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Virus-like particles (VLPs) are self-assembling protein nanoparticles that have great promise as vectors for drug delivery. VLPs are derived from viruses but retain none of their infection or replication capabilities. These protein particles have defined surface chemistries, uniform sizes, and stability properties that make them attractive starting points for drug-delivery scaffolds. Here, we review recent advances in tailoring VLPs for drug-delivery applications, including VLP platform engineering approaches as well as methods for cargo loading, activation, and release. Finally, we highlight several successes using VLPs for drug delivery in model systems. • Virus-like particles (VLPs) are versatile structuresfor drug-delivery applications. • Mutations to VLP structural proteins can improve VLP drug delivery. • VLP–cargo loading, release, and activation are key traits for drug delivery. [ABSTRACT FROM AUTHOR]

Small molecule production in microbial hosts is limited by the accumulation of the product inside the cell. Efflux transporters show promise as a solution to removal of the often-toxic products. Recent advances in transporter identification through expression profiling, heterologous expression, and knockout studies have identified transporters capable of secreting compounds of biotechnological interest. In addition, engineering of well-studied transporters has shown that substrate specificity in these transporters is malleable. Future work in identification, engineering, and expression of small molecule exporters can be instrumental in expanding the biocatalysis portfolio. [ABSTRACT FROM AUTHOR]

Highlights: ▶ Enzyme engineering enables the development of green industrial processes. ▶ The conversion of biomass to chemicals relies heavily on engineered enzymes. ▶ In vitro catalytic processes employ engineered enzymes to produce key intermediates. [Copyright &y& Elsevier]

Advances in metabolic engineering have given rise to the biological production of novel fuels and chemicals, but yields are often low without significant optimization. One generalizable solution is to create a specialized organelle for the sequestration of engineered metabolic pathways. Bacterial microcompartments are an excellent scaffold for such an organelle. These compartments consist of a porous protein shell that encapsulates enzymes. To repurpose these structures, researchers have begun to determine how the protein shell is assembled, how pores may be used to control small molecule transport across the protein shell, and how to target heterologous enzymes to the compartment interior. With these advances, it will soon be possible to use engineered forms of these protein shells to create designer organelles. [ABSTRACT FROM AUTHOR]

The Escherichia coli genome encodes at least 29 putative signal peptides containing a twin arginine motif characteristic of proteins exported via the twin arginine translocation (Tat) pathway. Fusions of the putative Tat signal peptides plus six to eight amino acids of the mature proteins to three reporter proteins (short-lived green fluorescent protein, maltose-binding protein (MBP), and alkaline phosphatase) and also data from the cell localization of epitope-tagged full-length proteins were employed to determine the ability of the 29 signal peptides to direct export through the Tat pathway, through the general secretory pathway (Sec), or through both. 27/29 putative signal peptides could export one or more reporter proteins through Tat. Of these, 11 signal peptides displayed Tat specificity in that they could not direct the export of Sec-only reporter proteins. The rest (16/27) were promiscuous and were capable of directing export of the appropriate reporter either via Tat (green fluorescent protein, MBP) or via Sec (PhoA, MBP). Mutations that conferred a ≥ + 1 charge to the N terminus of the mature protein abolished or drastically reduced routing through the Sec pathway without affecting the ability to export via the Tat pathway. These experiments demonstrate that the charge of the mature protein N terminus affects export promiscuity, independent of the effect of the folding state of the mature protein. [ABSTRACT FROM AUTHOR]

[Display omitted] • MCPs are organelles uniquely suited for metabolic engineering applications. • MCPs increase flux while protecting cells from toxic intermediates. • Insights into MCP structure and function have enabled engineering efforts. • Shell proteins assemble into large structures that can be used as designer scaffolds. Organization of metabolic processes within the space of a cell is critical for the survival of many organisms. In bacteria, spatial organization is achieved via proteinaceous organelles called bacterial microcompartments, which encapsulate pathway enzymes, substrates, and co-factors to drive the safe and efficient metabolism of niche carbon sources. Microcompartments are self-assembled from shell proteins that encapsulate a core comprising various enzymes. This review discusses how recent advances in understanding microcompartment structure and assembly have informed engineering efforts to repurpose compartments and compartment-based structures for non-native functions. These advances, both in understanding of the native structure and function of compartments, as well as in the engineering of new functions, will pave the way for the use of these structures in bacterial cell factories. [ABSTRACT FROM AUTHOR]

• Pdu MCPs are promising scaffolds for the encapsulation of non-native pathways. • Multiple non-native cargo proteins can be encoded on the genome and co-encapsulated. • Cargo encapsulation levels vary with different tags and genomic loci. • Cargo encapsulation is independent of expression for genomically expressed proteins. 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. [ABSTRACT FROM AUTHOR]

Various bacteria localize metabolic pathways to proteinaceous organelles known as bacterial microcompartments (MCPs), enabling the metabolism of carbon sources to enhance survival and pathogenicity in the gut. There is considerable interest in exploiting bacterial MCPs for metabolic engineering applications, but little is known about the interactions between MCP signal sequences and the protein shells of different MCP systems. We found that the N-terminal sequences from the ethanolamine utilization (Eut) and glycyl radical-generating protein MCPs are able to target reporter proteins to the 1,2-propanediol utilization (Pdu) MCP, and that this localization is mediated by a conserved hydrophobic residue motif. Recapitulation of this motif by the addition of a single amino acid conferred targeting function on an N-terminal sequence from the ethanol utilization MCP system that previously did not act as a Pdu signal sequence. Moreover, the Pdu-localized signal sequences competed with native Pdu targeting sequences for encapsulation in the Pdu MCP. Salmonella enterica natively possesses both the Pdu and Eut operons, and our results suggest that Eut proteins might be localized to the Pdu MCP in vivo. We further demonstrate that S. enterica LT2 retained the ability to grow on 1,2-propanediol as the sole carbon source when a Pdu enzyme was replaced with its Eut homolog. Although the relevance of this finding to the native system remains to be explored, we show that the Pdu-localized signal sequences described herein allow control over the ratio of heterologous proteins encapsulated within Pdu MCPs. [ABSTRACT FROM AUTHOR]