assembly-quantification-co-cultures-combining-heterotrophic-yeast

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria

With the increasing demand for sustainable biotechnologies, mixed consortia containing a phototrophic microbe and heterotrophic partner species are being ...

Heinrich Heine University Düsseldorf

Michigan State University

In plants, a limited capacity to utilize or export the end-products of the Calvin-Benson cycle (CB) from photosynthetically active source cells to non-photosynthetic sink cells can result in reduced carbon capture and photosynthetic electron transport (PET), and lowered photochemical efficiency. The down-regulation of photosynthesis caused by reduced capacity to utilize photosynthate has been termed 'sink limitation'. Recently, several cyanobacterial and algal strains engineered to overproduce target metabolites have exhibited increased photochemistry, suggesting that possible source-sink regulatory mechanisms may be involved. We directly examined photochemical properties following induction of a heterologous sucrose 'sink' in the unicellular cyanobacterium Synechococcus elongatus PCC 7942. We show that total photochemistry increases proportionally to the experimentally controlled rate of sucrose export. Importantly, the quantum yield of PSII (ΦII) increases in response to sucrose export while the PET chain becomes more oxidized from less PSI acceptor-side limitation, suggesting increased CB activity and a decrease in sink limitation. Enhanced photosynthetic activity and linear electron flow are detectable within hours of induction of the heterologous sink and are independent of pigmentation alterations or the ionic/osmotic effects of the induction system. These observations provide direct evidence that secretion of heterologous carbon bioproducts can be used as an alternative approach to improve photosynthetic efficiency, presumably by by-passing sink limitation. Our results also suggest that engineered microalgal production strains are valuable alternative models for examining photosynthetic sink limitation because they enable greater control and monitoring of metabolite fluxes relative to plants.

Increased Photochemical Efficiency in Cyanobacteria via an Engineered Sucrose Sink.

Cyanobacteria are emerging as alternative crop species for the production of fuels, chemicals, and biomass. Yet, the success of these microbes depends on the development of cost-effective technologies that permit scaled cultivation and cell harvesting. Here, we investigate the feasibility of engineering cell morphology to improve biomass recovery and decrease energetic costs associated with lysing cyanobacterial cells. Specifically, we modify the levels of Min system proteins in  PCC 7942. The Min system has established functions in controlling cell division by regulating the assembly of FtsZ, a tubulin-like protein required for defining the bacterial division plane. We show that altering the expression of two FtsZ-regulatory proteins, MinC and Cdv3, enables control over cell morphology by disrupting FtsZ localization and cell division without preventing continued cell growth. By varying the expression of these proteins, we can tune the lengths of cyanobacterial cells across a broad dynamic range, anywhere from an ∼20% increased length (relative to the wild type) to near-millimeter lengths. Highly elongated cells exhibit increased rates of sedimentation under low centrifugal forces or by gravity-assisted settling. Furthermore, hyperelongated cells are also more susceptible to lysis through the application of mild physical stress. Collectively, these results demonstrate a novel approach toward decreasing harvesting and processing costs associated with mass cyanobacterial cultivation by altering morphology at the cellular level. We show that the cell length of a model cyanobacterial species can be programmed by rationally manipulating the expression of protein factors that suppress cell division. In some instances, we can increase the size of these cells to near-millimeter lengths with this approach. The resulting elongated cells have favorable properties with regard to cell harvesting and lysis. Furthermore, cells treated in this manner continue to grow rapidly at time scales similar to those of uninduced controls. To our knowledge, this is the first reported example of engineering the cell morphology of cyanobacteria or algae to make them more compatible with downstream processing steps that present economic barriers to their use as alternative crop species. Therefore, our results are a promising proof-of-principle for the use of morphology engineering to increase the cost-effectiveness of the mass cultivation of cyanobacteria for various sustainability initiatives.

Engineering Cyanobacterial Cell Morphology for Enhanced Recovery and Processing of Biomass.

We previously reported that Synechococcus elongatus PCC 7942, engineered with the sucrose transporter CscB, can export up to 85% of its photosynthetically-fixed carbon as sucrose and shows considerable promise as an alternative carbohydrate source. One approach to effectively utilize this cyanobacterium is to generate synthetic, light-driven consortia in which sucrose-metabolizing heterotrophs catalyze the conversion of the low-value carbohydrate into higher-value compounds in co-culture. Here, we report an improved synthetic photoautotroph/chemoheterotroph consortial design in which sucrose secreted by S. elongatus CscB directly supports the bacterium Halomonas boliviensis, a natural producer of the bioplastic precursor, PHB. We show that alginate encapsulation of S. elongatus CscB enhances sucrose-export rates ~2-fold within 66h, to ~290mg sucrose Ld OD and enhances the co-culture stability. Consortial H. boliviensis accumulate up to 31% of their dry-weight as PHB, reaching productivities up to 28.3mg PHB Ld. This light-driven, alginate-partitioned co-culture platform achieves PHB productivities that match or exceed those of traditionally engineered cyanobacterial monocultures. Importantly, S. elongatus CscB/H. boliviensis co-cultures were continuously productive for over 5 months and resisted invasive microbial species without the application of antibiotics or other chemical selection agents.

A synthetic, light-driven consortium of cyanobacteria and heterotrophic bacteria enables stable polyhydroxybutyrate production.

Cyanobacteria must prevent imbalances between absorbed light energy (source) and the metabolic capacity (sink) to utilize it to protect their photosynthetic apparatus against damage. A number of photoprotective mechanisms assist in dissipating excess absorbed energy, including respiratory terminal oxidases and flavodiiron proteins, but inherently reduce photosynthetic efficiency. Recently, it has been hypothesized that some engineered metabolic pathways may improve photosynthetic performance by correcting source/sink imbalances. In the context of this subject, we explored the interconnectivity between endogenous electron valves, and the activation of one or more heterologous metabolic sinks. We coexpressed two heterologous metabolic pathways that have been previously shown to positively impact photosynthetic activity in cyanobacteria, a sucrose production pathway (consuming ATP and reductant) and a reductant-only consuming cytochrome P450. Sucrose export was associated with improved quantum yield of phtotosystem II (PSII) and enhanced electron transport chain flux, especially at lower illumination levels, while cytochrome P450 activity led to photosynthetic enhancements primarily observed under high light. Moreover, coexpression of these two heterologous sinks showed additive impacts on photosynthesis, indicating that neither sink alone was capable of utilizing the full "overcapacity" of the electron transport chain. We find that heterologous sinks may partially compensate for the loss of photosystem I (PSI) oxidizing mechanisms even under rapid illumination changes, although this compensation is incomplete. Our results provide support for the theory that heterologous metabolism can act as a photosynthetic sink and exhibit some overlapping functionality with photoprotective mechanisms, while potentially conserving energy within useful metabolic products that might otherwise be "lost."

Improved photosynthetic capacity and photosystem I oxidation via heterologous metabolism engineering in cyanobacteria.

Co-cultivation of an autotrophic species with one or more heterotrophic microbes is a strategy for photobiological production of high-value compounds and is relatively underexplored in comparison to cyanobacterial or microalgal monocultures. Long-term stability of such consortia is required for useful collaboration between the partners, and this property can be increased by encapsulation of phototrophic partners within a hydrogel. Encapsulated cyanobacteria have advantages relative to planktonic cultures that may be useful to explore the potential for artificial microbial communities for targeted biomolecule synthesis, such as increased control over population sizes and reduced liquid handling requirements. In this chapter, we describe a method for encapsulation of genetically modified cyanobacterial strain (Synechococcus elongatus PCC 7942, CscB+) into a sodium alginate matrix, and the utilization of these encapsulated cells to construct stable, artificial autotroph/heterotroph co-cultures. This method has applications for the study of phototroph-based synthetic microbial consortia, and multi-species photobiological production.

Generation of Stable, Light-Driven Co-cultures of Cyanobacteria with Heterotrophic Microbes.

Switching off the lights to illuminate a photosynthetic brake in cyanobacteria.

Photosynthetic organisms need to balance the rate of photosynthesis with the utilization of photosynthetic products by downstream reactions. While such "source/sink" pathways are well-interrogated in plants, analogous regulatory systems are unknown or poorly studied in single-celled algal and cyanobacterial species. Towards the identification of energy/sugar sensors in cyanobacteria, we utilized an engineered strain of Synechococcus elongatus PCC 7942 that allows experimental manipulation of carbon status. We conducted a screening of all two-component systems (TCS) and serine/threonine kinases (STKs) encoded in S. elongatus PCC 7942 by analyzing phenotypes consistent with sucrose-induced relaxation of sink inhibition. We narrowed the candidate sensor proteins by analyzing changes observed after sucrose feeding. We show that a clustered TCS network containing RpaA, CikB, ManS and NblS are involved in the regulation of genes related to photosynthesis, pigment synthesis, and Rubisco concentration in response to sucrose. Altogether, these results highlight a regulatory TCS group that may play under-appreciated functions in carbon partitioning and energy balancing in cyanobacteria.

Coordination of carbon partitioning and photosynthesis by a two-component signaling network in Synechococcus elongatus PCC 7942.

Synthetic biology conceptualizes biological complexity as a network of biological parts, devices, and systems with predetermined functionalities and has had a revolutionary impact on fundamental and applied research. With the unprecedented ability to synthesize and transfer any DNA and RNA across organisms, the scope of synthetic biology is expanding and being recreated in previously unimaginable ways. The field has matured to a level where highly complex networks, such as artificial communities of synthetic organisms, can be constructed. In parallel, computational biology became an integral part of biological studies, with computational models aiding the unravelling of the escalating complexity and emerging properties of biological phenomena. However, there is still a vast untapped potential for the complete integration of modelling into the synthetic design process, presenting exciting opportunities for scientific advancements. Here, we first highlight the most recent advances in computer-aided design of microbial communities. Next, we propose that such a design can benefit from an organism-free modular modelling approach that places its emphasis on modules of organismal function towards the design of multispecies communities. We argue for a shift in perspective from single organism-centred approaches to emphasizing the functional contributions of organisms within the community. By assembling synthetic biological systems using modular computational models with mathematical descriptions of parts and circuits, we can tailor organisms to fulfil specific functional roles within the community. This approach aligns with synthetic biology strategies and presents exciting possibilities for the design of artificial communities. .

Daniel C. Ducat

Institute for Synthetic Microbiology

Heinrich Heine University Düsseldorf

Assembly and Quantification of Co-Cultures Combining Heterotrophic Yeast with Phototrophic Sugar-Secreting Cyanobacteria