Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii.

BACKGROUND: Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii. RESULTS: In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system. CONCLUSIONS: In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.

Evaluation of reference genes for transcript analyses in Komagataella phaffii (Pichia pastoris).

BACKGROUND: The yeast Komagataella phaffii (Pichia pastoris) is routinely used for heterologous protein expression and is suggested as a model organism for yeast. Despite its importance and application potential, no reference gene for transcript analysis via RT-qPCR assays has been evaluated to date. In this study, we searched publicly available RNASeq data for stably expressed genes to find potential reference genes for relative transcript analysis by RT-qPCR in K. phaffii. To evaluate the applicability of these genes, we used a diverse set of samples from three different strains and a broad range of cultivation conditions. The transcript levels of 9 genes were measured and compared using commonly applied bioinformatic tools. RESULTS: We could demonstrate that the often-used reference gene ACT1 is not very stably expressed and could identify two genes with outstandingly low transcript level fluctuations. Consequently, we suggest the two genes, RSC1, and TAF10 to be simultaneously used as reference genes in transcript analyses by RT-qPCR in K. phaffii in future RT-qPCR assays. CONCLUSION: The usage of ACT1 as a reference gene in RT-qPCR analysis might lead to distorted results due to the instability of its transcript levels. In this study, we evaluated the transcript levels of several genes and found RSC1 and TAF10 to be extremely stable. Using these genes holds the promise for reliable RT-qPCR results.

Recombinant Protein L: Production, Purification and Characterization of a Universal Binding Ligand.

Protein L (PpL) is a universal binding ligand that can be used for the detection and purification of antibodies and antibody fragments. Due to the unique interaction with immunoglobulin light chains, it differs from other affinity ligands, like protein A or G. However, due to its current higher market price, PpL is still scarce in applications. In this study, we investigated the recombinant production and purification of PpL and characterized the product in detail. We present a comprehensive roadmap for the production of the versatile protein PpL in E. coli.

Cascaded processing enables continuous upstream processing with E. coli BL21(DE3).

In many industrial sectors continuous processing is already the golden standard to maximize productivity. However, when working with living cells, subpopulation formation causes instabilities in long-term cultivations. In cascaded continuous cultivation, biomass formation and recombinant protein expression can be spatially separated. This cultivation mode was found to facilitate stable protein expression using microbial hosts, however mechanistic knowledge of this cultivation strategy is scarce. In this contribution we present a method workflow to reduce workload and accelerate the establishment of stable continuous processes with E. coli BL21(DE3) exclusively based on bioengineering methods.