3 Alternative Feeding Strategies to Improve Cell Line Performance

revised December 16, 2022

In addition to the multitude of different biological pathways and organism physiologies that can be leveraged to produce bioproducts, process feeding strategies like those that utilize multiple feeds are also important for improving microbe performance.

While glucose is one of the most common substrates utilized in commercial fermentation processes, a number of other substrates can be used to improve strain performance in a variety of feeding strategies. All of these feeding strategies should be thoroughly vetted before being implemented in development efforts. A techno-economic analysis (TEA) can help ensure that the proposed strategy will be commercially viable, and that the components can be sourced at a commercial scale. An assessment of the infrastructure required to execute the proposed bioprocess at scale should also be performed to ensure that it is feasible from both a safety and reactor engineering perspective. Once these criteria have been met, the proposed feeding strategies can be implemented into research and development efforts. Some of the examples described below may not be commercially viable processes, but nonetheless demonstrate the concepts of these different feeding strategies.

1. Alternative carbon sources 

Based on the physiology of an organism, carbon sources other than the commonly used glucose may be better suited to the production of specific molecules. Saccharomyces cerevisiae, for example, can metabolize fermentable carbon sources such as glucose, fructose, and galactose as well as non-fermentable carbon sources such as ethanol and glycerol (Casanabra et al, 2021). Utilizing different carbon sources to promote respiratory or fermentative metabolism also results in different carbon fluxes in the cell. Wehrs et al (2018) demonstrated this concept where the production of the blue pigment indigoidine was linked to the respiratory metabolic state of S. cerevisiae. In this respiratory metabolic state, significantly increased flux through the TCA (tricarboxylic acid) cycle increases the precursor pools needed for indigoidine production. Thus, when respiratory metabolism is driven by glycerol or ethanol, the production of indigoidine is increased. In contrast, under conditions of excess glucose and fermentative metabolism, little indigoidine production was observed. It was a clean demonstration of the link between the respiratory state of the organism and, consequently, the production of indigoidine. Depending on the physiology of the organism, product of interest, and the availability, cost, and productivity supported by alternative substrates, alternative carbon sources can provide an interesting approach to boost production. 

2. Diauxic production bioprocesses

Diauxic bioprocesses involve altering carbon sources at some point in the process to support robust production. One of the best-known diauxic process systems is one using the methylotrophic yeast, Pichia pastoris. This yeast, which can assimilate methanol as the sole carbon and energy sources, has been used extensively for protein production. In addition to its utilization of methanol, an alcohol oxidase (AOX) expression system was developed for use in Pichia. This system relies on the AOX1 promoter, which is repressed in the presence of glucose or glycerol and highly expressed in the presence of methanol (Couderc and Baratti, J.,1980, Cregg et al, 1993). This allows for the separation of growth and production phases by using glucose or glycerol for growth and adding methanol as a carbon source for strong pathway induction and protein production.

Saccharomyces cerevisiae has also been used in diauxic production processes, including several in the production of terpenoids (Carsanaba et al, 2021). Saccharomyces has been used to produce a number of different terpenoids partially because the pathway precursors are synthesized from mevalonate, and Saccharomyces possesses an endogenous mevalonate pathway where Acetyl-CoA is upstream of mevalonate. Several groups have leveraged this phenomenon and developed diauxic bioprocesses with a growth phase on glucose followed by production on ethanol. Ethanol, when metabolized, is directed towards Acetyl-CoA and increases upstream pathway flux, which can translate to higher productivity. Different groups have leveraged this phenomenon in different ways. Some have fed ethanol as a substrate in a diauxic process. Others have developed glucose pulse-feeding strategies to create diauxic fermentation processes that take advantage of the microbes’ production of ethanol under glucose excess (Wehrs et al, 2017). Upon depletion of the glucose, ethanol will be consumed which presumably boosts flux through Acetyl-CoA and towards the product of interest.

3. Bioconversion processes

Bioconversion processes depend on the activity of enzymes during the production phase of a molecule of interest. The enzymes, which may be either native to the organism or heterologously expressed, are harnessed to drive conversion or incorporation of an added precursor molecule into the desired product. 

Novel natural products produced via secondary metabolism, where organisms produce metabolites that confer a biological advantage over competing organisms, may require an organism to go through a series of developmental stages under specific environmental induction conditions in order to produce the molecule of interest. While complex, many important secondary metabolites are produced in this manner. In fact, under specific conditions, many Streptomyces species may each have the capability to produce up to 30 different secondary metabolites (Craney et al, 2013). These cryptic pathways, equipped with enzymes with novel functions, can be leveraged in bioconversion processes to produce novel molecules. For example, Restaino et al (2016) developed a fed-batch process where the bioconversion of hydrocortisone into 16-hydroxy hydrocortisone was facilitated by a hydroxylation reaction using enzymes native to Streptomyces roseochromogenes. 16-hydroxy hydrocortisone is, in turn, a precursor for an anti-inflammatory drug. In an optimized fermentation process, bioconversion of hydrocortisone was on the order of 80%, with high product purity observed. This bioconversion process was an attractive alternative to the chemical synthesis of the same molecule. Overall, bioconversions offer a means to produce difficult-to-synthesize or novel molecules via a biological route. 

Feed strategies supported at Culture

Culture’s cloud bioreactor systems support a diversity of feed strategies. Each reactor is equipped with up to five individual feeds, all with mass-based feedback control and correction for substrate evaporation for ensuring precise substrate delivery. The capability to support up to five different feeds allows a multitude of different substrate feeding strategies; including alternative substrates, various diauxic strategies, or bioconversions. Culture’s recipe-based programming system allows feeds to be delivered precisely at the intended stage, whether based on timing or dynamically based on physiological cues from the culture. The flexibility to explore different feed strategies can help identify optimized bioprocesses to maximize product formation and reach project goals faster. 

For more on feed strategies for process development, including key challenges to keep in mind and best practices, check out this resource.

References

1. Wehrs M, Prahl JP, Moon J, Li Y, Tanjore D, Keasling JD, Pray T, Mukhopadhyay A. Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae. Microb Cell Fact. 2018 Dec 13;17(1):193. doi: 10.1186/s12934-018-1045-1. Erratum in: Microb Cell Fact. 2019 Dec 29;18(1):218. PMID: 30545355; PMCID: PMC6293659.

2. Couderc, R., & Baratti, J. (1980). Oxidation of Methanol by the Yeast,Pichia pastoris. Purification and Properties of the Alcohol Oxidase. Agricultural and Biological Chemistry, 44(10), 2279–2289. doi:10.1080/00021369.1980.10864320 

3. Cregg, J. M., Vedvick, T. S., & Raschke, W. C. (1993). Recent Advances in the Expression of Foreign Genes in Pichia pastoris. Nature Biotechnology, 11(8), 905–910. doi:10.1038/nbt0893-905 

4. Craney A, Ahmed S, Nodwell J. Towards a new science of secondary metabolism. J Antibiot (Tokyo). 2013 Jul;66(7):387-400. doi: 10.1038/ja.2013.25. Epub 2013 Apr 24. PMID: 23612726.

5. Restaino O, Marseglia M, Diana P, Borzacchiello M, Finamore R, Vitiello M, D’Agostino A,  De Rosa M, Schiraldi C (2016). Advances in the 16α-hydroxy transformation of hydrocortisone by Streptomyces roseochromogenes. Process Biochemistry, 51(1), 1-8. https://doi.org/10.1016/j.procbio.2015.11.009.