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Plant Cell Reports

, Volume 37, Issue 10, pp 1383–1399 | Cite as

Advanced genetic tools enable synthetic biology in the oleaginous microalgae Nannochloropsis sp.

  • Eric Poliner
  • Eva M. Farré
  • Christoph Benning
Review

Abstract

Nannochloropsis is a genus of fast-growing microalgae that are regularly used for biotechnology applications. Nannochloropsis species have a high triacylglycerol content and their polar lipids are rich in the omega-3 long-chain polyunsaturated fatty acid, eicosapentaenoic acid. Placed in the heterokont lineage, the Nannochloropsis genus has a complex evolutionary history. Genome sequences are available for several species, and a number of transcriptomic datasets have been produced, making this genus a facile model for comparative genomics. There is a growing interest in Nannochloropsis species as models for the study of microalga lipid metabolism and as a chassis for synthetic biology. Recently, techniques for gene stacking, and targeted gene disruption and repression in the Nannochloropsis genus have been developed. These tools enable gene-specific, mechanistic studies and have already allowed the engineering of improved Nannochloropsis strains with superior growth, or greater bioproduction.

Keywords

Nannochloropsis Algal biotechnology Marker-free engineering Gene stacking Synthetic biology Episomes 

Introduction

Algae are highly efficient at turning solar energy into biomass and are sources of unique bioproducts, such as omega-3 fatty acids (Mühlroth et al. 2013; Zou et al. 2000), carotenoids (Yaakob et al. 2014), and interesting polysaccharides, such as agarose, alginate, and β-1,3-glucans (Chew et al. 2017; Sheehan et al. 1998; Yaakob et al. 2014). Several groups have screened algae for productivity and production of valuable compounds (Rodolfi et al. 2009; Sheehan et al. 1998; Unkefer et al. 2017). Nannochloropsis was identified as a genus with rapid growth and high lipid content, including triacylglycerol (TAG) (Rodolfi et al. 2009) and the omega-3 (ω3) long-chain polyunsaturated fatty acid, eicosapentaenoic acid (EPA) (Zou et al. 2000). Under nutrient-replete conditions Nannochloropsis species have a lipid content of approximately 25–30% of dry weight (Jia et al. 2015; Meng et al. 2015; Rodolfi et al. 2009; Xiao et al. 2015). Abiotic stresses, such as high light or nutrient deprivation, in particular nitrogen (N) deprivation, cause microalgae to pause growth and accumulate storage compounds. Under these conditions, Nannochloropsis species accumulate high quantities of TAG, up to 60% of biomass (Jia et al. 2015; Meng et al. 2015; Rodolfi et al. 2009; Simionato et al. 2013; Vieler et al. 2012; Xiao et al. 2015). In recent years, genomes for several Nannochloropsis species have become available (Table 1) (Corteggiani Carpinelli et al. 2013; Radakovits et al. 2012; Vieler et al. 2012; Wang et al. 2014) and molecular tools have been developed (Tables 2, 3, 4) (Ajjawi et al. 2017; Kilian et al. 2011; Poliner et al. 2017; Wei et al. 2017c), making species in this genus excellent microalgal models for comparative genomics (Hu et al. 2014; Wang et al. 2014).

Table 1

Publicly available whole-genome datasets produced in Nannochloropsis species

Citations

Strains

Datasets

Public access

Radakovits et al. (2012)

N. gaditana CCMP526

Genome assembly

http://www.nannochloropsis

genomeprojectsolutions-databases.com/

Corteggiani Carpinelli et al. (2013)

N. gaditana B-31

Genome browser and BLAST server

http://www.nannochloropsis.org

Wang et al. (2014)

N. oceanica IMET1 and CCMP531, N. granulata CCMP529, N. oculata CCMP525, N. salina CCMP537, N. gaditana CCMP526

Genome browser and BLAST server

http://www.bioenergychina.org:8989/

Vieler et al. (2012)

N. oceanica CCMP1779

Genome browser and BLAST server

https://genome.jgi.doe.gov/Nanoce1779/

Nanoce1779.home.html

Poliner et al. (2015)

N. oceanica CCMP1779

RNA-Seq during light:dark cycles

https://www.ncbi.nlm.nih.gov/geo/query/

acc.cgi?acc = GSE69460

Mühlroth et al. (2017)

N. oceanica CCMP1779

RNA-Seq under phosphate deprivation

https://www.ncbi.nlm.nih.gov/geo/query/

acc.cgi?acc = GSE95774

Hu et al. (2014)

N. oceanica IMET1 and CCMP531, N. granulata CCMP529, N. oculata CCMP525, N. salina CCMP537, N. gaditana CCMP526

Predicted regulatory connections of lipid biosynthetic genes

http://www.singlecellcenter.org

/en/NannoRegulationDatabase/Download.htm

Table 2

Genomic transformation protocols developed for the Nannochloropsis genus

Citations

Strain(s)

Reporter or epitope

Promoter(s)

Selection agent(s)

Vieler et al. (2012)

N. oceanica CCMP1779

 

LDSP

Hygromycin

Radakovits et al. (2012)

N. gaditana CCMP526

 

β-tub, UEP, HSP

Zeocin

Li et al. (2014a)

N. oceanica PP983 and MBIC10090, N. granulata MBIC10054, N. salina MBIC10063, N. gaditana CCAP849/5, N. oculata CCAP 849/1, N. limnetica KR1998/3

GUS

β-tub

Zeocin

Cha et al. (2011)

N. sp. UMT-M3

 

CMV 35S

Hygromycin

Ma et al. (2016)

N. oculata CS-179

 

C. reinhardtii HSP70A::RBCS2

Zeocin

Moog et al. (2015)

N. oceanica CCMP1779

Green FP

VCP

Zeocin

Shih et al. (2015)

N. oculata NIES-2146

shCP

C. reinhardtii HSP70A::RBCS2

 

Kang et al. (2015a)

N. salina CCMP1776

sfCherry

β-tub and UEP

Zeocin

Chen et al. (2008)

N. oculata

 

C. reinhardtii HSP70A::RBCS2

 

Kilian et al. (2011)

N. oceanica W2J3B

 

VCP

Zeocin, Hygromycin, Blasticidin

Reporter or epitope indicates if a protein tag was included, reporter abbreviations are FP fluorescent protein, CP chromoprotein, and GUS-β glucuronidase. Promoters used to drive transcription of transgenes in vivo are listed; the species of origin for non-Nannochloropsis promoters are indicated in italics. Endogenous promoter abbreviations are LDSP lipid droplet surface protein, β-tub β-tubulin, UEP ubiquitin extension protein, HSP heat shock protein, and VCP violaxanthin–chlorophyll a binding protein. Selection agent refers to antibiotic selection used to isolate Nannochloropsis transformants

Table 3

Overexpression studies conducted in the Nannochloropsis genus

Citations

Method(s)

Strain

Target gene(s)

Reporter(s) or epitope(s)

Promoter(s)

Selection agent(s)

Poliner et al. (2017)

Multi-gene overexpression

N. oceanica CCMP1779

delta-9, delta-12, delta-5 fatty acid desaturases

Firefly luciferase, NanoLuciferase, Cyan FP (Cerulean variant)

EF, LDSP, Ribi

Hygromycin, Zeocin

Zienkiewicz et al. (2017)

Overexpression

N. oceanica CCMP1779

diacylglycerol acyltransferase type 2–5

Yellow FP (Venus variant)

EF

Hygromycin

Kaye et al. (2015)

Overexpression

N. oceanica CCMP1779

delta-12 fatty acid desaturase

 

LDSP

Hygromycin

Li et al. (2016a)

Overexpression

N. oceanica CCMP1779

diacylglycerol acyltransferase type 2–7

Flag tag

Hsp20

Zeocin

Kang et al. (2015b)

Overexpression

N. salina CCMP1776

basic helix loop helix 2

Flag tag

β-tub, UEP

Zeocin

Beacham and Ali (2016)

Overexpression

N. salina CCAP 849/3

S. cerevisiae DGA1

 

CMV Tef, 35S

Hygromycin

Chen et al. (2017)

Overexpression

N. oceanica IMET1

malonyl CoA-acyl carrier protein transacylase

Flag tag

HSP20

Zeocin

Wei et al. (2017b)

Overexpression

N. oceanica IMET1

RuBisCO activase

 

β-tub, HSP70

Zeocin

Iwai et al. (2015)

Overexpression, inducible expression

N. oceanica NIES-2145

C. reinhardtii diacylglycerol acyltransferase type 2–4

 

C. reinhardtii SQD, VCP

Zeocin

Kang et al. (2017)

Overexpression

N. salina CCMP1776

A. thaliana WRINKLED1

Flag tag

β-tub, UEP

Zeocin

Kwon et al. (2017)

Overexpression

N. salina CCMP1776

basic leucine zipper domain 1

Flag tag

β-tub, UEP

Zeocin

Methods describe the genetic techniques used in the study. Strains refers to the Nannochloropsis subspecies. The target gene refers to the function of the primary gene manipulated. Reporter or epitope indicates if a protein tag was included. Promoters used to drive transcription of transgenes in vivo are listed; the species of origin for non-Nannochloropsis promoters are indicated in italics. Endogenous promoter abbreviations are LDSP lipid droplet surface protein, EF elongation factor, Ribi ribosomal subunit bidirectional, β-tub β-tubulin, UEP ubiquitin extension protein, HSP heat shock protein, and VCP violaxanthin–chlorophyll α binding protein. Selection agent refers to antibiotic selection used to isolate Nannochloropsis transformants

Table 4

Gene inactivation or repression studies conducted in the Nannochloropsis genus

Citations

Method(s)

Strain(s)

Target gene(s)

Reporter(s) or epitope(s)

Promoter(s)

Selection agent(s)

Kilian et al. (2011)

Disruption by HR

N. oceanica W2J3B

nitrate reductase

 

VCP

Zeocin, Hygromycin, Blasticidin

Wang et al. (2016)

Disruption by CRISPR/Cas9

N. oceanica IMET1

nitrate reductase

 

VCP, β-tub, V-ATPase

Hygromycin

Ajjawi et al. (2017)

Disruption by CRISPR/Cas9, Repression by CRISPR/Cas9, RNAi

N. gaditana CCMP1894

ZnCys

Green FP, Flag tag

initiation factor 4AIII, 60S ribosomal protein L24, initiation factor 3, TCT

Hygromycin, Blasticidin

Wei et al. (2017c)

Repression by RNAi

N. oceanica IMET1 and CCMP1779

carbonic anhydrase

 

β-tub

Zeocin

Dolch et al. (2017)

Disruption by HR

N. gaditana CCMP526

palmitate elongase

 

UEP

Zeocin

Perin et al. (2015)

Insertional mutagenesis

N. gaditana CCAP 849/5

  

UEP

Zeocin

Wei et al. (2017a)

Overexpression and RNAi

N. oceanica IMET1

diacylglycerol acyltransferase type 1-1A

Green FP

VCP, UEP, β-tub

Zeocin

Gee and Niyogi (2017)

Disruption by HR, complementation

N. oceanica CCMP1779

carbonic anhydrase

Flag tag, Yellow FP (Venus variant)

UEP

Zeocin

Nobusawa et al. (2017)

Overexpression and disruption by HR

N. oceanica NIES-2145

lysophosphatidic acid acyltransferase 1–4

Green FP, Yellow FP (Venus variant)

LDSP

Zeocin, Hygromycin

Ma et al. (2017)

Overexpression and repression by RNAI

N. salina CCMP537

pyruvate dehydrogenase kinase

Green FP

β-tub, VCP

Zeocin

Strain refers to the Nannochloropsis subspecies. Methods describe the genetic techniques used in the study. The target gene refers to the function of the primary gene manipulated. Reporter or epitope indicates if a protein tag was included, FP indicates fluorescent protein. Promoters used to drive transcription of transgenes in vivo are listed; the origin of transgenic promoters are indicated in italics. Endogenous promoter abbreviations are LDSP lipid droplet surface protein, β-tub β-tubulin, UEP ubiquitin extension protein, HSP heat shock protein, and VCP violaxanthin–chlorophyll a binding protein. Selection agent refers to antibiotic selection used to isolate Nannochloropsis transformants

Synthetic biology is an emerging field based on rationally designing biological systems (Andrianantoandro et al. 2006). To develop systems that behave as desired, an approach described as design, build, test is used to iteratively test refinements and determine how elements of the system influence the outcome (Agapakis 2014). The design phase is often based on information drawn from genome-wide data and databases of related systems. Organisms with high-quality genome-wide data and advanced genetic engineering tools that can be redesigned are known as chassis organisms. Metabolic maps are built using genome assemblies, functional annotation, and databases of known enzymatic pathways. Regulatory networks are coming into focus through integrating RNA-seq, chromatin immunoprecipitation DNA sequencing (CHIP-Seq), and databases of transcription factors and their target DNA motifs. To build or refine biosynthetic pathways or develop chassis organisms, several molecular tools are needed to modify the genome of an organism. While molecular tools, such as mutant libraries, transgenic overexpression, and reporter protein fusions, are instrumental in gaining a molecular understanding of biological processes, they are by themselves insufficient to create optimized biological systems. Redesigned algae will require a new generation of tools that enable precise and marker-free knockout mutants, and high-capacity gene stacking systems that can robustly and predictably express multiple genes. Finally, to test the synthesized system, highly facile methods to select or screen for the desired modifications as quickly as possible post-transformation are needed.

The Nannochloropsis genus is also an emerging algal model for genetic engineering of lipid accumulation (Ajjawi et al. 2017; Hu et al. 2014; Li et al. 2014b). Several Nannochloropsis species seem particularly amenable to transgene expression, with a moderate GC (guanine cytosine) content and simple gene structure facilitating genetic engineering (Jinkerson et al. 2013; Vieler et al. 2012; Wang et al. 2014). Several endogenous promoters and terminators are in use, including bidirectional promoters that are helpful in stacking transgenes, i.e., expressing multiple genes (Jinkerson et al. 2013; Kilian et al. 2011; Moog et al. 2015; Nobusawa et al. 2017; Poliner et al. 2017). Methods exist for targeted DNA insertion by homologous recombination into the genome (Dolch et al. 2017; Kilian et al. 2011; Nobusawa et al. 2017). CRISPR (clustered regularly interspaced short palindromic repeats)-based methods have been developed, making targeted gene disruption and editing possible (Ajjawi et al. 2017; Wang et al. 2016). Genetic engineering toolkits are becoming publicly available and should accelerate development of Nannochloropsis species as chassis organisms.

Taxomony and evolution of the Nannochloropsis genus

Nannochloropsis is a genus in the heterokont phylum. Photosynthetic heterokonts are secondary endosymbionts originating from a unicellular heterotrophic eukaryotic cell that engulfed a red alga, which, over time, became a plastid. The evolutionary relationships of organisms in this clade are complex and still not well defined. The red algal-type plastid shows several signatures of its origin distinct from green lineage plastids (Janouskovec et al. 2010; Keeling 2009; Wei et al. 2013). Brown algae and diatoms are also algae of the heterokont lineage that shares 57% and 51% of genes with N. gaditana CCMP526 genome, respectively (Radakovits et al. 2012).

The Nannochloropsis genus was established by Hibberd based on morphological characteristics (Hibberd 1981; Lubián 1982) and constituent species identified (oceanica, granulata, limnetica, salina, gaditana) based on the 18S ribosomal RNA (18S rDNA) and/or the plastid genome RuBisCO large subunit-encoding gene (rbcL) (Andersen et al. 1998; Fawley et al. 2015; Suda et al. 2002; Vieler et al. 2012). Whole genome phylogeny of six species of Nannochloropsis showed N. gaditana and N. salina to be closely related and separate from N. oceanica, N. granulata, and N. oculata (Wang et al. 2014). Fawley et al. proposed a separate genus (Microchloropsis) for N. gaditana and N. salina (Fawley et al. 2015); however in this review, the Nannochloropsis genus will be inclusive to N. salina and N. gaditana.

The Nannochloropsis genus seems to possess a number of nuclear genes derived from endosymbiotic gene transfer, particularly in lipid biosynthetic and carbohydrate degradation pathways, e.g., glycosyl hydrolases (Wang et al. 2014). The diverse genetic background of the Nannochloropsis genus may have contributed to its oleaginousness, with a particularly large set of putative lipid biosynthetic genes. For example, Nannochloropsis oceanica possess 11 type-2 diacylglycerol acyltransferases (DGATs) referred to also as DGTTs (Vieler et al. 2012; Wang et al. 2014; Xin et al. 2017; Zienkiewicz et al. 2017), and a high copy number of other predicted lipid biosynthetic genes such as: enoyl-ACP reductase (ENR), ketoacyl-ACP synthase (KAS), ketoacyl-ACP reductase (KAR), acyl-ACP thioesterase (TE), long-chain fatty acyl-CoA synthetase (LC-FACS), phosphatidic acid phosphatase (PAP), and lysophosphatidyl acyltransferase (LPAT) (Wang et al. 2014). Of these, only DGTTs (Li et al. 2016a; Wei et al. 2017a; Xin et al. 2017; Zienkiewicz et al. 2017) and LPATs (Nobusawa et al. 2017) have been characterized. It has been proposed that the ancestral heterotroph, the endosymbiotic red alga, and additional horizontal gene transfer contributed to the present genome (Wang et al. 2014).

The secondary endosymbiosis event also led to interesting cellular structure characteristics, such as four membranes surrounding the plastid, complicating intracellular trafficking (Keeling 2009; Kroth and Strotmann 1999; Murakami and Hashimoto 2009). Trafficking of nuclear encoded proteins into the plastid has been extensively studied in diatoms (Bolte et al. 2009), which led to the specialized protein location prediction software HECTAR (Gschloessl et al. 2008). While in several Nannochloropsis species, examples of protein localization by fluorescent protein (FP) fusions have been published, including to the plastid (Moog et al. 2015), endoplasmic reticulum (ER) (Gee and Niyogi 2017; Poliner et al. 2017), mitochondria (Ma et al. 2017), and lipid droplets (Nobusawa et al. 2017; Zienkiewicz et al. 2017), there have not been investigations into the signals and mechanisms of protein localization. The transport of metabolites across subcellular compartments also has not been studied in the Nannochloropsis genus, although it represents a plausible target for optimizing metabolite production (Loira et al. 2017).

Genetic diversity has also arisen frequently by horizontal gene transfer during algae evolution (Bowler et al. 2008; Diner et al. 2017) and has enabled adaptation to unique environments or metabolic niches (Schonknecht et al. 2013). Several lipid biosynthetic genes appear to be related to bacterial homologs and were likely acquired by horizontal gene transfer, including KAR, PAP, ENR, KAS, TE, and LC-FACS genes (Wang et al. 2014). An operon encoding proteins specialized in hydrogen generation possibly derived from bacteria has also been identified in N. oceanica (Vieler et al. 2012). Transkingdom gene transfer by bacterial conjugation to diatoms and the capacity of heterokonts to maintain episomal DNA indicates a possible route of gene acquisition (Diner et al. 2017; Karas et al. 2015). This evolutionary diversity of the Nannochloropsis genus and the genetic plasticity of the heterokonts provide an interesting model for symbiotic evolution and may lead to a chassis organism that can be robustly adapted for genetic engineering.

Genomes and transcriptomes across the Nannochloropsis genus

A complete genome sequence forms the foundation for gene-specific studies, and is a prerequisite for the drafting of metabolic and regulatory maps. Several genome assemblies have been generated for different species of Nannochloropsis, including multiple strains within a species, such as N. oceanica CCMP1779 (Vieler et al. 2012) and IMET1 (Wang et al. 2014), N. gaditana CCMP526 (Radakovits et al. 2012; Wang et al. 2016) and B-31 (Corteggiani Carpinelli et al. 2013), N. salina CCMP537, N. oculata CCMP525 (Wang et al. 2014), and N. granulata CCMP529 (Table 1) (Wang et al. 2014). The genomes of the examined Nannochloropsis species are approximately 30 megabases and contain 7–11,000 genes each (Radakovits et al. 2012; Vieler et al. 2012; Wang et al. 2014). The N. gaditana B-31 genome is estimated to be distributed over 30 chromosomes (Corteggiani Carpinelli et al. 2013), and the presumed N. oceanica IMET1 chromosomes separated by pulse-field gel electrophoresis as 22 individual genome fragments (Wang et al. 2014).

The plastid and mitochondrial genomes of representatives from five Nannochloropsis species were used to produce a pangenome of each organelle (Wei et al. 2013). The N. oceanica IMET1 plastid genome is 117,548 basepairs (bp) and contains 160 genes consisting of 126 protein-coding genes, and 34 RNA genes, and the mitochondrial genome is 38,057 bp and contains 63 genes consisting of 35 protein-coding genes and 28 RNA genes (Wei et al. 2013). The Nannochloropsis genus plastid pangenome contains signatures of a red algal origin, including red algal-type Rubisco and Rubisco activase genes (Starkenburg et al. 2014).

Extensive transcriptome data based on RNA sequencing of cells grown under different conditions reveal characteristic transcriptional changes, providing a whole-genome view of possible adjustments to maintaining homeostasis. Examined conditions include phosphorus (Mühlroth et al. 2017) and N deprivation (Corteggiani Carpinelli et al. 2013; Li et al. 2014b; Vieler et al. 2012), alternating light:dark cycles (Poliner et al. 2015), varying light intensities (Alboresi et al. 2016), and different growth phases of batch cultures (Radakovits et al. 2012) for various Nannochloropsis species (Table 1). These datasets suggest that different aspects of metabolism and other cellular processes, such as the cell cycle, are coordinated on a transcriptional level in response to environmental conditions. For example, N deprivation, which generally leads to a transition from the normal cell division cycle to quiescence, also causes transcriptional downregulation of photosynthesis and protein production, while lipid biosynthesis is upregulated as was observed for Nannochloropsis species (Corteggiani Carpinelli et al. 2013; Li et al. 2014b; Radakovits et al. 2012) and Chlamydomonas (Miller et al. 2010; Tsai et al. 2014). When N. oceanica is grown in a light:dark cycle, there is phased expression of certain genes at different times of day, including those involved in cell division at night, and anabolic processes during the day (Poliner et al. 2015). A majority (64%) of the DNA-binding transcription factors and 56% of other transcriptional regulators have phased expression during the light:dark cycles. These genome-wide datasets are an asset for further studies into metabolic adjustments occurring in response to environmental changes and the underlying regulation. Links to currently published genome-wide datasets are listed in Table 1.

Light regulation and photosynthesis

In heterokont algae, light, either by capture and conversion of solar energy during photosynthesis or by perception through photosensory regulatory proteins, affects metabolite levels (pigments, lipids, and carbohydrates) (Chauton et al. 2013; Poliner et al. 2015), coordinates the cell cycle (Ashworth et al. 2013; Chauton et al. 2013; Huysman et al. 2013, 2010; Poliner et al. 2015), and may entrain a circadian clock (Braun et al. 2014). In Nannochloropsis species, high-intensity light results in accumulation of TAG and a decrease in plastid size, thus maximizing energy conversion while avoiding photodamage (Alboresi et al. 2016; Sukenik et al. 1989; Xiao et al. 2015). The day:night cycle influences most organisms to coordinate behavior and/or metabolism with either phase (Ashworth et al. 2013; Chauton et al. 2013; Poliner et al. 2015). The transitory storage compounds used by N. oceanica during a light:dark cycle are under study with TAG and carbohydrates (measured in the form of hexoses) oscillating throughout a light:dark cycle (Fábregas et al. 2002; Poliner et al. 2015; Sukenik and Carmeli 1990). Nannochloropsis species accumulate TAG (Sukenik and Carmeli 1990) and carbohydrates during the day, which are both metabolized during the night, in accordance with transcriptional changes in genes encoding enzymes of the respective biosynthesis and utilization pathways (Poliner et al. 2015).

Nannochloropsis species are studied as a model for photosynthesis in secondary endosymbionts. The Nannochloropsis genus is notable for only possessing chlorophyll a, the unusual carotenoids violaxanthin and vaucheriaxanthin ester, and a xanthophyll cycle utilizing violaxanthin, antheraxanthin, and zeaxanthin (Alboresi et al. 2017; Cao et al. 2013; Chukhutsina et al. 2017). Red algae and their derived endosymbionts contain LHCr-type antenna proteins that link core complex pigment protein components and participate in energy transfer and photoprotection (Alboresi et al. 2017; Cao et al. 2013; Umetani et al. 2017). Characterization of the photosystem II (PSII) of N. gaditana identified the light harvesting complex proteins of the classes LHCx, LHCf, Red-CLH-like LHC, and LHCr that are characteristic of the red alga-type plastid (Umetani et al. 2017). Characterization of the N. gaditana photosystem I (PSI) discovered the absence of several subunits (PsaH, PsaK, PsaG) that are typically present in land plants, and identified the light harvesting complex proteins of the classes LHCr, LHCf, and LHCx associated with PSI (Alboresi et al. 2017).

Lipid and carbon metabolism

Lipid biosynthesis is the best characterized metabolic pathway in Nannochloropsis species, in particular the production of TAG and EPA. The Nannochloropsis genus is hypothesized to possess a cytosolic type-I fatty acid synthases (FAS) in addition to the plastid type-II FAS complex, but further studies to corroborate this hypothesis are needed (Alboresi et al. 2016; Poliner et al. 2015; Vieler et al. 2012). The TAG biosynthetic pathway involves the transfer of acyl chains to a glycerol backbone by the sequential action of glycerolphosphate acyltransferase (GPAT), LPAT, and DGAT, in addition phospholipidiacylglycerol acyltransferases (PDATs) have also been identified in Nannochloropsis species (Vieler et al. 2012). The four LPATs of N. oceanica have been investigated for their roles in membrane lipid and TAG biosynthesis with LPAT1 and LPAT4 having primary roles in each process, respectively, and LPAT2 and LPAT3 possibly playing roles in both processes (Nobusawa et al. 2017). Of the 13 DGAT-encoding genes, 6 are upregulated during N deprivation, a condition that also favors TAG accumulation (Zienkiewicz et al. 2017). A particularly robust DGAT of Nannochloropsis that functions in many different hosts has been identified (Zienkiewicz et al. 2017).

The Nannochloropsis genus contains the omega-3 fatty acid EPA in its membrane lipids [15–30% total fatty acids (TFA)] (Schneider et al. 1995; Schneider and Roessler 1994; Vieler et al. 2012; Zou et al. 2000). Reconstruction of the N. oceanica EPA biosynthetic pathway in S. cerevisiae by introducing four LC-PUFA fatty acid desaturases (FADs) and a fatty acid elongase (FAE) resulted in the production of EPA (0.1% TFA) (Poliner et al. 2017). FADs are named for the double bond introduced, a specific number of carbons from either the carboxyl (Δ, delta-) or methyl (ω, omega-) end of a fatty acid chain. Thus, omega-3 and delta-6 FADS act on the third carbon from the methyl end and the sixth carbon from the carboxyl end, respectively. The FADs of N. oceanica resemble those of other heterokont algae with their histidine box motifs for coordinating a diiron center and contain in two cases (delta-5 and delta-6) a cytochrome b domain (Poliner et al. 2017). Eleven fatty acid elongases have been identified in N. oceanica (Vieler et al. 2012) and the delta-6 and palmitic fatty acid-specific elongases from N. oceanica and N. gaditana, respectively, have been characterized in some detail. The palmitic acid elongase controls flux into the EPA pathway by conversion of 16:0 to 18:0 (Dolch et al. 2017), while the delta-6 elongase converts 18:3 to 20:3, two intermediates with low in vivo abundance (Poliner et al. 2017). EPA is likely produced in the ER but accumulates on diacylglyceryl-trimethylhomoserine (DGTS) and monogalactosyl diacylglycerol (MGDG), and to a lesser extent on digalactosyldiacylglycerol (DGDG) and phosphatidylglycerol (PG) (Simionato et al. 2013; Vieler et al. 2012). It has been proposed that EPA is imported into the plastid by a DGTS-mediated transport (Schneider and Roessler 1994).

Carbohydrates play structural, storage, and osmoprotectant roles in Nannochloropsis species. In N. oceanica, glucose is the predominant hexose in the total complex carbohydrate fraction, which contains smaller amounts of mannose and trace amounts of rhamnose, fucose, arabinose, xylose, and galactose (Vieler et al. 2012). Marine Nannochloropsis species reduce their level of the sugar alcohol mannitol and the disaccharide trehalose content in response to low-salt stress consistent with a role of these carbohydrates in osmoprotection (Pal et al. 2013). Heterokont algae lack starch, but produce β-1,3 linked polysaccharides (chrysolamarinin), by the activity of a β-1,3-glucan synthase which is predicted to be encoded in the genome of Nannochloropsis species (Corteggiani Carpinelli et al. 2013; Vieler et al. 2012). Approximately, 20% of alcohol-insoluble polysaccharides are in this form in N. oceanica (Vieler et al. 2012). Chrysolamarinin is also a storage compound in diatoms (Chauton et al. 2013; Hildebrand et al. 2017) and has been suggested to have a similar role in Nannochloropsis species (Arnold et al. 2015; Li et al. 2014b; Xiao et al. 2015), but further studies are needed to confirm this hypothesis. Cellulose is a major polysaccharide in Nannochloropsis species, with approximately 80% of the alcohol-insoluble polysaccharides in this form in N. oceanica (Vieler et al. 2012). Cellulose serves as a major component of the cell wall (Jeong et al. 2017; Scholz et al. 2014; Vieler et al. 2012), but the cell wall is quite complex in Nannochloropsis species (Scholz et al. 2014). Four putative cellulose synthase-encoding genes have been identified (Jeong et al. 2017; Scholz et al. 2014). A large number of carbohydrate-degrading enzymes, 48–49 glycosyl hydrolases, with very diverse taxonomic relations, are found encoded across the pangenome (Wang et al. 2014). The complete repertoire of carbohydrate metabolism in the Nannochloropsis genus is yet to be fully established.

To understand the metabolic networks of Nannochloropsis species, a summary of possible chemical reactions in the form of a metabolic map has been generated. A mass-balanced metabolic map for N. salina CCMP537 has been produced by Loira and colleagues taking into account nine organelles (as well as the plastid lumen) of the cell (Loira et al. 2017). The model was validated by modeling different growth conditions and comparing it to in vivo data. The conditions of N and phosphate deprivation were used to maximize lipid production and determine essential nutrients, respectively. The iNS934 map-based model indicated several genes whose disruption may result in increased TAG.

Transcriptional regulation

The dynamic control of metabolism in response to environmental changes and intracellular cues is multilayered, but inevitably involves transcription factors (TF, possess DNA binding activity) and transcriptional regulators (TR, regulators of TF activity) that modulate the gene expression. Databases of known TFs and corresponding position-weighted matrices (PWM) (Stormo 2000) can be used to systematically determine potential interacting TF-DNA sequences. Having a large number of closely related organisms is a valuable resource for in silico predictions of conserved regulatory DNA motifs (Hu et al. 2014). Genome sequencing and cataloging of TFs as a first step have been undertaken for the Nannochloropsis genus (Hu et al. 2014; Vieler et al. 2012). Comparative genomic studies of the heterokont lineage have identified TF signatures based upon organismal lifestyle (autotrophic, parasitic), multicellularity, or life cycle stages (Buitrago-Flórez et al. 2014; Rayko et al. 2010; Thiriet-Rupert et al. 2016). TF prediction of N. oceanica implied the presence of 115 putative TFs and 109 putative TRs, which combined represent about 2% of the predicted proteins encoded in the genome (Vieler et al. 2012). The Nannochloropsis genus has a reduced number of TF and TR families (20–26) compared to land plants and green algae, possibly due to a simpler life cycle and its unicellularity (Hu et al. 2014; Vieler et al. 2012). Putative TFs of the Myb family (29–35 members), a TF family known to regulate growth and metabolism in other organisms, are enriched in the Nannochloropsis genus (Hu et al. 2014; Vieler et al. 2012). Several TFs in Nannochloropsis species have been investigated, but studies of their targets are only beginning. Despite the large amounts of predicted TFs and TRs, their roles need to be experimentally corroborated.

Several approaches have been used to identify regulators of lipid biosynthesis in Nannochloropsis species. Hu and colleagues (Hu et al. 2014) took advantage of the extensive genome sequences available to predict conserved transcription factor binding sites (TFBS). They determined the enrichment of gene ontology (GO) terms associated with each motif, and enrichment of motifs associated with lipid biosynthetic genes. Using a TF catalog and RNA-seq during N deprivation, Hu et al. identified TFs that showed positive or negative co-expression with lipid biosynthetic genes. Finally, they predicted putative connections between TFs and lipid biosynthetic gene promoters based on a TF-DNA motif database (Wingender 2008). One of these predicted lipid biosynthesis regulating TFs (bZIP1) was recently investigated (Kwon et al. 2017). In addition, Ajjawi and colleagues (Ajjawi et al. 2017) identified 20 TFs possibly involved in TAG accumulation based on changes in expression under N deprivation, and used CRISPR/Cas9 disruption to assess the predictions.

As a photosynthetic organism, light sensing is likely important for tuning metabolism in Nannochloropsis species. Aureochromes are heterokont-specific photosensitive transcription activators, with a bZIP DNA binding domain and a photosensing dimerization LOV domain (Vieler et al. 2012). In diatoms the aureochromes are implicated in regulating several processes, including cell cycle and light acclimation (Huysman et al. 2013; Mann et al. 2017; Schellenberger Costa et al. 2013). Cryptochromes are blue light photoreceptors derived from DNA repair enzymes, which have been characterized in diatoms (Coesel et al. 2009) and found to oscillate during light:dark cycles in diatoms (Ashworth et al. 2013) and N. oceanica (Poliner et al. 2015). Recently, the Chlamydomonas reinhardtii animal-like cryptochrome was reported to be involved in regulating the cell cycle and the circadian clock (Müller et al. 2017; Zou et al. 2017). Finally, although phytochromes were thought to be absent in most heterokonts, they have recently been identified in diatoms (Fortunato et al. 2016). N. oceanica possesses three aureochromes, an animal-like cryptochrome, and lacks phytochromes, but these proteins have not been functionally characterized (Vieler et al. 2012).

Transformation and gene expression platforms

The most widely adapted method for transformation of Nannochloropsis species is by electroporation (Kilian et al. 2011; Radakovits et al. 2012; Vieler et al. 2012), but other protocols based on biolistics (Kang et al. 2015a, b) or agrobacterium have been developed (Beacham and Ali 2016; Cha et al. 2011) (Table 2). For insertion of a transgene into the genome by electroporation, a linear piece of DNA is required; a constructed DNA (construct) is therefore digested with restriction enzymes or PCR-amplified (Kilian et al. 2011; Li et al. 2014a; Poliner et al. 2017; Vieler et al. 2012). Each transformant is likely to have a distinct insertion site, and therefore may display different phenotypes due to genome context-specific regulation of the transgene’s expression or disruption of endogenous genes by transgene insertion (Cha et al. 2011).

Introduction of circular DNA has so far had mixed success in producing transformants (Kilian et al. 2011; Li et al. 2014a; Vieler et al. 2012). Synthetic Genomics Inc. has described plasmid/episome maintenance in algae by use of autonomous replication sequences from a Nannochloropsis species (Dehoff et al. 2014) and the utilization of an S. cerevisiae centromere (autonomous replication sequence, CEN/ARS) region in pennate and centric diatoms (Diner et al. 2016a, 2017; Karas et al. 2015). In diatoms, episomes are maintained under antibiotic selection, but are gradually lost without selection pressure (Diner et al. 2016a, 2017; Karas et al. 2015). The transgene expression levels from episomes are more uniform compared to genome-integrated constructs between independent transformants, likely due to the absence of insertion site-specific effects using this approach (Diner et al. 2016a; Karas et al. 2015). The capacity of episomes for maintenance of foreign DNA have been reported to be up to 94 kb in diatoms (Karas et al. 2015).

Antibiotic resistance marker genes

Several antibiotics are effective depending on the Nannochloropsis strain, including hygromycin, zeocin, and blastidicin, and genes conferring resistance are used to isolate Nannochloropsis transformants (Ajjawi et al. 2017; Kilian et al. 2011; Nobusawa et al. 2017; Radakovits et al. 2012; Vieler et al. 2012). Zeocin in combination with its respective marker gene is the most widely used selection in Nannochloropsis species due to its stringency at low concentrations (Tables 2, 3, 4). However, it is mutagenic and can lead to secondary mutations (Lin et al. 2017). In diatoms, the selection agents nourseothricin and G418 are frequently utilized with their respective antibiotic resistance genes (Hildebrand et al. 2017; Karas et al. 2015; Zaslavskaia et al. 2001). Mutated endogenous proteins in conjunction with competitive inhibitors, such as phytoene desaturase and the inhibitor norflurazon can also be used as a selection marker in some algae (Huang et al. 2008). We have made a number of vectors containing N. oceanica-adapted antibiotic selection marker genes that will be available on Addgene (http://www.addgene.com). When several selection agents and resistance genes are available, multiple transgenic tools can be used in conjunction in one transgenic line (Fig. 1a). However, techniques for generation of transgenic algae without antibiotic resistance markers are necessary for deployment into open ponds. The removal of an antibiotic resistance marker gene could be achieved by the use of a recombinase or endogenous homologous recombination (Cheah et al. 2013). The cotransformation of an episome carrying an antibiotic resistance gene with an insertion construct without a selection marker may also enable generation of marker-free mutants after episome loss in the absence of selection pressure.

Fig. 1

Gene stacking strategies in N. oceanica. Diagrams represent configuration for two protein-coding transgenes. Gene1 represented by GFP is shown in green. Gene2 represented by NanoLuciferase is shown in blue. Promoters are indicated by arrows and terminators by a T (Synthetic Biology Open Language standard) (Galdzicki et al. 2014). Resistance markers are indicated as R’ (red). Nannochloropsis expression cassettes without a plasmid backbone are shown. a Constructs with unique selection markers (indicated as R’1 and R’2, red) can be introduced into one line. b Bidirectional promoters regulate transcription on both DNA strands and express two transcripts. c Sequences encoding 2A peptides are placed between two protein-encoding genes. A peptide bond is not formed between the two final amino acids (*) during translation. Two discrete proteins are produced, the N’ terminal protein (green) contains the majority of the 2A peptide (yellow) and the C terminal protein (blue) contains the last amino acid of the 2A peptide (not shown). d Assembly of multiple expression cassettes into a single construct to produce multiple transcripts from different promoters. The image of the GFP protein was obtained from the NIH Image Gallery (https://www.flickr.com/photos/nihgov/) and is adapted under the terms of CC BY-NC 2.0. The NanoLuciferase image is adapted from Hall et al. with permission (Hall et al. 2012)

Transgenic expression in Nannochloropsis species

A fundamental technique for genetic engineering and synthetic biology is the overexpression of target genes by increasing transcriptional and/or translational efficiency. Most often, strong promoters that mediate high transcription rates are utilized to express heterologous or endogenous genes at elevated levels. Several endogenous promoters from a variety of Nannochloropsis species have been isolated and applied to transgenic expression (Tables 2, 3, 4), including those driving the genes encoding ubiquitin extension protein (UEP) (Dolch et al. 2017; Kang et al. 2015a; Radakovits et al. 2012; Wei et al. 2017a), β-tubulin (β-tub) (Kang et al. 2015a; Li et al. 2014a; Ma et al. 2017; Radakovits et al. 2012; Wang et al. 2016), lipid droplet surface protein (LDSP) (Kaye et al. 2015; Nobusawa et al. 2017; Poliner et al. 2017; Vieler et al. 2012), and elongation factor (EF) (Poliner et al. 2017; Zienkiewicz et al. 2017). Several bidirectional promoters are utilized for transgenic expression, including those driving the expression of the genes encoding violaxanthin chlorophyll binding proteins (VCP) (Kilian et al. 2011; Ma et al. 2017; Moog et al. 2015) or ribosomal subunits (Ribi) (Fig. 1b) (Poliner et al. 2017). To enhance translational efficiency of transgenes, a 5′ UTR can include a consensus Kozak sequence (Dehoff and Soriaga 2014) or leader-enhancing sequence (Gallie et al. 1987; Li et al. 2016a; Xue et al. 2015).

Reporter genes are useful for evaluating transgenic strategies and understanding gene/promoter function. In several Nannochloropsis species, members of the classes of the fluorescent- (FP), luminescent- (lux), and chromoproteins (CP) are used (Tables 2, 3, 4). Green fluorescent protein (GFP) is the most widely reported fluorescent protein and has been utilized for subcellular localization of fusion proteins throughout Nannochloropsis cells (Ma et al. 2017; Moog et al. 2015; Nobusawa et al. 2017). Other fluorescent proteins such as a red fluorescent protein (RFP, sfCherry) (Kang et al. 2015a), yellow FP (YFP, Venus variant) (Gee and Niyogi 2017; Nobusawa et al. 2017; Zienkiewicz et al. 2017), and cyan FP (CFP, Cerulean variant) (Poliner et al. 2017) are employed in different Nannochloropsis species. Luciferases have the advantage of a high signal to noise ratio, and specific substrates allow their use in combination. Codon-optimized firefly luciferase (Flux) and the ultra-bright NanoLuciferase (Nlux) for in vivo assays in N. oceanica are in use (Poliner et al. 2017). The ultra-bright Nlux allows detection of very low protein quantities and is an effective photon donor for bioluminescence resonance energy transfer (BRET) (Hall et al. 2012; Suzuki et al. 2016). Chromoproteins are colored and do not need a substrate, while the β-glucuronidase (GUS) reporter is an enzyme that produces a blue stain after conversion of 5-bromo-4-chloro-3-indolyl-beta-d-glucuronic acid (X-Gluc). A purple chromoprotein (shPCP) from the sea anemone Stichodacyla haddoni was successfully produced in N. oculata and used for screening of transformants (Shih et al. 2015), while GUS was utilized in N. salina (Li et al. 2014a).

Virus-derived 2A peptides allow production of two discrete proteins from a single transcript by preventing the formation of a peptide bond (peptide bond “skipping”) (Fig. 1c). The nascent protein interacts with the ribosome causing it to stall, then during the release of the ribosome a peptide bond is “skipped” between the final two amino acids (Sharma et al. 2012). 2A peptides are widely applied to different hosts and the efficiency of skipping depends on the peptide variant and host. After screening several variants and lengths in N. oceanica, the P2A peptide of 60 amino acids was found to be most efficient (Poliner et al. 2017). The F2A peptide has also been used in N. salina (Unkefer et al. 2017) and Chlamydomonas (Plucinak et al. 2015). To facilitate multigene expression, bidirectional promoters with P2A peptide sequences were assembled in the pNOC-stacked vector series and will be made available through Addgene (Poliner et al. 2017). Toolkits for assembly of multiple transgenic expression cassettes into a single vector have been developed for some algae (Hamilton et al. 2014; Jia et al. 2016) and would further facilitate gene stacking in Nannochloropsis species (Fig. 1d).

Generation of targeted gene disruption and transcriptional repression

RNA interference (RNAi) is a powerful technique that can suppress gene expression to varying degrees. RNAi by antisense or double-stranded RNA has been developed in a number of algae including Chlamydomonas (Moellering and Benning 2010; Rohr et al. 2004), diatoms (De Riso et al. 2009; Hildebrand et al. 2017; Schellenberger Costa et al. 2013), and several Nannochloropsis species (Ajjawi et al. 2017; Ma et al. 2017; Wei et al. 2017a, c). RNAi using an inverted repeat of regions of a target gene has been found to be effective throughout the Nannochloropsis genus (Fig. 2a) (Table 4) (Ajjawi et al. 2017; Ma et al. 2017; Wei et al. 2017a, c). To generate a strong and stable repression effect, several strategies are available including fusing the interfering RNA to an antibiotic resistance gene (De Riso et al. 2009; Wei et al. 2017c), co-silencing a gene that can be counter selected (Rohr et al. 2004), or expression of the interfering RNA from a bidirectional promoter that also drives expression of an antibiotic resistance gene. In case of essential genes, full disruption of the target gene can result in slow growth or lethality, while transcriptional repression targeting the same gene may result in a moderate phenotype (Ajjawi et al. 2017).

Fig. 2

Gene repression and inactivation techniques in the Nannochloropsis genus. Promoters are indicated by arrows and terminators by a T (Synthetic Biology Open Language standard) (Galdzicki et al. 2014). Resistance markers are indicated as R’ (red). Extended DNA is indicated by an overhanging backbone, and free ends of DNA are indicated by blunt ends. a RNA interference by expression of sense (blue) and antisense (green) sequences of a target gene at the 3′ end of a resistance gene. The sense and antisense sequences form an inverted repeat on the resistance marker transcript. b Homology arms are the sequences flanking a desired insertion site (green). A resistance cassette with flanking homology arms to a target region is introduced by transformation. Homologous recombination between the homology arms of the resistance cassette and the target region results in a disrupted gene. c CRISPR/Cas9 techniques utilize the Cas9 nuclease and an sgRNA, forming ribonucleoprotein complex (yellow). The PAM site (blue) and the final 3′ nucleotide of the guide sequence (green) are indicated. Double stranded cuts are produced in the 3′ end of the target region. Incorrect repair of the strand break prevents further Cas9 action. Insertion or deletion mutations are most likely to occur. d Insertional mutagenesis by transformation with a resistance cassette. The resistance cassette is randomly inserted throughout the genome, resulting in mutant lines with unique insertion sites. Desired mutants (green) are identified by a phenotype screen, target gene screen, or from a mutant library

Homologous recombination has been demonstrated in several Nannochloropsis species for insertion of an antibiotic resistance marker with flanking regions of 1 kb identical to the insertion site (Fig. 2b). Targeting efficiency has been reported to be high when transforming low-density cultures (Kilian et al. 2011). Several groups have used this technique for gene disruption (Table 4) (Dolch et al. 2017; Gee and Niyogi 2017; Kilian et al. 2011; Nobusawa et al. 2017). Homologous recombination is adaptable to several purposes, such as, insertion of protein tags, insertion into neutral sites, or replacement of genes with altered functionality.

CRISPR/Cas9 is an RNA-guided nuclease-based approach that is dramatically expanding the capabilities of biologists to modify genomes, particularly through the ability to disrupt specific genes or perform precise gene editing (Fig. 2c) (Cong et al. 2013; Ran et al. 2013). In this system, two components have to be localized to the nucleus, a single-guide RNA (sgRNA) and the Cas9 nuclease, which together form a ribonucleoprotein complex. The sgRNA requires production without extraneous sequences or modifications on the termini; strategies for sgRNA production include the use of RNA polymerase III-driven promoters (most often the U6 promoter) (Nymark et al. 2016), direct introduction into the cell (Ajjawi et al. 2017), expression of modified tRNAs containing the sgRNA in a spliced region (Cermak et al. 2017), co-expression of a ribonuclease and sgRNA with cleavage sites (Cermak et al. 2017), and use of self-cleaving ribozymes (Cermak et al. 2017). The U6 promoter from diatoms appear to be active and suitable for sgRNA production (Nymark et al. 2016). However, there have been no reports of successful U6 promoter use in the Nannochloropsis genus, and two publications utilized alternative strategies (Table 4). Wang and colleagues (Wang et al. 2016) expressed the sgRNA from a V-ATPase promoter and had a low mutational efficiency. In a strategy developed by Ajjawi and coworkers (Ajjawi et al. 2017), the sgRNA is synthesized and introduced by transformation into a Nannochloropsis strain expressing Cas9. The generation of off-target mutations is an unresolved issue for CRISPR-based gene editing, which has not been examined in the Nannochloropsis genus. Several strategies exist to reduce the number of potential off-targets, including transient expression of one or both components (Ajjawi et al. 2017; Baek et al. 2016; Xie et al. 2017), paired nickase Cas9 (Ran et al. 2013), or high-fidelity Cas9 enzymes (Kleinstiver et al. 2016; Slaymaker et al. 2016).

Insertional mutatagenesis, whereby an antibiotic resistance gene is randomly integrated into a genome, results in gene disruptions and gene deletions (Fig. 2d) (Li et al. 2016b; Tsai et al. 2014). Insertional mutagenesis conducted in N. gaditana produced mutants with a variety of growth and photosynthetic phenotypes, the screening of which identified lines with enhanced light-use efficiency (Perin et al. 2015). While insertional mutagenesis is an efficient method for forward-genetic screens, ready-to-use lines with a disruption of a desired gene requires a mutant library that takes a significant investment to establish (Li et al. 2016b).

Altering metabolism in Nannochloropsis species by protein engineering

The usefulness of the aforementioned genetic engineering tools has been demonstrated by modifying different aspects of metabolism. The majority of studies have targeted lipid biosynthesis, either to enhance TAG or EPA production (Tables 2, 3, 4). In several cases endogenous or heterologous (from S. cerevisiae or C. reinhardtii) DGATs have been overproduced in different Nannochloropsis species (Beacham and Ali 2016; Iwai et al. 2015; Li et al. 2016a; Wei et al. 2017a; Xin et al. 2017; Zienkiewicz et al. 2017). Overexpression of the endogenous DGAT1a-encoding gene in N. oceanica resulted in a 39% increase in TAG content per cell and RNAi repression resulted in a 20% decrease in TAG content per cell following N deprivation (Wei et al. 2017a). Overexpression of the endogenous DGGT5-encoding cDNA in N. oceanica resulted in a 3.5-fold increase in TAG (as %TFA) (Zienkiewicz et al. 2017). Furthermore, the DGTT7-encoding cDNA has also been overexpressed in N. oceanica IMET1, resulting in 69 and 129% increase in neutral lipid (% dry weight) content under N-replete and N-deprivation conditions (Li et al. 2016a). The malonyl-CoA transacylase of N. oceanica IMET1, which loads the malonyl group onto the acyl-carrier protein for fatty acid synthesis, has been characterized (Tian et al. 2013) and its overproduction resulted in a 36% (% dry weight) increase in lipids without compromised growth (Chen et al. 2017). In N. salina, RNAi conducted against the pyruvate dehydrogenase complex (PDC) kinase (PDCK), to increase acetyl-CoA levels for fatty acid production (21), resulted in enhanced TAG content at the expense of protein (Ma et al. 2017). Increased expression of the desaturase genes in the EPA pathway using gene stacking techniques achieved up to a 25% increase in EPA (as %TFA) (Poliner et al. 2017). Engineering efforts are only beginning and the enhanced productivity of modified Nannochloropsis strains will require further identification of targets to affect metabolite partitioning into the desired pathways, and/or the reduction of final product turnover.

Altering metabolism in Nannochloropsis species by regulatory engineering

Manipulation of entire metabolic pathways on a greater scale could be accomplished by TF engineering. Recently, TF overproduction, or inactivation (CRISPR) and repression (RNAi), of a TF encoding gene has been used in various Nannochloropsis species with the goal of increasing biomass and/or lipid production (Tables 3, 4) (Ajjawi et al. 2017; Kang et al. 2015b, 2017; Kwon et al. 2017). Overproduction of the bHLH2 TF in N. salina resulted in an increased growth rate and a greater biomass productivity, although the transcriptional reprogramming was not described (Kang et al. 2015b). Overproduction of the bZIP1 TF, a predicted lipid biosynthetic TF (Hu et al. 2014), in N. salina resulted in enhanced growth, and under stress conditions enhanced lipid content (Kwon et al. 2017). In the bZIP1 overexpressor lines, the expression of putative target genes involved in lipid biosynthesis (Hu et al. 2014) was increased under normal conditions and more dramatically under stress conditions (Kwon et al. 2017). Incredibly, the introduction of the Arabidopsis thaliana WRINKLED1 TF (WRI1), a regulator of seed oil production, into N. salina enhanced lipid accumulation, possibly by upregulating lipid biosynthetic genes containing the WRI1 motifs in the promoter (Kang et al. 2017). CRISPR inactivation of the ZnCys TF, likely involved in N assimilation, resulted in a large increase in TAG content in N. gaditana, but a strong reduction of growth. An optimization of the balance between growth versus lipid accumulation was achieved through decreased expression of ZnCys by RNAi or by the CRISPR/Cas9-mediated insertion of an antibiotic resistance cassette into the 3′ UTR of this gene (Ajjawi et al. 2017).

Additional challenges for the development of improved Nannochloropsis strains

To develop improved strains optimized for biosynthetic yield, either by increasing flux into the target pathway or by disruption of competing pathways, multiple specific modifications are likely to be required. For example, stacking gene modifications will be necessary to introduce new pathways or for the optimization of existing pathways. Furthermore, the development of marker-free strategies or the utilization of auxotrophic markers for selection of genetically modified strains are necessary for deployment of engineered strains into open ponds exposed to the environment.

Disrupting competing metabolic pathways may yield enhanced productivity of certain bioproducts. Carbohydrates are a competing sink for lipid production and polysaccharide biosynthetic genes have been targeted for repression in green algae, and diatoms to increase lipid accumulation (Daboussi et al. 2014; Hildebrand et al. 2017; Work et al. 2010). The tough cell wall of the Nannochloropsis genus takes considerable cellular resources to construct and is an impediment to efficient processing of cells to obtain bioproducts. Therefore, Nannochloropsis strains with weakened walls may be superior for bioproduction. Reducing the turnover of desired products may enhance their accumulation, and identification of the genes involved in product degradation will be an avenue toward enhancing the productivity of algae (Trentacoste et al. 2013; Xue et al. 2013).

The successful adoption of gene disruption technology will facilitate disruption of biosynthetic genes for essential metabolites, generating strains that require supplementation or gene complementation (auxotrophy). Nitrate reductase (NR)-deficient strains of Chlamydomonas strains can be complemented with a wild-type NR gene and selected on nitrate media (Kindle et al. 1989). The NR gene has been targeted in several studies in various Nannochloropsis species (Ajjawi et al. 2017; Kilian et al. 2011; Wang et al. 2016) and diatoms (Diner et al. 2016b; McCarthy et al. 2017). Auxotrophic selection based on the NR-disrupted strains may be particularly useful when paired with episomal artificial chromosomes, enabling a nutrient selection pressure on episome maintenance.

Call for an open alga

Establishing a model organism (or bioproduct chassis) requires dissemination of the skills and tools developed to a wide network of scientists. A niche is developing for third-party repositories (TPR) that facilitate the maintenance of accumulated biological materials and are making these materials more accessible than ever before. However, for a TPR to be sustainable, innovators must be willing to transfer their materials and utilize the TPR as part of their own workflow. A notable nonprofit TPR for plasmid and strain dissemination is Addgene that we are collaborating with by depositing the collection of Nannochloropsis engineering vectors developed in our studies. Some of the model Nannochoropsis species such as CCMP1779 (N. oceanica), CCMP526 (N. gaditana), and CCMP537 (N. salina) are publicly available from algae culture collections (NCMA, https://ncma.bigelow.org/). We are making an engineered NR knockout strain publicly available through NCMA, as well. As transgenic tools and knockout and chassis strains are produced, deposition with TPRs will accelerate innovation, allow researchers to do more with less, build genome-wide overexpression and knockout strain libraries, and establish quality controls and standardization in the field.

Author contribution statement

EP, EF, and CB wrote the manuscript. All authors read and approved the manuscript.

Notes

Acknowledgements

This work was supported by a National Science Foundation grant (IOS-1354721) to EF. In addition, parts of this work were supported by the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of the United States Department of Energy (DE-FG02-91ER20021) and MSU-AgBioResearch to CB.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Cell and Molecular Biology ProgramMichigan State UniversityEast LansingUSA
  2. 2.MSU-DOE Plant Research LaboratoryMichigan State UniversityEast LansingUSA
  3. 3.Department of Plant BiologyMichigan State UniversityEast LansingUSA
  4. 4.Department of Biochemistry and Molecular BiologyMichigan State UniversityEast LansingUSA

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