Lorenzo L. Taping IIIBS Biology

Disruption of PMR1 in Kluyveromyces lactisimproves secretion of

calf prochymosin

Chymosin

• Also known as rennin

• Main coagulating enzyme found in rennet which is used extensively in cheese production.

• hydrolyses a specific bond (Phe105–Met106) in kappa-casein of milk

• kappa-casein acts as micelle stabilizer

• Precipitation of insoluble constituents

Chymosin

Cheese curd

Whey

Chymosin

• Calf abomasums as the traditional principal source

Abomasum

Omasum

Reticulum

Rumen

Search for Alternative Rennet

• increasing demand • shortage of calf stomachs • ethical issues with animal

slaughtering

Kluyveromyces lactis

Increase chymosin production has been made through expression of calf chymosin gene in recombinant K. lactis.

Advantages of K. lactis

• Nontoxicogenic GRAS microorganism approved by US FDA

• Unlike P. pastoris, it does not require methanol to induce protein secretion

• Unlike E. coli, it doesn’t secrete the expressed protein enclosed in inclusion bodies

Further Improvements

Disruption of PMR1 in other yeast species (S. cerevisiae, P. pastoris, and H. polymorpha) increase the secretion of heterologous protein leading to the hypothesis that disrupting PMR1 in K. lactis would have the same effect.

PMR1• encodes for a P-type Ca2+-ATPase

localized in the Golgi apparatus• provides the yeast secretory

pathway with Ca2+ and Mn2+ required for glycosylation, sorting and endoplasmic reticulum-associated degradation of tagged proteins

• Improper sorting of synthesized proteins

• secretion of partially folded proteins• increase in the secretion of

heterologous proteins normally destined for degradation in the lysosome.

Defective PMR1

Experimentation

Microbial Strains K. lactis GG799 (type strain) E. coli JM109

Media Yeast Potato Dextrose for yeast Luria-Bertani for bacterium

Construction of K. lactis GG799 (pmr1∆)

Construction of expression vector

pKLAC1

BlnI SalI

pUC18-prochymosin

SalI BlnI

ligation

pKLAC1-N-prochymosin

Transformed into E. coli JM109

Prochymosin gene

Transformation of K. lactis GG799

SacII SacIILinearised cassette

Linearised cassette

K. lactis GG799

Transformed K. lactis GG799

transformants were selected using Yeast Carbon Base agar containing acetamide

Protein expression and chymosin activity assays

• Incubated in shake flasks containing YPD

• culture supernatants was added to 5mL of a suspension of non-fat milk powder and incubated until a clot formed

• One unit of chymosin activity was defined as the amount of active chymosin required to produce a clot in 40 min