Enhanced Genetic Tools for Engineering Multigene Traits into Green Algae

Rasala, Beth A.; Chao, Syh-Shiuan; Pier, Matthew M.; Barrera, Daniel J.; Mayfield, Stephen P.

doi:10.1371/journal.pone.0094028

Cited by 115 publications

(70 citation statements)

References 43 publications

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“…Constructs were also designed which contained the PsaD or AtpA chloroplast or mitochondrial targeting peptides (Lauersen et al, 2015b;Rasala et al, 2014), respectively, in order to determine where subcellular pools of FPP may be within the cell (Sup. Fig.…”

Section: Chlorophyll Fluorescence Measurementsmentioning

confidence: 99%

“…One strategy in over expression of target proteins is gene-loading, involving either high copy plasmids or multiple copies of a transgene expression cassette introduced into the genome (Aw and Polizzi, 2013). The only examples of multiple transgene expression from the nuclear genome of C. reinhardtii have been through double transformations with selectable markers (Lauersen et al, 2015b), mating of two separately expressing strains (Rasala et al, 2014), or using viral 2A peptides for cleavage (Rasala et al, 2014).…”

Section: Increasing Sesquiterpene Synthase Titre Can Increase Relativmentioning

confidence: 99%

“…4B, iii+ix). Variable expression after secondary transformation for C. reinhardtii has been previously described (Lauersen et al, 2015b;Rasala et al, 2014). To circumvent these limitations, we next constructed repetitive fusions of the PcPs in two different orientations in vectors with paromomycin resistance: PcPs-YFP-PcPs and PcPs-PcPs-YFP (Fig.…”

Section: Increasing Sesquiterpene Synthase Titre Can Increase Relativmentioning

confidence: 99%

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

Lauersen

Baier

Wichmann

et al. 2016

Metabolic Engineering

122

231

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Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii, Metabolic Engineering, http://dx.doi.org/10.1016/j.ymben.2016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. olaf.kruse@uni-bielefeld.de AbstractThe heterologous expression of terpene synthases in microbial hosts has opened numerous possibilities for bioproduction of desirable metabolites. Photosynthetic microbial hosts present a sustainable alternative to traditional fermentative systems, using freely available (sun)light and carbon dioxide as inputs for bio-production. Here, we report the expression of a patchoulol synthase from Pogostemon cablin Benth in the model green microalga Chlamydomonas reinhardtii. The sesquiterpenoid patchoulol was produced from the alga and was used as a marker of sesquiterpenoid production capacity. A novel strategy for gene loading was employed and patchoulol was produced up to 922 ± 242 µg g -1 CDW in six days. We additionally investigated the effect of carbon source on sesquiterpenoid productivity from C. reinhardtii in scale-up batch cultivations. It was determined that up to 1.03 mg L -1 sesquiterpenoid products could be produced in completely photoautotrophic conditions and that the alga exhibited altered sesquiterpenoid production metabolism related to carbon source.

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Section: Chlorophyll Fluorescence Measurementsmentioning

confidence: 99%

Section: Increasing Sesquiterpene Synthase Titre Can Increase Relativmentioning

confidence: 99%

Section: Increasing Sesquiterpene Synthase Titre Can Increase Relativmentioning

confidence: 99%

See 1 more Smart Citation

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

Lauersen

Baier

Wichmann

et al. 2016

Metabolic Engineering

122

231

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show abstract

“…However, the development of a genetically engineered inducible lysis system for microalgae has limitations in that these microorganisms are eukaryotic and genomic sequences of most microalgae are not available yet. Nevertheless, new molecular biology tools have emerged in the last decade (Potvin and Zhang 2010;Radakovits et al 2010Radakovits et al , 2011Rasala et al 2014) and can help to overcome this limitation. Further inquiry into the development of inducible system for microalgae cell disruption is necessary.…”

Section: Accepted Manuscriptmentioning

confidence: 99%

Algicidal microorganisms and secreted algicides: New tools to induce microalgal cell disruption

Demuez

González‐Fernández

Ballesteros

2015

Biotechnology Advances

126

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“…As an alternative, Rasala et al employed viral 2A peptides to express multi-cistronic transgenes (Rasala et al 2014;Rasala et al 2012), representing an important step towards more complex metabolic engineering approaches.…”

Section: Transformation Markers and Reportersmentioning

confidence: 99%

Genetic tools and techniques for Chlamydomonas reinhardtii

Mussgnug

2015

Appl Microbiol Biotechnol

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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 "genetic 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-the-art techniques for targeted nuclear genome editing and high-throughput screening approaches.

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Enhanced Genetic Tools for Engineering Multigene Traits into Green Algae

Cited by 115 publications

References 43 publications

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

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

Algicidal microorganisms and secreted algicides: New tools to induce microalgal cell disruption

Genetic tools and techniques for Chlamydomonas reinhardtii

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