Manuel Serif

Bio: Manuel Serif is an academic researcher from University of Konstanz. The author has contributed to research in topics: Phaeodactylum tricornutum & Thylakoid. The author has an hindex of 7, co-authored 12 publications receiving 241 citations. Previous affiliations of Manuel Serif include Norwegian University of Science and Technology & Scottish Association for Marine Science.

One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing.

Abstract: Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest

A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum

Abstract: Reverse genetics techniques are powerful tools for studying gene functions. In the model diatom Phaeodactylum tricornutum , RNAi-mediated knockdown of genes still is the most commonly used reverse genetics technique. Due to the diploidic life cycle missing reproduction in lab cultures, many commonly used techniques to create knockout instead of knockdown lines are not applicable in P. tricornutum . These limitations can be overcome by using genome editing approaches like TALEN (Transcription activator-like effector nucleases), and/or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats), allowing the introduction of targeted mutagenesis events. Both techniques have recently been adapted exemplarily for diatoms, however, no concise guidelines exist yet for routine utilization of these tools and the subsequent characterization of the mutants. We therefore have adapted a cost-effective TALEN generation system previously established for mammalian cells for the use in P. tricornutum, allowing the assembly of TALENs in about two weeks. We further provide protocols for: a) choosing a TALEN target site in order to avoid potentially ineffective and/or off-target prone TALEN constructs, b) efficient transformation of P. tricornutum with both TALEN constructs, utilizing two antibiotics resistance markers, c) effective screening of the transformants. In order to test our system we chose the blue-light dependent transcription factor Aureochrome 1a ( PtAureo1a ) as a target gene due to the known phenotype of previously characterized P. tricornutum RNAi knockdown strains. Our TALEN approach appears to be highly efficient: targeted mutation events were detected in 50% of all transformants obtained, whereas 21% of the transformants were found to be bi-allelic knockout lines. Furthermore, most TALEN transformed cell lines were found to be genetically homogeneous without the need for re-plating, which greatly facilitates the screening process.

Lhcx proteins provide photoprotection via thermal dissipation of absorbed light in the diatom Phaeodactylum tricornutum

Abstract: Diatoms possess an impressive capacity for rapidly inducible thermal dissipation of excess absorbed energy (qE), provided by the xanthophyll diatoxanthin and Lhcx proteins. By knocking out the Lhcx1 and Lhcx2 genes individually in Phaeodactylum tricornutum strain 4 and complementing the knockout lines with different Lhcx proteins, multiple mutants with varying qE capacities are obtained, ranging from zero to high values. We demonstrate that qE is entirely dependent on the concerted action of diatoxanthin and Lhcx proteins, with Lhcx1, Lhcx2 and Lhcx3 having similar functions. Moreover, we establish a clear link between Lhcx1/2/3 mediated inducible thermal energy dissipation and a reduction in the functional absorption cross-section of photosystem II. This regulation of the functional absorption cross-section can be tuned by altered Lhcx protein expression in response to environmental conditions. Our results provide a holistic understanding of the rapidly inducible thermal energy dissipation process and its mechanistic implications in diatoms.

Genome editing in diatoms: achievements and goals

Abstract: Diatoms are major components of phytoplankton and play a key role in the ecology of aquatic ecosystems. These algae are of great scientific importance for a wide variety of research areas, ranging from marine ecology and oceanography to biotechnology. During the last 20 years, the availability of genomic information on selected diatom species and a substantial progress in genetic manipulation, strongly contributed to establishing diatoms as molecular model organisms for marine biology research. Recently, tailored TALEN endonucleases and the CRISPR/Cas9 system were utilized in diatoms, allowing targeted genetic modifications and the generation of knockout strains. These approaches are extremely valuable for diatom research because breeding, forward genetic screens by random insertion, and chemical mutagenesis are not applicable to the available model species Phaeodactylum tricornutum and Thalassiosira pseudonana, which do not cross sexually in the lab. Here, we provide an overview of the genetic toolbox that is currently available for performing stable genetic modifications in diatoms. We also discuss novel challenges that need to be addressed to fully exploit the potential of these technologies for the characterization of diatom biology and for metabolic engineering.

Allosteric communication between DNA-binding and light-responsive domains of diatom class I aureochromes

Abstract: The modular architecture of aureochrome blue light receptors, found in several algal groups including diatoms, is unique by having the LOV-type photoreceptor domain fused to the C-terminus of its putative effector, an N-terminal DNA-binding bZIP module. The structural and functional understanding of aureochromes' light-dependent signaling mechanism is limited, despite their promise as an optogenetic tool. We show that class I aureochromes 1a and 1c from the diatom Phaeodactylum tricornutum are regulated in a light-independent circadian rhythm. These aureochromes are capable to form functional homo- and heterodimers, which recognize the ACGT core sequence within the canonical 'aureo box', TGACGT, in a light-independent manner. The bZIP domain holds a more folded and less flexible but extended conformation in the duplex DNA-bound state. FT-IR spectroscopy in the absence and the presence of DNA shows light-dependent helix unfolding in the LOV domain, which leads to conformational changes in the bZIP region. The solution structure of DNA bound to aureochrome points to a tilted orientation that was further validated by molecular dynamics simulations. We propose that aureochrome signaling relies on an allosteric pathway from LOV to bZIP that results in conformational changes near the bZIP-DNA interface without major effects on the binding affinity.

Genetic compensation induced by deleterious mutations but not gene knockdowns

Abstract: Cells sense their environment and adapt to it by fine-tuning their transcriptome. Wired into this network of gene expression control are mechanisms to compensate for gene dosage. The increasing use of reverse genetics in zebrafish, and other model systems, has revealed profound differences between the phenotypes caused by genetic mutations and those caused by gene knockdowns at many loci, an observation previously reported in mouse and Arabidopsis. To identify the reasons underlying the phenotypic differences between mutants and knockdowns, we generated mutations in zebrafish egfl7, an endothelial extracellular matrix gene of therapeutic interest, as well as in vegfaa. Here we show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl7 morpholino (morphants) exhibit severe vascular defects. We further observe that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose. Comparing egfl7 mutant and morphant proteomes and transcriptomes, we identify a set of proteins and genes that are upregulated in mutants but not in morphants. Among them are extracellular matrix genes that can rescue egfl7 morphants, indicating that they could be compensating for the loss of Egfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPR interference, which obstructs transcript elongation and causes severe vascular defects, does not cause the upregulation of these genes. Similarly, vegfaa mutants but not morphants show an upregulation of vegfab. Taken together, these data reveal the activation of a compensatory network to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown.

Parameters of photosynthesis: definitions, theroy and interpretation of results

Abstract: A global assessment of carbon flux in the world ocean is one of the major undertakings of the Joint Global Ocean Flux Study (JGOFS). This has to be undertaken using historical in situ data of primary productivity. As required by the temporal and spatial scales involved in a global study, it can be conveniently done by combining, through appropriate models, remotely sensed information (chlorophyll a, temperature) with basic information about the parameters related to the carbon uptake by phytoplanktonic algae. This requires a better understanding as well as a more extended knowledge of these parameters which govern the radiative energy absorption and utilization by algae in photosynthesis. The measurement of the photosynthetic response of algae [the photosynthesis (P) versus in-adiance (£) curves], besides being less shiptime consuming than in situ primary production experiments, allows the needed parameters to be derived and systematically studied as a function of the physical, chemical and ecological conditions. The aim of the present paper is to review the significance of these parameters, especially in view of their introduction into models, to analyze the causes of their variations in the light of physiological considerations, and finally to provide methodological recommendations for meaningful determinations, and interpretation, of the data resulting from /"versus £ determinations. Of main concern are the available and usable irradiance, the chlorophyll a-specific absorption capabilities of the algae, the maximum light utilization coefficient (a), the maximum quantum yield (4>m), the maximum photosynthetic rate (Pm) and the light saturation index (£k). The potential of other, non-intrusive, approaches, such as the stimulated variable fluorescence, or the sun-induced natural fluorescence techniques is also examined.

Fourth generation biofuel: a review on risks and mitigation strategies

Abstract: Fourth generation biofuel (FGB) uses genetically modified (GM) algae to enhance biofuel production. Although GM algae biofuel is a well-known alternative to fossil fuels, the potential environmental and health-related risks are still of great concern. An evaluation of these concerns and accordingly devising appropriate mitigation strategies to deal with them are important to a successful commercialized production of FGB. While extensive research has been carried out on genetic modification and other technologies that aim to increase the productivity of algae strains, only a handful of them deal with the legislative limitations imposed on exploiting and processing GM algae. This paper examines this legislation and the mitigation strategies to meet potential risks associated with the exploitation and processing of FGB. Open-pond system is an economic solution for large-scale cultivation of microalgae; however, the concern regarding the health and environmental risk of cultivating GM algae and the associated stringent regulations is considered as the main barrier of FGB production. Disposal of the residue is another important issue that should be considered in FGB production. The byproducts obtained from energy extraction step and residual water from the harvesting process may contain plasmid or chromosomal DNA that may cause the risk of lateral gene transfer. Hence an appropriate mitigation practices should be used for replacement of the hazardous water residue and by-products with more environmentally friendly alternatives. The results obtained from several field testing projects for open-environment exploitation of GM algae show that under the various conditions used, there was no apparent proof to support possible horizontal gene transfer in release of GM algae.

Emerging Technologies in Algal Biotechnology: Toward the Establishment of a Sustainable, Algae-Based Bioeconomy.

Abstract: Mankind has recognized the value of land plants as renewable sources of food, medicine, and materials for millennia. Throughout human history, agricultural methods were continuously modified and improved to meet the changing needs of civilization. Today, our rapidly growing population requires further innovation to address the practical limitations and serious environmental concerns associated with current industrial and agricultural practices. Microalgae are a diverse group of unicellular photosynthetic organisms that are emerging as next-generation resources with the potential to address urgent industrial and agricultural demands. The extensive biological diversity of algae can be leveraged to produce a wealth of valuable bioproducts, either naturally or via genetic manipulation. Microalgae additionally possess a set of intrinsic advantages, such as low production costs, no requirement for arable land, and the capacity to grow rapidly in both large-scale outdoor systems and scalable, fully contained photobioreactors. Here, we review technical advancements, novel fields of application, and products in the field of algal biotechnology to illustrate how algae could present high-tech, low-cost, and environmentally friendly solutions to many current and future needs of our society. We discuss how emerging technologies such as synthetic biology, high-throughput phenomics, and the application of internet of things (IoT) automation to algal manufacturing technology can advance the understanding of algal biology and, ultimately, drive the establishment of an algal-based bioeconomy.

Strategies in the delivery of Cas9 ribonucleoprotein for CRISPR/Cas9 genome editing.

Abstract: CRISPR/Cas9 genome editing has gained rapidly increasing attentions in recent years, however, the translation of this biotechnology into therapy has been hindered by efficient delivery of CRISPR/Cas9 materials into target cells. Direct delivery of CRISPR/Cas9 system as a ribonucleoprotein (RNP) complex consisting of Cas9 protein and single guide RNA (sgRNA) has emerged as a powerful and widespread method for genome editing due to its advantages of transient genome editing and reduced off-target effects. In this review, we summarized the current Cas9 RNP delivery systems including physical approaches and synthetic carriers. The mechanisms and beneficial roles of these strategies in intracellular Cas9 RNP delivery were reviewed. Examples in the development of stimuli-responsive and targeted carriers for RNP delivery are highlighted. Finally, the challenges of current Cas9 RNP delivery systems and perspectives in rational design of next generation materials for this promising field will be discussed.