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The push for industrial sustainability benefits from the use of enzymes as a replacement for traditional chemistry. Biological catalysts, especially those that have been engineered for increased activity, stability, or novel function, and are often greener than alternative chemical approaches. This Review highlights the role of engineered enzymes (and identifies directions for further engineering efforts) in the application areas of greenhouse gas sequestration, fuel production, bioremediation, and degradation of plastic wastes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) functions in the shikimate pathway which is responsible for the production of aromatic amino acids and precursors of other essential secondary metabolites in all plant species. EPSPS is also the molecular target of the herbicide glyphosate. While some plant EPSPS variants have been characterized with reduced glyphosate sensitivity and have been used in biotechnology, the glyphosate insensitivity typically comes with a cost to catalytic efficiency. Thus, there exists a need to generate additional EPSPS variants that maintain both high catalytic efficiency and high glyphosate tolerance. Here, we create a synthetic yeast system to rapidly study and evolve heterologous EPSP synthases for these dual traits. Using known EPSPS variants, we first validate that our synthetic yeast system is capable of recapitulating growth characteristics observed in plants grown in varying levels of glyphosate. Next, we demonstrate that variants from mutagenesis libraries with distinct phenotypic traits can be isolated depending on the selection criteria applied. By applying strong dual-trait selection pressure, we identify a notable EPSPS mutant after just a single round of evolution that displays robust glyphosate tolerance (K i of nearly 1 mM) and improved enzymatic efficiency over the starting point (~2.5 fold). Finally, we show the crystal structure of corn EPSPS and the top resulting mutants and demonstrate that certain mutants have the potential to outperform previously reported glyphosate-resistant EPSPS mutants, such as T102I and P106S (denoted as TIPS), in whole-plant testing. Altogether, this platform helps explore the trade-off between glyphosate resistance and enzymatic efficiency., Competing Interests: Competing interests statement:A patent application has been filed relating to the mutants and selection system developed in this study. C.T.L. and S.G. are employees of Bayer Crop Science, a manufacturer of crop protection products. This is listed in the conflict of interest statement.

Electroactive organisms contribute to metal cycling, pollutant removal, and other redox-driven environmental processes. Studying this phenomenon in high-throughput is challenging since extracellular reduction cannot easily be traced back to its cell of origin within a mixed population. Here, we describe the development of a microdroplet emulsion system to enrich EET-capable organisms. We validated our system using the model electroactive organism S. oneidensis and describe the tooling of a benchtop microfluidic system for oxygen-limited processes. We demonstrated enrichment of EET-capable phenotypes from a mixed wild-type and EET-knockout population. As a proof-of-concept application, bacteria were collected from iron sedimentation from Town Lake (Austin, TX) and subjected to microdroplet enrichment. We observed an increase in EET-capable organisms in the sorted population that was distinct when compared to a population enriched in a bulk culture more closely akin to traditional techniques for discovering EET-capable bacteria. Finally, two bacterial species, C. sakazakii and V. fessus not previously shown to be electroactive, were further cultured and characterized for their ability to reduce channel conductance in an organic electrochemical transistor (OECT) and to reduce soluble Fe(III). We characterized two bacterial species not previously shown to exhibit electrogenic behavior. Our results demonstrate the utility of a microdroplet emulsions for identifying putative EET-capable bacteria and how this technology can be leveraged in tandem with existing methods., Competing Interests: Competing Interests The authors declare no competing interests.

Plastic waste is an environmental challenge, but also presents a biotechnological opportunity as a unique carbon substrate. With modern biotechnological tools, it is possible to enable both recycling and upcycling. To realize a plastics bioeconomy, significant intrinsic barriers must be overcome using a combination of enzyme, strain, and process engineering. This article highlights advances, challenges, and opportunities for a variety of common plastics., (© 2024. The Author(s).)

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., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

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., (© 2023 Wiley‐VCH GmbH.)

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.

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., (© 2024. The Author(s).)

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., Competing Interests: HA is an Associate Editor for Synthetic and Systems Biotechnology and was not involved in the editorial review or the decision to publish this article. All authors declare that there are no competing interests., (© 2024 The Authors.)

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 (EC 50  = 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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2023 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

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., (© 2023. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interests The authors declare no competing interests.

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.

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., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: James M. Carothers is an advisor to Wayfinder Biosciences., (© 2023 Published by Elsevier Ltd.)

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.

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.

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2022 Wiley Periodicals LLC.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

β-alanine is an important biomolecule used in nutraceuticals, pharmaceuticals, and chemical synthesis. The relatively eco-friendly bioproduction of β-alanine has recently attracted more interest than petroleum-based chemical synthesis. In this work, we developed two types of in vivo high-throughput screening platforms, wherein one was utilized to identify a novel target ribonuclease E (encoded by rne) as well as a redox-cofactor balancing module that can enhance de novo β-alanine biosynthesis from glucose, and the other was employed for screening fermentation conditions. When combining these approaches with rational upstream and downstream module engineering, an engineered E. coli producer was developed that exhibited 3.4- and 6.6-fold improvement in β-alanine yield (0.85 mol β-alanine/mole glucose) and specific β-alanine production (0.74 g/L/OD 600 ), respectively, compared to the parental strain in a minimal medium. Across all of the strains constructed, the best yielding strain exhibited 1.08 mol β-alanine/mole glucose (equivalent to 81.2% of theoretic yield). The final engineered strain produced 6.98 g/L β-alanine in a batch-mode bioreactor and 34.8 g/L through a whole-cell catalysis. This approach demonstrates the utility of biosensor-enabled high-throughput screening for the production of β-alanine., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

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 yeast Saccharomyces 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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cheng, Wang, Sun, Tang, Bai, Alper and Zhao.)

A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can improve yield. The facile identification of genetically encoded biosensors can overcome this limitation and become part of a general method for engineering scaled production. We have developed a combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors; using RamR as a starting point, we evolve highly specific (>100-fold preference) and sensitive (half-maximum effective concentration (EC 50 ) < 30 μM) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine and noscapine. High-resolution structures reveal multiple evolutionary avenues for the malleable effector-binding site and the creation of new pockets for different chemical moieties. These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine, a precursor to four modern pharmaceuticals, collapsing multiple methylation steps into a single evolved enzyme. Our methods for evolving biosensors enable the rapid engineering of pathways for therapeutic alkaloids., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The growing burden of waste disposal coupled with natural resource scarcity has renewed interest in the remediation, valorization, and/or repurposing of waste. Traditional approaches such as composting, anaerobic digestion, use in fertilizers or animal feed, or incineration for energy production extract very little value out of these waste streams. In contrast, waste valorization into fuels and other biochemicals via microbial fermentation is an area of growing interest. In this review, we discuss microbial valorization of nonconventional, aqueous waste streams such as food processing effluents, wastewater streams, and other industrial wastes. We categorize these waste streams as carbohydrate-rich food wastes, lipid-rich wastes, and other industrial wastes. Recent advances in microbial valorization of these nonconventional waste streams are highlighted, along with a discussion of the specific challenges and opportunities associated with impurities, nitrogen content, toxicity, and low productivity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Plastic waste poses an ecological challenge 1-3 and enzymatic degradation offers one, potentially green and scalable, route for polyesters waste recycling 4 . Poly(ethylene terephthalate) (PET) accounts for 12% of global solid waste 5 , and a circular carbon economy for PET is theoretically attainable through rapid enzymatic depolymerization followed by repolymerization or conversion/valorization into other products 6-10 . Application of PET hydrolases, however, has been hampered by their lack of robustness to pH and temperature ranges, slow reaction rates and inability to directly use untreated postconsumer plastics 11 . Here, we use a structure-based, machine learning algorithm to engineer a robust and active PET hydrolase. Our mutant and scaffold combination (FAST-PETase: functional, active, stable and tolerant PETase) contains five mutations compared to wild-type PETase (N233K/R224Q/S121E from prediction and D186H/R280A from scaffold) and shows superior PET-hydrolytic activity relative to both wild-type and engineered alternatives 12 between 30 and 50 °C and a range of pH levels. We demonstrate that untreated, postconsumer-PET from 51 different thermoformed products can all be almost completely degraded by FAST-PETase in 1 week. FAST-PETase can also depolymerize untreated, amorphous portions of a commercial water bottle and an entire thermally pretreated water bottle at 50 ºC. Finally, we demonstrate a closed-loop PET recycling process by using FAST-PETase and resynthesizing PET from the recovered monomers. Collectively, our results demonstrate a viable route for enzymatic plastic recycling at the industrial scale., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

A variety of yeast species have been considered ideal hosts for metabolic engineering to produce value-added chemicals, including the model organism Saccharomyces cerevisiae , as well as non-conventional yeasts including Yarrowia lipolytica , Kluyveromyces marxianus , and Pichia pastoris . However, the metabolic capacity of these microbes is not simply dictated or implied by genus or species alone. Within the same species, yeast strains can display distinct variations in their phenotypes and metabolism, which affect the performance of introduced pathways and the production of interesting compounds. Moreover, it is unclear how this metabolic potential corresponds to function upon rewiring these organisms. These reports thus point out a new consideration for successful metabolic engineering, specifically: what are the best strains to utilize and how does one achieve effective metabolic engineering? Understanding such questions will accelerate the host selection and optimization process for generating yeast cell factories. In this review, we survey recent advances in studying yeast strain variations and utilizing non-type strains in pathway production and metabolic engineering applications. Additionally, we highlight the importance of employing portable methods for metabolic rewiring to best access this metabolic diversity. Finally, we conclude by highlighting the importance of considering strain diversity in metabolic engineering applications.

Microbial fermentation offers a sustainable source of fuels, commodity chemicals, and pharmaceuticals, yet strain performance is influenced greatly by the growth media selected. Specifically, trace metals (e.g., iron, copper, manganese, zinc, and others) are critical for proper growth and enzymatic function within microorganisms yet are non-standardized across media formulation. In this work, the effect of trace metal supplementation on the lipid production profile of Yarrowia lipolytica was explored using tube scale fermentation followed by biomass and lipid characterization. Addition of iron (II) to the chemically defined Yeast Synthetic Complete (YSC) medium increased final optical density nearly twofold and lipid production threefold, while addition of copper (II) had no impact. Additionally, dose-responsive changes in lipid distribution were observed, with the percent of oleic acid increasing and stearic acid decreasing as initial iron concentration increased. These changes were reversible with subsequent iron-selective chelation. Use of rich Yeast Peptone Dextrose (YPD) medium enabled further increases in the production of two specialty oleochemicals ultimately reaching 63 and 47% of the lipid pool as α-linolenic acid and cyclopropane fatty acid, respectively, compared to YSC medium. Selective removal of iron (II) natively present in YPD medium decreased this oleochemical production, ultimately aligning the lipid profile with that of non-supplemented YSC medium. These results provide further insight into the proposed mechanisms for iron regulation in yeasts especially as these productions strains contain a mutant allele of the iron regulator, mga2. The work presented here also suggests a non-genetic method for control of the lipid profile in Y. lipolytica for use in diverse applications. KEY POINTS: • Iron supplementation increases cell density and lipid titer in Yarrowia lipolytica. • Iron addition reversibly alters lipid portfolio increasing linolenic acid. • Removal of iron from YPD media provides a link to enhanced oleochemical production., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Prokaryotic transcription factors can be repurposed as analytical and synthetic tools for precise chemical measurement and regulation. Monoterpenes encompass a broad chemical family that are commercially valuable as flavors, cosmetics, and fragrances, but have proven difficult to measure, especially in cells. Herein, we develop genetically encoded, generalist monoterpene biosensors by using directed evolution to expand the effector specificity of the camphor-responsive TetR-family regulator CamR from Pseudomonas putida . Using a novel negative selection coupled with a high-throughput positive screen (Seamless Enrichment of Ligand-Inducible Sensors, SELIS), we evolve CamR biosensors that can recognize four distinct monoterpenes: borneol, fenchol, eucalyptol, and camphene. Different evolutionary trajectories surprisingly yielded common mutations, emphasizing the utility of CamR as a platform for creating generalist biosensors. Systematic promoter optimization driving the reporter increased the system's signal-to-noise ratio to 150-fold. These sensors can serve as a starting point for the high-throughput screening and dynamic regulation of bicyclic monoterpene production strains.

Commercial-scale research translation has been muted.

Enzymes are represented across a vast space of protein sequences and structural forms and have activities that far exceed the best chemical catalysts; however, engineering them to have novel or enhanced activity is limited by technologies for sensing product formation. Here, we describe a general and scalable approach for characterizing enzyme activity that uses the metabolism of the host cell as a biosensor by which to infer product formation. Since different products consume different molecules in their synthesis, they perturb host metabolism in unique ways that can be measured by mass spectrometry. This provides a general way by which to sense product formation, to discover unexpected products and map the effects of mutagenesis., (© 2021. The Author(s).)

Sorting large libraries of cells for improved small molecule secretion is throughput limited. Here, we combine producer/secretor cell libraries with whole-cell biosensors using a microfluidic-based screening workflow. This approach enables a mix-and-match capability using off-the-shelf biosensors through either coencapsulation or pico-injection. We demonstrate the cell type and library agnostic nature of this workflow by utilizing single-guide RNA, transposon, and ethyl-methyl sulfonate mutagenesis libraries across three distinct microbes ( Escherichia coli , Saccharomyces cerevisiae , and Yarrowia lipolytica ), biosensors from two organisms ( E. coli and S. cerevisiae ), and three products (triacetic acid lactone, naringenin, and L-DOPA) to identify targets improving production/secretion., Competing Interests: Competing interest statement: X.L. and F.F.C. are employees of Sphere Fluidics Limited.

Metabolic heterogeneity and dynamic changes in metabolic fluxes are two inherent characteristics of microbial fermentation that limit the precise control of metabolisms, often leading to impaired cell growth and low productivity. Dynamic metabolic engineering addresses these challenges through the design of multi-layered and multi-genetic dynamic regulation network (DRN) that allow a single cell to autonomously adjust metabolic flux in response to its growth and metabolite accumulation conditions. Here, we developed a growth coupled NCOMB (Naringenin-Coumaric acid-Malonyl-CoA-Balanced) DRN with systematic optimization of (2S)-naringenin and p-coumaric acid-responsive regulation pathways for real-time control of intracellular supply of malonyl-CoA. In this scenario, the acyl carrier protein was used as a novel critical node for fine-tuning malonyl-CoA consumption instead of direct repression of fatty acid synthase commonly employed in previous studies. To do so, we first engineered a multi-layered DRN enabling single cells to concurrently regulate acpH, acpS, acpT, acs, and ACC in malonyl-CoA catabolic and anabolic pathways. Next, the NCOMB DRN was optimized to enhance the synergies between different dynamic regulation layers via a biosensor-based directed evolution strategy. Finally, a high producer obtained from NCOMB DRN approach yielded a 8.7-fold improvement in (2S)-naringenin production (523.7 ± 51.8 mg/L) with a concomitant 20% increase in cell growth compared to the base strain using static strain engineering approach, thus demonstrating the high efficiency of this system for improving pathway production., (Copyright © 2021 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Cell-free systems using crude cell extracts present appealing opportunities for designing biosynthetic pathways and enabling sustainable chemical synthesis. However, the lack of tools to effectively manipulate the underlying host metabolism in vitro limits the potential of these systems. Here, we create an integrated framework to address this gap that leverages cell extracts from host strains genetically rewired by multiplexed CRISPR-dCas9 modulation and other metabolic engineering techniques. As a model, we explore conversion of glucose to 2,3-butanediol in extracts from flux-enhanced Saccharomyces cerevisiae strains. We show that cellular flux rewiring in several strains of S. cerevisiae combined with systematic optimization of the cell-free reaction environment significantly increases 2,3-butanediol titers and volumetric productivities, reaching productivities greater than 0.9 g/L-h. We then show the generalizability of the framework by improving cell-free itaconic acid and glycerol biosynthesis. Our coupled in vivo/in vitro metabolic engineering approach opens opportunities for synthetic biology prototyping efforts and cell-free biomanufacturing., (© 2021. The Author(s).)

Since long ago, pelagic Sargassum mats have been known to be abundant in the Sargasso Sea, where they provide habitat to diverse organisms. However, over the last few years, massive amounts of pelagic Sargassum have reached the coast of several countries in the Caribbean and West Africa, causing economic and environmental problems. Aiming for lessening the impacts of the blooms, governments and private companies remove the seaweeds from the shore, but this process results expensive. The valorization of this abundant biomass can render Sargassum tides into an economic opportunity and concurrently solve their associated environmental problems. Despite the diverse fields where algae have found applications and the relevance of this recurrent situation, Sargassum biomass remains without large scale applications. Therefore, this review aims to present the potential uses of these algae, identifying the limitations that must be assessed to effectively valorize this bioresource. Due to the constraints identified for each of the presented applications, it is concluded that a biorefinery approach should be developed to effectively valorize this abundant biomass. However, there is an urgent need for investigations focusing on holopelagic Sargassum to be able to truly valorize this seaweed., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Synthetic biology holds great promise for addressing global needs. However, most current developments are not immediately translatable to 'outside-the-lab' scenarios that differ from controlled laboratory settings. Challenges include enabling long-term storage stability as well as operating in resource-limited and off-the-grid scenarios using autonomous function. Here we analyze recent advances in developing synthetic biological platforms for outside-the-lab scenarios with a focus on three major application spaces: bioproduction, biosensing, and closed-loop therapeutic and probiotic delivery. Across the Perspective, we highlight recent advances, areas for further development, possibilities for future applications, and the needs for innovation at the interface of other disciplines.

Food additives and colorants are extensively used in the food industry to improve food quality and safety during processing, storage and packing. Sourcing of these molecules is predominately through three means: extraction from natural sources, chemical synthesis, and bio-production, with the first two being the most utilized. However, growing demands for sustainability, safety and "natural" products have renewed interest in using bio-based production methods. Likewise, the move to more cultured foods and meat alternatives requires the production of new additives and colorants. The production of bio-based food additives and colorants is an interdisciplinary research endeavor and represents a growing trend in future food. To highlight the potential of microbial hosts for food additive and colorant production, we focus on current advances for example molecules based on their utilization stage and bio-production yield as follows: (I) approved and industrially produced with high titers; (II) approved and produced with decent titers (in the g/L range), but requiring further engineering to reduce production costs; (III) approved and produced with very early stage titers (in the mg/L range); and (IV) new/potential candidates that have not been approved but can be sourced through microbes. Promising approaches, as well as current challenges and future directions will also be thoroughly discussed for the bioproduction of these food additives and colorants., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Traditional production of industrial and therapeutic proteins by eukaryotic cells typically requires large-scale fermentation capacity. As a result, these systems are not easily portable or reusable for on-demand protein production applications. In this study, we employ Bioproduced Proteins On Demand (Bio-POD), a F127-bisurethane methacrylate hydrogel-based technique that immobilizes engineered Pichia pastoris for preservable, on-demand production and secretion of medium- and high-molecular weight proteins (in this case, SEAP, α-amylase, and anti-HER2). The gel samples containing encapsulated-yeast demonstrated sustained protein production and exhibited productivity immediately after lyophilization and rehydration. The hydrogel platform described here is the first hydrogel immobilization using a P. pastoris system to produce recombinant proteins of this breadth. These results highlight the potential of this formulation to establish a cost-effective bioprocessing strategy for on-demand protein production., Competing Interests: None of the authors have any conflicts of interest with the work presented., (© 2021 [The Author/The Authors].)

A mutant excision + /integration - piggyBac transposase can be used to seamlessly excise a chromosomally integrated, piggyBac-compatible selection marker cassette from the Yarrowia lipolytica genome. This piggyBac transposase-based genome engineering process allows for both positive selection of targeted homologous recombination events and scarless or footprint-free genome modifications after precise marker recovery. Residual non-native sequences left in the genome after marker excision can be minimized (0-4 nucleotides) or customized (user-defined except for a TTAA tetranucleotide). Both of these options reduce the risk of unintended homologous recombination events in strains with multiple genomic edits. A suite of dual positive/negative selection marker pairs flanked by piggyBac inverted terminal repeats (ITRs) have been constructed and are available for precise genome engineering in Y. lipolytica using this method. This protocol specifically describes the split marker homologous recombination-based disruption of Y. lipolytica ADE2 with a piggyBac ITR-flanked URA3 cassette, followed by piggyBac transposase-mediated excision of the URA3 marker to leave a 50 nucleotide synthetic barcode at the ADE2 locus. The resulting ade2 strain is auxotrophic for adenine, which enables the use of ADE2 as a selectable marker for further strain engineering.

Biotechnology offers a green alternative for the production of fuels and chemicals using microbes. Although traditional model hosts such as Escherichia coli and Saccharomyces cerevisiae have been widely studied and used, they may not be the best hosts for industrial application. In this review, we explore recent advances in the use of nonconventional hosts for the production of a variety of fuel, cosmetics, perfumes, food, and pharmaceuticals. Specifically, we highlight twenty-seven popular molecules with a special focus on recent progress and metabolic engineering strategies to enable improved production of fuels and chemicals. These examples demonstrate the promise of nonconventional host engineering., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Lactams, cyclic carboxamide acids, are important building blocks as monomers for the manufacture of polyamides (nylons), with a market of millions of tons per year. Likewise, their non-natural building blocks, straight chain ω-amino acids, also have a wide range of applications as pharmaceuticals, therapeutic agents, and precursors to other platform chemicals. Current industrial lactam production requires petrochemically-derived routes that involve the use of harsh chemicals and reaction conditions. Microbial production provides a more sustainable method for production, from cost effective renewable resources. This review provides an extensive overview of progress toward the microbial production of lactams, particularly 4C butyrolactam, 5C valerolactam and 6C caprolactam, and their ω-amino acid precursors. Additionally, recent advances in the field as well as proposed microbial production pathways will be discussed, as well as future perspectives for the production of these important bulk chemicals., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Genetically encoded biosensors are extensively utilized in synthetic biology and metabolic engineering. However, reported xylose biosensors are far too sensitive with a limited operating range to be useful for most sensing applications. In this study, we describe directed evolution of Escherichia coli XylR, and construction of biosensors based on XylR and the corresponding operator xylO . The operating range of biosensors containing the mutant XylR was increased by nearly 10-fold comparing with the control. Two individual amino acid mutations (either L73P or N220T) in XylR were sufficient to extend the linear response range to upward of 10 g/L xylose. The evolved biosensors described here are well suited for developing whole-cell biosensors for detecting varying xylose concentrations across an expanded range. As an alternative use of this system, we also demonstrate the utility of XylR and xylO as a xylose inducible system to enable graded gene expression through testing with β-galactosidase gene and the lycopene synthetic pathway. This evolution strategy identified a less-sensitive biosensor for real applications, thus providing new insights into strategies for expanding operating ranges of other biosensors for synthetic biology applications.

Hydrothermal liquefaction is a promising technology to upgrade wet organic waste into a biocrude oil for diesel or jet fuel; however, this process generates an acid-rich aqueous phase which poses disposal issues. This hydrothermal liquefaction aqueous phase (HTL-AP) contains organic acids, phenol, and other toxins. This work demonstrates that Y. lipolytica as a unique host to valorize HTL-AP into a variety of co-products. Specifically, strains of Y. lipolytica can tolerate HTL-AP at 10% in defined media and 25% in rich media. The addition of HTL-AP enhances production of the polymer precursor itaconic acid by 3-fold and the polyketide triacetic acid lactone at least 2-fold. Additional co-products (lipids and citric acid) were produced in these fermentations. Finally, bioreactor cultivation enabled 21.6 g/L triacetic acid lactone from 20% HTL-AP in mixed sugar hydrolysate. These results demonstrate the first use of Y. lipolytica in HTL-AP valorization toward production of a portfolio of value-added compounds., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Most classic genetic approaches utilize binary modifications that preclude the identification of key knockdowns for essential genes or other targets that only require moderate modulation. As a complementary approach to these classic genetic methods, we describe a plasmid-based library methodology that affords bidirectional, graded modulation of gene expression enabled by tiling the promoter regions of all 969 genes that comprise the ito977 model of Saccharomyces cerevisiae' s metabolic network. When coupled with a CRISPR-dCas9-based modulation and next-generation sequencing, this method affords a library-based, bidirection titration of gene expression across all major metabolic genes. We utilized this approach in two case studies: growth enrichment on alternative sugars, glycerol and galactose, and chemical overproduction of betaxanthins, leading to the identification of unique gene targets. In particular, we identify essential genes and other targets that were missed by classic genetic approaches., Competing Interests: The authors declare no competing interest.

Background: Resveratrol is a plant secondary metabolite with diverse, potential health-promoting benefits. Due to its nutraceutical merit, bioproduction of resveratrol via microbial engineering has gained increasing attention and provides an alternative to unsustainable chemical synthesis and straight extraction from plants. However, many studies on microbial resveratrol production were implemented with the addition of water-insoluble phenylalanine or tyrosine-based precursors to the medium, limiting in the sustainable development of bioproduction., Results: Here we present a novel coculture platform where two distinct metabolic background species were modularly engineered for the combined total and de novo biosynthesis of resveratrol. In this scenario, the upstream Escherichia coli module is capable of excreting p-coumaric acid into the surrounding culture media through constitutive overexpression of codon-optimized tyrosine ammonia lyase from Trichosporon cutaneum (TAL), feedback-inhibition-resistant 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroG fbr ) and chorismate mutase/prephenate dehydrogenase (tyrA fbr ) in a transcriptional regulator tyrR knockout strain. Next, to enhance the precursor malonyl-CoA supply, an inactivation-resistant version of acetyl-CoA carboxylase (ACC1 S659A,S1157A ) was introduced into the downstream Saccharomyces cerevisiae module constitutively expressing codon-optimized 4-coumarate-CoA ligase from Arabidopsis thaliana (4CL) and resveratrol synthase from Vitis vinifera (STS), and thus further improve the conversion of p-coumaric acid-to-resveratrol. Upon optimization of the initial inoculation ratio of two populations, fermentation temperature, and culture time, this co-culture system yielded 28.5 mg/L resveratrol from glucose in flasks. In further optimization by increasing initial net cells density at a test tube scale, a final resveratrol titer of 36 mg/L was achieved., Conclusions: This is first study that demonstrates the use of a synthetic E. coli-S. cerevisiae consortium for de novo resveratrol biosynthesis, which highlights its potential for production of other p-coumaric-acid or resveratrol derived biochemicals.