A Host-Vector System for the Expression of a Thermostable Bacterial Lipase in a Locally Isolated Meyerozyma guilliermondii SMB
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Plasmid
2.2. Identification of Locally Isolated Yeast Strain SMB
2.3. Screening of Native Lipase in Strain SMB
2.4. Determination of Antibiotic Selection Markers
2.5. Amplification of Methanol-Inducible Promoters
2.6. Construction of Recombinant pFLDhα/L2 Lipase
2.7. Expression of L2 Lipase in Recombinant M. guilliermondii SMB
2.8. Optimization of L2 Lipase Production
3. Results and Discussion
3.1. Identification and Characterization of Strain SMB
3.2. Screening of Strain SMB Native Lipase
3.3. Determination of Antibiotic Selection Marker
3.4. Screening of Methanol-Inducible Promoters
3.5. Optimization of L2 Lipase Production in Strain SMB
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Targeted Regions | Primers | Nucleotide Sequence (5′→3′) | TA (°C) | Reference |
---|---|---|---|---|
Identification of isolates | ||||
rRNA ITS | fITS1 rITS4 | TCCGTAGGTGAACCTGCGG TCCTCCGCTTATTGATATGC | 60.0 | [10] |
rRNA 18S | f18S r18S | AACCTGGGTTGATCCTGCCAGT TGATCCTTCTGCAGGTTCACCTAC | 65.0 | [11] |
rRNA 25S | f25S r25S | TCATGAGACTACTGGCAGGATCAAC GGATCCGTTTAGACCGTCGTGAGA | 64.7 | [12] |
Promoters | ||||
AOX 1 | AOX1f AOX1r | GACTGGTTCCAATTGACAGC GCAAATGGCATTCTGACATCC | 51 | [13] |
FLD 1 | FLD1f FLD1r | CGGGATCCGCATGCAGGAATCTCTGGA CGCAATTGTGTGAATATCAAGAATTG | 50 | Invitrogen, USA |
Vector modification | ||||
pFLDα-Z | CYC1 HindIII F pTEF1 PstI R | ATACAAGCTTCACGTCCGACGCGGCCCGACGGGT TATACTGCAGCCGCCCTTAGATTAGATTGCTATGCTTTCT | 66 | This study |
Hygromycin gene | hyg-cmvF hyg-cmvR | TTACCTGCAGGCGTTACATAACTTACGGTAAATGG AATCAAGCTTTCATTCCTTTGCCCTCGGACGAGT | 64 | This study |
Cloning of L2 lipase | ||||
Mature L2 lipase | fSfiI L2 rKpnI L2 | AATTGGCCCAGCCGGCCAGCATCCCTA TTCTAGGTACCAGGGAGCAAGCTTGCCAA | 65 | This study |
α-factor/3′AOX1 | α-factor 3′ AOX1 | TACTATTGCCAGCATTGCTGC GCAAATGGCATTCTGACATCC | 60 | Invitrogen, USA |
Markers | Concentration (μg/mL) | Growth |
---|---|---|
Blasticidin | 50 | +++ |
Puromycin | 25 | +++ |
Geneticin | 400 | +++ |
Phleomycin | 25 | +++ |
Zeocin | 500 | + |
Hygromycin | 50 | - |
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Salleh, A.B.; Baharuddin, S.M.; Rahman, R.N.Z.R.A.; Leow, T.C.; Basri, M.; Oslan, S.N. A Host-Vector System for the Expression of a Thermostable Bacterial Lipase in a Locally Isolated Meyerozyma guilliermondii SMB. Microorganisms 2020, 8, 1738. https://doi.org/10.3390/microorganisms8111738
Salleh AB, Baharuddin SM, Rahman RNZRA, Leow TC, Basri M, Oslan SN. A Host-Vector System for the Expression of a Thermostable Bacterial Lipase in a Locally Isolated Meyerozyma guilliermondii SMB. Microorganisms. 2020; 8(11):1738. https://doi.org/10.3390/microorganisms8111738
Chicago/Turabian StyleSalleh, Abu Bakar, Siti Marha Baharuddin, Raja Noor Zaliha Raja Abd Rahman, Thean Chor Leow, Mahiran Basri, and Siti Nurbaya Oslan. 2020. "A Host-Vector System for the Expression of a Thermostable Bacterial Lipase in a Locally Isolated Meyerozyma guilliermondii SMB" Microorganisms 8, no. 11: 1738. https://doi.org/10.3390/microorganisms8111738
APA StyleSalleh, A. B., Baharuddin, S. M., Rahman, R. N. Z. R. A., Leow, T. C., Basri, M., & Oslan, S. N. (2020). A Host-Vector System for the Expression of a Thermostable Bacterial Lipase in a Locally Isolated Meyerozyma guilliermondii SMB. Microorganisms, 8(11), 1738. https://doi.org/10.3390/microorganisms8111738