Smith, O. B. | Search bioRxiv

bioRxiv2024

High-throughput optimisation of protein secretion in yeast via an engineered biosensor.

Secretion of high value proteins and enzymes is fundamental to the synthetic biology economy; allowing continuous fermentation during production and protein purification without cell lysis. Most eukaryotic protein secretion is encoded by an N-terminal signal peptide; however, the strong impact of signal peptide sequence variation on the secretion efficiency of a given protein is not well defined. Despite high natural signal peptide sequence diversity, most recombinant protein secretion systems employ only a few well characterised signal peptides. Additionally, the selection of promoters and terminators can significantly affect secretion efficiency, yet screening numerous genetic constructs for optimal sequences remains inefficient. Here, we have adapted a yeast G-protein coupled receptor biosensor, to measure the concentration of a peptide tag that is co-secreted with any protein of interest. Protein secretion efficiency can thus be quantified via the induction of a fluorescent reporter that is upregulated downstream of receptor activation. This enables high-throughput screening of over 6000 combinations of promoters, signal peptides and terminators, assembled using one-pot Combinatorial Golden Gate cloning. We demonstrate this biosensor can quickly identify best combinations for secretion and quantify secretion levels. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=188 HEIGHT=200 SRC="FIGDIR/small/594099v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@87d612org.highwire.dtl.DTLVardef@1f73d24org.highwire.dtl.DTLVardef@cf35a7org.highwire.dtl.DTLVardef@1cb8a3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Identification and characterization of a bacterial periplasmic solute binding protein that binds L-amino acid amides.

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds O_SCPLOWLC_SCPLOW-amino acid amides but not the corresponding O_SCPLOWLC_SCPLOW-amino acids. An X-ray crystal structure of MeAmi_SBP bound to O_SCPLOWLC_SCPLOW-serinamide highlights the residues that impart distinct specificity for O_SCPLOWLC_SCPLOW-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved amongst the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake and metabolism of O_SCPLOWLC_SCPLOW-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with O_SCPLOWLC_SCPLOW-amino acid amides and bacterial nitrogen utilization.

biochemistry↗