Gentic Manipulation Of Fungi

Question 1

Why are fungi genetically manipulated?

Answer 1

To enhance their production of metabolites

Question 2

What has science innovations allowed us to do with fungi?

Answer 2

Genomic alterations have allowed increased yields of useful metabolites and create novel biosubstances

Question 3

3 techniques of fungal genetic manipulation

Answer 3

Gene editing, transformation and metabolic engineering.

Question 4

2 methods of genetic transformation

Answer 4

Protoplast transformation and agrobacterium mediated transformation (AMT)

Question 5

Steps of protoplast transformation

Answer 5

1. Enzymatically remove cell walls
2. Insert foreign DNA with PEG and calcium chloride.
3. Cell walls are regenerated, new genes expressed.

Question 6

Enzymes involved in protoplast transformation

Answer 6

Chitinase and glucanase

Question 7

What chemicals are needed in protoplast transformation?

Answer 7

Polyethylene glycol (PEG) and calcium chloride. They change the plasma membrane to allow DNA uptake.

Question 8

Example of protoplast transformation

Answer 8

Aspergillus niger has been enhanced to increase yields of glucoamylase and protease enzymes.

Question 9

Steps on agrobacterium mediated transformation

Answer 9

1. Bacteria agrobacterium tumafaciens engineered to carry target gene.
2. This bacteria naturally attacks filamentous fungi.
3. Target gene integrates into fungal genome and is replicated.

Question 10

Example for agrobacterium mediated transformation

Answer 10

Trichoderma Reesei was modified to enhance its cellulose fermentation processes, leading to better biofuels from waste products.

Question 11

Steps in gene editing

Answer 11

1.CRISPR gRNA guides enzymes to the target sequence
2. Cas9 puts a double stranded break in the fungal DNA.
3. New DNA is incorporated in repair process.
4. Target gene expressed.

Question 12

Methods of DNA repair in CRISPR

Answer 12

Homology directed repair and non-homologous end joining

Question 13

Example of gene editing in fungi

Answer 13

Penicillium chrysogenum now makes up to 1.7x penicillin thanks to CRISPR, by editing genes pcbAB and penDE

Question 14

Metabolic engineering

Answer 14

Genetically engineered fungi that overexpress rate-limiting enzymes or delete metabolic pathways, making the fungi stuck in a state where target metabolites are produced.

Question 15

Examples for metabolic engineering

Answer 15

1. Saccharomyces cerevisiae, enzymes in shikimote pathway are overexpressed leading to rapid production of aromatic compounds such as vanillin.
2. Aspergillus oryzae expresses steptomyces genes to produce erythromycin antibiotics.

Question 16

Regulators of gene expression

Answer 16

1. AlcA is used to promote enzyme production in aspergillus species.
2. GFP (green fluorescent protein) is used to monitor expression in target genes.

Question 17

Commercial applications of GM fungi

Answer 17

1. Aspergillus terreus is used to produce lovastatin for high cholesterol patients
2. Saccharomyces cerevisiae is used to produce ethanol in high quantities.