A crewed mission to and from Mars may include an exciting array of enabling biotechnologies that leverage inherent mass, power, and volume advantages over traditional abiotic approaches. In this perspective, we articulate the scientific and engineering goals and constraints, along with example systems, that guide the design of a surface biomanufactory. Extending past arguments for exploiting stand-alone elements of biology, we argue for an integrated biomanufacturing plant replete with modules for microbial in situ resource utilization, production, and recycling of food, pharmaceuticals, and biomaterials required for sustaining future intrepid astronauts. We also discuss aspirational technology trends in each of these target areas in the context of human and robotic exploration missions.

We develop fully glycosylated computational models of ACE2-Fc fusion proteins which are promising targets for a COVID-19 therapeutic. These models are tested in their interaction with a fragment of the receptor-binding domain (RBD) of the Spike Protein S of the SARS-CoV-2 virus, via atomistic molecular dynamics simulations. We see that some ACE2 glycans interact with the S fragments, and glycans are influencing the conformation of the ACE2 receptor. Additionally, we optimize algorithms for protein glycosylation modelling in order to expedite future model development. All models and algorithms are openly available.

Protein N-glycosylation is an important post-translational modification and has influences on a variety of biological processes at the cellular and molecular level, making glycosylation a major study aspect for glycoprotein-based therapeutics. To achieve a comprehensive understanding on how N-glycosylation impacts protein properties, an Fc-fusion anthrax decoy protein, viz rCMG2-Fc, was expressed in Nicotiana benthamiana plant with three types of N-glycosylation profiles. Three variants were produced by targeting protein to plant apoplast (APO), endoplasmic reticulum (ER) or removing the N-glycosylation site by a point mutation (Agly). Both the APO and ER variants had a complex-type N-glycan (GnGnXF) as their predominant glycans. In addition, ER variant had a higher concentration of mannose-type N-glycans (50%). The decoy protein binds to the protective antigen (PA) of anthrax through its CMG2 domain and inhibits toxin endocytosis. The protein expression, sequence, N-glycosylation profile, binding kinetics to PA, toxin neutralization efficiency, and thermostability were determined experimentally. In parallel, we performed molecular dynamics (MD) simulations of the predominant full-length rCMG2-Fc glycoform for each of the three N-glycosylation profiles to understand the effects of glycosylation at the molecular level. The MAN8 glycoform from the ER variant was additionally simulated to resolve differences between the APO and ER variants. Glycosylation showed strong stabilizing effects on rCMG2-Fc during in planta accumulation, evidenced by the over 2-fold higher expression and less protein degradation observed for glycosylated variants compared to the Agly variant. Protein function was confirmed by toxin neutralization assay (TNA), with effective concentration (EC 50 ) rankings from low to high of 67.6 ng/ml (APO), 83.15 ng/ml (Agly), and 128.9 ng/ml (ER). The binding kinetics between rCMG2-Fc and PA were measured with bio-layer interferometry (BLI), giving sub-nanomolar affinities regardless of protein glycosylation and temperatures (25 and 37°C). The protein thermostability was examined utilizing the PA binding ELISA to provide information on EC 50 differences. The fraction of functional ER variant decayed after overnight incubation at 37°C, and no significant change was observed for APO or Agly variants. In MD simulations, the MAN8 glycoform exhibits quantitatively higher distance between the CMG2 and Fc domains, as well as higher hydrophobic solvent accessible surface areas (SASA), indicating a possibly higher aggregation tendency of the ER variant. This study highlights the impacts of N-glycosylation on protein properties and provides insight into the effects of glycosylation on protein molecular dynamics.

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A crewed mission to and from Mars may include an exciting array of enabling biotechnologies that leverage inherent mass, power, and volume advantages over traditional abiotic approaches. In this perspective, we articulate the scientific and engineering goals and constraints, along with example systems, that guide the design of a surface biomanufactory. Extending past arguments for exploiting stand-alone elements of biology, we argue for an integrated biomanufacturing plant replete with modules for microbial \textit{in situ} resource utilization, production, and recycling of food, pharmaceuticals, and biomaterials required for sustaining future intrepid astronauts. We also discuss aspirational technology trends in each of these target areas in the context of human and robotic exploration missions in the coming century.

Kifunensine, a potent and selective inhibitor of class I α-mannosidases, prevents α-mannosidases I from trimming mannose residues on glycoproteins, thus resulting in oligomannose-type glycans. We report for the first time that through one-time vacuum infiltration of kifunensine in plant tissue, N-linked glycosylation of a recombinant protein transiently produced in whole-plants shifted completely from complex-type to oligomannose-type. Fc-fused capillary morphogenesis protein 2 (CMG2-Fc) containing one N-glycosylation site on the Fc domain, produced in Nicotiana benthamiana whole plants, served as a model protein. The CMG2-Fc fusion protein was produced transiently through vacuum agroinfiltration, with and without kifunensine at a concentration of 5.4 µM in the agroinfiltration suspension. The CMG2-Fc N-glycan profile was determined using LC-MS/MS with a targeted dynamic multiple reaction monitoring (MRM) method. The CMG2-Fc expression level in the infiltrated plant tissue and the percentage of oligomannose-type N-glycans for kifunensine treated plants was 874 mg/kg leaf fresh weight (FW) and 98.2%, respectively, compared to 717 mg/kg leaf FW and 2.3% for untreated plants. Oligomannose glycans are amenable to in vitro enzymatic modification to produce more human-like N-glycan structures that are preferred for the production of HIV-1 viral vaccine and certain monoclonal antibodies. This method allows glycan modifications using a bioprocessing approach without compromising protein yield or modification of the primary sequence, and could be expanded to other small molecule inhibitors of glycan-processing enzymes. For recombinant protein targeted for secretion, kifunensine treatment allows collection of glycoform-modified target protein from apoplast wash fluid (AWF) with minimal plant-specific complex N-glycan at higher starting purity and concentration than in whole-leaf extract, thus simplifying the downstream processing.

We develop fully glycosylated computational models of ACE2-Fc fusion proteins which are promising targets for a COVID-19 therapeutic. These models are tested in their interaction with fragments of the binding domain of the Spike Protein S1 of the SARS-CoV-2 virus. We see that some ACE2 glycans interact with S1 and glycans are influencing the conformation of the ACE2 receptor. Additionally, we optimize algorithms for protein glycosylation modelling in order to expedite future model development. All models and algorithms are openly available.

Space missions have always assumed that the risk of spacecraft malfunction far outweighs the risk of human system failure. This assumption breaks down for longer duration exploration missions and exposes vulnerabilities in space medical system. Space agencies can no longer buy down the majority of human system risk through the crew member selection process and emergency re-supply or evacuation. No mature medical solutions exist to close the risk gap. With recent advances in biotechnology, there is promise in augmenting a space pharmacy with a biologically-based space foundry for on-demand manufacturing of high-value medical products. Here we review the challenges and opportunities of molecular pharming, the production of pharmaceuticals in plants, as the basis of a space medical foundry to close the risk gap in current space medical systems. Plants have long been considered an important life support object in space and can now also be viewed as programmable factories in space. Advances in molecular pharming-based space foundries will have widespread application in promoting simple and accessible pharmaceutical manufacturing on Earth.