scholarly journals Controlling Unconventional Secretion for Production of Heterologous Proteins in Ustilago maydis through Transcriptional Regulation and Chemical Inhibition of the Kinase Don3

2021 ◽  
Vol 7 (3) ◽  
pp. 179
Author(s):  
Kai P. Hussnaetter ◽  
Magnus Philipp ◽  
Kira Müntjes ◽  
Michael Feldbrügge ◽  
Kerstin Schipper
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Ustilago Maydis ◽  
Downstream Processing ◽  
Culture Broth ◽  
Yeast Cells ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Regulatory Systems ◽  
Unconventional Secretion

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream processing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. The kinase Don3 is essential for functional assembly of the fragmentation zone and hence, for release of Cts1-fusion proteins. Here, we are first to develop regulatory systems for unconventional protein secretion using Don3 as a gatekeeper to control when export occurs. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional don3 regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

scholarly journals Establishing regulated unconventional secretion for production of heterologous proteins

2021 ◽  
Author(s):  
Kai P. Hussnaetter ◽  
Magnus Philipp ◽  
Kira Müntjes ◽  
Michael Feldbrügge ◽  
Kerstin Schipper
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Culture Broth ◽  
Yeast Cells ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Regulatory Systems ◽  
Production Platform ◽  
Unconventional Secretion ◽  
Different Levels

AbstractHeterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream pro-cessing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. Here, we are first to develop regulatory systems for unconventional protein secretion. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

scholarly journals Enhanced Recombinant Protein Production Under Special Environmental Stress

2021 ◽  
Vol 12 ◽  
Author(s):  
Xinyi Chen ◽  
Chun Li ◽  
Hu Liu
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Synthesis Process ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Eukaryotic Microorganism ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Environment Stress ◽  
Extracellular Environment

Regardless of bacteria or eukaryotic microorganism hosts, improving their ability to express heterologous proteins is always a goal worthy of elaborate study. In addition to traditional methods including intracellular synthesis process regulation and extracellular environment optimization, some special or extreme conditions can also be employed to create an enhancing effect on heterologous protein production. In this review, we summarize some extreme environmental factors used for the improvement of heterologous protein expression, including low temperature, hypoxia, microgravity and high osmolality. The applications of these strategies are elaborated with examples of well-documented studies. We also demonstrated the confirmed or hypothetical mechanisms of environment stress affecting the host behaviors. In addition, multi-omics techniques driving the stress-responsive research for construction of efficient microbial cell factories are also prospected at the end.

scholarly journals Secretion of Human Serum Albumin by Kluyveromyces lactis Overexpressing KlPDI1 and KlERO1

2005 ◽  
Vol 71 (8) ◽  
pp. 4359-4363 ◽  
Author(s):  
Tiziana Lodi ◽  
Barbara Neglia ◽  
Claudia Donnini
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Protein Conformation ◽  
Protein Production ◽  
Heterologous Protein ◽  
Kluyveromyces Lactis ◽  
Heterologous Proteins ◽  
Disulfide Bridges ◽  
Genes Encoding

ABSTRACT The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast Kluyveromyces lactis and evaluated the secretion of heterologous proteins. KlERO1, an orthologue of Saccharomyces cerevisiae ERO1, was cloned by functional complementation of the ts phenotype of an Scero1 mutant. The expression of KlERO1 was induced by treatment of the cells with dithiothreitol and by overexpression of human serum albumin (HSA), a disulfide bond-rich protein. Duplication of either PDI1 or ERO1 led to a similar increase in HSA yield. Duplication of both genes accelerated the secretion of HSA and improved cell growth rate and yield. Increasing the dosage of KlERO1 did not affect the production of human interleukin 1β, a protein that has no disulfide bridges. The results confirm that the ERO1 genes of S. cerevisiae and K. lactis are functionally similar even though portions of their coding sequence are quite different and the phenotypes of mutants overexpressing the genes differ. The marked effects of KlERO1 copy number on the expression of heterologous proteins with a high number of disulfide bridges suggests that control of KlERO1 and KlPDI1 is important for the production of high levels of heterologous proteins of this type.

scholarly journals Engineering Biology to Construct Microbial Chassis for the Production of Difficult-to-Express Proteins

2020 ◽  
Vol 21 (3) ◽  
pp. 990 ◽  
Author(s):  
Kangsan Kim ◽  
Donghui Choe ◽  
Dae-Hee Lee ◽  
Byung-Kwan Cho
Keyword(s):  
Recombinant Protein ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Heterologous Protein ◽  
Recombinant Protein Expression ◽  
Cost Effective ◽  
Protein Yield ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Expression Pathways

A large proportion of the recombinant proteins manufactured today rely on microbe-based expression systems owing to their relatively simple and cost-effective production schemes. However, several issues in microbial protein expression, including formation of insoluble aggregates, low protein yield, and cell death are still highly recursive and tricky to optimize. These obstacles are usually rooted in the metabolic capacity of the expression host, limitation of cellular translational machineries, or genetic instability. To this end, several microbial strains having precisely designed genomes have been suggested as a way around the recurrent problems in recombinant protein expression. Already, a growing number of prokaryotic chassis strains have been genome-streamlined to attain superior cellular fitness, recombinant protein yield, and stability of the exogenous expression pathways. In this review, we outline challenges associated with heterologous protein expression, some examples of microbial chassis engineered for the production of recombinant proteins, and emerging tools to optimize the expression of heterologous proteins. In particular, we discuss the synthetic biology approaches to design and build and test genome-reduced microbial chassis that carry desirable characteristics for heterologous protein expression.

scholarly journals Induction by Hypoxia of Heterologous-Protein Production with the KlPDC1 Promoter in Yeasts

2006 ◽  
Vol 73 (3) ◽  
pp. 922-929 ◽  
Author(s):  
Andrea Camattari ◽  
Michele M. Bianchi ◽  
Paola Branduardi ◽  
Danilo Porro ◽  
Luca Brambilla
Keyword(s):  
Protein Production ◽  
Heterologous Protein ◽  
Heterologous Gene Expression ◽  
Kluyveromyces Lactis ◽  
Expression System ◽  
Promoter Sequence ◽  
Heterologous Protein Production ◽  
Heterologous Proteins ◽  
Hypoxic Induction ◽  
Production Ratio

ABSTRACT The control of promoter activity by oxygen availability appears to be an intriguing system for heterologous protein production. In fact, during cell growth in a bioreactor, an oxygen shortage is easily obtained simply by interrupting the air supply. The purpose of our work was to explore the possible use of hypoxic induction of the KlPDC1 promoter to direct heterologous gene expression in yeast. In the present study, an expression system based on the KlPDC1 promoter was developed and characterized. Several heterologous proteins, differing in size, origin, localization, and posttranslational modification, were successfully expressed in Kluyveromyces lactis under the control of the wild type or a modified promoter sequence, with a production ratio between 4 and more than 100. Yields were further optimized by a more accurate control of hypoxic physiological conditions. Production of as high as 180 mg/liter of human interleukin-1β was obtained, representing the highest value obtained with yeasts in a lab-scale bioreactor to date. Moreover, the transferability of our system to related yeasts was assessed. The lacZ gene from Escherichia coli was cloned downstream of the KlPDC1 promoter in order to get β-galactosidase activity in response to induction of the promoter. A centromeric vector harboring this expression cassette was introduced in Saccharomyces cerevisiae and in Zygosaccharomyces bailii, and effects of hypoxic induction were measured and compared to those already observed in K. lactis cells. Interestingly, we found that the induction still worked in Z. bailii; thus, this promotor constitutes a possible inducible system for this new nonconventional host.

CRABTREE POZİTİF VE CRABTREE NEGATİF MAYA TÜRLERİNDE GCR1 GENİNİN IN SILICO ANALİZİ

2021 ◽  
Vol 8 (17) ◽  
pp. 207-216
Author(s):  
Tülay TURGUT GENÇ ◽  
Ataberk ÇAKAN ◽  
Melih GÜNAY
Keyword(s):  
In Silico ◽  
Protein Production ◽  
Heterologous Protein ◽  
Yeast Species ◽  
Yeast Cells ◽  
Crabtree Effect ◽  
Promoter Regions ◽  
Yeast Strains ◽  
Genes Encoding ◽  
Chromatin Folding

The use of fermentation in the presence of oxygen and at high glucose concentrations is referred to as the Crabtree effect. Yeast species that have the Crabtree effect are called Crabtree positive, and yeast species that do not have the Crabtree effect are called Crabtree negative. While Crabtree negative yeast strains are mostly used for heterologous protein production in the industrial field, Crabtree positive yeast strains are used to understand metabolic events in cancer cells. The genes encoding the enzymes involved in the glycolytic pathway in S. cerevisiae yeast cells are controlled by Gcr1p. Gcr1p binds to CT elements located in the promoter regions of glycolytic genes and activates their transcription. In our study, Crabtree positive and negative yeast strains containing Sc-Gcr1p similar proteins were determined, and protein similarity analyzes and promoter analyzes of genes encoding the relevant proteins in these yeast strains were compared in silico using different databases and analysis programs. For this purpose, SGD, UNIPROT, NCBI-Genome and Yeastract databases and BLASTp-NCBI, MEGA-X and Chromatin Folding V2 programs were used. Using the SGD database, 32 different yeast strains were identified that matched with Sc-Gcr1p. Five different Crabtree positive and 5 different Crabtree negative yeast strains were selected from these yeast strains and in silico analyzes were performed using these yeast strains. After protein analysis and promoter analysis, it was determined that the similarities and differences between yeast species were not specific for Crabtree positive and Crabtree negative yeast species, but varied between species.

Recombinant Protein Production and Plasmid Stability in Escherichia coli Biofilms

2020 ◽  
Author(s):  
Luciana C. Gomes ◽  
Gabriel A. Monteiro ◽  
Filipe J. Mergulhão
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Heterologous Protein ◽  
Plasmid Stability ◽  
Gene Dosage ◽  
Heterologous Protein Expression ◽  
E Coli ◽  
The Impact

<p><em>Escherichia coli</em> biofilms have a great biotechnological potential since this organism has been one of the preferred hosts for recombinant protein production for the past decades and it has been successfully used in metabolic engineering for the production of high-value compounds.</p> <p>In a previous study, we have demonstrated that the non-induced enhanced green fluorescent protein (eGFP) expression from <em>E. coli</em> biofilm cells was 30-fold higher than in the planktonic state without any optimization of cultivation parameters [1]. The aim of the present work was to evaluate the effect of chemical induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) on the expression of eGFP by planktonic and biofilm cells of <em>E. coli</em> JM109(DE3) transformed with a plasmid containing a T7 promoter.</p> <p>It was shown that induction negatively affected the growth and viability of planktonic cultures, and eGFP production did not increase. Recombinant protein production was not limited by gene dosage or by transcriptional activity. Results suggest that plasmid maintenance at high copy number imposes a metabolic burden that precludes high level expression of the recombinant protein. In biofilm cells, the inducer avoided the overall decrease in the amount of expressed eGFP, although this was not correlated with the gene dosage. Higher specific production levels were always attained with biofilm cells and it seems that while induction of biofilm cells shifts their metabolism towards the maintenance of recombinant protein concentration, in planktonic cells the cellular resources are directed towards plasmid replication and growth [2].</p> <p>It is expected that this work will be of great value to elucidate the mechanisms of induction on recombinant protein production, especially in biofilm cells which have shown potential to be used as protein factories.</p> <p> </p> <p> </p> <p>References:</p> <p>[1] Gomes, L.C., & Mergulhão, F.J. (2017) Heterologous protein production in <em>Escherichia coli</em> biofilms: A non-conventional form of high cell density cultivation. <em>Process Biochemistry, 57, 1-8</em>. https://doi.org/10.1016/j.procbio.2017.03.018</p> <p>[2] Gomes, L., Monteiro, G., & Mergulhão, F. (2020). The Impact of IPTG Induction on Plasmid Stability and Heterologous Protein Expression by <em>Escherichia coli</em> Biofilms. <em>International Journal of Molecular Sciences, 21(2), 576</em>. https://doi.org/10.3390/ijms21020576</p>

scholarly journals Transformation of, and Heterologous Protein Expression in,Lactobacillus agilisandLactobacillus vaginalisIsolates from the Chicken Gastrointestinal Tract

2010 ◽  
Vol 77 (1) ◽  
pp. 220-228 ◽  
Author(s):  
David P. Stephenson ◽  
Robert J. Moore ◽  
Gwen E. Allison
Keyword(s):  
Gastrointestinal Tract ◽  
Protein Expression ◽  
Reporter Gene ◽  
Heterologous Protein ◽  
High Protein Diet ◽  
Plasmid Vector ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins

ABSTRACTLactobacilli are naturally found in the gastrointestinal tract of chickens, and there is interest in utilizing autochthonous strains for the delivery of therapeutic proteins. Previously we identified three chicken-derivedLactobacillusstrains,Lactobacillus agilisLa3,Lactobacillus vaginalisLv5, andLactobacillus crispatusLc9, which persist in the gastrointestinal tract of chickens fed either a commercial or high-protein diet. In the current study, we investigated the ability to electrotransform these strains, determined plasmid vector stability, and compared reporter gene expression directed by several different promoters. The La3 and Lv5 strains were reproducibly transformed with efficiencies of 108and 106transformants per microgram of plasmid DNA, respectively. The third strain tested,L. crispatusLc9, was recalcitrant to all transformation protocols examined. The plasmid vectors pTRK563 and pTRKH2 were maintained over 100 generations in La3 and Lv5, respectively. The ability of La3 and Lv5 to express the heterologous reporter genegfpwas analyzed using heterologous and homologous promoters. Transformants of both La3 and Lv5 containing the La3ldhLpromoter were the most fluorescent. To our knowledge, this is the first report of successful transformation and heterologous protein expression inL. agilisandL. vaginalis. The ability of these strains to express heterologous proteinsin vitroindicates their potential utility asin vivodelivery vectors for therapeutic peptides to the chicken gastrointestinal tract.

scholarly journals Modulation of heterologous protein secretion in the thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 by CRISPR-Cas9 system

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0258005
Author(s):  
Worarat Kruasuwan ◽  
Aekkachai Puseenam ◽  
Chitwadee Phithakrotchanakoon ◽  
Sutipa Tanapongpipat ◽  
Niran Roongsawang
Keyword(s):  
Er Stress ◽  
Protein Secretion ◽  
Protein Production ◽  
Secretory Pathway ◽  
Heterologous Protein ◽  
Methylotrophic Yeast ◽  
Heterologous Proteins ◽  
Loss Of Function ◽  
Heterologous Protein Secretion ◽  
Model Protein

The thermotolerant methylotrophic yeast Ogataea thermomethanolica TBRC 656 is a potential host strain for industrial protein production. Heterologous proteins are often retained intracellularly in yeast resulting in endoplasmic reticulum (ER) stress and poor secretion, and despite efforts to engineer protein secretory pathways, heterologous protein production is often lower than expected. We hypothesized that activation of genes involved in the secretory pathway could mitigate ER stress. In this study, we created mutants defective in protein secretory-related functions using clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) tools. Secretion of the model protein xylanase was significantly decreased in loss of function mutants for oxidative stress (sod1Δ) and vacuolar and protein sorting (vps1Δ and ypt7Δ) genes. However, xylanase secretion was unaffected in an autophagy related atg12Δ mutant. Then, we developed a system for sequence-specific activation of target gene expression (CRISPRa) in O. thermomethanolica and used it to activate SOD1, VPS1 and YPT7 genes. Production of both non-glycosylated xylanase and glycosylated phytase was enhanced in the gene activated mutants, demonstrating that CRISPR-Cas9 systems can be used as tools for understanding O. thermomethanolica genes involved in protein secretion, which could be applied for increasing heterologous protein secretion in this yeast.

scholarly journals A Novel Potent Carrier for Unconventional Protein Export in Ustilago maydis

2022 ◽  
Vol 9 ◽  
Author(s):  
Magnus Philipp ◽  
Kai P. Hussnaetter ◽  
Michèle Reindl ◽  
Kira Müntjes ◽  
Michael Feldbrügge ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Full Range ◽  
Ustilago Maydis ◽  
Firefly Luciferase ◽  
Protein Export ◽  
Spike Protein ◽  
Heterologous Proteins ◽  
Reporter Activity ◽  
Unconventional Secretion ◽  
Protein Functions

Recombinant proteins are ubiquitously applied in fields like research, pharma, diagnostics or the chemical industry. To provide the full range of useful proteins, novel expression hosts need to be established for proteins that are not sufficiently produced by the standard platform organisms. Unconventional secretion in the fungal model Ustilago maydis is an attractive novel option for export of heterologous proteins without N-glycosylation using chitinase Cts1 as a carrier. Recently, a novel factor essential for unconventional Cts1 secretion termed Jps1 was identified. Here, we show that Jps1 is unconventionally secreted using a fusion to bacterial β-glucuronidase as an established reporter. Interestingly, the experiment also demonstrates that the protein functions as an alternative carrier for heterologous proteins, showing about 2-fold higher reporter activity than the Cts1 fusion in the supernatant. In addition, Jps1-mediated secretion even allowed for efficient export of functional firefly luciferase as a novel secretion target which could not be achieved with Cts1. As an application for a relevant pharmaceutical target, export of functional bi-specific synthetic nanobodies directed against the SARS-CoV2 spike protein was demonstrated. The establishment of an alternative efficient carrier thus constitutes an excellent expansion of the existing secretion platform.

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