Science is a contributive effort. Described below is my contribution to the collaborative exploration of the genetic regulation pathways involved in beta carotene synthesis. This work was done in my Yeast Metabolic Engineering course within the Biotechnology department at North Carolina State University in the spring of 2021.
Described below are the steps taken to accomplish this exploration.
The Experimental Goal
This experiment had a goal of enhancing beta carotene production in Saccharomyces cerevisiae and analyzing a pooled deletion library to infer which gene deletion impacted the beta carotene regulatory pathways. This can be accomplished by co-opting genes from other organisms, Specifically, three genes from carotenoid- producing Xanthophyllomyces dendrohous, crtYB, crtI, and crtE were required to increase native production of beta carotene precursor, GGPP. Additionally, a 4th gene, HMG-CoA reductase, needed to be over expressed to increase an ergosterol precursor.
The First Steps
Making functional transcription units
A concept called yeast Golden Gate was used to assemble multiple transcription units (TUs) which each consist of a promoter, the coding sequence, and a terminator. Each TU is flanked by a restriction site that later allows the TUs to be stitched together. A process called VEGAS was then used to connect each TU into a full genetic pathway that could then be transformed into yeast cells. Each TU was flanked by unique adaptor sequences called VAs. These provide terminal homology which allows the units to be aligned by yeast's homologous recombination strategies.
Transformation into Yeast Cells
First, the TUs needed to be amplified by PCR to ensure a high enough quantity for downstream use. The TUs and linearized vectors were transformed into two yeast strains. The Wild Type strain, BY4741 is the most commonly used strain in the laboratory. They were also transformed into a pooled deletion library, where each transformant had a different gene deleted. Utilizing a pooled deletion library allowed for identification of genetic changes that impact beta carotene production. Stains that differ in only a single gene are called isogenic. Each isogenic strain also contained unique barcodes that later allowed for determination of which gene had been deleted.
Transformant Selection
Transformants with TUs inserted and a random gene deleted were plated, as well as Wild Type transformants with no genes deleted and TUs incorporated. Two colonies were selected based on phenotypes that appeared to have a resulting effect from beta carotene pathway alteration. I selected a colony that appeared bright orange, indicating high beta carotene production, and another that was bright white, indicating a lack of beta carotene production. Those colonies were then sequenced so the barcodes could be isolated. This allowed it to be known which gene had been deleted in that specific transformant. This was followed by analysis of the gene that had been deleted, to determine if it had a known relation to the beta carotene pathway. If the phenotype displayed a resulting impact due to the deletion of that gene, but the gene had no known connection to the beta carotene regulatory pathways, that indicated discovery of a novel beta carotene regulator!
My Colonies
I selected two colonies based on their extreme appearing phenotypes, then analyzed the genotype of the deleted gene in relation to beta carotene production.
The first colony I selected had a bright white phenotype, indicating a failure to produce beta carotene. It can be seen on the left side of the picture to the right of the text. This indicated a positive regulator had been deleted, rendering the pathway nonfunctional. The colony was missing a gene called KSS1, or kinase suppressor of Sst2 mutation, which codes for a mitogen-activate protein kinase involved in signal transduction pathways that control filamentous growth and pheromone response. I could not determine previous knowledge of this gene being related to beta carotene regulatory networks, which indicates I had discovered a novel regulator of beta carotene production pathway. This deletion strain should be studied further to ensure my results and determine its specific relation and effect on the beta carotene pathway.
My second colony was selected due to its bright orange appearance, indicating successful beta carotene production. It can be seen on the right side of the photo attached to the right of the text. This meant the gene deleted did not impact the regulatory pathways of interest. The gene deleted was identified as YDR282C. All that is known of this gene is that it codes for a mitochondrial inner membrane protein with an unknown function. However, the function appeared to not related to beta carotene production, so further study would not be done on this deletion strain.
