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 Ca2+ and chitosan, sodium alginate crosslinked with Ca2+ , Protanal alginate crosslinked with Ca2+ and chitosan, Protanal alginate crosslinked with Ca2+ , with the last of these consistently demonstrating the highest values. Young's 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.

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 metazoans. 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.

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 interindividual 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, although 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.

Systematic Discovery of Archaeal Transcription Factor Functions in Regulatory Networks through Quantitative Phenotyping Analysis.

Gene regulatory networks (GRNs) are critical for dynamic transcriptional responses to environmental stress. However, the mechanisms by which GRN regulation adjusts physiology to enable stress survival remain unclear. Here we investigate the functions of transcription factors (TFs) within the global GRN of the stress-tolerant archaeal microorganism Halobacterium salinarum. We measured growth phenotypes of a panel of TF deletion mutants in high temporal resolution under heat shock, oxidative stress, and low-salinity conditions. To quantitate the noncanonical functional forms of the growth trajectories observed for these mutants, we developed a novel modeling framework based on Gaussian process regression and functional analysis of variance (FANOVA). We employ unique statistical tests to determine the significance of differential growth relative to the growth of the control strain. This analysis recapitulated known TF functions, revealed novel functions, and identified surprising secondary functions for characterized TFs. Strikingly, we observed that the majority of the TFs studied were required for growth under multiple stress conditions, pinpointing regulatory connections between the conditions tested. Correlations between quantitative phenotype trajectories of mutants are predictive of TF-TF connections within the GRN. These phenotypes are strongly concordant with predictions from statistical GRN models inferred from gene expression data alone. With genome-wide and targeted data sets, we provide detailed functional validation of novel TFs required for extreme oxidative stress and heat shock survival. Together, results presented in this study suggest that many TFs function under multiple conditions, thereby revealing high interconnectivity within the GRN and identifying the specific TFs required for communication between networks responding to disparate stressors. IMPORTANCE To ensure survival in the face of stress, microorganisms employ inducible damage repair pathways regulated by extensive and complex gene networks. Many archaea, microorganisms of the third domain of life, persist under extremes of temperature, salinity, and pH and under other conditions. In order to understand the cause-effect relationships between the dynamic function of the stress network and ultimate physiological consequences, this study characterized the physiological role of nearly one-third of all regulatory proteins known as transcription factors (TFs) in an archaeal organism. Using a unique quantitative phenotyping approach, we discovered functions for many novel TFs and revealed important secondary functions for known TFs. Surprisingly, many TFs are required for resisting multiple stressors, suggesting cross-regulation of stress responses. Through extensive validation experiments, we map the physiological roles of these novel TFs in stress response back to their position in the regulatory network wiring. This study advances understanding of the mechanisms underlying how microorganisms resist extreme stress. Given the generality of the methods employed, we expect that this study will enable future studies on how regulatory networks adjust cellular physiology in a diversity of organisms.

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.