بسم اهللا الرحمن الرحيم

Chloroplast Genome

Engineering

Biology and Biotechnology

Seyed Javad DavarpanahFaculty of Bioscience, Shahid Beheshti University

June 19, 2010

A 50-290 kb double stranded circular molecule

A pair of 20-30 kb inverted repeat (IR) sequence

Prokaryotic protein synthesis machinery

100 chloroplasts per mesophyll cell and 100 genome copies per chloroplast (100 x 100 = 10,000 genome copies per cell)

Chloroplast genetic system

Chloroplast Genome Structure

Typical Chloroplast Genome Exception

Euglena

Pea

Higher plants plastome structure

Minicircle Structure in Dinoflagellates

Typical coding minicircles

Circular DNA molecules ranging insize between 2.2-3.8 kb

Around 14 genes –Mostly one gene in a minicircle (one gene - one circle or two genes - one circle )

250-500 bp non-coding core region

Gene (s) always in the same orientationregarding the core region

Core region may function as replication origin or promoter

Other Minicircles:Empty, Chimeric minicircles, Jumbled minicircles and Microcircles

Nuclear transformation

Biosafetyrisk of gene flow to the environmentsuperweed productionpollen poisoning for non-target insects

Stability of expression of transgenetransgene silencing (TGS) and (PTGS)

Expression level of foreign genes is higher than nuclear transformation; 5–80 (Chlamydomonas) or 500–10,000 (Nicotiana) DNA copies per cell

Multiple genes can be introduced as an operon

No risk of transgene escape – environmentally friendly

No position effect

No transgene silencing

Sequestration of foreign proteins in the organelle

Chloroplast transformation

Advantages of transplastomic plants

Transgenic pollen toxic to non-target insects of 60 major crop plants, only 11 have no wild relatives

No gene escape to WT (exceptions being alfalfa and possibly rice and pea => No WT insensitiveness to herbicides

Introgression of WT genes to transplastomic is in general in unusual

introgression of the common weed Raphanus raphanistruminto Brassica napus (oilseed rape) occurred at higher rates than the reciprocal cross of Brassica napus pollen into Raphanus raphanistrum.

• Transformation of plastids has already been achieved for tobacco , Arabidopsis, soybean , cotton, lettuce, cauliflower, poplar and potato

• The cereal crops rice, maize and wheat continue to be recalcitrant

• plastid-mediated molecular pharming will lead to the biofabrication of a range of biopolymers and pharmaceutical proteins

Stable transplastomic plants

Plastid transformed plants

Basics of Chloroplast Transformation

Chloroplast Transgenic Production Homologous Recombination Homoplasmy Process

Chloroplast transformation techniques

Biolistic delivery systemsPolyethylene glycol (PEG) treatment of protoplast• For unknown reasons, the technique has a lower success rate

than biolistic bombardment• long selection times required after initial DNA delivery• technically demanding and requires specialized tissue culture

skillsFemtoinjection technique: injection of DNA material into

chloroplasts using syringes with extremely narrow tipsAgrobacterium-mediated plastid transformation:• Two preliminary and thus far unconfirmed reports

Particle Delivery System

Advantages and disadvantages of biolistic method

Relatively high efficiency

Technical simplicity

Potential for mechanical shearing of large plasmidsduring particle preparation or delivery

Chemical attack by tungsten (a reactive transition metal) which can promote modifications or cleavage of DNA

Advantages of femtoinjection technique

Cells survive the injection

Transformed cell can be spotted easily

Cellular context remains intact

• The fate of the inserted gene or gene products to be followed.

Galinstan Expansion Femtosyringe (GEF)

Ex: Phormidium laminosum, bla gene: spectinomycingfp gene under the control of a chloroplast rRNA promoter

Chl autofluorescence GFP fluorescence overlay of both channels

marginal mesophyll cells of tobacco leaf

Reporter gene strategies

• Gene coding for the green fluorescent protein (GFP)• Resistance genes against lethal agents (e.g. spectinomycin and

streptomycin)• disadvantage of resistance marker genes: transformed cells must be

traced by stringent methods• Vectors carrying the bacterial gene aphA-6, coding for an

aminoglycoside phosphotransferase that detoxifies kanamycin or amikacin

• FLARE-S system, the aminoglycoside 3′′ adenyltransferase (aadAgene), which confers resistance against spectomycin and streptomycin,is translationally fused to the gfp gene of Aequoreavictoria

• In the case of an optical marker like GFP, difficulties arise with the regeneration of a plant from a single GFP-expressing cell

Reporter gene strategy: genetic contamination problems

Reasons to produce marker-free transplastomic plants

Potential metabolic burden imposed by high levels of marker gene expressionhomoplastomic state :the marker gene product 5% to 18% of the total cellular soluble protein

Shortage of primary plastid selective markersonly genes that confer resistance to spectinomycin and streptomycin (aadA) or kanamycin (neo or kan and aphA-6

Opposition to having any unnecessary DNA in transgenic crops, especially antibiotic resistance genes

Approaches for production of marker free transplastomic plants

Homology-based excision via directly repeated sequences

Excision by phage site-specific recombinanses

Transient co-integration of the marker gene

Cotransformation-segregation approach

Homology-based excision of Marker gene via directly repeated sequences

Recognition sequence of site-specific recombinanse

Marker gene excision by phage site-specific recombinanses

Marker gene excision by phage site-specific recombinanses

1-transplastomics carry marker gene flanked by two directly oriented recombinase target sites

2-removal of marker gene by introduction of a gene encoding a plastid-targeted recombinase in the plant nucleus

• recombinases (Cre and Int)• absence of homology between the attB and attP sites

and the absence of pseudo-att sites in ptDNA=> Intbetter than Cre

Transient cointegration of the marker gene to obtain marker-free plants

Cotransformation-segregation

New marker genes applying RNA editing in plastids

conversion of specific C nucleotides to U in plastids

Mediated by a nuclear encoded complex

Some plastid genes (e.g., psbL, ndhD, rpl2) the start codon is

encoded as ACG and must be edited to AUG

=>constructing new selectable marker gene only expressible

when integrated into the plastome

System Overall cost

Production timescale

scale-up capacity

Product quality

Glycosylation Contamination risks

Storage cost

Bacteria Low Short High Low None Endotoxins Moderate

Yeast Medium Medium High Medium Incorrect Low risk Moderate

Mammalian cell culture

High Long Very low Very high

Correct Viruses, prions and oncogenic DNA

Expensive

Transgenic animals

High Very long Low Very high

Correct Viruses, prions and oncogenic DNA

Expensive

Plant cell cultures

Medium Medium Medium High Minor differences

Low risk Moderate

Transgenic plants

Very low Long Very high

High Minor differences

Low risk Inexpensive

Comparison of Systems for Production of Heterologous Protein

Heterologous genes expressed stably in plastids of tobacco

Production of various protein classes

• expression of genes coding for insecticidal proteins or allowing for herbicide resistance

Bacillus thuringiensis (Bt) toxin: the gene (cry1A) coding for the Bt toxin Cry1A(c)

cry2Aa2 Bt geneNucleus: suboptimal production of toxin=> toxin

resistanceChloroplast:100% mortality of resistant insects 20-30 fold higher Bt prototoxin production

Oxyfluorfen resistance

• plastomic insertion of the Bacillus subtilis gene encoding protoporphyrinogen oxidase (protox)

• a diphenyl herbicide resistant

• higher degree of oxyfluorfen resistance

Glyphosate resistance

• EPSPS: a nuclear encoded, plastid targeted enzyme

• Integration of the petunia EPSPS (5-enol-pyruvyl shikimate-3-phosphate synthase) gene into the tobacco plastome

• Overproduction of EPSPS

• Glyphosate resistance

• production of a human somatropin in a soluble biologically active form

• biodegradable protein-based polymers in tobacco• introduce into plants a set of bacterial genes for the

biosynthesis of polyhydroxyalkanoates (PHAs)• PHAs: a class of biodegradable polymers • fermentative production has proven too costly for large-scale

production • Targeting of PHA biosynthetic genes from Ralstonia eutropha• Proteins involved in the metabolic pathways of plastidsRubisco, Reaction Center proteinsrbcL of Synechococcus: mRNA production but no protein or

enzyme activity

Engineering of plastid metabolism

Site-directed mutagenesis of Rubiscoo deletion of rbcL, replacement with chimeric plastid targeted LSUo rbcL replacement with cyanobacterial

homologues: no translation Plastid reverse genetics• function of several chloroplastic open reading frames (ORFs)ycf1,ycf2,ycf9 transplastomics: all lines heteroplasmicycf9 ORF: stabilisation of LHCycf6: involved in construction of cyt b6f complex• functioning of plastidic RNAfunctioning of plastidic RNA endonuclease• chloroplast structure and physiology only partly suffered from knocking

out plastid-encoded RNA polymerase

Requirements for widespread application of chloroplast engineering

the number of plant species to which plastome technology is applicable needs to be increased considerably

the success rate of gene insertion into the plastome has to be increased

the screening protocols must be simplified and become applicable to a large range of plant species

Thanks for your patience