Scaling eukaryotic cell-free protein synthesis achieved with the versatile and high-yielding tobacco BY-2 cell lysate.

Eukaryotic cell-free protein synthesis (CFPS) can accelerate expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing. Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N-glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale-up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre-scale reaction in a rocking-type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields. Production of multimeric virus-like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult-to-express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS-CoV-2 receptor-binding domain; a human growth factor; and a G protein-coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in-depth PTM characterization of purified proteins through disulfide bond and N-glycan analysis. Taken together, BYL is a promising end-to-end R&D to manufacturing platform with the potential to significantly reduce the time-to-market for high value proteins and biologics.

ALiCE ® : A versatile, high yielding and scalable eukaryotic cell-free protein synthesis (CFPS) system.

Eukaryotic cell-free protein synthesis (CFPS) systems have the potential to simplify and speed up the expression and high-throughput analysis of complex proteins with functionally relevant post-translational modifications (PTMs). However, low yields and the inability to scale such systems have so far prevented their widespread adoption in protein research and manufacturing. Here, we present a detailed demonstration for the capabilities of a CFPS system derived from Nicotiana tabacum BY-2 cell culture (BY-2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in under 48 hours, complete with native disulfide bonds and N-glycosylation. An optimised version of the technology is commercialised as 'ALiCE ® ', engineered for high yields of up to 3 mg/mL. Recent advances in the scaling of BYL production methodologies have allowed scaling of the CFPS reaction. We show simple, linear scale-up of batch mode reporter proten expression from a 100 µL microtiter plate format to 10 mL and 100 mL volumes in standard Erlenmeyer flasks, culminating in preliminary data from 1 L reactions in a CELL-tainer® CT20 rocking motion bioreactor. As such, these works represent the first published example of a eukaryotic CFPS reaction scaled past the 10 mL level by several orders of magnitude. We show the ability of BYL to produce the simple reporter protein eYFP and large, multimeric virus-like particles directly in the cytosolic fraction. Complex proteins are processed using the native microsomes of BYL and functional expression of multiple classes of complex, difficult-to-express proteins is demonstrated, specifically: a dimeric, glycoprotein enzyme, glucose oxidase; the monoclonal antibody adalimumab; the SARS-Cov-2 receptor-binding domain; human epidermal growth factor; and a G protein-coupled receptor membrane protein, cannabinoid receptor type 2. Functional binding and activity are shown using a combination of surface plasmon resonance techniques, a serology-based ELISA method and a G protein activation assay. Finally, in-depth post-translational modification (PTM) characterisation of purified proteins through disulfide bond and N-glycan analysis is also revealed - previously difficult in the eukaryotic CFPS space due to limitations in reaction volumes and yields. Taken together, BYL provides a real opportunity for screening of complex proteins at the microscale with subsequent amplification to manufacturing-ready levels using off-the-shelf protocols. This end-to-end platform suggests the potential to significantly reduce cost and the time-to-market for high value proteins and biologics.

Contribution of Complex I NADH Dehydrogenase to Respiratory Energy Coupling in Glucose-Grown Cultures of Ogataea parapolymorpha.

The thermotolerant yeast Ogataea parapolymorpha (formerly Hansenula polymorpha) is an industrially relevant production host that exhibits a fully respiratory sugar metabolism in aerobic batch cultures. NADH-derived electrons can enter its mitochondrial respiratory chain either via a proton-translocating complex I NADH-dehydrogenase or via three putative alternative NADH dehydrogenases. This respiratory entry point affects the amount of ATP produced per NADH/O2 consumed and therefore impacts the maximum yield of biomass and/or cellular products from a given amount of substrate. To investigate the physiological importance of complex I, a wild-type O. parapolymorpha strain and a congenic complex I-deficient mutant were grown on glucose in aerobic batch, chemostat, and retentostat cultures in bioreactors. In batch cultures, the two strains exhibited a fully respiratory metabolism and showed the same growth rates and biomass yields, indicating that, under these conditions, the contribution of NADH oxidation via complex I was negligible. Both strains also exhibited a respiratory metabolism in glucose-limited chemostat cultures, but the complex I-deficient mutant showed considerably reduced biomass yields on substrate and oxygen, consistent with a lower efficiency of respiratory energy coupling. In glucose-limited retentostat cultures at specific growth rates down to ∼0.001 h-1, both O. parapolymorpha strains showed high viability. Maintenance energy requirements at these extremely low growth rates were approximately 3-fold lower than estimated from faster-growing chemostat cultures, indicating a stringent-response-like behavior. Quantitative transcriptome and proteome analyses indicated condition-dependent expression patterns of complex I subunits and of alternative NADH dehydrogenases that were consistent with physiological observations.IMPORTANCE Since popular microbial cell factories have typically not been selected for efficient respiratory energy coupling, their ATP yields from sugar catabolism are often suboptimal. In aerobic industrial processes, suboptimal energy coupling results in reduced product yields on sugar, increased process costs for oxygen transfer, and volumetric productivity limitations due to limitations in gas transfer and cooling. This study provides insights into the contribution of mechanisms of respiratory energy coupling in the yeast cell factory Ogataea parapolymorpha under different growth conditions and provides a basis for rational improvement of energy coupling in yeast cell factories. Analysis of energy metabolism of O. parapolymorpha at extremely low specific growth rates indicated that this yeast reduces its energy requirements for cellular maintenance under extreme energy limitation. Exploration of the mechanisms for this increased energetic efficiency may contribute to an optimization of the performance of industrial processes with slow-growing eukaryotic cell factories.

Biological Parts for Kluyveromyces marxianus Synthetic Biology.

Kluyveromyces marxianus is a non-conventional yeast whose physiology and metabolism lends itself to diverse biotechnological applications. While the wild-type yeast is already in use for producing fragrances and fermented products, the lack of standardised tools for its genetic and metabolic engineering prevent it from being used as a next-generation cell factory for bio-based chemicals. In this paper, we bring together and characterise a set of native K. marxianus parts for the expression of multiple genes for metabolic engineering and synthetic biology. All parts are cloned and stored according to the MoClo/Yeast Tool Kit standard for quick sharing and rapid construction. Using available genomic and transcriptomic data, we have selected promoters and terminators to fine-tune constitutive and inducible gene expression. The collection includes a number of known centromeres and autonomously replication sequences (ARS). We also provide a number of chromosomal integration sites selected for efficiency or visible phenotypes for rapid screening. Finally, we provide a single-plasmid CRISPR/Cas9 platform for genome engineering and facilitated gene targeting, and rationally create auxotrophic strains to expand the common range of selection markers available to K. marxianus. The curated and characterised tools we have provided in this kit will serve as a base to efficiently build next-generation cell factories from this alternative yeast. Plasmids containing all parts are available at Addgene for public distribution.

Evaluation of a novel cloud-based software platform for structured experiment design and linked data analytics.

Open data in science requires precise definition of experimental procedures used in data generation, but traditional practices for sharing protocols and data cannot provide the required data contextualization. Here, we explore implementation, in an academic research setting, of a novel cloud-based software system designed to address this challenge. The software supports systematic definition of experimental procedures as visual processes, acquisition and analysis of primary data, and linking of data and procedures in machine-computable form. The software was tested on a set of quantitative microbial-physiology experiments. Though time-intensive, definition of experimental procedures in the software enabled much more precise, unambiguous definitions of experiments than conventional protocols. Once defined, processes were easily reusable and composable into more complex experimental flows. Automatic coupling of process definitions to experimental data enables immediate identification of correlations between procedural details, intended and unintended experimental perturbations, and experimental outcomes. Software-based experiment descriptions could ultimately replace terse and ambiguous 'Materials and Methods' sections in scientific journals, thus promoting reproducibility and reusability of published studies.

Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid.

While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade- mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible in Kluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.