BackgroundThe methylotrophic yeast Pichia pastoris is a common host for the production of recombinant proteins. However, hypermannosylation hinders the use of recombinant proteins from yeast in most biopharmaceutical applications. Glyco-engineered yeast strains produce more homogeneously glycosylated proteins, but can be physiologically impaired and show tendencies for cellular agglomeration, hence are hard to cultivate. Further, comprehensive data regarding growth, physiology and recombinant protein production in the controlled environment of a bioreactor are scarce.ResultsA Man5GlcNAc2 glycosylating and a Man8–10GlcNAc2 glycosylating strain showed similar morphological traits during methanol induced shake-flask cultivations to produce the recombinant model protein HRP C1A. Both glyco-engineered strains displayed larger single and budding cells than a wild type strain as well as strong cellular agglomeration. The cores of these agglomerates appeared to be less viable. Despite agglomeration, the Man5GlcNAc2 glycosylating strain showed superior growth, physiology and HRP C1A productivity compared to the Man8–10GlcNAc2 glycosylating strain in shake-flasks and in the bioreactor. Conducting dynamic methanol pulsing revealed that HRP C1A productivity of the Man5GlcNAc2 glycosylating strain is best at a temperature of 30 °C.ConclusionThis study provides the first comprehensive evaluation of growth, physiology and recombinant protein production of a Man5GlcNAc2 glycosylating strain in the controlled environment of a bioreactor. Furthermore, it is evident that cellular agglomeration is likely triggered by a reduced glycan length of cell surface glycans, but does not necessarily lead to lower metabolic activity and recombinant protein production. Man5GlcNAc2 glycosylated HRP C1A production is feasible, yields active protein similar to the wild type strain, but thermal stability of HRP C1A is negatively affected by reduced glycosylation.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1032-6) contains supplementary material, which is available to authorized users.

The magnetization of non-magnetic cells has great potential to aid various processes in medicine, but also in bioprocess engineering. Current approaches to magnetize cells with magnetic nanoparticles (MNPs) require cellular uptake or adsorption through in vitro manipulation of cells. A relatively new field of research is "magnetogenetics" which focuses on in vivo production and accumulation of magnetic material. Natural intrinsically magnetic cells (IMCs) produce intracellular, MNPs, and are called magnetotactic bacteria (MTB). In recent years, researchers have unraveled function and structure of numerous proteins from MTB. Furthermore, protein engineering studies on such MTB proteins and other potentially magnetic proteins, like ferritins, highlight that in vivo magnetization of non-magnetic hosts is a thriving field of research. This review summarizes current knowledge on recombinant IMC generation and highlights future steps that can be taken to succeed in transforming non-magnetic cells to IMCs.

In biotechnological processes, technical failures in the upstream process often lead to batch loss. It is of great interest to investigate the empirical impact of technical failures to understand and mitigate their impact accurately and reduce economic damage. We investigated the impact in the upstream and downstream of a recombinant antibody fragment inclusion body production process chain to provide integrated empirical data and knowledge. First, we provided a reproducible process chain that yielded high inclusion body content, high specific product titer, and a refolding yield of 30%. The inclusion body downstream proved to be of high reproducibility. Through the intended introduction of technical failures, we were not only able to shed more light on the empirical responses in the upstream and downstream, but also on process-boosting parameters that would have been neglected. Herein, a short increase in temperature during the cultivation clearly increased the refolding yield.Electronic supplementary materialThe online version of this article (10.1007/s00449-019-02158-x) contains supplementary material, which is available to authorized users.

The REACH regulation stands for “Registration, Evaluation, Authorization and Restriction of Chemicals” and defines certain substances as harmful to human health and the environment. This urges manufacturers to adapt production processes. Boric acid and cobalt dichloride represent such harmful ingredients, but are commonly used in yeast cultivation media. The yeast Komagataella phaffii (Pichia pastoris) is an important host for heterologous protein production and compliance with the REACH regulation is desirable. Boric acid and cobalt dichloride are used as boron and cobalt sources, respectively. Boron and cobalt support growth and productivity and a number of cobalt-containing enzymes exist. Therefore, depletion of boric acid and cobalt dichloride could have various negative effects, but knowledge is currently scarce. Herein, we provide an insight into the impact of boric acid and cobalt depletion on recombinant protein production with K. phaffii and additionally show how different vessel materials affect cultivation media compositions through leaking elements. We found that boric acid could be substituted through boron leakiness from borosilicate glassware. Furthermore, depletion of boric acid and cobalt dichloride neither affected high cell density cultivation nor cell morphology and viability on methanol. However, final protein quality of three different industrially relevant enzymes was affected in various ways.

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Recent findings have sparked great interest in the putative magnetic receptor protein MagR. However, in vivo experiments have revealed no magnetic moment of MagR at room temperature. Nevertheless, the interaction of MagR and MagR fusion proteins with silica-coated magnetite beads have proven useful for protein purification. In this study, we recombinantly produced two different MagR proteins in Escherichia coli BL21(DE3) to (1) expand earlier protein purification studies, (2) test if MagR can magnetize whole E. coli cells once it is expressed to a high cytosolic, soluble titer, and (3) investigate the MagR-expressing E. coli cells’ magnetic properties at low temperatures. Our results show that MagR induces no measurable, permanent magnetic moment in cells at low temperatures, indicating no usability for cell magnetization. Furthermore, we show the limited usability for magnetic bead-based protein purification, thus closing the current knowledge gap between theoretical considerations and empirical data on the MagR protein.