermentation Performance Validation: Test the final engineered strain in fed-batch fermentation to assess 7-DHC titer, yield, and purity relative to other sterols.
• Final Conversion Test: Conduct a small-scale UV irradiation step on the purified 7-DHC to confirm efficient conversion to Vitamin D3 (Cholecalciferol).
• Result Report Output: Compile a detailed Experimental Report including gene modification data, metabolic flux analysis, and fermentation metrics (titer and purity) , supporting IND/commercial filing.

Technical communication is maintained throughout the process, focusing on timely feedback regarding yield and sterol purity.

Explore the potential for a high-quality, bio-based Vitamin D3 supply. CD Biosynsis provides customized strain engineering solutions:

• Detailed Sterol Profile Analysis Report , demonstrating the successful accumulation and purity of 7-DHC via GC-MS or HPLC analysis.
• Consultation on optimized extraction methods for efficient 7-DHC recovery from the yeast biomass.
• Experimental reports include complete raw data on carbon yield (g 7-DHC/g sugar) and strain stability over multiple generations.