METHODS DEVELOPMENT FOR THE ANALYSIS OF BIOMOLECULES IN CHALLENGING PLANT AND MICROBIAL MATRICES

Kaylie Barton

doi:10.7273/000007321

Reliable and reproducible method development and data analysis are crucial for biotechnological research. Focusing on two studies, we optimized the analytical workflows for gene expression and metabolite quantitation across distinct biological systems. One study focused on the quantitation of specific isoforms ferredoxin (Fd) and ferredoxin-NADP+ reductase (FNR) in peppermint (Mentha × piperita L.) across various peppermint organs and cell types, both of which have been determined to have essential roles in the reductive steps in the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway of terpenoid biosynthesis in glandular trichomes (GTs). We recognized that a single reference gene approach for normalization was inadequate and determined that the utilization of a GPU-accelerated tool, NormiRazor, to validate a three-gene normalization strategy for reference gene stability was necessary. Based on the results from the expression patterns of each isoform, we determined that Fd II was highly expressed in GTs, with moderate expression in the roots, compared to the other isoform, Fd III, which had significantly less expression in the roots and the GTs. These results suggest that the high transcript abundance of Fd II is responsible for the flux through the MEP pathway rather than the biochemical differences in enzymes encoded by the Fd II and Fd III gene transcripts. The second study centered around genetically modified yeast strains (Saccharomyces cerevisiae Y55-1034 and Saccharomyces boulardii (SB)2022) from the AMES NASA BioNutrients-1 project for the biomanufacturing of essential carotenoids, β-carotene and zeaxanthin, for astronauts during long-term spaceflight. Due to the sensitive nature of carotenoids, which degrade due to temperature, light, and oxygen, robust methods for carotenoid extraction, as well as standard curve generation, were developed. First, stability analysis of carotenoids, specifically β-carotene, revealed that after short-term storage of samples, degradation of the carotenoid is observed, quantified by an increase in cleavage product peak areas and a decrease of the β-carotene peak area (one exhibiting a 51% decrease). Similarly, the method for carotenoid extraction optimization was crucial for ensuring complete recovery of the target compound. Adding a mechanical agitator significantly improved yeast cell lysis and β-carotene yield in the same experimental groups. In one case, the utilization of a mechanical agitator recovered enough β-carotene (95.5 μg/ g culture biomass) for HPLC instrument detection; compared to extraction without, there was no β-carotene detected. The standard curves were tightly correlated around their best-fit lines, validating the method for the development of reproducible and accurate standard curves. Together, these studies revolve around the critical need for method development to enable the accurate quantitation of biological molecules in challenging plant and microbial systems.

METHODS DEVELOPMENT FOR THE ANALYSIS OF BIOMOLECULES IN CHALLENGING PLANT AND MICROBIAL MATRICES

Files and links (1)

Abstract

Metrics

Details