Toward consistent and productive complex media for industrial fermentations: Studies on yeast extract for a recombinant yeast fermentation process

Abstract

Yeast extract (YE) is commonly used as a key component in the complex media for industrial fermentations. However, the lot-to-lot variation of this raw material frequently requires extensive "use testing" of many lots to identify only the few that support desired fermentation performance. Through extensive fermentation studies and chemical analyses, we have identified adenine and two metabolizable carbon sources, trehalose and lactate, as the principle components in YE that affect the production of a recombinant protein antigen by a yeast strain. Adenine is required for culture growth and the relationship between biomass and measured adenine can be expressed by a Michaelis-Menten model, while the slowly metabolized trehalose serves to maintain the energy supply to the continued antigen synthesis. The rapidly utilized lactate exerts an indirect positive effect by sparing some of the accumulated ethanol from being consumed for growth to being utilized in the product formation. The effects of these YE components are mutually dependent. Based on the database generated from 40 lots at laboratory scale, a relatively high level of carbon sources in YE (trehalose plus lactate, >9.5% w/w) and an intermediate level of adenine (0.14-0.24% w/w) appear to be the minimal requirement of a good lot for this recombinant yeast fermentation. Many poor lots were improved in lab fermenters by rational supplementation of trehalose, lactate, or adenine to compensate for their insufficiencies. At the large production scale, predictions based on adenine and trehalose/lactate contents in various YE lots used correlated reasonably well with culture growth and antigen yield, illustrating the feasibility of such a simple chemical/biochemical analysis as a rapid and reliable initial screening tool. Without incurring any compositional change to an established manufacturing medium, this study demonstrates an effective approach to achieve consistency in fermentations employing complex nutrients and to improve fermentation productivities supported by suboptimal lots of raw material.

Simplified feeding strategies for the fed-batch cultivation of Kluyveromyces lactis GG799 for enhanced recombinant xylanase production

“…In contrast to glucose, nitrogen depletion results in an increase in pmtease activity, which can be avoided by increasing initial nitrogen concentrations. Protein yields were shown to improve in several cultures grown on complex or amino acid-enriched media [9]. Complex or enriched media not only prevent nutrient limitation but may also inhibit protcase activity by providing competing enzymatic subsftates.…”

Characterization of yeastolate fractions that promote insect cell growth and recombinant protein production

“…Yeastolate has been commonly used as a key medium component for microbial fermentations for many decades (Bridson and Brecker 1970). The activity of yeastolate varies from lot-to-lot due to less controlled fermentation conditions and downstream processes (Zhang et al 2003). Lots from the same manufacturing process could vary by almost 50% in biomass and growth promoting activity (Potvin et al 1997).…”

“…Lots from the same manufacturing process could vary by almost 50% in biomass and growth promoting activity (Potvin et al 1997). Thus, extensive ''use testing'' of several lots is required to identify a few lots that promote cell growth (Zhang et al 2003). Alternatively, there are desires to replace yeastolate with defined active substances (Wu and Lee 1998).…”

“…Many efforts have been dedicated to screen yeastolate and to identify active components which support the cell growth and fermentation. The experimental approaches include testing known components (Smith et al 1975;Wu and Lee 1998), finding deficient or limiting components in yeastolate by supplementing potential limiting components in bad lots of yeastolate (Zhang et al 2003), and correlating the yield of fermentation product and the composition of yeastolate as characterised by techniques such as automated turbidimetry system (Potvin et al 1997) and nearinfrared spectroscopy (Kasprow et al 1998). In these studies, it is not completely understood what components or any components other than the limiting ones are required for promoting microbial and cell growth.…”

Metabolomic study of baculovirus infected insect cells to identify metabolite based strategies for improving virus yields

“…5.2 b). The presence of undefined growth promoters in the protein hydrolysate (yeast extract) together with other essential substrates including peptides, carbohydrates, vitamins and minerals in the Sf900™III medium (Ikonomou et al, 2001;Siemensma et al, 2010;Sommer, 1996;Zhang et al, 2003) possibly explain the extra cell growth of the Sf900™III medium. The viability of Sf9 cells propagated in CDM was similar to that in Sf900™III (Fig.…”