Improving plastic degrading enzymes via directed evolution.

Plastic degrading enzymes have immense potential for use in industrial applications. Protein engineering efforts over the last decade have resulted in considerable enhancement of many properties of these enzymes. Directed evolution, a protein engineering approach that mimics the natural process of evolution in a laboratory, has been particularly useful in overcoming some of the challenges of structure-based protein engineering. For example, directed evolution has been used to improve the catalytic activity and thermostability of polyethylene terephthalate (PET)-degrading enzymes, although its use for the improvement of other desirable properties, such as solvent tolerance, has been less studied. In this review, we aim to identify some of the knowledge gaps and current challenges, and highlight recent studies related to the directed evolution of plastic-degrading enzymes.

Multiple classes and isoforms of the RNA polymerase recycling motor protein HelD.

Efficient control of transcription is essential in all organisms. In bacteria, where DNA replication and transcription occur simultaneously, the replication machinery is at risk of colliding with highly abundant transcription complexes. This can be exacerbated by the fact that transcription complexes pause frequently. When pauses are long-lasting, the stalled complexes must be removed to prevent collisions with either another transcription complex or the replication machinery. HelD is a protein that represents a new class of ATP-dependent motor proteins distantly related to helicases. It was first identified in the model Gram-positive bacterium Bacillus subtilis and is involved in removing and recycling stalled transcription complexes. To date, two classes of HelD have been identified: one in the low G+C and the other in the high G+C Gram-positive bacteria. In this work, we have undertaken the first comprehensive investigation of the phylogenetic diversity of HelD proteins. We show that genes in certain bacterial classes have been inherited by horizontal gene transfer, many organisms contain multiple expressed isoforms of HelD, some of which are associated with antibiotic resistance, and that there is a third class of HelD protein found in Gram-negative bacteria. In summary, HelD proteins represent an important new class of transcription factors associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.

Glycoengineering design options for IgG1 in CHO cells using precise gene editing.

Precise gene editing technologies are providing new opportunities to stably engineer host cells for recombinant production of therapeutic glycoproteins with different glycan structures. The glycosylation of recombinant therapeutics has long been a focus for both quality and consistency of products and for optimizing and improving pharmacokinetic properties as well as bioactivity. Structures of glycans on therapeutic glycoproteins are important for circulation, biodistribution and bioactivity. In particular, the latter has been demonstrated for therapeutic IgG1 antibodies where the core α1,6Fucose on the conserved N-glycan at Asn297 have remarkable dampening effects on antibody effector functions. We previously explored precise gene engineering and design options for N-glycosylation in CHO cells, and here we focus on engineering options possible for N-glycans on human IgG1. We demonstrate stable precise gene engineering of rather homogenous biantennary N-glycans with and without galactose (G0F, G2F) as well as the α2,6-linked monosialylated (G2FS1) glycoform. We were unable to introduce substantial disialylated glycoforms. Instead we engineered a novel monoantennary homogeneous N-glycan design with complete α2,6-linked sialic acid capping. All N-glycoforms may be engineered with and without core α1,6Fucose. The stably engineered design options enable production of human IgG antibodies with an array of distinct glycoforms for testing and selection of optimal design for different therapeutic applications.