Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds.

Liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here, we introduce an oligomerizing biomimetic system, "Corelets," and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs and the transition mechanism, in a protein sequence dependent manner. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes. VIDEO ABSTRACT.

Optogenetic regulation of engineered cellular metabolism for microbial chemical production.

The optimization of engineered metabolic pathways requires careful control over the levels and timing of metabolic enzyme expression. Optogenetic tools are ideal for achieving such precise control, as light can be applied and removed instantly without complex media changes. Here we show that light-controlled transcription can be used to enhance the biosynthesis of valuable products in engineered Saccharomyces cerevisiae. We introduce new optogenetic circuits to shift cells from a light-induced growth phase to a darkness-induced production phase, which allows us to control fermentation with only light. Furthermore, optogenetic control of engineered pathways enables a new mode of bioreactor operation using periodic light pulses to tune enzyme expression during the production phase of fermentation to increase yields. Using these advances, we control the mitochondrial isobutanol pathway to produce up to 8.49 ± 0.31 g l-1 of isobutanol and 2.38 ± 0.06 g l-1 of 2-methyl-1-butanol micro-aerobically from glucose. These results make a compelling case for the application of optogenetics to metabolic engineering for the production of valuable products.

Optogenetic control of the lac operon for bacterial chemical and protein production.

Control of the lac operon with isopropyl ß-D-1-thiogalactopyranoside (IPTG) has been used to regulate gene expression in Escherichia coli for countless applications, including metabolic engineering and recombinant protein production. However, optogenetics offers unique capabilities, such as easy tunability, reversibility, dynamic induction strength and spatial control, that are difficult to obtain with chemical inducers. We have developed a series of circuits for optogenetic regulation of the lac operon, which we call OptoLAC, to control gene expression from various IPTG-inducible promoters using only blue light. Applying them to metabolic engineering improves mevalonate and isobutanol production by 24% and 27% respectively, compared to IPTG induction, in light-controlled fermentations scalable to at least two-litre bioreactors. Furthermore, OptoLAC circuits enable control of recombinant protein production, reaching yields comparable to IPTG induction but with easier tunability of expression. OptoLAC circuits are potentially useful to confer light control over other cell functions originally designed to be IPTG-inducible.

COVID-19: Clinical Presentation and Prognostic Factors of Severe Disease and Mortality in the Oldest-Old Population: A Cohort Study.

INTRODUCTION: The oldest-old population (80 years or older) has the highest lethality from COVID-19. There is little information on the clinical presentation and specific prognostic factors for this group. This trial evaluated the clinical presentation and prognostic factors of severe disease and mortality in the oldest-old population. METHODS: This is an ambispective cohort study of oldest-old patients hospitalized for respiratory infection associated with COVID-19 and with a positive test by RT-PCR. The clinical presentation and the factors associated with severe disease and mortality were evaluated (logistic regression). All patients were followed up until discharge or death. RESULTS: A total of 103 patients (59.2% female) were included. The most frequent symptoms were fever (68.9%), dyspnoea (60.2%), and cough (39.8%), and 11.7% presented confusion. Fifty-nine patients (57.3%) presented severe disease, and 59 died, with 43 patients (41.7%) presenting both of these. In the multivariate analysis, female sex (odds ratio [OR] 0.31, 95% confidence interval [95% CI] 0.13-0.73, p 0.0074) and serum lactate dehydrogenase (LDH) (OR 2.55, 95% CI 1.21-5.37, p 0.0139) were associated with severe disease, and serum sodium was associated with mortality (OR 3.12, 95% CI 1.18-8.26, p 0.0222). No chronic disease or pharmacological treatment was associated with worse outcomes. CONCLUSIONS: The typical presenting symptoms of respiratory infection in COVID-19 are less frequent in the oldest-old population. Male sex and LDH level are associated with severe disease, and the serum sodium level is associated with mortality in this population.

Light-based control of metabolic flux through assembly of synthetic organelles.

To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.

Engineering acetyl-CoA supply and ERG9 repression to enhance mevalonate production in Saccharomyces cerevisiae.

Mevalonate is a key precursor in isoprenoid biosynthesis and a promising commodity chemical. Although mevalonate is a native metabolite in Saccharomyces cerevisiae, its production is challenged by the relatively low flux toward acetyl-CoA in this yeast. In this study we explore different approaches to increase acetyl-CoA supply in S. cerevisiae to boost mevalonate production. Stable integration of a feedback-insensitive acetyl-CoA synthetase (Se-acsL641P) from Salmonella enterica and the mevalonate pathway from Enterococcus faecalis results in the production of 1,390 ± 10 mg/l of mevalonate from glucose. While bifid shunt enzymes failed to improve titers in high-producing strains, inhibition of squalene synthase (ERG9) results in a significant enhancement. Finally, increasing coenzyme A (CoA) biosynthesis by overexpression of pantothenate kinase (CAB1) and pantothenate supplementation further increased production to 3,830 ± 120 mg/l. Using strains that combine these strategies in lab-scale bioreactors results in the production of 13.3 ± 0.5 g/l, which is ∼360-fold higher than previously reported mevalonate titers in yeast. This study demonstrates the feasibility of engineering S. cerevisiae for high-level mevalonate production.

¡Viva la mitochondria!: harnessing yeast mitochondria for chemical production.

The mitochondria, often referred to as the powerhouse of the cell, offer a unique physicochemical environment enriched with a distinct set of enzymes, metabolites and cofactors ready to be exploited for metabolic engineering. In this review, we discuss how the mitochondrion has been engineered in the traditional sense of metabolic engineering or completely bypassed for chemical production. We then describe the more recent approach of harnessing the mitochondria to compartmentalize engineered metabolic pathways, including for the production of alcohols, terpenoids, sterols, organic acids and other valuable products. We explain the different mechanisms by which mitochondrial compartmentalization benefits engineered metabolic pathways to boost chemical production. Finally, we discuss the key challenges that need to be overcome to expand the applicability of mitochondrial engineering and reach the full potential of this emerging field.

Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae.

Bioconversion of xylose-the second most abundant sugar in nature-into high-value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long-term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially-targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed-batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value-added products.

Harnessing yeast organelles for metabolic engineering.

Each subcellular compartment in yeast offers a unique physiochemical environment and metabolite, enzyme, and cofactor composition. While yeast metabolic engineering has focused on assembling pathways in the cell cytosol, there is growing interest in embracing subcellular compartmentalization. Beyond harnessing distinct organelle properties, physical separation of organelles from the cytosol has the potential to eliminate metabolic crosstalk and enhance compartmentalized pathway efficiency. In this Perspective we review the state of the art in yeast subcellular engineering, highlighting the benefits of targeting biosynthetic pathways to subcellular compartments, including mitochondria, peroxisomes, the ER and/or Golgi, vacuoles, and the cell wall, in different yeast species. We compare the performances of strains developed with subcellular engineering to those of native producers or yeast strains previously engineered with cytosolic pathways. We also identify important challenges that lie ahead, which need to be addressed for organelle engineering to become as mainstream as cytosolic engineering in academia and industry.

Uncovering the role of branched-chain amino acid transaminases in Saccharomyces cerevisiae isobutanol biosynthesis.

Isobutanol and other branched-chain higher alcohols (BCHAs) are promising advanced biofuels derived from the degradation of branched-chain amino acids (BCAAs). The yeast Saccharomyces cerevisiae is a particularly attractive host for the production of BCHAs due to its high tolerance to alcohols and prevalent use in the bioethanol industry. Degradation of BCAAs begins with transamination reactions, catalyzed by branched-chain amino acid transaminases (BCATs) located in the mitochondria (Bat1p) and cytosol (Bat2p). However, the roles that these transaminases play in isobutanol production remain poorly understood and obscured by conflicting reports in the literature. In this work, we elucidate the influence of BCATs on isobutanol production in two genetic backgrounds (CEN.PK2-1C and BY4741). In the process, we uncover and characterize two competing isobutanol pathways, which can be manipulated by overexpressing or deleting BAT1 or BAT2, and adding or removing valine from the fermentation media. We show that deletion of BAT1 alone increases isobutanol production by 14.2-fold over wild type strains in media lacking valine, and examine how interactions between valine and the regulatory protein Ilv6p affect isobutanol production. Compartmentalizing the five-gene isobutanol biosynthetic pathway in mitochondria of BAT1 deletion strains results in an additional 2.1-fold increase in isobutanol production in the absence of valine. While valine inhibits isobutanol production, it boosts 2-methyl-1-butanol production. This work clarifies the role of transamination activity in BCHA biosynthesis, and develops valuable strategies and strains for future optimization of isobutanol production.

Mevalonate secretion is not mediated by a singular non-essential transporter in Saccharomyces cerevisiae.

Isoprenoids are highly valued targets for microbial chemical production, allowing the creation of fragrances, biofuels, and pharmaceuticals from renewable carbon feedstocks. To increase isoprenoid production, previous efforts have manipulated pyruvate dehydrogenase (PDH) bypass pathway flux to increase cytosolic acetyl-coA; however, this results in mevalonate secretion and does not necessarily translate into higher isoprenoid production. Identification and disruption of the transporter mediating mevalonate secretion would allow us to determine whether increasing PDH bypass activity in the absence of secretion improves conversion of mevalonate into downstream isoprenoids. Attempted identification of the mevalonate transporter was accomplished using a pooled CRISPR library targeting all nonessential transporters and two different screening methods. Using a high throughput screen, based on growth of a mevalonate auxotrophic Escherichia coli strain, it was found that ZRT3 disruption largely abolished accumulation of extracellular mevalonate. However, disruption of ZRT3 was found to lower overall mevalonate pathway activity, rather than prevent secretion, indicating a previously unreported interaction between zinc availability and the mevalonate pathway. In a second screen, significant differences in PDR5/15 and QDR1/2 library representation were found between wild-type and mevalonate secreting Saccharomyces cerevisiae strains. However, no single deletion (or selected pair of double deletions) abolishes mevalonate secretion, indicating that this process appears to be mediated through multiple redundant transporters.

Asymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility.

Endogenous biomolecular condensates, composed of a multitude of proteins and RNAs, can organize into multiphasic structures with compositionally distinct phases. This multiphasic organization is generally understood to be critical for facilitating their proper biological function. However, the biophysical principles driving multiphase formation are not completely understood. Here we use in vivo condensate reconstitution experiments and coarse-grained molecular simulations to investigate how oligomerization and sequence interactions modulate multiphase organization in biomolecular condensates. We demonstrate that increasing the oligomerization state of an intrinsically disordered protein results in enhanced immiscibility and multiphase formation. Interestingly, we find that oligomerization tunes the miscibility of intrinsically disordered proteins in an asymmetric manner, with the effect being more pronounced when the intrinsically disordered protein, exhibiting stronger homotypic interactions, is oligomerized. Our findings suggest that oligomerization is a flexible biophysical mechanism that cells can exploit to tune the internal organization of biomolecular condensates and their associated biological functions.

Using MitER for 3D analysis of mitochondrial morphology and ER contacts.

We have developed an open-source workflow that allows for quantitative single-cell analysis of organelle morphology, distribution, and inter-organelle contacts with an emphasis on the analysis of mitochondria and mitochondria-endoplasmic reticulum (mito-ER) contact sites. As the importance of inter-organelle contacts becomes more widely recognized, there is a concomitant increase in demand for tools to analyze subcellular architecture. Here, we describe a workflow we call MitER (pronounced "mightier"), which allows for automated calculation of organelle morphology, distribution, and inter-organelle contacts from 3D renderings by employing the animation software Blender. We then use MitER to quantify the variations in the mito-ER networks of Saccharomyces cerevisiae, revealing significantly more mito-ER contacts within respiring cells compared to fermenting cells. We then demonstrate how this workflow can be applied to mammalian systems and used to monitor mitochondrial dynamics and inter-organelle contact in time-lapse studies.

High-throughput feedback-enabled optogenetic stimulation and spectroscopy in microwell plates.

The ability to perform sophisticated, high-throughput optogenetic experiments has been greatly enhanced by recent open-source illumination devices that allow independent programming of light patterns in single wells of microwell plates. However, there is currently a lack of instrumentation to monitor such experiments in real time, necessitating repeated transfers of the samples to stand-alone analytical instruments, thus limiting the types of experiments that could be performed. Here we address this gap with the development of the optoPlateReader (oPR), an open-source, solid-state, compact device that allows automated optogenetic stimulation and spectroscopy in each well of a 96-well plate. The oPR integrates an optoPlate illumination module with a module called the optoReader, an array of 96 photodiodes and LEDs that allows 96 parallel light measurements. The oPR was optimized for stimulation with blue light and for measurements of optical density and fluorescence. After calibration of all device components, we used the oPR to measure growth and to induce and measure fluorescent protein expression in E. coli. We further demonstrated how the optical read/write capabilities of the oPR permit computer-in-the-loop feedback control, where the current state of the sample can be used to adjust the optical stimulation parameters of the sample according to pre-defined feedback algorithms. The oPR will thus help realize an untapped potential for optogenetic experiments by enabling automated reading, writing, and feedback in microwell plates through open-source hardware that is accessible, customizable, and inexpensive.

Light-Controlled Fermentations for Microbial Chemical and Protein Production.

Microbial cell factories offer a sustainable alternative for producing chemicals and recombinant proteins from renewable feedstocks. However, overburdening a microorganism with genetic modifications can reduce host fitness and productivity. This problem can be overcome by using dynamic control: inducible expression of enzymes and pathways, typically using chemical- or nutrient-based additives, to balance cellular growth and production. Optogenetics offers a non-invasive, highly tunable, and reversible method of dynamically regulating gene expression. Here, we describe how to set up light-controlled fermentations of engineered Escherichia coli and Saccharomyces cerevisiae for the production of chemicals or recombinant proteins. We discuss how to apply light at selected times and dosages to decouple microbial growth and production for improved fermentation control and productivity, as well as the key optimization considerations for best results. Additionally, we describe how to implement light controls for lab-scale bioreactor experiments. These protocols facilitate the adoption of optogenetic controls in engineered microorganisms for improved fermentation performance.

Physiological limitations and opportunities in microbial metabolic engineering.

Metabolic engineering can have a pivotal role in increasing the environmental sustainability of the transportation and chemical manufacturing sectors. The field has already developed engineered microorganisms that are currently being used in industrial-scale processes. However, it is often challenging to achieve the titres, yields and productivities required for commercial viability. The efficiency of microbial chemical production is usually dependent on the physiological traits of the host organism, which may either impose limitations on engineered biosynthetic pathways or, conversely, boost their performance. In this Review, we discuss different aspects of microbial physiology that often create obstacles for metabolic engineering, and present solutions to overcome them. We also describe various instances in which natural or engineered physiological traits in host organisms have been harnessed to benefit engineered metabolic pathways for chemical production.