Lecture 11: Sustainable phytochemical engineering biology

Question 1

Lecture outline

Answer 1

Phytochemical & metabolic diversity
Programming plant processes
Expanding trait variability & availability

Sustainable Phytochemical Engineering Biology

Crop Future Strategies: Expand trait variability & availability/ Sustainable approaches

Question 2

Key milestones in humans’ exploration of plant chemistry and biochemistry using modern scientific methods:

Answer 2

-2010 and onward Renaissance of phytochemistry
. Pathway elucidation in diverse nonmodel plants
. Link between chemistry and biological functions
. Engineering plant natural product biosynthesis

2006
Production of artemisinic acid in engineered yeast
1990s
Adoption of model plants for phytochemistry research e.g. Arabidopsis & rice

1970s
Discovery of antimalarial drug
artemisinin from sweet wormwood

1960s
Plant biochemistry using crude methods

1940s
Semisynthesis of progesterone from
diosgenin isolated from Mexican yams

1920
Birth of phytochemistry - structural elucidation of quinine and vitamin c

1820
Isolation and crystalisation of quinine from
quinine from cinchona tree

1747
First clinical trial using citrus against scurvy

1663
Antimalarial properties of cinchona tree recorded

Question 3

Phytochemical example: Arugula (rocket)

Answer 3

Rocket, arugula, salad rocket or garden rocket

‘Eruca sativa’, mustard (Brassicaceae) family
Latin words ‘uro’ or ‘urere’, which translates to burn

Phytochemicals:

Glucosinolates, such as glucosativin, glucoraphanin & glucoerucin

They are broken down into isothiocyanates & other compounds that show biological activity.

Flavonols, mainly quercetin and kaempferol.

Nitrate, which is proposed to increase plasma nitrate and nitrite and help reduce blood pressure

Dietary fibres

Micronutrients, especially calcium, iron and vitamins A, C & K

See video by Dr Michael Rotter

Question 4

Phytochemical diversity

Answer 4

Specialised metabolites:
*essential for sessile living, structurally & functionally diverse
*Adaptive chemicals and compounds utilised throughout plant lifecycles

Indispensable:
*essential for long-term survival of humans and other animals
*Food, feed, energy, medicine, industrial use, renewables climate maintenance etc.

Relevance to module:
*loss of bio and genetic diversity due to intensive agro and enviro deterioration
*Bio based production, relevant to farming and combined breeding practices

Question 5

Phytochemicals are chemicals of plant origin

Answer 5

What are they?
*A vast array of natural chemicals essential for sessile lifestyle

Why are they there?
*Allow plants to adapt to constantly changing environment
*Survive & communicate in complex and constantly changing ecological environment

By contributing to:
*plant growth or defense against competitors, pathogens, or predators
*Resistance and tolerance to biotic and abiotic stresses

Where do they come from?
Products of primary & Secondary metabolism:
*Biological activity
*Growth & Cellular replenishment
*Variety occurs due to plant chemodiversity and metabolic plasticity

Question 6

Phytochemicals: Primary metabolites - Used for immediate survival, conserved in species
necessary for basic physiological mechanisms:

Answer 6

Growth, development, reproduction, photosynthesis, nutrient assimilation, solute transport and respiration

These include:
*Amino acids, proteins, nucleic acids, carbohydrates, lipids, organic acids, phosphorylated compounds

Phytochemicals: Secondary metabolites – many functions – many still unknown!

Aka ‘specialised metabolites (SM)’ ‘natural products’

Plant chemodiversity:
*Diversity of specialised metabolites (SMs) plants produce at different levels of organisation
– from single organs to entire communities (Wetzel and Whitehead, 2020)

SMs are classified according to structure and function:
*Terpenoids
*phenolic compounds (flavanoids)
*nitrogen containing compounds (alkaloids)
*Sulphur containing compounds

Chemotype: Metabolite class – composition differences within species
^ Defined by biological activity

Question 7

How many phytochemicals exist and what do they do?

Answer 7

Collective metabolite and chemodiversity > 100.000 to 1 million

Single plant species produces only subset ~5000 – tens of thousands

Each food plant_unique combination of phytochemicals (dozens – 100’s)

In total estimation ~ 1,500 to 2,000 phytochemicals present in our diet

50,000 - 130,000 phytochemicals have been discovered

PhytoHub database inventoried > 1,350 food phytochemical (https://phytohub.eu)

ONLY 15% of 350,000 plant species investigated for chemical constituents
A priority: Exploring, mining & harnessing plant chemo diversity

Question 8

Phytochemicals and us

Answer 8

Nutrition - Essential & non-essential bioactivities in the human body

Organoleptic characteristics - Colour, flavour and aroma

Phytochemicals & health benefits - Potent antioxidant properties that protect cells from free
radicals - mitigating aging and disease

Industry / Economy - Phytochemicals as commodities

Enormous variety in complex phytochemical structures reflected in many uses

Question 9

Pharmaceutical health and nutraceuticals

Answer 9

Since humans have looked to nature to provide cures for various ailments and diseases:

Herbs, Seeds, Leaves, Fruits, Bark and Roots have roles in medicine, ceremonials, hallucinogenic & other areas:
➢ 30% of our drugs ➢ 60% drugs introduced in last 20 yrs based on plants extracts or derivatives
- DRUG Discovery Inspiration for more synthetic drugs & medicine

Question 10

Industrial applications:

Answer 10

Food additives: technological characteristics are improved in foods by the use of food additives/ingredients:

*Flavourings, Sweeteners and sugar substitutes, colouring substances
* Antimicrobial agents and Antioxidants
* Stabilisation of fats, frying oils and fried products
* Stabilisation and development of other food products
* Anti-browning in foods, Colour stabilisation in meat products
* Antimicrobials to extend shelf life.
* Cosmetic applications e.g. essential oils and stabilisers/shelf life extenders
* Bio-pesticides

Question 11

Health benefits of phytochemicals

Answer 11

Carotenoids: B-carotene, lutein, lycopene – act as anti-oxidant to free radicals
-Yellow, orange, red fruits and vegetables

Phytoestrogens: Flavonoids, Isoflavones, Lignans - ‘Detoxify’ carcinogens, Estrogen antagonists
-Legumes, Soybeans, Whole grains

Phenols: Anthocyanin, Capsaicin, Polyphenol - Antioxidant to free radicals, Induce detoxification enzymes
- Blueberries, Grapes, Raspberries, Peppers

Terpenes: Limonene, perillyl alcohol – anti-cancer effects
- Citrus oils, cherries, garlic

Phytosterols: Campesterol, Stigmasterol - Decrease colon cancer promoters & Decrease cholesterol
-Vegetable oils, nuts, seeds

Question 12

Health benefits associated with particular plants, fruits and vegetables:

Answer 12

Apples: Anti-cancer, anti-tumour, antioxidant activity, anti-diabetic and weight-loss associated

Oranges: antioxidant and anti-inflammatory

Grapes: anti-oxidant, anti-platelet, anti-tumour and antidiabetic

Potato? : anti-oxidant

Leafy - herb? : anti-oxidant, anti-inflammatory, anti-obesity, anti-diabetic

Leafy – veg? : anti-oxidant, anti –cancer, anti- inflammatory, antidiabetic

Question 13

Phytochemical diversity an important dimension of Biodiversity - Why so many?
Functions of plant specialized metabolites in plant defence and other roles:

Answer 13

Mutualistic & Antagonistic - Defence Hypothesis
➢ Roles in plant growth or defence against competitors, pathogens, predators
➢ Developmentally and temporally regulated
➢ Resistance and tolerance to biotic and abiotic stresses

Ecological
➢ Roles in plant communications and interactions in natural ecosystems and their adaptation to climate changes
➢ Phytochemical diversity is multidimensional and multiscale, and can be quantified using species diversity metrics
Richness; evenness, Functional diversity, alpha, gamma, beta diversity
➢ Models: Gradient Boost Model (GBM)

Linear mixed effect models (LMM)
Generalized Additive Model (GAM)

Other roles - Fitness
➢ pollinator attraction
➢ microbiome management
➢ inter- and intra-plant signalling - mediated by volatiles (indirect)
➢ protection against oxidants

Question 14

Common features of phytochemical SMs

Answer 14

primary metabolites contribute to immediate survival:
*growth, development and reproduction
*Proteins, carbohydrates and lipids
*Necessary for photosynthesis, respiration, nutrient assimilation, and solute transport.

Secondary Metabolites aka specialised metabolites contribute to ecology and fitness:
*2 or more metabolic pathways overlap / ‘cooperate’
^ Flavonoids: Shikimic & Malonic pathways

*Biosynthesis steps expressed in different tissues
^ Alkaloid vinblastine: Epidermis, parenchyma, idioblasts, laticifers

*Produced / Stored in specialized cell types or structures:
^ Terpenes: Glandular trichomes, resin ducts
^ Bisindole alkaloids: Cytosol, vacuole, chloroplast, nucleus, endoplasmic reticulum

*Modulated by biotic & abiotic signals:
^Phytohormones: JA. SA, ETH, AA & NO
^ROS & NOS

in response to:
- herbivore and pathogen attacks,
- drought, flooding, extreme temperatures
- excess irradiance, UV stress
- salinity, and mineral imbalance

Question 15

Secondary/Specialised metabolyte organisation

Answer 15

Accumulation patterns – 3 major types:

1. Constitutive
   accumulated metabolites present as basal chemical constituents defence activity against pathogens and herbivores
   o phenolic acids & flavonols
2. Preformed
   accumulated metabolites often in non-toxic forms in vacuolar storage become bioactive upon physical damage (by herbivores or necrotrophic pathogens)
   ^ glucosinolates/cyanogenic glucosides form & isothiocyanates and cyanide

3 Inducible
Absent or present in low concentrations prior to stress - biosynthesis is
activated
^ Terpenes and alkaloids

Post-translational modifications regulate accumulation of secondary metabolites
➢ signaling pathways of plant secondary metabolism regulation

Question 16

Common features: Organisation Genomic Level

Answer 16

➢Clusters of related gene sequences
Biosynthetic Gene Clusters (BGC)
-arrays, pairs and clusters - paralogs and non-paralogs
-gene duplications and genomic rearrangements
They facilitate co-expression, coinheritance, assembly of metabolic channels, regulatory chromatin changes

Example 1:
nepetalactone biosynthesis in catnip

Example 2:
-genomic and biosynthetic origins of noscapine and morphinans in Papaver somniferum (2 BGC’s)

➢Proteins arranged in multi-enzyme complexes or metabolons
*acceleration of biochemical reactions reduced dilution of metabolites
*improved flux control coefficient of pathways
*easier metabolic flow of relatively insoluble or unstable intermediates
*less undesired effects of toxic intermediates
*facilitated allocation of metabolites to appropriate storage compartments
*improved metabolic cross-talk and redirection

see figure in notes: transient multi-protein complexes of sequential enzymes that mediate substrate channeling –efficient catalysis process to convert intermediates to final product – reducing effects of toxic intermediates

Question 17

Rise of plant chemodiversity

Answer 17

Mechanisms contributing to the diversity of plant specialized metabolites:

*Derived from primary metabolic precursors
*Enzyme promiscuity
*Metabolite modifications and gene duplication
*Subfunctionalization or neofunctionalization

Question 18

Primordial metabolism

Answer 18

• Smaller number of multifunctional catalytic proteins, peptides, and/or RNAs
• Primary metabolic enzymes under strict natural selection
  
  • constrained chemical mechanism

Evolving increasing order and catalytic efficiency

Secondary/specialized metabolic enzymes
*Gene duplications, mutations, Gene clusters
*Catalytic/mechanistic elasticity, Expanded substrate recognition
*Multiple products from single enzyme

Question 19

^ Catalytic promiscuity & Functional innovation through natural selection 3

Answer 19

Examples
A: Substrate permissiveness - HCT

B: Refinement of generalist ancestor to catalytic specialist – two catalytic roles - parallel pathway
e.g. DFR Anthocyanin biosynthesis in nightshades

C: Relaxed specificity – HPS/TEAS subfamily – multitude of products from single enzyme

Question 20

4 main classes of specialised phytochemical metabolites

Answer 20

Terpenes: Monoterpenes, sesquiterpines, diterpines, triterpines, tetraterpenes (carotenoids)

Nitrogen containing: many drugs of powerful therapeutic interest (e.g. morphine), alkaloids, glucosinolates, isothiocyanates, amines and amino acids

Polyphenols: flavanoids, lignans, ellagitanins, coumarins, phenolic acids, stilbenes

Miscellaneous: Sulfur-containing and many other compounds that belong to smaller families
e.g. phytoprostanes or thiosulfinates

Question 21

Plant SMs, numbers and pathways >200,000

Answer 21

Class 1: Terpenoids/Isoprenoids - largest class >30,000

• Monoterpenes, sesquiterpines, diterpines, triterpines, tetraterpenes (carotenoids)
• mevalonate (MVA) pathway
• glyceraldehyde phosphate-pyruvic acid pathway

Class 2: Nitrogen containing – Alkaloids >21000
- water soluble, basic, 1 or more N, usually ring, activity
- used in defence pathway, many pharmacological properties
- many drugs of powerful therapeutic interest (e.g. morphine),
- alkaloids, glucosinolates, isothiocyanates, amines and amino acids

Class 3 : phenolic compounds – Polyphenols - ~5000 flavanoids and tannins

• flavanoids, lignans, ellagitanins, coumarins, phenolic acids, stilbenes
• 1 or more OH and one or more organic ring
• Shikimate and phenylpropanoid pathways (AA to PAL/flavanoids)

Class 4: Sulphur containing and miscellaneous
- Sulfur-containing and many other compounds that belong to smaller families
- e.g. phytoprostanes or thiosulfinates

Question 22

The chemistry behind the scent of lavender

Answer 22

A combination of 3 chemicals:

1. linalool - a terpene alcohol
   *floral and sweet notes
   *therapuetic - calming and destressing

2 terpinen-4-ol: Natures antiseptic
*anti-bacterial and antifungal
w/medicinal application

3. Camphor
   *refreshing with pleasant contrast to linalool

Question 23

The chemistry behind the scent of citronella

Answer 23

Citronella oil is an essential oil from leaves and stems of lemongrass species

The scent is due to the cyclic monoterpene (citral) which has a lemon-like odour

Chemicals such as citronellal, citronellol, and geraniol are used in soap, candles and incense, perfumery, cosmetic, and flavouring industries

Citronella oil is a plant-based insect repellent
+ it has strong antifungal properties

Question 24

Phytochemical classification

Answer 24

Polyphenols, carotenoids, alkaloids, gluconsinolates, polyacetylenes, polysaccharides, allium compounds, lectins, terpenes, capsaicinoids, betalains and chlorophyll

^see notes for key examples of all groups

Question 25

Interface primary – SMs in plant metabolism

Answer 25

Metabolic flow from primary to SMs, occurring in distinct cell compartments with extensive trafficking of metabolic intermediates involved Shikimic acid pathway: -metabolism of carbohydrates and aromatic amino acids -one of the important entrances for plant SM biosynthesis -high-flux bearing pathway in land plants ->30% of all fixed carbon is directed through this pathway Isoprenoids: derived from two isomeric 5 carbon units: Isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) 2 different pathways: 1) mevalonate (MVA) pathway (Non-MEP) 2) 2-C-methyl-D-erithrytol 4 Phosphate (MEP) Phenylpropanoid metabolism: lignols, flavanoids, isoflavones, anthocyanins, stilbenes, coumarins and lignins Lipid pathways: Fatty acid, glycerolipid and triacylglycerol metabolism

Question 26

Interplay of metabolic pathway and sub cellular compartments

Answer 26

see figure in notes: Depicted are the flavanoid, lipid and acetyl-CoA metabolism in Arabidopsis see also figure below: Metabolic connections – interlinking 2 primary and a secondary pathway Depicted - Shikimic acid pathway - mevalonate (MVA) and non-mevalonate (MEP)and phenylpropanoid pathways

Question 27

Metabolic engineering of phytochemical natural products or non-natural products

Answer 27

SMs from plants are high-value: *medicine and pharmaceuticals, bulk chemicals, biofuels, food derivatives, nutraceuticals (health/therapeutic/supplement) We can potentially expand biodiversity by increasing chemodiversity develop: 'new' crops, green or microbial factories, by expanding traits – de novo domestication Resource limitations and sustainability: * develop sustainable routes to produce SM in plants or microbes *replace or supplement direct extraction from plants (medicinal or other) Multi - 'omics' *Innovations in technological tools for comprehensive studies *Major branches: genomics, proteomics, metabolomics, cytomics, transcriptomics, glycomics Metabolic engineering *Engineering and optimisation of biological systems and cellular processes by manipulating flux towards desired products, preferrably cheap and simple "the direct improvement of product formation through the modification of specific biochemical reactions or the introduction of new ones" Synthetic biology Design and creation of new biological pathways to biosynthesise novel compounds in organisms see figure for omics tools

Question 28

Strategies for Metabolic Engineering (ME)

Answer 28

*Production of (phyto-) chemicals & drugs, food, non-natural *Re-programme or develop new crops with improved or novel traits *Environmental resilience & reduce CO2 emissions Plant, microbial, algal 'production platforms' *Optimize existing pathway towards natural SM in planta *'nativeʼ plant (transformation/cultivation systems availability) where the substance already occurs *Yield, quality, localization Engineer existing pathway towards natural SM in planta or Non-natural SM in planta *Natural: Alternative or related plant species *Non-natural: by adjusting nuclear pathways Engineer or manipulate pathway towards Natural or non-natural SM in microbe/algae *Natural: N/A? *More commonly done *non-natural: bioplastics or polymer/nylon intermediates, fuel, drugs, herbicides, flavourings & fragrances

Question 29

Metabolic Engineering Approaches & Requirements

Answer 29

5 Approaches: 1) Introduce/overexpress endogenous genes (from plant) or heterologous genes (from another plant or organism) 2) Over express, ectopic expression or down regulation transcription factors (TF) 3) Downregulate or block competing metabolic reactions 4) Engineer an enzyme which is not found in nature (mutant/directed evolution) 5) Engineer (novel minimal pathway) in heterologous host

Question 30

Knowledge and technological/skill requirement

Answer 30

1. Knowledge of biosynthetic pathway(s) of the chemical to be produced 2. Genes encoding the related enzymes 3. Metabolic flux analysis, 'omics' & modelling 4. Compartmentalisation 5. Protein organisation & interactors 6. Regulation of such genes & enzymes [Regulation (TF); Feedback; Signalling; post translational epigenomic] 7. Gene editing in vivo / in vitro to alter properties of the encoded enzyme 8. Ability to transfer and express or suppress the required genes in (host) organism 9. Assemble an array of genes for their expression inside the host cells

Question 31

Overview of different levels of metabolic engineering

Answer 31

Levels of engineering in diagram (left): 1- introduce new biosynthetic genes 2- introduce inducing transcription factors (TF) 3- enhance/change the flux of substrates, energy and reducing power Example: multilevel engineering of Genistein in tomato fruit Genistein = natural compound, isoflavone – angiogenesis inhibitpr and phytoestrogen Levels (diagram centre): introduction of new biosynthetic gene, LjIFS, TF AtMYB12 activate genes of phenylpropanoid metabolism increase supply of substrates via activation of ENO and DAHPS by AtMYB12, & increase energy & reducing power via increased flux through glycolysis, the TCA cycle, & the oxidative pentose phosphate pathway. Additionally: use fruit-specific E8 promoter for expression of LjIFS and AtMYB12 & f3h mutant introduced to block the competing synthesis of flavonols

Question 32

Tomato engineering for sweetness

Answer 32

Crop improvement is essential to produce nutritious plants & feed a growing global population Improvement strategies hindered by metabolic and genetic trade-offs Nutritional / Morphological & Organoleptic / Technofunctional adaptation e.g.: Size: ❖ Wild relatives produce tiny, cherry-sized fruit ❖ Breeders have prioritized fruit size (10-100 x larger) Sweetness ❖ Some tastier varieties (sweet tomoatoes) ❖ Tomato fruit quality component = soluble solids content (SSC) - Sugars, mainly fructose and glucose - ~55% to 65% of the total SSC fraction - 50% of the total solids in the tomato fruit

Question 33

Releasing a sugar brake generates sweeter tomato without yield penalty

Answer 33

Genome-wide association study (GWAS) 1. associated loci for tomato sugar accumulation Metabolomics: *To identify differences in SSC between the two *gas chromatography with mass spectrometry (MS) *liquid chromatography with MS/MS *Wild type compared to domesticated cultivars. Genomic regions - Sugar accumulation: 2. Solyc11g065660, encoding SICDPK27 negatively correlated expression pattern with SSC accumulation *High SSC accumulation /Sweetness *Low S/CDPK27 expression *SICDPK27 ; a 'sink gene' - regulates ability of the fruit to store sugars not affecting the fruit weight 3.SICDPK27 expression increases during fruit ripening 4 Genome editing of SICDPK27 & SICDPK26 -> create knock out mutants CRISPR–Cas9. Three null mutants MM-CDPK27-CR1, MM-CDPK27-CR2 & M82-CDPK27-CR3 5. How does SlCDPK27 regulate sugar accumulation Identify interacting protein (immunoprecipitation/MS) - SlSUS3 -> Sucrose synthase (SUS) see next slide for more info on SISUS3 Outcome : - Increased glucose and fructose contents < 30% - NO fruit weight or yield penalty

Question 34

Overview: an approach to engineer high-yield tomatoes that are super sweet

Answer 34

Domesticated (less sweet) SlSUS3 converts sucrose into glucose & fructose (‘sweetness’) SlCDPK27/SlCDPK26 phosphorylate SlSUS3 -> Inhibit SlSUS3 Not (too) sweet ^these inactivate SISUS3 Mutant (Sweet) no phosphorylation MM SlCDPK27 (knockout) SlSUS3 Active -> SlSUS3 Sweet

Question 35

Plant metabolic engineering for nutraceuticals (adjusting accumulation of beneficial SMs)

Answer 35

Nutraceutical = Food component or compound with clinical, health or therapeutic benefits, including prevention and treatment of disease Antioxidant: protection against certain cancers, cardiovascular disease, age-related degenerative diseases. Anti-inflammatory activity, promote visual acuity, & hinder obesity and diabetes. Bioactive pigments: Anthocyanins: Among polyphenols, anthocyanins are a class of water-soluble flavanoids present in fruits, foliage, fruit and veg and flowers (>6000 flavanoids have been identified) Dietary sources of anthocyanins include red and purple berries, grapes, apples, plums, cabbage, or foods containing high levels of natural colorants Cyanidin, delphinidin, malvidin, peonidin, petunidin, and pelargonidin are 6 common anthocyanidins Anthocyanin levels in most commonly eaten fruits and vegetables may be inadequate to confer optimal benefits Ways to increase anthocyanin (or any SM) production: Metabolic engineering pathway in native OR heterologous plant crop Metabolic engineering in microorganisms Biofortification through plant metabolic engineering

Question 36

Metabolic engineering of tomato fruit to increase anthocyanin content by expression of transcription factors

Answer 36

Two Anthocyanin gene transcription regulators in snapdragon Antirrhinum majus Delila (Del); a basic helix-loop-helix TF Rosea1 (Ros1): MYB-related TF Express in fruit of transgenic tomatoes (Solanum lycopersicum cv. MicroTom)) Plants accumulate anthocyanins at high concentrations so that their fruits (tomatoes) have a level of anthocyanin comparable to those found in blackberries and blueberries Pilot test: cancer-susceptible Trp53-/- mice fed a diet supplemented with high-anthocyanin tomatoes showed a significant extension of life span

Question 37

Plant lipid metabolic engineering

Answer 37

Lipids = water-insoluble, esters of fatty acids and glycerols Essential biological functions: *Sphingolipids-lipid bilayer *Interface cell/tissue & atmosphere *Cuticle protects from water loss *Signalling apoptosis/ pollen *Plant-microbe interactions *Response biotic and abiotic stress *Germination Different crops have different oil composition either because they have been farmed for that trait or due to local social and climactic reasons Domesticated Oilseed Crops used for: *Food, Feed, Forage *Human & Animal Health *Fuel *Industrial Applications Metabolic Engineering for higher lipid content: *Nutritional applications: Improving / modifying Composition / Yield *Spare marine life by reducing nutritional dependence on fish oil for nutritionally & medically important longer polyunsaturated fatty acids [PUFA] Renewable resource for industrial applications: *Unusual fatty acids in storage oils *chemical properties industrial processes *substitute for petrochemicals

Question 38

Unusual plant fatty acids are highly valuable

Answer 38

>300 'unusual' FA Storage lipids: Tri Acyl Glycerol (TAG) Platforms: oilseed, plant vegetative tissue and algae Approaches: *Metabolic pathway econstruction -> Nutraceuticals, EPA, DHA & c-7 monounsaturated FAs *Introduction of foreign genes and silencing of endogenous competing pathways -> Industrial oils, Hydroxy FAs, Epoxy FAs, o- t-dacids, 1-7 monounsaturated FAs, Sulfonated FAs, Acetyl TAGs and Wax esters Genetic engineering: Pathway introduction, Silencing -> Biofuel, TAGS, Acetyl TAGs. Alkanes, Wax esters

Question 39

Organisation of TAG biosynthesis through TAG remodelling

Answer 39

Metabolic flux: rate of molecular turnover through a metabolic pathway Metabolic engineering: Determining factors for storage oil TAG: *Fatty acid biosynthesis *FA selectivity of acyltransferases *Acyl flux (or delivery) *Assembly enzymes Which of the 9 genes are responsible for creating storage lipids? How many pathways involved/ which are competing? Post-translational processes and unexpected metabolic processes ^Silent constraints: the hidden challenges faced in plant metabolic engineering

Answer 40

Hydroxylated FAs – Ricinoleic acid – castor oil for making nylon 11/ polyamide 11 Castor bean is the only commercial source of ricinoleic acid Ricinoleic acid is a petrochemical replacement in a variety of industrial processes Low temperature viscosity properties High temperature lubrication, Hydraulic fluid pipes in engines Transportation, Cosmetics, Pharmaceutical & Manufacturing Highly solvent-resistant polymer; Nylon-11 [N11] 300 000 tonnes of castor bean harvested per year >100 different chemical processes demand growing fluctuations in the supply, price and quality of the oil Not agronomic: Castor bean is not suited to large-scale agricultural production *Seeds must be harvested by hand *Economic and political instabilities *Contains ricin, a highly poisonous protein *Allergens, health problems Oilseed metabolic engineering: *Agronomically suitable plant crops: Temperate crops; Brassica, Camelina, Arabidopsis *Microorganism as model or test system (Oleaginous yeast or Algae)

Question 41

Plant metabolic engineering for hydroxylated FA (HFA) - ricinoleic acid – A multigene approach:

Answer 41

Check recording for 3 step process Challenges: Which Pathway is important? Multigene gene families (e.g. one enzyme reaction could be encoded by 9 genes) Which Isoform is the right one? Differences in plant species ? Precise gene function elusive Competing reactions & Acyl Flux Heterologous plants often not as good as native plants for producing SM see: https://www.youtube.com/watch?v=tFW8gPLyL9I

Question 42

Systems metabolic engineering for microbial cell factories: The Ultimate goal of Metabolic Engineering

Answer 42

Produce valuable substances on an industrial scale in a cost-effective manner Rationale for Metabolic Engineering ^ Find ways that are renewable ^ Overcome toxic/unfavorable substrates/intermediates ^ Overcome inhibition of target product biosynthesis ^ Streamline metabolic pathways ^ Efficiently utilize feedstocks Why Metabolic Engineering and microorganisms: *Climate change *Population growth/limited resources (fossil fuels) *Easy to grow and engineer *Renewable / Fast ^ Traditional chemical synthesis & PLANT Metabolic Engineering is technically more challenging Type of Microorganisms, Knowledge & tools *Host selection (Workhorses & Specialized Hosts): fundamental for microbial synthesis, *key determinant of pathway performance *access to a complete genome sequence *availability of genetic tools (CRISPR/HomRec)

Question 43

Drivers of systems metabolic engineering

Answer 43

Advances in synthetic biology: synthesis of natural and non-natural products Enabling construction: new artificial metabolic pathways/modulation of existing ones Advancing bioinformatics tools Expanding omics databases: libraries of candidate gap-filling enzymes compiled by querying (plant) transcriptome databases*, Resolution of enzyme gaps resolve single/multienzyme gaps in plant metabolic pathways and in heterologous microbial host pathways Precursor availability (e.g. Shikimate) sidestepping the need to rewire core metabolism DNA synthesis: no physical plant material or cDNA, genes are codon-optimised and synthesises, expressed in microbe see figure in notes: synthetic biology strategies for microbial biosynthesis of plant natural products

Question 44

Microbial cell factories case example:

Answer 44

Sustainable production of ‘Plant Natural Products’ (PNP) & Other Chemicals Establishing a coumarin production platform by protein and metabolic engineering Coumarins (1,2-benzopyrones) *Structurally diverse family of natural products *Diverse & unique biological & optical properties Industries *Agrochemical *Pharmaceuticals *Food *Cosmetic *Photochemical Environmental stress *Toxic raw materials / concentrated acids High production costs *LOW CONCENTRATIONS IN PLANTA - No crop-based manufacturing *Structural / stereochemical COMPLEXITY - Complex or no chemical synthesis Synthetic biology *Interfacing plant secondary pathways with core host metabolism *Synthesis in tractable laboratory microbes Biosynthesis *Sustainable and economical production of coumarins

Question 45

Biosynthetic pathways of simple coumarins in plants, fungi and microorganisms

Answer 45

shikimate pathway (aromatic amino acids) spontaneous process (ex. umbelliferone) 2GT, 2-coumarate O-β-glucosyltransferase 2OG, 2-oxoglutarate 4CL, 4-coumarate CoA ligase 4HPA3H, 4-hydroxyphenylacetic acid 3-hydroxylase BGLU, β-glucosidase C2′H, cinnamate 2-hydroxylase C3′H: p-coumaroylshikimate/quinate 3-hydroxylase C4H, cinnamate 4-hydroxylase CCoAOMT, caffeoyl-CoA O-methyltransferase CSE, caffeoyl shikimate esterase F6′H, feruloyl coenzyme A 6′-hydroxylase HCT, hydroxycinnamoyl transferase PAL, phenylalanine ammonia-lyase S8H, scopoletin 8-hydroxylase TAL, tyrosine ammonia-lyase. Coumarin: Flavoring, Fragrance, Anti-inflammatory, Photochemotherapy, Chemical structure of coumarins & their pharmacological activities: Umbelliferone: Absorbs UV light, Fluorescent (sunscreens), Pharmacological Antibacterial and antifungal, Anti-biofilm formation Esculetin: Antioxidant, Anti-inflammatory, Antiviral, Antibacterial, Antifungal Scopoletin: medicinal and edible plants, therapeutic and chemopreventive agent Fraxetin: Anti-Cancer /-Diabetes/Anti-Inflammation, Neuroprotection, Antibacterial, Antioxidant, Liver /Heart/Bone protection E. coli platform for de novo biosynthesis

Question 46

Establishing an E. coli platform for de novo biosynthesis of coumarins

Answer 46

➢ Combining protein and metabolic engineering PPP, Pentose phosphate pathway E4P, erythrose 4-phosphate; PEP, phosphoenolpyruvate FEC, feruloyl-CoA; CAC, caffeoyl-CoA;COC, coumaroyl-CoA DAHP, 3-deoxy-D-arabino-heptulosonic acid 7-phosphate See: Lin et al., 2013b & Xie et al. Met. Engin.2024 Pathway construction in E.coli (& yeast) *Low coumarin yield *Poor activity and stability of CCHs Cinnamyl-CoA ortho-hydroxylases (CCHs) *Gateway and Rate-limiting step *AtF6'H = CCH Protein Engineering CCHs *Primary target is catalytic pocket * Catalytic activity and substrate specificity

Question 47

Enzyme/ organism of origin list

Answer 47

RgTAL, tyrosine ammonla lyase - Rhodotorula glutinis (an aerobic pink yeast) KphpaBC,4-hydroxyphenylacrtate-3-hydroxylase - Klebsiella pneumoniae (intestinal bacteria) Fre, NAD(P)H-flavin reductase - E. coli AtCOMT, caffeate 3-O-methyltransferase - Arabidopsis thaliana At4CL, 4-coumarate:CoA ligase - Arabidopsis thaliana AtF6'H. feruloyl-CoA 6'-hydroxylase - Arabidopsis thaliana IbF6'H, coumaroyl-CoA 2'-hydroxylase - Ipomoea batatas (sweet potato) AtCOSY, coumarin synthase - Arabidopsis thaliana AtS8H, scopoletin 8-hydroxylase - Arabidopsis thaliana UGT72E1, fraxetin 8-O-glucosyltransferase - Arabidopsis thaliana TOGT, scopoletin 7-0-glucosyltransferase - Nicotiana tabacum UGT92G7, esculetin 6-O-glucosyltransferase - Aesculus chinensis (chinese horse chestnut)

Question 48

Protein Engineering: Directed Evolution of AtF6′H

Answer 48

Directed evolution, Semi-rational design, Rational Design ^ strategies for protein engineering Gene/Protein of interest = AtF6′H Arabidopsis Cinnamyl-CoA ortho-hydroxylase (CCH) Directed evolution and structure-guided engineering - Improved activity/increased catalytic efficiency Fluorescence-based screening method - Feruloyl-CoA (FEC) as the substrate Transplantable surface mutations Substrate-specific pocket mutations Improve the performance of AtF6′H(a CCH)

Question 49

Surface mutations improve enzyme performance of AtF6′H

Answer 49

A: Fluorescence screen variants B: Positive changes random mutagenesis C: Relative activity of AtF6′H-WT IbF6′H-WT and variants towards FEC D: Relative activity CAC E: Sequence D5, AtF6′H and IbF6′H F: Relative activity COC G: Stability analysis of AtF6′H-WT and D5, ^ improved titers by 5 to 22- fold Introduction glycosylation modules ➢ 4 coumarin glucosides o titer of aesculin increased 15.7-fold o 3 g/L in scale-up fermentation

Question 50

World grappling with challenges in relation to crop production: benefits and disadvantages of monocultures

Answer 50

- a growing population, climate change, diminishing natural resources etc. -adjustments and solutions necessary Monoculture - cultivation of a single crop variety over large areas -common agricultural practice that can lead to genetic erosion Genetic erosion -gradual loss of genetic diversity within a population or species Loss of bio and genetic diversity per area: intensive agriculture, environmental deterioration Monoculture advantages: * Increased Productivity And Efficiency * New Technologies - Crop Monitoring * Specialized Production * Yields Maximization * Easier To Manage * Higher Revenue Monoculture disadvantages: * Vulnerability to pests Pest management (Higher pesticide use) * Vulnerability to diseases & Environmental changes * Entire population susceptible to particular threat (Economic risk) * Soil degradation & Fertility loss (Higher fertiliser use) * Higher water use * Environmental impact (Exploitative)

Question 51

Crop future strategies -> sustainably maintain and expand yield and trait variability

Answer 51

Three main technological revolutions in agriculture: 1. Agricultural machinery revolution 2. GMO and green revolution 3. Digital agricultural revolution Sustainable bioeconomy Increase product diversity, resilience & sustainability & Production of chemicals, drugs & biologics Biofortified Crops, Climate-Resilient Crops, Pest and Disease Resistance Nutritional Enhancement, Enhanced yield, Less or no input chemicals/fertiliser

Question 52

Uniting Breeding Technologies & Industries for a Sustainable Agricultural Revolution

Answer 52

Doesn't have to be bioengineering Farming & combined breeding practices: *Diversified Cropping Systems (Inter cropping) *In Situ Conservation -natural habitats or within traditional farming system -protect local varieties /unique traits adapted to specific environments *Ex Situ Conservation -collection and preservation of seeds, plant tissues, or whole plants outside their natural habitat *Participatory Plant Breeding -involves collaboration between farmers, researchers, & breeders in the selection and development of new crop varieties Technological innovations & Research: *Harnessing Biotechnological Tools for Speed Breeding *Non-biotechnological approach complementing biotechnological tools *Significant shortening time to produce new varieties *Manipulating environmental conditions such as light, temperature, and day length *Accelerate plant's growth cycle & shorten generation times Explore & understand metabolic diversity-Profiling technologies *Research Epigenetic Influence Gene Expression *DNA methylation, histone modification, and small RNA molecules Synthetic, Metabolic, Engineering biology, Bio-based production *Metabolically engineer new capabilities in planta ('traits', diversity) *Engineer plant pathways or use plant genes in microbial systems Developing overlooked and new crops *Plant Molecular F/Pharming *De novo domestication alternative crops *Nitrogen fixation by crop - Less fertiliser input

Question 53

De novo domestication; an alternative route towards new crops for the future

Answer 53

'Domestication Syndrome' ^ Changes to plant architecture and morphology demanded by agronomic practices: *Height *Branching *Growth habit *Increased yield *Greater seed size *Easier harvesting discovery of multiple key domestication genes and development of technologies for manipulation target genes simultaneously

Question 54

Genome editing key genes for De novo domestication & agronomic traits case studies

Answer 54

Case 1: Wild allotetraploid rice can be rapidly domesticated into new-type rice cultivars that have strong environmental adaptability and high grain yield Case 2: Maize Domestication Genes and Phenotypes *Four major genes are essential for the transition from teosinte to maize morphology *tb1 (teosinte branched1) regulates tga1 (teosinte glume architecture) *tru1 (tassels replace upper ears1) control glume softness and aerial branch fates activates gt1 (grassy tillers) ^ to repress vegetative tillering and prolificacy - suppression of axillary branches and prolificacy - promotion of lateral branch sex identity

Question 55

Solutions for global food, resource and energy security: Plant Molecular F/Pharming

Answer 55

The process of molecular farming: 1. Plant modification *Introduce target protein to plant with instructions to replicate (example: whey or casein) 2. Grow modified plants *Traditional growth in a field, greenhouse, or vertical farm 3. Downstream processing * Recovery and purification of target ingredient Applications of molecular farming: *Production of protein & valuable compounds in transgenic plants & (green) algae on an agricultural scale * Organs, tissues, cells, cell cultures * Green bio reactors, sustainable chemical factories for proteins, antibodies, vaccines, and other therapeutics. (+ usage in non food, feed, fiber)

Question 56

Definitions molecular farming vs pharming

Answer 56

Molecular Farming: Using GMO's as production platform for renewable raw materials, fine chemicals and dietary supplements Molecular Pharming: Using GMO's as production platform for active pharmaceutical substances such as valuable diagnostic proteins, vaccines & antibodies

Question 57

why plant molecular F/Pharming

Answer 57

Scalable, cost effective and sustainable: *Green biotech <-> process economics *Emerging subdivision in biopharmaceutical industry Protein integrity & functionality *Higher plants generally synthesize proteins from eukaryotes with correct folding, glycosylation & activity Lower production costs of biomass *Compared to transgenic animals, ‘precision’ fermentation or bioreactors (microbes) Infrastructure & expertise *Exists for planting, harvesting & processing of plant material Scalability - Plants can be grown in open fields, avoiding scaling challenges of large bioreactors Speed -Transient expression systems can produce proteins in a few days Safety -Plants do not contain known human pathogens (prions, virions, etc) Sustainability -Plants are biodegradable and single-use bioreactors Flexibility - Plant molecular farming can be used to produce a wide range of products Production platform for: *Pharmaceutical products Antibodies, therapeutics, and vaccines *Industrial enzymes Variety of industrial processes *Biofuels Help to reduce carbon dioxide emissions *Alternative proteins Improve nutrition of plants and animals

Question 58

Timeline of the development of plant molecular farming

Answer 58

A few examples of what has been produced 'Plantibodies' Antigens from different pathogens expressed in plants: - rabies virus, hepatitis B, rotavirus, -HIV, and other pathogens Monoclonal antibodies -Immunoglobulin G (IgG) and Immunoglobulin A (IgA), -IgA and IgG shimmer molecules, IgG and IgA secreted molecules -single-Chain variable fragment, fragment antigen-binding -second variable of heavy and light chains Pharmaceutical proteins -erythropoietin, interferon, hirudin, aprotinin, insuline -Leu-enkephalin, somatotropin of human growth hormone Non-pharmaceutical proteins or industrial proteins - avidin, trypsin, aprotinin, ß-glucocerebrosidase, peroxidase and cellulose, etc. - hemicellulase, xylanase, and particularly ligninase (for biofuel industry respecting cellulosic ethanol) Plant-derived anticancer agents ^ A green anticancer approach

Question 59

Case example: Comparison of microbial and transient expression (tobacco plants and plant‐cell packs) for the production and purification of the anticancer mistletoe lectin viscumin

Answer 59

Carbohydrate-binding lectins Cytotoxic protein (ribosome inactivating protein) that binds to galactose residues of cell surface glycoproteins and may be internalised by endocytosis Strongly inhibits protein synthesis by inactivating the 60 S ribosomal subunit Mistletoe, the natural source of viscumin Up to 20 years to grow in to a bush with a diameter of 1 m An obligate parasite, so needs host plants -Increases the cultivation effort -Limits the cultivation density -Automation potential, containment, & product flexibility Viscumin is a heterodimer synthesized as a single polypeptide precursor Activated by proteolytically removing a central amino acid linker sequence The active form of the protein comprises an A chain rich in α-helices with N-glycosidase activity & a glycosylated B chain mostly composed of β-sheets, which binds to carbohydrates Viscumin cannot be produced in mammalian cells, due to its toxicity Nonglycosylated recombinant viscumin from Escherichia coli forms inclusion bodies resolubilization and refolding of proteins laborious and inefficient Can we achieve higher yield & purification of potent viscumin effectively in heterologous plant source? 1. Tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) plant-cell packs (PCPs) 2. Intact Nicotiana benthamiana plants

Question 60

Viscumin is purified to 0.54 g/g TSP in a single lactosyl-Sepharose affinity chromatography step

Answer 60

Observations & Conclusions Isolation of viscumin from a heterologous source achieves higher yields than native mistletoe Plant-derived recombinant viscumin is glycosylated - threefold more toxic than viscumin purified from E. coli beneficial in terms of production process design The expression of viscumin in plants has potential to reduce production costs by ~80% Native Mistletoe (Viscum album) compared to Heterologous plant expression: *Reduced yield time by several years *Increased containment *Increased space-time yield *Facilitated targeted product modification

Question 61

Summary

Answer 61

Domestication, intensive agriculture and other human activity has led to diminished genetic and biodiversity Bio & phytochemical diversity needs to be protected and 'even' expanded Immense phytochemicals diversity with enormous utility and only a small fraction has been explored Dietary adjustments, uniting breeding technologies and industries for a sustainable agricultural revolution Technological advancements and knowledge of biochemistry, metabolism, genetics and physiology is critical for effective breeding and engineering biology