Recombinant Protein Expression and Its Biotechnological Applications in Chlorella spp.
Abstract
:1. Introduction
2. Genetic Elements for Protein Expression in Chlorella spp.
2.1. Transformation Methods
2.2. Selectable Markers
2.3. Promoters and Terminators
2.4. Enhancers, Introns, and Signal Peptides
3. Applications for Recombinant Protein Expression
3.1. Model Proteins
Protein (Gene) | Selection | Transfection Method | Expression Elements | Notes | Ref. |
---|---|---|---|---|---|
GUS | Hygromycin B | EP | PCaMV35S-gusA-TNOS | C. vulgaris | [76] |
GUS | Kanamycin | MBP | PCaMV35S-gusA-TNOS | C. vulgaris | [77] |
GUS | Geneticin | AMT | PCaMV35S-gus-TCaMV35S | C. vulgaris Contains IV2 intron from ST-LS1 | [48] |
GUS | n.a. | EP | PNR-gus:NR-TNOS | C. ellipsoidea NR-GUS fusion protein Transient expression | [78] |
GUS | Hygromycin B | AMT | PCaMV35S-gus-TNOS | C. sorokiniana | [79] |
GUS | Phleomycin | PP + PEG | PUbi-gus-TNOS | C. ellipsoidea Stable for 10 months | [60] |
GUS | n.a. | MPB | PCaMV35S-gus-TNOS | C. ellipsoidea Transient expression | [34] |
GUS | n.a. | PP + EP | PCaMV35S-gus-TNOS | C. saccharophila Transient expression | [80] |
GUS | Geneticin | MPB | PCaMV35S-gus-TNOS | C. kessleri | [81] |
GUS | n.a. | EP | PCaMV35S-gus-TNOS | Chlorella sp. MACC/C95 Transient expression | [82] |
GFP-GUS fusion | Hygromycin B | AMT | PCaMV35S-gfp:gusA-TNOS | C. vulgaris | [25] |
Enhanced GFP (egfp) | Geneticin | PP + PEG | PCaMV35S-egfp-TNOS PNOS-gusA-TNOS | C. vulgaris | [31] |
Cyan fluorescent protein (cfp), GFP (mgfp5) | Hygromycin B or Geneticin | PP + EP | PCaMV35S-mgfp5-TNOS PHSP70-cfp-TNOS | C. vulgaris | [49] |
EGFP | Hygromycin B | HIV-TAT peptide + Triton X-100 | PCaMV35S-egfp-TNOS | C. vulgaris Used cell-penetrating peptide for transformation | [43] |
GFP | Hygromycin B | EP | PCaMV35S-mgfp5-TNOS PCaMV35S-zCas9-NLS-TNOS | C. vulgaris sgRNA expressed using U6 promoter directed toward FAD Enhance lipid production | [41] |
EGFP | Hygromycin B | AMT | PCaMV35S-mgfp5-TNOS | C. vulgaris Video method | [55] |
EGFP | Geneticin | EP | PUbi1-egfp-TNOS | C. pyrenoidosa | [74] |
mCherry EGFP AMP MSI99 | Phleomycin | EP | P3843-ble:mCherry-T8657 P8657-egfp-T8655 P3843-ble-2A-MSI99-2A-mCherry | Chlorella sp. MEM25 Used transcriptomics to design native promoter and terminator | [65] |
Luciferase | n.a. | PP + PEG | PCaMV35S-luc-TNOS | C. ellipsoidea Transient expression | [75] |
Luciferase | KClO3 | EP | PSIP-luc-TRbcS2 | C. vulgaris Identified new salt inducible promoter (SIP) | [46] |
3.2. Recombinant Protein Production
Protein (Gene) | Expression Elements | Notes | Ref. |
---|---|---|---|
Human growth hormone (hGh) | PCaMV35S-hGH PRbcS2-hGH PCaMV35S+RbcS2-hGH | C. vulgaris Yield: 200–600 µg/L Extracellular, transient expression Also tested a Chlorella virus promoter and RbcS2 intron 1. | [67] |
GUS, Neutrophil peptide-1 (NP1) | PCaMV35S-gus-TNOS PUbi-gus-TNOS PCaMV35S+Ubi-gus-TNOS PUbi-NP1-TNOS | C. ellipsoidea Enhancer (TMV Ω 5′UTR) doubled GUS activity | [68] |
Flounder growth hormone (fGH) | PCaMV35S-fGH | C. ellipsoidea Yield: 400 µg/L Oral growth supplement for flounder | [32] |
Trypsin-modulating oostatic factor (tmfA) | PCaMV35S-tmfA-TRbcS | C. dessicata Yield: 17–20 µg/3 × 108 cells Stable > 3 months | [84] |
Infectious bursal disease virus protein 2 (IBDV vp2) | PCaMV35S-vp2-TOCS | C. pyrenoidosa Edible chicken vaccine | [26] |
Human granulocyte colony-stimulating factor (hG-CSF) | PCvNDI1-hG-CSF-TRAmy3D | C. vulgaris Transit peptides from highly secreted proteins Nitrogen deficiency inducible promoter (CvNDI1) | [56] |
SARS-CoV-2 receptor-binding domain (RBD), Basic fibroblast growth factor (bFGF) | PCaMV35S-RBD-TExt3 PCaMV35S-bFGF-TExt3 | C. vulgaris Yield: 1.14 µg/g RBD Yield: 1.61 ng/g bFGF Transient expression using geminiviral system Dual CaMV35S promoter Tobacco extension 3′UTR NbPsalK2T1-63 5′UTR, | [24] |
bFGF | Prrn-bFGF-RBS-aph6-TpsbA | C. vulgaris Chloroplast integration using HR with 16S-trn1 and trnA-23S region C. reinhartii ribosomal RNA (rrn) promoter | [29] |
Viral hemorrhagic septicemia virus glycoprotein (VHSV G) | PCaMV35S-VHSVG-TRbcS2 | C. vulgaris KClO3—Selection for NR KO Stable > 1 year Used triple HR | [47] |
VHSV G | PSIP-VHSVG-TRbcS2 | C. vulgaris KClO3—Selection for NR KO Yield: 41.1 mg/10 g wet biomass Used to vaccinate fish | [45] |
White spot syndrome virus (WSSV) VP28 | PCaMV35S-RBD-TRbcS | C. vulgaris KClO3—Selection for NR KO Used to vaccinate shrimp Used triple HR | [28] |
3.3. Metabolic Engineering
4. Conclusions
Author Contributions
Funding
Conflicts of Interest
Abbreviations
AMPs | Antimicrobial peptides |
AMT | Agrobacterium mediate transformation |
APT | Adenine phosphoribosyltransferase |
bFGF | Basic fibroblast growth factor |
bZIP | Basic leucine zipper |
CaMV35S | Cauliflower mosaic virus 35S |
Cas9 | CRIPSR-associated protein 9 |
CHO | Chinese hamster ovary |
CP | Chloroplast |
CRISPR | Clustered regularly interspaced short palindromic repeatsCTP—Chloroplast transit peptide |
DGA | Diacylglycerol acyltransferase |
EGFP | Enhanced green fluorescent protein |
ELISA | enzyme-linked immunosorbent assay |
EP | Electroporation |
ER | Endoplasmic reticulum |
Ext3 | Tobacco extension 3′UTR |
FAD | ω-3 fatty acid desaturase |
FBA | Fructose 1,6-bisphosphate adolase |
fGH | Flounder growth hormone |
GFP | Green fluorescent proteinGUS—β-glucuronidase |
HEK | Human embryonic kidney |
Hg-CSF | Human granulocyte colony-stimulating factor |
hGH | Human growth factor |
HIV | Human immunodeficiency virus |
HSP70 | Heat shock protein 70 |
HR | Homologous recombination |
IBDV | Infectious bursal disease virus |
KO | Knockout |
MBP | Microparticle bombardment |
mGFP5 | Modified green fluorescent protein 5 |
MT | Mitochondria |
NLS | Nuclear localization signal |
NOS | Nopaline synthase |
NP1 | Neutrophil peptide-1 |
NR | Nitrate reductase |
OCS | Octopine synthase |
RbcS/L | Ribulose-1,5-bisphosphate carboxylase/oxygenase small/large subunit |
RBD | Receptor binding domain |
PDS | Phytoene desaturase |
PP | Protoplasting |
PEG | Polyethylene glycol transformation |
SARS-CoV-2 | Severe acute respiratory syndrome coronavirus 2 |
SDS-PAGE | Sodium dodecyl sulfate–polyacrylamide gel electrophoresis |
Sf9/21 | Spodoptera frugiperda Sf9/21 cell |
SIP | Salt inducible promoter |
SV40 | Simian vacuolating virus 40 |
Tag | T antigen |
TAT | Transactivator of transcription |
TF | Transcription factor |
TMV | Tobacco mosaic virus |
TMOF | Trypsin-modulating oostatic factor |
Ubi | Maize ubiquitin |
UTR | Untranslated region |
VHSV | Viral hemorrhagic septicemia virus |
WSSV | White spot syndrome virus |
ZCD1 | Carotenoid cleavage dioxygenase |
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Species | Genome Size (Mb) | Chromosomal Arrangement (Ploidy of Vegetative Cells) | Ref. |
---|---|---|---|
E. coli K12 | 4.6 | 1 chromosome (n = 1) | [15] |
S. cerevisiae | 12.1 | 16 chromosomes + MT, (mostly n = 1) | [16] |
Cricetulus griseus (CHO-K1) | 2450 | 21 chromosomes + MT (n = 2) | [17] |
N. tabacum | 3600 | 24 chromosomes + MT + CP, (n = 2) | [18] |
C. reinhardtii | 111 | 17 chromosomes + MT + CP, (mostly n = 1) | [19,20] |
P. tricornutum | 27.5 | 25 chromosomes + MT + CP, (n = 2) | [21,22] |
C. vulgaris | 40.2 | 14 scaffolds + MT + CP, (n = 1) | [23] |
C. sorokiniana | 38.6 | 13 chromosomes + MT + CP, (n = 1) | GCA_025917655.1 |
Antibiotic/Herbicide (Genes) | Conc. | C. vulgaris | C. ellipsoidae | C. sorokiniana |
---|---|---|---|---|
Geneticin/G418 (nptII) | 15–500 µg/mL | + | + | + |
Hygromycin B (hpt) | 15–500 µg/mL | + | n.d. | + |
Kanamycin (nptII) | 15–50 µg/mL | + | n.d. | n.d. |
Chloramphenicol (cat) | 200 µg/mL | + | n.d. | n.d. |
Phleomycin or Zeocin (ble) | 1–10 µg/mL | + | + | n.d. |
Paromomycin (nptII) | 10 µg/mL | + | n.d. | n.d. |
Protein (Gene) | Expression Elements | Notes | Ref. |
---|---|---|---|
YoeB toxin GFP fusion (yeoB:gfp) PezT toxin GFP fusion (pezT:gfp) | POlexA-yeoB:gfp-TT3A POlexA-pezT:gfp-TT3A | C. vulgaris XVE/OlexA estrogen inducible promoter system Stable for > 1 year | [71] |
NZ2114 (ant1), piscidin-4 (ant-2) | PrbcL-ant1-RBS-ant2-TpsbA | C. vulgaris Chloroplast integration using HR with 16S-trn1 and trnA-23S region | [33] |
Heliomicin (HeM) | PCaMV35S-HeM-TNOS | C. ellipsoidea MNP—Magnetic nanoparticle-mediated transformation | [42] |
Hepcidin (hepc), scygonadin (scy), and fusion (scy:hepc) | PCaMV35S-hepc-TNOS PCaMV35S-scy-TNOS PCaMV35S-hepc:scy-TNOS | Chlorella sp. Production of AMPs for animal feed | [85] |
Proteins (Genes) | Expression Elements | Notes | Ref. |
---|---|---|---|
Lipid accumulation-associated enzymes | PCaMV35S-gene-TNOS PRbcS2-gene-TNOS | C. minutissima Include homology regions Up to 5 genes expressed together | [86] |
Vitreoscilla hemoglobin (vgb) | PCaMV35S-vgb-TCYC1 | C. vulgaris Increase cell respiration efficiency | [89] |
TF GmDof4 | PUbi-GmDof4-TNOS | C. ellipsoidea Enhance lipid production | [27] |
Lipid accumulation-associated enzymes | TCaMV35S | Chlorella sp. Promoter not specified | [87] |
β-carotene hydroxylase 1 (crtRB) Carotenoid cleavage dioxygenase (ZCD1) | PCaMV35S-crtRB-TNOS & PUbi-ZCD1-TNOS | C. vulgaris Produce crocetin from saffron crocus | [90] |
EGFP (egfp), Fructose 1,6-bisphosphate aldolase (fba) | PCaMV35S-egfp-TNOS PCaMV35S-fba-TNOS | C. vulgaris Chloroplast localization Increase phototrophic growth rate | [30] |
Carbonic anhydrase (Mica) | PCaMV35S-Mica-TNOS | C. sorokiniana Improve CO2 capture | [91] |
Omega-3 desaturase (FAD), GFP/GUS fusion | PFAD-ω-3 FAD-TFAD PCaMV35S-mgfp5:gusA-TFAD | C. vulgaris Enhance lipid production | [69] |
Carbonic anhydrase | PCaMV35S-Mica-TNOS | C. vulgaris Improve CO2 capture | [91] |
DNA binding with one finger (DOF)-type TF | PHSP+RbcS-ble-2A-DOF-TRbcS | C. vulgaris Enhance lipid production | [59] |
Malic enzyme (me) | PCaMV35S-g-TNOS | C. protothecoides Enhance lipid production | [88] |
Basic leucine zipper (bZIP)-TF (ZIP1) | PHSP70-ZIP1-TNOS | Chlorella sp. HS2 C-terminal FLAG tag Enhance lipid production | [92] |
TF Lec1 | PUbi-Lec1-TNOS | C. ellipsoidea Enhance lipid production | [93] |
TF Lec2 | PCaMV35S-Lec2 | C. sorokiniana Increase relative electron transfer rate Enhance lipid production | [94] |
Diacylglycerol acyltransferase (DGA) | PHsp70-RbcS2-dga-TNOS | C. sorokiniana Enhance lipid production | [35] |
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Chen, C.; Ward, V.C.A. Recombinant Protein Expression and Its Biotechnological Applications in Chlorella spp. SynBio 2024, 2, 223-239. https://doi.org/10.3390/synbio2020013
Chen C, Ward VCA. Recombinant Protein Expression and Its Biotechnological Applications in Chlorella spp. SynBio. 2024; 2(2):223-239. https://doi.org/10.3390/synbio2020013
Chicago/Turabian StyleChen, Chuchi, and Valerie C. A. Ward. 2024. "Recombinant Protein Expression and Its Biotechnological Applications in Chlorella spp." SynBio 2, no. 2: 223-239. https://doi.org/10.3390/synbio2020013
APA StyleChen, C., & Ward, V. C. A. (2024). Recombinant Protein Expression and Its Biotechnological Applications in Chlorella spp. SynBio, 2(2), 223-239. https://doi.org/10.3390/synbio2020013