Evaluation of secretion reporters to microalgae biotechnology: Blue to red fluorescent proteins
Graphical abstract
Introduction
Fluorescent proteins (FPs) emit light when excited by certain electromagnetic waves. One such molecule, green FP (GFP), was first discovered and identified in a jellyfish, Aequorea victoria [1]. However, biotechnological applications, such as the expression of GFP in Escherichia coli and Caenorhabditis elegans, began to be implemented only in the 1990s [2]. GFP is the first in a family of proteins that have been either discovered or developed with fluorescence properties, applied in numerous fields of study, and assisted evaluation of intra- and extracellular molecular phenomena. Currently, >90 types of proteins are available for numerous biotechnological applications [3]. The useful spectrum, initially restricted to the green region, was expanded, ranging from proteins, such as Sirius, fluorescing in the UV region [4] to proteins, such as iRFP720, fluorescing in the near infrared region [5]. However, FPs not only have specific excitation and emission wavelengths, but also have properties that help elucidate several cellular mechanisms. For example, these proteins can possibly help establish the location of protein synthesis [4], define expression levels [2,6], obtain temporal information from cellular events [7], estimate the pH in organelles [8], and determine the proximity between different molecules within the cells [9]. Their successful application in the life sciences is related to their remarkable ability to fluoresce after translation, without the addition of cofactors or chaperones [10], being extremely straightforward experimentally. Moreover, this field is constantly developing, and even greater diversity could be observed in the next decade in experiments involving FPs, such as the use of fluorescent timers [11]. In addition, the development of low-cost DNA synthesis techniques allowed the discovery and development of protein sequences globally, which could reach the research laboratories worldwide spreading the latest developments immediately.
Practically, the only limitation in the use of these proteins is the capacity of genetic manipulation of the target organism and molecular characteristics of the host system. Nevertheless, despite the extensive use of FPs across species, they were mostly developed in the cytoplasm of bacteria, which poses the need to test various FPs and define the best suited for an intended application [12]. For example, GFP directed towards the secretory pathway in the endocrine cells fluoresced partially, although it was fully active in the cytoplasmic environment [13]. The literature lacks studies comparing different FPs in C. reinhardtii, despite its importance as a model organism in different research areas, such as photosynthesis [14], cell flagella [15], circadian rhythm [16], and more recently in biotechnological application as a heterologous protein expression system [17]. In a major advancement in 2013, Rasala et al. evaluated six FPs in C. reinhardtii, comprehensively assessing their application in observation of cytoplasmic expression [18]. Nevertheless, even though a direct comparison of FPs was presented, it was restricted to the cytoplasmic compartment, limiting its extrapolation to other cellular compartments. In a previous study, Lauersen et al. evaluated four FPs, targeted towards different organelles and the secretory pathway, expanding our understanding of the applicability of FPs in C. reinhardtii [19]. Unfortunately, the study did not report a comprehensive evaluation of the four FPs for the secretory pathway, as it was not the focus of the study.
Despite the wide use of FPs in C. reinhardtii, there is a lack of direct comparison between the target of FPs and the secretory pathway, even though this process holds great potential in the exploitation of microalgae as a heterologous protein expression system. Studying efficient protein secretion is the key to microalgae technology, as it enables bioprocess development to expand cell viability inside reactors, impacting protein production outcomes [20]. Secretion of proteins can lead to a reduction in cost in downstream processing, by reducing the matrix solution complexity in which the product is recovered [21]. Downstream processing can constitute up to 80% of the complete process costs [22]. Therefore, efficient protein reporters are central in the development of efficient secretion platforms, from the screening of signal peptides to the bioprocess development applications. Among known reporters, FPs are suitable candidates, but as fluorescence is the emission of light, their use can be hampered by various photosynthetic pigments, as well as deleterious compartment-related modifications [13].
This study aimed to evaluate the application of FPs in secretion pathway in C. reinhardtii and to determine the most suitable FP for protein secretion studies in this microalga. In this study, we evaluated the secretion of seven FPs, demonstrating the successful secretion of six FPs that cover different regions of the visual spectrum (cyan: mCerulean; green: GFP and Emerald; orange: cOFP, tdTomato; and red mCherry). Every FP was expressed in the cytoplasm and secreted, and the fluorescence output was compared among themselves and with that of the wild type. Finally, the FP-secreting strains were assessed by fluorescence microscopy.
Section snippets
Plasmid construction
The pAH04 vector is similar to previously published pBR9 vector [18], except that pAH04 contains three RBCS2 introns incorporated into the vector in the natural order found in RBCS2 gene, as described in Fig. 1. pAH04 was constructed in pBlueScript II (pBSII). To generate pJP22 and pJP23, SLiCE reactions [23] were performed in the pAH04 vector by seamlessly incorporating ARS1 signal peptide into the Ble-2A fusion peptide, maintaining XhoI restriction site immediately after the signal peptide
Fluorescence measurement
As protein secretion is an important process carried out by cells and its implications in biotechnology are well known [20], the use of FPs as reporters in this process was analyzed in C. reinhardtii. Fluorescence signals can be influenced by multiple factors, including cell content, FP surrounding, misfolding intensity, FP maturation time, and auto-fluorescence [18]. To evaluate the suitability of FP for secretion studies, three general constructs—a cytosolic expressing vector, pAH04, and two
Discussion
Reliable protein reporters are highly useful in the studies on cell biology processes and have a great impact on the outcomes of research results. Currently, available reporters were evaluated for cytosolic and cellular compartment studies using C. reinhardtii [20]. However, protein reporter secretion was not extensively evaluated, leaving the most suitable reporter for protein secretion studies in C. reinhardtii unclear. Identification of reliable, inexpensive, and high-throughput protein
Declaration of interest
We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
Acknowledgements
We thank Austin Hallgren for assistance with pAH04 construction and Beth Rasala, PhD. for fluorescence microscopy instruction.
Funding
This work was supported in part by U.S. Department of Energy grant DE-EE0008246 and by the grant 2013/18224-2, São Paulo Research Foundation (FAPESP). The funding source had no involvement in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.
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