Organizing heterologous biosyntheses inside bacterial cells can alleviate common problems owing to toxicity, poor kinetic performance, and cofactor imbalances. A subcellular organelle known as a bacterial microcompartment, such as the 1,2-propanediol utilization microcompartment of Salmonella, is a promising chassis for this strategy. Here we demonstrate de novo design of the N-terminal signal sequences used to direct cargo to these microcompartment organelles. We expand the native repertoire of signal sequences using rational and library-based approaches and show that a canonical leucine-zipper motif can function as a signal sequence for microcompartment localization. Our strategy can be applied to generate new signal sequences localizing arbitrary cargo proteins to the 1,2-propanediol utilization microcompartments.
Biotechnological processes use microbes to convert abundant molecules, such as glucose, into high-value products, such as pharmaceuticals, commodity and fine chemicals, and energy. However, from the outset of the development of a new bioprocess, it is difficult to determine the feasibility, expected yields, and targets for engineering. In this review, we describe a methodology that uses rough estimates to assess the feasibility of a process, approximate the expected product titer of a biological system, and identify variables to manipulate in order to achieve the desired performance. This methodology uses estimates from literature and biological intuition, and can be applied in the early stages of a project to help plan future engineering. We highlight recent literature examples, as well as two case studies from our own work, to demonstrate the use and power of rough estimates. Describing and predicting biological function using estimates guides the research and development phase of new bioprocesses and is a useful first step to understand and build a new microbial factory.
Bacterial microcompartments are a class of proteinaceous organelles comprising a 12 characteristic protein shell enclosing a set of enzymes. Compartmentalization can prevent escape of 13 volatile or toxic intermediates, prevent off-pathway reactions, and create private cofactor pools. 14 Encapsulation in synthetic microcompartment organelles will enhance the function of heterologous 15 pathways, but to do so, it is critical to understand how to control diffusion in and out of the 16 microcompartment organelle. To this end, we explored how small differences in the shell protein structure 17 result in changes in the diffusion of metabolites through the shell. We found that the ethanolamine 18 utilization (Eut) protein EutM properly incorporates into the 1,2-propanediol utilization (Pdu) 19 microcompartment, altering native metabolite accumulation and the resulting growth on 1,2-propanediol 20 as the sole carbon source. Further, we identified a single pore-lining residue mutation that confers the 21 same phenotype as substitution of the full EutM protein, indicating that small molecule diffusion through 22 the shell is the cause of growth enhancement. Finally, we show that the hydropathy index and charge of 23 pore amino acids are important indicators to predict how pore mutations will affect growth on 1,2-24
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.