Genetic tools and techniques for Chlamydomonas reinhardtii

Mussgnug JH. Genetic tools and techniques for Chlamydomonas reinhardtii. Applied Microbiology and Biotechnology. 2015;99(13):5407-5418.The development of tools has always been a major driving force for the advancement of science. Optical microscopes were the first instruments that allowed discovery and descriptive studies of the subcellular features of microorganisms. Although optical and electron microscopes remained at the forefront of microbiological research tools since their inventions, the advent of molecular genetics brought about questions which had to be addressed with new Bgenetic tools^. The unicellular green microalgal genus Chlamydomonas, especially the most prominent species C. reinhardtii, has become a frequently used model organism for many diverse fields of research and molecular genetic analyses of C. reinhardtii, as well as the available genetic tools and techniques, have become increasingly sophisticated throughout the last decades. The aim of this review is to provide an overview of the molecular key features of C. reinhardtii and summarize the progress related to the development of tools and techniques for genetic engineering of this organism, from pioneering DNA transformation experiments to state-of-theart techniques for targeted nuclear genome editing and highthroughput screening approaches

Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit

Lauersen KJ, Kruse O, Mussgnug JH. Targeted expression of nuclear transgenes in Chlamydomonas reinhardtii with a versatile, modular vector toolkit. Applied Microbiology and Biotechnology. 2015;99(8):3491-3503

A novel one-stage cultivation/fermentation strategy for improved biogas production with microalgal biomass

Klassen V, Blifernez-Klassen O, Hoekzema Y, Mussgnug JH, Kruse O. A novel one-stage cultivation/fermentation strategy for improved biogas production with microalgal biomass. Journal of Biotechnology. 2015;215:44-51

Reconstruction of the lipid metabolism for the microalga Monoraphidium neglectum from its genome sequence reveals characteristics suitable for biofuel production

Bogen C, Al-Dilaimi A, Albersmeier A, et al. Reconstruction of the lipid metabolism for the microalga Monoraphidium neglectum from its genome sequence reveals characteristics suitable for biofuel production. BMC Genomics. 2013;14(1): 926.BACKGROUND: Microalgae are gaining importance as sustainable production hosts in the fields of biotechnology and bioenergy. A robust biomass accumulating strainof the genus Monoraphidium (SAG 48.87) was investigated in this work as apotential feedstock for biofuel production. The genome was sequenced, annotated, and key enzymes for triacylglycerol formation were elucidated. RESULTS: Monoraphidium neglectum was identified as an oleaginous species with favourable growth characteristics as well as a high potential for crude oil production, based on neutral lipid contents of approximately 21% (dry weight) under nitrogen starvation, composed of predominantly C18:1 and C16:0 fatty acids. Further characterization revealed growth in a relatively wide pH range and salt concentrations of up to 1.0% NaCl, in which the cells exhibited larger structures. This first full genome sequencing of a member of the Selenastraceae revealed a diploid, approximately 68 Mbp genome with a G + C content of 64.7%. The circular chloroplast genome was assembled to a 135,362 bp single contig, containing 67 protein-coding genes. The assembly of the mitochondrial genome resulted in two contigs with an approximate total size of 94 kb, the largest known mitochondrial genome within algae. 16,761 protein-coding genes were assigned to the nuclear genome. Comparison of gene sets with respect to functional categories revealed a higher gene number assigned to the category "carbohydrate metabolic process" and in "fatty acid biosynthetic process" in M. neglectum when compared to Chlamydomonas reinhardtii and Nannochloropsis gaditana, indicating a higher metabolic diversity for applications in carbohydrate conversions of biotechnological relevance. CONCLUSIONS: The genome of M. neglectum, as well as the metabolic reconstruction of crucial lipid pathways, provides new insights into the diversity of the lipid metabolism in microalgae. The results of this work provide a platform to encourage the development of this strain for biotechnological applications and production concepts

Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii

Lauersen KJ, Baier T, Wichmann J, et al. Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii. Metabolic Engineering. 2016;38:331-343

Phosphorus and nitrogen starvation reveal life-cycle specific responses in the metabolome of Emiliania huxleyi (Haptophyta)

The coccolithophore Emiliania huxleyi is a microalga with biogeochemical and biotechnological relevance, due to its high abundance in the ocean and its ability to form intricate calcium carbonate structures. Depletion of macronutrients in oceanic waters is very common and will likely enhance with advancing climate change. We present the first comprehensive metabolome study analyzing the effect of phosphorus (P) and nitrogen (N) starvation on the diploid and haploid life-cycle stage, applying various metabolome analysis methods to gain new insights in intracellular mechanisms to cope with nutrient starvation. P-starvation led to an accumulation of many generic and especially N-rich metabolites, including lipids, osmolytes, and pigments. This suggests that P-starvation primarily arrests cell-cycling due to lacking P for nucleic acid synthesis, but that enzymatic functionality is widely preserved. Also, the de-epoxidation ratio of the xanthophyll cycle was upregulated in the diploid stage under P-starvation, indicating increased nonphotochemical quenching, a response typically observed under high light stress. In contrast, N-starvation resulted in a decrease of most central metabolites, also P-containing ones, especially in the diploid stage, indicating that most enzymatic functionality ceased. The two investigated nutrient starvation conditions caused significantly different responses, contrary to previous assumptions derived from transcriptomic studies. Data highlight that instantaneous biochemical flux is a more dominant driver of the metabolome than the transcriptomically rearranged pathway patterns. Due to the fundamental nature of the observed responses it may be speculated that microalgae with similar nutrient requirements can cope better with P-starvation than with N-starvation

Investigating the dynamics of recombinant protein secretion from a microalgal host

Lauersen KJ, Huber I, Wichmann J, et al. Investigating the dynamics of recombinant protein secretion from a microalgal host. Journal of Biotechnology. 2015;215:62-71

Systematische molekulargenetische Identifizierung von TAK Insertionsmutanten der einzelligen Grünalge Chlamydomonas reinhardtii

Mussgnug JH. Systematische molekulargenetische Identifizierung von TAK Insertionsmutanten der einzelligen Grünalge Chlamydomonas reinhardtii. Diploma work. Bielefeld University: Bielefeld University; 2000

Optimizing microalgal biomass for biofuel production: the impact of glucose feeding on lipid and fatty acid profiles in Chlorella zofingiensis

Mussgnug JH. Optimizing microalgal biomass for biofuel production: the impact of glucose feeding on lipid and fatty acid profiles in Chlorella zofingiensis. Biofuels. 2010;1(6):831-833

Molekulargenetische und biochemische Charakterisierungen redox-regulierter plastidärer Signal-Transduktionsmechanismen in der einzelligen Grünalge Chlamydomonas reinhardtii

Mussgnug JH. Molekulargenetische und biochemische Charakterisierungen redox-regulierter plastidärer Signal-Transduktionsmechanismen in der einzelligen Grünalge Chlamydomonas reinhardtii. Bielefeld University: Bielefeld University; 2004