Solution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol Bioreactor

Abstract

Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains containing the modified BMCs produce elevated levels of alcohol.

Localization of Proteins to the 1,2-Propanediol Utilization Microcompartment by Non-native Signal Sequences Is Mediated by a Common Hydrophobic Motif

“…Further investigation by NMR confirmed that the PduP N-terminal signal sequence adopts an ␣-helical conformation, providing support for this model of signal sequence-shell protein interaction (3). Only two signal sequences (both from natively encapsulated Pdu enzymes) have been identified and shown experimentally to localize proteins to the Pdu MCP, however, and a more diverse set of signal sequences with a variety of encapsulation levels is desirable for the encapsulation of heterologous pathways to allow tuning of the loading and stoichiometry of multiple heterologous enzymes in the Pdu MCP.…”

“…To observe Eut MCP formation, we used a fluorescent reporter of encapsulation in which EutC 1-20 is fused to GFP followed by a C-terminal ssrA tag, which mediates degradation of the fluorophore by the ClpXP protease in the cytosol (encapsulation in an MCP thus rescues the reporter from proteolysis). When expression of the encapsulation reporter construct pBADeutC [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] -gfp-ssrA was induced by the addition of 0.02% arabinose, fluorescent puncta were not observed in a ⌬eutR::FRT strain upon the addition of 30 mM ethanolamine and 150 nM vitamin B 12 , but puncta were observed in the ⌬pocR::FRT strain under the same conditions (Figs. 2 and 3).…”

“…EutC [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and EutE [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Direct Heterologous Proteins to the Pdu MCP-We next explored whether two signals sequences from the Eut MCP are promiscuous in their localization of heterologous proteins, that is, whether the N-terminal signal sequences EutC [1][2][3][4][5][6][7][8][9][10][11]…”

“…There is increasing interest in applying these organelles to the encapsulation of engineered enzymatic pathways to enhance pathway flux (3). Heterologous proteins can be directed to the 1,2-propanediol utilization (Pdu) MCPs, for example, by fusion of the N-terminal signal sequences from the natively encapsulated PduP and PduD proteins to the proteins of interest (4,5).…”

Facile methods for heterologous production of bacterial microcompartments in diverse host species

“…Among these, MCPs provide natural starting points for applications aimed at organizing metabolic processes (Kim and Tullman‐Ercek, 2013; Lawrence et al ., 2014). They are especially attractive for applications where limited molecular exchange between different subcellular regions is desirable, either to improve channelling of substrates through multiple enzymatic reactions, or to limit the escape of toxic metabolites or even toxic proteins (Yung et al ., 2017).…”