Key Applications of Plant Metabolic Engineering

Mirza Faisal Qaseem

08-arid-876

PhD scholar (Bot)

PMAS- Arid Agriculture University

Rawalpindi, Pakistan

Contents

• Challenges in plant metabolic engineering

• Plants That Can Fix Their Own Nitrogen

• Crop Plants with Altered Nutrient Content

• Enhancing Photosynthetic Efficiency

• TALENs

• CRISPRs

Plants

• Derive energy entirely from the sun

• Carbon from CO2

• Defend themselves from pests and predators

• Complex symbioses

• Can survive extremes of

– temperature

– nutrient

– water availability

Challenges in plantmetabolic engineering

• Four long-standing grand challenges in plant metabolic engineering

• Two deal with important applications in food and energy

• Two are of general utility in improving plant fitness

• Solution

– Subjecting plant genome to deletion editing and insertions

Plants That Can Fix Their OwnNitrogen

• 180 million tons of nitrogen fertilizer is used every year in industrial farming

• Disadvantages – Substantial cost– Deleterious effects

• Soil • Surrounding environment• Water

• Plants able to make their own nitrogenTransform agriculture by reducing or eliminating

this enormous dependence on fertilizer

Plant engineering using Bacteria

• First method takes advantage of the fact that some bacteria carry out their own version of the Haber-Bosch process—reducing atmospheric N2 into a more bio available form, NH3—using the enzyme nitrogenase

Nitrogenase

• Complex enzyme

– multiple metalloclusters

• Require

– large quantity of biochemical energy

• Transfer the electrons needed to activate the exceptionally stable N2 triple bond.

• By expressing nitrogenase, plants would be able to fix their own nitrogen.

• Nitrogen fixed by a plant could be used immediately to generate amino acid and nucleic acid monomers

• Transport them to neighboring cells

• Disadvantage

– Process induce a metabolic cost

• Can be regulated by the endogenous level of nitrogen to maximize its efficiency

Challenges

• Eighteen gene products (in Klebsiella oxytoca)are necessary and sufficient for theproduction of nitrogenase and its complexiron-molybdenum cofactor.

• Biosynthetic gene cluster for nitrogenase has been refactored—taken apart, recoded, and put back together using known components—and shown to be active in its new host

The successful transfer of other large geneclusters from one microbe to another suggeststhat the process of functionalizing microbes isundergoing a dramatic improvement

Challenges

• 18 components of the nitrogenasebiosynthetic apparatus would need to beexpressed simultaneously in plants andfunction in concert

• Since plants are eukaryotic and multicellular,where in the plant cell should the genes beexpressed and in which cell types of the plant

• Nitrogenase which is poisoned by oxygen and must therefore be expressed under anaerobic conditions.

• Tools that enable organelle and cell-type specific expression will be of great utility here and in other plant engineering efforts.

Second method of increasing N uptake

• Engineer a rhizosphere symbiosis between a nitrogen-fixing microorganism and a plant host

• Primary advantages

i. It uncouples the difficulties of utilizingnitrogenase from the biology of the plant host

ii. Outsources the demanding chemistry involvedto a bacterial strain better suited to the task.

ii. The well-studied symbioses between legumesand their nitrogen-fixing, rootnodulatingbacterial symbionts prove that a bacterialmutualist can satisfy the nitrogen needs of aplant host

• Nitrogen-fixing bacteria in the rhizosphere opensthe possibility that symbioses of this sort are amuch more widely distributed phenomenon

Challenges

• Enabling efficient nutrient exchange

• Maintaining specificity of the host-microbepair– Both could take years to develop and are likely to

require not just plant but also microbial metabolicengineering.

– Advanced molecular breeding tools that enableaccess to natural variation in a plant’s wildancestors are a promising alternative approach toincreasing crop plant yields

Crop Plants with AlteredNutrient Content

• Golden rice proves the concept that the nutrient content of a crop plant can be improved by metabolic engineering

• Nutrient levels of certain plants was enhancedusing breeding – rice

– maize

– wheat

– Tomatoes

• Golden rice

– adding the beta-carotene pathway to rice, to produce rice with higher levels of vitamin A

• Targets for nutrient engineering

– Metabolic pathways

– alter the level of a nutrient in its native host

Engineering of Metabolic pathways

• Those pathways that produce

– Phytoalexins

– Flavonoids and other molecules

• Play a role in the chemopreventive properties of vegetables and fruits.

Alteration of a nutrient level

• Not require knowing the genes in its biosynthetic pathway

• Example

• Expressing two transcription factors

from snapdragon in tomato

• Levels of the flavonoid anthocyanin have been increased 3-fold

• Flavonoid confer improved chemopreventiveproperties in cancer susceptible mice

Expression of Health promoting molecule in a new host.

• Golden rice is example of this strategy

• Only two genes were required for the production of beta-carotene

• A more ambitious prospect would be to transfer the 13-gene glucoraphanin pathway to a widely consumed crop such as rice, wheat, or maize

• Require

– New approaches for discovering the genes

– new tools for site-selective genome editing

• Introducing or increasing the level of anutrient compound could also alter the tasteof a plant, potentially impacting itspalatability.

– since small molecule metabolites make an important contribution to flavor.

• Notable targets in this area include the steviolglycosides and the mixed esters that givestrawberry plants their distinctive flavor

Engineering Crops for BiofuelProduction

• Plants are ideal invention• Combat the dual challenges

– Rising greenhouse gases– Used for green energy

• They capture CO2 from the air and turn it into sugar, the ideal substrate for biofuel production.

• Plants protect this energy rich polymer• Most of the carbon is stored as dehydrated

crystalline cellulose, wrapped in a meshwork of crosslinked phenylpropanoids, lignin

• Cellulose presents a challenge in itself.

• The beta-1,4-linked chains of cellulose can pack tightly together, excluding water in a way that prevents glycosidic enzymes from releasing its constituent sugar monomers

• Cellulose co purifies with lignin which inhabit the action of cellulases

• So there is need for costly and energy intensive pretreatment to separate the cellulose from lignin

• Lignin could be degraded into valuable aromatic monomers either

• Chemically

• Enzymes found in

– White rot fungi

– Microorganisms

• Neither has been shown to work in a real-world setting

• Lignin biosynthesis can be geneticallymodified to change its chemical compositionor to reduce its content in plant tissues toimprove the processing of polysaccharides.

• Arabidopsis thaliana knockout mutant of caffeoyl shikimate esterase

– Gave a 4-fold increase in the efficiency ofsaccharification

• In Lignin modified plants Discernible effects on plant growth and development

– Transgenics contained 25% less cellulose content

– 40% lighter and smaller than wild-type

• Protein engineering and expression of a 4-O-methyltransferase in A. thaliana substantially reduced lignin content

• Interestingly, no significant changes in growth phenotype were observed and saccharification yields improved by 25%.

• Ideal scenario would be for plants to degrade their own lignin, releasing pure cellulose that could be more easily degraded into glucose

• For that matter, the plant could be engineered to break down its own cellulose on demand, releasing fermentation- ready sugars for biofuel production.

Challenges

• Feasibility of enzymatically degrading lignin toliberate cellulose

• Although this would undoubtedly be a difficulttask, the ability of white rot fungi to degradelignin proves the concept that there existenzymes (e.g., lignin peroxidases) that cancleave the lignin meshwork into monomersand smaller polymers

• Heavily crosslink (and consolidate) the lignin

– causing it to precipitate and making it easier to separate from cellulose.

• Process likely to be carried out by suites of degrading enzymes in rot fungi, a critical step would be to first identify sets of enzymes that could be coexpressed to make the necessary modifications to lignin.

Enhancing PhotosyntheticEfficiency

• Rubisco is the enzyme that catalyzes the firstkey step in CO2 fixation as part of the CalvinCycle

• Its low turnover rate and ability to also use oxygen as a substrate in photorespiration make it notoriously inefficient.

• Plants make more Rubisco than any other protein

Alternative carbon fixation systems

• Improve the efficiency of photosynthesis by actively concentrating CO2 and reducing the oxygenase activity of Rubisco

• Plants have evolved two systems to improve photosynthesis efficiency

• C4

• Crassulacean acid metabolism (CAM)

C3, C4 and CAM plants

• C3

• the process of fixing carbon into C3 acids occurs in the same cell type

• C4

• Evolved to separate the Calvin cycle and carbon capture into different cell types.

– CO2 is first captured within mesophyll cells to produce C4 acids

– Which diffuse to bundle sheath cells where theyare decarboxylated and concentrated to maximizeRubisco’s carboxylating efficiency.

• CAM plants

• Photosynthesis and carbon capture are separated temporally– capture CO2 at night

– Close their stomata during the day

– C4 acids generated by CAM photosynthesis are decarboxylated and concentrated to enhance Rubisco’s efficiency

• All the enzymes of the C4 cycle are known andalready exist in C3 plants.

• However, expressing the enzymes of the C4 cyclealone will not be enough, as the plant’s anatomyis crucial for the success of the pathway

• Genes that are responsible for controlling C4 leafanatomy remain largely unknown and are beingidentified by mutant populations of model C4plants like Sorghum

• Cell-specific promoters will need to be identified to enable cell-type-specific expression in bundle-sheath or mesophyllcells.

• > 20 genes needed for the installation of C4 photosynthesis in C3 plant

• Sophisticated transformation

• Genome editing technologies

Technologies

• Transcriptomic and metabolomic analyses

• Genome editing tools– TALENs

– CRISPR/Cas9

• Synthetic biology parts list specific to plants– tissue-specific promoters,

– Transporters

– multi-gene expression constructs

– biosynthetic enzymes

Transcription activator-like effectornucleases TALENs

• Artificial restriction enzymes

• Generated by fusing a

• TAL effector DNA binding domain

• DNA cleavage domain.

• TAL effectors

– are proteins secreted by Xanthomonas bacteriawhen they infect various plant species.

• TALEs can be quickly engineered to bind practically any desired DNA sequence

• Combining such an engineered TALE with a DNA cleavage domain (which cuts DNA strands), one can engineer restriction enzymes that are specific for any desired DNA sequence.

• When these restriction enzymes are introduced into cells, they can be used for genome editing in situ

Applications

– Elucidating basic function and regulating a gene

– Study of metabolic pathways

– Embryonic stem cell research

– Research on disease model

– Provide therapeutic avenues for genetic disorders including monogenic diseases

– Method of the Year” for 2011 by Nature Methods

Problems

• Non specific

• off-target cleavage may result

• lead to the production of enough double-strand breaks to overwhelm the repair machinery.

• consequently yield chromosomal rearrangements and/or cell death.

CRISPRs (clustered regularly interspaced short palindromic repeats)

• DNA loci containing short repetitions of base sequences

• Repetition is followed by short segments of "spacer DNA“

• CRISPR loci range in size from 24 to 48 base pairs

• Spacer DNA are regions of non-coding DNA between tandemly repeated genes.

• CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea

• CRISPR/Cas system is a prokaryotic immune system

• Confers resistance to foreign genetic elements such as plasmid and phages and provides a form of acquired immunity.

• CRISPR spacers recognize and cut these exogenous genetic elements

• Since 2013, the CRISPR/Cas system has been used for gene editing and gene regulation

Applications

• Artificial immunization against phage by introductionof engineered CRISPR loci in industrially importantbacteria, including those used in food production andlarge-scale fermentation

• Genome engineering at cellular or organismic level byreprogramming a CRISPR/Cas system to achieve RNA-guided genome engineering.

• Discrimination of bacterial strains by comparison ofspacer sequences

Reference

• Lau W, Fischbach MA, Osbourn A, Sattely ES (2014) Key Applications of Plant Metabolic Engineering. PLoS Biol 12(6): e1001879. doi:10.1371/journal.pbio.1001879