Dissertation
METABOLIC ENGINEERING OF YARROWIA LIPOLYTICA FOR SYNTHESIS OF VALUE ADDED CHEMICALS AND BIOFERTILIZER PRODUCTION FROM AZOTOBACTER VINELANDII
Doctor of Philosophy (PhD), Washington State University
01/2017
Handle:
https://hdl.handle.net/2376/111090
Abstract
Oleaginous yeast Y. lipolytica has been studied extensively over the last few decades as a production host for industrial applications. The goal of this work is to address some scientific hurdles and construct new strains with improved yield of value-added chemicals. In the first study, a robust strain that can accumulate large amounts of free fatty acid was developed. Three metabolic pathways of Y. lipolytica were engineered: the fatty acid activation pathway, β oxidation pathway and lipid biosynthesis pathway. Results show that combinatorial blocking of fatty acid activation and β oxidation improved free fatty acid accumulation. Free fatty acid accumulation was also enhanced by strengthening precursor synthesis, nutrient utilization and thioesterase expression. The strain with blocked pathways and expression of the E. coli thioesterase gene had the highest titer of 2.3 g/L in Y. lipolytica. In the second study, a strain was constructed with an improved ability to produce fatty alcohol by utilizing a lipid precursor. The lipid biosynthesis was interrupted and the fatty alcohol synthesis pathway was introduced by expressing the fatty acyl-CoA reductase gene. This resulted in a fatty alcohol producing strain with a yield of 0.03 g/g glucose. To relieve metabolic stress by the pathway engineering, the bacterial hemoglobin gene responsible for promoting oxygen transfer and energy metabolism was expressed. This resulted in a strain with a maximum yield of 0.05 g/g of glucose and a titer of 1 g/L.
In the final study, ways to produce biofertilizer was explored by utilizing the waste stream from anaerobic digestion and cellulosic sugars from biomass. Findings reveal that the bacteria Azotobacter Vinelandii can utilize glucose obtained from the enzymatic saccharification of wheat straw. Results also show that nutrient-recovered anaerobic digestion effluent, when supplemented with glucose, support growth. In this study, the effluent accumulated 6.8 g/L biomass. Residual nitrogen level in the effluent decreased by >70 % following the growth of A. vinelandii. This resulted in a cleaner effluent stream. Production of biofertilizer provides a potential solution to mitigate soil and water quality degradation arising from removal of agricultural residue for biorefineries and direct disposal of anaerobic digestion effluent.
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Details
- Title
- METABOLIC ENGINEERING OF YARROWIA LIPOLYTICA FOR SYNTHESIS OF VALUE ADDED CHEMICALS AND BIOFERTILIZER PRODUCTION FROM AZOTOBACTER VINELANDII
- Creators
- Rishikesh Janardhan Ghogare
- Contributors
- Shulin Chen (Advisor)Manuel Garcia-Perez (Committee Member)Amit Dhingra (Committee Member)Xaiochao Xiong (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Biological Systems Engineering, Department of
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 168
- Identifiers
- 99900581428301842
- Language
- English
- Resource Type
- Dissertation