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Sustainable aviation fuel (SAF) will significantly impact global warming in the aviation sector, and important SAF targets are emerging. Isoprenol is a precursor for a promising SAF compound DMCO (1,4-dimethylcyclooctane) and has been produced in several engineered microorganisms. Recently, Pseudomonas putida has gained interest as a future host for isoprenol bioproduction as it can utilize carbon sources from inexpensive plant biomass. Here, we engineer metabolically versatile host P. putida for isoprenol production. We employ two computational modeling approaches (Bilevel optimization and Constrained Minimal Cut Sets) to predict gene knockout targets and optimize the "IPP-bypass" pathway in P. putida to maximize isoprenol production. Altogether, the highest isoprenol production titer from P. putida was achieved at 3.5 g/L under fed-batch conditions. This combination of computational modeling and strain engineering on P. putida for an advanced biofuels production has vital significance in enabling a bioproduction process that can use renewable carbon streams.

Sustainably grown biomass is a promising alternative to produce fuels and chemicals and reduce the dependency on fossil energy sources. However, the efficient conversion of lignocellulosic biomass into biofuels and bioproducts often requires extensive testing of components and reaction conditions used in the pretreatment, saccharification, and bioconversion steps. This restriction can result in a significant and unwieldy number of combinations of biomass types, solvents, microbial strains, and operational parameters that need to be characterized, turning these efforts into a daunting and time-consuming task. Here we developed a high-throughput feedstocks-to-fuels screening platform to address these challenges. The result is a miniaturized semi-automated platform that leverages the capabilities of a solid handling robot, a liquid handling robot, analytical instruments, and a centralized data repository, adapted to operate as an ionic-liquid-based biomass conversion pipeline. The pipeline was tested by using sorghum as feedstock, the biocompatible ionic liquid cholinium phosphate as pretreatment solvent, a "one-pot" process configuration that does not require ionic liquid removal after pretreatment, and an engineered strain of the yeast Rhodosporidium toruloides that produces the jet-fuel precursor bisabolene as a conversion microbe. By the simultaneous processing of 48 samples, we show that this configuration and reaction conditions result in sugar yields (~70%) and bisabolene titers (~1500 mg/L) that are comparable to the efficiencies observed at larger scales but require only a fraction of the time. We expect that this Feedstocks-to-Fuels pipeline will become an effective tool to screen thousands of bioenergy crop and feedstock samples and assist process optimization efforts and the development of predictive deconstruction approaches.

Engineering metabolism to efficiently produce chemicals from multi-step pathways requires optimizing multi-gene expression programs to achieve enzyme balance. CRISPR-Cas transcriptional control systems are emerging as important tools for programming multi-gene expression, but poor predictability of guide RNA folding can disrupt expression control. Here, we correlate efficacy of modified guide RNAs (scRNAs) for CRISPR activation (CRISPRa) in E. coli with a computational kinetic parameter describing scRNA folding rate into the active structure (rS = 0.8). This parameter also enables forward design of scRNAs, allowing us to design a system of three synthetic CRISPRa promoters that can orthogonally activate (>35-fold) expression of chosen outputs. Through combinatorial activation tuning, we profile a three-dimensional design space expressing two different biosynthetic pathways, demonstrating variable production of pteridine and human milk oligosaccharide products. This RNA design approach aids combinatorial optimization of metabolic pathways and may accelerate routine design of effective multi-gene regulation programs in bacterial hosts.

Self-driving labs (SDLs) combine fully automated experiments with artificial intelligence (AI) that decides the next set of experiments. Taken to their ultimate expression, SDLs could usher a new paradigm of scientific research, where the world is probed, interpreted, and explained by machines for human benefit. While there are functioning SDLs in the fields of chemistry and materials science, we contend that synthetic biology provides a unique opportunity since the genome provides a single target for affecting the incredibly wide repertoire of biological cell behavior. However, the level of investment required for the creation of biological SDLs is only warranted if directed toward solving difficult and enabling biological questions. Here, we discuss challenges and opportunities in creating SDLs for synthetic biology.

Megasynthase enzymes such as type I modular polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) play a central role in microbial chemical warfare because they can evolve rapidly by shuffling parts (catalytic domains) to produce novel chemicals. If we can understand the design rules to reshuffle these parts, PKSs and NRPSs will provide a systematic and modular way to synthesize millions of molecules including pharmaceuticals, biomaterials, and biofuels. However, PKS and NRPS engineering remains difficult due to a limited understanding of the determinants of PKS and NRPS fold and function. We developed ClusterCAD to streamline and simplify the process of designing and testing engineered PKS variants. Here, we present the highly improved ClusterCAD 2.0 release, available at https://clustercad.jbei.org. ClusterCAD 2.0 boasts support for PKS-NRPS hybrid and NRPS clusters in addition to PKS clusters; a vastly enlarged database of curated PKS, PKS-NRPS hybrid, and NRPS clusters; a diverse set of chemical 'starters' and loading modules; the new Domain Architecture Cluster Search Tool; and an offline Jupyter Notebook workspace, among other improvements. Together these features massively expand the chemical space that can be accessed by enzymes engineered with ClusterCAD.

Metabolic fluxes, the number of metabolites traversing each biochemical reaction in a cell per unit time, are crucial for assessing and understanding cell function. 13C Metabolic Flux Analysis (13C MFA) is considered to be the gold standard for measuring metabolic fluxes. 13C MFA typically works by leveraging extracellular exchange fluxes as well as data from 13C labeling experiments to calculate the flux profile which best fit the data for a small, central carbon, metabolic model. However, the nonlinear nature of the 13C MFA fitting procedure means that several flux profiles fit the experimental data within the experimental error, and traditional optimization methods offer only a partial or skewed picture, especially in "non-gaussian" situations where multiple very distinct flux regions fit the data equally well. Here, we present a method for flux space sampling through Bayesian inference (BayFlux), that identifies the full distribution of fluxes compatible with experimental data for a comprehensive genome-scale model. This Bayesian approach allows us to accurately quantify uncertainty in calculated fluxes. We also find that, surprisingly, the genome-scale model of metabolism produces narrower flux distributions (reduced uncertainty) than the small core metabolic models traditionally used in 13C MFA. The different results for some reactions when using genome-scale models vs core metabolic models advise caution in assuming strong inferences from 13C MFA since the results may depend significantly on the completeness of the model used. Based on BayFlux, we developed and evaluated novel methods (P-13C MOMA and P-13C ROOM) to predict the biological results of a gene knockout, that improve on the traditional MOMA and ROOM methods by quantifying prediction uncertainty.

The growing capabilities of synthetic biology and organic chemistry demand tools to guide syntheses toward useful molecules. Here, we present Molecular AutoenCoding Auto-Workaround (MACAW), a tool that uses a novel approach to generate molecules predicted to meet a desired property specification (e.g., a binding affinity of 50 nM or an octane number of 90). MACAW describes molecules by embedding them into a smooth multidimensional numerical space, avoiding uninformative dimensions that previous methods often introduce. The coordinates in this embedding provide a natural choice of features for accurately predicting molecular properties, which we demonstrate with examples for cetane and octane numbers, flash points, and histamine H1 receptor binding affinity. The approach is computationally efficient and well-suited to the small- and medium-size datasets commonly used in biosciences. We showcase the utility of MACAW for virtual screening by identifying molecules with high predicted binding affinity to the histamine H1 receptor and limited affinity to the muscarinic M2 receptor, which are targets of medicinal relevance. Combining these predictive capabilities with a novel generative algorithm for molecules allows us to recommend molecules with a desired property value (i.e., inverse molecular design). We demonstrate this capability by recommending molecules with predicted octane numbers of 40, 80, and 120, which is an important characteristic of biofuels. Thus, MACAW augments classical retrosynthesis tools by providing recommendations for molecules on specification.

Researchers from across the four United States Department of Energy Bioenergy Research Centers (BRCs) engaged in a microbiome workshop that focused on identifying challenges and collaboration opportunities to better understand bioenergy-relevant plant–microbe interactions. The virtual workshop included hands-on educational sessions and a keynote address on current best practices in microbiome science and community microbiome standards, as well as breakout sessions aimed at identifying microbiome-related data and measurements that should be prioritized, opportunities for and barriers to integrating plant metabolites to microbiome research, and strategies for more effectively integrating microbiome data and processes into existing models. Based on participant discussion, key findings of the workshop were the need to prioritize scaling data sharing across BRCs and the broader research community and securing collaborative infrastructure in the areas of microbiome-ecosystem modeling and molecular plant–microbe interactions. This workshop review highlights additional main findings from this event, to encourage cross-site and more holistic metaanalyses while promoting wide scientific community engagement across plant microbiome sciences.

We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on a chip. The microfluidic device contains a 10 × 10 element array, and each element contains sets of electrodes for two electric field-actuated operations: electrowetting for merging droplets to mix reagents and electroporation for transformation. This device can perform up to 100 genetic modification reactions in parallel, providing a scalable platform for generating the large number of engineered strains required for the combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the system's capabilities through the CRISPR-based engineering of two test cases: (1) disruption of the function of the enzyme galactokinase (galK) in E. coli and (2) targeted engineering of the glutamine synthetase gene (glnA) and the blue-pigment synthetase gene (bpsA) to improve indigoidine production in E. coli.

Concerns over climate change have necessitated a rethinking of our transportation infrastructure. One possible alternative to carbon-polluting fossil fuels is biofuels produced by engineered microorganisms that use a renewable carbon source. Two biofuels, ethanol and biodiesel, have made inroads in displacing petroleum-based fuels, but their uptake has been limited by the amounts that can be used in conventional engines and by their cost. Advanced biofuels that mimic petroleum-based fuels are not limited by the amounts that can be used in existing transportation infrastructure but have had limited uptake due to costs. In this Review, we discuss engineering metabolic pathways to produce advanced biofuels, challenges with substrate and product toxicity with regard to host microorganisms and methods to engineer tolerance, and the use of functional genomics and machine learning approaches to produce advanced biofuels and prospects for reducing their costs.

Biology has changed radically in the past two decades, growing from a purely descriptive science into also a design science. The availability of tools that enable the precise modification of cells, as well as the ability to collect large amounts of multimodal data, open the possibility of sophisticated bioengineering to produce fuels, specialty and commodity chemicals, materials, and other renewable bioproducts. However, despite new tools and exponentially increasing data volumes, synthetic biology cannot yet fulfill its true potential due to our inability to predict the behavior of biological systems. Here, we showcase a set of computational tools that, combined, provide the ability to store, visualize, and leverage multiomics data to predict the outcome of bioengineering efforts. We show how to upload, visualize, and output multiomics data, as well as strain information, into online repositories for several isoprenol-producing strain designs. We then use these data to train machine learning algorithms that recommend new strain designs that are correctly predicted to improve isoprenol production by 23%. This demonstration is done by using synthetic data, as provided by a novel library, that can produce credible multiomics data for testing algorithms and computational tools. In short, this paper provides a step-by-step tutorial to leverage these computational tools to improve production in bioengineered strains.

Machine learning provides researchers a unique opportunity to make metabolic engineering more predictable. In this review, we offer an introduction to this discipline in terms that are relatable to metabolic engineers, as well as providing in-depth illustrative examples leveraging omics data and improving production. We also include practical advice for the practitioner in terms of data management, algorithm libraries, computational resources, and important non-technical issues. A variety of applications ranging from pathway construction and optimization, to genetic editing optimization, cell factory testing, and production scale-up are discussed. Moreover, the promising relationship between machine learning and mechanistic models is thoroughly reviewed. Finally, the future perspectives and most promising directions for this combination of disciplines are examined.

Through advanced mechanistic modeling and the generation of large high-quality datasets, machine learning is becoming an integral part of understanding and engineering living systems. Here we show that mechanistic and machine learning models can be combined to enable accurate genotype-to-phenotype predictions. We use a genome-scale model to pinpoint engineering targets, efficient library construction of metabolic pathway designs, and high-throughput biosensor-enabled screening for training diverse machine learning algorithms. From a single data-generation cycle, this enables successful forward engineering of complex aromatic amino acid metabolism in yeast, with the best machine learning-guided design recommendations improving tryptophan titer and productivity by up to 74 and 43%, respectively, compared to the best designs used for algorithm training. Thus, this study highlights the power of combining mechanistic and machine learning models to effectively direct metabolic engineering efforts.

Synthetic biology allows us to bioengineer cells to synthesize novel valuable molecules such as renewable biofuels or anticancer drugs. However, traditional synthetic biology approaches involve ad-hoc engineering practices, which lead to long development times. Here, we present the Automated Recommendation Tool (ART), a tool that leverages machine learning and probabilistic modeling techniques to guide synthetic biology in a systematic fashion, without the need for a full mechanistic understanding of the biological system. Using sampling-based optimization, ART provides a set of recommended strains to be built in the next engineering cycle, alongside probabilistic predictions of their production levels. We demonstrate the capabilities of ART on simulated data sets, as well as experimental data from real metabolic engineering projects producing renewable biofuels, hoppy flavored beer without hops, fatty acids, and tryptophan. Finally, we discuss the limitations of this approach, and the practical consequences of the underlying assumptions failing.

Polyketide synthase (PKS) engineering is an attractive method to generate new molecules such as commodity, fine and specialty chemicals. A significant challenge is re-engineering a partially reductive PKS module to produce a saturated β-carbon through a reductive loop (RL) exchange. In this work, we sought to establish that chemoinformatics, a field traditionally used in drug discovery, offers a viable strategy for RL exchanges. We first introduced a set of donor RLs of diverse genetic origin and chemical substrates  into the first extension module of the lipomycin PKS (LipPKS1). Product titers of these engineered unimodular PKSs correlated with chemical structure similarity between the substrate of the donor RLs and recipient LipPKS1, reaching a titer of 165 mg/L of short-chain fatty acids produced by the host Streptomyces albus J1074. Expanding this method to larger intermediates that require bimodular communication, we introduced RLs of divergent chemosimilarity into LipPKS2 and determined triketide lactone production. Collectively, we observed a statistically significant correlation between atom pair chemosimilarity and production, establishing a new chemoinformatic method that may aid in the engineering of PKSs to produce desired, unnatural products.

Caprolactam is an important polymer precursor to nylon traditionally derived from petroleum and produced on a scale of 5 million tons per year. Current biological pathways for the production of caprolactam are inefficient with titers not exceeding 2 mg/L, necessitating novel pathways for its production. As development of novel metabolic routes often require thousands of designs and result in low product titers, a highly sensitive biosensor for the final product has the potential to rapidly speed up development times. Here we report a highly sensitive biosensor for valerolactam and caprolactam from Pseudomonas putida KT2440 which is >1000× more sensitive to an exogenous ligand than previously reported sensors. Manipulating the expression of the sensor oplR (PP_3516) substantially altered the sensing parameters, with various vectors showing Kd values ranging from 700 nM (79.1 μg/L) to 1.2 mM (135.6 mg/L). Our most sensitive construct was able to detect in vivo production of caprolactam above background at ∼6 μg/L. The high sensitivity and range of OplR is a powerful tool toward the development of novel routes to the biological synthesis of caprolactam.

The Design-Build-Test-Learn (DBTL) cycle, facilitated by exponentially improving capabilities in synthetic biology, is an increasingly adopted metabolic engineering framework that represents a more systematic and efficient approach to strain development than historical efforts in biofuels and biobased products. Here, we report on implementation of two DBTL cycles to optimize 1-dodecanol production from glucose using 60 engineered Escherichia coli MG1655 strains. The first DBTL cycle employed a simple strategy to learn efficiently from a relatively small number of strains (36), wherein only the choice of ribosome-binding sites and an acyl-ACP/acyl-CoA reductase were modulated in a single pathway operon including genes encoding a thioesterase (UcFatB1), an acyl-ACP/acyl-CoA reductase (Maqu_2507, Maqu_2220, or Acr1), and an acyl-CoA synthetase (FadD). Measured variables included concentrations of dodecanol and all proteins in the engineered pathway. We used the data produced in the first DBTL cycle to train several machine-learning algorithms and to suggest protein profiles for the second DBTL cycle that would increase production. These strategies resulted in a 21% increase in dodecanol titer in Cycle 2 (up to 0.83 g/L, which is more than 6-fold greater than previously reported batch values for minimal medium). Beyond specific lessons learned about optimizing dodecanol titer in E. coli, this study had findings of broader relevance across synthetic biology applications, such as the importance of sequencing checks on plasmids in production strains as well as in cloning strains, and the critical need for more accurate protein expression predictive tools.

Metabolic models can estimate intrinsic product yields for microbial factories, but such frameworks struggle to predict cell performance (including product titer or rate) under suboptimal metabolism and complex bioprocess conditions. On the other hand, machine learning, complementary to metabolic modeling necessitates large amounts of data. Building such a database for metabolic engineering designs requires significant manpower and is prone to human errors and bias. We propose an approach to integrate data-driven methods with genome scale metabolic model for assessment of microbial bio-production (yield, titer and rate). Using engineered E. coli as an example, we manually extracted and curated a data set comprising about 1200 experimentally realized cell factories from ~100 papers. We furthermore augmented the key design features (e.g., genetic modifications and bioprocess variables) extracted from literature with additional features derived from running the genome-scale metabolic model iML1515 simulations with constraints that match the experimental data. Then, data augmentation and ensemble learning (e.g., support vector machines, gradient boosted trees, and neural networks in a stacked regressor model) are employed to alleviate the challenges of sparse, non-standardized, and incomplete data sets, while multiple correspondence analysis/principal component analysis are used to rank influential factors on bio-production. The hybrid framework demonstrates a reasonably high cross-validation accuracy for prediction of E.coli factory performance metrics under presumed bioprocess and pathway conditions (Pearson correlation coefficients between 0.8 and 0.93 on new data not seen by the model).

Robust fermentation of biomass-derived sugars into bioproducts demands the reliable microbial expression of metabolic pathways. Plasmid-based expression systems may suffer from instability and result in highly variable titers, rates, and yields. An established mitigation approach, chemical induced chromosomal expansion (CIChE), expands a singly integrated pathway to plasmid-like copy numbers while maintaining stability in the absence of antibiotic selection pressure. Here, we report parallel integration and chromosomal expansion (PIACE), extensions to CIChE that enable independent expansions of pathway components across multiple loci, use suicide vectors to achieve high-efficiency site-specific integration of sequence-validated multigene components, and introduce a heat-curable plasmid to obviate recA deletion post pathway expansion. We applied PIACE to stabilize an isopentenol pathway across three loci in E. coli DH1 and then generate libraries of pathway component copy number variants to screen for improved titers. Polynomial regressor statistical modeling of the production screening data suggests that increasing copy numbers of all isopentenol pathway components would further improve titers.

Genome scale modeling (GSM) predicts the performance of microbial workhorses and helps identify beneficial gene targets. GSM integrated with intracellular flux dynamics, omics, and thermodynamics have shown remarkable progress in both elucidating complex cellular phenomena and computational strain design (CSD). Nonetheless, these models still show high uncertainty due to a poor understanding of innate pathway regulations, metabolic burdens, and other factors (such as stress tolerance and metabolite channeling). Besides, the engineered hosts may have genetic mutations or non-genetic variations in bioreactor conditions and thus CSD rarely foresees fermentation rate and titer. Metabolic models play important role in design-build-test-learn cycles for strain improvement, and machine learning (ML) may provide a viable complementary approach for driving strain design and deciphering cellular processes. In order to develop quality ML models, knowledge engineering leverages and standardizes the wealth of information in literature (e.g., genomic/phenomic data, synthetic biology strategies, and bioprocess variables). Data driven frameworks can offer new constraints for mechanistic models to describe cellular regulations, to design pathways, to search gene targets, and to estimate fermentation titer/rate/yield under specified growth conditions (e.g., mixing, nutrients, and O2). This review highlights the scope of information collections, database constructions, and machine learning techniques (such as deep learning and transfer learning), which may facilitate "Learn and Design" for strain development.

Flowers of the hop plant provide both bitterness and "hoppy" flavor to beer. Hops are, however, both a water and energy intensive crop and vary considerably in essential oil content, making it challenging to achieve a consistent hoppy taste in beer. Here, we report that brewer's yeast can be engineered to biosynthesize aromatic monoterpene molecules that impart hoppy flavor to beer by incorporating recombinant DNA derived from yeast, mint, and basil. Whereas metabolic engineering of biosynthetic pathways is commonly enlisted to maximize product titers, tuning expression of pathway enzymes to affect target production levels of multiple commercially important metabolites without major collateral metabolic changes represents a unique challenge. By applying state-of-the-art engineering techniques and a framework to guide iterative improvement, strains are generated with target performance characteristics. Beers produced using these strains are perceived as hoppier than traditionally hopped beers by a sensory panel in a double-blind tasting.

We present a novel automated flow-based/digital microfluidic platform integrated with onsite electroporation function. In addition to high-throughput arraying of microdroplets and mixing of DNA parts and cells, proposed platform is capable of multiplexed electroporation process and dual optical detection of expressed fluorescence on chip. Unlike conventional microtiter plate based reactions, our platform would allow completely automated and robust genetic engineering steps using drastically smaller amounts of reagents and can be useful for gene editing processes such as CRISPR/Cas9 for synthetic biology applications.

Accelerating the Design-Build-Test-Learn (DBTL) cycle in synthetic biology is critical to achieving rapid and facile bioengineering of organisms for the production of, e.g., biofuels and other chemicals. The Learn phase involves using data obtained from the Test phase to inform the next Design phase. As part of the Learn phase, mathematical models of metabolic fluxes give a mechanistic level of comprehension to cellular metabolism, isolating the principle drivers of metabolic behavior from the peripheral ones, and directing future experimental designs and engineering methodologies. Furthermore, the measurement of intracellular metabolic fluxes is specifically noteworthy as providing a rapid and easy-to-understand picture of how carbon and energy flow throughout the cell. Here, we present a detailed guide to performing metabolic flux analysis in the Learn phase of the DBTL cycle, where we show how one can take the isotope labeling data from a 13C labeling experiment and immediately turn it into a determination of cellular fluxes that points in the direction of genetic engineering strategies that will advance the metabolic engineering process.For our modeling purposes we use the Joint BioEnergy Institute (JBEI) Quantitative Metabolic Modeling (jQMM) library, which provides an open-source, python-based framework for modeling internal metabolic fluxes and making actionable predictions on how to modify cellular metabolism for specific bioengineering goals. It presents a complete toolbox for performing different types of flux analysis such as Flux Balance Analysis, 13C Metabolic Flux Analysis, and it introduces the capability to use 13C labeling experimental data to constrain comprehensive genome-scale models through a technique called two-scale 13C Metabolic Flux Analysis (2S-13C MFA) [1]. In addition to several other capabilities, the jQMM is also able to predict the effects of knockouts using the MoMA and ROOM methodologies. The use of the jQMM library is illustrated through a step-by-step demonstration, which is also contained in a digital Jupyter Notebook format that enhances reproducibility and provides the capability to be adopted to the user's specific needs. As an open-source software project, users can modify and extend the code base and make improvements at will, providing a base for future modeling efforts.

New synthetic biology capabilities hold the promise of dramatically improving our ability to engineer biological systems. However, a fundamental hurdle in realizing this potential is our inability to accurately predict biological behavior after modifying the corresponding genotype. Kinetic models have traditionally been used to predict pathway dynamics in bioengineered systems, but they take significant time to develop, and rely heavily on domain expertise. Here, we show that the combination of machine learning and abundant multiomics data (proteomics and metabolomics) can be used to effectively predict pathway dynamics in an automated fashion. The new method outperforms a classical kinetic model, and produces qualitative and quantitative predictions that can be used to productively guide bioengineering efforts. This method systematically leverages arbitrary amounts of new data to improve predictions, and does not assume any particular interactions, but rather implicitly chooses the most predictive ones.

Although recent advances in synthetic biology allow us to produce biological designs more efficiently than ever, our ability to predict the end result of these designs is still nascent. Predictive models require large amounts of high-quality data to be parametrized and tested, which are not generally available. Here, we present the Experiment Data Depot (EDD), an online tool designed as a repository of experimental data and metadata. EDD provides a convenient way to upload a variety of data types, visualize these data, and export them in a standardized fashion for use with predictive algorithms. In this paper, we describe EDD and showcase its utility for three different use cases: storage of characterized synthetic biology parts, leveraging proteomics data to improve biofuel yield, and the use of extracellular metabolite concentrations to predict intracellular metabolic fluxes.

This study investigates the alignment of research on small island developing states (SIDSs) with sustainable development goals (SDGs), identifying key focus areas, interdisciplinary connections, research gaps, thematic clusters, and insights from case studies. By employing a mixed methods research design that combines quantitative scientometric analysis with qualitative case studies, this unique study examines the nexus between SIDS research and its contribution to SDGs. The findings reveal that SDG 13 (climate action) and SDG 14 (life below water) dominate SIDS research, emphasizing the need to address climate challenges and marine biodiversity. Economic aspects are underscored by SDG 8 (decent work and economic growth) and SDG 12 (responsible consumption and production), while urban development and inequality are addressed through SDG 11 (sustainable cities and communities) and SDG 10 (reduced inequalities). Renewable energy and food security are focal points in SDG 7 (affordable and clean energy) and SDG 2 (zero hunger). Moderate research outputs in SDG 3 (good health and well-being) and SDG 15 (life on land) suggest a holistic approach, while lower activity in SDGs 16 (peace), 6 (clean water), 4 (education), and 5 (gender) presents opportunities for further exploration. SIDS research is highly interdisciplinary, as seen through three distinct SDG networks: foundational social goals, economic and environmental objectives, and environmental preservation. The thematic clusters in SIDS research include environmental challenges and adaptation, public health, and socioeconomic development, and energy policy and environmental economics. Case studies from various SIDSs illustrate the complex interplay between environmental sustainability, economic development, and social well-being, highlighting the importance of tailored approaches, international cooperation, and local community engagement. These findings underscore the need for integrated research strategies to address the complex challenges of SIDSs, emphasizing climate action, sustainable economic practices, and comprehensive development approaches to achieve the SDGs effectively. [ABSTRACT FROM AUTHOR]

BackgroundGlycolysis breakdowns glucose into essential building blocks and ATP/NAD(P)H for the cell, occupying a central role in its growth and bio-production. Among glycolytic pathways, the Entner Doudoroff pathway (EDP) is a more thermodynamically favorable pathway with fewer enzymatic steps than either the Embden-Meyerhof-Parnas pathway (EMPP) or the oxidative pentose phosphate pathway (OPPP). However, Escherichia coli do not use their native EDP for glucose metabolism.ResultsOverexpression of edd and eda in E. coli to enhance EDP activity resulted in only a small shift in the flux directed through the EDP (~20 % of glycolysis flux). Disrupting the EMPP by phosphofructokinase I (pfkA) knockout increased flux through OPPP (~60 % of glycolysis flux) and the native EDP (~14 % of glycolysis flux), while overexpressing edd and eda in this ΔpfkA mutant directed ~70 % of glycolytic flux through the EDP. The downregulation of EMPP via the pfkA deletion significantly decreased the growth rate, while EDP overexpression in the ΔpfkA mutant failed to improve its growth rates due to metabolic burden. However, the reorganization of E. coli glycolytic strategies did reduce glucose catabolite repression. The ΔpfkA mutant in glucose medium was able to cometabolize acetate via the citric acid cycle and gluconeogenesis, while EDP overexpression in the ΔpfkA mutant repressed acetate flux toward gluconeogenesis. Moreover, 13C-pulse experiments in the ΔpfkA mutants showed unsequential labeling dynamics in glycolysis intermediates, possibly suggesting metabolite channeling (metabolites in glycolysis are pass from enzyme to enzyme without fully equilibrating within the cytosol medium).ConclusionsWe engineered E. coli to redistribute its native glycolytic flux. The replacement of EMPP by EDP did not improve E. coli glucose utilization or biomass growth, but alleviated catabolite repression. More importantly, our results supported the hypothesis of channeling in the glycolytic pathways, a potentially overlooked mechanism for regulating glucose catabolism and coutilization of other substrates. The presence of channeling in native pathways, if proven true, would affect synthetic biology applications and metabolic modeling.

The collaboration between JBEI and Abengoa will most likely lead to new metabolically engineered microorganisms and perhaps even novel fuel molecules. If the features of these new molecules suggest potential improvements, the next step would be to scale-up the production in a pilot plant and later in a demonstration facility affiliated with Abengoa Bioenergy NT in different US locations to demonstrate the feasibility of the commercial process. It then intends to implement these solutions on a commercial scale where economically promising in the US. Both steps would lead to the creation of new farming, construction, supply chain, and plant operation jobs in the US.

Understanding the complex interactions that occur between heterologous and native biochemical pathways represents a major challenge in metabolic engineering and synthetic biology. We present a workflow that integrates metabolomics, proteomics, and genome-scale models of Escherichia coli metabolism to study the effects of introducing a heterologous pathway into a microbial host. This workflow incorporates complementary approaches from computational systems biology, metabolic engineering, and synthetic biology; provides molecular insight into how the host organism microenvironment changes due to pathway engineering; and demonstrates how biological mechanisms underlying strain variation can be exploited as an engineering strategy to increase product yield. As a proof of concept, we present the analysis of eight engineered strains producing three biofuels: isopentenol, limonene, and bisabolene. Application of this workflow identified the roles of candidate genes, pathways, and biochemical reactions in observed experimental phenomena and facilitated the construction of a mutant strain with improved productivity. The contributed workflow is available as an open-source tool in the form of iPython notebooks.