Nourseothricin N-acetyl transferase (NAT), a new selectable marker for nuclear gene expression in Chlamydomonas
Chlamydomonas reinhardtii is a unicellular green alga, which is a most commonly used model organism for basic research and biotechnological applications. Generation of transgenic strains, which usually requires selectable markers, is instrumental in such studies/applications. Compared to other organisms, the number of selectable markers is limited in this organism. Nourseothricin (NTC) N-acetyl transferase (NAT) has been reported as a selectable marker in a variety of organisms but not including C. reinhardtii. Thus, we investigated whether NAT was useful and effective for selection of transgenic strains in C. reinhardtii. The successful use of NAT would provide alterative choice for selectable markers in this organism and likely in other microalgae.
C. reinhardtii was sensitive to NTC at concentrations as low as 5 µg/ml. There was no cross-resistance to nourseothricin in strains that had been transformed with hygromycin B and/or paromomycin resistance genes. A codon-optimized NAT from Streptomyces noursei was synthesized and assembled into different expression vectors followed by transformation into Chlamydomonas. Around 500 transformants could be obtained by using 50 ng DNA on selection with 10 µg/ml NTC. The transformants exhibited normal growth rate and were stable at least for 10 months on conditions even without selection. We successfully tested that NAT could be used as a selectable marker for ectopic expression of IFT54-HA in strains with paromomycin and hygromycin B resistance markers. We further showed that the selection rate for IFT54-HA positive clones was greatly increased by fusing IFT54-HA to NAT and processing with the FMDV 2A peptide.
This work represents the first demonstration of stable expression of NAT in the nuclear genome of C. reinhardtii and provides evidence that NAT can be used as an effective selectable marker for transgenic strains. It provides alterative choice for selectable markers in C. reinhardtii. NAT is compatible with paromomycin and hygromycin B resistance genes, which allows for multiple selections.
KeywordsChlamydomonas Nourseothricin N-acetyl transferase Transformation Selectable marker Genetic engineering
nourseothricin (NTC) N-acetyl transferase
- FMDV 2A
intraflagellar transport 54
calcium dependent kinase 3
differential interference contrast
Chlamydomonas reinhardtii (C. reinhardtii), a unicellular green alga, is a widely used model organism for basic scientific research as well as biotechnological applications . Generation of transgenic strains plays a critical role in our deeper understanding of molecular mechanisms involved in various cellular processes and genetic engineering for producing valuable products [2, 3].
Because of low efficiency of transformation, a selectable marker is usually needed for selection of transgenic strains. Currently, there are three types of selections used in nuclear transformation of C. reinhardtii: auxotrophy rescue, herbicide resistance and antibiotic resistance [1, 2, 3]. Auxotrophy rescue involves using parental strains with mutations thus limiting its application. For example, Nit1, a gene encoding nitrate reductase, can only be used in transformation of strains with defects in Nit1 . Several herbicide resistance markers have been reported [5, 6, 7]. The herbicides used include dichlorophenyl dimethyl urea (DCMU), norflurazon, oxyfluorfen, glyphosate and sulfadiazine. For reasons unknown, the herbicide resistance markers are rarely adopted in the community. It is likely due to high dose application of herbicide, poor transformation efficiency and/or other reasons. Six antibiotics have been used in C. reinhardtii for selection of transgenic strains transformed with corresponding selectable markers [1, 3]. The antibiotics used include paromomycin, zeocin, spectinomycin, hygromycin B, kanamycin and tetracycline. According to our understanding, only paromomycin, hygromycin B and zeocin resistance genes are commonly used as selectable markers [8, 9, 10]. Zeocin for selecting of Ble gene transformants is light sensitive and may induce genomic damages even in cells harboring the selection marker . Compared to higher number of selectable markers in higher plant and mammalian cells [12, 13], the number of effective selectable markers is limited in C. reinhardtii. Therefore, availability of additional selectable markers in C. reinhardtii will enable complex experimental design, for example triple or more selection for transgenic strains.
Nourseothricin (NTC), a metabolite produced by Streptomyces noursei, belongs to streptothricin-class aminoglycoside antibiotics that inhibit protein synthesis . NTC N-acetyl transferase (NAT) derived from S. noursei inactivates NTC by acetylating the beta-amino group of the beta-lysine residue . NTC is highly soluble in water (1 g/ml) and stable for 2 years even in solution. NAT has been used as a selectable marker in a variety of organisms including bacteria, fungi, plant and mammalian cells (https://www.jenabioscience.com/images/741d0cd7d0/NTC-Flyer.pdf). However, NAT has been used in diatoms but not in other microalgae including C. reinhardtii .
In this report, we have shown that NAT is an effective selectable marker for nuclear transformation of C. reinhardtii. NTC, as low as 5 µg/ml, effectively kills or suppresses the growth of C. reinhardtii wild type cells as well as strains harboring paromomycin and/or hygromycin B resistant genes. Codon-optimized NAT from S. noursei is expressible in C. reinhardtii and confers cell resistance to NTC. We further show that NAT can be used as a selectable marker for transgenic strains even in strains harboring paromomycin and/or hygromycin B resistant genes. Furthermore, by fusing of a target gene to NAT and processing with the FMDV 2A peptides, the selection efficiency for targeted transgenic transformants is dramatically increased.
Wild type C. reinhardtii strain is sensitive to nourseothricin
NTC is compatible with paromomycin and hygromycin B selections
For studies involved with transgenic strains, multiple selections are usually required. For example, rescue of insertional mutants generated by using antibiotic resistance genes requires another antibiotic selectable maker. Commonly used antibiotics for selection of transgenic strains of C. reinhardtii include paromomycin and hygromycin B . We wondered whether NTC is compatible with these two antibiotics for selection in C. reinhardtii. NTC, paromomycin and hygromycin B all inhibit protein synthesis, but their working mechanisms are different. Paromomycin inhibits initiation of translation or earlier steps of elongation while hygromycin B potently inhibits elongation . NTC inhibits protein synthesis with miscoding activity . It has been shown in mammalian cells and fungus that NTC is compatible with selections with hygromycin B . Whether it is compatible with selection with paromomycin and/or hygromycin B in Chlamydomonas cells is not known.
Expression of codon optimized NAT gene confers resistance to NTC of C. reinhardtii
To examine the sensitivity of the transgenic strains with different levels of NAT expression, cells of strains with higher expression (strains #1 and #6) and lower expression (strain #2) were grown on agar plates supplemented with various concentrations of NTC (Fig. 4d). Consistent with results shown above, wild type cells were killed at 10 µg/ml NTC while the transformants grew normally. However, at higher concentrations of NTC, the transformants showed different extent of growth. For strain #2, which had lower expression of NAT, strong growth inhibition was observed at 50 µg/ml of NTC. In contrast, for strains #1 and #6, which had relatively higher expression of NAT, strong inhibition was observed at 100 µg/ml of NTC. These data further demonstrate that the tolerance to NTC is conferred by the expression of NAT and reveal that the extent of tolerance is correlated with the levels of NAT expression.
NAT can be used as a selectable marker for transgenic strains
Next, we examined whether NAT can be used as a selectable marker in strains with both paromomycin and hygromycin B resistance. The pPSAD-IFT54-HA(NAT+) was transformed into a strain with paromomycin and hygromycin B resistance. 7 out of 116 (6.03%) colonies grown on the NTC selection plates expressed IFT54-HA as examined by immunoblotting (Fig. 5d and data not shown). Taken together, we have shown that NAT is an efficient selectable marker, which is compatible with paromomycin and/or hygromycin B resistance genes.
Fusion of a target gene IFT54-HA to NAT and processing with the FMDV 2A peptide increases gene expression efficiency
The ability to generate transgenic cells is crucial for genetic engineering widely used in basic research as well as in biotechnological applications. As a model organism, C. reinhardtii is widely used for exploration of basic cellular processes such as cilia biogenesis and photosynthesis and for producing commercially valuable products as a cell factory [1, 2, 3]. Although a few selectable markers have been developed in this organism, few of them have been widely used. An ideal selectable marker may possess the following properties: (1) high stability, aqueous solubility and low dosage of the selection reagents; (2) non-toxicity of the selection reagents in the presence of a selectable marker; (3) non-toxicity of the selectable markers; 4) high efficiency of transformation; (5) compatibility with other selectable makers and 6) no genotype requirement for the parental strains.
We have shown that NAT is an effective and stable selectable marker in C. reinhardtii that confers resistance to NTC. C. reinhardtii is very sensitive to NTC. No viable colonies were observed even in the presence of 5 µg/ml NTC though we have used 10 µg/ml for the selection. NTC is soluble in water and highly stable. The transformation efficiency of NAT is high. Around 500 cfu were routinely obtained by using 50 ng plasmid DNA for transformation. Expression of NAT was stable even in the absence of NTC. We have not observed any growth defects in NAT transgenic strains. As NTC is an antibiotic, it does not require strains with specific genotype. Thus, NAT provides an alternative choice for selectable markers in C. reinhardtii. Random insertion of foreign DNA into the genome of C. reinhardtii occurs during transformation and this property has been used to generate insertional mutants [26, 27]. Though we have not examined the patterns of integration of NAT into the genome of C. reinhardtii, NAT is expected to behave as other foreign DNA fragments. Thus, NAT may be used for generation of insertional mutants from which desired functional genes can be cloned.
We have tested using NAT as a selectable marker for transgenic expression of a target gene IFT54. We have used parental strains that had been previously transformed with paromomycin and/or hygromycin B resistant genes. Around 6.5% IFT54 transgenic strains were obtained from NTC resistant colonies, demonstrating that NAT can be used as a selectable marker. These data also indicate that NAT is compatible with hygromycin B and paromomycin resistant genes, which allow for multiple selections. We have developed a construct by fusing the target gene IFT54 to NAT and processing with FMDV 2A peptide. Compared to the non-fusion construct, the efficiency of expression of IFT54-HA has increased around ninefold. Thus, this fusion expression system can increase selection efficiency of transgenic strains. Because the NTC resistance is correlated with the expression levels of NAT, this system may also be used for obtaining strains with higher expression of target genes by selection at higher concentrations of NTC.
NAT as a selectable marker has been used in microalgae but so far only in marine diatoms including Chaetoceros gracilis, P. tricornutum and T. pseudonana [16, 28, 29]. Our demonstration that NAT can be used a selectable marker in a fresh water green alga, opening a promising prospect in using NAT in other microalgae, especially in those algae with fewer choices for selectable markers. For example, Dunaliella, a saline green alga, is a popular model organism for the study of adaptation of eukaryotic cells to high salt concentrations and some Dunaliella species are of economic value for producing beta-carotene . However, Dunaliella is resistant to paromomycin, hygromycin B, spectinomycin and kanamycin . Thus, NTC resistance needs to be tested in Dunaliella before the NAT/NTC selection system can be used.
We have developed a new stable selectable marker for selection of transgenic strains in C. reinhardtii that confers resistance to NTC, which provides an alternative choice for selectable markers. In addition, NAT is compatible with paromomycin and hygromycin B resistance genes, two most commonly used selectable markers in C. reinhardtii, which allows combination of multiple selectable markers in transgenic studies.
Strains and culture
Chlamydomonas reinhardtii wild type strain 21gr (CC-1690, mt+) was from the Chlamydomonas Resource Center. ift54 (a paromomycin resistant strain) , lf4::LF4-HA (a hygromycin B resistant strain)  and wdr92::WDR92-YFP (a paromomycin and hygromycin B double resistant strain)  were generated in our own lab. Unless otherwise stated, cells were grown at 23 °C in M liquid medium in a 14/10 light/dark cycle . Cells used for transformation were grown at 23 °C in liquid TAP medium under continuous light .
Paromomycin and hygromycin B were purchased from Merck Millipore, USA, while NTC was obtained from Jena biosciences, Germany. The antibiotics were solubilized in water and sterilized by filtration. The concentrations used for selection for paromomycin, hygromycin B and NTC were 10, 20 and 10 µg/ml, respectively.
Drug sensitivity assay
To determine the sensitivity of C. reinhardtii to NTC, 1 × 106 of wild type cells were spotted on 1.5% TAP agar plates supplemented with various concentrations of NTC (0, 2.5, 5, 10 and 40 µg/ml) and incubated for 4 days at 23 °C in a 14/10 light/dark cycle. To test whether strains with paromomycin and/or hygromycin B resistant genes are sensitive to NTC, 1 × 106 cells of wild type and strains with various resistant genes were grown on 1.5% TAP agar plates supplemented with different antibiotics as indicated in the text.
Construction of the transformation vectors
To generate a construct for expressing NAT, the coding region of NAT from S. noursei was codon optimized for C. reinhardtii and chemically synthesized (Genscript, China). Codon optimized NAT tagged with 3× HA tag at the 3′ end driven by HSP70a/RBCS2 and terminated by RBCS2 terminator was cloned into ZT4-blunt vector. The HSP70a/RBCS2 promoter and RBCS2 terminator were cloned from pCB740 . The 3× HA tag was cloned from pKL-3XHA . The final construct was termed pHR-NAT-HA. To enable expression of IFT54-HA in C. reinhardtii with NAT as a selectable marker, the expression cassette for hygromycin B in the vector pPSAD-IFT54-HA-Hyg was replaced with the NAT expression cassette in pHR-NAT-HA with 3xHA being removed . The resulting construct was termed pPSAD-IFT54-HA(NAT+). To generate construct pHR-NAT-2A-IFT54-HA for fusion of IFT54-HA to NAT and processing by FMDV 2A peptide, the sequence for Ble and GFP-tubulin in the vector pBR25-sfGFP-TUA were replaced by NAT and IFT54-HA, respectively . All the constructs were verified by sequencing.
Transformation of Chlamydomonas was performed by electroporation using BTX ECM630 (Harvard Apparatus Inc, USA) following a previously published protocol . For each transformation, 5 × 107 cells were mixed with 50 ng plasmid DNA linearized by AclI. After electroporation, the transformation mixture was diluted in 10 ml TAP + 50 mM sorbitol and kept away from light for 8 h. Transformants were selected on agar plates supplemented with 10 µg/ml NTC.
SDS-PAGE and immunoblotting
SDS-PAGE and immunoblotting analysis were performed as described previously . Briefly, cells were lysed with Buffer A (50 mM Tris–HCl pH 7.5, 10 mM MgCl2, 1 mM EDTA, and 1 mM DTT) containing protease inhibitor cocktail (Roche, Switzerland) and boiled for 10 min in 1× SDS loading buffer. The proteins were separated in 10% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, USA) and probed with the indicated antibodies. The primary antibodies used include the following: rat anti-HA (Roche, Switzerland), 1:3000; mouse anti-α-tubulin (Sigma-Aldrich, USA), 1: 3000; rabbit anti-IFT54, 1:3000  and rabbit anti-CDPK3, 1: 5000 .
After cell fixation in 1% glutaraldehyde, DIC images were captured by Zeiss Axio Observer Z1 microscope (Carl Zeiss, Germany) equipped with a CCD camera (QuantEM512SC, Photometrics, USA). The images were processed in Photoshop and/or Illustrator (Adobe, USA).
We would like to thank Dr. Karl F. Lechtreck (University of Georgia) for providing plasmids pBR25-sfGFP-TUA.
XJY and JLP performed the experiments. XJY and JMP analyzed the data. JMP provided reagents and supervised the project. JMP and XJY wrote the paper. All authors read and approved the final manuscript.
This work was supported by the National Key R&D Program of China (2018YFA0902500) and the National Natural Science Foundation of China (31671387) (to J.P.)
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The authors declare that they have no competing interests.
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