Zedler, J. A. Z. | Search bioRxiv

bioRxiv2022

Droplet-based microfluidics as a media optimization tool for cyanobacteria

The urgent need to increase sustainability in biotechnology has led to an increased interest in photosynthetic production platforms. Cyanobacteria are particularly attractive for their fast photoautotrophic growth and genetic accessibility. However, the lack of systematic strain optimization is holding back progress towards industrialization. To overcome this bottleneck, here we introduce a droplet-based microfluidics platform capable of one- (1D) and two-dimension (2D) screening of key parameters in cyanobacterial cultivation. We successfully grew three different unicellular, biotechnologically relevant cyanobacteria: Synechocystis sp. PCC 6803, Synechococcus elongatus UTEX 2973 and Synechococcus sp. UTEX 3154. Highly-resolved 1D screening of nitrate, phosphate, carbonate, and salt content show that nitrate and/or phosphate can be limiting in standard cultivation media. Finally, we show that 2D screening results from our microfluidic setup translate well to a laboratory scale high-density cultivation setup. This study demonstrates that droplet-based microfluidics by micro segmented-flow are a powerful tool for high-throughput optimization of cyanobacterial cultivation.

microbiology↗

bioRxiv2021

Development of a highly sensitive luciferase-based reporter system to study two-step protein secretion in cyanobacteria

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multi-protein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however little is known regarding their function, regulation and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of non-pilin proteins, we developed a NanoLuc-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. PCC 6803. The NLuc reporter presented a wide dynamic range with at least one order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that two-step protein secretion in cyanobacteria is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to explore protein secretion in cyanobacteria further. ImportanceProtein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here we introduce a NanoLuc (NLuc)-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge on cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

microbiology↗