Table 2 Engineering the chloroplast genome for biotechnology applications

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]

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]

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]