TY - JOUR
T1 - A genomic integration platform for heterologous cargo encapsulation in 1,2-propanediol utilization bacterial microcompartments
AU - Nichols, Taylor M.
AU - Kennedy, Nolan W.
AU - Tullman-Ercek, Danielle
N1 - Funding Information:
The authors would like to thank the Tullman-Ercek group, and specifically Svetlana Ikonomova, Carolyn Mills, and Han Teng Wong, for helpful comments and discussions during the preparation of this manuscript. Thanks also to Christopher Jakobson for helpful discussions for the design and construction of strains. This work was supported by the Army Research Office (grant W911NF-16-1-0169 to DTE) and the Department of Energy (grant DE-SC0019337 to DTE). TMN and NWK were supported by the National Science Foundation Graduate Research Fellowship Program (grant DGE-1842165). NWK was supported in part by the National Institutes of Health Training Grant (T32GM008449) through Northwestern University's Biotechnology Training Program. This work made use of the EPIC facility of Northwestern University's NUANCE Center, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205); the MRSEC program (NSF DMR-1720139) at the Materials Research Center; the International Institute for Nanotechnology (IIN); the Keck Foundation; and the State of Illinois, through the IIN.
Funding Information:
The authors would like to thank the Tullman-Ercek group, and specifically Svetlana Ikonomova, Carolyn Mills, and Han Teng Wong, for helpful comments and discussions during the preparation of this manuscript. Thanks also to Christopher Jakobson for helpful discussions for the design and construction of strains. This work was supported by the Army Research Office (grant W911NF-16-1-0169 to DTE ) and the Department of Energy (grant DE- SC0019337 to DTE ). TMN and NWK were supported by the National Science Foundation Graduate Research Fellowship Program (grant DGE-1842165 ). NWK was supported in part by the National Institutes of Health Training Grant ( T32GM008449 ) through Northwestern University's Biotechnology Training Program. This work made use of the EPIC facility of Northwestern University’s NUANCE Center, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205); the MRSEC program (NSF DMR-1720139) at the Materials Research Center; the International Institute for Nanotechnology (IIN); the Keck Foundation; and the State of Illinois, through the IIN.
Publisher Copyright:
© 2020
PY - 2020/4/15
Y1 - 2020/4/15
N2 - Bacterial microcompartments (MCPs) are protein structures that encapsulate specific metabolic pathways in bacteria. The 1,2-propanediol utilization (Pdu) MCP in Salmonella enterica serovar Typhimurium LT2 encapsulates the pathway for 1,2-propanediol degradation to sequester a toxic intermediate, enable cofactor recycling, and enhance pathway flux. The Pdu MCP has potential as an enclosed scaffold for metabolic engineering applications. To successfully use Pdu MCPs for this purpose, however, methods to enable and control heterologous cargo encapsulation are critical. To this end, we here developed a genomic expression platform for cargo encapsulation in Pdu MCPs. We integrated signal sequence-tagged fluorescent reporters into the pdu operon in place of native Pdu enzymes and evaluated the resulting expression and encapsulation levels. We found that fluorescent reporters were successfully co-encapsulated, with varying relative encapsulation levels achieved when using different integration locus and signal sequence combinations. We also observed that the native Pdu signal sequences mediated different encapsulation efficiencies independent of expression levels. This work establishes the genomic integration platform as a viable method for controlling cargo encapsulation, expanding the toolkit toward engineering the Pdu MCP as a tunable nanobioreactor.
AB - Bacterial microcompartments (MCPs) are protein structures that encapsulate specific metabolic pathways in bacteria. The 1,2-propanediol utilization (Pdu) MCP in Salmonella enterica serovar Typhimurium LT2 encapsulates the pathway for 1,2-propanediol degradation to sequester a toxic intermediate, enable cofactor recycling, and enhance pathway flux. The Pdu MCP has potential as an enclosed scaffold for metabolic engineering applications. To successfully use Pdu MCPs for this purpose, however, methods to enable and control heterologous cargo encapsulation are critical. To this end, we here developed a genomic expression platform for cargo encapsulation in Pdu MCPs. We integrated signal sequence-tagged fluorescent reporters into the pdu operon in place of native Pdu enzymes and evaluated the resulting expression and encapsulation levels. We found that fluorescent reporters were successfully co-encapsulated, with varying relative encapsulation levels achieved when using different integration locus and signal sequence combinations. We also observed that the native Pdu signal sequences mediated different encapsulation efficiencies independent of expression levels. This work establishes the genomic integration platform as a viable method for controlling cargo encapsulation, expanding the toolkit toward engineering the Pdu MCP as a tunable nanobioreactor.
KW - Bacterial microcompartments
KW - Cargo encapsulation
KW - Genomic integration
KW - Metabolic engineering
KW - Pdu
KW - Salmonella
UR - http://www.scopus.com/inward/record.url?scp=85078681326&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85078681326&partnerID=8YFLogxK
U2 - 10.1016/j.bej.2020.107496
DO - 10.1016/j.bej.2020.107496
M3 - Article
AN - SCOPUS:85078681326
VL - 156
JO - Biochemical Engineering Journal
JF - Biochemical Engineering Journal
SN - 1369-703X
M1 - 107496
ER -