Biotech manufacturing products
acronym BRAVABS
Biopolymers (eg Xanthan gum)
REs
AAs (Cyst, Val)
vitamins (eg ascorbic acid Vit C)
Antibiotics
Bioplastics (eg poly-B-hydroxybutyrate)
Small molecs (eg lycopene)
biotech manufacturing - Biopolymers
eg Xanthan gum
modify Xanthanomas camestris w/Lac Z and Lac Y genes –> can metabolize lactose or Whey into xanthan
Why? because whey is a really abundant/cheap waste product from cheese industry
biotech manufacturing - REs
- isolated from Ecoli periplasm (space between outter and inner membrane) –> prevent foreign DNA from entering cytoplasm
- DdeI RE found in same loci as methylase –> recall methylation of DNA prevents self-cleavage
- place loci on plasmid w/DdeI cut site
- if loci is inserted –> methylase protects the plasmid –> Ecoli will overexpress DdeI from extra copy
- if loci fails to insert –> wt DdeI will cleave plasmid therefore degraded
biotech manufacturing - Amino acids
- Serine to cysteine –> controled by negative feedback loop + exces Cyst is degraded. Therefore do mutagenic KO of feedback loop + KO of degradation –> therefore allow mass Cst accumualtion
- Glucose to valine –> feed backloop control + side rxns w/intermediates (therefore less valine yield) –> KO of side rxns + KO of control regulation + upregulate gluc-val enzymes
basically: KO regulation pathways, KO intermediate side rxns, KO product degradation pathways, upregulate enzymes
biotech manufacturing - vitamins (Reminder: what modifications were made to the transgene?)
eg ascorbic acid (vit C)
glucose –> 2,5-DKG –> 2-KLG –> vit C
Erwinia bacteria unable to form 2-KLG –> provide transgenic 2,5-DKG reductase isolated from corynebacterium
- reductase modified w/ Glu192 Arg mutation –> 1.8x stronger activity + 0.75x Km coefficient
- reductase active site gene shuffling –> further 75x stronger activity
biotech manufacturing - Antibiotics
- Most Abs isolated from Streptococcus
- undecyle prodigiosin - unique reg coloured antibiotic –> easily identified in certain colonies
- eg polyketide antibiotics
Poly ketide antibiotics
- eg Erythromycin
- Enzymatic activity comes from 1 massive peptide w/many sites or many peptides working together
- Features core polyketide synthase backbone –> everything else can be gene shuffled to create new antibiotics
S.aureus and cholesterol
SA produces staphyloxanthin using same precursor as human cholesterol
Staphyloxanthin used for ROS detox to protect SA
therefore inhibit precursor synthesis in humans –> SA susceptible to ROS
PT can’t produce cholesterol –> can survive by consuming it in foods
biotech manufacturing - bioplastics - What organism was modified + what additional transgenes required?
- Synthesis of bioplastic by Alcaligenes eutrophs –> slow growing therefore low yield –> need alternative
- transform various biosynth pathways to Ecoli –> combined transgenic ability to synth bioplastics
- stabilize plasmid using Par B gene so it is retained in daughter cells
- provide transgenic fadR + atoC egnes –> ensures enough precursor is made to ensure constant bioplastic synthesis without interruption
Increasing Ethanol production - core principles
increased EtOH tolerance
in vivo starch degradation using transgenic amylase + glucoamylase
stronger glucoamylase affinity to allow fermentation of insoluble starch
increasing ethanol production - details
- Recombination of yeast strains + mutagenize SPT15 TF to enhance [EtOH] tolerance
- transgenic amylase + selection on KanR –> in vivo starch degradation to enhance glucose fermentation (Grow on iodine media –> reacts with starch –> clearing = transformants)
- transgenic glucoamylase –>in vivo starch breakdown –> integration to genome for transient expression (Modified glucoamylase w/excretion domain + stronger starch affinity –> breakdown insoluble starch)
Pseudomonas superbug for oil degradation
- many strains can degrade various oil xenobiotics –> recombine into a superbug to degrade all of them
- conjugation with pychrophiles to allow low T degradation
- Note: Xylene operon - Xylene S and E
Oil degradation - Xyl S
- Xylene S binds substrate to activate the operon –> modify Xyl S to also use/metabolize 4-ethylbenzoate (expanded ease of operon activation
- Selection using TetR controlled by mutant Xyl S –> grow on tet media w/4-ethylbenzoate –> transformants will use 4-ethylbenzoate to express TetR
Oil degradation - Xyl E
- related to xylene degrdation/metabolism –> can be inhibited by 4-ethylcatechol
- mutagenize Xyl E so it can’t interact with 4-ethylcatechol
- selection on 4-ethylbenzoate media in presence of 4-ethylcatechol –> transformants can still metabolize 4-EB in presence of 4-EC
Xenobiotics - Organophosphates - What gene encodes their degradation? how was it modified? effects of modification?
- Ecoli modified with transgenic OPD gene = organophosphate degradation
- OPD fused with OmpA transmembrane –> enzyme is embedded to CM
- EC is able to extracelll digest organophosphates –> 7x more activity than cytosolic digestion
Xenobiotics - Nitroaromatics - What enzyme was modified? what are the new nitroaromatic substrates?
- nitroaromatics = carcinogens
- Burkholderia bacteria carries 4-methyl-5-nitrocatechol monooxygenase
- unable to natively degrade 4-nitrophenol or 3-methyl-4-nitrophenol
- error prone PCR to modify 4-methyl-5-nitrocatechol MOase –> able to degrade the new substrates + enhanced degradation of original substrate range
xenobiotics - cellulosomes - what is it? what enzymes involved? how to detect these enzymes?
- cellulosomes = protein complexes on surface of microbes carrying cellulolytic domains
- cell deposits cellulosomes onto plant/fungi –> extracell digestion
- endoglucanse = initial degradation (detectable by CMC media + congo red stai –> forms yellow spot)
- exoglucanase = cellulo bi/triose degradation (mAb detection)
- beta glucosidase= final degradation to glucose
transfection by Agrobacterium
- carries Ti plasmid w/T-DNA –> encodes various hormones to induce plant synthesis of food
- T-DNA is flanked by left/right border sequences
- T-DNA excised and transfected using Vir proteins
Insert GOI into T-DNA + marker for delivery –> Random integration to host
BT pesticide toxin - how it works
- encoded by cry genes
- Rxn with enzymes in insect gut –> activation –> form gut leakage of ATP and nutrients = death
- no rxn in human gut –> harmless
recombination with various BT genes = stronger/new toxin
BT toxin - transfection method into plants + specific delivery within cell (why there?)
agrobacterium using 2 plasmids - 1 carrying GOI (strong P35S promoter) + NPT (KanR), 1 carrying Vir
plasmids have homology region –> recombine to make armed plasmid for actual transfection
Delivery to Chl, NOT nucleus
- chl protein mods similar to bacteria –> easier to make fxnal proteins
- maternally inherited genes (in egg) –> GOI is not HGT’d in pollen
cowpea trypsin inhibitor
trypsin allows insects to hydrolize plant proteins –> inhibition = death
transfect w/ P35S strong promoter + GOi + NPT (KanR) on 2 Ti plasmids –> recombine into fully armed plasmid
Misc pesticides
cholesterol oxidase - disrupt midgut membrane
VIP proteins - Gene shuffled to strongest variant (VIP3AcAa) –> transfected with BT to make evolved resistance harder
Cytochrom P450 - neutralizes Gossypol pesticides –> RNAi P450 to make susceptible to Gossypol
RNAi - How to impact insects but not the crop?
RNAi delivery to chlorophyll –> isolated from nucelus –> no inhibition of cytosolic RNA
injested by insect –> released from chl –> RNAi to insect
Plant protection from viruses - what methods?
transgenic coat proteins
CRISPR - rolling replication dsDNA can be cleaved by cas9
scFab/FV targetting - recall cancer targetted treatment –> same idea
