Doctor of Philosophy
Chemical and Biochemical Engineering
Dr. Amarjeet Bassi
Dr. Lars Rehmann
The field of biochemical engineering has made substantial progress through major advances in genetic and metabolic engineering with applications in various sectors such as energy, food science, pharmaceuticals, etc. The hosts used for this work are constantly broadening. A host particularly important for energy applications are microalgae. The potential to enhance microalgae genetically for energy applications is not well explored and was therefore investigated in this thesis. Non-photosynthetic micro-organisms and photosynthetic microalgae offer a potential approach to enhance sustainable biochemical production. In this study expression vectors for Escherichia coli (E. coli) and Chlorella vulgaris (C. vulgaris) were constructed for the expression of two enzymes: exo-inulinase enzyme and β-glucuronidase (GUS reporter) reporter protein. The expressed enzymes were characterized for their activity. The study involved three phases.
In the first phase of the research, the inulin hydrolyzing enzyme exo-inulinase was expressed in E. coli strain Rosetta-gami B(DE3). For this purpose, first the exo-inulinase gene from Aspergillus niger 12 (A. niger 12) was isolated by reverse transcription polymerase chain reaction (RT-PCR) and further an expression vector pET32a+EX-INU was constructed. The recombinant exo-inulinase was then expressed in E. coli strain Rosetta-gami B(DE3). The recombinant exo-inulinase was purified, and characterized for its activity. The molecular weight of the recombinant exo-inulinase was 81 Da (similar to native exo-inulinase). The Km and Vmax toward inulin were 5.3 ± 1.1 mM and 402.1 ± 53.1 µmol min-1 mg-1 protein respectively and the optimum temperature and pH for maximum enzyme activity were 55 0Cand 5.0 respectively.
In the second phase of the research, the 4-Methylumbelliferyl-β-D-glucuronide (4-MUG) hydrolyzing enzyme β-glucuronidase was expressed in the photosynthetic microalgae Chlorella vulgaris (C. vulgaris). The expression vector pBIN+TetR+TetO was first constructed and transformed into C. vulgaris (UTEX 2714) by co-cultivation with Agrobacterium tumefaciens LBA 4404 (A. tumefaciens). For intact cell screening of high expression level of β-glucuronidase in transgenic C. vulgaris, a new high throughput screening (HTS) method was proposed, developed and evaluated. A recombinant micro-algal isolate (1 of 32 transgenic C. vulgaris) gave the highest florescence intensity of 16,988 ± 1168 by expression of the GUS reporter enzyme. In a lysed cell study, enzyme kinetic analysis for the expressed phenotype was also carried out. The values of Km and Vmax of the recombinant GUS enzyme were 0.1304 ± 0.0101 mM and 0.35 ± 0.004 pmol 4-MU/min/ml of crude cell lysate respectively.
In the third and final phase of the research, the proposed HTS method developed in the previous study was applied to a second microalgal system. First, 4-MUG hydrolyzing GUS enzyme was expressed in Chlamydomonas reinhardtii CC1690 (C. reinhardtii). The transgenic C. reinhardtii expressing GUS enzyme was developed by A. tumefaciens transformation techniques. The expression vector used in this work was pBIN-Hyg-Tx–GUS-INT. High throughput screening of transgenic colonies expressing β-glucuronidase (GUS activity) was carried out directly from agar plates. 1 out of 126 transgenic C. reinhardtii colony, showed the highest fluorescence intensity 1,113. This study confirmed the application of the HTS method to microalgal systems. This is a new tool which can be applied for fast screening of genetic transformations and expression in microalgal systems.
Yedahalli, Shreyas S., "Genetic Engineering Studies of Escherichia coli and Microalgae for Expression of Hydrolytic Enzymes and Development of High Throughput Screening Technique" (2017). Electronic Thesis and Dissertation Repository. 4706.