Synthetic Bio

What is synthetic biology?

Engineering new biological parts, pathways, or systems, or redesigning existing ones.

What is pathway engineering?

Introducing or optimising metabolic pathways using genes from one or more organisms.

Compare yeast and Zymomonas as ethanol fermenters.

Yeast = slower but high ethanol tolerance.

Zymomonas = faster but low ethanol tolerance.

Which enzymes convert xylose into glycolytic intermediates?

xylA (isomerase), xylB (kinase), tktA & tal (PPP enzymes).

Why engineer yeast with starch‑degrading enzymes?

To allow one organism to convert raw starch → glucose → ethanol. (i.e. to combine starch breakdown and fermentation in one organism)

What is HFCS?

High Fructose Corn Syrup

List the advantages and disadvantages of HFCS

Advantages - its sweeter, more cost-effective than granulated sugar, better stability, texture, colour and consistency

Disadvantages - Dangerous for health can cause weight gain, Type II diabetes, potentially cancer

What limits cellulose degradation, and how can it be improved?

Cellobiose inhibition by glucose; Improved by increasing β‑glucosidase expression.

What are 1st gen bio fuels

Food crops (edible feedstocks)

What are the advantages and limitations of 1st‑gen biofuels?

Advantages: simple conversion

Limitations: Food competition + high land/water use.

What are 2nd gen bio fuels and their advantages?

Lignocellulosic biomass (waste plant materials)

Advantage: avoids food conflict

Why are 2nd‑gen biofuels harder to process?

Lignin & hemicellulose require complex pretreatment.

What are 3rd gen bio-fuels and their limitations

Microalgae

Limitation: expensive harvesting, poor economics

What makes 3rd‑gen biofuels attractive?

Microalgae use sunlight & CO₂, grows on non-arable land and have high lipid content.

What are 4th gen biofuels and their advantages?

GM microalgae

Advantage: engineered for higher productivity & CO2 fixation

What is the major concern with 4th‑gen biofuels?

GMO release and ecological risk requiring strict regulation

What is biodiesel?

Generated from plant oils and animal fats

Biodiesel production: oils/fats + methanol + NaOH → "FAME (biodiesel)" + glycerol + water.

What does FAME and FAR stand for

FAME = Fatty Acid Methyl Esters

FAR = Fatty Acid Reductase

Engineering E. coli to produce hydrocarbons: what is the main goal of this synthetic biology system in E. coli?

To engineer E. coli to produce hydrocarbons and biodiesel by increasing free fatty acid production and converting fatty acids into branched hydrocarbons.

Engineering E. coli to produce hydrocarbons: What is the purpose of adding branched chains to fatty acids?

To produce branched hydrocarbons, which improve fuel properties (e.g., lower freezing point).

Engineering E. coli to produce hydrocarbons: What are the two major engineering strategies in this system?

1. Increase free fatty acid production

2. Convert fatty acids into branched hydrocarbons

Engineering E. coli to produce hydrocarbons: Why use genes from Bacillus subtilis?

They naturally produce branched-chain fatty acids, enabling branched hydrocarbon synthesis.

Engineering E. coli to produce hydrocarbons: Why is FAR important in the pathway?

FAR (fatty acid reductase) from Photorhabdus luminnescens reduces fatty acyl‑CoA to fatty aldehydes, a key intermediate for hydrocarbon formation.

Engineering E. coli to produce hydrocarbons: What is the final product of aldehyde decarbonylase?

Aldehyde decarbonylase from Nostoc punctiforme convert to hydrocarbons (alkanes/alkenes)

Why is sulfur in fossil fuels a problem?

Burning sulfur-containing compounds produces SO₂, which leads to acid rain.

What are the major organic sulfur compounds in fossil fuels?

Thiophene and Dibenzothiophene (DBT).

Which organism naturally removes sulfur from DBT? And what is this pathway called?

Rhodococcus; 4S pathway

What operon in Rhodococcus performs sulfur removal?

The dszABC operon.

Which enzymes are encoded in the dsz operon?

DszC, DszA, DszB, and DszD.

What is the final product of DBT desulfurization?

2‑Hydroxybiphenyl (sulfur-free).

What happens to the sulfur removed from DBT?

It is released as sulfite.

Why is Rhodococcus not ideal for industrial biorefining? And what organism is used instead?

1. It is poorly characterized.

2. It cannot grow to high density.

3. The dsz operon is repressed by organic and inorganic sulfur.

E.coli

How do engineers prevent sulfur repression of the dsz operon in E. coli?

By cloning dszABC under the tac promoter, which is IPTG-inducible.

What is the limiting step in the natural pathway?

FMN reduction by DszD.

How is the FMN reduction bottleneck solved in engineered E. coli?

Replace DszD with HpaC, an E. coli flavin reductase.

Why is HpaC better than DszD?

Provides faser FMNH2 supply, increasing pathway efficiency

Why are micro algae useful for biofuel production?

Naturally accumulate lipids

What type of organism is Chlorella?

A single‑celled, photosynthetic green alga without flagella (2-10 μm diameter).

Why is Chlorella useful for biomineralization?

It can precipitate metal ions (including lithium) into carbonate forms.

Why is lithium carbonate important?

It is a key material for lithium‑ion battery production.

What is the purpose of the lithium biomineralization tank system?

To extract leftover lithium from industrial waste and convert it into lithium carbonate.

What happens to the lithium after biomineralization?

It is collected as Li₂CO₃ and can be reused in battery manufacturing.

What organism produces ice‑nucleation proteins (INPs)?

Pseudomonas syringae

What are INPs? And what do they do?

Ice-Nucleation Proteins

Trigger ice formation by acting as templates for ice crystals.

How does removing ice‑forming bacteria help crops?

Allows water to supercool, reducing frost damage.

What gene encodes INP?

inaZ

What is the other use of InaZ (truncated INP)?

The ice nucleation protein, InaZ, has been used to display other proteins on the surface of bacteria such as E. coli

Applications of INP surface display?

Lignocellulose enzyme screening, virus capture, vaccine antigen display, biosensing.

How does INP anchor in E. coli?

N-terminus anchors to outer membrane; central repeat domains act as templates for ice crystal formations

Which fungi naturally produce β‑lactam antibiotics?

Penicillium species.

β‑Lactam Antibiotic Biosynthesis: How us industrial production improved and what does it enable?

Engineering P. chrysogenum to express both enzymes → direct 7‑ACA biosynthesis.

Diversification of β‑lactams, including carbapenems like thienamycin.

Complete Opium Biosynthesis by Yeast is significant because?

Shows that complex plant alkaloids can be fully synthesized microbially.

Complete Cannabinoids Biosynthesis by Yeast is important becuase?

Enables cannabinoid production without growing cannabis plants.

What is chondroitin sulfate used for?

Joint health, cartilage elasticity, osteoarthritis supplements.

What is the traditional source of chondroitin sulfate?

Cow trachea

What did engineered E. coli achieve and why is it important?

Full biosynthesis of chondroitin‑4‑sulfate from glucose.

Microbial production of complex human biomolecules without animals.

What is heparin and its traditional source? Why is it important?

A highly sulfated anticoagulant drug.

Pig intestine

Modern synthetic biology can now reconstruct highly complex human drug biosynthesis

What are PHAs? List some examples.

PHAs are biodegradable polyesters produced by bacteria.

E.g.:

- PHB = polyhydroxybutyrate (4C)

- PHV = polyhydroxyvalerate (5C)

- PHBV = copolymer of PHB + PHV (improves flexibility)

- PHH = p-hexanoate (6C)

- PHO = p-octanoate(8C)

What is Biopol? What are some limitations?

A commercial PHB/PHV bioplastic produced by Alcaligenes eutrophus

Fully biodegradable but expensive due to fermentation + purification (endotoxin issue).

What organism degrades PET?

deonella sakaiensis

What enzyme breaks PET?

PETase → produces MHET, BHET, TPA.

What enzyme breaks MHET?

MHETase → TPA + EG.

How was PETase improved?

Machine learning + structure‑guided engineering.

What properties of PETase were enhanced?

Thermostability

Active‑site geometry

Activity at ambient temperature

pH tolerance

How is engineered PETase used industrially? And what are the key challanges

Enzymatically depolymerizes PET → TPA + EG → repolymerized into virgin PET.

Scaling enzyme production, reactor design, biosafety

Why are PE & PP hard to degrade?

Hydrophobic + C‑C backbone.

What organisms degrade PS?

Mealworms, superworms, Exiguobacterium.

What degrades PU? And what is the limitation

Some Pseudomonas species.

Limitation: Slow, partial degradation — not scalable.

What role do insects play in plastic degradation? Who does the chemical depolymerisation? Limitations

Mechanical shredding + gut conditions

Gut microbes

Limitations: Slow rates, partial mineralisation

xDNA vs XNA

xDNA: DNA with expanded bases (xA, xT, xG, xC) — wider helix, fluorescent.

XNA: Nucleic acids with modified sugar backbones (not DNA).

Key properties of XNA?

Binds DNA/RNA

Resistant to nucleases

Requires engineered polymerases

Why is XNA important?

Enables synthetic genetics, new therapeutics, and orthogonal life systems

What is the 6-letter Genetic Alphabet?

A system using A,T,G,C + synthetic bases X and Y

What synthetic base pair expands the genetic alphabet?

dNaM-d5SICS, which pair via hydrophobic packing, not hydrogen bonds.

How many codons exist in the natural genetic code? How many codons exist with a six‑letter alphabet?

64 codons (4³).

216 codons (6³).

What does an expanded genetic alphabet enable?

Incorporation of unnatural amino acids into proteins.

What is a Semi‑Synthetic Organism (SSO)?

A cell that can replicate DNA containing X-Y bases, transcribe them into RNA, and translate proteins containing unnatural amino acids.

What is Hachimoji DNA/RNA? and why is it significant?

An eight‑letter genetic system with 4 orthogonal base pairs.

It forms stable, predictable duplexes and expands the chemical space of genetic information.

What is the natural "5th base" found in some viruses?

2‑aminoadenine (Z).

How does Z-T pairing differ from A-T? How do Z‑phages ensure Z incorporation?

Z-T forms 3 hydrogen bonds, making it more stable.

Their polymerase selectively incorporates Z and excludes A.