Unlocking the potential of microbial synergy: Multi-OMICs insights into the co-culture of cyanobacteria and Pseudomonas putida

Unlocking the potential of microbial synergy: Multi-OMICs insights into the co-culture of cyanobacteria and Pseudomonas putida
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Pseudomonas putida as saviour for troubled Synechococcus elongatus in a synthetic co-culture – interaction studies based on a multi-OMICs approach - Communications Biology

In their natural habitats, microbes rarely exist in isolation; instead, they thrive in consortia, where various interactions occur. In this study, a defined synthetic co-culture of the cyanobacterium S. elongatus cscB, which supplies sucrose to the heterotrophic P. putida cscRABY, is investigated to identify potential interactions. Initial experiments reveal a remarkable growth-promoting effect of the heterotrophic partner on the cyanobacterium, resulting in an up to 80% increase in the growth rate and enhanced photosynthetic capacity. Vice versa, the presence of the cyanobacterium has a neutral effect on P. putida cscRABY, highlighting the resilience of pseudomonads against stress and their potential as co-culture partners. Next, a suitable reference process reinforcing the growth-promoting effect is established in a parallel photobioreactor system, which sets the basis for the analysis of the co-culture at the transcriptome, proteome, and metabolome levels. In addition to several moderate changes, including alterations in the metabolism and stress response in both microbes, this comprehensive multi-OMICs approach strongly hints towards the exchange of further molecules beyond the unidirectional feeding with sucrose. Taken together, these findings provide valuable insights into the complex dynamics between both co-culture partners, indicating multi-level interactions, which can be employed for further streamlining of the co-cultivation system.

It might be crystal clear for a microbial ecologist that microbes rarely exist in isolation but instead are embedded in a complex web of multiple partnerships. However, this understanding only begins to trickle into the mindset of pure biotechnologists, generally having their ideal process (or experiment) to be “sterile” from other microorganisms. Yet, acknowledging the natural lifestyle of microbes expands the bioproduction of value-added compounds through novel pathways and strategies [1, 2]. This means that investing co-cultures can enable us to understand synergistic effects and uncover otherwise hidden behaviours, thus allowing for targeted utilization of their capabilities. However, to really become an alternative to single or so-called axenic cultures, co-cultures must be as well-defined and controllable as such.

The design of applicable co-cultures is not a trivial task, and in the best-case scenario, the co-cultures should align with a sustainable bioprocess. In most cases, it is intended that the microbes live under the premise of the division of labour, which allows different traits of microbes to complement each other in a profitable way [3, 4]. One example of doing so is pairing heterotrophs with phototrophs [5]. This combination of microbes is ubiquitous in the ocean, as can be seen exemplarily in microbial mats [6], where the photoautotrophic member, such as cyanobacteria or algae, uses solar power to fix CO2 into organic carbon, a portion of which then is accessible to the heterotrophic members of the community. In the case of synthetic co-cultures, this concept is taken a step further by employing the heterotrophic partner to convert the carbon source provided into value-added products by engineering and optimizing metabolic pathways.

The co-culture journey in our lab started by combining two genetically modified bacteria to build up a synthetic co-culture of a phototrophic and a heterotrophic partner. The cyanobacterium Synechochoccus elongatus cscB was utilized, as it naturally produces sucrose to protect against high salt levels. Thanks to the heterologously expressed CscB sucrose permease, this sugar is secreted into the culture supernatant [7], where it serves then as the sole carbon source for the partner Pseudomonas putida cscRABY, a derivative modified for the uptake and metabolization of sucrose [8].

In our previous work, we demonstrated a high level of controllability in the co-culture while producing the bioplastic mcl-polyhydroxyalkanoate (PHA) [9, 10]. Following this, we asked ourselves: Is there potential feedback between the two microbes beyond the unidirectional synthetic interaction we have integrated? Given that interactions between phototrophs and heterotrophs are prevalent in ocean ecosystems, we were intrigued by how deeply ingrained these interactions might be through evolution, considering that P. putida is associated with soil rather than with marine environments.

To explore this in detail, we conducted a series of growth experiments under different environmental conditions, varying the induction of sucrose secretion, light exposure, and inoculation cell ratios to determine the phenomenological effect of co-culturing. Across different scales, it became evident that co-cultivating the cyanobacterium with P. putida cscRABY, especially in stressful conditions, restored and even increased viability. For instance, the growth rate of S. elongatus cscB increased up to 80% in co-culture grown cells as compared to axenic cultures with induced sucrose secretion. Furthermore, the photosynthetic capacity, estimated in terms of fixed carbon, increased in the total culture when paired with the co-culture partner.

In collaboration with the Pandhal lab from Sheffield University, UK, and the BayBioMS center from TUM Freising, Germany, we conducted an in-depth investigation of the co-culture at a transcriptome, proteome, and metabolome level. To this end, we set up an experiment comparing the co-culture to the respective axenic cultures grown under comparable conditions. For the heterotrophic partner, a fed-batch mode was chosen to mimic the feed of S. elongatus cscB. This enabled a reproducible and reliable experiment to harvest samples for OMICs analysis.

In summary, the multi-OMICs analysis of the co-culture provided us with a detailed snapshot of the cellular status at the moment of sampling, revealing a complex network of small to moderate changes. For example, we identified differentially regulated transporter genes alongside variations in the core carbon metabolism, indicating potential additional exchanges between the partners. Thus, in addition to the synthetic connection by sucrose, more links connecting the co-culture partners seem to be present. We also identified signs of competition for shared resources, such as medium components, including citrate and various salts. Furthermore, ion homeostasis was unbalanced in both organisms, suggesting limitations or reduced accessibility of ions due to potential advanced scavenging strategies of the respective co-culture partner. These findings underscore the importance of careful medium optimisation in co-cultures in general. Unexpectedly, despite the overall robust growth of the co-culture, stress signals were higher in the co-culture compared to the axenic culture. This suggests that co-culturing, while beneficial for growth, may introduce additional stressors that need to be addressed to further optimize co-culture conditions.

The primary challenge now lies in determining whether these exchanges are targeted processes, undirected responses, or an inherent feature of cell death in co-cultures. So, what’s the plan for moving forward? Armed with the insights gained, our next step involves developing a combined Flux Balance Analysis (FBA) model of the co-culture partners to computationally elucidate the exchange of different carbon sources between them. Our goal is to gain a deeper understanding of the interaction structure, which will need to be confirmed through experimental validation. Additionally, we are focussing on optimizing the co-culture itself by introducing an improved cyanobacterial partner. As biotechnologists, we are actively participating in the shift from axenic cultures to the application of well-defined co-cultures by enhancing the understanding of the complex network of inter-species interactions in synthetic communities.

 

  1. Nai, C. & Meyer, V. From axenic to mixed multures: Technological advances accelerating a paradigm shift in microbiology. Trends Microbiol. 26, 538–554 (2018).
  2. Peng, X.-Y. et al. Co-culture: stimulate the metabolic potential and explore the molecular diversity of natural products from microorganisms. Mar. life Sci. Technol. 3, 363–374 (2021).
  3. Diender, M., Parera Olm, I. & Sousa, D. Z. Synthetic co-cultures: novel avenues for bio-based processes. Curr. Opin. Biotechnol. 67, 72–79 (2021).
  4. Duncker, K. E., Holmes, Z. A. & You, L. Engineered microbial consortia: strategies and applications. Microb. Cell Fact. 20, 211 (2021).
  5. Zuñiga, C. et al. Synthetic microbial communities of heterotrophs and phototrophs facilitate sustainable growth. Nat. Commun.11, 3803 (2020).
  6. Cole, J. et al. Phototrophic biofilm assembly in microbial-mat-derived unicyanobacterial consortia: model systems for the study of autotroph-heterotroph interactions. Front. Microbiol. 5, (2014).
  7. Ducat, D. C., Avelar-Rivas, J. A., Way, J. C. & Silver, P. A. Rerouting carbon flux to enhance photosynthetic productivity. Appl. Environ. Microbiol. 78, 2660–2668 (2012).
  8. Löwe, H., Sinner, P., Kremling, A. & Pflüger-Grau, K. Engineering sucrose metabolism in Pseudomonas putida highlights the importance of porins. Microb. Biotechnol. 13, 97–106 (2020).
  9. Löwe, H., Hobmeier, K., Moos, M., Kremling, A. & Pflüger-Grau, K. Photoautotrophic production of polyhydroxyalkanoates in a synthetic mixed culture of Synechococcus elongatus cscB and Pseudomonas putida cscAB. Biotechnol. Biofuels 10, 190 (2017).
  10. Kratzl, F., Andreas, K. & Pflüger-Grau, K. Streamlining of a synthetic co-culture towards an individually controllable one-pot process for polyhydroxyalkanoate production from light and CO2. Eng. Life Sci. 23, (2023).

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