High Carotenoid Mutants of Chlorella vulgaris Show Enhanced Biomass Yield under High Irradiance
Abstract
:1. Introduction
2. Results
2.1. Isolation of NFR-3 and NFR-13, High Car Content Mutants of Chlorella vulgaris
2.2. Stoichiometry of Pigment–Protein Complexes and Photosynthetic Efficiency
2.3. Phototolerance of WT and NFR Strains during EL Exposure
2.4. Biomass Productivity of WT and NFR Strains in Laboratory-Scale PBR
2.5. Lipid Productivity under Nitrogen Starvation
2.6. Genotypic Characterization of NFR Strains by Whole-Genome Sequencing
3. Discussion
3.1. Optimization of Light-Use Efficiency Enhances Biomass Yield
3.2. Influence of ROS-Resistance Traits on Stress-Induced Lipid Production
3.3. NFR as Promising Car Producer Strains for Commercial Applications
3.4. Whole-Genome Sequencing to Identify Gene(s) Responsible for the Enhanced Light-Use Efficiency in C. vulgaris
- Glycerol-3-phosphate acyltransferase 3. A transversion (G to C) was observed at nucleotide 534 on the 6th exon. This results in missense amino acid substitution of a glutamic acid into an aspartic acid, a mutation shared by both NFR mutants, affecting a subunit involved in neutral lipid metabolism. Glutamic acid and aspartic acid are both acidic amino acids with side chains containing a carboxylic acid group which carries a full negative charge. In terms of interaction modes, both amino acids include ionic, van der Waals and H-bonds. However, glutamic acid differs from aspartic acid only in that its side chain is longer by one methylene group. This results in higher side chain flexibility in glutamic acid compared to aspartic acid whose side chain flexibility is moderate. Transmembrane topology prediction indicates that the site of mutation is at the boundary between cytoplasmic domain (residues 169–179) and the consecutive transmembrane region (residues 180–199). Multiple sequence alignments of top BLAST hits indicate that the last two residues of the cytoplasmic domain (E178 and R179) are conserved among green cut species (Figure S7). Glycerol-3-phosphate acyltransferase 3 is a key component of the diglyceride-3-phosphate synthesis pathway. Consistently, its activity has been reported to be directly correlated to the amount of glycerophospholipids in E. coli cells [82] and used as a target for membrane engineering to enhance production and accumulation of ß-carotene [83].
- E3 Ubiquitin-protein Ligase SP1-related (PTHR47568:SF2) encoding sequence contains a splice donor variant and is shared by both mutants, NFR-3 and NFR-13. A transition (G to A) occurs at the first nucleotide of the second intron affecting canonical GT-AG splice site pairs. Group of E3 ubiquitin–protein ligases, including SP1 from Arabidopsis (UniProtKB: Q8L7N4), are involved in regulation of plastid’s proteome via ubiquitination and subsequent degradation of translocon at the outer envelope membrane of chloroplasts (TOC) complexes [84]. In addition, it promotes stress tolerance by depleting the chloroplast protein import apparatus, which limits photosystem assembly and the potential for ROS formation [85]. Formighieri et al. [86] demonstrated that absence of ARSA1, a protein localized in the C. reinhardtii cytosol, led to a strongly decreased Chl content per cell; ARSA1 was shown to be required for optimal biogenesis of photosystems due to its involvement in the accumulation of TOC34, a component of the outer chloroplast membrane translocon complex. Interestingly, in both species, a mutation targeting a component interacting with TOC is associated with a pale green phenotype.
- Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A contains variation observed in NFR-13 mutant only. Transition mutation (C to T) results in substitution of an arginine by cysteine. 2A protein phosphatases are evolutionarily conserved and carry out multiple functions such as growth- and stress-related signaling, cell cycle regulation, vesicle trafficking, as well as regulation of the activities of a number of enzymes involved in key metabolic pathways [87]. The mutant 2A protein phosphatase in NFR-13 contains fatty acid desaturase domain (IPR005804) which is involved in the lipid metabolic process (GO:0006629).
- Both NFR-3 and NFR-13 share an intronic variation on Allophanate hydrolase (g7748). In Chlamydomonas reinhardtii, allophanate hydrolase is involved in urea hydrolysis to produce ammonium depending on the source of nitrogen available [88].
- Another exonic mutation found only in NFR-3 targets biosynthetic arginine decarboxylase (g9015) involved in the polyamine biosynthesis pathway. In plants, polyamine accumulation was shown to correlate with Car content [89].
- A missense amino acid substitution (Arg233Cys) was identified on S49 family peptidase (g6424) in NFR-3. The predicted protein contains peptidase_S49 domain (IPR002142) from residues 20–236. InterPro entry (IPR002142) describes proteolytic enzymes that exploit serine in their catalytic activity. The sequences of these ubiquitous enzymes are variously annotated in different taxonomic groups. In plants, these proteolytic sequences are annotated as Signal peptide peptidase A (SppA; protease IV; MEROPS identifier S49.001) which are involved in cleavage of signal peptides.
- NFR-13 mutant contains a point mutation resulting in Ala80Thr on Acylamino-acid-releasing enzyme-like (g7937). Acylamino-acid-releasing enzymes have been identified and characterized in plants. They are found to be mainly localized in the stroma of chloroplasts, and are possibly involved in degradation of glycated/oxidized proteins, such as glycated RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), and thus contribute to sustain the chloroplast antioxidative system [90].
4. Conclusions
5. Materials and Methods
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Genotype | Chl/Cell (pg) | Car/Cell (fg) | Chl a/b | Fv/Fm | PSII Antenna Size (T2/3−1·103, ms−1) |
---|---|---|---|---|---|
WT | 0.26 ± 0.03 a | 57 ± 3 a | 2.67 ± 0.22 a | 0.67 ± 0.03 a | 6.74 ± 0.49 a |
NFR-3 | 0.11 ± 0.02 b | 71 ± 3 b | 4.14 ± 0.35 b | 0.66 ± 0.03 a | 4.32 ± 0.22 b |
NFR-13 | 0.07 ± 0.01 c | 65 ± 2 c | 4.92 ± 0.46 c | 0.65 ± 0.04 a | 3.19 ± 0.42 c |
mol/100 mol Chl | |||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Genotype | Car/Cell (fg) | Chl a/b | Chl/Car | Neo | Viola | Anthera | Lute | Zea | α-Car | β-Car | |
Control | WT | 57 ± 3 a | 2.7 ± 0.2 a | 4.3 ± 0.1 a | 2.8 ± 0.8 a | 2.8 ± 0.7 a | 0.1 ± 0.1 a | 13.5 ± 1.7 a | 1.3 ± 0.1 a | 0.5 ± 0.1 a | 1.9 ± 0.3 a |
NFR-3 | 71 ± 3 b | 4.1 ± 0.3 b | 1.6 ± 0.1 c | 5.3 ± 1.3 b | 8.9 ± 1.7 b | 1.4 ± 0.2 c | 37.3 ± 8.7 c | 5.8 ± 1.1 c | 0.8 ± 0.2 a | 4.9 ± 1.0 b,c | |
NFR-13 | 65 ± 2 c | 4.9 ± 0.4 b | 1.0 ± 0.3 d | 4.6 ± 0.5 b | 15.5 ± 3.0 c | 2.0 ± 0.5 c,d | 54.9 ± 5.0 d | 7.9 ± 1.6 c | 1.5 ± 0.2 b | 9.5 ± 1.6 c,d | |
N-depleted | WT | 89 ± 4 d | 2.8 ± 0.1 a | 2.8 ± 0.1 e | 3.9 ± 0.5 b | 3.1 ± 0.4 a | 1.1 ± 0.2 c | 21.5 ± 0.3 b | 2.9 ± 0.3 b | 0.7 ± 0.1 a | 2.9 ± 0.1 b |
NFR-3 | 121 ± 2 e | 4.1 ± 0.1 b | 0.9 ± 0.1 d | 14.7 ± 1.1 d | 18.8 ± 3.9 e | 3.1 ± 0.8 d | 52.9 ± 1.1 d | 11.2 ± 0.8 d | 1.8 ± 0.1 b | 8.4 ± 0.1 d | |
NFR-13 | 160 ± 8 f | 4.8 ± 0.2 b | 0.4 ± 0.1 d | 27.7 ± 7.1 e | 40.0 ± 0.6 f | 4.7 ± 1.7 d | 116.3 ± 2.6 e | 21.9 ± 0.4 e | 2.9 ± 0.2 c | 19.8 ± 0.2 e |
Parameters. | WT | NFR-3 | NFR-13 |
---|---|---|---|
Half-saturation intensity (µmol photons m−2 s−1) | 110 ± 24 a | 128 ± 12 a | 241 ± 73 b |
Pmax (µmol O2 mg Chl−1 h−1) | 96 ± 5 a | 225 ± 2 b | 323 ± 45 c |
Respiration (µmol O2 mg Chl−1 h−1) | 25 ± 3 a | 52 ± 7 b | 73 ± 6 c |
Respiration (fmol oxygen cell−1 h−1) | 6.4 ± 0.8 a | 5.7 ± 0.7 a | 5.1 ± 0.4 a |
Pmax/respiration (relative units) | 3.9 ± 0.4 a | 4.4 ± 0.5 b | 4.4 ± 0.4 b |
Genotype | Mean Increase of Biomass (g L−1 day−1) | µ (day−1) |
---|---|---|
WT | 0.43 ± 0.03 a | 1.87 ± 0.08 a |
NFR-3 | 0.56 ± 0.03 b | 1.99 ± 0.04 b |
NFR-13 | 0.56 ± 0.01 b | 1.99 ± 0.02 b |
Sample | Total Number of Reads Produced | Total Number of Reads after Trimming | Mapped Reads | Mean Fold Coverage |
---|---|---|---|---|
WT | 38,406,276 | 25,308,558 | 25,157,713 | 65.9× |
NFR-3 | 37,378,884 | 28,743,282 | 20,555,916 | 55.2× |
NFR-13 | 17,628,716 | 11,931,178 | 7,249,676 | 18.6× |
Mutant | Variant pos. | Ref/Alt | Region | CDS Effect [SIFT Prediction] | TargetP | ChloroP | Putative Gene Function (Gene ID) | |
---|---|---|---|---|---|---|---|---|
NFR-13 | SS_1:3929152 | G | A | Exon | Missense sub. (Arg598Gln) [SIFT: Tolerated] | noTP | - | Predicted protein (g380) |
NFR-13 | SS_2:14375 | G | A | Exon | Missense sub. (Arg150Cys) [SIFT: Deleterious] | noTP | - | RNA helicase, activating signal cointegrator 1 (g5804) |
NFR-13/NFR-3 | SS_2:5043375 | G | C | Exon | Missense sub. (Gln67His) [SIFT: Tolerated] | noTP | - | Predicted protein (g2438) |
NFR-13 | SS_3:393571 | G | A | Exon | Missense sub. (Arg470Cys) [SIFT: Deleterious *] | noTP | - | Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A beta isoform-like (g2544) |
NFR-13/NFR-3 | SS_5:2569375 | G | C | Exon | Missense sub. (Glu178Asp) [SIFT: Deleterious] | noTP | cTP | Glycerol-3-phosphate acyltransferase 3 (g4271) |
NFR-13 | SS_5:2624939 | C | T | Exon | Missense sub. (Ser119Leu) [SIFT: Deleterious] | SP | - | Serine threonine- kinase receptor R831 (g4284) |
NFR-3 | SS_10:492953 | G | A | Exon | Missense sub. (Arg233Cys) [SIFT: Deleterious] | noTP | - | S49 family peptidase (g6424) |
NFR-13 | SS_13:1005023 | G | A | Exon | Missense sub. (Ala80Thr) [SIFT: Tolerated] | mTP | cTP | Acylamino-acid-releasing enzyme-like (g7937) |
NFR-3 | SS_14:552899 | G | A | Exon | Missense sub. (Gly391Asp) [SIFT: Deleterious] | noTP | - | Biosynthetic arginine decarboxylase (g9015) |
NFR-13 | SS_14:1569725 | C | T | Exon | Missense sub. (Ala6Thr) [SIFT: Deleterious *] | noTP | - | Hypothetical protein (g8357) |
NFR-13/NFR-3 | SS_18:1233529 | G | A | Exon | Missense sub. (Gly1235Arg) [SIFT: Deleterious *] | noTP | - | Hypothetical protein (g9528) |
NFR-13/NFR-3 | SS_1:4651287 | G | A | Exon | Synonymous sub. | noTP | - | cold shock domain-containing protein (g211) |
NFR-13/NFR-3 | SS_2:4256144 | C | T | Exon | Synonymous sub. | noTP | - | haloalkane dehalogenase (g2220) |
NFR-13/NFR-3 | SS_2:4392343 | C | G | Exon | Synonymous sub. | noTP | - | solute carrier family 25 member 44 (g2251) |
NFR-13/NFR-3 | SS_5:1173035 | G | A | Exon | Synonymous sub. | noTP | - | Large subunit GTPase 1 (g3878) |
NFR-13/NFR-3 | PO_26F:57866 | C | T | Exon | Synonymous sub. | noTP | - | Predicted protein (g10631) |
NFR-3 | SS _1:3944123 | C | T | Intron | No | mTP | cTP | Methionyl-tRNA synthetase (g376) |
NFR-13/NFR-3 | SS_1:5342472 | G | A | Intron | No | noTP | cTP | Cleavage and polyadenylation specificity factor subunit 1 (g15) |
NFR-13/NFR-3 | SS_2:4545066 | G | A | Intron | No | noTP | - | Putative Xaa-Pro aminopeptidase 3 (g2293) |
NFR-13/NFR-3 | SS_2:4545075 | G | A | Intron | No | noTP | - | Putative Xaa-Pro aminopeptidase 3 (g2293) |
NFR-13/NFR-3 | SS _3:1002678 | A | G | Intron | No | SP | - | Hypothetical protein (g2664) |
NFR-13/NFR-3 | SS_5: 1971361 | AG | A | Intron | No | noTP | - | MAU2 chromatid cohesion factor-like protein (g4126) |
NFR-3 | SS _5:2687539 | G | C | Intron | No | noTP | cTP | Serine/threonine-protein kinase sepA (g4294) |
NFR-13/NFR-3 | SS_6: 1727681 | TC | T | Intron | No | cTP | cTP | Water chloroplastic-like (g4763) |
NFR-13/NFR-3 | SS _13:303225 | G | A | Intron | No | noTP | cTP | Allophanate hydrolase (g7748) |
NFR-3 | SS_13:1920503 | G | A | Intron | No | noTP | - | U3 small nucleolar ribonucleoprotein protein IMP4 (g8749) |
NFR-3 | SS_7:737237 | G | C | Intron | No | noTP | - | Sodium:proline symporter (g5015) |
NFR-3 | SS_8:332225 | C | T | Intron | No | noTP | cTP | MFS general substrate transporter (g5411) |
NFR-13 | SS_1:269018 | C | A | Splice region | No | noTP | - | Preprotein translocase subunit SecA (g1481) |
NFR-13/NFR-3 | SS_3:344676 | G | A | Splice donor | Yes | noTP | - | E3 Ubiquitin-protein Ligase SP1 related [PTHR47568:SF2] (g2532) |
NFR-13/NFR-3 | SS_13:2646657 | G | A | Splice region | No | cTP | cTP | S1 motif domain-containing protein (g8549) |
NFR-13 | SS_1:2991332 | G | T | Intergenic | No | noTP | - | Upstream of: Nuclear/nucleolar GTPase 2 (g672); Nuclear transport receptor (g671) |
NFR-13/NFR-3 | SS_5:1304829 | C | T | Intergenic | No | noTP | - | Upstream of predicted protein ( g3913). Downstream of predicted protein (g3914) |
NFR-3 | SS1:3529923 | A | AT | Intergenic | No | noTP | - | Downstream of transcription initiation factor IIB-2 (g501). Downstream of putative phosphatase 2C 35 (g500) |
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Guardini, Z.; Dall’Osto, L.; Barera, S.; Jaberi, M.; Cazzaniga, S.; Vitulo, N.; Bassi, R. High Carotenoid Mutants of Chlorella vulgaris Show Enhanced Biomass Yield under High Irradiance. Plants 2021, 10, 911. https://doi.org/10.3390/plants10050911
Guardini Z, Dall’Osto L, Barera S, Jaberi M, Cazzaniga S, Vitulo N, Bassi R. High Carotenoid Mutants of Chlorella vulgaris Show Enhanced Biomass Yield under High Irradiance. Plants. 2021; 10(5):911. https://doi.org/10.3390/plants10050911
Chicago/Turabian StyleGuardini, Zeno, Luca Dall’Osto, Simone Barera, Mehrdad Jaberi, Stefano Cazzaniga, Nicola Vitulo, and Roberto Bassi. 2021. "High Carotenoid Mutants of Chlorella vulgaris Show Enhanced Biomass Yield under High Irradiance" Plants 10, no. 5: 911. https://doi.org/10.3390/plants10050911