One-loop fermion determinant with explicit chiral symmetry breaking

We use a proper-time regularization to define the one-loop fermion determinant for the case in which explicit chiral symmetry breaking takes place. We show how to obtain the polynomial by which the standard definition of lndetD needs to be modified in order to arrive at the fermion determinant whose transformation properties are consistent with the general symmetry requirements of the basic Lagrangian. As an example it is shown how the fundamental symmetries and the explicit chiral symmetry breaking pattern associated with the ENJL model are preserved in a consistent way.Comment: 7 pages, plain LaTe

Investigating the role of oxidative stress response genes in protein secretion using a novel combinatorial Golden Gate based approach

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Impact of oxygen availability on organelle-specific redox potentials and stress in recombinant protein producing Pichia pastoris

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Going beyond the limit: impact of increasing global translation activity on the productivity of recombinant secreted proteins in Pichia pastoris

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Established tools and emerging trends for the production of recombinant proteins and metabolites in Pichia pastoris

Besides bakers' yeast, the methylotrophic yeast Komagataella phaffii (also known as Pichia pastoris) has been developed into the most popular yeast cell factory for the production of heterologous proteins. Strong promoters, stable genetic constructs and a growing collection of freely available strains, tools and protocols have boosted this development equally as thorough genetic and cell biological characterization. This review provides an overview of state-of-the-art tools and techniques for working with P. pastoris, as well as guidelines for the production of recombinant proteins with a focus on small-scale production for biochemical studies and protein characterization. The growing applications of P. pastoris for in vivo biotransformation and metabolic pathway engineering for the production of bulk and specialty chemicals are highlighted as well

Versatile modeling and optimization of fed batch processes for the production of secreted heterologous proteins with Pichia pastoris

BACKGROUND: Secretion of heterologous proteins depends both on biomass concentration and on the specific product secretion rate, which in turn is not constant at varying specific growth rates. As fed batch processes usually do not maintain a steady state throughout the feed phase, it is not trivial to model and optimize such a process by mathematical means. RESULTS: We have developed a model for product accumulation in fed batch based on iterative calculation in Microsoft Excel spreadsheets, and used the Solver software to optimize the time course of the media feed in order to maximize the volumetric productivity. The optimum feed phase consisted of an exponential feed at maximum specific growth rate, followed by a phase with linearly increasing feed rate and consequently steadily decreasing specific growth rate. The latter phase could be modeled also by exact mathematical treatment by the calculus of variations, yielding the explicit shape of the growth function, however, with certain indeterminate parameters. To evaluate the latter, one needs a numerical optimum search algorithm. The explicit shape of the growth function provides additional evidence that the Excel model results in correct data. Experimental evaluation in two independent fed batch cultures resulted in a good correlation to the optimized model data, and a 2.2 fold improvement of the volumetric productivity. CONCLUSION: The advantages of the procedure we describe here are the ease of use and the flexibility, applying software familiar to every scientist and engineer, and rapid calculation which makes predictions extremely easy, so that many options can be tested in silico quickly. Additional options like further biological and technological constraints or different functions for specific productivity and biomass yield can easily be integrated

Flo8 – A versatile regulator for improving recombinant protein production in Pichia pastoris

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Slow growth rate triggered transition to a pseudohyphal lifestyle of the protein production host Pichia pastoris

Specific growth rate is an important process control parameter for industrial protein production. In the widely used yeast protein production host Pichia pastoris, growth rate is known to significantly impact protein expression and secretion [1]. In that regard, glucose-limited chemostat cultivations carried out over a wide range of specific growth rates have revealed that slow growth rates can trigger a pseudohyphal phenotype in P. pastoris [2]. Such phenotypes are undesirable during large-scale protein production processes since they can lead to foam production. In Saccharomyces cerevisiae pseudohyphal growth is controlled by FLO11, a member of the FLO gene family, which is a group of genes encoding cell surface proteins responsible for conferring a diverse array of adhesion-related phenotypes and reported to be controlled by epigenetic mechanisms. P. pastoris also carries a number of FLO genes but their functions and regulatory patterns are yet unknown. Thus, we set out to investigate this gene family to shed some light on how pseudohyphal growth and other adhesion phenotypes are triggered and regulated in P. pastoris. Please click Additional Files below to see the full abstract

Unfolded protein response biosensors for recombinant protein expression

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Engineering vacuolar sorting pathways for efficient secretion of recombinant proteins

Recombinant protein production is an expanding branch of biotechnology with increasing economic importance. Currently, 20% of biopharmaceutical proteins and approximately half of the industrial enzymes are produced in yeasts. Many proteins are efficiently secreted by yeast systems, reaching product titers in the g L-1 range. The expression of more complex proteins, however, may overwhelm the folding and secretion capacity of the host cells. This triggers the unfolded protein response (UPR), which aims at restoring endoplasmic reticulum (ER) homeostasis. The UPR, in turn, is thought to activate ER-associated protein degradation (ERAD). Alternatively, trafficking of correctly folded proteins can be hampered on their way to the cell exterior leading e.g. to missorting and subsequent degradation in the vacuole. The methylotrophic yeast Pichia pastoris (Komagataella spp.) is a popular microbial host for the production of recombinant proteins. Vacuolar protein sorting has not been investigated in detail so far in P. pastoris, although there were a few indications that vacuolar mistargeting of recombinant products might occur also in this yeast. Thus we engineered the vacuolar sorting pathways in P. pastoris and investigated their impact on extracellular product titers as well as intracellular localization of the recombinant secretory product. Thereby, differences between vps (vacuolar protein sorting) mutant strains disrupted in genes involved either in the CORVET or the HOPS tethering complexes became obvious. Moreover, we were able to show that engineering of the vacuolar sorting pathways has a positive impact on heterologous protein secretion, however, in some cases simultaneous inactivation of specific vacuolar proteases was necessary. Taken together, these studies allowed us to gain deeper insight into the pathways leading to intracellular degradation of recombinant secretory proteins. Based on these findings, approaches how to efficiently adapt the host cell’s secretion capacity will be presented, which confirm that impairment of vacuolar protein sorting is an effective means of enhancing secretion of heterologous proteins