One-Step In Vivo Assembly of Multiple DNA Fragments and Genomic Integration in Komagataella phaffii

The methylotrophic yeast species Komagataella phaffii (synonym: Pichia pastoris) is widely used as a host for recombinant protein production. Although several genetic engineering techniques are being employed on K. phaffii, advanced methods such as in vivo DNA assembly in this yeast species are required for synthetic biology applications. In this study, we established a technique for accomplishing one-step in vivo assembly of multiple DNA fragments and genomic integration in K. phaffii.
To concurrently achieve an accurate multiple DNA assembly and a high-efficient integration into the target genomic locus in vivo, a K. phaffii strain, lacking a non-homologous end joining-related protein, DNA ligase IV (Dnl4p), that has been reported to improve gene targeting efficiency by homologous recombination, was used. Using green fluorescent protein along with the lycopene biosynthesis, we showed that our method that included a Dnl4p-defective strain permits direct and easy engineering of K. phaffii strains.

Nitroreduction of flutamide by Cunninghamella elegans NADPH: Cytochrome P450 reductase

The microbial model of mammalian drug metabolism, Cunninghamella elegans, has three cytochrome P450 reductase genes in its genome: g1631 (CPR_A), g4301 (CPR_B), and g7609 (CPR_C). The nitroreductase activity of the encoded enzymes was investigated via expression of the genes in the yeast Pichia pastoris X33. Whole cell assays with the recombinant yeast demonstrated that the reductases converted the joplink Recombinant Human Tumor necrosis factor anticancer drug flutamide to the nitroreduced metabolite that was also produced from the same substrate when incubated with human NADPH: cytochrome P450 reductase.
The nitroreductase activity extended to other substrates such as the related drug nilutamide and the environmental contaminants 1-nitronaphthalene and 1,3-dinitronaphthalene. Comparative experiments with cell lysates of recombinant yeast were conducted under aerobic and reduced oxygen conditions and demonstrated that the reductases are oxygen sensitive.

Genome-scale modeling of yeast metabolism: retrospectives and perspectives

Yeasts have been widely used for the production of bread, beer and wine as well as for production of bioethanol, but they have also been designed as cell factories to produce various chemicals, advanced biofuels and recombinant proteins. To systematically understand and rationally engineer yeast metabolism, genome-scale metabolic models (GEMs) have been reconstructed for the model yeast Saccharomyces cerevisiae and non-conventional yeasts.
Here we review the historical development of yeast GEMs together with their recent applications including metabolic flux prediction, cell factory design, culture condition optimization and multi-yeast comparative analysis. Furthermore, we present an emerging effort, namely the integration of proteome constraints into yeast GEMs, resulting in models with improved performance. At last, we discuss challenges and perspectives on the development of yeast GEMs and the integration of proteome constraints.

Going beyond the limit: Increasing global translation activity leads to increased productivity of recombinant secreted proteins in Pichia pastoris

Yeasts are widely used cell factories for commercial heterologous protein production, however, specific productivities are usually tightly coupled to biomass formation. This greatly impacts production processes, which are commonly not run at the maximum growth rate, thereby resulting in suboptimal productivities. To tackle this issue, we evaluated transcriptomics datasets of the yeast Pichia pastoris (syn. Komagataella phaffii), which is known for its high secretory efficiency and biomass yield.
These showed a clear downregulation of genes related to protein translation with decreasing growth rates, thus revealing the yeast translation machinery as cellular engineering target. By overexpressing selected differentially expressed translation factors, translation initiation was identified to be the main rate-limiting step. Specifically, overexpression of factors associated with the closed-loop conformation, a structure that increases stability and rates of translation initiation before start codon scanning is initiated, showed the strongest effects.
Overexpression of closed-loop factors alone or in combination increased titers of different heterologous proteins by up to 3-fold in fed-batch processes. Furthermore, translation activity, correlating to the obtained secreted recombinant protein yields, selected transcript levels and total protein content were higher in the engineered cells. Hence, translation factor overexpression, globally affects the cell. Together with the observed impact on the transcriptome and total protein content, our results indicate that the capacity of P. pastoris for protein production is not at its limit yet.

High Cell-Density Expression System: Yeast Cells in a Phalanx Efficiently Produce a Certain Range of “Difficult-to-Express” Secretory Recombinant Proteins

Yeast’s extracellular expression provides a cost-efficient means of producing recombinant proteins of academic or commercial interests. However, depending on the protein to be expressed, the production occasionally results in a poor yield, which is frequently accompanied with a deteriorated growth of the host. Here we describe our simple approach, high cell-density expression, to circumvent the cellular toxicity and achieve the production of a certain range of “difficult-to-express” secretory protein in preparative amount.
The system features an ease of performing: (a) pre-cultivate yeast cells to the stationary phase in non-inducing condition, (b) suspend the cells to a small aliquot of inducing medium to form a high cell-density suspension or “a phalanx,” then (c) give a sufficient aeration to the phalanx. Factors and pitfalls that affect the system’s performance are also described.

Protection of Mice against Experimental Cryptococcosis by Synthesized Peptides Delivered in Glucan Particles

The high global burden of cryptococcosis has made development of a protective vaccine a public health priority. We previously demonstrated that a vaccine composed of recombinant Cryptococcus neoformans chitin deacetylase 2 (Cda2) delivered in glucan particles (GPs) protects BALB/c and C57BL/6 mice from an otherwise lethal challenge with a highly virulent C. neoformans strain. An immunoinformatic analysis of Cda2 revealed a peptide sequence predicted to have strong binding to the major histocompatibility complex class II (MHC II) H2-IAd allele found in BALB/c mice.
BALB/c mice vaccinated with GPs containing a 32-amino-acid peptide (Cda2-Pep1) that included this strong binding region were protected from cryptococcosis. Protection was lost with GP-based vaccines containing versions of recombinant Cda2 protein and Cda2-Pep1 with mutations predicted to greatly diminish MHC II binding. Cda2 has homology to the three other C. neoformans chitin deacetylases, Cda1, Cda3, and Fpd1, in the high-MHC II-binding region.
GPs loaded with homologous peptides of Cda1, Cda3, and Fpd1 protected BALB/c mice from experimental cryptococcosis, albeit not as robustly as the Cda2-Pep1 vaccine. Finally, seven other peptides were synthesized based on regions in Cda2 predicted to contain promising CD4+ T cell epitopes in BALB/c or C57BL/6 mice.
While five peptide vaccines significantly protected BALB/c mice, only one protected C57BL/6 mice. Thus, GP-based vaccines containing a single peptide can protect mice against cryptococcosis. However, given the diversity of human MHC II alleles, a peptide-based Cryptococcus vaccine for use in humans would be challenging and likely need to contain multiple peptide sequences.
 IMPORTANCE Cryptococcosis, due to infection by fungi of the Cryptococcus neoformans species complex, is responsible for substantial morbidity and mortality in immunocompromised persons, particularly those with AIDS. Cryptococcal vaccines are a public health priority yet are not available for human use. We previously demonstrated mice could be protected from experimental cryptococcosis with vaccines composed of recombinant cryptococcal proteins encased in hollow highly purified yeast cell walls (glucan particles).
In this study, we examined one such protective protein, Cda2, and using bioinformatics, we identified a region predicted to stimulate strong T cell responses. A peptide containing this region formulated in glucan particle-based vaccines protected mice as well as the recombinant protein. Other peptide vaccines also protected, including peptides containing sequences from proteins homologous to Cda2. These preclinical mouse studies provide a proof of principle that peptides can be effective as vaccines to protect against cryptococcosis and that bioinformatic approaches can guide peptide selection.

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