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.

Cyanobacteria are prolific producers of natural products, including polyketides and hybrid compounds thereof. Type III polyketide synthases (PKSs) are of particular interest, due to their wide substrate specificity and simple reaction mechanism, compared to both type I and type II PKSs. Surprisingly, only two type III PKS products, hierridins and (7.7)paracyclophanes, have been isolated from cyanobacteria. Here we report the mining of 517 cyanobacterial genomes for type III PKS biosynthesis gene clusters. Approximately 17% of the genomes analysed encoded one or more type III PKSs. Together with already characterised type III PKSs, the phylogeny of this group of enzymes was investigated. Our analysis showed that type III PKSs in cyanobacteria evolved into three major lineages, including enzymes associated with (i) (7.7)paracyclophane-like biosynthesis gene clusters, (ii) hierridin-like biosynthesis gene clusters, and (iii) cytochrome b5 genes. The evolutionary history of these enzymes is complex, with some sequences partitioning primarily according to speciation and others putatively according to their reaction type. Protein modelling showed that cyanobacterial type III PKSs generally have a smaller active site cavity (mean = 109.035 Å3) compared to enzymes from other organisms. The size of the active site did not correlate well with substrate size, however, the ‘Gatekeeper’ amino acid residues within the active site were strongly correlated to enzyme phylogeny. Our study provides unprecedented insight into the distribution, diversity and molecular evolution of cyanobacterial type III PKSs, which could facilitate the discovery, characterisation and exploitation of novel enzymes, biochemical pathways, and specialised metabolites from this biosynthetically talented clade of microorganisms.

Transcription elongation is punctuated by pauses that serve important functions in permitting correct folding of structural RNA, efficient coupling of transcription and translation, and ensuring efficient transcription termination at the correct site (Saba et al., 2019). Whilst most pausing events serve an important function, on occasion RNA polymerase (RNAP) is unable to restart transcription and must be removed from the DNA to prevent damaging collisions with the DNA replication machinery or other transcription

Protein N-glycosylation is an essential and highly conserved posttranslational modification found in all eukaryotic cells. Yeast, plants and mammalian cells, however, produce N-glycans with distinct structural features. These species-specific features not only pose challenges in selecting host cells for production of recombinant therapeutics for human medical use but also provide opportunities to explore and utilize species-specific glycosylation in design of vaccines. Here, we used reverse cross-species engineering to stably introduce plant core α3fucose (α3Fuc) and β2xylose (β2Xyl) N-glycosylation epitopes in the mammalian Chinese hamster ovary (CHO) cell line. We used directed knockin of plant core fucosylation and xylosylation genes (AtFucTA/AtFucTB and AtXylT) and targeted knockout of endogenous genes for core fucosylation (fut8) and elongation (B4galt1), for establishing CHO cells with plant N-glycosylation capacities. The engineering was evaluated through coexpression of two human therapeutic N-glycoproteins, erythropoietin (EPO) and an immunoglobulin G (IgG) antibody. Full conversion to the plant-type α3Fuc/β2Xyl bi-antennary agalactosylated N-glycosylation (G0FX) was demonstrated for the IgG1 produced in CHO cells. These results demonstrate that N-glycosylation in mammalian cells is amenable for extensive cross-kingdom engineering and that engineered CHO cells may be used to produce glycoproteins with plant glycosylation.

SUMMARYEfficient 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 protein 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. Therefore, HelD proteins represent an important new class of transcription factor associated with genome maintenance and antibiotic resistance that are conserved across the Eubacterial kingdom.