Engineering cyanobacteria as photosynthetic feedstock factories

Hays, Stephanie G.; Ducat, Daniel C.

doi:10.1007/s11120-014-9980-0

Cited by 99 publications

(74 citation statements)

References 89 publications

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“…While much research focuses on engineering cyanobacterial metabolism towards the synthesis of end products (e.g., biofuels), cyanobacteria are also under consideration for the production of carbohydrate feedstocks to support fermentative bioindustrial processes [1]. In this approach, cyanobacterial biomass is processed to provide organic carbon [2–6], or cyanobacterial cells are manipulated to secrete simple fermentable sugars [7–12].…”

Section: Introductionmentioning

confidence: 99%

“…To provide organic carbon to engineered consortia, we use a Synechococcus elongatus PCC 7942 strain previously engineered to export up to 85% of the carbon it fixes in the form of sucrose [12], a simple sugar also produced by plant-based feedstocks (e.g., sugarcane) that is readily consumed by many microbes. S. elongatus naturally accumulates sucrose as a compatible solute [1, 19], and can export this carbohydrate through heterologous expression of the proton/sucrose symporter, cscB (hereafter cscB + )[12, 20]. Under mild osmotic shock and slightly alkaline conditions, cscB + S. elongatus continuously generate sucrose at up to 36 mg sucrose L −1 hr −1 , a rate predicted to substantially exceed that of sugarcane, if successfully cultivated to scale [12].…”

Section: Introductionmentioning

confidence: 99%

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Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction

et al. 2017

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BackgroundMicrobial consortia composed of autotrophic and heterotrophic species abound in nature, yet examples of synthetic communities with mixed metabolism are limited in the laboratory. We previously engineered a model cyanobacterium, Synechococcus elongatus PCC 7942, to secrete the bulk of the carbon it fixes as sucrose, a carbohydrate that can be utilized by many other microbes. Here, we tested the capability of sucrose-secreting cyanobacteria to act as a flexible platform for the construction of synthetic, light-driven consortia by pairing them with three disparate heterotrophs: Bacillus subtilis, Escherichia coli, or Saccharomyces cerevisiae. The comparison of these different co-culture dyads reveals general design principles for the construction of robust autotroph/heterotroph consortia.ResultsWe observed heterotrophic growth dependent upon cyanobacterial photosynthate in each co-culture pair. Furthermore, these synthetic consortia could be stabilized over the long-term (weeks to months) and both species could persist when challenged with specific perturbations. Stability and productivity of autotroph/heterotroph co-cultures was dependent on heterotroph sucrose utilization, as well as other species-independent interactions that we observed across all dyads. One destabilizing interaction we observed was that non-sucrose byproducts of oxygenic photosynthesis negatively impacted heterotroph growth. Conversely, inoculation of each heterotrophic species enhanced cyanobacterial growth in comparison to axenic cultures. Finally, these consortia can be flexibly programmed for photoproduction of target compounds and proteins; by changing the heterotroph in co-culture to specialized strains of B. subtilis or E. coli we demonstrate production of alpha-amylase and polyhydroxybutyrate, respectively.ConclusionsEnabled by the unprecedented flexibility of this consortia design, we uncover species-independent design principles that influence cyanobacteria/heterotroph consortia robustness. The modular nature of these communities and their unusual robustness exhibits promise as a platform for highly-versatile photoproduction strategies that capitalize on multi-species interactions and could be utilized as a tool for the study of nascent symbioses. Further consortia improvements via engineered interventions beyond those we show here (i.e., increased efficiency growing on sucrose) could improve these communities as production platforms.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-017-0048-5) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction

et al. 2017

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“…Recent studies have demonstrated the feasibility of converting energy from sunlight and carbon from CO 2 directly into biofuels using photosynthetic microorganisms (Hays and Ducat, 2015; Oliver and Atsumi, 2014). Cyanobacteria have evolved efficient metabolic processes for harvesting light energy to produce organic molecules, and they can be cultivated in locations that do not compete with food production for land resources (Melis, 2009; Zhu et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production

Jazmin

Cheah

et al. 2017

Metabolic Engineering

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We applied isotopically nonstationary 13C metabolic flux analysis (INST-MFA) to compare the pathway fluxes of wild-type (WT) Synechococcus elongatus PCC 7942 to an engineered strain (SA590) that produces isobutyraldehyde (IBA). The flux maps revealed a potential bottleneck at the pyruvate kinase (PK) reaction step that was associated with diversion of flux into a three-step PK bypass pathway involving the enzymes PEP carboxylase (PEPC), malate dehydrogenase (MDH), and malic enzyme (ME). Overexpression of pk in SA590 led to a significant improvement in IBA specific productivity. Single-gene overexpression of the three enzymes in the proposed PK bypass pathway also led to improvements in IBA production, although to a lesser extent than pk overexpression. Combinatorial overexpression of two of the three genes in the proposed PK bypass pathway (mdh and me) led to improvements in specific productivity that were similar to those achieved by single-gene pk overexpression. Our work demonstrates how 13C flux analysis can be used to identify potential metabolic bottlenecks and novel metabolic routes, and how these findings can guide rational metabolic engineering of cyanobacteria for increased production of desired molecules.

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“…In this explanation, photosynthetic carbon and energy assimilation that can no longer be directed to growth when population increase is inhibited by nutrient deficiency or other stresses results in overflow products, particularly TAG (Du and Benning, 2016). The concept of overflow metabolism has traditionally been associated with the export of metabolites by heterotrophic microbes (Neijssel and Tempest, 1975) and has more recently also been applied to photosynthetic metabolism in cyanobacteria (Hays and Ducat, 2015;Courchesne et al, 2009;Gründel et al, 2012) and higher plants (Weise et al, 2011). In microalgal work, the idea of photosynthetic overflow (excess photosynthetic energy and carbon) as the driver of oil accumulation has become a widely accepted explanation (Hu et al, 2008;Li et al, 2012;Solovchenko, 2012;Li et al, 2013;Klok et al, 2014;Du and Benning, 2016).…”

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confidence: 99%

The Relationship of Triacylglycerol and Starch Accumulation to Carbon and Energy Flows During Nutrient Deprivation in Chlamydomonas.

Juergens

Disbrow

2016

Plant Physiol.

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ORCID IDs: 0000-0001-5136-9025 (M.T.J.); 0000-0001-5136-9025 (Y.S.-H.).Because of the potential importance of algae for green biotechnology, considerable effort has been invested in understanding their responses to nitrogen deprivation. The most frequently invoked reasons proposed for the accumulation of high cellular levels of triacylglycerol (TAG) and starch are variants of what may be termed the "overflow hypothesis." According to this, growth inhibition results in the rate of photosynthetic energy and/or carbon input exceeding cellular needs; the excess input is directed into the accumulation of TAG and/or starch to prevent damage. This study was aimed at providing a quantitative dataset and analysis of the main energy and carbon flows before and during nitrogen deprivation in a model system to assess alternative explanations. Cellular growth, biomass, starch, and lipid levels as well as several measures of photosynthetic function were recorded for cells of Chlamydomonas reinhardtii cultured under nine different autotrophic, mixotrophic, and heterotrophic conditions during nutrient-replete growth and for the first 4 d of nitrogen deprivation. The results of a 13 C labeling time course indicated that in mixotrophic culture, starch is predominantly made from CO 2 and fatty acid synthesis is largely supplied by exogenous acetate, with considerable turnover of membrane lipids, so that total lipid rather than TAG is the appropriate measure of product accumulation. Heterotrophic cultures accumulated TAG and starch during N deprivation, showing that these are not dependent on photosynthesis. We conclude that the overflow hypothesis is insufficient and suggest that storage may be a more universally important reason for carbon compound accumulation during nutrient deprivation.

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Engineering cyanobacteria as photosynthetic feedstock factories

Cited by 99 publications

References 89 publications

Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction

Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction

Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production

The Relationship of Triacylglycerol and Starch Accumulation to Carbon and Energy Flows During Nutrient Deprivation in Chlamydomonas.

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