Yeast strain and enzyme engineering

Question 1

Describe the ‘renaissance of yeasts as microbial factories’.

Answer 1

• Genetic engineering to produce microorganisms with industrial applications

Question 2

Describe yeast-based biomanufacturing. [3]

Answer 2

1. Yeast strain is engineered using genes derived from any organism.
2. A process is optimized where a feedstock (e.g., sugar) is used to grow yeast in a bioreactor.
3. The engineered yeast produces the protein or molecule of interest.

Question 3

Give examples of animal feed enzymes produced by DuPont.

Answer 3

• Danisco Xylanase targets arabinoxylans in dietary fiber to mitigate their negative effects and generate additional energy. It helps to release valuable nutrients, reduce digesta viscosity, and improve litter quality as well as faecal consistency.
• Porzyme tp100 for starter pigs - an enzyme blend that optimizes digestibility in diets based on corn, broken rice, wheat, as well as sorghum and grain by-products
• Porzyme 9300 for grower-finisher pigs - a single activity enzyme that maximizes performance from grower-finisher pigs based on corn, broken rice, sorghum, wheat, rye, triticale, and grain by-products. Multiple trials show that it can improve bodyweight gain by 6.7% and feed conversion rate by 6%. While application in corn-soy diets for lactating sows increases weight gain per piglet by 14% compared to control.

Question 4

Describe how the Impossible Burger is made.

It ‘bleeds’ although it is completely vegan.

Answer 4

• In animals, the hemoglobin molecule contains four globin proteins bound to a pigment molecule called heme, which contains an ion of iron.
• The heme protein leghemoglobin (legHB) in soybean, provides oxygen to its symbiotic bacteria colonized in its roots.

Question 5

What is synthetic biology?

Answer 5

• A field of science that involves redesigning organisms for useful purposes by engineering them to have new abilities.
• Leveraging the power of DNA/RNA sequencing and cheaper DNA synthesis to create novel biological processes or optimize existing ones.

Question 6

What is the core of synthetic biology?

Answer 6

• The DBTL cycle
• Yeast factories to produce a desired product are engineered following the design, build, test, and learn principles of bioengineering. Synthetic DNA encoding enzymes needed for production are introduced into yeasts. The yeast strains consume the feedstock and the product is then “grown” by fermentation

Question 7

Describe the synthetic biology toolkit.

Answer 7

• DNA sequencing/synthesis
• CRISPR-Cas9 mediated genome engineering

Question 8

Describe enzymes & active sites.

Answer 8

Question 9

What is amylase?

Answer 9

• An enzyme that catalyzes the hydrolysis of starch into sugars
• alpha- and beta-amylases are important in brewing beer and liquor made from sugars which are derived from starch.
• The sugars are then consumed by yeasts and produce alcohol

Alpha- and beta-amylases have different preferred temperatures for optimal activity.

Question 10

Give an overview of brewing beer.

Answer 10

Question 11

Describe differences between alpha- and beta-amylase. [2]

Answer 11

• In barley malt they hydrolyze starch in different ways.
• They also have different preferred temperatures for optimal activity, beta-amylase prefers a lower temperature than alpha-amylase.

Note the 'brewer's window' at ~67 degrees C.

Question 12

So what if we wanted to have an amylase enzyme that works best at higher temperatures like 100 degrees C?

Answer 12

• Looking at natural diversity is just one approach
• Using the full power of synthetic biology you can develop enzymes with any mutation in any position in any combination
• Screening these enzymes for functioning in your conditions (e.g., elevated temperatures) will enable you to identify the variants you want!

Question 13

Describe a case study for temperature stable enzyme engineering.

Answer 13

• Carbonic anhydrase is one of the fastest enzymes known. The active site can turnover carbon dioxide and water to bicarbonate and a proton up to a million times per second, and are used by almost every living organism to maintain pH balance and transport carbon dioxide.
• Technical hurdle: Evolve a CA enzyme that functions at temperatures of up to 107 C for extended periods of time (e.g. weeks) and in a 4.2 M Nmethyldiethanolamine solvent (MDEA) @pH 10.

Question 14

What is saturation mutagenesis?

Answer 14

• A single codon or set of codons is substituted with all possible amino acids at a position.
• Can make these libraries yourself with PCR or send out to synthetic DNA companies

Diversity generation!

Question 15

What is compounded fold improvement?

Answer 15

• After each round of evolution, the relative fold improvement for each round is shown in red, whereas the temperature at which half-life was determined is shown in black above each bar.

Question 16

Describe how with a relatively small number of changes you can make a BIG difference in enzyme function.

Answer 16

• Mutations accumulated in nine rounds of evolution of DvCA are shown in orange with the corresponding residues of the wild-type enzyme shown above

Question 17

How can we make beer brewing more efficient?

Answer 17

• Making engineered alpha- and beta-amylases.