Designer diatom episomes delivered by bacterial conjugation

Abstract

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.

Nitrate Reductase Knockout Uncouples Nitrate Transport from Nitrate Assimilation and Drives Repartitioning of Carbon Flux in a Model Pennate Diatom

“…In keeping with traditional genetic studies, P. tricornutum NR-KO lines 9 and 14 were complemented by a conjugative plasmid containing the native NR gene fused to YFP under the control of the native ProNR and TermNR; the plasmid was delivered by Escherichia coli (Karas et al, 2015). Sixteen transformants from each KO line were screened on NO 3 2 for viability, and all grew on solid and liquid media amended with NO 3 2 .…”

“…Two P. tricornutum NR-KO lines, 9 and 14, were complemented by a conjugative plasmid delivered into P. tricornutum by E. coli as described by Karas et al (2015). The plasmid was constructed to contain yeast replication origins and endogenous P. tricornutum NR fused to YFP under the control of the native NRprom and NRterm, as previously described (Karas et al, 2013).…”

Additional conjugation systems for inter-domain plasmid transfer to the diatom Phaeodactylum tricornutum

“…Cargo plasmid pPt-Cargo1383a was constructed using the pRL1383a (22) vector 115 derived from plasmid RSF1010 (23) that included the IncQ-MobA relaxase and transfer origin (oriT) to complement the knocked out IncP-TraI relaxase on pTA-MOB. A CenArsHis (containing the yeast-derived CenArsHis region that allows for centromeric replication in P. tricornutum (4,6) and the ShBle cassette that permits growth in the presence of phleomycin (24) were added to allow for episomal maintenance and selection of the cargo plasmid in P. 120 tricornutum. To make plasmid pPt-Cargo1383a, the CenArsHis-ShBle cassette was amplified using primers 1383a-insert-1 and 1383a-insert-2 (Table 1) from template plasmid pPtPBR1.…”

“…Conjugation between E. coli donor cells and P. tricornutum recipients was then tested using the same four sets of donor cells with the protocol that was previously developed for IncPbased conjugation between E. coli and P. tricornutum (6). P. tricornutum colonies were patched onto L1-Phelo20 plates and were tested for the presence of bacteria by streaking on LB medium.…”

“…The integrity of constructed pPT-Agro vectors were subsequently validated with colony PCR (primers Pt-AgroCheck1and SeqTest4-R, We developed a dual plasmid system to test whether the plasmid RSF1010 was able to be mobilized to the diatom nucleus by conjugation. We first constructed an RSF1010-based cargo plasmid, pPt-Cargo1383a, that contained a bleomycin selectable marker for P. 205 tricornutum (ShBle) and a centromeric maintenance region (6,24). Mobilization of RSF1010 requires the MPF functions of a conjugative plasmid such as RP4/RK2 plasmid.…”