scholarly journals A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression inKluyveromyces lactis

2019 ◽  
Vol 85 (14) ◽  
Author(s):  
Hassan Sakhtah ◽  
Juliane Behler ◽  
Alana Ali-Reynolds ◽  
Thomas B. Causey ◽  
Saulius Vainauskas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Carbon Source ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Heterologous Protein ◽  
Kluyveromyces Lactis ◽  
Heterologous Protein Expression ◽  
Heterologous Proteins ◽  
Content Type ◽  
Hybrid Promoter

ABSTRACTThe yeastKluyveromyces lactishas been a successful host for the production of heterologous proteins for over 30 years. Currently, the galactose-/lactose-inducible and glucose-repressibleLAC4promoter (PLAC4) is the most widely used promoter to drive recombinant protein expression inK. lactis. However, PLAC4is not fully repressed in the presence of glucose and significant protein expression still occurs. Thus, PLAC4is not suitable in processes where tight regulation of heterologous gene expression is required. In this study, we devised a novelK. lactispromoter system that is both strong and tightly controllable. We first tested several different endogenousK. lactispromoters for their ability to express recombinant proteins. A novel hybrid promoter (termed P350) was created by combining segments of twoK. lactispromoters, namely, the strong constitutive PGAP1promoter and the carbon source-sensitive PICL1promoter. We demonstrate that P350is tightly repressed in the presence of glucose or glycerol and becomes derepressed upon depletion of these compounds by the growing cells. We further illustrate the utility of P350-controlled protein expression in shake flask and high-cell-density bioreactor cultivation strategies. The P350hybrid promoter is a strong derepressible promoter for use in autoinduction of one-step fermentation processes for the production of heterologous proteins inK. lactis.IMPORTANCEThe yeastKluyveromyces lactisis an important host for the expression of recombinant proteins at both laboratory and industrial scales. However, the system lacks a tightly regulated promoter that permits controlled expression of heterologous proteins. In this study, we report the engineering of a highly regulated strong hybrid promoter (termed P350) for use inK. lactis. P350is tightly repressed by glucose or glycerol in the medium but strongly promotes gene expression once the carbon source has been consumed by the cells. This feature permits heterologous protein expression to be “autoinduced” at any scale without the addition of a gratuitous inducer molecule or changing feed solutions.

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 Complete Genome Sequence of Kluyveromyces lactis Strain GG799, a Common Yeast Host for Heterologous Protein Expression

2017 ◽  
Vol 5 (30) ◽  
Author(s):  
Léa Chuzel ◽  
Mehul B. Ganatra ◽  
Kelly M. Schermerhorn ◽  
Andrew F. Gardner ◽  
Brian P. Anton ◽  
...  
Keyword(s):  
Protein Expression ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Heterologous Protein ◽  
Kluyveromyces Lactis ◽  
Heterologous Protein Expression ◽  
Content Type ◽  
The Pacific ◽  
Lactis Strain

ABSTRACT We report the genome sequence of the dairy yeast Kluyveromyces lactis strain GG799 obtained using the Pacific Biosciences RS II platform. K. lactis strain GG799 is a common host for the expression of proteins at both laboratory and industrial scales.

scholarly journals Fungal Light-Oxygen-Voltage Domains for Optogenetic Control of Gene Expression and Flocculation in Yeast

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Francisco Salinas ◽  
Vicente Rojas ◽  
Verónica Delgado ◽  
Javiera López ◽  
Eduardo Agosin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Dynamic Range ◽  
Industrial Applications ◽  
Yeast Cells ◽  
Heterologous Protein Expression ◽  
Control Of Gene Expression ◽  
Content Type ◽  
Chemical Inducers

ABSTRACT Optogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers, and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV, an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (light-oxygen-voltage) blue-light photoreceptors from the fungus Neurospora crassa. When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution and a broad dynamic range of over 1,300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical-inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin-encoding gene FLO1, by the FUN-LOV switch, yielded flocculation in light (FIL), whereas the light-controlled expression of the corepressor TUP1 provided flocculation in darkness (FID). Altogether, the results reveal the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV’s ability to accurately manipulate gene expression, with a high temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms. IMPORTANCE Optogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Here, we report a novel optogenetic switch (FUN-LOV) based on the LOV domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa. In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes, heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.

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 Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

2021 ◽  
Author(s):  
Richard Nelson Hall ◽  
Uri Weill ◽  
Margarita Khariton ◽  
Sergio Leal-Ortiz ◽  
Leonard Drees ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Untranslated Region ◽  
Adult Stem Cells ◽  
Heterologous Protein ◽  
Heterologous Protein Expression ◽  
Reporter Expression ◽  
Mrna Transfection ◽  
Genetic Constructs ◽  
Exogenous Gene

Planarians have long been studied for their regenerative abilities, but they possess limited genetic tools due to challenges in gene delivery, expression, and detection, despite decades of work. We developed a toolbox for heterologous protein expression in planarian cells and in live animals. Specifically, we identified and optimized nanotechnological and chemical transfection methods to efficiently deliver mRNA encoding nanoluciferase into somatic cells, including planarian adult stem cells (neoblasts). The use of a luminescent reporter allowed us to quantitatively measure protein expression through spectroscopy and microscopy, thus overcoming the strong autofluorescent background of planarian tissues. Using this platform, we investigated the use of endogenous untranslated region (UTR) sequences and codon usage bias to post-transcriptionally alter gene expression. Our work provides a strong foundation for advancing exogenous gene expression and for the rapid prototyping of genetic constructs to accelerate the development of transgenic techniques in planarians.

scholarly journals Overcoming codon-usage bias in heterologous protein expression in Streptococcus gordonii

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3581-3588 ◽  
Author(s):  
Song F. Lee ◽  
Yi-Jing Li ◽  
Scott A. Halperin
Keyword(s):  
Protein Expression ◽  
Codon Usage ◽  
Codon Usage Bias ◽  
Heterologous Protein ◽  
Trna Gene ◽  
Heterologous Protein Expression ◽  
Streptococcus Gordonii ◽  
Heterologous Proteins ◽  
Single Chain ◽  
Rare Codons

One of the limitations facing the development of Streptococcus gordonii into a successful vaccine vector is the inability of this bacterium to express high levels of heterologous proteins. In the present study, we have identified 12 codons deemed as rare codons in S. gordonii and seven other streptococcal species. tRNA genes encoding 10 of the 12 rare codons were cloned into a plasmid. The plasmid was transformed into strains of S. gordonii expressing the fusion protein SpaP/S1, the anti-complement receptor 1 (CR1) single-chain variable fragment (scFv) antibody, or the Toxoplasma gondii cyclophilin C18 protein. These three heterologous proteins contained high percentages of amino acids encoded by rare codons. The results showed that the production of SpaP/S1, anti-CR1 scFv and C18 increased by 2.7-, 120- and 10-fold, respectively, over the control strains. In contrast, the production of the streptococcal SpaP protein without the pertussis toxin S1 fragment was not affected by tRNA gene supplementation, indicating that the increased production of SpaP/S1 protein was due to the ability to overcome the limitation caused by rare codons required for the S1 fragment. The increase in anti-CR1 scFv production was also observed in Streptococcus mutans following tRNA gene supplementation. Collectively, the findings in the present study demonstrate for the first time, to the best of our knowledge, that codon-usage bias exists in Streptococcus spp. and the limitation of heterologous protein expression caused by codon-usage bias can be overcome by tRNA supplementation.

scholarly journals FUN-LOV: Fungal LOV domains for optogenetic control of gene expression and flocculation in yeast

2018 ◽  
Author(s):  
Francisco Salinas ◽  
Vicente Rojas ◽  
Verónica Delgado ◽  
Javiera López ◽  
Eduardo Agosin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Heterologous Protein ◽  
Dynamic Range ◽  
Industrial Applications ◽  
Yeast Cells ◽  
Heterologous Protein Expression ◽  
Control Of Gene Expression ◽  
Chemical Inducers ◽  
Cellular Processes

AbstractOptogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV; an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (Light Oxygen Voltage) blue-light photoreceptors from the fungus Neurospora crassa. When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution, and a broad dynamic range of over 1300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin encoding gene FLO1, by the FUN-LOV switch, yielded Flocculation in Light (FIL), whereas the light-controlled expression of the co-repressor TUP1 provided Flocculation in Darkness (FID). Overall, the results revealed the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV’ s ability to accurately manipulate gene expression, with a high-temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.ImportanceOptogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Herein, we report a novel optogenetic switch (FUN-LOV) based on the LOV-domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa. In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes: heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.

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.

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