A toolkit for Nannochloropsis oceanica CCMP1779 enables gene stacking and genetic engineering of the eicosapentaenoic acid pathway for enhanced long‐chain polyunsaturated fatty acid production

Abstract

Summary Nannochloropsis oceanica is an oleaginous microalga rich in ω3 long‐chain polyunsaturated fatty acids (LC‐PUFAs) content, in the form of eicosapentaenoic acid (EPA). We identified the enzymes involved in LC‐PUFA biosynthesis in N. oceanica CCMP1779 and generated multigene expression vectors aiming at increasing LC‐PUFA content in vivo. We isolated the cDNAs encoding four fatty acid desaturases (FAD) and determined their function by heterologous expression in S. cerevisiae. To increase the expression of multiple fatty acid desaturases in N. oceanica CCMP1779, we developed a genetic engineering toolkit that includes an endogenous bidirectional promoter and optimized peptide bond skipping 2A peptides. The toolkit also includes multiple epitopes for tagged fusion protein production and two antibiotic resistance genes. We applied this toolkit, towards building a gene stacking system for N. oceanica that consists of two vector series, pNOC‐OX and pNOC‐stacked. These tools for genetic engineering were employed to test the effects of the overproduction of one, two or three desaturase‐encoding cDNAs in N. oceanica CCMP1779 and prove the feasibility of gene stacking in this genetically tractable oleaginous microalga. All FAD overexpressing lines had considerable increases in the proportion of LC‐PUFAs, with the overexpression of Δ12 and Δ5 FAD encoding sequences leading to an increase in the final ω3 product, EPA.

Identification of circadian rhythms inNannochloropsisspecies using bioluminescence reporter lines

“…The LDSP terminator (CCMP1779_4188) was PCR amplified from N. oceanica CCMP1779 genome using the primers LDSP term sac F+ and LDSP term afl R-(Figure S16), and digested with SacI and AflII to replace the 35S terminator as the terminator for the luciferase gene. The LUC+HA gene was then replaced by either N. oceanica CCMP1779 codon optimized coding sequences for firefly luciferase (FLUC) and, nanoluciferase (NLUC) (Hall et al , 2012, Poliner et al , 2018b) or a Chlamydomonas codon optimized renilla luciferase (RLUC) (Ferrante et al , 2008) to form pNOC-Fluc, pNOC-Nluc, and pNOC-Rluc respectively using AscI and SacI and the primers listed in Figure S16. The pNOC vectors were used to transform N. oceanica CCMP1779.…”

“…This cassette, which includes the CCMP537 EF promoter (NODE_7115_length_93819_cov_37.515949:78382..79716), the zeocin resistance gene and the CCMP1779 LDSP terminator, from N. salina selection construct pNSA-511 were cloned by restriction digest (AhdI/SbfI) generating the pNSA-FLUC and vector (Dataset 1). The pNSA-511 construct was generated by cloning the CCMP537 EF promoter into pNOC-411 using SnabI and XhoI (Poliner et al , 2018b). The promoters of the cellulose synthase (NannoCCMP1779|5780, NODE_11401_length_57650_cov_57.237450:4263..6099), LHC1 (NODE_14101_length_22726_cov_35.737923:17540..18097), and LHC8 (NannoCCMP1779|6809, NODE_11394_length_43512_cov_40.897591:1551..2663) promoters were amplified from the N. oceanica CCMP1779 and N. salina CCMP537 genomes using primers listed in Figure S16 and inserted into pENTR-D-Topo (Invitrogen).…”

“…Several Nannochloropsis strains from different worldwide locations have been sequenced and current data suggests that Nannochloropsis species are haploid with genome sizes of ~30 Mb, containing from ~6,500 to ~12,000 genes (Corteggiani Carpinelli et al , 2014, Radakovits et al , 2012, Schwartz et al , 2018, Vieler et al , 2012, Wang et al , 2014). Recent years have seen the development of a comprehensive set of molecular tools for working with these algae, such as homologous gene replacement, overexpression, gene silencing and CRISPR/Cas9 based genome editing (Kilian et al , 2011, Poliner et al , 2018a, Poliner et al , 2018b, Poliner et al , 2018c, Verruto et al , 2018, Wang et al , 2016, Wei et al , 2017). In this study, we describe the development of a bioluminescence reporter system for the long-term in vivo measurement of gene expression in two Nannochloropsis species.…”

Identification of circadian rhythms in Nannochloropsis species using bioluminescence reporter lines

“…This cassette, which includes the CCMP537 EF promoter (NODE_7115_length_93819_cov_37.515949:78382..79716), the zeocin resistance gene and the CCMP1779 LDSP terminator from N. salina selection construct pNSA-511 were cloned by restriction digest (AhdI/SbfI), generating the pNSA-FLUC and the vector (Dataset S1). The pNSA-511 construct was generated by cloning the CCMP537 EF promoter into pNOC-411 using SnabI and XhoI (Poliner et al, 2018b). The promoters of the cellulose synthase (NannoCCMP1779|5780, NODE_11401_length_57650_ cov_57.237450:4263..6099), LHC1 (NODE_14101_length_22726_ cov_35.737923:17540..18097) and LHC8 (NannoCCMP1779|6809, NODE_11394_length_43512_cov_40.897591:1551..2663) promoters were amplified from the N. oceanica CCMP1779 and N. salina CCMP537 genomes using the primers listed in Figure S16 and inserted into pENTR-D-Topo (Invitrogen, now ThermoFisher Scientific, https://www.thermofisher.com).…”

Optimized methods of chromatin immunoprecipitation for profiling histone modifications in industrial microalgae Nannochloropsis spp.

“…Moreover, they are excellent research models for microalgal systems and synthetic biology, due to their small genome size and simple gene structure (Vieler et al 2012, Jinkerson et al 2013, Wei et al 2013, Corteggiani et al 2014). Moreover, a comprehensive set of genetic tools has been recently established that includes homologous recombination (Kilian et al 2011, Gee andNiyogi 2017), gene knockdown (Wei et al 2017b, Xin et al 2017, gene editing (Wang et al 2016, Ajjawi et al 2017) and overexpression (Kang et al 2015, Li et al 2016, Nobusawa et al 2017, Xin et al 2017, Poliner et al 2018. Thus, Nannochloropsis spp.…”