Abstract
BackgroundPhotosynthetic cyanobacteria are attractive for a range of biotechnological applications including biofuel production. However, due to slow growth, screening of mutant libraries using microtiter plates is not feasible.ResultsWe present a method for high-throughput, single-cell analysis and sorting of genetically engineered l-lactate-producing strains of Synechocystis sp. PCC6803. A microfluidic device is used to encapsulate single cells in picoliter droplets, assay the droplets for l-lactate production, and sort strains with high productivity. We demonstrate the separation of low- and high-producing reference strains, as well as enrichment of a more productive l-lactate-synthesizing population after UV-induced mutagenesis. The droplet platform also revealed population heterogeneity in photosynthetic growth and lactate production, as well as the presence of metabolically stalled cells.ConclusionsThe workflow will facilitate metabolic engineering and directed evolution studies and will be useful in studies of cyanobacteria biochemistry and physiology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0380-2) contains supplementary material, which is available to authorized users.
Highlights
Photosynthetic cyanobacteria are attractive for a range of biotechnological applications including biofuel production
Picoinjection is preferable to co-encapsulation of cells with assay solution because it decouples photoautotrophic lactate production (6 h) from the lactate assay (30 min), allowing the use of assay reagents that may be transported out of droplets on a longer timescale or that are light sensitive
Calibration assays using pure lactate showed a linear response from 10 to 200 μM lactate, which is comparable to a 50 μL microtiter plate assay using the same reagents (Fig. 1b; Additional file 1: Fig. S1)
Summary
Photosynthetic cyanobacteria are attractive for a range of biotechnological applications including biofuel production. Cyanobacteria are model organisms for the study of biological light harvesting [1], photosynthesis [2], and circadian gene regulation [3]. Their minimal nutrient requirements and an expanding genetic toolbox have made cyanobacteria attractive for a range of biotechnological applications, such as hosts for next-generation biofuel [4, 5] and fine chemical production [6]. We used the droplet platform to measure population heterogeneities in Synechocystis growth and l-lactate production and their dependence on a circadian dark– light cycle
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