Genetically-encoded biosensors for analyzing and controlling cellular process in yeast
Introduction
Yeast has been a robust workhorse to manufacture bioproducts and enable the expression of a broad range of heterologous pathways for various biotechnological applications. This is partly because of the existence of the spatially separated organelles to allow for gene expression and specialized metabolic activity taking place at various cellular locations and time-scales. For example, gene transcription and mRNA translation are separated by nucleus membrane. Catabolic and anabolic metabolism are distinctly localized in mitochondria, peroxisome and cytoplasm et al. The separation and compartmentalization of different genetic and metabolic events presents us the opportunity to precisely control and program gene expression for more advanced biological functions. Other industrially-relevant traits include its tolerance to a number of biotic or abiotic stress factors, such as acids, reactive oxygen species, carbon or nitrogen starvations, osmotic pressure, and mechanical shear force. These genetic and phenotypic advantage have made yeast a powerful host organism for production of biofuels [1, 2, 3], commodity chemicals [4], natural products [5] and pharmaceuticals [6, 7, 8].
As for fermentation process, we have been able to monitor cell metabolism by tracking cell growth, pH, oxygen uptake rate, the CO2 emission rate, respiratory quotient and a number of nutrients including glucose, glutamate, lactic acid, ammonia et al. These parameters allow us to precisely interpret cell physiology and predict the microbial process kinetics as well as apply control strategies to improve the economics of industrial fermentation [9,10]. As we move forward in the post-genomic era, one area that is largely underexplored is to integrate genetically encoded biosensors with hardwired optical sensors or electrochemical signals that may translate the metabolic activity into readable output, permitting us to rapidly screen mutant strains and seamlessly optimize fermentation process [11•]. It is hoped that sensors, or ‘electrodes’ that could be installed inside the cell to forecast or troubleshoot the complicated cellular process.
Biosensing in eukaryote, like yeast, is more complicated compared to simple prokaryotic organisms like bacteria. Gene transcription involves the recruitment of multiple transcriptional factors and associated proteins into the nucleus; signal relay in yeast requires the activity of multiple phosphorylase and dephosphorylase to bridge the gap between receptor and the actuator proteins. An essential feature of eukaryote biosensing is the cooperative assembly and de-assembly of multiple regulatory proteins leading to the complex/nonlinear signal processing and cellular decision-making functions [12••]. The dynamic response, sensitivity and operational range of these genetically encoded sensors are determined by the molecular structure and interactions of the related proteins and DNAs [13••]. In this short review, we will cover the design principles of engineering yeast-based allosteric transcriptional factors, and novel biosensors including GPCR-based sensors and optogenetic-based sensors in yeast (Figure 1).
Section snippets
Allosteric transcriptional factor-based biosensors in yeast
Allosteric transcriptional factors (aTF) consist of a DNA binding domain (DBD) and an effector-binding domain (EBD). The DBD of aTF will specifically interact with a cis-regulatory DNA sequence (generally called operator or enhancer) adjacent to the promoter to restrict or enhance the access of RNA polymerase (RNAP), thus repressing or activating gene transcription [9,14]. The EBD of aTF is the sensor domain that can bind with small molecules or environmental stress factors (heavy metals,
GPCR-based biosensors in yeast
G-protein coupled receptors (GPCRs) are large and diverse family of cell surface receptors that respond to an enormous amount of cell signals, including neurotransmitters, hormones, carbon/nitrogen sources and sensations (tastes and odors) [30] et al. Binding of the signaling molecules or the ligand to the GPCRs result in G-protein activation which then triggers the production of other secondary messengers. These molecular systems allow the rapid and dynamic transmission of biochemical
Optogenetics-based biochemical control in yeast
The use of chemical inducer as input signal is powerful to control cellular regulatory networks and reprogram gene expression. But these chemical inducers are less hardwired with our commonly used optoelectrical devise that could give us direct signal to initiate any feedback control. Light-controlled gene expression and protein assembly have been suggested as an extremely useful solution to dissect and analyze cellular network function. It will improve our ability to interrogate and understand
Conclusions and perspectives
For the purpose of pathway engineering, the expression of heterologous pathway to redirect carbon flux toward the targeted metabolism is a challenging task. This may compete with the native metabolism, and the buildup of nonnative metabolites may elicit cellular stress response. It is important to install sensors to control the dosage and temporospatial expression of enzyme [41]. Yeast has been an excellent platform that allows for temporospatial separation of genetic and metabolic events. The
Conflicts of interest statement
Nothing declared.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This work was supported by the Cellular & Biochem Engineering Program of the National Science Foundation under grant no.1805139 and Bill & Melinda Gates Foundation (grant number OPP1188443). The authors would also like to acknowledge the Department of Chemical, Biochemical and Environmental Engineering at University of Maryland Baltimore County for funding support.
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These authors contributed equally to this article.