Enhancing the Heterologous Fructosyltransferase Activity of Kluyveromyces lactis: Developing a Scaled-Up Process and Abolishing Invertase by CRISPR/Cas9 Genome Editing
| Autor: | Dustin Eckhardt, Peter Czermak, Markus Baas, Jan Philipp Burghardt, Doreen Gerlach, Rong Fan |
|---|---|
| Přispěvatelé: | Publica |
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
Histology lcsh:Biotechnology Mutant Biomedical Engineering Heterologous Bioengineering 01 natural sciences chemistry.chemical_compound 0404 agricultural biotechnology 010608 biotechnology lcsh:TP248.13-248.65 Transferase chemically-defined fermentation medium CRISPR/Cas9 Original Research Kluyveromyces lactis fructosyltransferase (FFT) high-cell-density cultivation biology Chemistry Fructosyltransferase activity Oxygen transport Bioengineering and Biotechnology 04 agricultural and veterinary sciences Kluyveromyces lactis GG799 biology.organism_classification 040401 food science Invertase Biochemistry Galactose fructo-oligosaccharides (FOS) prebiotic Biotechnology |
| Zdroj: | Frontiers in Bioengineering and Biotechnology, Vol 8 (2020) Frontiers in Bioengineering and Biotechnology |
| ISSN: | 2296-4185 |
| DOI: | 10.3389/fbioe.2020.607507 |
| Popis: | The enzymatic production of prebiotic fructo-oligosaccharides (FOS) from sucrose involves fructosyltransferases (FFTs) and invertases, both of which catalyze forward (transferase) and reverse (hydrolysis) reactions. FOS yields can therefore be increased by favoring the forward reaction. We investigated process conditions that favored transferase activity in the yeast strain Kluyveromyces lactis GG799, which expresses a native invertase and a heterologous FFT from Aspergillus terreus. To maximize transferase activity while minimizing native invertase activity in a scaled-up process, we evaluated two reactor systems in terms of oxygen input capacity in relation to the cell dry weight. In the 0.5-L reactor, we found that galactose was superior to lactose for the induction of the LAC4 promoter, and we optimized the induction time and induction to carbon source ratio using a response surface model. Based on the critical parameter of oxygen supply, we scaled up the process to 7 L using geometric similarity and a higher oxygen transport rate, which boosted the transferase activity by 159%. To favor the forward reaction even more, we deleted the native invertase gene by CRISPR/Cas9 genome editing and compared the ΔInv mutant to the original production strain in batch and fed-batch reactions. In fed-batch mode, we found that the ΔInv mutant increased the transferase activity by a further 66.9%. The enhanced mutant strain therefore provides the basis for a highly efficient and scalable fed-batch process for the production of FOS. |
| Databáze: | OpenAIRE |
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