Henriquez Morales, Maria-Jose;
(2018)
Synthetic Biology Routes To Production Of Chiral Amino-alcohols In Pichia Pastoris.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Pichia pastoris (P. pastoris) is an attractive industrial host cell due to its ability to grow up to 60% wet cell weight (WCW) by volume, a far higher level of biomass than the typical values reached by Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae). This thesis seeks to explore how the genetic tractability and high cell densities characteristic of P. pastoris can be exploited to intensify whole-cell biocatalysis. Chiral amino alcohols such as 2-amino-1,3,4-butanetriol (ABT) are key building blocks of small molecule pharmaceuticals and have previously been produced by whole-cell biocatalysis using cells engineered to express a de novo enzyme pathway consisting of transketolase and transaminase. Within this work, native and foreign P. pastoris transaminases were characterised with respect to their biocatalytic potential. Genomic data mining was performed to explore the GS115 strain genome, allowing the selection of three putative Class III transaminase genes and the construction of overexpressor strains PpTAm107, PpTAm677 and PpTAm410. The well-studied ω-transaminase CV2025 from Chromobacterium violaceum (C. violaceum) was also successfully engineered to generate two strains; PpTAmCV708 for single expression of CV2025, and PpTAm-TK16 strain for CV2025 co-expression alongside a native transketolase previously characterised for L-erythrulose production. The rapid growth and high biomass characteristics of P. pastoris were successfully exploited for production of ABT by whole-cell biocatalysis. At high cell density, the best performance for the de novo pathway was obtained with the engineered PpTAm-TK16 strain, which tolerated high concentration of substrates to achieve STY 0.29 g L-1 h-1 of ACP, 49-fold higher than levels previously achieved with E. coli in the presence of the same substrates.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Synthetic Biology Routes To Production Of Chiral Amino-alcohols In Pichia Pastoris |
| Event: | UCL |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| UCL classification: | UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > UCL BEAMS UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10050823 |
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