Addressing amino acid-derived inhibitory metabolites and enhancing CHO cell culture performance through DOE-guided media modifications.

Previously, we identified six inhibitory metabolites (IMs) accumulating in Chinese hamster ovary (CHO) cultures using AMBIC 1.0 community reference medium that negatively impacted culture performance. The goal of the current study was to modify the medium to control IM accumulation through design of experiments (DOE). Initial over-supplementation of precursor amino acids (AAs) by 100% to 200% in the culture medium revealed positive correlations between initial AA concentrations and IM levels. A screening design identified 5 AA targets, Lys, Ile, Trp, Leu, Arg, as key contributors to IMs. Response surface design analysis was used to reduce initial AA levels between 13% and 33%, and these were then evaluated in batch and fed-batch cultures. Lowering AAs in basal and feed medium and reducing feed rate from 10% to 5% reduced inhibitory metabolites HICA and NAP by up to 50%, MSA by 30%, and CMP by 15%. These reductions were accompanied by a 13% to 40% improvement in peak viable cell densities and 7% to 50% enhancement in IgG production in batch and fed-batch processes, respectively. This study demonstrates the value of tuning specific AA levels in reference basal and feed media using statistical design methodologies to lower problematic IMs.

Chemical speciation of trace metals in mammalian cell culture media: looking under the hood to boost cellular performance and product quality.

Upstream process development seeks to optimize media formulations to promote robust cell culture conditions and regulate product quality attributes such as glycosylation, aggregation, and charge variants. Transition metal ions Mn, Fe, Cu, and Zn present in cell culture media have a significant impact on cell growth, metabolism and product quality. These metals and other media components can have different chemical associations or speciation in media that are poorly characterized but may significantly impact their properties and effect on cellular performance. Computer-based equilibrium models are a good starting point for exploring metal speciation, bioavailability and conditions where precipitation may occur. However, some equilibrium constants, especially for newly introduced medium components, have not been experimentally determined. Owing to concurrent physical and biological processes, speciation may also be controlled by reaction kinetics rather than by equilibrium. These factors highlight the importance of analytically interrogating medium speciation to gain insights into the complex interconnections between media components and bioprocess performance.

Partners for life: building microbial consortia for the future.

New technologies have allowed researchers to better design, build, and analyze complex consortia. These developments are fueling a wider implementation of consortium-based bioprocessing by leveraging synthetic biology, delivering on the field's multitudinous promises of higher efficiencies, superior resiliency, augmented capabilities, and modular bioprocessing. Here we chronicle current progress by presenting a range of screening, computational, and biomolecular tools enabling robust population control, efficient division of labor, and programmatic spatial organization; furthermore, we detail corresponding advancements in areas including machine learning, biocontainment, and standardization. Additionally, we show applications in myriad sectors, including medicine, energy and waste sustainability, chemical production, agriculture, and biosensors. Concluding remarks outline areas of growth that will promote the utilization of complex community structures across the biotechnology spectrum.

Synthetic microbial communities of heterotrophs and phototrophs facilitate sustainable growth.

Microbial communities comprised of phototrophs and heterotrophs hold great promise for sustainable biotechnology. Successful application of these communities relies on the selection of appropriate partners. Here we construct four community metabolic models to guide strain selection, pairing phototrophic, sucrose-secreting Synechococcus elongatus with heterotrophic Escherichia coli K-12, Escherichia coli W, Yarrowia lipolytica, or Bacillus subtilis. Model simulations reveae metabolic exchanges that sustain the heterotrophs in minimal media devoid of any organic carbon source, pointing to S. elongatus-E. coli K-12 as the most active community. Experimental validation of flux predictions for this pair confirms metabolic interactions and potential production capabilities. Synthetic communities bypass member-specific metabolic bottlenecks (e.g. histidine- and transport-related reactions) and compensate for lethal genetic traits, achieving up to 27% recovery from lethal knockouts. The study provides a robust modelling framework for the rational design of synthetic communities with optimized growth sustainability using phototrophic partners.

Evidence for a mutualistic relationship between the cyanobacteria Nostoc and fungi Aspergilli in different environments.

Symbiotic partnerships are widespread in nature and in industrial applications yet there are limited examples of laboratory communities. Therefore, using common photobionts and mycobionts similar to those in natural lichens, we create an artificial lichen-like symbiosis. While Aspergillus nidulans and Aspergillus niger could not obtain nutrients from the green algae, Chlorella, and Scenedesmus, the cyanobacteria Nostoc sp. PCC 6720 was able to support fungal growth and also elevated the accumulation of total biomass. The Nostoc-Aspergillus co-cultures grew on light and CO2 in an inorganic BG11 liquid medium without any external organic carbon and fungal mycelia were observed to peripherally contact with the Nostoc cells in liquid and on solid media at lower cell densities. Overall biomass levels were reduced after implementing physical barriers to indicate that physical contact between cyanobacteria and heterotrophic microbes may promote symbiotic growth. The synthetic Nostoc-Aspergillus nidulans co-cultures also exhibited robust growth and stability when cultivated in wastewater over days to weeks in a semi-continuous manner when compared with axenic cultivation of either species. These Nostoc-Aspergillus consortia reveal species-dependent and mutually beneficial design principles that can yield stable lichen-like co-cultures and provide insights into microbial communities that can facilitate sustainability studies and broader applications in the future. KEY POINTS: • Artificial lichen-like symbiosis was built with wild-type cyanobacteria and fungi. • Physical barriers decreased biomass production from artificial lichen co-cultures. • Artificial lichen adapted to grow and survive in wastewater for 5 weeks.

Filamentous cyanobacteria triples oil production in seawater-based medium supplemented with industrial waste: monosodium glutamate residue.

BACKGROUND: To overcome the daunting technical and economic barriers of algal biofuels, we evaluated whether seawater can be a viable medium for economically producing filamentous Spirulina subsalsa as feedstock, using monosodium glutamate residue (MSGR) produced by the glutamate extraction process as an inexpensive nutrient source. RESULTS: Spirulina subsalsa cannot grow in pure seawater, but exhibited faster biomass accumulation in seawater supplemented with MSGR than in freshwater medium (modified Zarrouk medium). Introducing seawater into media ensured this cyanobacterium obtained high lipid productivity (120 mg/L/day) and suffered limited bacterial infections during growth. Moreover, the yields of protein, carotenoids and phytols were also improved in seawater mixed with MSGR. S. subsalsa exhibited high biomass and lipid productivity in bag bioreactors with 5- and 10-L medium, demonstrating the potential of this cultivation method for scaling up. Moreover, seawater can produce more biomass through medium reuse. Reused seawater medium yielded 72% of lipid content compared to pristine medium. The reason that S. subsalsa grew well in seawater with MSGR is its proficient adaptation to salinity, which included elongation and desaturation of fatty acids, accumulation of lysine and methionine, and secretion of sodium. The nutrients provided by MSGR, like organic materials, played an important role in these responses. CONCLUSION: Spirulina subsalsa has an efficient system to adapt to saline ambiance in seawater. When supplemented with MSGR, seawater is a great potential medium to produce S. subsalsa in large scale as biofuel feedstock. Meanwhile, value-added products can be derived from the ample protein and pigments that can broaden the range of biomass application and improve this biorefinery economics.

One primer to rule them all: universal primer that adds BBa_B0034 ribosomal binding site to any coding standard 10 BioBrick.

Here, we present a universal, simple, efficient, and reliable way to add small BioBrick parts to any BioBrick via PCR that is compatible with BioBrick assembly standard 10. As a proof of principle, we have designed a universal primer, rbs_B0034, that contains a ribosomal binding site (RBS; BBa_B0034) and that can be used in PCR to amplify any coding BioBrick that starts with ATG. We performed test PCRs with rbs_B0034 on 31 different targets and found it to be 93.6% efficient. Moreover, when supplemented with a complementary primer, addition of RBS can be accomplished via whole plasmid site-directed mutagenesis, thus reducing the time required for further assembly of composite parts. The described method brings simplicity to the addition of small parts, such as regulatory elements to existing BioBricks. The final product of the PCR assembly is indistinguishable from the standard or 3A BioBrick assembly.