Genetic Engineering for plant secondary metabolites. Agrobacterium Tumifaciens, Agrobacterium Rhizogenes

1. PLANT SEONDARY METABOLITES
Under supervision of: Dr. ND KANNAN

2. Introduction
 Plants are capable of producing a myriad of chemical compounds.
 While these compounds serve specific functions in the plant, many
have surprising effects on the human body, often with positive action
against diseases.
 These compounds are often difficult to synthesize ex vivo and
require the coordinated and compartmentalized action of enzymes in
living organisms.
 However, the amounts produced in whole plants are often small and
restricted to single tissues of the plant or even cellular organelles,
making their extraction an expensive process.
 Metabolic genetic engineering is introduced in light of physiological
and genetic methods to enhance production of high-value plant
secondary metabolites.

3. Commercialisation
There are as many as about 23,000 reported secondary metabolites biologically
produced by microorganisms, of which only 150 are used in pharmaceutical,
agricultural, or other fields.
About 45% of all biologically active microbial metabolites are known, and 80% of these
known compounds are used in practical applications.
More than 10,000 of these compounds are extracted from actinomycetes.
Approximately 7600 compounds are produced by Streptomyces species which falls under
actinomycetes .
Drugs are produced by chemical synthesis or semiindustrial processes for commercial
uses. These microbial strains require sophisticated production methods to separate
the compounds through fermentation.

4. Cost Restrictions
Due to these reasons, the production costs involved in the isolation and
extraction of these compounds are high, resulting in a limited extraction of
secondary metabolites from the naturally available fungi.
It clearly shows that the cost cannot be lowered unless the production is
increased and it actually increases if a new compound is processed.
After the discovery of antibiotics in 1940, the excessive production of
pharmaceuticals was the basis for the increase in microorganism
fermentation.
Processes using the usual “mutate-and-screen” method was advanced for
penicillin strains, and the current production of penicillin from Penicillium
chrysogenum stands 1000 times more than 70 g/l, which which was earlier
only 60 mg/l .
The protein production of Pseudomonas denitrificans, 100,000 times the more
soluble, of vitamin B2 of through Ashbya gossypii, and this is 40,000 times
more soluble than the water-soluble vitamins, and this is another way to heal
with the usage of microbe strain.

5. GENETIC ENGINEERING OF THE SECONDARY METABOLIC
PATHWAY IN PLANTS
 The first genetic engineering was used in the flavonoid and
anthocyanin .
 The various experiments carried out involved excessive
expression of diverse pathways of genes, for instance, to
produce new flower colors using new plant compounds.
 Due to their antioxidant nature, external surfaces in food
additives and plant pigments have a structure based on or
similar to that of flavone which are a significance role in diet.
 An alternative imperative drug group is an important
secondary plant metabolite, consisting isoquinoline alkaloids,
which include significant drugs such as morphine and codeine.
Majority of secondary substances are terpenoids by far.

6. Regulation of Quantity of secondary
metabolites
 Genetic engineering of secondary metabolites is a way to
increase or reduce the quantity of a specific compound or
a group of target compounds.
 Reduction of the production of unwanted (group of)
combination(s) can be achieved by:
 By reducing the level of compatible RNA templates through
antisense methods,
 crystallization or ribonucleic acid interposition procedures,
 through the excessive expression of antibodies, contrary to
the enzymatic stage in the pathway, can be eliminated.
 The change of color of flowers has been effectively
achieved by antisense gene method.

7. To increase the production
 Flux deviation to a competitive pathway or increase in
catabolism are the next goals to be achieved.
 Transferring pathways to other plant species or natural
plant species or microorganisms are often the goal to
cumulate the efficacy of specific compounds.
 Moreover, new compounds that are yet to be discovered
follow two general approaches to increase the production
of these compounds.
 Initially, one or more genes were used to overcome the
specific steps limiting the speed of the pathway, blocking
competitive pathways, and reducing catabolism.
 The second approach attempts to control the multiple
biosynthesis genes to alter the expression of regulatory
genes.

8. Genetic control of secondary
metabolites production
Types of bacteria:
1. Agrobacterium tumefaciens:
Cause crown gall tumor.
2. Agrobacterium rhizogenes:
Cause Hairy Root (HR) tumor or
root tratoma.
A crown gall
Source:January 31,
2020/in Plant Science
Research Weekly /by Mary
Williams

9. Agrobacterium tumefaciens
 Agrobacterium tumefaciens is a soil phytopathogen that naturally infects
plant wound sites and causes crown gall disease via delivery of transferred
(T)-DNA from bacterial cells into host plant cells through a bacterial type IV
secretion system (T4SS).
 Through the advancement and innovation of molecular biology technology
during the past few decades, various important bacterial and plant genes
involved in tumorigenesis were identified.
 With the help of more comprehensive knowledge of how A.
tumefaciens interacts with host cells, A. tumefaciens has become the most
popular plant transformation tool to date.
 Any gene of interest can now easily be used to replace the oncogenes in the
T-DNA region of various types of binary vectors to perform plant genetic
transformation with A. tumefaciens.
 Arabidopsis, the most-studied model plant with powerful genetic and
genomic resources, is readily transformable by A. tumefaciens for stable and
transient transformation in several ecotypes tested, although variable
transformation efficiencies in different accessions were observed.

10. Agrobacterium tumefaciens
Entry: enters plant via wound
sites.
Spread: Widespread soil
bacterium
Disease: Crown gall disease
(causes cancerous-like
tumors).
Gall grows and divides (even if
bacteria dies):
 1. Gall tissues have high [CK]
and [IAA]
 2. Opines(derivatives of aa)
made (C source)
Crown Gall growing on root Crown Galls
on the root. Because A. tumefaciens
dwells in the soil, this is one of the
typical locations they attack.
From: Clemson University - USDA
Cooperative Extension Slide Series

11. Agrobacterium tumefaciens
 Plasmid transfer: Agrobacterium stably transfers its plasmid into plant DNA by Interkingdom
horizontal DNA transfer.
 Plasmid types: Specific plasmid known as Ti-plasmid [tumor-inducing plasmid] contain T-DNA
that integrated in the DNA of the plant cell leads to crown gall tumor.
 Virulent native promoter: Constructed virulent A. tumefaciens (which is lysogenized with
Cauliflower mosaic virus 35 S):
 1. Ca MV-35 S
 2. Ca MV-19 S So, we have 3 strains of A. tumefaciense.
 Response to plasmid strains: is independent & give different results according to
type of the plant & no ideal but logic they are causing CGT but some cause HRT as A.
rhizogenes where:
 if constructed with Ca-MV-35 :tumor morphology [shooty tratoma] & tms plasmid formed
 if constructed with Ca-MV-19 : tumor morphology [rooty tratoma] & tmr plasmid formed.

12. Results of bacteria infection leads to:
1. Repression of (IaaM) & (IaaH) genes.
2. Activation of IPT gene so leads to crown gall
tumor.

13. Agrobacterium rhizogenes
Agrobacterium rhizogenes is a soil-borne gram-negative bacterium that
belongs to the Rhizobiacea family.
It causes hairy root disease in infected dicotyledonous plants.
Agrobacterium, also known as natural genetic engineers, have begun to
attract interest in gene transfer to plants in the mid-1980s.
The integration of the T-DNA region of the Ri (root-inducing) plasmid of A.
rhizogenes into the plant DNA results in infection in plants and reveals the
hairy root phenotype.
Hairy roots have some characteristic features, such as frequent branching, high
density of capillary roots, high biomass production potential, and rapid
development. Agrobacterium is routinely used in gene transfer studies
because of its ease of application, economic viability, high rate of
transformation, and rapid root growth. In hairy root cultures , secondary
metabolite production rates can be as high as that of the parent plant.

14.  Basically, the genetic transformation can be divided into following steps:
 Agrobacterium sensing phenolic compounds released by plant roots,
 which triggers attachment of the bacteria to the root;
 Processing the T-DNA into Agrobacterium cells and T complex formation
(mechanism similar to those of A. tumefaciens); transfer of T complexes
from the bacteria to the host plant genome.
 The A. rhizogenes T-DNA has two independent sequences:
 TL (left)
 TR (right) borders.
 TL-DNA and TR-DNA are generally independently transferred and integrated
into the host plant genome, however only the TL-DNA is essential (and
hence sufficient) to induce hairy roots.
 Sequence analysis of TL-DNA revealed four open reading frames that are
essential for hairy roots induction (rolA, B, C, and D).
 RolB gene appears to be the most important in hairy roots induction,
since loss-of-function mutation at this locus renders the plasmid as avirulent.

15. Why Hairy Roots?
 Allows genetic manipulations to be performed in the metabolic pathway of
the synthesis.
 The reason for choosing hairy roots is to provide rapid development without
the need for any auxin source from outside.
 No light is needed for incubation.
 Metabolite production is stable along with the genetic stability.

16. Because of all these advantages, many root-derived plant products that are thought not
to be suitable for cell culture have been poduced via hairy root culture technology
(Giri and Narasu, 2000).
Reports on hairy root cultures established for secondary metabolite production in 2016
are listed in Table.
Source:Pinar Nartop, in Plant Metabolites and Regulation Under Environmental Stress,
2018

17. Pictorial representation of Genetic manipulation in hairy root culture for
secondary metabolite production using Agrobacterium rhizogenes

18. Conclusion
 Nowadays, hairy root culture systems are considered as green
factories for mass production.
 Examples of pharmaceutically relevant molecules produced by
transformed root culture include
 anticancer (e.g., paclitaxel and camptothecin),
 antimalarial (e.g., artemisinin; its discovery was awarded Nobel
Prize in Physiology and Medicine in 2015),
 anti-inflammatory (verbascoside) compounds.
 A. rhizogenes–mediated transformations are utilized to
elucidate
 biosynthetic pathways and physiological processes,
 generate recombinant therapeutic proteins,
 assist molecular breeding,
 enhance phytoremediation efforts.

19. References
 PUBMED: Introduction to metabolic genetic engineering for the production of valuable
secondary metabolites in in vivo and in vitro plant systems (Vagner A Benedito, Luzia V
Modolo )
 https://www.researchgate.net/publication/333827490_Genetic_Manipulation_of_Secondary_Metab
olites_Producers
 https://bioone.org/journals/the-arabidopsis-book/volume-2017/issue-15
 Science Direct: Pinar Nartop, in Plant Metabolites and Regulation Under Environmental Stress,
2018
 Z.P. Yordanova, M.I. Georgiev, in Encyclopedia of Applied Plant Sciences (Second Edition), 2017

20. Presented By
AMREEN KHAN
20BIT38