ABSTRACT
BACKGROUND: Metabolic rewiring in microbes is an economical and sustainable strategy for synthesizing valuable natural terpenes. Terpenes are the largest class of nature-derived specialized metabolites, and many have valuable pharmaceutical or biological activity. Squalene, a medicinal terpene, is used as a vaccine adjuvant to improve the efficacy of vaccines, including pandemic coronavirus disease 2019 (COVID-19) vaccines, and plays diverse biological roles as an antioxidant and anticancer agent. However, metabolic rewiring interferes with inherent metabolic pathways, often in a way that impairs the cellular growth and fitness of the microbial host. In particular, as the key starting molecule for producing various compounds including squalene, acetyl-CoA is involved in numerous biological processes with tight regulation to maintain metabolic homeostasis, which limits redirection of metabolic fluxes toward desired products. RESULTS: In this study, focusing on the recycling of surplus metabolic energy stored in lipid droplets, we show that the metabolic recycling of the surplus energy to acetyl-CoA can increase squalene production in yeast, concomitant with minimizing the metabolic interferences in inherent pathways. Moreover, by integrating multiple copies of the rate-limiting enzyme and implementing N-degron-dependent protein degradation to downregulate the competing pathway, we systematically rewired the metabolic flux toward squalene, enabling remarkable squalene production (1024.88 mg/L in a shake flask). Ultimately, further optimization of the fed-batch fermentation process enabled remarkable squalene production of 6.53 g/L. CONCLUSIONS: Our demonstration of squalene production via engineered yeast suggests that plant- or animal-based supplies of medicinal squalene can potentially be complemented or replaced by industrial fermentation. This approach will also provide a universal strategy for the more stable and sustainable production of high-value terpenes.
ABSTRACT
BACKGROUND: In Korea, there were issues regarding the use of immunoassays for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies to detect infection. So, we compared antibody results of eight kinds of commercial immunoassays using clinical remnant specimens. METHODS: We compared the results of several immunoassay kits tested on 40 serum samples from 15 confirmed patients and 86 remnant serum samples from clinical laboratory. Eight kinds of IVD kits-four enzyme-linked immunosorbent assay, two lateral flow rapid immunochromatographic assays, and two chemiluminescent immunoassays with one RUO kit were tested. RESULTS: Among 40 serum samples from 15 coronavirus disease 2019 (COVID-19) patients, 35 yielded at least one positive result for detecting antibodies in the combined assessment. There were inconsistent results in 12 (28%) samples by single immunoassay. Forty samples collected in 2019 before the first COVID-19 Korean case showed negative results except for one equivocal result. CONCLUSION: The discrepant results obtained with different immunoassay kits in this study show that serological assessment of SARS-CoV-2 by a single immunoassay requires caution not only in detecting infection but also in assessing immunologic status.