A modular and synthetic biosynthesis platform for de novo production of diverse halogenated tryptophan-derived molecules.

Halogen-containing molecules are ubiquitous in modern society and present unique chemical possibilities. As a whole, de novo fermentation and synthetic pathway construction for these molecules remain relatively underexplored and could unlock molecules with exciting new applications in industries ranging from textiles to agrochemicals to pharmaceuticals. Here, we report a mix-and-match co-culture platform to de novo generate a large array of halogenated tryptophan derivatives in Escherichia coli from glucose. First, we engineer E. coli to produce between 300 and 700 mg/L of six different halogenated tryptophan precursors. Second, we harness the native promiscuity of multiple downstream enzymes to access unexplored regions of metabolism. Finally, through modular co-culture fermentations, we demonstrate a plug-and-play bioproduction platform, culminating in the generation of 26 distinct halogenated molecules produced de novo including precursors to prodrugs 4-chloro- and 4-bromo-kynurenine and new-to-nature halogenated beta carbolines.

Design and synthesis of synthetic promoters for consistency of gene expression across growth phases and scale in S. cerevisiae.

Metabolic engineering and synthetic biology endeavors benefit from promoters that perform consistently (or robustly) with respect to cellular growth phase (exponential and stationary) and fermentation scale (microtiter plates, tubes, flasks, and bioreactors). However, nearly all endogenous promoters (especially in Saccharomyces cerevisiae) do not perform in this manner. In this work, a hybrid promoter engineering strategy is leveraged to create novel synthetic promoters with robustness across these conditions. Using a multi-dimensional RNA-seq dataset, promoters with specific phase dependencies were identified. Fragments enriched with functional transcription factors were identified using MEME suite. These motif-containing fragments could impart activity dependence in the opposing condition. Specifically, we obtain two new promoters with high and consistent expression across both phases by increasing the exponential phase activity of the starting stationary-phase scaffold by 38 and 23-fold respectively. Further, we show that these promoters function consistently across various laboratory growth scales over time in a microtiter plate and in flasks. Overall, this work presents and validates a new strategy for engineering promoters in S. cerevisiae with high levels of expression that are robust to cellular growth phase and the scale of the culture.

Leveraging a Y. lipolytica naringenin chassis for biosynthesis of apigenin and associated glucoside.

Flavonoids are a diverse set of natural products with promising bioactivities including anti-inflammatory, anti-cancer, and neuroprotective properties. Previously, the oleaginous host Yarrowia lipolytica has been engineered to produce high titers of the base flavonoid naringenin. Here, we leverage this host along with a set of E. coli bioconversion strains to produce the flavone apigenin and its glycosylated derivative isovitexin, two potential nutraceutical and pharmaceutical candidates. Through downstream strain selection, co-culture optimization, media composition, and mutant isolation, we were able to produce168 mg/L of apigenin, representing a 46% conversion rate of 2-(R/S)-naringenin to apigenin. This apigenin platform was modularly extended to produce isovitexin by addition of a second bioconversion strain. Together, these results demonstrate the promise of microbial production and modular bioconversion to access diversified flavonoids.

Biosensor and machine learning-aided engineering of an amaryllidaceae enzyme.

A major challenge to achieving industry-scale biomanufacturing of therapeutic alkaloids is the slow process of biocatalyst engineering. Amaryllidaceae alkaloids, such as the Alzheimer's medication galantamine, are complex plant secondary metabolites with recognized therapeutic value. Due to their difficult synthesis they are regularly sourced by extraction and purification from the low-yielding daffodil Narcissus pseudonarcissus. Here, we propose an efficient biosensor-machine learning technology stack for biocatalyst development, which we apply to engineer an Amaryllidaceae enzyme in Escherichia coli. Directed evolution is used to develop a highly sensitive (EC50 = 20 µM) and specific biosensor for the key Amaryllidaceae alkaloid branchpoint 4'-O-methylnorbelladine. A structure-based residual neural network (MutComputeX) is subsequently developed and used to generate activity-enriched variants of a plant methyltransferase, which are rapidly screened with the biosensor. Functional enzyme variants are identified that yield a 60% improvement in product titer, 2-fold higher catalytic activity, and 3-fold lower off-product regioisomer formation. A solved crystal structure elucidates the mechanism behind key beneficial mutations.

Fluorescence-Based Screens for Engineering Enzymes Linked to Halogenated Tryptophan.

Directed evolution is often limited by the throughput of accurate screening methods. Here we demonstrate the feasibility of utilizing a singular transcription factor (TF)-system that can be refactored in two ways (both as an activator and repressor). Specifically, we showcase the use of previously evolved 5-halo- or 6-halo-tryptophan-specific TF biosensors suitable for the detection of a halogenated tryptophan molecule in vivo. We subsequently validate the biosensor's utility for two halogenase-specific halo-tryptophan accumulation screens. First, we isolated 5-tryptophan-halogenase, XsHal, from a mixed pool of halogenases with 100% efficiency. Thereafter, we generated a targeted library of the catalytic residue of 6-tryptophan halogenase, Th-Hal, and isolated functioning halogenases with 100% efficiency. Lastly, we refactor the TF circuit to respond to the depletion of halogenated tryptophan and prototype a high-throughput biosensor-directed evolution scheme to screen for downstream enzyme variants capable of promiscuously converting halogenated tryptophan. Altogether, this work takes a significant step toward the rapid and higher throughput screening of halogenases and halo-tryptophan converting enzymes to further reinforce efforts to enable high-level bioproduction of halogenated chemicals.

Engineered Living Material Bioreactors with Tunable Mechanical Properties using Vat Photopolymerization.

3D-printed engineered living materials (ELM) are promising bioproduction platforms for agriculture, biotechnology, sustainable energy, and green technology applications. However, the design of these platforms faces several challenges, such as the processability of these materials into complex form factors and control over their mechanical properties. Herein, ELM are presented as 3D-printed bioreactors with arbitrary shape geometries and tunable mechanical properties (moduli and toughness). Poly(ethylene glycol) diacrylate (PEGDA) is used as the precursor to create polymer networks that encapsulate the microorganisms during the vat photopolymerization process. A major limitation of PEGDA networks is their propensity to swell and fracture when submerged in water. The authors overcame this issue by adding glycerol to the resin formulation to 3D print mechanically tough ELM hydrogels. While polymer concentration affects the modulus and reduces bioproduction, ELM bioreactors still maintain their metabolic activity regardless of polymer concentration. These ELM bioreactors have the potential to be used in different applications for sustainable architecture, food production, and biomedical devices that require different mechanical properties from soft to stiff.

Advances in enzymatic and organismal technologies for the recycling and upcycling of petroleum-derived plastic waste.

Biological catalysts are emerging with the capability to depolymerize a wide variety of plastics. Improving and discovering these catalysts has leveraged a range of tools, including microbial ecology studies, high-throughput selections, and computationally guided mutational studies. In this review, we discuss the prospects for biological solutions to plastic recycling and upcycling with a focus on major advances in polyethylene terephthalate depolymerization, expanding the range of polymers with known biological catalysts, and the utilization of derived products. We highlight several recent improvements in enzymes and reaction properties, the discovery of a wide variety of novel plastic-depolymerizing biocatalysts, and how depolymerization products can be utilized in recycling and upcycling.

Establishing a versatile toolkit of flux enhanced strains and cell extracts for pathway prototyping.

Building and optimizing biosynthetic pathways in engineered cells holds promise to address societal needs in energy, materials, and medicine, but it is often time-consuming. Cell-free synthetic biology has emerged as a powerful tool to accelerate design-build-test-learn cycles for pathway engineering with increased tolerance to toxic compounds. However, most cell-free pathway prototyping to date has been performed in extracts from wildtype cells which often do not have sufficient flux towards the pathways of interest, which can be enhanced by engineering. Here, to address this gap, we create a set of engineered Escherichia coli and Saccharomyces cerevisiae strains rewired via CRISPR-dCas9 to achieve high-flux toward key metabolic precursors; namely, acetyl-CoA, shikimate, triose-phosphate, oxaloacetate, α-ketoglutarate, and glucose-6-phosphate. Cell-free extracts generated from these strains are used for targeted enzyme screening in vitro. As model systems, we assess in vivo and in vitro production of triacetic acid lactone from acetyl-CoA and muconic acid from the shikimate pathway. The need for these platforms is exemplified by the fact that muconic acid cannot be detected in wildtype extracts provided with the same biosynthetic enzymes. We also perform metabolomic comparison to understand biochemical differences between the cellular and cell-free muconic acid synthesis systems (E. coli and S. cerevisiae cells and cell extracts with and without metabolic rewiring). While any given pathway has different interfaces with metabolism, we anticipate that this set of pre-optimized, flux enhanced cell extracts will enable prototyping efforts for new biosynthetic pathways and the discovery of biochemical functions of enzymes.

Additive Manufacturing of Engineered Living Materials with Bio-augmented Mechanical Properties and Resistance to Degradation.

Engineered living materials (ELMs) combine living cells with polymeric matrices to yield unique materials with programmable functions. While the cellular platform and the polymer network determine the material properties and applications, there are still gaps in our ability to seamlessly integrate the biotic (cellular) and abiotic (polymer) components into singular material, then assemble them into devices and machines. Herein, we demonstrated the additive-manufacturing of ELMs wherein bioproduction of metabolites from the encapsulated cells enhanced the properties of the surrounding matrix. First, we developed aqueous resins comprising bovine serum albumin (BSA) and poly(ethylene glycol diacrylate) (PEGDA) with engineered microbes for vat photopolymerization to create objects with a wide array of 3D form factors. The BSA-PEGDA matrix afforded hydrogels that were mechanically stiff and tough for use in load-bearing applications. Second, we demonstrated the continuous in situ production of L-DOPA, naringenin, and betaxanthins from the engineered cells encapsulated within the BSA-PEGDA matrix. These microbial metabolites bioaugmented the properties of the BSA-PEGDA matrix by enhancing the stiffness (L-DOPA) or resistance to enzymatic degradation (betaxanthin). Finally, we demonstrated the assembly of the 3D printed ELM components into mechanically functional bolts and gears to showcase the potential to create functional ELMs for synthetic living machines.

A tripartite microbial co-culture system for de novo biosynthesis of diverse plant phenylpropanoids.

Plant-derived phenylpropanoids, in particular phenylpropenes, have diverse industrial applications ranging from flavors and fragrances to polymers and pharmaceuticals. Heterologous biosynthesis of these products has the potential to address low, seasonally dependent yields hindering ease of widespread manufacturing. However, previous efforts have been hindered by the inherent pathway promiscuity and the microbial toxicity of key pathway intermediates. Here, in this study, we establish the propensity of a tripartite microbial co-culture to overcome these limitations and demonstrate to our knowledge the first reported de novo phenylpropene production from simple sugar starting materials. After initially designing the system to accumulate eugenol, the platform modularity and downstream enzyme promiscuity was leveraged to quickly create avenues for hydroxychavicol and chavicol production. The consortia was found to be compatible with Engineered Living Material production platforms that allow for reusable, cold-chain-independent distributed manufacturing. This work lays the foundation for further deployment of modular microbial approaches to produce plant secondary metabolites.

Gene expression dynamics in input-responsive engineered living materials programmed for bioproduction.

Engineered living materials (ELMs) fabricated by encapsulating microbes in hydrogels have great potential as bioreactors for sustained bioproduction. While long-term metabolic activity has been demonstrated in these systems, the capacity and dynamics of gene expression over time is not well understood. Thus, we investigate the long-term gene expression dynamics in microbial ELMs constructed using different microbes and hydrogel matrices. Through direct gene expression measurements of engineered E. coli in F127-bisurethane methacrylate (F127-BUM) hydrogels, we show that inducible, input-responsive genetic programs in ELMs can be activated multiple times and maintained for multiple weeks. Interestingly, the encapsulated bacteria sustain inducible gene expression almost 10 times longer than free-floating, planktonic cells. These ELMs exhibit dynamic responsiveness to repeated induction cycles, with up to 97% of the initial gene expression capacity retained following a subsequent induction event. We demonstrate multi-week bioproduction cycling by implementing inducible CRISPR transcriptional activation (CRISPRa) programs that regulate the expression of enzymes in a pteridine biosynthesis pathway. ELMs fabricated from engineered S. cerevisiae in bovine serum albumin (BSA) - polyethylene glycol diacrylate (PEGDA) hydrogels were programmed to express two different proteins, each under the control of a different chemical inducer. We observed scheduled bioproduction switching between betaxanthin pigment molecules and proteinase A in S. cerevisiae ELMs over the course of 27 days under continuous cultivation. Overall, these results suggest that the capacity for long-term genetic expression may be a general property of microbial ELMs. This work establishes approaches for implementing dynamic, input-responsive genetic programs to tailor ELM functions for a wide range of advanced applications.

Temporal sorting of microdroplets can identify productivity differences of itaconic acid from libraries of Yarrowia lipolytica.

Microdroplet screening of microorganisms can improve the rate of strain selection and characterization within the canonical design-build-test paradigm. However, a full analysis of the microdroplet environment and how well these conditions translate to culturing conditions and techniques is lacking in the field. Quantification of three different biosensor/analyte combinations at 12 hour timepoints reveals the potential for extended dose-response ranges as compared to traditional in vitro conditions. Using these dynamics, we present an application and analysis of microfluidic droplet screening utilizing whole-cell biosensors, ultimately identifying an altered productivity profile of itaconic acid in a Yarrowia lipolytica-based piggyBac transposon library. Specifically, we demonstrate that the timepoint for microdroplet selection can influence the outcome of the selection and thus shift the identified strain productivity and final titer. In this case, strains selected at earlier timepoints showed increased early productivity in flask scale, with the converse true as well. Differences in response indicate microdroplet assays require tailored development to more accurately sort for phenotypes that are scalable to larger incubation volumes. Likewise, these results further highlight that screening conditions are critical parameters for success in high-throughput applications.

Using oils and fats to replace sugars as feedstocks for biomanufacturing: Challenges and opportunities for the yeast Yarrowia lipolytica.

More than 200 million tons of plant oils and animal fats are produced annually worldwide from oil, crops, and the rendered animal fat industry. Triacylglycerol, an abundant energy-dense compound, is the major form of lipid in oils and fats. While oils or fats are very important raw materials and functional ingredients for food or related products, a significant portion is currently diverted to or recovered as waste. To significantly increase the value of waste oils or fats and expand their applications with a minimal environmental footprint, microbial biomanufacturing is presented as an effective strategy for adding value. Though both bacteria and yeast can be engineered to use oils or fats as the biomanufacturing feedstocks, the yeast Yarrowia lipolytica is presented as one of the most attractive platforms. Y. lipolytica is oleaginous, generally regarded as safe, demonstrated as a promising industrial producer, and has unique capabilities for efficient catabolism and bioconversion of lipid substrates. This review summarizes the major challenges and opportunities for Y. lipolytica as a new biomanufacturing platform for the production of value-added products from oils and fats. This review also discusses relevant cellular and metabolic engineering strategies such as fatty acid transport, fatty acid catabolism and bioconversion, redox balances and energy yield, cell morphology and stress response, and bioreaction engineering. Finally, this review highlights specific product classes including long-chain diacids, wax esters, terpenes, and carotenoids with unique synthesis opportunities from oils and fats in Y. lipolytica.

Cell Extracts from Bacteria and Yeast Retain Metabolic Activity after Extended Storage and Repeated Thawing.

Cell-free synthetic biology enables rapid prototyping of biological parts and synthesis of proteins or metabolites in the absence of cell growth constraints. Cell-free systems are frequently made from crude cell extracts, where composition and activity can vary significantly based on source strain, preparation and processing, reagents, and other considerations. This variability can cause extracts to be treated as black boxes for which empirical observations guide practical laboratory practices, including a hesitance to use dated or previously thawed extracts. To better understand the robustness of cell extracts over time, we assessed the activity of cell-free metabolism during storage. As a model, we studied conversion of glucose to 2,3-butanediol. We found that cell extracts from Escherichia coli and Saccharomyces cerevisiae subjected to an 18-month storage period and repeated freeze-thaw cycles retain consistent metabolic activity. This work gives users of cell-free systems a better understanding of the impacts of storage on extract behavior.

Evolving tolerance of Yarrowia lipolytica to hydrothermal liquefaction aqueous phase waste.

Hydrothermal liquefaction (HTL) is an emerging method for thermochemical conversion of wet organic waste and biomass into renewable biocrude. HTL also produces an aqueous phase (HTL-AP) side stream containing 2-4% light organic compounds that require treatment. Although anaerobic digestion (AD) of HTL-AP has shown promise, lengthy time periods were required for AD microbial communities to adapt to metabolic inhibitors in HTL-AP. An alternative for HTL-AP valorization was recently demonstrated using two engineered strains of Yarrowia lipolytica, E26 and Diploid TAL, for the overproduction of lipids and the polyketide triacetic acid lactone (TAL) respectively. These strains tolerated up to 10% HTL-AP (v/v) in defined media and up to 25% (v/v) HTL-AP in rich media. In this work, adaptive laboratory evolution (ALE) of these strains increased the bulk population tolerance for HTL-AP to up to 30% (v/v) in defined media and up to 35% (v/v) for individual isolates in rich media. The predominate organic acids within HTL-AP (acetic, butyric, and propionic) were rapidly consumed by the evolved Y. lipolytica strains. A TAL-producing isolate (strain 144-3) achieved a nearly 3-fold increase in TAL titer over the parent strain while simultaneously reducing the chemical oxygen demand (COD) of HTL-AP containing media. Fermentation with HTL-AP as the sole nutrient source demonstrated direct conversion of waste into TAL at 10% theoretical yield. Potential genetic mutations of evolved TAL production strains that could be imparting tolerance were explored. This work advances the potential of Y. lipolytica to biologically treat and simultaneously extract value from HTL wastewater. KEY POINTS: • Adaptive evolution of two Y. lipolytica strains enhanced their tolerance to waste. • Y. lipolytica reduces chemical oxygen demand in media containing waste. • Y. lipolytica can produce triacetic acid lactone directly from wastewater.

Intracellular biosensor-based dynamic regulation to manipulate gene expression at the spatiotemporal level.

The use of intracellular, biosensor-based dynamic regulation strategies to regulate and improve the production of useful compounds have progressed significantly over previous decades. By employing such an approach, it is possible to simultaneously realize high productivity and optimum growth states. However, industrial fermentation conditions contain a mixture of high- and low-performance non-genetic variants, as well as young and aged cells at all growth phases. Such significant individual variations would hinder the precise controlling of metabolic flux at the single-cell level to achieve high productivity at the macroscopic population level. Intracellular biosensors, as the regulatory centers of metabolic networks, can real-time sense intra- and extracellular conditions and, thus, could be synthetically adapted to balance the biomass formation and overproduction of compounds by individual cells. Herein, we highlight advances in the designing and engineering approaches to intracellular biosensors. Then, the spatiotemporal properties of biosensors associated with the distribution of inducers are compared. Also discussed is the use of such biosensors to dynamically control the cellular metabolic flux. Such biosensors could achieve single-cell regulation or collective regulation goals, depending on whether or not the inducer distribution is only intracellular.

Enhancing long-term storage and stability of engineered living materials through desiccant storage and trehalose treatment.

Engineered living materials (ELMs) have broad applications for enabling on-demand bioproduction of compounds ranging from small molecules to large proteins. However, most formulations and reports lack the capacity for storage beyond a few months. In this study, we develop an optimized procedure to maximize stress resilience of yeast-laden ELMs through the use of desiccant storage and 10% trehalose incubation before lyophilization. This approach led to over 1-year room temperature storage stability across a range of strain genotypes. In particular, we highlight the superiority of exogenously added trehalose over endogenous, engineered production in yielding robust preservation resilience that is independent of cell state. This simple, effective protocol enables sufficient accumulation of intracellular trehalose over a short period of contact time across a range of strain backgrounds without requiring the overexpression of a trehalose importer. A variety of microscopic analysis including µ-CT and confocal microscopy indicate that cells form spherical colonies within F127-BUM ELMs that have variable viability upon storage. The robustness of the overall procedure developed here highlights the potential for widespread deployment to enable on-demand, cold-chain independent bioproduction.

Modular, Synthetic Boolean Logic Gates Enabled in Saccharomyces cerevisiae through T7 Polymerases/CRISPR dCas9 Designs.

Synthetic control of gene expression, whether simply promoter selection or higher-order Boolean-style logic, is an important tool for metabolic engineering and synthetic biology. This work develops a suite of orthogonal T7 RNA polymerase systems capable of exerting AND/OR switchlike control over transcription in the yeastSaccharomyces cerevisiae. When linked with CRISPR dCas9-based regulation systems, more complex circuitry is possible including AND/OR/NAND/NOR style control in response to combinations of extracellular copper and galactose. Additionally, we demonstrate that these T7 system designs are modular and can accommodate alternative stimuli sensing as demonstrated through blue light induction. These designs should greatly reduce the time and labor necessary for developing Boolean gene circuits in yeast with novel applications including metabolic pathway control in the future.

Valorization of Caribbean Sargassum biomass as a source of alginate and sugars for de novo biodiesel production.

Since 2011, a massive influx of pelagic brown algae Sargassum has invaded coastlines causing environmental and economic disaster. Valorizing this plentiful macroalgae can present much needed economic relief to the areas affected. Here the production of biodiesel and a high-value alginate stream using Sargassum biomass collected from the coast of Quintana Roo, Mexico is reported. Biomass was pretreated via AEA (Alginate Extraction Autohydrolysis) and enzymatic saccharification via fungal Solid State Fermentation, releasing 7 g/L total sugars. The sugar mixture was fermented using engineered Yarrowia lipolytica resulting in 0.35 g/L total lipid titer at the lab tube scale. Additionally, the capability of extracting 0.3875 g/g DW of a high-value, purified alginate stream from this material is demonstrated. The findings presented here are promising and suggest an opportunity for the optimization and scale up of a biodiesel production biorefinery for utilization of Sargassum seaweeds during seasons of high invasion.

Deletion of NGG1 in a recombinant Saccharomyces cerevisiae improved xylose utilization and affected transcription of genes related to amino acid metabolism.

Production of biofuels and biochemicals from xylose using yeast cell factory is of great interest for lignocellulosic biorefinery. Our previous studies revealed that a natural yeast isolate Saccharomyces cerevisiae YB-2625 has superior xylose-fermenting ability. Through integrative omics analysis, NGG1, which encodes a transcription regulator as well as a subunit of chromatin modifying histone acetyltransferase complexes was revealed to regulate xylose metabolism. Deletion of NGG1 in S. cerevisiae YRH396h, which is the haploid version of the recombinant yeast using S. cerevisiae YB-2625 as the host strain, improved xylose consumption by 28.6%. Comparative transcriptome analysis revealed that NGG1 deletion down-regulated genes related to mitochondrial function, TCA cycle, ATP biosynthesis, respiration, as well as NADH generation. In addition, the NGG1 deletion mutant also showed transcriptional changes in amino acid biosynthesis genes. Further analysis of intracellular amino acid content confirmed the effect of NGG1 on amino acid accumulation during xylose utilization. Our results indicated that NGG1 is one of the core nodes for coordinated regulation of carbon and nitrogen metabolism in the recombinant S. cerevisiae. This work reveals novel function of Ngg1p in yeast metabolism and provides basis for developing robust yeast strains to produce ethanol and biochemicals using lignocellulosic biomass.