Precision fermentation


Precision fermentation is a production process in which specific biological molecules are manufactured using microorganisms. It can be used to produce food ingredients that are conventionally sourced from animals and plants, including proteins, lipids, carbohydrates, and other metabolites. Bacteria, yeast, or other cell types produce large quantities of a specific compound which is then extracted and purified from the fermentation broth or cell lysate. For example, milk or egg proteins, dairy fats, functional oligosaccharides, flavour and colour compounds or vitamins.
Precision fermentation is different from other forms of fermentation used in the food industry because it produces a single target molecule with high precision and purity. In other approaches like traditional fermentation or biomass fermentation, the product contains a mix of fermentation outputs, biomass, and substrates. This precision is achieved by optimising both the culture conditions and the microbial strains used in the process. While it is possible to use organisms that naturally produce useful ingredients, in some cases engineered strains are developed using adaptative laboratory evolution, mutagenesis, or by introducing of specific gene sequences. The purification and extraction process ensures that genetically engineered organisms are not present in any final products.
Although the term "precision fermentation" is relatively new, the underlying technologies have been used since the 1980s. Proteins like insulin for diabetes treatment or chymosin for cheese manufacturing have been produced by these techniques for decades and are well integrated in the market. Precision fermentation incorporates genetic tools, synthetic biology approaches and strain engineering techniques, and it has a promising role in future biobased food production systems. It is expected to become an essential technology in a global shift towards more sustainable food systems, in the context of climate change and in areas where the availability of agricultural land is limited.

Principles of precision fermentation

A fermentation bioprocess encompasses all the steps necessary to transform a raw feedstock into the desired molecule of interest, including the choice of the microbial strain that will be performing the transformation and the conditions in which it will be growing.

Feedstocks

Feedstocks generally refer to the predominant raw materials used as a source of carbon, nitrogen, and energy for microorganisms to grow and produce end products. The choice of feedstocks for precision fermentation is critical, as it significantly impacts the cost and sustainability of the products.
  • First generation sugars. Currently, the majority of precision fermentation processes are conducted using refined glucose derived from food crops. These sugars support robust microbial growth and are safe for use in food production; however, they have drawbacks such as higher costs and competition with the food supply.
  • Second generation sugars. These are fermentable sugars obtained from non-food, lignocellulosic biomass such as agricultural residues. This reduces competition with food production and improves the sustainability of fermentation-based products.
  • C1 feedstocks. Carbon dioxide, methane, formate, and methanol are considered highly sustainable for microbial growth and product synthesis, as they help minimise environmental impact. Although significant achievements have been made in this field, major challenges still remain for the effective utilisation of C1 feedstocks in mainstream manufacturing processes.
  • Food industry sidestreams. Food manufacturing wastewaters or byproducts from food processing can be used as inexpensive feedstocks in a circular bioeconomy approach and can be converted into fermentable sugars to serve as substrates for precision fermentation.

    Cell factories

A cell factory is a biological system that transform substrates into high-value biological products, such as proteins, enzymes, vitamins, pharmaceuticals and biomaterials under controlled conditions. Microbial strains commonly used include Bacillus subtilis, Corynebacterium glutamicum, Escherichia coli, Komagataella phaffii, Saccharomyces cerevisiae, and Yarrowia lipolytica. Each of these hosts possesses unique genetic and metabolic characteristics that make them suitable for defined production purposes in precision fermentation. The selection of a microbial host is based on various factors, including the presence of native biosynthetic pathways for the desired product, the capacity for efficient expression of heterologous pathways, the safety profile of the organism, and its compatibility with different cultivation conditions.
  • B. subtilis is a widely studied Gram-positive bacterium, known as a plant growth-promoting rhizobacterium. It can adapt to harsh growth conditions, make use of low-cost substrates, grow rapidly, and possesses good genetic stability along with advantages in expression systems. B. subtilis is considered safe and can be used to produce a variety of biomolecules.
  • C. glutamicum is a fast-growing, facultative aerobic, Gram-positive bacterium. It can utilize a variety of sugars, organic acids, and alcohols as sole or mixed carbon sources. Known for its excellent amino acid production capabilities, C. glutamicum is also used in the industrial production of other high-value compounds, including organic acids and terpenoids.
  • E. coli is a rod-shaped Gram-negative bacterium. A widespread and non-pathogenic strain of this bacterium a commonly used host strain to produce various valuable products. This expression system has the advantages of convenient genetic manipulation, simple transformation process, low cost, and efficient production.
  • K. phaffii is a non-conventional yeast with advantages in natural product biosynthesis. It is easier to genetically modify and is well-suited for protein expression.
  • S. cerevisiae exhibits rapid growth kinetics, high product yields, and good tolerance to environmental stresses such as low pH and oxygen limitation. Its GRAS status and the availability of advanced genetic tools make it valuable for industrial applications.
  • Y. lipolytica is a non-conventional, oleaginous yeast species. It can metabolise a wide range of substrates and shows strong environmental adaptability. It is regarded as a promising host for the production of proteins, lipids, flavour compounds, and pigments.

    Metabolic engineering

Microbial strains often undergo a process of engineering and optimisation to improve the production of a target molecule. Metabolic engineering encompasses a range of strategies to study and optimise the biochemical reactions happening in the cell to increase the synthesis rate of a specific molecule.
Typically, the target molecule will be a lipid, carbohydrate or other metabolites that is synthesis inside the cell following a succession of enzymatic reactions. These sequentially transform the molecules obtained from the feedstock into the product of interest through a series of intermediate compounds. This may involve reactions and intermediates that are natively part of the host's metabolism, or heterologous enzymes from other organisms. Metabolic engineering designs modifications both in the native metabolic network and in heterologous reactions that maximise product synthesis. For example, in order to increase metabolic flux towards the desired product and reduce the formation of undesired by-products, key enzymes in the host's metabolism can be up-regulated, down-regulated or knocked out. Synthetic biology provides tools to fine-tune expression levels, such as libraries of promoters of different strengths. Computational and mathematical methods are used to model the metabolic network across the cell and predict the effect of these modifications.
In some cases, the target molecule is a protein itself. In that case, genetic strategies are often used to ensure it is expressed at high levels, which may involve the use of strong promoters, or timing the expression with an inducible promoter. Strategies may also be used to force this protein to be secreted outside the cell, facilitating its recovery.

Process optimisation

Process optimisation in precision fermentation aims to improve productivity, sustainability, and efficiency of producing target compounds. This targets various aspects of the fermentation process, such as the culture medium, feeding strategy, bioreactor, and the fermentation conditions.
The composition of the culture medium plays a critical role in process optimisation. Culture media typically consist of a carbon source, a nitrogen source, minerals, and water. Depending on the characteristics of the cell factory, additional nutrients such as growth factors and vitamins may be required to support optimal production. Alternative carbon sources can be derived from agricultural by-products or industrial residues, which can be used as substrates for cell factories to convert waste into high-value products.
Feeding strategies include batch, fed batch, and continuous modes. Each has its own advantages and limitations. Selecting the appropriate feeding mode based on the characteristics of the fermentation product can improve production efficiency.
Different bioreactors, such as stirred tank, airlift, wave, and membrane types, have different features related to mixing, oxygen transfer, shear stress, scalability, and cost. Selecting a suitable bioreactor based on cell type and product needs is key to efficient and stable production.
Fermentation conditions such as pH, temperature, agitation, aeration rate, inoculum size and age, can be improved during the production process. With the development of AI, predictive models can be established to optimise and control these parameters, thereby simplifying the precision fermentation process, improving efficiency, and reducing manual intervention.