Emerging enzyme surface display systems for waste resource recovery.

The current century marks an inflection point for human progress, as the developed world increasingly comes to recognize that the ecological and socioeconomic impacts of resource extraction must be balanced with more sustainable modes of growth that are less reliant on non-renewable sources of energy and materials. This has opened a window of opportunity for cross-sector development of biotechnologies that harness the metabolic problem-solving power of microbial communities. In this context, recovery has emerged as an organizing principal to create value from industrial and municipal waste streams, and the search is on for new enzymes and platforms that can be used for waste resource recovery at scale. Enzyme surface display on cells or functionalized materials has emerged as a promising platform for waste valorization. Typically, surface display involves the use of substrate binding or catalytic domains of interest translationally fused with extracellular membrane proteins in a microbial chassis. Novel display systems with improved performance features include S-layer display with increased protein density, spore display with increased resistance to harsh conditions, and intracellular inclusions including DNA-free cells or nanoparticles with improved social licence for in situ applications. Combining these display systems with advances in bioprinting, electrospinning and high-throughput functional screening have potential to transform outmoded extractive paradigms into 'trans-metabolic" processes for remediation and waste resource recovery within an emerging circular bioeconomy.

Reducing metabolic burden in the PACEmid evolver system by remastering high-copy phagemid vectors.

Orthogonal or non-cross-reacting transcription factors are used in synthetic biology as components of genetic circuits. Brödel et al. (2016) engineered 12 such cIλ transcription factor variants using a directed evolution 'PACEmid' system. The variants operate as dual activator/repressors and expand gene circuit construction possibilities. However, the high-copy phagemid vectors carrying the cIλ variants imposed high metabolic burden upon cells. Here, the authors 'remaster' the phagemid backbones to relieve their burden substantially, exhibited by a recovery in Escherichia coli growth. The remastered phagemids' ability to function within the PACEmid evolver system is maintained, as is the cIλ transcription factors' activity within these vectors. The low-burden phagemid versions are more suitable for use in PACEmid experiments and synthetic gene circuits; the authors have, therefore, replaced the original high-burden phagemids on the Addgene repository. The authors' work emphasises the importance of understanding metabolic burden and incorporating it into design steps in future synthetic biology ventures.