Glycans and glycosylated biomolecules are directly involved in almost every biological process as well as the etiology of most major diseases. Hence, glycoscience knowledge is essential to efforts aimed at addressing fundamental challenges in understanding and improving human health, protecting the environment and enhancing energy security, and developing renewable and sustainable resources that can serve as the source of next-generation materials. While much progress has been made, there remains an urgent need for new tools that can overexpress structurally uniform glycans and glycoconjugates in the quantities needed for characterization and that can be used to mechanistically dissect the enzymatic reactions and multi-enzyme assembly lines that promote their construction. To address this technology gap, cell-free synthetic glycobiology has emerged as a simplified and highly modular framework to investigate, prototype, and engineer pathways for glycan biosynthesis and biomolecule glycosylation outside the confines of living cells. From nucleotide sugars to complex glycoproteins, we summarize here recent efforts that harness the power of cell-free approaches to design, build, test, and utilize glyco-enzyme reaction networks that produce desired glycomolecules in a predictable and controllable manner. We also highlight novel cell-free methods for shedding light on poorly understood aspects of diverse glycosylation processes and engineering these processes toward desired outcomes. Taken together, cell-free synthetic glycobiology represents a promising set of tools and techniques for accelerating basic glycoscience research (e.g., deciphering the “glycan code”) and its application (e.g., biomanufacturing high-value glycomolecules on demand).

Summary Few studies have described chromosomal dynamics in bacterial cells with more than two complete chromosome copies or described changes with respect to development in polyploid cells. We examined the arrangement of chromosomal loci in the very large, highly polyploid, uncultivated intestinal symbiont Epulopiscium sp. type B using fluorescent in situ hybridization. We found that in new offspring, chromosome replication origins (oriCs) are arranged in a three‐dimensional array throughout the cytoplasm. As development progresses, most oriCs become peripherally located. Siblings within a mother cell have similar numbers of oriCs. When chromosome orientation was assessed in situ by labeling two chromosomal regions, no specific pattern was detected. The Epulopiscium genome codes for many of the conserved positional guide proteins used for chromosome segregation in bacteria. Based on this study, we present a model that conserved chromosomal maintenance proteins, combined with entropic demixing, provide the forces necessary for distributing oriCs. Without the positional regulation afforded by radial confinement, chromosomes are more randomly oriented in Epulopiscium than in most small rod‐shaped cells. Furthermore, we suggest that the random orientation of individual chromosomes in large polyploid cells would not hamper reproductive success as it would in smaller cells with more limited genomic resources.

Bradyrhizobium japonicum is a Gram-negative soil bacterium that can fix nitrogen into ammonia by developing a symbiotic relationship with the soybean plant. MocR proteins make up a subfamily of GntR superfamily, one of the most widely distributed and prolific groups of the helix-turn-helix transcription factors. In this study, we constructed a mutant strain for mocR (blr6977) to investigate its role in cellular processes and symbiosis in B. japonicum. Although growth rate and morphology of the mutant were indistinguishable from those of the wild type, the mutant showed significant differences in motility and attachment (i.e., biofilm formation) from the wild type. The mutant displayed a decrease in biofilm formation, but was more motile than the wild type. The inactivation of mocR did not affect the number of nodules on soybean roots, but caused delayed nodulation. Delayed nodulation intrigued us to study competitiveness of the mutant infecting soybeans. The mutant was less competitive than the wild type, indicating that delayed nodulation might be due to competitiveness. Gene expressions of other MocR subfamily members were also compared between the wild type and mutant strains. None of the mocR-like genes examined in this study were differentially expressed between both strains.

Escherichia coli remains one of the preferred hosts for biotechnological protein production due to its robust growth in culture and ease of genetic manipulation. It is often desirable to export recombinant proteins into the periplasmic space for reasons related to proper disulfide bond formation, prevention of aggregation and proteolytic degradation, and ease of purification. One such system for expressing heterologous secreted proteins is the twin-arginine translocation (Tat) pathway, which has the unique advantage of delivering correctly folded proteins into the periplasm. However, transit times for proteins through the Tat translocase, comprised of the TatABC proteins, are much longer than for passage through the SecYEG pore, the translocase associated with the more widely utilized Sec pathway. To date, a high protein flux through the Tat pathway has yet to be demonstrated. To address this shortcoming, we employed a directed coevolution strategy to isolate mutant Tat translocases for their ability to deliver higher quantities of heterologous proteins into the periplasm. Three supersecreting translocases were selected that each exported a panel of recombinant proteins at levels that were significantly greater than those observed for wild-type TatABC or SecYEG translocases. Interestingly, all three of the evolved Tat translocases exhibited quality control suppression, suggesting that increased translocation flux was gained by relaxation of substrate proofreading. Overall, our discovery of more efficient translocase variants paves the way for the use of the Tat system as a powerful complement to the Sec pathway for secreted production of both commodity and high value-added proteins.

19The twin-arginine translocation (Tat) pathway involves an inbuilt quality control (QC) 20 system that synchronizes proofreading of substrate protein folding with lipid bilayer 21 transport. However, the molecular details of this QC mechanism remain poorly 22 its irreversible aggregation and stabilizing the active species. Collectively, these results 1 suggest that the Tat translocase may use chaperone-like client recognition to monitor the 2 conformational status of its substrates. 3 4