Recombinant DNA technology is major DNA-based tool that has gained popular attention in the past decade. Significant advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of yeast species have led the recombinant yeast technology a robust tool for both designed catalysts and new biologicals. Yeast combines molecular genetic manipulations and growth characteristics of prokaryotic organisms together with the sub-cellular machinery for performing post-translational protein modifications (O and N- linked glycosylation, disulphide bond formation) and secretion of protein (Intracellularly or extracellularly). A large number of yeast hosts (Saccharomyces cerevisiae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Yarrowia lipolytica, etc) are available for heterologous protein expression. The methylotrophic yeast, Pichia species is the most highly developed one among a small group of alternative yeast species chosen for their perceived advantages over S. cerevisiae as a expression host for the generation of recombinant protein of commercial interest. Advantages of the system include the AOX1 promoter (alcohol oxidase) and other alternate promoters (GAP, FLD1, PEX8, and YPT1), with transcription characteristics that are useful for regulating heterologous protein expression. Auxotrophic mutants (MutS and Mut+) and a new set of biosynthetic markers such as ADE1, ARG4 and URA 3 have been used successfully for better selection of transformed host. Protease deficient hosts and site specific integration of expression vectors into Pichia genome result into high expression of gene of interest. Additional features that are present in certain P. pastoris expression vectors serve as tools for specialized functions. The availability of the expression system as a commercially available kit (Invitrogen) extends the usefulness of system. Several different secretion signal sequences including the native secretion signal or secretion signal sequences from S. cerevisiae such as µ factor prepro peptide causes the protein to be secreted into the growth medium, which greatly facilitates subsequent protein purification. The P. pastoris expression platform is now well developed, as proven by multiple products used in human and veterinary medicine and in industry. A better understanding of secretion signals, glycosylation, and endogenous yeast proteases would be extremely helpful in developing and improving the yeast heterologous expression system.

1. Recombinant Yeast Technology at the Cutting
Edge: Robust Tools for both Designed
Catalysts and New Biologicals
Presented by:
Navprabhjot Kaur
Ph. D Microbiology
L-2010-BS-63-D

2. Contents
• Recombinant Yeast Technology
• Yeast Expression Systems
• Problems Associated with Saccharomyces cerevisae
• Cloning into Yeast: Pichia pastoris
• Features of Pichia Technology
• Expression Vectors, Promoters, Selectable markers and Host
strains
• Site-Specific Integration
• Post-translational modification
• Heterologous proteins expressed in Yeast Expression Systems
• Limitations
• Conclusions

3. Recombinant DNA Technology

4. Steps in Recombinant DNA Technology

5. Expression Systems
5

6. Recombinant Yeast Technology

7. Yeast Expression Systems
7

8. Saccharomyces cerevisiae

9. Problems Associated With S. cerevisae

10.  Like Saccharomyces cerevisiae:
 Easy to manipulate
 Faster, easier, less expensive than other eukaryotic
systems
 Advantage over Saccharmoyces cerevisiae:
 10-100 fold higher heterologous protein expression
levels!!
10
Major Breakthrough in Recombinant Yeast Technology
Cloning into Yeast: Pichia pastoris
ichia is a methylotrophic yeast(can metabolize MeOH)
HCHO
O2 H2O2
CH3OH
AOX

11. Pichia pastoris as an experimental organism

12. Features of Pichia Technology

13. Continued…..
Synthetic promoters for fine tuned, both methanol-induced or
methanol-free Gene expression

14. Pichia pastoris as a Methylotrophic Yeast

15. Alcohol Oxidase Proteins
AOX2: Very low level of alcohal oxidase activity

16. Phenotype of aox1 mutants

17. Construction of Expression Strains

18. Selecting a Pichia Expression Vector

19.  EPISOMAL or Plasmid – over expression
 High copy (20-100 copies per cell)
 Two origin of replication
 INTEGRATIVE – introduce gene into yeast chromosome
 Single copy, aids understanding protein function/
role in pathway
 CENTROMERIC –low copy (YAC)
19
Cloning into Yeast: Choice of Vector

20. Alternative Promoters

21. PGAP

22. PFLD1 (Glutathione-dependent Formaldehyde Hydrogenase)

23. PPEX8 and PYPT1

24. Selectable markers

25. Host strains

26. Protease-deficient Host Strains

27. P. pastoris host strains

28. Integration of expression vectors into the P. pastoris genome

29. Gene Insertion at AOX I or aoxI:: ARG4
His+
Mut+
(GS115), His+
Muts
(KM71)

30. Gene Insertion Events at HIS 4

31. Multiple Gene Insertion Events

32. Gene replacement event at AOX I locus
His+
Muts
(GS115)

33. Transformation

34. Post-translational Modification of Secreted Proteins

35. Intracellular and Secretory Protein Expression

36. Secretion Signal Selection

37. Glycosylation

38. Posttranslational Modifications in comparison to S. cerevisiae

39. Heterologous proteins expressed in P. pastoris

41. Other Yeast Expression Systems

42. Continued….

43. Heterologous Proteins Expressed in Other Yeast Host

44. Limitations

45. Conclusions
 As a unicellular eukaryote, yeast is quick, easy and
inexpensive to genetically manipulate and culture
 Yeast share many conserved pathways with higher eukaryotes
making it an excellent platform for studying protein function
 As well, the wealth of knowledge and set of tools available for
Yeast species, make it a very powerful genetic tool for
studying protein function
 High protein yield makes yeast strains useful for
pharmaceutical protein production
 A better understanding of secretion signals, glycosylation, and
endogenous yeast proteases would be extremely helpful in
developing and improving the yeast heterologous expression
system.

46. Thank You