Novel aspects of ethylene glycol catabolism.

Ethylene glycol (EG) is an industrially important two-carbon diol used as a solvent, antifreeze agent, and building block of polymers such as poly(ethylene terephthalate) (PET). Recently, the use of EG as a starting material for the production of bio-fuels or bio-chemicals is gaining attention as a sustainable process since EG can be derived from materials not competing with human food stocks including CO2, syngas, lignocellulolytic biomass, and PET waste. In order to design and construct microbial process for the conversion of EG to value-added chemicals, microbes capable of catabolizing EG such as Escherichia coli, Pseudomonas putida, Rhodococcus jostii, Ideonella sakaiensis, Paracoccus denitrificans, and Acetobacterium woodii are candidates of chassis for the construction of synthetic pathways. In this mini-review, we describe EG catabolic pathways and catabolic enzymes in these microbes, and further review recent advances in microbial conversion of EG to value-added chemicals by means of metabolic engineering. KEY POINTS: • Ethylene glycol is a potential next-generation feedstock for sustainable industry. • Microbial conversion of ethylene glycol to value-added chemicals is gaining attention. • Ethylene glycol-utilizing microbes are useful as chassis for synthetic pathways.

Pseudomonas putida Dynamics of Adaptation under Prolonged Resource Exhaustion.

Many nonsporulating bacterial species survive prolonged resource exhaustion, by entering a state termed long-term stationary phase. Here, we performed long-term stationary phase evolutionary experiments on the bacterium Pseudomonas putida, followed by whole-genome sequencing of evolved clones. We show that P. putida is able to persist and adapt genetically under long-term stationary phase. We observed an accumulation of mutations within the evolving P. putida populations. Within each population, independently evolving lineages are established early on and persist throughout the 4-month-long experiment. Mutations accumulate in a highly convergent manner, with similar loci being mutated across independently evolving populations. Across populations, mutators emerge, that due to mutations within mismatch repair genes developed a much higher rate of mutation than other clones with which they coexisted within their respective populations. While these general dynamics of the adaptive process are quite similar to those we previously observed in the model bacterium Escherichia coli, the specific loci that are involved in adaptation only partially overlap between P. putida and E. coli.

ATP biosensor reveals microbial energetic dynamics and facilitates bioproduction.

Adenosine-5'-triphosphate (ATP), the primary energy currency in cellular processes, drives metabolic activities and biosynthesis. Despite its importance, understanding intracellular ATP dynamics' impact on bioproduction and exploiting it for enhanced bioproduction remains largely unexplored. Here, we harness an ATP biosensor to dissect ATP dynamics across different growth phases and carbon sources in multiple microbial strains. We find transient ATP accumulations during the transition from exponential to stationary growth phases in various conditions, coinciding with fatty acid (FA) and polyhydroxyalkanoate (PHA) production in Escherichia coli and Pseudomonas putida, respectively. We identify carbon sources (acetate for E. coli, oleate for P. putida) that elevate steady-state ATP levels and boost FA and PHA production. Moreover, we employ ATP dynamics as a diagnostic tool to assess metabolic burden, revealing bottlenecks that limit limonene bioproduction. Our results not only elucidate the relationship between ATP dynamics and bioproduction but also showcase its value in enhancing bioproduction in various microbial species.

Inactivation of Pseudomonas putida KT2440 pyruvate dehydrogenase relieves catabolite repression and improves the usefulness of this strain for degrading aromatic compounds.

Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl-CoA, which feeds the tricarboxylic acid cycle. We investigated how the loss of PDH affects metabolism in Pseudomonas putida. PDH inactivation resulted in a strain unable to utilize compounds whose assimilation converges at pyruvate, including sugars and several amino acids, whereas compounds that generate acetyl-CoA supported growth. PDH inactivation also resulted in the loss of carbon catabolite repression (CCR), which inhibits the assimilation of non-preferred compounds in the presence of other preferred compounds. Pseudomonas putida can degrade many aromatic compounds, most of which produce acetyl-CoA, making it useful for biotransformation and bioremediation. However, the genes involved in these metabolic pathways are often inhibited by CCR when glucose or amino acids are also present. Our results demonstrate that the PDH-null strain can efficiently degrade aromatic compounds even in the presence of other preferred substrates, which the wild-type strain does inefficiently, or not at all. As the loss of PDH limits the assimilation of many sugars and amino acids and relieves the CCR, the PDH-null strain could be useful in biotransformation or bioremediation processes that require growth with mixtures of preferred substrates and aromatic compounds.

Disproportionate Carbon Dioxide Efflux in Bacterial Metabolic Pathways for Different Organic Substrates Leads to Variable Contribution to Carbon-Use Efficiency.

Microbial organic matter turnover is an important contributor to the terrestrial carbon dioxide (CO2) budget. Partitioning of organic carbons into biomass relative to CO2 efflux, termed carbon-use efficiency (CUE), is widely used to characterize organic carbon cycling by soil microorganisms. Recent studies challenge proposals of CUE dependence on the oxidation state of the substrate carbon and implicate instead metabolic strategies. Still unknown are the metabolic mechanisms underlying variability in CUE. We performed a multiomics investigation of these mechanisms in Pseudomonas putida, a versatile soil bacterium of the Gammaproteobacteria, processing a mixture of plant matter derivatives. Our 13C-metabolomics data captured substrate carbons into different metabolic pathways: cellulose-derived sugar carbons in glycolytic and pentose-phosphate pathways; lignin-related aromatic carbons in the tricarboxylic acid cycle. Subsequent 13C-metabolic flux analysis revealed a 3-fold lower investment of sugar carbons in CO2 efflux compared to aromatic carbons, in agreement with reported substrate-dependent CUE. Proteomics analysis revealed enzyme-level regulation only for substrate uptake and initial catabolism, which dictated downstream fluxes through CO2-producing versus biomass-synthesizing reactions. Metabolic partitioning as shown here explained the substrate-dependent CUE calculated from reported metabolic flux analyses of other bacteria, further supporting a metabolism-guided perspective for predicting the microbial conversion of accessible organic matter to CO2 efflux.

Evaluation of the benefits of plant growth-promoting rhizobacteria and mycorrhizal fungi on biochemical and morphophysiological traits of Aloe barbadensis Mill under water deficit stress.

Aloe barbadensis is a drought-tolerant perennial medicinal plant with both nutritional and cosmetic uses. Drought is one of the main abiotic stresses limiting plant growth and development. However, the use of drought-resistant plants combined with beneficial soil micro-organisms could improve the effectiveness of biological methods to mitigate drought damage. This research aims to evaluate the effects of Funneliformis mosseae (MF), plant growth-promoting rhizobacteria (PGPR) (including Pseudomonas putida and Pantoea agglomerans), and their co-inoculation on the macronutrient status, antioxidant enzyme activities, and other morphophysiological traits of A. barbadensis under four irrigation regimes [25%, 50%, 75% and 100% of water requirement (WR)]. Three harvests were conducted, revealing that inoculation enhanced the survival rate and shoot fresh weight (SFW) compared to the control plants. However, at 25% WR, the SFW was reduced by 43% more than the control. across all harvests, while the PGPR + MF treatment showed increases of more than 19%, 11%, and 17% compared to the control, MF, and PGPR treatments, respectively. The results also showed that A. barbadensis exhibited innate drought tolerance up to a 50% WR level by enhancing physiological defenses, such as antioxidant enzyme activity. Inoculation increased the macronutrient status of the plant at all levels of irrigation regimes especially under severe drought conditions. The highest levels of nitrogen (N) (16.24 mg g-1 DW) and phosphorus (P) (11.29 mg g-1 DW) were observed in the PGPR + MF treatment at 100% WR. The maximum relative water content under MF inoculation and 75% WR (98.24%) (98.24%) was reached. PGPR + MF treatment alleviated drought-induced osmotic stress, as indicated by reduced antioxidant enzyme activities and electrolyte leakage. However, P. putida and P. agglomerans strains alone or in combination with F. mosseae increased plant yield, macronutrient uptake and antioxidant enzyme activity. This study underscores the potential of these PGPR and MF strains as invaluable biological tools for the cultivation of A. barbadensis in regions with severe drought stress.

Biochar supported Pseudomonas putida based globules for effective removal of Bisphenol A with a practical approach.

The widespread and inevitable use of plastic has led to prospective ecological problems through Bisphenol A (BPA), a synthetic chemical in plastic manufacturing. The present study addresses a unique methodology for eliminating BPA using the assistance of Pseudomonas putida. In the present work, biomass was torrefied to generate biochar with highly porous networks that could accommodate the bacterial species for effective colonization and multiplication. The designed biochar-bacterial globules demonstrated the ability to effectively remove BPA (96.88%) at a concentration of up to 2 g/L. The biochar-bacterial globules could effectively adsorb BPA at a low concentration of 20 mg/L. The alteration in pH did not impact the globule's performance, providing additional support for the practical utilization of these globules in polluted water bodies. In addition, the biochar-bacterial globules exhibited superior effectiveness in degradation compared to the standard levels, particularly in saline conditions. The simplicity and effectiveness of the approach make it promising for real-world implementation in addressing ecological problems associated with BPA contamination.

Isolation and characterization of a phage collection against Pseudomonas putida.

The environmental bacterium, Pseudomonas putida, possesses a broad spectrum of metabolic pathways. This makes it highly promising for use in biotechnological production as a cell factory, as well as in bioremediation strategies to degrade various aromatic pollutants. For P. putida to flourish in its environment, it must withstand the continuous threats posed by bacteriophages. Interestingly, until now, only a handful of phages have been isolated for the commonly used laboratory strain, P. putida KT2440, and no phage defence mechanisms have been characterized. In this study, we present a new Collection of Environmental P. putida Phages from Estonia, or CEPEST. This collection comprises 67 double-stranded DNA phages, which belong to 22 phage species and 9 phage genera. Our findings reveal that most phages in the CEPEST collection are more infectious at lower temperatures, have a narrow host range, and require an intact lipopolysaccharide for P. putida infection. Furthermore, we show that cryptic prophages present in the P. putida chromosome provide strong protection against the infection of many phages. However, the chromosomal toxin-antitoxin systems do not play a role in the phage defence of P. putida. This research provides valuable insights into the interactions between P. putida and bacteriophages, which could have significant implications for biotechnological and environmental applications.

XTT assay for detection of bacterial metabolic activity in water-based polyester polyurethane.

Cellular metabolic activity can be detected by tetrazolium-based colorimetric assays, which rely on dehydrogenase enzymes from living cells to reduce tetrazolium compounds into colored formazan products. Although these methods have been used in different fields of microbiology, their application to the detection of bacteria with plastic-degrading activity has not been well documented. Here, we report a microplate-adapted method for the detection of bacteria metabolically active on the commercial polyester polyurethane (PU) Impranil®DLN using the tetrazolium salt 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT). Bacterial cells that are active on PU reduce XTT to a water-soluble orange dye, which can be quantitatively measured using a microplate reader. We used the Pseudomonas putida KT2440 strain as a study model. Its metabolic activity on Impranil detected by our novel method was further verified by Fourier-transform infrared spectroscopy (FTIR) analyses. Measurements of the absorbance of reduced XTT at 470 nm in microplate wells were not affected by the colloidal properties of Impranil or cell density. In summary, we provide here an easy and high-throughput method for screening bacteria active on PU that can be adapted to other plastic substrates.

Comprehensive Proteomics Analysis of Polyhydroxyalkanoate (PHA) Biology in Pseudomonas putida KT2440: The Outer Membrane Lipoprotein OprL is a Newly Identified Phasin.

Pseudomonas putida KT2440 is an important bioplastic-producing industrial microorganism capable of synthesizing the polymeric carbon-rich storage material, polyhydroxyalkanoate (PHA). PHA is sequestered in discrete PHA granules, or carbonosomes, and accumulates under conditions of stress, for example, low levels of available nitrogen. The pha locus responsible for PHA metabolism encodes both anabolic and catabolic enzymes, a transcription factor, and carbonosome-localized proteins termed phasins. The functions of phasins are incompletely understood but genetic disruption of their function causes PHA-related phenotypes. To improve our understanding of these proteins, we investigated the PHA pathways of P.putida KT2440 using three types of experiments. First, we profiled cells grown in nitrogen-limited and nitrogen-excess media using global expression proteomics, identifying sets of proteins found to coordinately increase or decrease within clustered pathways. Next, we analyzed the protein composition of isolated carbonosomes, identifying two new putative components. We carried out physical interaction screens focused on PHA-related proteins, generating a protein-protein network comprising 434 connected proteins. Finally, we confirmed that the outer membrane protein OprL (the Pal component of the Pal-Tol system) localizes to the carbonosome and shows a PHA-related phenotype and therefore is a novel phasin. The combined datasets represent a valuable overview of the protein components of the PHA system in P.putida highlighting the complex nature of regulatory interactions responsive to nutrient stress.

Pseudomonas putida as a platform for medium-chain length α,ω-diol production: Opportunities and challenges.

Medium-chain-length α,ω-diols (mcl-diols) play an important role in polymer production, traditionally depending on energy-intensive chemical processes. Microbial cell factories offer an alternative, but conventional strains like Escherichia coli and Saccharomyces cerevisiae face challenges in mcl-diol production due to the toxicity of intermediates such as alcohols and acids. Metabolic engineering and synthetic biology enable the engineering of non-model strains for such purposes with P. putida emerging as a promising microbial platform. This study reviews the advancement in diol production using P. putida and proposes a four-module approach for the sustainable production of diols. Despite progress, challenges persist, and this study discusses current obstacles and future opportunities for leveraging P. putida as a microbial cell factory for mcl-diol production. Furthermore, this study highlights the potential of using P. putida as an efficient chassis for diol synthesis.

Enhanced chaotrope tolerance and (S)-2-hydroxypropiophenone production by recombinant Pseudomonas putida engineered with Pprl from Deinococcus radiodurans.

Pseudomonas putida is a soil bacterium with multiple uses in fermentation and biotransformation processes. P. putida ATCC 12633 can biotransform benzaldehyde and other aldehydes into valuable α-hydroxyketones, such as (S)-2-hydroxypropiophenone. However, poor tolerance of this strain toward chaotropic aldehydes hampers efficient biotransformation processes. To circumvent this problem, we expressed the gene encoding the global regulator PprI from Deinococcus radiodurans, an inducer of pleiotropic proteins promoting DNA repair, in P. putida. Fine-tuned gene expression was achieved using an expression plasmid under the control of the LacIQ /Ptrc system, and the cross-protective role of PprI was assessed against multiple stress treatments. Moreover, the stress-tolerant P. putida strain was tested for 2-hydroxypropiophenone production using whole resting cells in the presence of relevant aldehyde substrates. P. putida cells harbouring the global transcriptional regulator exhibited high tolerance toward benzaldehyde, acetaldehyde, ethanol, butanol, NaCl, H2 O2 and thermal stress, thereby reflecting the multistress protection profile conferred by PprI. Additionally, the engineered cells converted aldehydes to 2-hydroxypropiophenone more efficiently than the parental P. putida strain. 2-Hydroxypropiophenone concentration reached 1.6 g L-1 upon a 3-h incubation under optimized conditions, at a cell concentration of 0.033 g wet cell weight mL-1 in the presence of 20 mM benzaldehyde and 600 mM acetaldehyde. Product yield and productivity were 0.74 g 2-HPP g-1 benzaldehyde and 0.089 g 2-HPP g cell dry weight-1 h-1 , respectively, 35% higher than the control experiments. Taken together, these results demonstrate that introducing PprI from D. radiodurans enhances chaotrope tolerance and 2-HPP production in P. putida ATCC 12633.

A model-driven approach to upcycling recalcitrant feedstocks in Pseudomonas putida by decoupling PHA production from nutrient limitation.

Bacterial polyhydroxyalkanoates (PHAs) have emerged as promising eco-friendly alternatives to petroleum-based plastics since they are synthesized from renewable resources and offer exceptional properties. However, their production is limited to the stationary growth phase under nutrient-limited conditions, requiring customized strategies and costly two-phase bioprocesses. In this study, we tackle these challenges by employing a model-driven approach to reroute carbon flux and remove regulatory constraints using synthetic biology. We construct a collection of Pseudomonas putida-overproducing strains at the expense of plastics and lignin-related compounds using growth-coupling approaches. PHA production was successfully achieved during growth phase, resulting in the production of up to 46% PHA/cell dry weight while maintaining a balanced carbon-to-nitrogen ratio. Our strains are additionally validated under an upcycling scenario using enzymatically hydrolyzed polyethylene terephthalate as a feedstock. These findings have the potential to revolutionize PHA production and address the global plastic crisis by overcoming the complexities of traditional PHA production bioprocesses.

Genetic Code Expansion in Pseudomonas putida KT2440.

Emerging microorganism Pseudomonas putida KT2440 is utilized for the synthesis of biobased chemicals from renewable feedstocks and for bioremediation. However, the methods for analyzing, engineering, and regulating the biosynthetic enzymes and protein complexes in this organism remain underdeveloped.Such attempts can be advanced by the genetic code expansion-enabled incorporation of noncanonical amino acids (ncAAs) into proteins, which also enables further controls over the strain's biological processes. Here, we give a step-by-step account of the incorporation of two ncAAs into any protein of interest (POI) in response to a UAG stop codon by two commonly used orthogonal archaeal tRNA synthetase and tRNA pairs. Using superfolder green fluorescent protein (sfGFP) as an example, this method lays down a solid foundation for future work to study and enhance the biological functions of KT2440.

Bio-upcycling of even and uneven medium-chain-length diols and dicarboxylates to polyhydroxyalkanoates using engineered Pseudomonas putida.

Bio-upcycling of plastics is an emerging alternative process that focuses on extracting value from a wide range of plastic waste streams. Such streams are typically too contaminated to be effectively processed using traditional recycling technologies. Medium-chain-length (mcl) diols and dicarboxylates (DCA) are major products of chemically or enzymatically depolymerized plastics, such as polyesters or polyethers. In this study, we enabled the efficient metabolism of mcl-diols and -DCA in engineered Pseudomonas putida as a prerequisite for subsequent bio-upcycling. We identified the transcriptional regulator GcdR as target for enabling metabolism of uneven mcl-DCA such as pimelate, and uncovered amino acid substitutions that lead to an increased coupling between the heterologous ß-oxidation of mcl-DCA and the native degradation of short-chain-length DCA. Adaptive laboratory evolution and subsequent reverse engineering unravelled two distinct pathways for mcl-diol metabolism in P. putida, namely via the hydroxy acid and subsequent native ß-oxidation or via full oxidation to the dicarboxylic acid that is further metabolized by heterologous ß-oxidation. Furthermore, we demonstrated the production of polyhydroxyalkanoates from mcl-diols and -DCA by a single strain combining all required metabolic features. Overall, this study provides a powerful platform strain for the bio-upcycling of complex plastic hydrolysates to polyhydroxyalkanoates and leads the path for future yield optimizations.

Bacteria use a catabolic patchwork pathway of apparently recent origin for degradation of the synthetic buffer compound TRIS.

The synthetic buffer compound TRIS (2-amino-2-(hydroxymethyl)propane-1,3-diol) is used in countless applications, and no detailed information on its degradation has been published so far. Herein, we describe the discovery of a complete bacterial degradation pathway for TRIS. By serendipity, a Pseudomonas strain was isolated from sewage sludge that was able to grow with TRIS as only carbon and nitrogen source. Genome and transcriptome analyses revealed two adjacent gene clusters embedded in a mobile genetic element on a conjugative plasmid to be involved in TRIS degradation. Heterologous gene expression revealed cluster I to encode a TRIS uptake protein, a TRIS alcohol dehydrogenase, and a TRIS aldehyde dehydrogenase, catalyzing the oxidation of TRIS into 2-hydroxymethylserine. Gene cluster II encodes a methylserine hydroxymethyltransferase (mSHMT) and a d-serine dehydratase that plausibly catalyze the conversion of 2-hydroxymethylserine into pyruvate. Conjugational plasmid transfer into Pseudomonas putida KT2440 enabled this strain to grow with TRIS and with 2-hydromethylserine, demonstrating that the complete TRIS degradation pathway can be transmitted by horizontal gene transfer. Subsequent enrichments from wastewater purification systems led to the isolation of further TRIS-degrading bacteria from the Pseudomonas and Shinella genera carrying highly similar TRIS degradation gene clusters. Our data indicate that TRIS degradation evolved recently via gene recruitment and enzyme adaptation from multiple independent metabolic pathways, and database searches suggest that the TRIS degradation pathway is now globally distributed. Overall, our study illustrates how engineered environments can enhance the emergence of new microbial metabolic pathways in short evolutionary time scales.

Engineering styrene biosynthesis: designing a functional trans-cinnamic acid decarboxylase in Pseudomonas.

We are interested in converting second generation feedstocks into styrene, a valuable chemical compound, using the solvent-tolerant Pseudomonas putida DOT-T1E as a chassis. Styrene biosynthesis takes place from L-phenylalanine in two steps: firstly, L-phenylalanine is converted into trans-cinnamic acid (tCA) by PAL enzymes and secondly, a decarboxylase yields styrene. This study focuses on designing and synthesizing a functional trans-cinnamic acid decarboxylase in Pseudomonas putida. To achieve this, we utilized the "wholesale" method, involving deriving two consensus sequences from multi-alignments of homologous yeast ferulate decarboxylase FDC1 sequences with > 60% and > 50% identity, respectively. These consensus sequences were used to design Pseudomonas codon-optimized genes named psc1 and psd1 and assays were conducted to test the activity in P. putida. Our results show that the PSC1 enzyme effectively decarboxylates tCA into styrene, whilst the PSD1 enzyme does not. The optimal conditions for the PSC1 enzyme, including pH and temperature were determined. The L-phenylalanine DOT-T1E derivative Pseudomonas putida CM12-5 that overproduces L-phenylalanine was used as the host for expression of pal/psc1 genes to efficiently convert L-phenylalanine into tCA, and the aromatic carboxylic acid into styrene. The highest styrene production was achieved when the pal and psc1 genes were co-expressed as an operon in P. putida CM12-5. This construction yielded styrene production exceeding 220 mg L-1. This study serves as a successful demonstration of our strategy to tailor functional enzymes for novel host organisms, thereby broadening their metabolic capabilities. This breakthrough opens the doors to the synthesis of aromatic hydrocarbons using Pseudomonas putida as a versatile biofactory.

Metabolic Engineering of Pseudomonas putida KT2440 for De Novo Biosynthesis of Vanillic Acid.

Vanillic acid (VA), as a plant-derived phenolic acid compound, has widespread applications and good market prospects. However, the traditional production process cannot meet market demand. In this study, Pseudomonas putida KT2440 was used for de novo biosynthesis of VA. Multiple metabolic engineering strategies were applied to construct these P. putida-based cell factories, including the introduction of a Hs-OMTopt, engineering the cofactor S-adenosylmethionine supply pathway through the overexpression of metX and metH, reforming solubility of Hs-OMTopt, increasing a second copy of Hs-OMTopt, and the optimization of the fermentation medium. The resulting strain, XCS17, de novo biosynthesized 5.4 g/L VA from glucose in a fed-batch fermentation system; this is the highest VA production titer reported up to recently. This study showed that P. putida KT2440 is a robust platform for achieving the effective production of phenolic acids.

Diversity in fatty acid elongation enzymes: The FabB long-chain ß-ketoacyl-ACP synthase I initiates fatty acid synthesis in Pseudomonas putida F1.

The condensation of acetyl-CoA with malonyl-acyl carrier protein (ACP) by ß-ketoacyl-ACP synthase III (KAS III, FabH) and decarboxylation of malonyl-ACP by malonyl-ACP decarboxylase are the two pathways that initiate bacterial fatty acid synthesis (FAS) in Escherichia coli. In addition to these two routes, we report that Pseudomonas putida F1 ß-ketoacyl-ACP synthase I (FabB), in addition to playing a key role in fatty acid elongation, also initiates FAS in vivo. We report that although two P. putida F1 fabH genes (PpfabH1 and PpfabH2) both encode functional KAS III enzymes, neither is essential for growth. PpFabH1 is a canonical KAS III similar to E. coli FabH whereas PpFabH2 catalyzes condensation of malonyl-ACP with short- and medium-chain length acyl-CoAs. Since these two KAS III enzymes are not essential for FAS in P. putida F1, we sought the P. putida initiation enzyme and unexpectedly found that it was FabB, the elongation enzyme of the oxygen-independent unsaturated fatty acid pathway. P. putida FabB decarboxylates malonyl-ACP and condenses the acetyl-ACP product with malonyl-ACP for initiation of FAS. These data show that P. putida FabB, unlike the paradigm E. coli FabB, can catalyze the initiation reaction in FAS.

Genome-scale and pathway engineering for the sustainable aviation fuel precursor isoprenol production in Pseudomonas putida.

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.