Display of Microbial Glucose Dehydrogenase and Cholesterol Oxidase on the Yeast Cell Surface for the Detection of Blood Biochemical Parameters

Abstract

High levels of blood glucose are always associated with numerous complications including cholesterol abnormalities. Therefore, it is important to simultaneously monitor blood glucose and cholesterol levels in patients with diabetes during the management of chronic diseases. In this study, a glucose dehydrogenase from Aspergillus oryzae TI and a cholesterol oxidase from Chromobacterium sp. DS-1 were displayed on the surface of Saccharomyces cerevisiae, respectively, using the yeast surface display system at a high copy number. In addition, two whole-cell biosensors were constructed through the immobilization of the above yeast cells on electrodes, for electrochemical detection of glucose and cholesterol. The assay time was 8.5 s for the glucose biosensors and 30 s for the cholesterol biosensors. Under optimal conditions, the cholesterol biosensor exhibited a linear range from 2 to 6 mmol·L−1. The glucose biosensor responded efficiently to the presence of glucose at a concentration range of 20–600 mg·dL−1 (1.4–33.3 mmol·L−1) and showed excellent anti-xylose interference properties. Both biosensors exhibited good performance at room temperature and remained stable over a three-week storage period.

Perspective—Surface-display Techniques in Electrochemical Biosensor Designs for Health Monitoring

“…62 Among the most frequently used yeast for enzyme surface display is Saccharomyces cerevisiae. [63][64][65][66] While the utilization of yeast for surface display provides advantages, it has certain drawbacks, specifically (1) yeast cell growth is significantly slower than that of most bacteria, and (2) yeast surface display has low transformation efficiency limiting the enzyme mutant library size of directed-evolution-based engineering strategies in yeast. On the other hand, prokaryotic surface-display systems most commonly use E.coli as a host for cell surface display of enzymes due to its well-established genetic toolbox and its ability to achieve highdensity surface display of full-length recombinant proteins.…”

Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications

“…Both constitutive and inducible promoters have been used for displaying proteins. The most common promoters used are the galactose-promoter (GAL1/GAL10) for expression in S. cerevisiae ( Schröter et al, 2018 ; Zhao et al, 2020b ), GAP and AOX1 promoters for expression in Pichia pastoris ( Yang et al, 2017 ; Li et al, 2019 ), and TEF1 and hp4d promoters for expression in Y. lipolytica ( Yuzbasheva et al, 2011 ; Moon et al, 2013 ). Recently, GAPDH, GPD or stress-induced (SED1) promoters have been used for protein expression in S. cerevisiae ( Inokuma et al, 2014 ; Zhang et al, 2019 ; Nguyen et al, 2020 ), proving to be appropriate alternatives to the galactose-induced promoter.…”

“…Animal vaccines have been developed which include vaccines against Toxoplasma gondii ( Wang et al, 2018 ), a vaccine against hemorrhagic disease of grass carp ( Luo et al, 2015 ) and an anti-tick vaccine ( Trentelman et al, 2021 ). Other relevant applications are the development of antibiotics ( Chun et al, 2020 ), biosensors to detect blood biochemical parameters ( Zhao et al, 2020b ) and the expression of hydrophobins ( Andreu et al, 2021 ).…”

Saccharomyces cerevisiae cell surface display technology: Strategies for improvement and applications

“…Cell surface engineering yeast can also be used in biosensors, protein library screening and other fields ( Han et al, 2018 ; Zhao et al, 2021 ). Cell surface display of fluorescent proteins exerts less metabolic stress on cells than intracellular expression.…”