Team:RUBochum/Results
Developing a cloning strategy for an industrial optimized process.
Bioinformatic planning
Protein Screening
For the first production of the variety of proteins in P. pastoris, we decided to use plasmids as extrachromosomal bodies. Our cloning strategy was based on the pPICZα A, which has the α secretion signal in front of the MCS and the BleoR gene which encodes for phleomycin D1.
The methanol-induced AOX1 promoter was used for the production of our proteins.
By creating two different pairs of primers for the Gibson assembly, we performed diverse insertions into the MCS.
Figure 1: Gibson cloning strategy for the pPICZα A. Genes can be inserted with a polyhistidine-tag and secretion signal.
All food proteins have been inserted with the α-factor secretion signal.
Proteins that are related to the overproduction of fatty acids were inserted without an affinity tag and secretion signal.
The monotreme lactation protein was additionallycoupled to the His-tag for further testing (Table 1).
Table1: Proteins and their features inside the MCS.
Proteins MCS Integration αs1 casein Bos taurus Secretion signal αs2 casein Bos taurus Secretion signal β casein Bos taurus Secretion signal κ casein Bos taurus Secretion signal αcasein Ornithorhynchus anatinus Secretion signal β casein Ornithorhynchus anatinus Secretion signal κ casein Ornithorhynchus anatinus Secretion signal Monotreme Lactation Protein Ornithorhynchus anatinus Secretion signal, His-Tag Brazzein Pentadiplandra brazzeana Secretion signal Acetyl-coA carborxylases complex E. coli k12 Only promoter Thioesterase II E. coli K12 Only promoter Aryl-Alcohol Oxidase Penicillium simplicissimum Only promoter
Improvement for an industrial process
The improved process is characterized by a joint synthesis of all products in one fermentor.
To simplify the production the number of genotypes needs to be reduced in an optimal manner to one.
Figure 2: Principle of the heterologous fermentation process. Starting from multiple genotypes producing the whole product, the amount of genotypes is reduced inside the fermentation due to genetic optimization.
For the genetic optimization, a chromosomal integration was planned with the GoldenPiCS system. Loci for the integration would have been RGI2, ENO1, NTS, and AOX1tt.
Those could have been further optimized with promoter shuffling.
Cloning
After our primers and genes arrived, the first step was to amplify those genes during PCR.
Figure 3: PCR of the pUCIDT and prepared E. coli samples.
Samples ordered from left to right:
Standard, 1. α-s1-casein bos taurus (67°C), 2. α-casein platypus (67°C), 3. β-casein platypus (67°C), 4. κ-casein platypus (67°C), 5. α-s1-casein bos taurus (67°C), 6. κ-casein platypus (67°C), 7 . Brazzein (67°C), 8. β-casein bos taurus (67°C), 9. Acetyl-coA carboxylase complex (AccA) (65°C), 10. AccB (65°C), 11. AccC (65°C), 12. AccD (65°C), 13. Thioesterase II (65°C), 14. BB pPICZα A casein (60°C), 15. BB pPICZα A Acc/Tes 60°C (58°C).
The annealing temperature varied between 58°C and 67°C. Amplification worked for the α-s1-casein bos taurus, α-casein platypus, β-casein platypus, α-s1-casein bos taurus, κ-casein platypus, β-casein bos taurus and AccD and the thioesterase II.
Figure 4: PCR of the pUCIDT and prepared E. coli samples.
Samples ordered from left to right:
Standard, 4. κ-casein platypus (65°C), 7 . Brazzein (65°C), 9. AccA (69°C) 10. AccB (69°C), 11. AccC (69°C), 14. BB pPICZα A casein (60°C).
Brazzein, AccA and the backbone for the insertion of the caseins were amplified.
Figure 5: PCR of the pUCIDT and prepared E. coli samples.
Samples ordered from left to right:
4:PCR of the pUCIDT and prepared E. coli samples. Samples ordered from left to right: Standard, 4. κ-casein platypus (55°C), 9. AccA (56°C) 10. AccB (69°C), 11. AccC (55°C),12. AccD (56°C), 13. Thioesterase II (55°C), 15. BB pPICZα A Acc/Tes (56°C), 17. BB pPICZα A MLP (57°C)
All Acc subunits, the thioesterase II and the MLP backbone were amplified.
Figure 6: PCR of the backbone.
Samples ordered from left to right:
Standard, 15. BB pPICZα A Acc/Tes 54°C, 55°C, 57°C, 59°C, 60°C.
All backbone samples were amplified.
Figure 7: PCR of the pUCIDT monotreme lactation protein.
Samples ordered from left to right:
Standard, 16. MLP (56°C)
Check PCR and Sequencing
After the amplification, the DpnI digestion was performed overnight. On the following day, Gibson assemblies were made which then were transformed. Subsequently, pre-cultures were grown, and the plasmids were purified.
Check PCRs were made before sequencing.
Figure 8: Example of an electropherogram. From β-casein bos taurus
Figure 9: Check PCR.
Arranged from left to right: Standard, 1. α-s1-casein bos taurus , 2. α-casein platypus , 3. β-casein platypus , 4. κ-casein platypus , 5. α-s1-casein Bos taurus, 6. κ-casein platypus, 7 . Brazzein, 8. 5.
The check PCR approved in its size α-s1-casein Bos taurus, α-casein platypus, κ-casein platypus, Brazzein, β-casein Bos taurus, which were later confirmed by sequencing.
Further check PCRs were made for the other proteins, which failed.
Heterologous overexpression
The approved constructs were successfully transformed into P. pastoris.
Figure 10: Example of P. pastoris transformants - 7. Brazzein, 8. β-casein Bos taurus
Figure 11: SDS-Page of the supernatant from the 72 h sample of
1. α-s1-casein Bos taurus , 2. α-casein platypus, 7. Brazzein, 8. β-casein Bos taurus
Due to a wrong sample storage method and a failed SDS-page, all our results of the overexpression got lost with the exception of the 72 h sample of the supernatant.
The repeated SDS-page of those samples showed no distinct bands, which might be related to a failed secretion or a failure of the staining solution.
Most casein proteins can show problems during secretion due to the post-translational modifications.
Figure 12: NanoDrop measurement of the 72h supernatant.
Ordered from right to left: 1. αs1 casein Bos taurus (26,3 mg/ml), 2. Brazzein (69,9 mg/ml), 3. β casein Ornithorhynchus anatinus (43,6 mg/ml)
The samples which were purified by size. Measured with the respective molecular weight and molar attenuation coefficient the protein yield was determined αs1 casein Bos taurus (26,3 mg/ml), Brazzein (69,9 mg/ml), β casein Ornithorhynchus anatinus (43,6 mg/ml)
).
Proteins couldn´t be detected on the SDS-page due to an unknown error source which might be related to the staining solution.
Summary and Outlook