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1. Parsed synthesis of pyocyanin via co-culture enables context-dependent intercellular redox communication

   https://doi.org/10.1186/s12934-021-01703-2  

   Chun, Kayla ; Stephens, Kristina ; Wang, Sally ; Tsao, Chen-Yu ; Payne, Gregory F. ; Bentley, William E. ( November 2021 , Microbial Cell Factories)

   Microbial co-cultures and consortia are of interest in cell-based molecular production and even as “smart” therapeutics in that one can take advantage of division of labor and specialization to expand both the range of available functions and mechanisms for control. The development of tools that enable coordination and modulation of consortia will be crucial for future application of multi-population cultures. In particular, these systems would benefit from an expanded toolset that enables orthogonal inter-strain communication.

   We created a co-culture for the synthesis of a redox-active phenazine signaling molecule, pyocyanin (PYO), by dividing its synthesis into the generation of its intermediate, phenazine carboxylic acid (PCA) from the first strain, followed by consumption of PCA and generation of PYO in a second strain. Interestingly, both PCA and PYO can be used to actuate gene expression in cells engineered with thesoxRSoxidative stress regulon, although importantly this signaling activity was found to depend on growth media. That is, like other signaling motifs in bacterial systems, the signaling activity is context dependent. We then used this co-culture’s phenazine signals in a tri-culture to modulate gene expression and production of three model products: quorum sensing molecule autoinducer-1 and two fluorescent marker proteins, eGFP and DsRed. We also showed how these redox-based signals could be intermingled with other quorum-sensing (QS) signals which are more commonly used in synthetic biology, to control complex behaviors. To provide control over product synthesis in the tri-cultures, we also showed how a QS-induced growth control module could guide metabolic flux in one population and at the same time guide overall tri-culture function. Specifically, we showed that phenazine signal recognition, enabled through the oxidative stress response regulonsoxRS,was dependent on media composition such that signal propagation within our parsed synthetic system could guide different desired outcomes based on the prevailing environment. In doing so, we expanded the range of signaling molecules available for coordination and the modes by which they can be utilized to influence overall function of a multi-population culture.

   Our results show that redox-based signaling can be intermingled with other quorum sensing signaling in ways that enable user-defined control of microbial consortia yielding various outcomes defined by culture medium. Further, we demonstrated the utility of our previously designed growth control module in influencing signal propagation and metabolic activity is unimpeded by orthogonal redox-based signaling. By exploring novel multi-modal strategies for guiding communication and consortia outcome, the concepts introduced here may prove to be useful for coordination of multiple populations within complex microbial systems.

    

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2. Longitudinal Single‐Cell Imaging of Engineered Strains with Stimulated Raman Scattering to Characterize Heterogeneity in Fatty Acid Production

   https://doi.org/10.1002/advs.202206519  

   Tague, Nathan ; Lin, Haonan ; Lugagne, Jean‐Baptiste ; O'Connor, Owen M. ; Burman, Deeya ; Wong, Wilson W. ; Cheng, Ji‐Xin ; Dunlop, Mary J. ( June 2023 , Advanced Science)

   Understanding metabolic heterogeneity is critical for optimizing microbial production of valuable chemicals, but requires tools that can quantify metabolites at the single‐cell level over time. Here, longitudinal hyperspectral stimulated Raman scattering (SRS) chemical imaging is developed to directly visualize free fatty acids in engineeredEscherichia coliover many cell cycles. Compositional analysis is also developed to estimate the chain length and unsaturation of the fatty acids in living cells. This method reveals substantial heterogeneity in fatty acid production among and within colonies that emerges over the course of many generations. Interestingly, the strains display distinct types of production heterogeneity in an enzyme‐dependent manner. By pairing time‐lapse and SRS imaging, the relationship between growth and production at the single‐cell level are examined. The results demonstrate that cell‐to‐cell production heterogeneity is pervasive and provides a means to link single‐cell and population‐level production.

    

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3. Chemotaxis of Pseudomonas putida F1 to Alcohols Is Mediated by the Carboxylic Acid Receptor McfP

   https://doi.org/10.1128/AEM.01625-19  

   Zhang, Xiangsheng ; Hughes, Jonathan G. ; Subuyuj, Gabriel A. ; Ditty, Jayna L. ; Parales, Rebecca E. ; Zhou, Ning-Yi ( November 2019 , Applied and Environmental Microbiology)

   ABSTRACT Although alcohols are toxic to many microorganisms, they are good carbon and energy sources for some bacteria, including many pseudomonads. However, most studies that have examined chemosensory responses to alcohols have reported that alcohols are sensed as repellents, which is consistent with their toxic properties. In this study, we examined the chemotaxis of Pseudomonas putida strain F1 to n -alcohols with chain lengths of 1 to 12 carbons. P. putida F1 was attracted to all n -alcohols that served as growth substrates (C 2 to C 12 ) for the strain, and the responses were induced when cells were grown in the presence of alcohols. By assaying mutant strains lacking single or multiple methyl-accepting chemotaxis proteins, the receptor mediating the response to C 2 to C 12 alcohols was identified as McfP, the ortholog of the P. putida strain KT2440 receptor for C 2 and C 3 carboxylic acids. Besides being a requirement for the response to n -alcohols, McfP was required for the response of P. putida F1 to pyruvate, l -lactate, acetate, and propionate, which are detected by the KT2440 receptor, and the medium- and long-chain carboxylic acids hexanoic acid and dodecanoic acid. β-Galactosidase assays of P. putida F1 carrying an mcfP-lacZ transcriptional fusion showed that the mcfP gene is not induced in response to alcohols. Together, our results are consistent with the idea that the carboxylic acids generated from the oxidation of alcohols are the actual attractants sensed by McfP in P. putida F1, rather than the alcohols themselves. IMPORTANCE Alcohols, released as fermentation products and produced as intermediates in the catabolism of many organic compounds, including hydrocarbons and fatty acids, are common components of the microbial food web in soil and sediments. Although they serve as good carbon and energy sources for many soil bacteria, alcohols have primarily been reported to be repellents rather than attractants for motile bacteria. Little is known about how alcohols are sensed by microbes in the environment. We report here that catabolizable n -alcohols with linear chains of up to 12 carbons serve as attractants for the soil bacterium Pseudomonas putida , and rather than being detected directly, alcohols appear to be catabolized to acetate, which is then sensed by a specific cell-surface chemoreceptor protein. 

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4. Metabolic engineering strategies to produce medium-chain oleochemicals via acyl-ACP:CoA transacylase activity

   https://doi.org/10.1038/s41467-022-29218-3  

   Yan, Qiang ; Cordell, William T. ; Jindra, Michael A. ; Courtney, Dylan K. ; Kuckuk, Madeline K. ; Chen, Xuanqi ; Pfleger, Brian F. ( March 2022 , Nature Communications)

   Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified inPseudomonads, that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA’s that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains ofEscherichia colicapable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones.

    

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5. 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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