Compartmentalization of designed metabolic pathways within protein based nanocompartments has the potential to increase reaction efficiency in multi-step biosynthetic reactions. We previously demonstrated proof-of-concept of this aim by targeting a functional enzyme to single cellular protein nanocompartments, which were formed upon recombinant expression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK in Escherichia coli. To optimize this system, increasing overall encapsulated enzyme reaction efficiency, factor(s) required for the production of more than one nanocompartment per cell must be identified. In this work we report that the cupin domain protein EutQ is required for assembly of more than one nanocompartment per cell. Overexpression of EutQ results in multiple nanocompartment assembly in our recombinant system. EutQ specifically interacts with the shell protein EutM in vitro via electrostatic interactions with the putative cytosolic face of EutM. These findings lead to the theory that EutQ could facilitate multiple nanocompartment biogenesis by serving as an assembly hub for shell proteins. This work offers insights into the biogenesis of Eut bacterial microcompartments, and also provides an improved platform for the production of protein based nanocompartments for targeted encapsulation of enzyme pathways.

Serofendic acid is a natural neuroprotective molecule found in fetal calf serum. It is able to protect neurons against mechanisms of cell death associated with neurodegenerative disease. Because only trace quantities are present in fetal calf serum and complete chemical syntheses are long and inefficient, its development as a therapeutic agent has been slow. We engineered a heterologous metabolic pathway in Streptomyces to produce a late-stage synthetic intermediate, ent-atiserenoic acid, at high titers. We completed the total synthesis of serofendic acid from this intermediate in four steps.

Advances in DNA synthesis and assembly technology allow for the high-throughput fabrication of hundreds to thousands of multi-part genetic constructs in a short time. This allows for rapid hypothesis-testing and genetic optimization in multi-gene biological systems. Here, we discuss key considerations to design and implement an algorithmic DNA assembly pipeline that provides the freedom to change nearly any design variable in a multi-gene system. In addition to considerations for pipeline design, we describe protocols for three useful molecular biology techniques in plasmid construction.