This document discusses key applications of plant metabolic engineering, including enabling plants to fix their own nitrogen, altering nutrient content of crop plants, enhancing photosynthetic efficiency, and using plants for biofuel production. Some of the challenges discussed are expressing the large number of genes required for nitrogen fixation in plants, developing efficient nutrient exchange in plant-microbe symbiosis, and modifying lignin in plants to more easily access cellulose for biofuels while avoiding negative impacts on growth.

1. Key Applications of Plant
Metabolic Engineering
Mirza Faisal Qaseem
08-arid-876
PhD scholar (Bot)
PMAS- Arid Agriculture
University
Rawalpindi, Pakistan

2. Contents
• Challenges in plant metabolic engineering
• Plants That Can Fix Their Own Nitrogen
• Crop Plants with Altered Nutrient Content
• Enhancing Photosynthetic Efficiency
• TALENs
• CRISPRs

3. 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

5. Challenges in plant
metabolic 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

6. Plants That Can Fix Their Own
Nitrogen
• 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 nitrogen
Transform agriculture by reducing or eliminating
this enormous dependence on fertilizer

7. 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

8. 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.

9. • 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

10. Challenges
• Eighteen gene products (in Klebsiella oxytoca)
are necessary and sufficient for the
production of nitrogenase and its complex
iron-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

11. The successful transfer of other large gene
clusters from one microbe to another suggests
that the process of functionalizing microbes is
undergoing a dramatic improvement

12. Challenges
• 18 components of the nitrogenase
biosynthetic apparatus would need to be
expressed simultaneously in plants and
function in concert
• Since plants are eukaryotic and multicellular,
where in the plant cell should the genes be
expressed and in which cell types of the plant

13. • 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.

14. 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 utilizing
nitrogenase from the biology of the plant host
ii. Outsources the demanding chemistry involved
to a bacterial strain better suited to the task.

15. ii. The well-studied symbioses between legumes
and their nitrogen-fixing, rootnodulating
bacterial symbionts prove that a bacterial
mutualist can satisfy the nitrogen needs of a
plant host
• Nitrogen-fixing bacteria in the rhizosphere opens
the possibility that symbioses of this sort are a
much more widely distributed phenomenon

17. Challenges
• Enabling efficient nutrient exchange
• Maintaining specificity of the host-microbe
pair
– Both could take years to develop and are likely to
require not just plant but also microbial metabolic
engineering.
– Advanced molecular breeding tools that enable
access to natural variation in a plant’s wild
ancestors are a promising alternative approach to
increasing crop plant yields

18. Crop Plants with Altered
Nutrient 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 enhanced
using breeding
– rice
– maize
– wheat
– Tomatoes

19. • 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

20. Engineering of Metabolic pathways
• Those pathways that produce
– Phytoalexins
– Flavonoids and other molecules
• Play a role in the chemopreventive properties
of vegetables and fruits.

21. 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 chemopreventive
properties in cancer susceptible mice

22. 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

24. • Introducing or increasing the level of a
nutrient compound could also alter the taste
of a plant, potentially impacting its
palatability.
– since small molecule metabolites make an
important contribution to flavor.
• Notable targets in this area include the steviol
glycosides and the mixed esters that give
strawberry plants their distinctive flavor

25. Engineering Crops for Biofuel
Production
• 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

26. • 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

27. • 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

28. • Lignin biosynthesis can be genetically
modified to change its chemical composition
or to reduce its content in plant tissues to
improve the processing of polysaccharides.
• Arabidopsis thaliana knockout mutant of
caffeoyl shikimate esterase
– Gave a 4-fold increase in the efficiency of
saccharification

29. • 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%.

30. • 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.

31. Challenges
• Feasibility of enzymatically degrading lignin to
liberate cellulose
• Although this would undoubtedly be a difficult
task, the ability of white rot fungi to degrade
lignin proves the concept that there exist
enzymes (e.g., lignin peroxidases) that can
cleave the lignin meshwork into monomers
and smaller polymers

32. • 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.

33. Enhancing Photosynthetic
Efficiency
• Rubisco is the enzyme that catalyzes the first
key step in CO2 fixation as part of the Calvin
Cycle
• 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

34. 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)

35. 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

36. – Which diffuse to bundle sheath cells where they
are decarboxylated and concentrated to maximize
Rubisco’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

37. • All the enzymes of the C4 cycle are known and
already exist in C3 plants.
• However, expressing the enzymes of the C4 cycle
alone will not be enough, as the plant’s anatomy
is crucial for the success of the pathway
• Genes that are responsible for controlling C4 leaf
anatomy remain largely unknown and are being
identified by mutant populations of model C4
plants like Sorghum

38. • Cell-specific promoters will need to be
identified to enable cell-type-specific
expression in bundle-sheath or mesophyll
cells.
• > 20 genes needed for the installation of C4
photosynthesis in C3 plant
• Sophisticated transformation
• Genome editing technologies

39. 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

40. Transcription activator-like effector
nucleases TALENs
• Artificial restriction enzymes
• Generated by fusing a
• TAL effector DNA binding domain
• DNA cleavage domain.
• TAL effectors
– are proteins secreted by Xanthomonas bacteria
when they infect various plant species.

41. • 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

42. 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

43. 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.

45. 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

46. • 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

47. Applications
• Artificial immunization against phage by introduction
of engineered CRISPR loci in industrially important
bacteria, including those used in food production and
large-scale fermentation
• Genome engineering at cellular or organismic level by
reprogramming a CRISPR/Cas system to achieve RNA-
guided genome engineering.
• Discrimination of bacterial strains by comparison of
spacer sequences

50. 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