Biology subjects

Avalos, J. L.

Publications and source records attributed to Avalos, J. L..

18 recordsLinked to original sources

bioRxiv2026

Multichromatic Dynamic Control of Multi-Membered Microbial Consortia Compositions for Chemical Production

Engineered microbial consortia offer a promising strategy for chemical production by distributing specialized functions among microbial strains, reducing metabolic burden, facilitating modular pathway optimization, reducing toxicity, and increasing strain stability. However, differences in growth rates can destabilize population composition, compromising productivity and limiting their applicability. Here, we developed a multichromatic optogenetic Toxin-Antitoxin (optogeneticTA) platform for dynamic control of Escherichia coli consortia of up to four members using blue, red, and near-infrared light and darkness. By varying light intensities or pulses, we precisely program and dynamically modulate the population composition of two-, three-, and four-membered consortia. We further developed a modular mathematical framework that captures and predicts population dynamics of these optogenetically controlled co-cultures. Applying dynamic control to a two-membered engineered consortium increased phenol production by ~69% relative to unregulated consortia. These results establish a programmable platform for stabilizing and dynamically optimizing microbial consortia, with potential applications across microbial biomanufacturing.

synthetic biology↗

bioRxiv2026

Heterologous iron-sulfur cluster biogenesis and delivery for cytosolic isobutanol and isopentanol production in Saccharomyces cerevisiae

Saccharomyces cerevisiae is an excellent microbial platform for sustainable production of next generation biofuels such as the branched chain higher alcohols (BCHAs) isobutanol and isopentanol. A cytosolic pathway for BCHA production is generated from expression of prokaryotic orthologs of branched-chain amino acid (BCAA) enzymes acetolactate synthase (ALS), mutant NADH-dependent ketol-acid reductoisomerase (KARIP2D1-A1), and dihydroxy-acid dehydratase (DHAD). The potential for this pathway has been hindered by the availability of iron-sulfur clusters, particularly the 2Fe-2S cluster, required for DHAD to function in the cytosol. ILV3, the endogenous yeast DHAD located in the mitochondria, can be deleted to create a valine auxotroph. In this study we use bioinformatics, heterologous gene library synthesis, and a valine complementation assay to find prokaryotic iron-sulfur cluster biosynthetic gene clusters (BGC) and accessory genes that aid DHAD function in the yeast cytosol. This work presents, to our knowledge, the first functional BGC that enhances the cytosolic activity of prokaryotic DHADs in S. cerevisiae. The SUF BGC from Bacillus subtilis combined with a ferritin-like protein (FTNB) from Escherichia coli and the Lactococcus lactis DHAD enhanced the production of BCHAs. Combined expression gave an average isobutanol titer of 412mg/L, 1.8-fold greater than L. lactis DHAD expressed alone. This work establishes a blueprint for better biofuel production by improving iron-sulfur cluster dependent enzyme activity in the yeast cytosol.

bioengineering↗

bioRxiv2026

Noise analysis of derivative-action biomolecular topologies

Temporal gradient sensing is a fundamental capability observed across diverse natural biological systems, contributing to the coordination of their functions. Harnessing this ability is also of significant interest in synthetic biology, particularly for sensing and control applications. In this work, we focus on a biomolecular topology that exemplifies a broader class of signal-differentiating architectures, while introducing a structural variant of it. We examine their behavior under both nominal and non-ideal conditions, accounting for stochastic noise arising from different sources. Our investigation includes scenarios where these topologies operate independently, as well as when embedded within minimal regulatory architectures based on negative as well as positive feedback. We analyze the stability of the resulting macroscopic dynamics--a prerequisite for practical deployment--and quantify stochastic fluctuations in system output, providing comparisons with the corresponding input/unregulated process. Importantly, our results demonstrate that signal differentiation can be effectively implemented in a biomolecular setting without incurring deleterious noise amplification--a major concern in the utilization of derivative action across disciplines.

synthetic biology↗

bioRxiv2026

Enhanced GPP synthesis by Erg20p-peptide fusions and biomolecular condensates boosts monoterpene production in yeast

Monoterpenes are a diverse class of natural products with broad industrial and pharmaceutical applications. While there is great interest in transitioning their production from chemical synthesis and natural source extraction to yeast bioprocesses, this approach remains limited by the dual functionality of the endogenous farnesyl diphosphate synthase Erg20p, which produces the monoterpene precursor geranyl diphosphate (GPP) but favors its subsequent conversion to farnesyl diphosphate (FPP). To address this limitation, we recruited Erg20p and monoterpene synthases into synthetic membraneless organelles, improving production. In doing so, we found that short C-terminal peptide fusions used for recruitment also significantly enhance GPP synthase activity relative to FPP synthase activity. The combined effects of metabolic spatial organization and GPP synthase activity enhancement significantly boost production of different monoterpenes, including geraniol titers exceeding 4 g/L. The strategies presented here can be readily integrated with other traditional metabolic engineering approaches to build yeast strains with high levels of monoterpene production.

bioengineering↗

bioRxiv2026

Spatial organization of enzymes and cofactors in synthetic membrane-associated condensates (sMACs)

Cells spatially organize metabolic enzymes to optimize flux through complex biochemical networks. Here, we engineered synthetic Membrane-Associated Condensates (sMACs), artificial assemblies that form on intracellular membranes through liquid-liquid phase separation, to couple metabolic compartmentalization with cofactor partitioning. Using intrinsically disordered regions (IDRs) characterized by simulations and validated by experiments, we identified an IDR sequence, Dbp1n, that selectively enrich NADH and NADPH, establishing distinct local redox microenvironments. At the endoplasmic reticulum (ER) membrane, NADPH-enriched sMACs enhanced cytochrome P450 activity by improving electron transfer and supporting in situ regeneration. At the plasma membrane, NADH-enriched sMACs provided scaffolds for a xylose uptake and assimilation pathway that exploits localized NADH/NAD recycling to improve flux. These results collectively demonstrated that condensate composition and membrane context can be engineered to enhance metabolic functions. sMACs thus provide a modular framework for creating programmable, membrane-associated microreactors, enabling new strategies for metabolic engineering and offering insights into how phase separation can be exploited in living cells.

synthetic biology↗

bioRxiv2026

Robust Multiplicative Control in Chemical Reaction Networks -Extended Version

Achieving complex multi-species control objectives is essential for engineering advanced autoregulated biomolecular devices. This paper addresses the problem of robust steady-state tracking for outputs defined as multiplicative combinations of biomolecular species concentrations. We first introduce a control architecture realized via chemical reaction networks that steers the product of two target species concentrations in the controlled network to a prescribed value. A robust stability analysis is provided for closed-loop system families with distinct structural characteristics. The proposed framework is also extended to a more general formulation capable of regulating arbitrary monomial outputs involving multiple species. Numerical simulations of representative examples corroborate the theoretical results and illustrate the effectiveness of our approach.

synthetic biology↗

bioRxiv2025

HP1-enhanced chromatin compaction stabilizes a synthetic metabolic circuit in yeast

Chromatin compaction defines genome topology, evolution, and function. The Saccharomycotina subphylum, including the fermenting yeast Saccharomyces cerevisiae have a decompacted genome, possibly because they lost two genes mediating a specific histone lysine methylation and histone binding protein heterochromatin protein 1 (HP1). This decompaction may result in the higher-than-expected mutation and meiotic recombination rates observed in this species. To test this hypothesis, we retro-engineered S. cerevisiae to compact the genome by expressing the HP1 homologue of Schizosaccharomyces pombe SpSwi6 and H3K9 methyltransferase SpClr4. The resulting strain had significantly more compact chromatin and reduced rates of mutation and meiotic recombination. The increased genomic stability significantly prolongs the optogenetic control of an engineered strain designed to grow only in blue light. This result demonstrates the potential of our approach to enhance the stability of strains for metabolic engineering and other synthetic biology applications, which are prone to lose activities due to genetic instability.

synthetic biology↗

bioRxiv2025

Balancing doses of EL222 and light improves optogenetic induction of protein production in Komagataella phaffii

Komagataella phaffii, also known as Pichia pastoris, is a powerful host for recombinant protein production, in part due to its exceptionally strong and tightly controlled PAOX1 promoter. Most K. phaffii bioprocesses for recombinant protein production rely on PAOX1 to achieve dynamic control in two-phase processes. Cells are first grown under conditions that repress PAOX1 (growth phase), followed by methanol-induced recombinant protein expression (production phase). In this study, we propose a methanol-free approach for dynamic metabolic control in K. phaffii using optogenetics, which can help enhance input tunability and flexibility in process optimization and control. The light-responsive transcription factor EL222 from Erythrobacter litoralis is used to regulate protein production from the PC120 promoter in K. phaffii with blue light. We used two system designs to explore the advantages and disadvantages of coupling or decoupling EL222 integration with that of the gene of interest. We investigate the relationship between EL222 gene copy number and light dosage to improve production efficiency for intracellular and secreted proteins. Experiments in lab-scale bioreactors demonstrate the feasibility of the outlined optogenetic systems as potential alternatives to conventional methanol-inducible bioprocesses using K. phaffii.

bioengineering↗

bioRxiv2024

Biochemical implementation of acceleration sensing and PIDA control

Designing dependable, self-regulated biochemical systems has long posed a challenge in the field of Synthetic Biology. Here, we propose a realization of a Proportional-Integral-Derivative-Acceleration (PIDA) control scheme as a Chemical Reaction Network (CRN) governed by mass action kinetics. A constituent element of this architecture is a speed and acceleration biosensing mechanism we introduce and, subsequently, place within a feedback configuration. Our control scheme provides enhanced dynamic performance and robust steady-state tracking. In addition to our theoretical analysis, this is practically highlighted in both the deterministic and stochastic settings by regulating a specific biochemical process in-silico and drawing comparisons with a simpler PID controller.

synthetic biology↗

bioRxiv2024

AC-BioSD : A biomolecular signal differentiator module with enhanced performance

Temporal gradient estimation is a pervasive phenomenon in natural biological systems and holds great promise for synthetic counterparts with broad-reaching applications. Here, we advance the concept of BioSD (Biomolecular Signal Differentiators) by introducing a novel biomolecular topology, termed Autocatalytic-BioSD or AC-BioSD. Its structure allows for insensitivity to input signal changes and high precision in terms of signal differentiation, even when operating far from nominal conditions. Concurrently, disruptive high-frequency signal components are effectively attenuated. In addition, the usefulness of our topology in biological regulation is highlighted via a PID (Proportional-Integral-Derivative) bio-control scheme with set point weighting and filtered derivative action in both the deterministic and stochastic domains.

synthetic biology↗

bioRxiv2023

A Modular Design for Synthetic Membraneless Organelles Enables Compositional and Functional Control

Living cells organize a wide variety of processes through compartmentalization into membraneless organelles, known as biomolecular condensates. Given their ubiquitous presence across a wide spectrum of different organisms and cell types, biomolecular condensates are increasingly considered to offer great potential for biotechnological applications. However, native condensates remain difficult to harness for engineering applications, both due to their intertwined mechanisms of assembly and compositional control, and potential disruptions to native cellular processes. Here, we demonstrate a modular framework for the formation of synthetic condensates designed to decouple cluster formation and protein recruitment. Synthetic condensates are built through constitutive oligomerization of intrinsically-disordered regions (IDRs), which drive the formation of condensates whose composition can be independently defined through fused interaction domains. The composition of the proteins driven to partition into the condensate can be quantitatively described using a binding equilibrium model, demonstrating predictive control of how component expression levels and interaction affinity determine the degree of protein recruitment. Finally, the engineered system is utilized to regulate protein interactions and metabolic flux by harnessing the systems compositional tunability.

synthetic biology↗

bioRxiv2023

Asymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility

Endogenous biomolecular condensates, comprised 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 utilize 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 region (IDR) results in enhanced immiscibility and multiphase formation. Interestingly, we found that oligomerization tunes the miscibility of IDRs in an asymmetric manner, with the effect being more pronounced when the IDR exhibiting stronger homotypic IDR interactions is oligomerized. Our findings suggest that oligomerization is a flexible biophysical mechanism which cells can exploit to tune the internal organization of biomolecular condensates and their associated biological functions.

biophysics↗

bioRxiv2022

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.

synthetic biology↗

bioRxiv2020

Optogenetic Amplification Circuits for Light-Induced Metabolic Control

Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 21.8-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light doses as low as ~1%, and strong homogeneous light-induction in bioreactors of at least 5L, with limited illumination at cell densities above 40 OD600. We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.

synthetic biology↗

bioRxiv2020

Genetically encoded biosensors for branched-chain amino acid metabolism to monitor mitochondrial and cytosolic production of isobutanol and isopentanol in yeast

Branched-chain amino acid (BCAA) metabolism can be harnessed to produce many valuable chemicals. Among these, isobutanol, which is derived from valine degradation, has received substantial attention due to its promise as an advanced biofuel. While Saccharomyces cerevisiae is the preferred organism for isobutanol production, the lack of isobutanol biosensors in this organism has limited the ability to screen strains at high throughput. Here, we use a transcriptional regulator of BCAA biosynthesis, Leu3p, to develop the first genetically encoded biosensor for isobutanol production in yeast. Small modifications allowed us to redeploy Leu3p in a second biosensor for isopentanol, another BCAA-derived product of interest. Each biosensor is highly specific to isobutanol or isopentanol, respectively, and was used to engineer metabolic enzymes to increase titers. The isobutanol biosensor was additionally employed to isolate high-producing strains, and guide the construction and enhancement of mitochondrial and cytosolic isobutanol biosynthetic pathways, including in combination with optogenetic actuators to enhance metabolic flux. These biosensors promise to accelerate the development of enzymes and strains for branched-chain higher alcohol production, and offer a blueprint to develop biosensors for other products derived from BCAA metabolism.

bioengineering↗

bioRxiv2020

Light-responsive monobodies for dynamic control of customizable protein binding

Customizable, high affinity protein-protein interactions, such as those mediated by antibodies and antibody-like molecules, are invaluable to basic and applied research and have become pillars for modern therapeutics. The ability to reversibly control the binding activity of these proteins to their targets on demand would significantly expand their applications in biotechnology, medicine, and research. Here we present, as proof-of-principle, a light-controlled monobody (OptoMB) that works in vitro and in vivo, whose affinity for its SH2-domain target exhibits a 300-fold shift in binding affinity upon illumination. We demonstrate that our SH2-OptoMB can be used to purify SH2-tagged proteins directly from crude E. coli extract, achieving 99.8% purity and over 40% yield in a single purification step. This OptoMB belongs to a new class of light-sensitive protein binders we call OptoBinders (OptoBNDRs) which, by virtue of their ability to be designed to bind any protein of interest, have the potential to find new powerful applications as light-switchable binders of untagged proteins with high affinity and selectivity, and with the temporal and spatial precision afforded by light.

biochemistry↗

bioRxiv2019

Optogenetic lac operon to control chemical and protein production in Escherichia coli with light

Control of the lac operon with IPTG has been used for decades to regulate gene expression in E. 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 developed an optogenetic lac operon in a series of circuits we call OptoLAC. With these circuits, we 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 2L bioreactors. Furthermore, OptoLAC circuits enable light control of recombinant protein production, reaching yields comparable to IPTG induction, but with enhanced tunability of expression and spatial control. OptoLAC circuits are potentially useful to confer light controls over other cell functions originally engineered to be IPTG-inducible.

synthetic biology↗

bioRxiv2019

Optogenetic control of protein binding using light-switchable nanobodies

A growing number of optogenetic tools have been developed to control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in vivo or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications: modulating intracellular protein localization and signaling pathway activity and controlling target protein binding to surfaces and in protein separation columns. This work represents a first step towards programmable photoswitchable regulation of untagged, endogenous target proteins.\n\nHighlightsO_LIOpto-Nanobodies (OptoNBs) enable light-regulated binding to a wide range of protein targets.\nC_LIO_LIWe identify an optimized LOV domain and two loop insertion sites for light-regulated binding.\nC_LIO_LIOptoNBs function in vivo: when expressed in cells and fused to signaling domains, OptoNBs enable light-activated and light-inhibited Ras/Erk signaling.\nC_LIO_LIOptoNBs function in vitro: Target proteins can be reversibly bound to OptoNB-coated beads and separated through size-exclusion chromatography.\nC_LI

biochemistry↗