Biology subjects

Sullivan, S. F.

Publications and source records attributed to Sullivan, S. F..

3 recordsLinked to original sources

bioRxiv2024

Microbial lysates as low-cost serum replacements in cellular agriculture media formulation

Cultivated meat, the process of generating meat in vitro without sacrificing animals, is a promising alternative to the traditional practice of livestock agriculture. However, the success of this field depends on finding sustainable and economical replacements for animal-derived and expensive fetal bovine serum (FBS) that is typically used in cell culture processes. Here, we outline an effective screening process to vet the suitability of microbial lysates to support the growth of immortalized bovine satellite cells (iBSCs) and mackerel (Mack1) cells. We show that easily producible, low-cost whole-cell lysates from Vibrio natriegens can be used to create serum-free media for the long-term growth of iBSCs. The optimized medium, named "VN40" (basal B8 media containing Vibrio natriegens lysate proteins at 40 {micro}g/mL), outperforms previously established serum-free media while maintaining cell phenotype and myogenicity. Overall, this study shows a novel approach to producing serum-free media for cultivated meat production using microbially-derived lysates.

bioengineering↗

bioRxiv2022

Towards universal synthetic heterotrophy using a metabolic coordinator

Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as potential inputs to biotechnological processes. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3pS25144.1), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3pMC (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies confirm that Gal3pMC occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3pMC to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3pMC-mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy.

synthetic biology↗

bioRxiv2022

Integration of metabolism and regulation reveals rapid adaptability to growth on non-native substrates.

Engineering synthetic heterotrophy (i.e., growth on non-native substrates) is key to the efficient bio-based valorization of renewable and waste substrates. Among these, engineering hemicellulosic pentose utilization has been well-explored in Saccharomyces cerevisiae (yeast) over several decades - yet the answer to what makes their utilization inherently recalcitrant remains elusive. Through implementation of a semi-synthetic regulon, we find that harmonizing cellular and engineering objectives are key to obtaining highest growth rates and yields with minimal metabolic engineering effort. Concurrently, results indicate that "extrinsic" factors - specifically, upstream genes that direct flux of pentoses into central carbon metabolism - are rate-limiting. We also reveal that yeast metabolism is innately highly adaptable to rapid growth on non-native substrates and that systems metabolic engineering (i.e., flux balancing, directed evolution, functional genomics, and network modeling) is largely unnecessary. We posit that the need for extensive engineering espoused by prior works is a consequence of unfortunate (albeit avoidable) antagonism between engineering and cellular objectives. We also found that deletion of endogenous genes to promote growth demonstrate inconsistent outcomes that are genetic-context- and condition-dependent. For the most part, these knockouts also lead to deleterious pleiotropic effects that decrease the robustness of strains against inhibitors and stressors associated with bioprocessing. Thus, at best, perturbation of "intrinsic" factors (e.g., native metabolic, regulatory genes) results in incremental and inconsistent benefits. At worst, they are detrimental. Overall, this work provides insight into the limitations and pitfalls to realizing efficient synthetic heterotrophy using traditional/systems metabolic engineering approaches, demonstrates the innate adaptability of yeast for metabolism of non-native substrates, and provides an alternate, novel, holistic (and yet minimalistic) approach based on integrating non-native metabolic genes with a native regulon system.

synthetic biology↗