Heterologous expression of cyanobacterial gas vesicle proteins in Saccharomyces cerevisiae

Abstract

Given the potential applications of gas vesicles (GVs) in multiple fields including antigen-displaying and imaging, heterologous reconstitution of synthetic GVs is an attractive and interesting study that has translational potential. Here, we attempted to express and assemble GV proteins (GVPs) into GVs using the model eukaryotic organism Saccharomyces cerevisiae. We first selected and expressed two core structural proteins, GvpA and GvpC from cyanobacteria Anabaena flos-aquae and Planktothrix rubescens, respectively. We then optimized the protein production conditions and validated GV assembly in the context of GV shapes. We found that when two copies of anaA were integrated into the genome, the chromosomal expression of AnaA resulted in GV production regardless of GvpC expression. Next, we co-expressed chaperone-RFP with the GFP-AnaA to aid the AnaA aggregation. The co-expression of individual chaperones (Hsp42, Sis1, Hsp104, and GvpN) with AnaA led to the formation of larger inclusions and enhanced the sequestration of AnaA into the perivacuolar site. To our knowledge, this represents the first study on reconstitution of GVs in S. cerevisiae. Our results could provide insights into optimizing conditions for heterologous protein production as well as the reconstitution of other synthetic microcompartments in yeast. K E Y W O R D Scellular aging, gas vesicle protein, gas vesicle, protein aggregation, spatial protein quality control, yeast INTRODUCTIONGas vesicles (GVs) are gas-filled proteinaceous intracellular compartments found in several microbes such as cyanobacteria. GVs are observed as spindle-and cylindrical-shapes which form small bicone Abbreviations: CFW, calcofluor white; GV, gas vesicle; GVP, gas vesicle protein; MRI, magnetic resonance imaging; RR, repeat region; RT, room temperature; SDD-AGE, semi-denaturing detergent agarose gel electrophoresis; TEM, transmission electron microscope; WT, wild-type structures which then extend to develop as mature GVs. GVs increase cellular buoyancy thus facilitating the upward movement in water columns. [1] The wall of GV is primarily formed by extremely high hydrophobic GV protein A or B (GvpA/B, 7-8 kDa), which is attached to the GvpC in some species to strengthen the GV structure. Specifically, an NMR study shows that the secondary structure of GvpA contains two α-helix separated by two antiparallel β-sheets forming an asymmetric dimer via its β-sheets of GvpA. This leads to the formation of

Elucidating the Assembly of Gas Vesicles by Systematic Protein-Protein Interaction Analysis

“…GvpF is the primary binding partner of GvpB (Figures 4D & 5A), an interaction that is superseded by the secondary binding of GvpG (Figures 3C & 5B). We propose that GvpF and GvpG are the chaperones of GvpB and prevent it from becoming thermodynamically trapped and forming proteotoxic amyloid species 43,54 . GvpF was observed to bind the first α-helix of haloarchaeal shell protein GvpA 41 , supporting a co-translational chaperoning.…”

“…First, we hypothesized that the number of E. coli transformants may reflect the cellular burden of the GV proteins being expressed, since leaky expression can still occur without chemical induction during the growth of cells on agar plates. Among GV proteins, the major shell protein, GvpB, has a highly hydrophobic surface, which is known to cause aggregation and proteotoxicity 43 . Indeed, we observed that every transformation that included expression of a fusion protein of GvpB led to a decrease in the total number of transformants (Figure 6A and Table S3).…”

Designing Intracellular Compartments for Efficient Engineered Microbial Cell Factories

“…There are also many prokaryotic organelles, such as gas vesicles, that have been artificially constructed in engineered strains such as Halobacterium salinarum , Escherichia coli, and Saccharomyces cerevisiae . Many synthetic scaffolds, such as nucleic acid interaction partners and protein interaction partners, have been developed to shorten the distance between enzymes by forming substrate delivery microcompartments and act as a controllable tool for constructing ASCs to promote the synthesis of target products . Since these ASCs have not been extensively exploited to promote metabolic synthesis in MCFs, we will not review them extensively here.…”

Transporter-Driven Engineering of a Genetic Biosensor for the Detection and Production of Short-Branched Chain Fatty Acids in Saccharomyces cerevisiae

“…For time course experiments, culture samples were collected every 12 h to determine the SBCFA levels and cell density (OD 600 ) was measured by a spectrophotometer BioPhotometer Plus (Eppendorf, Germany). The culture samples were diluted by SCD-U with KPB to adjust the cell concentration and the fluorescence intensity was measured on a Synergy H1 microplate reader and normalized to the cell density (Jung et al, 2021) as described above.…”