Nannochloropsis artificial chromosomes (NannoACs) loom on the horizon*

Li GUO, Guanpin YANG,2,3,**

1 College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China

2 Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China

3 Key Laboratory of Marine Genetics and Breeding of Ministry of Education, Ocean University of China, Qingdao 266003,China

Abstract Species in genus Nannochloropsis, especially N.oceanica and N.gaditana, have been evolving as the model microalgae for both application and theory studies.The position effect of genome integration, the carrying capability limitation of integrative vectors and the instability of non-integrative vectors have hindered Nannochloropsis genetic modification with concatenate genes and extremely long DNA fragments.The molecular tools including genetic transformation, homologous recombination, gene edition, gene stacking and episome vectors for transient gene expression and diverse reporters and selection markers have been rapidly developing in Nannochloropsis species.The construction of animal and plant artificial chromosomes with “top down” strategy has set fine examples for the construction of Nannochloropsis artificial chromosomes (NannoACs).It seems that the methods and materials to set the foundation for constructing NannoACs are at hands.In this review, we outlined the current status of transgenes in Nannochloropsis species, summarized the limitations of both integrative and non-integrative vectors, and proposed a tentative approach to construct NannoACs by doubling and stabilizing the genome first, and then truncating the natural chromosomes.NannoACs once constructed will facilitate transferring the desired traits and concatenate genes into Nannochloropsis genetic backgrounds, thus contributing towards its genetic improvement and synthetic biological studies.

Keyword: Nannochloropsis; artificial chromosome; microalgal genetic improvement; gene stacking;concatenate gene; trait transfer

1 INTRODUCTION

The genusNannochloropsiscomprises seven species that inhabit seawater, freshwater and brackish (Hibberd,1981; Fawley and Fawley, 2007).The genus has been split intoNannochloropsisandMicrochloropsisaccording to the differences of the concatenated 18S ribosomal RNA andrbcL genes (Fawley et al., 2015).Either inNannochloropsisorMicrochloropsis, all the species are assigned to class Eustigmatophyceae,phylum Ochrophyta, superphylum Heterokonta (or Stramenopiles), which are extremely small, nonmotile and spherical in shape (Bailey and Freshwater, 1997;Daugbjerg and Andersen, 1997; Andersen et al.,1998; Kandilian et al., 2013).These species are monoploidy and asexual (Galloway, 1990; Pan et al.,2011).Nannochloropsisspecies are able to accumulate high concentrations of pigments including astaxanthin,zeaxanthin, and canthaxanthin (Lubián et al., 2000)and have chlorophyllabut completely lack chlorophyllsbandc(Manning and Strain, 1943).Nannochloropsisspecies are mainly used as feed for fish larvae and rotifers and as food additives for human consumption.They also promise to be industrially utilized because they accumulate a large quantity of polyunsaturated fatty acids (Boussiba et al., 1987; Sukenik et al.,1989).

Species in genusNannochloropsishave been evolving into the models for both industrial applications and biological researches (Gee and Niyogi, 2017).The genomes of twoNannochloropsisspecies,N.gaditana(Radakovits et al., 2012;Carpinelli et al., 2014) andN.oceanica(Pan et al.,2011; Vieler et al., 2012; Liang et al., 2013; Guo et al., 2019; Gong et al., 2020), have been sequenced,which vary between 28.5 and 29.3 Mb in size.A phylogenetic analysis ofNannochloropsisspecies genomes revealed a significant dose expansion among lipid biosynthesis genes (Wang et al., 2014).The molecular tools including genetic transformation(Li et al., 2014; Chen and Hu, 2019; Abidin et al.,2020), targeted gene replacement by homologous recombination (Kilian et al., 2011), CRISPR/Cas9-based genome edition (Wang et al., 2016; Poliner et al.,2018c; Naduthodi et al., 2019; Zhang et al., 2019),gene stacking (Verruto et al., 2018; Poliner et al.,2020), transient expression of genes in episomes(Poliner et al., 2018b; Kurita et al., 2020; Wang et al.,2021), and sets of reporters and selection markers(Poliner et al., 2018b; Naduthodi et al., 2021b) have been rapidly developing inNannochloropsis.Unfortunately, the genes, either alone or being stacked, are not able to express sustainably if not integrated into genome and cause the position effect if integrated into genome.In addition, the genetic modification of microalgae, for example, the enhancement of productivity and lipid production(Muñoz et al., 2021; Naduthodi et al., 2021a;Zulaiha, 2021; Chen et al., 2022), cannot detour genetically engineering complex traits, metabolic pathways among others and combining genes locating separately in genome.Artificial chromosomes should aid to synthesize (integrate) separate genes and allow us to modify complex traits and metabolic pathways.The expressions of concatenate genes and the genes in large chromosomal fragments appreciate developingNannochloropsisartificial chromosomes (NannoACs).

2 CURRENT STATUS OF THE GENE ENGINEERING OF NANNOCHLOROPSIS

2.1 Expression of single genes

The expression of a single gene may modify the trait ofNannochloropsisspecies.For example, the NobZIP77 ofN.oceanicarepresses the transcription of a type-2 diacylgycerol acyltransferase gene under nitrogen-repletion condition.However, it relieves such repression under nitrogen-depletion condition.In addition, it senses blue light, and changes the growth rate and fatty acid productivity of the alga once being inactivated (Zhang et al., 2022).The expression of a transgene may be silenced due to its integration into the host genome (position effect).To detour the genomic integration, a “safe harboring”strategy has been established forNannochloropsis.The best integration position in genome is determined first according to the expression intensiveness of a reporter gene, and then the transgene is harbored(knocked in) this position with the aid of Cas9-mediated edition.Fatty acid desaturase (FAD12)gene encoding Δ12-fatty acid desaturase at this preferred locus significantly increased its expression and changed the fatty acid composition (Ryu et al.,2021).Recently, Südfeld et al.(2022) fortunately discovered a strong gene expression site withinNannochloropsisnucleolus where the cellular rRNA synthesis machinery is recruited for transcription.The exploited by the expression system include the highly efficient transcriptional activity of RNA polymerase I and an internal ribosome entry site for translation.Successful genetic/metabolic engineering ofNannochloropsisspecies has improved their phenotypes including, for example, violaxanthin yield (Park et al., 2021) and growth rate and lipid content (Ma et al., 2016).Unfortunately, the evaluation of influence-less position in genome is time consuming and labor intensive.It is hard to popularize such strategy in our routine trying to express foreign genes.

2.2 Stacking genes for metabolism pathways

The common situation we encounter often is that a metabolite synthesis pathway, a trait among others are run by a set of genes.Engineering these pathways and traits often requires the co-expression of multiple transgenes.To meet this requirement, a strategy called “gene stacking” has been established inN.oceanicaCCMP1779.A set of gateway entry and destination vectors are developed first, and then the entry cassette is stacked onto the destination vector via the phiC31-attintegration system (Reece-Hoyes and Walhout, 2018; Poliner et al., 2020).As an early trial, the enzymes involved in long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis inN.oceanicaCCMP1779 are identified and multigene expression vectors are generated to increase LC-PUFA content in vivo.The cDNAs encoding four fatty acid desaturases are isolated, which are then stacked together.The over expressions of these genes in different combinations lead to an increase in eicosapentaenoic acid (EPA) content(Poliner et al., 2018c).“Gene stacking” is not a new term and a new idea in plant breeding, which has been using in the context of genetically modified crops for many years (Halpin, 2005).Plant breeders always stack genes by crossing parents each carrying a desirable trait and then identifying offspring inheriting both of these desired traits.

Gene stacking appreciates the improvement of the gene carrying capability of vectors.The carrying capability of a plasmid vector cannot be expanded unlimitedly.One of the solutions is to adopt the bacterial artificial chromosome (BAC) which may carry a large number of genes in both fusion protein and concatenate gene cassettes and their expression controlling elements.A BAC is able to clone DNA sequences varying from 100 to about 300 kb(Shizuya and Kouros-Mehr, 2001; Zhang et al.,2021).Except for the limitation in the numbers of controlling elements, reporter genes and selection markers, the influence of integration position in the genome is also a challenge (Ryu et al., 2021).Plasmids are used in bacteria to build up gene constructs.Upon transformed into microalgae, they must integrate into the chromosomes, thus are called integrating vectors.Although they can deliver genetic materials to a target cell and enable long-term expression of transgenes,their integration derives a potential risk of causing insertional mutagenesis.In contrast, episomal vectors behave as separate extrachromosomal elements, thus may avoid these undesired side effects (Ehrhardt et al., 2008).The insertional mutation may disrupt the genes anywhere in host genome while the expression of genes on episomal vector does not interrupt any gene due to their independent extrachromosomal existence.Without selection pressure of antibiotic markers, it will disappear through biological dilution.

2.3 Non-integrative vector

Episomal vectors may aid to overcome the disadvantages of integrative ones.Very interestingly,foreignCENandARShave been proved to function inN.oceanica, and a one-vector CRISPR system for non-integrative gene disruption inN.oceanicahas been developed (Poliner et al., 2018c).A hygromycin B resistance cassette, a Cas9-3×glycine-serine linker-reporter fusion protein gene promoted by a bidirectional promoter and terminated by LDSP terminator and the sgRNA promoted by the same bidirectional promoter and terminated by a CS terminator are expressed simultaneously.The fusion protein is assigned into nucleus by placing SV40 nuclear localization signals at its N’ and C’ termini.The sgRNA is trimmed automatically after transcription by self-cleaving 3’ HDV and 5’ HH ribozymes.TheSaccharomyces cerevisiaeCEN/ARS6region is recombined into the episome to create an episomal CRISPR system which will lose when selection pressure is removed.This design assures the edited cells to be marker-free.The circular plasmids containingCEN/ARSare not inserted into the chromosome and can be removed after editing(Kurita et al., 2022).The nuclear episomal vector for gene cloning in diatomsPhaeodactylumtricornutumandThalassiosirapseudonanahas also been developed(Karas et al., 2015).The episome is not stable; it will be diluted when the antibiotics is removed away.The episome is the choice for gene edition;however, it is not appropriate for transgenes.

2.4 The prospect of Nannochloropsis YAC-like vectors looks bleak

A method similar to the construction of yeast artificial chromosome (YAC) could be used to develop microalgal artificial chromosomes (Fabris et al., 2020).Actually, only one-step ahead to evolve episome towards the YAC-like vectors inNannochloropsis.Four essential chromosomal elements,a yeast centromereCEN4, an autonomously replicating sequenceARS1and twoTetrahymenatelomeric sequencesTELs, function in yeast, making YAC replicate and vertically transfer from parents to daughter cells as a real yeast chromosome does.YAC can be modified thorough homologous recombination to carry additional selection markers and genes or delete the unwanted regions or recombine with other YAC (Burke et al., 1987; Arnak et al., 2012).

Embedding animal cells and plant protoplasts in agarose plugs and then extracting DNA within the agarose plugs minimize the risk of DNA breaking.The extracted DNA can be stored for long periods,partially fragmented enzymatically (Nair et al.,1999), and separated through pulsed-field gel electrophoresis (PFGE) (Hicks et al., 2018).Once being inserted into a YAC, the extra-long DNA fragments can be cloned and amplified in yeast.The spectrum of YAC applications is very wide, from constructing DNA libraries of the large genomes to identifying genes within a large chromosomal region.YAC can also be retrofitted with the desired genes and appropriate selective markers and transferred into animal cells, with the functions of genes within a large DNA region deciphered there.In addition,the selection markers of YAC are the normal genes of an auxotrophic mutant.

To build up a construct containingCEN,ARS,andTELcassettes may detour plasmid and bacteria by polishing (or blunting) and ligating DNA fragments and PCR products ofCEN,ARS, andTELelements, or via synthetic sequence-guided isothermal assembly (Torella et al., 2014; Kadkhodaei et al.,2016).As found in practice,CEN/ARSof yeast does not allow the episomal vectors ofNannochloropsisto have enough stability.Although not proved, YAC may not be transplanted intoNannochloropsis.We must consider the stability of the yeastCEN/ARS6region in episomal vectors ofNannochloropsis,since the vector may lose when selection pressure is removed (Kurita et al., 2022).

3 ANIMAL AND PLANT ARTIFICIAL CHROMOSOME EXAMPLES OF NANNOACS

In contrast, mammalian artificial chromosomes,for example, human artificial chromosomes (HACs)and mouse artificial chromosomes (MACs), can carry multiple genes or megabase-sized genomic loci and their associating regulatory elements, facilitate creating more useful and complex transgenic models for human disease studies and drug development(Moriwaki et al., 2020).HACs and MACs have set fine examples for the expecting NannoACs.Two strategies, “top down” and “bottom up”, have been used to develop HACs and MACs.

3.1 HAC and MAC constructions with “top down” strategy

The P1 bacteriophage cyclization recombination recombinase (Cre) mediates the recombination between pairedloxsites.Depending on the promoters and other regulatory controls,Crein bacteria, fungi,plants, and animals can be manipulated to synthesize Cre recombinase under certain conditions.Crecan also be expressed transiently by transfecting eukaryotic cells with bacterial plasmid containingCreand its controlling elements.The flankedloxsites integrated in eukaryotic chromosomes may cause deletions,inversions and translocations when Cre recombinase is available, which is called Cre-loxrecombination system.Similarly, FLP-frtsystem is becoming more frequently used in mouse-based researches, in which flippase (FLP) recombinase derived from the yeastS.cerevisiaerecognizes and recombines paired FLP recombinase target (frt) motifs that flank a genomic region of interest (Araki et al., 1997; Schweizer,2003).

The top-down construction of a HAC needs to reduce the size of a natural chromosome by targeted truncation.In the chicken DT40 cells allowing a high rate of homologous recombination, the transferred human chromosome 22 was truncated atLIFlocus and ligated with human telomeric repeats (TTAGGG)n(Kuroiwa et al., 1998).Homologous recombination based chromosomal truncation in eukaryotic cells provides the opportunity of constructing HACs with a “top-down” strategy and expressing genes in an extremely large genetic locus.The HAC is transferred into mouse embryo stem cells that are then injected into host embryo, leading to generate chimeric and germline transmissible mouse(Tomizuka et al., 1997).With the aids ofloxand Cre recombinase, defined regions of human chromosomes>one megabase in length can be cloned into a stable HAC in homologous recombinationproficient chicken DT40 cells (Kuroiwa et al.,2000).With the “top-down” strategy and the homologous recombination systems, a MAC with essential centromere and telomeres was constructed,which contains human Ig heavy chain locus (IGH)derived from hChr.14 and Ig kappa light chain locus(IGK) derived from hChr.2.It is maintained in transchromosomic mice which produces human antibody in the mouse Ig-knockout background(Satofuka et al., 2022).

With the “top-down” strategy, MACs from native mouse chromosomes 10 and 11 (10MAC and 11 MAC)have been constructed, which carry diverse markers like puromycin and neomycin resistances, enhanced green fluorescent protein (GFP) among others and Cre-loxDNA loading system (Takiguchi et al., 2014;Abe et al., 2021).HAC derived from native human chromosomes, independently and stably maintained without disrupting host chromosomes, have been also constructed, which carry recombination sites for loading genes, and are capable to transfer a large quantity of megabase sized genes (Oshimura et al., 2015).

3.2 HAC and MAC construction with “bottom up” strategy

In contrast to “top-down construction”, the “bottomup construction” strategy involves the de novo buildup of HACs and MACs by introducing DNA elements necessary for maintaining chromosome function into cells.A HAC is a mini-chromosome behaving as a stable chromosome in host nucleus using its own self-replicating and segregating systems.Using the “bottom-up” strategy, a HAC is constructed by transfecting a cultured human cell line (for example, HT1080) with cloned or synthetic centromeric alphoid DNA precursors with CENP-B boxes, where the precursors multimerize up to megabase size (Ikeno and Suzuki, 2011).Alphoid-BAC containing 50-kb alphoid DNA andlox-BAC containing the CAG/lox71/neo cassette with insulator elements, 5’HS5 and 3’HS1 at left and right ends are co-transfected into HT1080 cells, from them the cells with multimerized HAC are subsequently selected out according to neomycin resistance and through fluorescence in-situ hybridization (FISH) analysis.The HAC constructed can be transferred into mouse cells with microcell-mediated chromosome transfer method or transchromosomic technique.Interested genes or DNA fragments in plasmid may be inserted into HAC via Cre-loxsystem.

A tetracycline response element (TRE) is the 7 repeats of a 19 nucleotides tetracycline operator(tetO) sequence, and is recognized by the tetracycline repressor (TetR).A tetracycline-controlled transactivator (tTA) is created by fusing TetR with the C-terminal domain of virion protein 16 (VP16),an essential transcriptional activation domain from herpes simplex virus (HSV).Tetracycline or its analogs like doxycycline switches off the expression of the genes downstreamTREby binding to tTA(Gossen and Bujard, 1992).Later, the TetR is mutated into a reverse tetracycline repressor rTetR,which induces rather than impress the expression of the genes downstreamTREwhen tetracycline presents.The new reverse tetracycline-controlled transactivator(rtTA) is created by fusing rTetR with VP16.This bacterial system functions in mammalian cells,making the expression inducible and controllable.Alphoid DNA and its variant with CENP-B box replaced with the tetO motif (Ponomartsev et al.,2020) are circulated and amplified in a way of rolling PCR.The PCR product is inserted in between hooks of a plasmid and transferred into yeast where inserts are assembled by transformation-associated recombination into approximately a 50-kb synthetic alphoid array as a circular molecule.Recombination further multimerizes the array into 1.1 Mb in size,forming the alphoidtetO-HAC, which is proved to be a versatile tool for studying human chromosome transactions and structure as well as for genome and cancer studies (Kouprina et al., 2018).The alphoidtetO-HAC has also been modified to be applicable for assembling (iterate) genomic DNA fragments or genes with regulated kinetochore using three combinations of integrases (recombinase) and mediate motifs, Cre-lox, phiC31-att, and phiBT1-att, making cells capable of carrying multiple transgenes and megabase-sized genomic loci, thus allowing us to investigate complex biomedical pathways and gene regulation, and engineer synthetic chromosomes with a predetermined set of genes(Lee et al., 2018).

In essence, the HACs and MACs constructed with “bottom-up” strategy are the modified centromeres, into which genes can be integrated via the mediate motif and recombinase systems.They are circular DNA thus their sustainable inheritance does not need telomeres.These artificial chromosomes work; however, their replication effeciency is not determined.It is possible that such chromosome may not perform as good as the natural ones.In addition, the transformation efficiency may hinder effectively transferring large DNA constructs intoNannochloropsiscells.To these considerations, as the preliminary trials, it is better to construct NannoACs by truncating naturalNannochloropsischromosomes, i.e., with the top-down strategy.

3.3 Plant minichromosmes (artificial chromosomes)

The artificial chromosomes have also been constructed in plants.Although widely debated publicly, yet genetic engineering with just a few genes, most frequently the herbicide resistance gene for efficient weed control and theBacillus thuringiensis(Bt) toxin gene for insect resistance,have changed modern crop cultivation.Technical advancements provide more potentials for genetically engineering crops, which include plant artificial chromosome technology.It allows us to manage a large number of genes or extremely long DNA fragments and complex traits.Plant artificial chromosomes (PACs) or plant minichromosomes are usually small in size, which contain only essential chromosome components such as the centromere, telomeres and replication origins in order to maintain the normal chromosome function.Similar to animal artificial chromosomes, PACs can also be constructed with “bottom up” (de-novo assembling) and “top down (natural chromosome truncating) strategies.Comparatively, telomere mediated chromosomal truncation (TMCT) has meet more successes (Yu et al., 2016).TMCT depends on the transformation of a telomere-containing sequence into the genome where it replaces one of the old once integrated into the chromosome (Yu et al.,2006).Between T-DNA borders, bialophos resistance gene,loxandfrt, hygromycin B-resistance gene,GFP, red fluorescent protein gene, recombinase gene and telomere units isolated fromArabidopsisthalianawere arrayed in concatenation, and introduced into cells where it merged with a truncated chromosome with the centromere and the telomere at the distant end.Since the establishment of TMCT in maize (Yu et al., 2006), PACs have been constructed in many plant species including maize, rice,Arabidopsis,barley among others.

To manipulate genes or DNA fragments, PACs must contain different recombination systems, such as the Cre-lox(Abremski et al., 1983), FLP-frt(Golic and Lindquist, 1989), and the phiC31-att(Thorpe and Smith, 1998) available currently.With the aid of these recombination systems, the selection marker genes and interested genes can be exchanged between a PAC and the donor, and the desirable genes can be stacked on a PAC (Yu et al., 2016).Instead of artificial chromosome construction, a simple method is to insert a single-copy T-DNA from pDs-Lox into the centromere of anArabidopsischromosome.When Ac TPase gene is transformed into and transiently expressed in the plant genome, a cassette flanked by the inverted terminal region of maize Ds is transposed into right or left distant region of the chromosome.Mediated by Cre recombinase, the recombination betweenloxsites generated PACs into which a gene may be cloned at theloxsite (Murata et al., 2013).

4 CONSTRUCTION OF NANNOACS

4.1 Nannochloropsis species possess the prerequisites for NannoAC construction

Of the widely used three recombination systems,Cre-lox(Abremski et al., 1983), FLP-frt(Golic and Lindquist, 1989), and the phiC31-att(Thorpe and Smith, 1998), phiC31-attand Cre-loxsystems have been shown to work inNannochloropsis(Verruto et al., 2018; Poliner et al., 2020).Robust technical tools in microalgae includingNannochloropsisspecies are essential to manipulate both intrinsic(endogenous) and extrinsic (foreign) genes in their genomes for basic and applied studies.While Cas9-mediated gene editing has been developed inNannochloropsisspecies (Wang et al., 2016;Naduthodi et al., 2019, 2021b; Kurita et al., 2022),the molecular tools of knocking in or knocking out“traits” controlled by a group of genes within a large or among a group of DNA fragments are appreciated also.A concatenate construct, pSliceN’Excise,which containsCas9, nitrite inducibleCreand floxed (flanked byloxsites) blasticidin resistance(BSD) andGFPexpression cassettes, has been generated.After integration, the induced Cre eliminates the marker and the reporter, leaving a marker- and reporter-free genome constantly expressing Cas9 and expressing Cre upon induction.In this genome,the gRNA aids to knock out acyl-CoA oxidase gene(Aco1) by inserting the marker and reporter expression cassettes.Co-electroporating the specific gRNA with floxed marker/reporter cassette with additional sequence both sides cause insertion of the marker/reporter during DNA repairing at the Cas9 target site.After Cre induction, the marker/reporter cassette is removed and the additional sequence is stably integrated into and inactivated theZnCys.By employing Cas9 and Cre, stepwise iterative knocking out and deleting marker/reporter accumulatively modify the multiple genes of a trait (Verruto et al., 2018).

Today, groups of selection markers, reporters,and their expression controlling elements have been identified and used (Poliner et al., 2018a, b, 2020).With these foundations, we may try to construct NannoACs with both “bottom up” and “top down”strategies.As shown in episomal vector ofNannochloropsis, yeastCEN/ARSworks but may not assure the stable inheritance of the plasmid.Such scenario may not change when the telomeres are combined.The genomes ofNannochloropsishave been repeatedly sequenced and assembled(Guo et al., 2019; Gong et al., 2020); unfortunately,the telomere to telomere assembly is not available yet.The construction of NannoACs appreciate the sequence of centromeres.The ultralong reads degenerated by Nanopore sequencing make it possible to obtain the sequence of telomeres ofNannochloropsisand then construct NannoACs with bottom up strategy.The time has come to examine the complete sequence ofNannochloropsiscentromeres(Miga et al., 2020; Suzuki and Morishita, 2021).With the background information either available today or conveniently obtainable with the existing techniques, we may construct the NannoACs with both bottom-up strategy and top-down strategy that are feasible in both mammals and plants.

Adding an extra chromosome into the nuclei of diploidy animals and plants may not interfere the functioning of nuclei.However, when we construct NannoACs with top-down strategy, the truncation of a chromosome of the monoploidyNannochloropsismay be lethal.We have found that both mammals and plants tolerating chromosomal truncation are diploids, which allow one of the two homologous chromosomes to be truncated.

The genome ofNannochloropsisis monoploidy(Pan et al., 2011).Although some parts of the genome ofNannochloropsismay not absolutely necessary always (Wang et al., 2021), and plant genomes may allow deletion of some regions (Tan et al., 2020), it is imaginable that truncating any chromosome to some extent will be lethal to the cells.Colchicine induces chromosome doubling of plants (Manzoor et al., 2019).It also induces chromosomal doubling of microalgae (Nezhad and Mansouri, 2019; Szpyrka et al., 2020; Le-Feuvre et al., 2021; Mansouri et al., 2021).The diploid of doubled genomes has been traced for a certain amount of generations; however, whether the diploidy state can be maintained permanently is not sure.In cyanobacteria, the nuclear ploidy tightly associated with the environment (Zerulla et al., 2016; Riaz et al., 2021).It is reasonable to believe that the doubled genome is not permanently stable.As a preliminary trial, we are doubling the chromosomes ofN.oceanicaand trying to maintain its stability by Zeocin mutation that causes double strand breaks of DNA and introduces insertion and deletion during cellular DSB repairing (Yang et al., 2019).At a locus of doubled thus completely homologous chromosomes, a normal and a lethal allele may locate, surviving both chromosomes through functional complementation.Such scenario should aid to maintain the diploidy genome during cultivation.

Homologous recombination may destroy the maintenance of diploidy genome; however, homologous recombination may happen at an extremely low frequency in somatic cells; it takes place only when the alleles align together as are observed in meiosis.To this consideration, our strategy of diploid maintenance is the mutated nutrition alleles.As an alternative way of diploid maintenance, we may introduce different antibiotic markers into the doubled chromosomes, one each, and maintain the diploid ofNannochloropsiswith two antibiotics.Actually,Nannochloropsisspecies have been verified to perform double antibiotic resistance simultaneously(Poliner et al., 2018b).

4.2 NannoACs loom on the horizon

Nannochloropsisspecies are monoploidy (Pan et al., 2011).Some regions of their genomes including telomeres can be deleted (Wang et al., 2021);however, we prefer to believe that all chromosomal regions are necessary for their survival in all possible environments.Therefore, we may double their chromosomes with colchicine.It has been demonstrated that the doubled genome may maintain for a period (Nezhad and Mansouri, 2019;Szpyrka et al., 2020; Le-Feuvre et al., 2021; Mansouri et al., 2021); however, phosphate depletion will certainly reduce the ploidy of cyanobacteria (Zerulla et al., 2016; Riaz et al., 2021).We are endeavoring to mutate the genes essential forNannochloropsissurvival with Zeocin, a double strand breaker of DNA (Chankova et al., 2007; Yang et al., 2019),aiming to maintain the diploidy after chromosomal doubling.The mutated allele will stabilize the doubled chromosomes, allowing us to truncate one of the homologous chromosome pair.

To survive all possible environments, monoploidyNannochloropsisgenome should be doubled through colchicine treatment.The essential genes in doubled chromosomes may be mutated with zeocin so that the functionally complementary alleles can maintain the diploidy karyotype.If three mediate motifrecombinase systems work inNannochloropsis,constructing a NannoAC with three mediate motifs,one works alone and two work in combination, will be feasible.Simultaneous transformation of the episome carrying editing parts and the telomere constructs consisting of mediate motifs and telomere repeats will generate a NannoAC.With the aids of transiently expressed corresponding recombinases, we may integrate genes, concatenate genes and DNA fragments into NannoACs (see the main text for the details).

Fig.1 A proposed “top down” construction strategy of Nannochloropsis artificial chromosomes (NannoACs) with a high feasibility

Of three combinations of mediate motifrecombinase (Abremski et al., 1983; Golic and Lindquist, 1989; Thorpe and Smith, 1998), Cre-loxand phiC31-attsystems have been found to function well inNannochloropsis(Verruto et al., 2018;Poliner et al., 2020).Once all of the three systems work inNannochloropsis, it is feasible to construct a NannoAC with three mediate motifs, one works alone and two work in combination.To construct NannoAC with top-down strategy,Nannochloropsiscells with doubled genome are simultaneously transformed with the episome carrying sgRNA targeting selected chromosomal regions and the artificial telomere constructs consisting of mediate motifs andNannochloropsistelomere repeats (for example, Wang et al., 2021).The genomes ofNannochloropsisspecies have been sequenced repeatedly (Guo et al., 2019; Gong et al., 2020).With the telomere to telomere (T2T) sequencing technology (Miga et al., 2020), the precise sequences ofNannochloropsiscentromeres and telomeres will allow us to construct NannoACs with both “top down” and “bottom up” strategies.The applications of expected NannoACs are diverse.Either one mediate motif or two in combination can mediate the integration of concatenate genes (reporters,selection markers, the interested genes and their expression controlling elements) with the aid of cotransferred recombinase expression cassettes or even recombinases themselves.Reporters and selection markers should aid to screen out the transformants.However, multiplex PCR (Elnifro et al., 2000) and pooling design (Bruno et al., 1995) may accelerate the screening of transformants especially when the concatenate genes or DNA fragment are extremely long.Operating long concatenate genes and large DNA fragments and avoiding position effect are the advantages of artificial chromosomes over integrationbased transgene vectors while the stability of artificial chromosomes is the advantage over the episome.To both artificial chromosomes and integration-based vectors, bacterial plasmids and artificial chromosomes provide conveniences for handling genes and DNA fragments.Extralong DNA can be isolated from any cell wall free cells including animal cells and plant protoplasts (Nair et al., 1999) and separated through pulse field gel electrophoresis (Sharma-Kuinkel et al., 2016).Karyotyping microalgae may be difficult(Muravenko et al., 2001; Sánchez-Gárate et al.,2020).Fortunately, many pseudochromosomes (the chromosomal scale assemblies) ofNannochloropsisare < one megabase (Guo et al., 2019; Gong et al.,2020) and the isolation of microalgal protoplasts has been progressing (e.g., Echeverri et al., 2019; Guo et al., 2022), making pulse field gel electrophoresis separation ofNannochloropsischromosomes including artificial ones feasible.Such detection method is even easier than FISH that is widely used to visualize the animal and plant artificial chromosomes(Abe et al., 2021).In addition, the reference genome,the parts of artificial chromosomes and all relative elements are known.It is also convenient for us to prove our success in artificial chromosome construction and concatenate gene/DNA fragment insertion by resequencing at a low coverage (Ren et al., 2021).NannoACs loom on the horizon, which will appear in front of us soon (Fig.1).

5 PERSPECTIVE

The molecular tools including genetic transformation,homologous recombination, genome editing, gene stacking and episome vector and diverse reporters and selection markers have been rapidly developing inNannochloropsisspecies, the model microalgae for both applications and researches.Unfortunately,the position effect of integrative vectors and the instability of non-integrative ones have hindered genetically engineeringNannochloropsisspecies.Nannochloropsisartificial chromosomes (NannoACs)are highly appreciated; they should aid to express concatenate genes, stack genes, and transfer very large size DNA fragments which may control a complex trait.Currently, MACs and PACs have set fine examples for NannoACs.It has also been found that two mediate motif-recombinase systems function inNannochloropsis.The HACs and MACs constructed with “bottom-up” strategy are the circulated and modified centromeres in essence.As the preliminary trials, it is better to truncate natural chromosomes to construct NannoACs (top-down strategy).It seems that the methods and materials(information) to set the foundation for artificial chromosomes inNannochloropsisspecies are at hand.A possible and feasible approach to NannoAC construction with top-down strategy has been proposed in this review.NannoACs may contribute the genetic improvement of non-variationNannochloropsisspecies and their synthetic biological studies.

6 DATA AVAILABILITY STATEMENT

All data generated and/or analyzed during this study are available from the corresponding author on request.