Table 2.
Engineering the chloroplast genome for biotechnology applications
| Site of integration | Transgenes | Regulatory sequences | Efficiency of expression | Engineered traits or products | Reference(s) |
|---|---|---|---|---|---|
| Insect or pathogen tolerance | |||||
| trnI/trnA | Bgl-1 | 5′psbA/3′psbA | >160-fold enzyme | Resistance against whitefly and aphid | [141] |
| trnI/trnA | Pta | 5′psbA/3′psbA | 7.1–9.2 % TSP | Broad-spectrum resistance against aphid, whitefly, Lepidopteran insects, bacterial and viral pathogens | [142] |
| trnI/trnA | PelB1, PelD2 | 5′psbA/3′psbA | ~2.42 units mg−1 FW | Resistance against Erwinia soft rot | [150] |
| trnI/trnA | RC1011, PG12 | 5′psbA/3′ | 17–38 % TSP | Resistance to Erwinia soft rot and tobacco mosaic virus | [140] |
| trnI/trnA | cpo | Prrn/psbA/psbA | NR | Resistance to fungal pathogens in vitro (Aspergillus flavus, Fusarium verticillioides, and Verticillium dahliae) and in planta (Alternaria alternata) | [251] |
| trnI/trnA | Bt cry2Aa2 operon | Prrn/ggagg/psbA | 45.3 % TSP | 100 % mortality of cotton bollworm, beet armyworm; cuboidal Bt crystals formation | [137] |
| trnI/trnA | Bt cry9Aa2 | Prrn/ggagg/rbcL | ~10 % of TSP | Resistance to Phthorimaea operculella | [252] |
| trnI/trnA | msi-99 | Prrn/ggagg/psbA | 21–43 % TSP | Resistance to in planta challenge of Pseudomonas syringae, Aspergillus flavus, Fusarium moniliforme, Verticillium dahlia, and Colletotrichum destructivum | [253] |
| trnI/trnA | sporamin1, CeCPI2, and chitinase2 | Prrn/TpsbA | 0.85–1 % TSP | Resistance against Spodoptera litura and Spodoptera exigua leaf spot, as well as soft rot diseases | [254] |
| trnI/trnA | MSI-99 | Prrn/Trps16 | 89.75 μg g−1 FW | Resistance against rice blast fungus | [255] |
| trnV/rps12/7 | cry1A(c) | Prrn/rbcL/rps16 | 3–5 % of TSP | Resistance to larvae of Heliothis virescens, Helicoverpa zea, and Spodoptera exigua | [256] |
| trnV/rps12/7 | cry1Ab | Prrn/T7gene10/rbcL | NR | Resistance to caterpillar of Anticarsia gemmatalis | [145] |
| rbcL/accD | cry2Aa2 | Prrn/ggagg/psbA | 2–3 % of TSP | Resistance to Heliothis virescens, Helicoverpa zea, and Spodoptera exigua | [257] |
| Abiotic stress tolerance | |||||
| trnI/trnA | tps1 | Prrn/ggagg/psbA | >169-fold transcript | Drought tolerance: growth in 6 % polyethylene glycol and rehydration after 24 days of drought | [258] |
| trnI/trnA | merA/merB | Prrn/ggagg/psbA | NR | Phytoremediation: high level tolerance to the organomercurial compounds, up to 400 μM phenylmercuric acetate | [259] |
| trnI/trnA | badh | Prrn/T7 g10/rps16 | 93–101 μM g−1 FW | Salt tolerance: carrot plants survived up to 400 mM NaCl | [135] |
| trnI/trnA | γ-TMT | Prrn/T7g 10/TpsbA | >7.7 % TSP | Increased salt and heavy metal tolerance, enhanced accumulation of ɑ-tocopherol in seeds | [153] |
| trnI/trnA | mt1 | Prrn/T7 g10/Trps16 | NR | Phytoremediation: resistant to mercury, up to 20 μm | [260] |
| trnV/rps12/7 | b-bar1 | Prrn/TrbcL | >7 % TSP | Resistance to the herbicide phosphinothricin | [261] |
| trnV/rps7/12 | EPSPS | Prrn/Trps16 | >10 % TSP | Resistance to the herbicide glyphosate | [262] |
| rbcL/accD | EPSPS/aroA | Prrn/ggagg/psbA | NR | Resistance to glyphosate (>5 mM) | [129] |
| rbcL/accD | mALS | PpsbA/TpsbA | NR | Tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides | [263] |
| rbcL/accD | Bar | Prrn/rbcL/psbA | NR | Herbicide resistance: up to 25 μg ml−1 glufosinate | [264] |
| rbcL/rbcL | Hppd | psbA/psbA/3′rbcL | 5 % TSP | Resistance to herbicide | [265] |
| rbcL/accD | panD | Prrn/rbcL 3′ | >4-fold β-alanine | Tolerance to high-temperature stress | [266] |
| trnfM/trnG | lycopene β-cyclase | atpI/rps16 | 0.28 mg g−1 DW | Herbicide resistance and triggers conversion of lycopene | [133] |
| prs14/trnG | HTP, TCY, TMT | Prrn/T7 g10/TrbcL | NR | Increase in vitamin E in fruit; cold-stress tolerance | [267] |
| Other agronomic traits | |||||
| trnI/trnA | phaA | Prrn/psbA/psbA | 14.71β-ketothiolase mg−1 FW | Engineered cytoplasmic male sterility | [268] |
| trnI/trnA | RbcS | T7g10 or psbA | >150-fold RbcS transcript | Restoration of RuBisCO activity in rbcS mutants | [136] |
| rbcL/accD | TC, γ -TMT | PpsbA/Trsp16 | 3 nmol h−1 mg−1 FW | Vitamin E accumulation in tobacco and lettuce | [269] |
| rbcL/accD | CrtZ, CrtW | Prrn/Trps16 | NR | Accumulation of astaxanthin fatty acid esters in lettuce | [270] |
| trnV/orf708 | BicA | psbA/psbA/psbA | ~0.1 % TSP | CO2 capture within leaf chloroplasts | [271] |
| trnV/3′rps12 | Trx f, Trx m | prrn T7G10/rps12 | NR | Starch synthesis/chloroplast redox regulation | [272] |
| trnfM/trnG | CV-N | Prrn/T7g10/TatpA | ~0.3 % TSP | Increased mRNA stability and protein stability with the expression of CV-N in chloroplasts | [273] |
| trnI/trnA | Bgl-1 | 5′psbA/3′psbA | 44.4 units Bgl1 g−1 FW | β-Glucosidase increased enzyme cocktail efficiently to release sugar from paper, citrus peel, and wood | [141] |
| trnI/trnA | ubiC | 5′psbA/3′psbA | 25 % DW | 250-fold higher pHBA polymer accumulation than nuclear transgenic lines | [149] |
| trnI/trnA | man 1 | 5′psbA/3′psbA | 25 units g−1 FW | Mannanase increased enzyme cocktail released sugar from paper, citrus peel, and wood | [274] |
| trnI/trnA | cutinase or swoIlenin | 5′PsbA/3′PsbA | 47.7 % reduction of MGDG and DGDG in cutinase and 68.5 % in swollenin | Swollenin enlarged and irreversibly unwound cotton fiber; cutinase showed esterase and lipase activity; used in enzyme cocktails | [275] |
| trnI/trnA | bgl1 | 5′psbA/3′psbA | 14 units mg−1 FW | Enzyme cocktails produced glucose from filter paper, pine wood, or citrus peel | [150] |
| swo1 | NR | ||||
| xyn2 | 421 units mg−1 FW | ||||
| Acetyl sylan esterase | NR | ||||
| celD | 493 units mg−1 FW | ||||
| celO | 442 units mg−1 FW | ||||
| Lipase | NR | ||||
| Cutinase | 15 units mg−1 FW | ||||
| trnI/trnA | PMK, MVK, MDD, AACT, HMGS, HMGRt; IPP, FPP, ADS, CYP71AV1, AACPR | Prrn/PpsbA | 0.1 mg g−1 FW | Artemisinic acid for several isoprenoid products | [276] |
| trnI/trnA | Cel6A,Cel6B | Prrn/rbcL/rbcL | 2–4 % TSP | Hydrolyzed crystalline cellulose | [277] |
| trnfM/trnG | bgl1C, cel6B, cel9A, xeg74 | Prrn/T7g10/TrbcL | 5– 40 % TSP | Cell wall-degrading enzyme activity | [278] |
| rbcL/accD | phbC, phbA, phbB | Prrn/rbcL 3′ | 0.16 % DW | Polyhydroxybutyrate (PHB) accumulation in leaves | [279] |
| rbcL/accD | crtZ, crtW | Prrn/Trps16 | >0.5 % DW | Astaxanthin accumulation | [280] |
| trnV/rps7 | EGPh | psbA/psbA/Trps16 | 25 % TSP | Chloroplast-derived β-1,4-endoglucanase (EGPh) was recovered from dry leaves and digested carboxymethyl cellulose (CMC) substrate | [281] |
| trnI/trn A | EX4 | PpsbA/TpsbA | 14.3 % TSP | CTB–EX4 showed increased insulin secretion similar to the commercial injectable EX4 in pancreatic β-cells and in mice fed with cells expressing EX4 in chloroplasts | [160] |
| trnI/trn A | MBP | PpsbA/TpsbA | 2 % TSP | Amyloid loads were reduced in ex vivo studies in human Alzheimer’s brain and in vivo in Alzheimer’s mice fed with bio-encapsulated CTB–MBP. Abeta was also reduced in retinae and loss of retinal ganglion cells was prevented | [162] |
| trnI/trn A | FVIII | PpsbA/TpsbA | 370 mg g−1 FW | Feeding of the HC/C2 antigen mixture substantially suppressed T-helper cell responses and inhibitor formation against FVIII in hemophilia A mice | [282] |
| trnI/trn A | HSA | PpsbA/TpsbA | 26 % TSP | In vitro chaperone activity of Trx m and Trx f | [283] |
| trnI/trn A | EDA | PpsbA/TpsbA | 2.0 % TSP | The vaccine adjuvant EDA from fibronectin retains its proinflammatory properties when expressed in tobacco chloroplasts | [284] |
| trnI/trn A | Proinsulin | PpsbA/TpsbA | 47 % TSP in tobacco, 53 % TLP in lettuce | Oral delivery of proinsulin in plant cells lowered glucose levels comparably to injectable commercial insulin | [285] |
| trnI/trn A | HSA | psbA/psbA/psbA | ~11 % TSP | First report of human blood protein in chloroplasts; function not evaluated | [286] |
| trnI/trn A | IGF | psbA/psbA/psbA | 32.7 % TSP | Promoted growth of cultured HU-3 cells in a dose-dependent manner | [287] |
| trnI/trnA | FIX | PpsbA/TpsbA | 1 mg g−1 DW (0.56 % TLP) | Oral delivery of CTB-FIX lettuce cells suppressed inhibitor formation against FIX in hemophilia B mice | [6] |
| trnI/trnA | FIX | Ppsba/TpsbA | 3.8 % TSP; 0.4 mg g−1 FW | Tolerance induction via complex immune regulation, involving tolerogenic dendritic and T-cell subsets | [288] |
| trnI/trnA | GAA | Ppsba/TpsbA | 5.7 mg g−1 DW | Reduced toxic antibody responses in enzyme replacement therapy in Pompe mice | [289] |
| trnI/trnA |
ACE2
Ang-(1–7) |
PpsbA/TpsbA | CTB–ACE2: 2.14 % TLP CTB-Ang1–7: 8.7 % TLP |
Oral delivery of ACE2 and Ang (1–7) significantly improved cardiopulmonary structure and functions, decreased the elevated right ventricular systolic blood pressure and improved pulmonary blood flow in animals with induced pulmonary hypertension | [161] |
| trnI/trn A | BACE | Prrn/TpsbA | 2.0 % TSP | Immunogenic response against the BACE antigen in mice | [290] |
| trnI/trn A | IFNα2b | Prrn/TpsbA | 3 mg g−1 FW | Protected cells against VSV CPE and HIV; increased MHC I antibody on splenocytes and total number of natural killer cells and protected mice from a highly metastatic lung tumor | [291] |
| trnI/trn A | CTB-pins | Prrn/T7g10/TpsbA and rps16 | 16 % TSP in tobacco, 72 % TLP in lettuce | CTB-proinsulin-fed non-obese diabetic mice significantly decreased inflammation (insulitis); insulin-producing β cells in pancreatic islets were highly protected, increased in insulin production with lower blood or urine glucose levels; increased expression of immunosuppressive cytokines | [128, 292] |
| rbcL/accD | IFN-γ | PpsbA/TpsbA | 6 % TSP | Protection of human lung carcinoma cells against infection by encephalomyocarditis virus | [293] |
| rbcL/accD | hTrx | PpsbA/Trps16 | 1 % TSP | Protected mouse from hydrogen peroxide | [294] |
| rbcL/accD | A1AT | PpsbA/TrbcL | 2 % TSP | Binds to porcine pancreatic elastase | [295] |
| rbcL/accD | TGFβ3 | Prrn/T7g10/psbC | 12 % TLP | Inhibits mink lung epithelial cell proliferation | [296] |
| trnV/3′rps12 | hCT-1 | Prrn/G10L/Trps16 | 5 % TSP | Biologically active on human hepatocarcinoma cell line | [297] |
| trnV/rps7/12 | hST | PpsbA or Prrn/G10L/Trps16 | 0.2–7.0 % TSP | Promotes growth of Nb2 cells in a dose-dependent manner | [298] |
| trnfM/trnG | pal, cpl-1 | Prrn/T7g10/TpsbA | ~30 % TSP | Bacteriolytic activity and kills Streptococcus pneumoniae, the causative agent of pneumonia | [299] |
| trnI/trn A | ESAT-6 | 5′psbA/3′psbA | ~7.5 % TSP | Hemolysis of red blood cells and GM1 binding | [165] |
| trnI/trn A | AMA1 | 5′psbA/3′psbA | 7.3 % TSP in tobacco, 13.2 % TSP in lettuce | Long-term immunity against cholera challenge; inhibition of malarial parasite; protection correlated with IgA and IgG1 | [164] |
| trnI/trn A | MSP1 | 5′psbA/3′psbA | 10.1 % TSP in tobacco, 6.1 % TSP in lettuce | ||
| trnI/trn A | 2 L21 | 5′psbA/3′psbA | 6.0 % TSP | ||
| trnI/trn A | Pag | 5′psb/3′psbA | ~29.6 % TSP | Macrophage lysis assay, systemic immune response, toxin neutralization assay, mice survived (100 %) challenge with lethal doses of anthrax toxin | [300, 301] |
| trnI/trn A | L1 | PpsbA/TpsbA | 20–26 % TSP | Induced systemic immune response and produced neutralizing antibodies in mice | [302] |
| trnI/trnA | RA4 | PpsbA/T psbA | 0.2 % TLP | Oral administration elicited both mucosal and systemic Th1/Th2 responses to reduce Toxoplasma parasite load | [303] |
| trnI/trnA | rFaeG | PpsbA/TrbcL | >1 % DW | Transplastomic plants expressing the rFaeG protein could possibly be used for delivery of an oral vaccine against porcine F4+ ETEC infections | [304] |
| trnI/trn A | F1-V | Prrn/TpsbA | 14.8 % TSP | Orally immunized mice heavily challenged with plague (Yersinia pestis) were protected better than those given IP injections | [305] |
| trnI/trn A | CTB-2 L21 | PpsbA/TpsbA | 31.1 % TSP | Immunogenic in mice following IP or oral administration | [306] |
| trnI/trnA | VP8* | psbA/psbA/Trps16 | 600 μg g−1 FW | Induced strong immune response and virus neutralization | [307] |
| trnI/trn A | CtxB | Prrn/ggagg/TpsbA | 4.1 % TSP | Efficient GM1 ganglioside-binding | [308] |
| trnI/trn A | LTB | Prrn/ggagg/TpsbA | 2.5 % TSP | GM1 ganglioside-binding assay | [309] |
| trnI/trn A | LecA | Prrn/T7g10/TpsbA | 7 % TSP | Systemic immune response in mice | [310] |
| trnI/trn A | BACE | Prrn/TpsbA | 2.0 % TSP | Immunogenic response against the BACE antigen in mice | [290] |
| rbcL/accD | OspA, OspA-T | PpsbA/TpsbA | 1–10 % TSP | Systemic immune response and protection against Borrelia burgdorferi (Lyme disease) | [311] |
| trnN/trn R | LTB | Prrn/T7g10/TrbcL | 2.3 % TSP | GM1 ganglioside-binding assay; oral immunization partially protected mice from cholera toxin challenge | [312] |
| trnN/trnR | DPT | Prrn/T7g10/TrbcL | 0.8 % TSP | Immunogenic in orally inoculated mice with freeze-dried chloroplast-derived multi-epitope DPT protein | [313] |
| trnN/trnR | C4V3 | Prrn/T7g10/TrbcL | ~15 μg mg−1 DW | Plant-derived C4V3 has elicited both systemic and mucosal antibody responses in mice, as well as CD4+ T cell proliferation responses | [314] |
| trnN/trnR | L1 | Prrn/TrbcL | >2 % of TSP | Proper folding and display of conformational epitopes for L1 in the fusion protein by antigen capture ELISA | [315] |
| trnfM/trnG | p24 | Prrn/T7g10/TrbcL | ~4 % TSP | Induced strong CD4+ and CD8+ T-cell responses in mice | [316] |
| trnGtrnfM | HEV E2 | Prrn/psbA/TpsbA | 1.09 ng μg−1 TSP | Immune response in mice against hepatitis E virus | [317] |
| trnH/trnK | CSFV E2 | Prrn/TpsbA | 1–2 % TSP | Immune response in mice against swine fever | [318] |
| rrn16/rps12/7 | TetC | Prrn/T7 g10/TrbcL atpB/TrbcL |
10–25 % TSP | Mice developed systemic immune response and survived the tetanus toxin challenge | [319] |
| rrn16/trnI | E7 | PpsbA/Trps | 3–8 % TSP | Several therapeutic HPV-specific E7-based vaccine formulations have been tested in animal models and some have advanced into clinical trials | [320] |
Abbreviations: Ang (1–7) Angiotensin (1–7), BACE human b-site APP cleaving enzyme, Bgl β-glucosidase, CPE carbapenemase-producing Enterobacteriaceae, CTB cholera toxin B subunit, DGDG digalactosyldiacylglycerol, DPT diphteria, pertussis, tetanus, DW dry weight, EDA extra domain A-fibronectin, ELISA enzyme-linked immunosorbent assay, ETEC enterotoxigenic Escherichia coli, EX4 exendin-4, FVIII coagulation factor VIII, FW fresh weight, HPV human papilloma virus, IP intraperitoneal, MBP myelin basic protein, MGDG monogalactosyldiacylglycerol, NR not recorded, RbcS small subunit of RuBisCO, RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase, TLP total leaf protein, TSP total soluble protein, VSV vesicular stomatitis virus