Protein biopharmaceuticals play a key role in providing effective, targeted, and personalized therapies for diverse diseases, while also preventing and mitigating a broad range of infections. N-glycosylation is a key post-translational modification influencing the biological activity of many protein-based therapeutics, yet structure-function relationships of N-glycans remain poorly understood due to challenges in producing homogeneous glycoforms. Current go-to production hosts, mammalian and yeast cells, often yield heterogeneous glycan profiles and require extensive genetic manipulation. Alternative production hosts such as the plant Nicotiana benthamiana, provide more homogeneous glycosylation and flexibility through transient expression, but are limited in the generation of certain glycoforms. In vitro glycoengineering can overcome these limitations but is time consuming and requires expensive resources. In this study, we show that by combining in planta and in vitro glycoengineering strategies, we can quickly produce a wide range of homogeneous glycoforms of pharmaceutical proteins with high mannose, paucimannose, hybrid and complex N-glycan structures. Using N. benthamiana as a transient expression host, we produced two pharmaceutical glycoproteins - the monoclonal antibody rituximab and the helminth vaccine candidate OoASP-1 - and modified them in vitro using Escherichia coli produced glycoenzymes. The combination of these two glycoengineering systems minimizes the amount of time and resources required, while maintaining high glycan homogeneity. This scalable, flexible, and cost-effective platform opens the door to glycan structure-function relationship studies and can support rational design of next-generation biopharmaceuticals.

The green alga Chlamydomonas reinhardtii is an interesting organism for production of heterologous isoprenoid natural products through metabolic engineering as it can grow photosynthetically and represents a plant-like cell environment for synthase expression. Here, we investigated expression of the patchoulol synthase from Pogostemon cablin (Benth) (PcPS) to probe the production of sesquiterpenoids from different sub-cellular compartments of the algal cell. We observed that knock-down of squalene synthase (SQS) in the cytoplasm did not affect sesquiterpene production titers in the plastid, suggesting no backward movement of FPP from cytoplasmic pools. Fusion of PcPS with a farnesyl pyrophosphate synthase (FPPS) did not enhance patchoulol production in the cytoplasm but did in the plastid, as also reported elsewhere. Secondary transformations with cytoplasmic and chloroplast-localized PcPS constructs increased synthase titers and improved patchoulol yields from both compartments. In multi-parallel photobioreactor experiments with patchoulol extraction through two-phase solvent contact, different daily patchoulol production behaviors were observed based on subcellular localization of the PcPS and carbon source. Those with plastid localization of PcPS-FPPS exhibited continued production into later stages of cultivation, while cytoplasm-localized PcPS with SQS k.d. had the highest production rates in mid-logarithmic phase with low productivity in later growth stages. Cultivations in high-density photobioreactors with nutrient-enriched medium generated 15 mg patchoulol/L culture from CO2, the highest yield reported from the alga to date. The results indicate that the algal cell has flexible pools of isoprenoid precursors in different subcellular compartments from which the biotechnologist can design an expression strategy tailored to carbon-feeding regimes.

One of the most common post-translational modifications occurring in bacteria is proteolytic cleavage. Particularly, in secretory proteins this cleavage is observed for specific protein segments, named pro-peptides, which are frequently located between the signal peptide and the mature protein. Pro-peptides are known to serve two main functions, namely a chaperone function in protein folding and an enzyme-inhibiting function. To date, the contribution of pro-peptides to protein secretion is not fully understood. Therefore, the present study was aimed at assessing possible roles of the N-terminal pro-peptides from the S8 class subtilisin-like serine proteases Bpr, Vpr and WprA of Bacillus subtilis in protein secretion. To this end, the signal peptides of these proteases were fused to the alkaline phosphatase PhoA of Escherichia coli with or without the cognate pro-peptides. Subsequently, secretion of the PhoA protein and its activity were assessed in the B. subtilis type strain 168 or the genome-reduced strain IIG-Bs27-39 and correlated to the respective pro-peptide structure as predicted by AlphaFold. Altogether, the obtained results suggest that the contributions of the investigated pro-peptides to protein secretion in B. subtilis are related to a combination of their functions in protein folding, their own folding state, and the applied strain.

Saprobic fungi have important biotechnological applications, particularly in biorefinery processes in which their hydrolytic enzymes (CAZymes) convert the biomass into valuable products such as biofuels and biochemicals. Myceliophthora thermophila is a thermophilic fungus, which makes it an attractive source of enzymes for biorefinery. However, the regulatory system that controls the production of CAZymes in this species is only partially understood. This study focuses on understanding the role of ARA1 in M. thermophila, investigating its function beyond conventional L-arabinose/D-galactose utilization, by using deletion and overexpression strains for detailed transcriptomic and phenotypic analyses. The comparison with other fungi provides insights into ARA1's diverse functions within Sordariomycete fungi. Our results indicate that ARA1 is a key regulator in M. thermophila, as it controls the utilization of L-arabinose, D-galactose, xylan, cellulose and pectin, exhibiting a broader role compared to its orthologs in other Sordariomycetes. Moreover, co-regulation between XYR1 and ARA1, with significant overlap in target genes, was observed for the first time in M. thermophila. Constitutive overexpression of ara1 is identified as a promising approach for enhancing hydrolytic performance during M. thermophila growth on complex and recalcitrant substrates but is strongly dependent on the right promoter. In conclusion, our study identifies M. thermophila ARA1 as a key regulator in plant biomass utilization, although additional regulators, e.g., CLR1 and CLR2, remain to be investigated to fully elucidate the regulatory network governing this process. The observed diversity in the role of ARA1 among Sordariomycetes highlights the flexibility of transcriptional networks involved in biomass degradation and offers leads for improving biotechnological processes.

Fibroin, the primary component of silkworm silk, is a natural biocompatible and biodegradable material known for its exceptional mechanical properties, making it highly valued in the biomedical field. Traditional methods of extracting fibroin from Bombyx mori silk present challenges related to quality and purity of the product, as well as sustainability and ethics of the process. This underscores the need for a recombinant platform for producing high-quality, monodisperse vegan fibroin. The production of fibroin is particularly challenging due to its large and repetitive nature, also at the nucleic acid level. To address this challenge, we developed a DNA-mediated assembly method that utilizes a vector library offering flexibility to test the expression of recombinant proteins in different subcellular localizations in plants and the combination of various protein modules. This approach involves a series of Golden Gate cloning steps to gradually assemble the fibroin coding sequence, starting from the smallest repetitive unit. Using this method, we successfully produced recombinant fibroin polymers of up to 172 kDa in Nicotiana benthamiana via agroinfiltration. Additionally, we demonstrated the versatility of our vector library by assembling genetic constructs for another repetitive protein, resilin, and by creating chimeric proteins.