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1. Signal sequences target enzymes and structural proteins to bacterial microcompartments and are critical for microcompartment formation

   https://doi.org/10.1128/msphere.00962-24  

   Johnson, Elizabeth R; Kennedy, Nolan W; Mills, Carolyn E; Liang, Shiqi; Chandrasekar, Swetha; Nichols, Taylor M; Rybnicky, Grant A; Tullman-Ercek, Danielle (May 2025, mSphere)

   Ellermeier, Craig D (Ed.)

   ABSTRACT Spatial organization of pathway enzymes has emerged as a promising tool to address several challenges in metabolic engineering, such as flux imbalances and off-target product formation. Bacterial microcompartments (MCPs) are a spatial organization strategy used natively by many bacteria to encapsulate metabolic pathways that produce toxic, volatile intermediates. Several recent studies have focused on engineering MCPs to encapsulate heterologous pathways of interest, but how this engineering affects MCP assembly and function is poorly understood. In this study, we investigated the role of signal sequences, short domains that target proteins to the MCP core, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterized two novel Pdu signal sequences on the structural proteins PduM and PduB, which constitute the first report of metabolosome signal sequences on structural proteins rather than enzymes. We then explored the role of enzymatic and structural Pdu signal sequences on MCP assembly by deleting their encoding sequences from the genome alone and in combination. Deleting enzymatic signal sequences decreased the MCP formation, but this defect could be recovered in some cases by overexpressing genes encoding the knocked-out signal sequence fused to a heterologous protein. By contrast, deleting structural signal sequences caused similar defects to knocking out the genes encoding the full-length PduM and PduB proteins. Our results contribute to a growing understanding of how MCPs form and function in bacteria and provide strategies to mitigate assembly disruption when encapsulating heterologous pathways in MCPs.IMPORTANCESpatially organizing biosynthetic pathway enzymes is a promising strategy to increase pathway throughput and yield. Bacterial microcompartments (MCPs) are proteinaceous organelles that many bacteria natively use as a spatial organization strategy to encapsulate niche metabolic pathways, providing significant metabolic benefits. Encapsulating heterologous pathways of interest in MCPs could confer these benefits to industrially relevant pathways. Here, we investigate the role of signal sequences, short domains that target proteins for encapsulation in MCPs, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterize two novel signal sequences on structural proteins, constituting the first Pdu signal sequences found on structural proteins rather than enzymes, and perform knockout studies to compare the impacts of enzymatic and structural signal sequences on MCP assembly. Our results demonstrate that enzymatic and structural signal sequences play critical but distinct roles in Pdu MCP assembly and provide design rules for engineering MCPs while minimizing disruption to MCP assembly. 

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2. Microcompartment assembly around multicomponent fluid cargoes

   https://doi.org/10.1063/5.0089556  

   Tsidilkovski, Lev; Mohajerani, Farzaneh; Hagan, Michael_F (June 2022, The Journal of Chemical Physics)

   This article describes dynamical simulations of the assembly of an icosahedral protein shell around a bicomponent fluid cargo. Our simulations are motivated by bacterial microcompartments, which are protein shells found in bacteria that assemble around a complex of enzymes and other components involved in certain metabolic processes. The simulations demonstrate that the relative interaction strengths among the different cargo species play a key role in determining the amount of each species that is encapsulated, their spatial organization, and the nature of the shell assembly pathways. However, the shell protein–shell protein and shell protein–cargo component interactions that help drive assembly and encapsulation also influence cargo composition within certain parameter regimes. These behaviors are governed by a combination of thermodynamic and kinetic effects. In addition to elucidating how natural microcompartments encapsulate multiple components involved within reaction cascades, these results have implications for efforts in synthetic biology to colocalize alternative sets of molecules within microcompartments to accelerate specific reactions. More broadly, the results suggest that coupling between self-assembly and multicomponent liquid–liquid phase separation may play a role in the organization of the cellular cytoplasm. 

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3. Analogous Metabolic Decoupling in Pseudomonas putida and Comamonas testosteroni Implies Energetic Bypass to Facilitate Gluconeogenic Growth

   https://doi.org/10.1128/mbio.03259-21  

   Wilkes, Rebecca A.; Waldbauer, Jacob; Aristilde, Ludmilla (December 2021, mBio)

   Dunn, Anne K.; Ruby, Edward G. (Ed.)

   ABSTRACT Gluconeogenic carbon metabolism is not well understood, especially within the context of flux partitioning between energy generation and biomass production, despite the importance of gluconeogenic carbon substrates in natural and engineered carbon processing. Here, using multiple omics approaches, we elucidate the metabolic mechanisms that facilitate gluconeogenic fast-growth phenotypes in Pseudomonas putida and Comamonas testosteroni , two Proteobacteria species with distinct metabolic networks. In contrast to the genetic constraint of C. testosteroni , which lacks the enzymes required for both sugar uptake and a complete oxidative pentose phosphate (PP) pathway, sugar metabolism in P. putida is known to generate surplus NADPH by relying on the oxidative PP pathway within its characteristic cyclic connection between the Entner-Doudoroff (ED) and Embden-Meyerhoff-Parnas (EMP) pathways. Remarkably, similar to the genome-based metabolic decoupling in C. testosteroni , our 13 C-fluxomics reveals an inactive oxidative PP pathway and disconnected EMP and ED pathways in P. putida during gluconeogenic feeding, thus requiring transhydrogenase reactions to supply NADPH for anabolism in both species by leveraging the high tricarboxylic acid cycle flux during gluconeogenic growth. Furthermore, metabolomics and proteomics analyses of both species during gluconeogenic feeding, relative to glycolytic feeding, demonstrate a 5-fold depletion in phosphorylated metabolites and the absence of or up to a 17-fold decrease in proteins of the PP and ED pathways. Such metabolic remodeling, which is reportedly lacking in Escherichia coli exhibiting a gluconeogenic slow-growth phenotype, may serve to minimize futile carbon cycling while favoring the gluconeogenic metabolic regime in relevant proteobacterial species. IMPORTANCE Glycolytic metabolism of sugars is extensively studied in the Proteobacteria , but gluconeogenic carbon sources (e.g., organic acids, amino acids, aromatics) that feed into the tricarboxylic acid (TCA) cycle are widely reported to produce a fast-growth phenotype, particularly in species with biotechnological relevance. Much remains unknown about the importance of glycolysis-associated pathways in the metabolism of gluconeogenic carbon substrates. Here, we demonstrate that two distinct proteobacterial species, through genetic constraints or metabolic regulation at specific metabolic nodes, bypass the oxidative PP pathway during gluconeogenic growth and avoid unnecessary carbon fluxes by depleting protein investment into connected glycolysis pathways. Both species can leverage instead the high TCA cycle flux during gluconeogenic feeding to meet NADPH demand. Importantly, lack of a complete oxidative pentose phosphate pathway is a widespread metabolic trait in Proteobacteria with a gluconeogenic carbon preference, thus highlighting the important relevance of our findings toward elucidating the metabolic architecture in these bacteria. 

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4. Reverse β-oxidation pathways for efficient chemical production

   https://doi.org/10.1093/jimb/kuac003  

   Tarasava, Katia; Lee, Seung Hwan; Chen, Jing; Köpke, Michael; Jewett, Michael C; Gonzalez, Ramon (February 2022, Journal of Industrial Microbiology and Biotechnology)

   Abstract Microbial production of fuels, chemicals, and materials has the potential to reduce greenhouse gas emissions and contribute to a sustainable bioeconomy. While synthetic biology allows readjusting of native metabolic pathways for the synthesis of desired products, often these native pathways do not support maximum efficiency and are affected by complex regulatory mechanisms. A synthetic or engineered pathway that allows modular synthesis of versatile bioproducts with minimal enzyme requirement and regulation while achieving high carbon and energy efficiency could be an alternative solution to address these issues. The reverse β-oxidation (rBOX) pathways enable iterative non-decarboxylative elongation of carbon molecules of varying chain lengths and functional groups with only four core enzymes and no ATP requirement. Here, we describe recent developments in rBOX pathway engineering to produce alcohols and carboxylic acids with diverse functional groups, along with other commercially important molecules such as polyketides. We discuss the application of rBOX beyond the pathway itself by its interfacing with various carbon-utilization pathways and deployment in different organisms, which allows feedstock diversification from sugars to glycerol, carbon dioxide, methane, and other substrates. 

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5. Complete Biosynthesis of Anthocyanins Using E. coli Polycultures

   https://doi.org/10.1128/mBio.00621-17  

   Jones, J. Andrew; Vernacchio, Victoria R.; Collins, Shannon M.; Shirke, Abhijit N.; Xiu, Yu; Englaender, Jacob A.; Cress, Brady F.; McCutcheon, Catherine C.; Linhardt, Robert J.; Gross, Richard A.; et al (June 2017, mBio)

   ABSTRACT Fermentation-based chemical production strategies provide a feasible route for the rapid, safe, and sustainable production of a wide variety of important chemical products, ranging from fuels to pharmaceuticals. These strategies have yet to find wide industrial utilization due to their inability to economically compete with traditional extraction and chemical production methods. Here, we engineer for the first time the complex microbial biosynthesis of an anthocyanin plant natural product, starting from sugar. This was accomplished through the development of a synthetic, 4-strain Escherichia coli polyculture collectively expressing 15 exogenous or modified pathway enzymes from diverse plants and other microbes. This synthetic consortium-based approach enables the functional expression and connection of lengthy pathways while effectively managing the accompanying metabolic burden. The de novo production of specific anthocyanin molecules, such as calistephin, has been an elusive metabolic engineering target for over a decade. The utilization of our polyculture strategy affords milligram-per-liter production titers. This study also lays the groundwork for significant advances in strain and process design toward the development of cost-competitive biochemical production hosts through nontraditional methodologies. IMPORTANCE To efficiently express active extensive recombinant pathways with high flux in microbial hosts requires careful balance and allocation of metabolic resources such as ATP, reducing equivalents, and malonyl coenzyme A (malonyl-CoA), as well as various other pathway-dependent cofactors and precursors. To address this issue, we report the design, characterization, and implementation of the first synthetic 4-strain polyculture. Division of the overexpression of 15 enzymes and transcription factors over 4 independent strain modules allowed for the division of metabolic burden and for independent strain optimization for module-specific metabolite needs. This study represents the most complex synthetic consortia constructed to date for metabolic engineering applications and provides a new paradigm in metabolic engineering for the reconstitution of extensive metabolic pathways in nonnative hosts. 

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