This protocol details steps in high-throughput measurement of isoprenol (IP) with the Agilent RapidFire 400 coupled with Agilent 6460 QqQ system, and subsequent data processing using the Agilent Quantitative analysis software. This analytical assay provides accurate and high-throughput quantitation of IP from a broad variety of sample types, such as microbial fermentation cultures, chemical overlays, intracellular cell lysate, etc. The protocol provides flexible options for measurements of small number of samples or ultra high-throughput screening of thousands samples per day.

Polyketide synthases (PKSs) are modular enzymes with exceptional potential as biocatalysts for producing non-native compounds. Here, we report the first PKS-based pathway for adipic acid (AA), an industrial monomer for nylon production, by engineering one of the most extensively hybridized PKS systems to date. Using a retrobiosynthetic approach, we identified EtnB, a succinyl-CoA-loading module that uniquely retains the terminal carboxyl group, enabling access to dicarboxylic polyketide products, rarely produced by canonical PKSs. EtnB was coupled to a fully reducing extension module through an engineered communication linker, which improved ACP-KS interactions, enhanced titers, and demonstrated selective succinyl-CoA loading in vivo. This construct integrates genes from five organisms─with domains from seven PKS modules joined across six non-natural junctions─and functions in both Escherichia coli and Pseudomonas putida. Additional engineering that included AT domain exchanges, optimization of extender unit supply, and host strain metabolic rewiring further increased AA production. Together, this work demonstrates that highly chimeric PKSs can be rendered functional through rational design, expands the PKS toolkit with a carboxyl-retaining loading module, and establishes a versatile platform for engineering diacids and other noncanonical products through PKS pathways.

Groundwater acidification co-occurring with nitrate pollution is a common, global environmental health hazard. Denitrifying bacteria have been leveraged for the in situ removal of nitrate in groundwater. However, co-existing stressors-such as low pH-reduce the efficacy of biological removal processes. Castellaniella sp. str. MT123 is a complete denitrifier that was isolated from acidic, nitrate-contaminated groundwater. The strain grows robustly by nitrate respiration at pH < 6.0, completely reducing nitrate to dinitrogen gas. Genomic analyses of MT123 revealed few previously characterized acid tolerance genes. Thus, we utilized a combination of proteomics, metabolomics, and competitive mutant fitness to characterize the genetic mechanisms of MT123 acclimation to growth under mildly acidic conditions. We found that glutamate accumulation is critical in the acid acclimation of MT123, possibly through consumption of intracellular protons via glutamate decarboxylation to GABA. This is despite the fact that MT123 lacks the canonical glutamate decarboxylase-glutamate/GABA antiporter system implicated in acid tolerance in other bacteria. In contrast, branched-chain amino acid (BCAA) accumulation was detrimental to cell growth at lower pHs, possibly through indirect mechanisms impacting the cellular glutamate pool. Genetic analysis previously linked MT123 to a population of Castellaniella that bloomed-concurrent to nitrate removal-during a biostimulation effort to reduce groundwater nitrate concentrations at MT123's location of origin. Thus, our analyses provide novel insight into mechanisms of acclimation to acidic conditions in a strain with significant potential for nitrate bioremediation.IMPORTANCENitrate pollution in groundwater is a major threat to both environmental and human health. This nitrate pollution can come from a variety of sources, including farm fertilizers, sewage, animal waste, septic systems, and industrial discharge. Bacteria known as "denitrifiers" can convert this nitrate into harmless nitrogen gas, a process known as "denitrification." Denitrifiers can be used to clean up nitrate-contaminated groundwater. However, their ability to do this can be disrupted by changing environmental conditions. For example, groundwater that is polluted with nitrate is often acidic. Acidic conditions make it challenging for denitrifiers to survive, which results in less conversion of nitrate to nitrogen gas. In this study, we investigated how one denitrifying bacterium-originating from acidic, nitrate-contaminated groundwater-can cope with acidic conditions.

Reducing carbon emissions from aviation and long-distance transportation sectors requires the development of sustainable biofuels with suitable energy density, freezing point, and other physical properties. We previously demonstrated biological production of high energy polycyclopropanated fatty acids (POP-FAs, class I) using an iterative polyketide synthase (iPKS) pathway in a Streptomyces host. Here, we used a computational model of fuel properties to identify chain length and cyclopropanation control as critical steps to engineer this iPKS for biofuel applications. We next explored the natural diversity of POP biosynthesis by investigating homologous pathways. Then, by in vivo gene exchange, we determined cyclopropanase (CP) catalysis to be key for POP-FA engineering. Leveraging both natural and engineered pathway product diversity, we demonstrate targeted production of improved POP-FAs, namely shortened POP-FAs with predicted superior freezing point properties for aviation, as well as fully cyclopropane-saturated POP-FAs which should have superior energy-density. These precise and controllable modifications to POP-FA structure open the door for bioproduction of designer POP fuels.

This protocol details steps in high-throughput measurement of isoprenyl acetate (IPA) with the Agilent RapidFire 400 coupled with the Agilent 6460 QqQ system, and subsequent data processing using the Agilent Quantitative Analysis software. This analytical assay provides accurate and high-throughput quantitation of IPA from a wide variety of sample types, such as microbial fermentation cultures, chemical overlays, intracellular cell lysate, etc. The protocol provides flexible options for measurements of a small number of samples or ultra-high-throughput screening of thousands of samples per day.

Efficient co-utilization of hexose and pentose sugars from lignocellulose is essential for microbial production of bio-based chemicals, yet engineered non-native catabolic pathways can be suboptimal and evolutionarily unstable in complex resource environments. We used a Pseudomonas putida strain, previously engineered to catabolize xylose and arabinose to examine how resource abundance, temporal availability, and sub-culturing criteria shape evolutionary outcomes. Using an automated adaptive laboratory evolution (ALE) platform, we evolved the strain under static conditions with single selection pressures and dynamic regimes that imposed selection pressures on multiple sugars. These environments drove divergence between catabolic specialists and generalists. While selection regimes with weak or absent selection for xylose frequently resulted in loss of xylose catabolism, evolution under carbon-limited, mixed-sugar environments promoted stable retention and coordinated optimization of multiple catabolic pathways, increasing total sugar consumption in mixed-sugar conditions. Genomic, proteomic, and biochemical analyses showed that evolutionary stability was determined by pathway-specific fitness costs, leading to either pathway loss or cost-reducing refinement, depending on selection strength. An isolated generalist clone also exhibited improved indigoidine production from mixed sugars when compared to the parental strain. Together, these findings link resource dynamics to fitness landscapes that determine catabolic specialization, generalization, evolutionary trade-offs, and applicability to bioconversion. ### Competing Interest Statement The authors have declared no competing interest. Joint BioEnergy Institute, https://ror.org/03ww55028, DE-AC02-05CH11231 National Renewable Energy Laboratory, DE-AC36-08GO28308 Oak Ridge National Laboratory, DE-AC05-00OR22725 National Research Foundation of Korea, RS-2024-00334792, RS-2025-02215308

ABSTRACT Acetate is an attractive renewable two-carbon substrate for microbial biotechnology, but its toxicity limits growth and carbon-use efficiency at process-relevant concentrations. Here, we used adaptive laboratory evolution to improve acetate tolerance in a genome-reduced strain of Pseudomonas putida and combined whole-genome sequencing, reverse engineering, transcriptomics, proteomics, and 13 C-acetate fluxomics to resolve the underlying adaptation mechanisms. Evolution under increasing acetate concentrations selected recurrent mutations in gacA and fabB , which encode a global response regulator and a fatty acid biosynthesis enzyme, respectively. Reverse engineering of these mutations recovered most of the evolved phenotype, including shorter lag phase and substantially higher biomass yield from acetate. Multi-omic analyses showed repression of type VI secretion systems, carbohydrate storage functions, fatty acid metabolism, and oxidative stress-associated proteins, indicating resource reallocation away from costly stress and non-essential programs. Fluxomics further revealed reduced EDEMP cycling and increased glyoxylate shunt flux, consistent with improved acetate-carbon retention in biomass. These results establish acetate tolerance in P . putida as a resource-efficiency phenotype and identify gacA and fabB as actionable targets for acetate-based bioproduction.

The structural biology method of X-ray footprinting mass spectrometry (XFMS) is available at two national synchrotron beamlines in the USA: one at the Advanced Light Source (ALS) on the West Coast and the other at the National Synchrotron Light Source II on the East Coast. XFMS is a solution-state technique that utilizes oxidative modifications of proteins at micromolar concentrations in aqueous buffer to extract structural information. X-rays are employed to generate hydroxyl radicals in situ , which covalently modify specific protein side chains. These modifications are subsequently quantified using liquid chromatography and mass spectrometry. Ratiometric changes in modification levels between two protein states ( e.g. with and without ligand) generate a relative solvent accessibility map of the protein pairs, which serves to reveal structural features. Up until recently, the XFMS capability was available as part of a shared program at the ALS without a dedicated beamline. In this article, we describe the commissioning of ALS beamline 3.3.1, dedicated to XFMS, including the installation of a new focusing mirror, the design and construction of a new endstation with automated sample handling and exposure capabilities, and the use of accurate empirical dose calculations using Gafchromic film. Finally, we showcase the new beamline capabilities using two protein systems.

Abstract Efficient co-utilization of hexose and pentose sugars from lignocellulose is essential for microbial bioconversion, yet engineered catabolic pathways can be unstable or suboptimal in complex resource environments. Here, we use a Pseudomonas putida strain engineered to catabolize xylose and arabinose to examine how resource abundance, temporal availability, and subculturing shape evolutionary outcomes. Using an automated adaptive laboratory evolution (ALE) platform, we evolve the strain under simple single-substrate and complex multi-substrate selection pressures. These environments drive divergence between catabolic specialists and generalists. Weak or absent selection for xylose frequently leads to loss of xylose catabolism, whereas carbon-limited mixed-sugar environments promote stable retention and coordinated optimization of multiple catabolic pathways, enhancing growth and substrate utilization. Genomic, proteomic, and biochemical analyses show that pathway-specific fitness costs determine evolutionary stability. A generalist clone also shows improved indigoidine production from mixed sugars relative to the parental strain. Together, these findings show how resource dynamics shape fitness landscapes that govern catabolic specialization, generalization, evolutionary trade-offs, and engineering of bioconversion.

Artificial metalloenzymes (ArMs) expand the suite of synthetically valuable, new-to-nature biocatalytic reactions. Integrating these enzymes into biosynthetic pathways enables reactions not found in nature to occur in living cells with the intermediates or products of the metabolic pathways. However, the integration of reactions catalyzed by ArMs into complex metabolic pathways is constrained by the lack of methods to assemble these ArMs in organisms that are commonly used for metabolic engineering. We report the assembly of an iridium-containing artificial metalloenzyme (Ir-ArM) in Streptomyces albus, a Gram-positive bacterial chassis widely used for the heterologous expression of natural products. In this engineered organism, the Ir-ArM assembles in the cytoplasm and catalyzes abiological carbene transfer to the unactivated, disubstituted double bond of an exogenously added terpene with turnover numbers (TONs) that are two times higher than those for the same reaction catalyzed within E. coli cells harboring Ir-ArM and 20 times higher than the TONs for the same reaction catalyzed by the purified holoprotein itself.

Isoprenyl acetate, a volatile ester derived from isoprenol, is a key biosynthetic intermediate for the advanced aviation fuel candidate, 1,4-dimethylcyclooctane. Here, we engineered Pseudomonas putida KT2440 for the production of isoprenyl acetate from mixed sugar substrates. We first generated isoprenyl acetate by introducing a heterologous alcohol acetyltransferase (ATF1) and deleting three promiscuous native esterases to reduce product degradation. Then, we engineered efficient glucose and xylose co-utilization by integrating a heterologous xylose isomerase pathway and deleting global regulators crc and hexR to alleviate catabolite repression. Additionally, intracellular acetyl-CoA flux was reinforced through the expression of auxiliary carbon-conserving routes, including non-oxidative glycolysis and acetate assimilation. Culture conditions were systematically optimized by adjusting medium composition, induction, and overlay solvent to maximize product yields and titers. These cumulative efforts achieved isoprenyl acetate titers of 1.5 g/L in shake flasks and 1.9 g/L in fed-batch bioreactor cultures from mixed sugars, corresponding to a yield of 0.067 g/g of total sugar consumed. Our work demonstrates the potential of P. putida as a robust microbial chassis for scalable biosynthesis of ester-based biofuels from lignocellulosic feedstocks.

Metabolic engineering to produce molecules not naturally synthesized by the host often requires directed evolution to improve pathway enzyme performance. Growth-coupled selection can dramatically increase directed-evolution throughput, and manipulation of redox balance has proven effective for tying reductase fitness to microbial growth. However, most redox-balance selections require feeding the reductase substrate because of stoichiometric constraints. This is impractical for many biosynthetic pathways either due to practical limitations on cost or complexity of bulk substrate synthesis, or the lack of an ability to transport substrate into cells, for example intracellular acyl-CoA/ACP intermediates. Here we define stoichiometric constraints that make substrate feeding necessary for many acetyl-CoA-derived reduction pathways in NADPH-imbalanced hosts. We overcome these constraints with a dual-feedstock strategy in which glucose provides reducing power while acetate supplies additional acetyl-CoA without directly perturbing redox balance. In an engineered Escherichia coli selection strain, acetate co-feeding enabled growth coupling of acetaldehyde, 3-hydroxybutyrate, and mevalonate production and produced a linear correlation between product formation and growth. We then used this selection to evolve a class II HMG-CoA reductase (HMGR) from Delftia acidovorans toward NADPH utilization, enriching variants with improved NADPH-dependent activity. Finally, propionate co-feeding enabled growth coupling of propionyl-CoA reduction, supporting the generality of carbon co-feeding for selecting enzymes in pathways involving acyl-chain elongation and reduction.

Abstract Background To strengthen the national energy supply, there is an increasing demand for domestically generated aviation fuels. Bio-derived advanced aviation fuels offer the opportunity to meet this domestic need while presenting a unique opportunity to investigate the production of novel aviation fuels. Isoprenol, a chemical precursor to such novel fuels, has been shown to be a biologically producible compound in model organisms, but its bio-producibility needs to be further explored in organisms more compatible with industrial bioproduction. Results In this work, we evaluate isoprenol production using the promising bioproduction yeast, Rhodosporidium toruloides. First, we show successful isoprenol production using the IPP-bypass pathways most successful in laboratory strains of E. coli and S. cerevisiae. Next, we demonstrate that increased flux through the mevalonate pathway only modestly increases isoprenol titers. Using proteomics, we identified a potential bottleneck in production at the final step in the IPP-bypass pathway and explored alternative enzymes for this step. Finally, the top three strains of R. toruloides were evaluated in sorghum hydrolysates generated using cholinium lysinate. Through this work, 93.1 mg/L of isoprenol was produced in mock medium and 27.3 mg/L in sorghum hydrolysates. Conclusion Together these results lay the foundation for future work for the production of isoprenol from bioproduction crops.

Plastics derived from fossil feedstocks pose major recycling challenges, particularly crosslinked thermosets used in electronics, construction and composites. Polydiketoenamines (PDKs) are recyclable alternatives; however, monomers such as dimedone are petrochemical-derived and offer limited tunability. We computationally screened 144 β-keto-δ-lactones (BKDLs), identifying solvation free energy as the primary determinant of depolymerization temperature across a 20-60 °C range. We engineered hybrid type I polyketide synthases (PKSs) in Escherichia coli and Streptomyces hosts to biosynthesize BKDLs with diverse substituents and defined stereochemistry, reaching titers of 1.84 g L-1 in bioreactors. Polymerization of chemically synthesized BKDLs identical to PKS products confirmed tunable glass transition temperatures (53-98 °C) and temperature-gated depolymerization. Different BKDLs yielded PDKs with thermal, mechanical, solvent-resistance and optical properties governed by substituent and chirality. Technoeconomic and life-cycle analyses indicate that corn-stover-derived BKDLs can outperform petrochemical dimedone on cost and greenhouse gas emissions. This study demonstrates that engineered PKSs can produce monomers for recyclable plastics with programmable depolymerization behavior.

Abstract 2,4,6-Trinitrotoluene (TNT) is a recalcitrant and pervasive environmental pollutant. Although different environmental microbes have demonstrated their ability to degrade or transform TNT, the underlying genetic basis and cellular machinery remain unclear. In this study, we investigated bacterial strategies in response to TNT exposure in Pantoea sp. MT58 and P. putida KT2440 using proteomics and random barcode transposon-site sequencing (RB-TnSeq). Pantoea sp. MT58 was found to utilize TNT as a sole nitrogen source, whereas P. putida KT2440 exhibited only stress tolerance without assimilation. Pantoea sp. MT58 encodes multiple putative nitroreductases that were upregulated, yet deletion of these genes did not affect growth on TNT, revealing pathway redundancy. Furthermore, fitness profiling provided no evidence for genes involved in the canonical Meisenheimer-complex pathway associated with nitrite release. Instead, the data are most consistent with a sequential nitro-group reduction route in which nitrogen is ultimately recovered as ammonium, with nitrogen routed through the GS-GOGAT pathway with purine and urea pools as the candidate buffering architecture for TNT mineralization. Conversely, P. putida KT2440 relied on Ttg/RND efflux pumps and toluene tolerance proteins for survival without nitrogen assimilation from TNT. This work distinguishes routes for productive nitrogen assimilation from those involved in nitroaromatic tolerance, expanding the mechanistic understanding of anthropogenic compound metabolism to inform future bioremediation efforts.

Agrobacterium is not only a costly plant pathogen but is also an essential tool for plant transformation. Though Agrobacterium-mediated transformation (AMT) has been heavily studied, its polygenic nature and complex transcriptional regulation make identification of the genetic basis of transformational efficiency difficult through traditional genetic and bioinformatic approaches. Here, we use a bottom-up synthetic approach to systematically engineer the tumor-inducing plasmid (pTi), wherein the majority of virulence machinery is encoded. Using a validated toolkit to control Agrobacterium gene expression in planta, we perform a quantitative dissection of AMT to investigate the contributions of critical vir-genes at different expression levels. We construct a synthetic pTi capable of transient plant and stable fungal transformation and characterize bottlenecks and solutions for complex polygenic synthetic pTi designs. Our reductionist approach demonstrates how bottom-up engineering can be used to dissect and elucidate the genetic underpinnings of complex biological traits, laying the foundation for future engineering to establish full synthetic control over the critical process of AMT.

CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, its application is often constrained by two challenges: (i) rationally identifying effective gene targets for downregulation and (ii) efficiently constructing multiplexed CRISPRi systems. In this study, we address both challenges by integrating a computational prioritization tool with a versatile assembly method for building multiplexed CRISPRi systems. FluxRETAP (Flux-Reaction Target Prioritization) accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers in Pseudomonas putida KT2440, outperforming a conventional non-computational, pathway-guided target selection. The highest isoprenol titer of nearly 1.5 g/L was achieved by knocking down PP_4118 (a gene encoding α-ketoglutarate dehydrogenase). The use of VAMMPIRE (Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation) enabled accurate assembly of CRISPRi constructs containing up to five sgRNA arrays, reducing context dependency and achieving uniform, position-independent gene downregulation. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.

Clostridium butyricum (C. butyricum) is a notable butyrate-producing intestinal probiotic that has been utilized for the enhancement and treatment of various intestinal and extraintestinal diseases. Butyrate and acetate, as significant metabolites of this bacterium, are short-chain fatty acids (SCFAs) that play crucial roles in maintaining intestinal stability and safeguarding host health. However, the regulatory functions of lysine butyrylation (Kbu) and acetylation (Kac), mediated by these metabolites, remain inadequately understood. In this study, we performed a comprehensive dynamic multiomic analysis encompassing protein expression, Kbu, and Kac in C. butyricum. Our multiomic analysis identified a total of 2622 proteins, 1887 Kbu sites, and 2188 Kac sites. Subsequent bioinformatic analyses revealed that biological functions such as gene expression and energy metabolism exhibited dynamic changes throughout the growth cycle at both the proteome and post-translational modification levels. Enzymatic experiments demonstrated that Kbu modified key active sites of important substrates, including alanine-tRNA ligase at K794, GMP synthase at K384, and probable butyrate kinase at K269. This study enhances the current understanding of lysine acylations in bacteria and focuses on elucidating the finely tuned regulatory mechanisms of Kbu during the growth and development of C. butyricum.