USING CRISPR/Cas9 TO DELETE REDUCTASES IN YEAST TOWARDS IMPROVED CAPSAICINOID BIOSYNTHESIS

Abstract

The baker’s yeast, Saccharomyces cerevisiae (S. cerevisiae) was studied and genetically engineered. This was to enhance the transamination step of the biochemical reaction cascade towards the production of capsaicin. Inherent reductases were identified and twelve of them knocked out via homologous recombination. Using the CRISPR/Cas9 technology, the Green Fluorescence Protein (GFP) gene was introduced as a reporter molecule to monitor and determine the success of the knock-outs. Starting with a single copy number of GFP was a relevant step serving as a footprint in determining the approximate if not the exact copy number of aminotransaminases (ATAs) required for increased production of capsaicin in the long run. Further, methods for verifying and characterizing all knockout and GFP transformants included antibiotic selection pressure, Polymerase Chain Reactions (PCR), flow cytometry and HighPerformance Liquid Chromatography (HPLC). Flow cytometry was used to screen for GFP positive transformants under different growth conditions. The presence of vanillin and furfuraldehyde as inhibitors, are known to impair growth and induce reductases in yeast. Characterization by cell growth and bioconversion activity was determined in the transformants to ascertain the effect of each deletion mutation of these reductases in yeast. This exploratory study was performed to identify which gene improves capsaicinoid biosynthesis, using the CRISPR/Cas9 technique and GFP expression as a reporter marker. Knock-out and GFP strains were successfully constructed. However, verification of the knockout strains by PCR seemed unsuccessful. Characterizing these strains using HPLC to determine their bioconversion activity showed differences in activity among the strains. Also, in verifying the GFP strains, only two showed significant GFP expression. All other strains did not show improved green fluorescence signal.

Popular Abstract

IMPROVING CAPSAICINOID BIOSYNTHESIS IN YEAST USING CRISPR/Cas 9

Capsaicin is naturally produced in chilli pepper plants (Capsicum sp.) and this substance characterizes the strong burning sensation felt when consumed. It is well used for antimicrobial agents, cancer treatments, pharmaceuticals and medicated creams to relieve muscle or joint pain (Arce-Rodríguez and Ochoa-Alejo 2019). Biological production from renewable raw materials is therefore an attractive alternative to current production methods. There is a large potential to develop microbial systems that can be used to produce capsaicin and its derivatives allowing for combinatorial biosynthesis that can ultimately yield characteristics such as reduced side effects and improved analgesic properties.
The baker’s yeast, Saccharomyces cerevisiae (S. cerevisiae) is well known as an efficient eukaryotic cell factory due to its ability to grow anaerobically and tolerate low pH levels. Its uses include baking, wine making and production of bioethanol. It can be metabolically and genetically engineered through efficient transformation methodologies, to produce varieties of pharmaceuticals, food and other chemicals and in this case for the production of capsaicinoid.

Genetic engineering:
Biosynthesis of capsaicinoids requires the transamination of vanillin to vanillyl amine catalyzed by the enzyme vanillin aminotransferase. However, the presence of oxidoreductases results in conversion of vanillin to vanillyl alcohol, thereby lowering yield and productivity of capsaicinoids. Alcohol dehydrogenases (ADHs) encoded by genes such as ADH6, ADH7 and GRE2 are known to reduce vanillin to its corresponding alcohol which is strongly inhibitory to the survival and growth of yeast (Narayanan et al. 2017; Nguyen et al. 2015). However, other oxidoreductases are also involved and need to be identified and ultimately knocked-out to avoid formation of the by-product vanillyl alcohol.

The primary aim of this study was to identify which oxidoreductases are responsible for vanillin reduction, and to guide a future deletion strategy to construct an optimal platform strain for capsaicinoid biosynthesis. A second aim of the study was to construct GFP-based reporter systems to study regulation of oxidoreductase gene expression as a response to vanillin. For this purpose a new CRISPR/Cas9 strategy was applied. In the wild type yeast strain CEN.PK 113-7D, single deletions of genes encoding these reductases were done. Using the drug resistance cassette Nat-MX, these genes were knocked out by homologous recombination and by the CRISPR/Cas9 system, the knockout strains were transformed with GFP. Transformants were selected by their resistance to specific antibiotics in the growth media and further characterized by their ability to convert vanillin to vanillyl alcohol. This was done by high-performance liquid chromatography (HPLC).

Determination of gene(s) in the GFP strains that was upregulated to detoxify both vanillin and furfural were done by flow cytometry analysis. Observation made was the upregulation of ADH6 gene as the only gene that induced significant GFP expression in the strain. Thus, this gene was involved in detoxifying the inhibitors, contributing to the growth and survival of the strain. This deletion step of reductases therefore contributes a great deal in the biosynthetic cascade towards improved capsaicinoid production.

Master’s Degree Project in Molecular Biology 60 credits 2020
Department of Biology, Lund University

Advisors: Magnus Carlquist and Arne Hagman
Division of Applied Microbiology - Chemistry Department, Lund University.

• author: Abaka, Raymilda Ekua
• supervisor: Magnus Carlquist; Arne Hagman
• organization: Degree Projects in Molecular Biology
• course: MOBN03 20192
• year: 2020
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9022027
• date added to LUP: 2020-06-24 12:06:23
• date last changed: 2020-06-24 12:06:23