Encapsulation enhances protoplast fusant stability

A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing

Matrices (re)loaded: Durability, viability, and fermentative capacity of yeast encapsulated in beads of different composition during long-term fed-batch culture

Encapsulated microbes have been used for decades to produce commodities ranging from methyl ketone to beer. Encapsulated cells undergo limited replication, which enables them to more efficiently convert substrate to product than planktonic cells and which contributes to their stress resistance. To determine how encapsulated yeast supports long-term, repeated fed-batch ethanologenic fermentation, and whether different matrices influence that process, fermentation and indicators of matrix durability and cell viability were monitored in high-dextrose, fed-batch culture over 7 weeks. At most timepoints, ethanol yield (g/g) in encapsulated cultures exceeded that in planktonic cultures. And frequently, ethanol yield differed among the four matrices tested: sodium alginate crosslinked with Ca and chitosan, sodium alginate crosslinked with Ca , Protanal alginate crosslinked with Ca and chitosan, Protanal alginate crosslinked with Ca , with the last of these consistently demonstrating the highest values. Young\u27s modulus and viscosity were higher for matrices crosslinked with chitosan over the first week; thereafter values for both parameters declined and were indistinguishable among treatments. Encapsulated cells exhibited greater heat shock tolerance at 50°C than planktonic cells in either stationary or exponential phase, with similar thermotolerance observed across all four matrix types. Altogether, these data demonstrate the feasibility of re-using encapsulated yeast to convert dextrose to ethanol over at least 7 weeks. 2+ 2+ 2+ 2

Diverse conditions support near-zero growth in yeast: Implications for the study of cell lifespan

Baker’s yeast has a finite lifespan and ages in two ways: a mother cell can only divide so many times (its replicative lifespan), and a non-dividing cell can only live so long (its chronological lifespan). Wild and laboratory yeast strains exhibit natural variation for each type of lifespan, and the genetic basis for this variation has been generalized to other eukaryotes, including met-azoans. To date, yeast chronological lifespan has chiefly been studied in relation to the rate and mode of functional decline among non-dividing cells in nutrient-depleted batch culture. However, this culture method does not accurately capture two major classes of long-lived metazoan cells: cells that are terminally differentiated and metabolically active for periods that approximate animal lifespan (e.g. cardiac myocytes), and cells that are pluripotent and metabolically quiescent (e.g. stem cells). Here, we consider alternative ways of cultivating Saccharomyces cerevisiae so that these different metabolic states can be explored in non-dividing cells: (i) yeast cultured as giant colonies on semi-solid agar, (ii) yeast cultured in retentostats and provided sufficient nutrients to meet minimal energy requirements, and (iii) yeast encapsulated in a semisolid matrix and fed ad libitum in bioreactors. We review the physiology of yeast cultured under each of these conditions, and explore their potential to provide unique insights into determinants of chronological lifespan in the cells of higher eukaryotes

Densely packed yeast: A tool to study evolution of group dynamics and to enhance biomanufacturing

Yeast can thrive in dense assemblages of its own, or our making, resulting in patterns of behavior that differ markedly from that of single planktonic cells. We first studied unicellular yeast that create their own assemblage upon selection for rapid settling in liquid medium. In these experiments, large clonal clusters, composed of hundreds to thousands of cells, began to form even larger macroscopic aggregates composed of hundreds of potentially unrelated clusters. The matrix of these aggregates includes extracellular proteins, and is likely produced via apoptosis. We found that such aggregates increase survival in environments that favor fast settling, but are evolutionarily unstable because they do not discriminate between aggregate-producers and non-producers. Next, we examined unicellular yeast maintained at high density via immobilization in a Ca2+-alginate matrix. Immobilization uncouples reproduction from metabolism, resulting in metabolically active but growth-arrested cells. Because immobilized yeast allocates little substrate to biomass, we investigated its potential to enhance ethanol production under biorefinery-like conditions. We found that over a 7-week course of fed-batch culture immobilized yeast is able to produce more ethanol from the same amount of substrate than planktonic yeast. We further tested the resilience of different immobilization matrices to support long-term, fed-batch cultures, and determined that Protanal alginate has the lowest rates of cell escape and the highest ethanol yields. Lastly, we tested whether immobilization, by inducing replication arrest, could stabilize the genomes of dikaryons created by protoplast fusion of different yeast species: Saccharomyces cerevisiae and Pichia stipitis. In the absence of selection, planktonic dikaryons quickly revert to their parental genotypes via nuclear segregation during replication. We discovered that genome content of growth-arrested immobilized dikaryons is stable in 19-day, fed-batch cultures, and that stable dikaryons retain the metabolic capacity of different the fused species to ferment 6-carbon and 5-carbon sugars. Taken together, our results demonstrate the utility of densely-packed yeasts to address issues related to the evolution of group dynamics as well as to enhance efficiency and longevity in biomanufacturing.Ph.D

Data Files

Data File

Data from: Evolution of altruistic cooperation among nascent multicellular organisms

Cooperation is a classic solution to hostile environments that limit individual survival. In extreme cases this may lead to the evolution of new types of biological individuals (e.g., eusocial super-organisms). We examined the potential for inter-individual cooperation to evolve via experimental evolution, challenging nascent multicellular ‘snowflake yeast’ with an environment in which solitary multicellular clusters experienced low survival. In response, snowflake yeast evolved to form cooperative groups composed of thousands of multicellular clusters that typically survive selection. Group formation occurred through the creation of protein aggregates, only arising in strains with high (>2%) rates of cell death. Nonetheless, it was adaptive and repeatable, though ultimately evolutionarily unstable. Extracellular protein aggregates act as a common good, as they can be exploited by cheats that do not contribute to aggregate production. These results highlight the importance of group formation as a mechanism for surviving environmental stress, and underscore the remarkable ease with which even simple multicellular entities may evolve—and lose—novel social traits

Multi-Modal Brain Segmentation Using Hyper-Fused Convolutional Neural Network

Algorithms for fusing information acquired from different imaging modalities have shown to improve the segmentation results of various applications in the medical field. Motivated by recent successes achieved using densely connected fusion networks, we propose a new fusion architecture for the purpose of 3D segmentation in multi-modal brain MRI volumes. Based on a hyper-densely connected convolutional neural network, our network features in promoting a progressive information abstraction process, introducing a new module – ResFuse to merge and normalize features from different modalities and adopting combo loss for handing data imbalances. The proposed approach is evaluated on both an outsourced dataset for acute ischemic stroke lesion segmentation and a public dataset for infant brain segmentation (iSeg-17). The experiment results show our approach achieves superior performances for both datasets compared to the state-of-art fusion network

MidFusNet: Mid-Dense Fusion Network for Multi-Modal Brain MRI Segmentation

The fusion of multi-modality information has proved effective at improving the segmentation results of targeted regions (e.g., tumours, lesions or organs) of medical images. In particular, layer-level fusion represented by DenseNet has demonstrated a promising level of performance for various medical segmentation tasks. Using stroke and infant brain segmentation as example of ongoing challenging applications involving multi-modal images, we investigate whether it is possible to create a more effective of parsimonious fusion architecture based on the state-of-art fusion network - HyperDenseNet. Our hypothesis is that by fully fusing features throughout the entire network from different modalities, this not only increases network computation complexity but also interferes with the unique feature learning of each modality. Nine new network variants involving different fusion points and mechanisms are proposed. Their performances are evaluated on public datasets including iSeg-2017 and ISLES15-SSIS and an acute stroke lesion dataset collected by medical professionals. The experiment results show that of the nine proposed variants, the ‘mid-dense’ fusion network (named as MidFusNet) is able to achieve a performance comparable to the state-of-art fusion architecture, but with a much more parsimonious network (i.e., ~3.5 million parameters less compared to the baseline network for three modalities)

A regulatory hierarchy controls the dynamic transcriptional response to extreme oxidative stress in archaea.

Networks of interacting transcription factors are central to the regulation of cellular responses to abiotic stress. Although the architecture of many such networks has been mapped, their dynamic function remains unclear. Here we address this challenge in archaea, microorganisms possessing transcription factors that resemble those of both eukaryotes and bacteria. Using genome-wide DNA binding location analysis integrated with gene expression and cell physiological data, we demonstrate that a bacterial-type transcription factor (TF), called RosR, and five TFIIB proteins, homologs of eukaryotic TFs, combinatorially regulate over 100 target genes important for the response to extremely high levels of peroxide. These genes include 20 other transcription factors and oxidative damage repair genes. RosR promoter occupancy is surprisingly dynamic, with the pattern of target gene expression during the transition from rapid growth to stress correlating strongly with the pattern of dynamic binding. We conclude that a hierarchical regulatory network orchestrated by TFs of hybrid lineage enables dynamic response and survival under extreme stress in archaea. This raises questions regarding the evolutionary trajectory of gene networks in response to stress