Karas Bogumil J, Diner Rachel E, Lefebvre Stephane C, McQuaid Jeff, Phillips Alex P R, Noddings Chari M, Brunson John K, Valas Ruben E, Deerinck Thomas J, Jablanovic Jelena, Gillard Jeroen T F, Beeri Karen, Ellisman Mark H, Glass John I, Hutchison Clyde A, Smith Hamilton O, Venter J Craig, Allen Andrew E, Dupont Christopher L, Weyman Philip D
Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, California 92037, USA.
1] Microbial and Environmental Genomics Group, J. Craig Venter Institute, La Jolla, California 92037, USA [2] Integrative Oceanography Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92037, USA.
Nat Commun. 2015 Apr 21;6:6925. doi: 10.1038/ncomms7925.
Eukaryotic microalgae hold great promise for the bioproduction of fuels and higher value chemicals. However, compared with model genetic organisms such as Escherichia coli and Saccharomyces cerevisiae, characterization of the complex biology and biochemistry of algae and strain improvement has been hampered by the inefficient genetic tools. To date, many algal species are transformable only via particle bombardment, and the introduced DNA is integrated randomly into the nuclear genome. Here we describe the first nuclear episomal vector for diatoms and a plasmid delivery method via conjugation from Escherichia coli to the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. We identify a yeast-derived sequence that enables stable episome replication in these diatoms even in the absence of antibiotic selection and show that episomes are maintained as closed circles at copy number equivalent to native chromosomes. This highly efficient genetic system facilitates high-throughput functional characterization of algal genes and accelerates molecular phytoplankton research.
真核微藻在生物燃料和高价值化学品的生物生产方面具有巨大潜力。然而，与诸如大肠杆菌和酿酒酵母等模式遗传生物相比，藻类复杂生物学和生物化学特性的表征以及菌株改良一直受到低效遗传工具的阻碍。迄今为止，许多藻类物种仅能通过粒子轰击进行转化，且导入的DNA会随机整合到核基因组中。在此，我们描述了首个用于硅藻的核游离型载体以及一种通过从大肠杆菌接合转移至三角褐指藻和假微型海链藻的质粒递送方法。我们鉴定出一个酵母来源的序列，该序列能使这些硅藻中的游离型质粒即使在无抗生素选择的情况下也能稳定复制，并表明游离型质粒以闭环形式维持，其拷贝数与天然染色体相当。这种高效的遗传系统有助于对藻类基因进行高通量功能表征，并加速分子浮游植物研究。
