Glucaric acid production in yeast from sucrose
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Kemian tekniikan korkeakoulu |
Master's thesis
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Authors
Date
2021-12-14
Department
Major/Subject
Biotechnology
Mcode
Degree programme
Master's Programme in Chemical, Biochemical and Materials Engineering
Language
en
Pages
67+2
Series
Abstract
Production of biochemicals from simple sugars using biotechnological routes is of utmost demand to reduce our dependency on non-renewable resources and to achieve UN sustainability goals. One such compound of commercial interest is glucaric acid (GA), which is industrially produced by a chemical route involving toxic by-products, making it non-sustainable. Production of this compound from microbes have been reported earlier but suffers from a common issue of strains favouring cell growth over glucaric acid production. Hence, the aim of this work was to investigate whether an energy efficient phosphorolytic pathway in conversion of sucrose to glucaric acid could be efficient in production of glucaric acid by the yeast Saccharomyces cerevisiae. This thesis describes the production of glucaric acid from sucrose by S. cerevisiae using different sucrose metabolizing pathways – the native hydrolytic pathway and energy efficient heterologous phosphorolytic pathway. To achieve this, all the native sucrose hydrolyzing genes were deleted to construct sucrose negative strain H5787, which could not grow on sucrose. The native SUC2, MAL32 and heterologous sucrose phosphorylase encoding gene from Leuconostoc mesenteroides (SP) was integrated to the genome of the H5787 strain to construct three different sucrose metabolizing strains, followed by transformation of plasmid harbouring genes required for glucaric acid production. In addition, the PGI1 gene was deleted in the best production strains to test if this could improve glucaric acid yield. Our results demonstrate that the H5820 strain (H5787 + iSP + GA) having the energy efficient intracellular phosphorolytic pathway produces about 20 mg/L glucaric acid in shake flasks, which is twice as high as with the H5822 strain (H5787 + iMAL + GA) expressing native intracellular hydrolytic pathway. Moreover, the H5820 strain accumulates 500% more myo-inositol (30 mg/L), a precursor for GA synthesis, when compared with the H5822 strain (5 mg/L). Deletion of the PGI1 gene resulted in increase in GA production. The H5851 strain (H5787 + iSP + GA + ∆pgi) produced about 30 mg/L GA, which was 50% higher than the H5820 strain which has the PGI gene intact while maintaining similar myo-inositol levels (~30 mg/L). The increase in production levels after PGI1 deletion were more profound for the H5853 strain (H5787 + iMAL + GA + ∆pgi). This strain produced about 40 mg/L GA and 20 mg/L myo-inositol, which is 300% more than its counterpart H5822 (H5787 + iMAL + GA). However, the production levels are still low and further understanding and tuning of metabolism is necessary to improve GA yield.Description
Supervisor
Penttilä, MerjaThesis advisor
de Ruijter, JorgPylkkänen, Robert
Keywords
sucrose phosphorylase, glucaric acid, Saccharomyces cerevsiae, metabolic engineering, phosphoglucoisomerase, sucrose fermentation