scholarly journals The “beauty in the beast”—the multiple uses of Priestia megaterium in biotechnology

2021 ◽  
Author(s):  
Rebekka Biedendieck ◽  
Tobias Knuuti ◽  
Simon J. Moore ◽  
Dieter Jahn
Keyword(s):  
Small Molecules ◽  
Recombinant Proteins ◽  
Value Added ◽  
Expression Vectors ◽  
Translation System ◽  
Vitamin B ◽  
Cell Factory ◽  
Promising Alternative ◽  
In Vitro Synthesis ◽  
Whole Cell

Abstract Over 30 years, the Gram-positive bacterium Priestia megaterium (previously known as Bacillus megaterium) was systematically developed for biotechnological applications ranging from the production of small molecules like vitamin B12, over polymers like polyhydroxybutyrate (PHB) up to the in vivo and in vitro synthesis of multiple proteins and finally whole-cell applications. Here we describe the use of the natural vitamin B12 (cobalamin) producer P. megaterium for the elucidation of the biosynthetic pathway and the subsequent systematic knowledge-based development for production purposes. The formation of PHB, a natural product of P. megaterium and potential petro-plastic substitute, is covered and discussed. Further important biotechnological characteristics of P. megaterium for recombinant protein production including high protein secretion capacity and simple cultivation on value-added carbon sources are outlined. This includes the advanced system with almost 30 commercially available expression vectors for the intracellular and extracellular production of recombinant proteins at the g/L scale. We also revealed a novel P. megaterium transcription-translation system as a complementary and versatile biotechnological tool kit. As an impressive biotechnology application, the formation of various cytochrome P450 is also critically highlighted. Finally, whole cellular applications in plant protection are completing the overall picture of P. megaterium as a versatile giant cell factory. Key points • The use of Priestia megaterium for the biosynthesis of small molecules and recombinant proteins through to whole-cell applications is reviewed. • P. megaterium can act as a promising alternative host in biotechnological production processes.

scholarly journals Screening of CHO-K1 endogenous promoters for expressing recombinant proteins in mammalian cell cultures

2021 ◽  
Author(s):  
Ileana Tossolini ◽  
Agustina Gugliotta ◽  
Fernando Lopez Diaz ◽  
Ricardo Kratje ◽  
Claudio Prieto
Keyword(s):  
Recombinant Proteins ◽  
Mammalian Cells ◽  
High Strength ◽  
High Rate ◽  
High Expression ◽  
Expression Vectors ◽  
Cmv Promoter ◽  
Promising Alternative ◽  
Expression Levels ◽  
Synthetic Promoters

For the production of recombinant protein therapeutics in mammalian cells, a high rate of gene expression is desired and hence strong viral-derived promoters are commonly used. However, they usually induce cellular stress and can be susceptible to epigenetic silencing. Endogenous promoters, which coordinates their activity with cellular and bioprocess dynamics while at the same time they maintain high expression levels, may help to avoid such drawbacks. In this work, endogenous promoters were identified based on high expression levels in RNA-seq data of CHO-K1 cells cultured in high density. The promoters of Actb, Ctsz, Hmox1, Hspa5, Vim and Rps18 genes were selected for generating new expression vectors for the production of recombinant proteins in mammalian cells. The in silico derived promoter regions were experimentally verified and the majority showed transcriptional activity comparable or higher than CMV. Also, stable expression following a reduction of culture temperature was investigated. The characterized endogenous promoters (excluding Rps18) constitute a promising alternative to CMV promoter due to their high strength, long-term expression stability and integration into the regulatory network of the host cell. These promoters may also comprise an initial panel for designing cell engineering strategies and synthetic promoters, as well as for industrial cell line development.

scholarly journals Efficient expression vectors and host strain for the production of recombinant proteins by Yarrowia lipolytica in process conditions

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Young-Kyoung Park ◽  
Marie Vandermies ◽  
Paul Soudier ◽  
Samuel Telek ◽  
Stéphane Thomas ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Recombinant Proteins ◽  
Expression System ◽  
Host Strain ◽  
Expression Vectors ◽  
Process Conditions ◽  
Cell Factory ◽  
Batch Bioreactor ◽  
Model Protein ◽  
Extracellular Protein Production

Abstract Background The oleaginous yeast Yarrowia lipolytica is increasingly used as an alternative cell factory for the production of recombinant proteins. Recently, regulated promoters from genes EYK1 and EYD1, encoding an erythrulose kinase and an erythritol dehydrogenase, respectively, have been identified and characterized in this yeast. Hybrid promoters up-regulated by polyols such as erythritol and erythrulose have been developed based on tandem copies of upstream activating sequences from EYK1 (UAS1EYK1) and XPR2 (encoding extracellular protease, UAS1XPR2) promoters. Results The strength of native (pEYD1) and engineered promoters (pEYK1-3AB and pHU8EYK) was compared using the extracellular lipase CalB from Candida antarctica as a model protein and a novel dedicated host strain. This latter is engineered in polyol metabolism and allows targeted chromosomal integration. In process conditions, engineered promoters pEYK1-3AB and pHU8EYK yielded 2.8 and 2.5-fold higher protein productivity, respectively, as compared to the reference pTEF promoter. We also demonstrated the possibility of multicopy integration in the newly developed host strain. In batch bioreactor, the CalB multi-copy strain RIY406 led to a 1.6 fold increased lipase productivity (45,125 U mL−1) within 24 h as compared to the mono-copy strain. Conclusions The expression system described herein appears promising for recombinant extracellular protein production in Y. lipolytica.

State of the art in the production of transgenic goats

2004 ◽  
Vol 16 (4) ◽  
pp. 465 ◽  
Author(s):  
H. Baldassarre ◽  
B. Wang ◽  
C. L. Keefer ◽  
A. Lazaris ◽  
C. N. Karatzas
Keyword(s):  
Recombinant Proteins ◽  
Traditional Method ◽  
Nuclear Transfer ◽  
State Of The Art ◽  
Expression Vectors ◽  
Marketing Approval ◽  
Transfected Cells ◽  
Pronuclear Microinjection ◽  
Transgenic Goats

This review summarises recent advances in the field of transgenic goats for the purpose of producing recombinant proteins in their milk. Production of transgenic goats via pronuclear microinjection of DNA expression vectors has been the traditional method, but this results in low efficiencies. Somatic cell nuclear transfer has dramatically improved efficiencies in rates of transgenesis. Characterisation of transfected cells in vitro before use in nuclear transfer guarantees that kids born are transgenic and of predetermined gender. Using these platform technologies, several recombinant proteins of commercial interest have been produced, although none of them has yet gained marketing approval. Before these technologies are implemented in goat improvement programmes, efficiencies must be improved, costs reduced, and regulatory approval obtained for the marketing of food products derived from such animals.

scholarly journals Comparison of Expression Vectors for Transient Expression of Recombinant Proteins in Plants

2013 ◽  
Vol 31 (6) ◽  
pp. 1529-1538 ◽  
Author(s):  
Kausar Hussain Shah ◽  
Bachar Almaghrabi ◽  
Holger Bohlmann
Keyword(s):  
Transient Expression ◽  
Recombinant Proteins ◽  
Expression Vectors

scholarly journals Different expression levels of two KgmB-His fusion proteins

2005 ◽  
Vol 70 (12) ◽  
pp. 1401-1407 ◽  
Author(s):  
Sandra Markovic ◽  
Sandra Vojnovic ◽  
Milija Jovanovic ◽  
Branka Vasiljevic
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Kanamycin Resistance ◽  
Expression Vectors ◽  
E Coli ◽  
C Terminus ◽  
N Terminus ◽  
Different Levels ◽  
Streptomyces Tenebrarius

The KgmB methylase from Streptomyces tenebrarius was expressed and purified using the QIAexpress System. Two expression vectors were made: pQEK-N, which places a (His)6 tag at the N-terminus, and pQEK-C, which places a (His)6 tag at the C-terminus of the recombinant KgmB protein. Kanamycin resistance of the E. coli cells containing either the pQEK-N or the pQEK-C recombinant plasmids confirmed the functionality of both KgmB-His fusion proteins in vivo. Interestingly, different levels of expression were observed between these two recombinant proteins. Namely, KgmB methylase with the (His)6 tag at the N-terminus showed a higher level of expression. Purification of the (His)6-tagged proteins using Ni-NTA affinity chromatography was performed under native conditions and the KgmB methylase with (His)6 tag at the N-terminus was purified to homogeneity >95 %. The recombinant KgmB protein was detected on a Western blot using anti-Sgm antibodies.

Production of Brucella melitensis Omp16 protein fused to the human interleukin 2 in Lactococcus lactis MG1363 toward developing a Lactococcus-based vaccine against brucellosis

2020 ◽  
Vol 66 (1) ◽  
pp. 39-45
Author(s):  
Marzieh Rezaei ◽  
Mohammad Rabbani Khorasgani ◽  
Sayyed Hamid Zarkesh Esfahani ◽  
Rahman Emamzadeh ◽  
Hamid Abtahi
Keyword(s):  
Fusion Protein ◽  
Lactococcus Lactis ◽  
Fusion Gene ◽  
Brucella Melitensis ◽  
Expression System ◽  
Interleukin 2 ◽  
Cell Factory ◽  
Promising Alternative ◽  
Alternative Expression ◽  
Restriction Sites

The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransformation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis.

scholarly journals A human expression system based on HEK293 for the stable production of recombinant erythropoietin

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Christine Lin Chin ◽  
Justin Bryan Goh ◽  
Harini Srinivasan ◽  
Kaiwen Ivy Liu ◽  
Ali Gowher ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
High Titer ◽  
Expression System ◽  
Mass Spectrometry Analysis ◽  
Hek293 Cells ◽  
Expression Vectors ◽  
Methionine Sulfoximine ◽  
Mammalian Host ◽  
Recombinant Erythropoietin ◽  
Cellular Expression

AbstractMammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins.

Expression Vectors for the Rapid Purification of Recombinant Proteins in Bacillus subtilis

2007 ◽  
Vol 55 (2) ◽  
pp. 89-93 ◽  
Author(s):  
Hoang Duc Nguyen ◽  
Trang Thi Phuong Phan ◽  
Wolfgang Schumann
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Proteins ◽  
Expression Vectors ◽  
Rapid Purification

Pharmacotherapeutics of Biotechnology-Derived Products

1998 ◽  
Vol 11 (1) ◽  
pp. 54-71
Author(s):  
Peggy Bush
Keyword(s):  
Gene Therapy ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Small Molecules ◽  
Orphan Drug ◽  
Recombinant Proteins ◽  
Human Disease ◽  
Drug Status ◽  
Small Patient ◽  
Biotechnology Products

Biotechnology has contributed to important advances in the healthcare field. Products include various hormones, enzymes, cytokines, vaccines, and monoclonal antibodies, with use in diverse therapeutic areas. The majority of approved biotechnology-derived therapeutic products are recombinant proteins. Many have orphan drug status and, therefore, are used in relatively small patient populations. Newer generation biotechnology products are likely to include small molecules, gene therapy products, and increased numbers of vaccines and monoclonal antibody products. Biotechnology provides the means to develop diverse, innovative, and effective approaches to the prevention, treatment, and cure of human disease.

scholarly journals Optimizing mevalonate pathway for squalene production in Yarrowia lipolytica

2020 ◽  
Author(s):  
Huan Liu ◽  
Fang Wang ◽  
Li Deng ◽  
Peng Xu
Keyword(s):  
Mevalonate Pathway ◽  
Cell Metabolism ◽  
Fold Increase ◽  
Cell Factory ◽  
Promising Alternative ◽  
Plant Extraction ◽  
Cost Efficient ◽  
Skin Compatibility ◽  
Coa Reductase ◽  
Engineered Strain

AbstractSqualene is the gateway molecule for triterpene-based natural products and steroids-based pharmaceuticals. As a super lubricant, it has been used widely in health care industry due to its skin compatibility and thermostability. Squalene is traditionally sourced from shark-hunting or oil plant extraction, which is cost-prohibitive and not sustainable. Reconstitution of squalene biosynthetic pathway in microbial hosts is considered as a promising alternative for cost-efficient and scalable synthesis of squalene. In this work, we reported the engineering of the oleaginous yeast, Y. lipolytica, as a potential host for squalene production. We systematically identified the bottleneck of the pathway and discovered that the native HMG-CoA reductase led to the highest squalene improvement. With the recycling of NADPH from the mannitol cycle, the engineered strain produced about 180.3 mg/l and 188.2 mg/L squalene from glucose or acetate minimal media, respectively. By optimizing the C/N ratio, controlling the media pH and mitigating the acetyl-CoA flux competition from lipogenesis, the engineered strain produced about 502.7 mg/L squalene in shake flaks, a 28-fold increase compared to the parental strain (17.2 mg/L). We also profiled the metabolic byproducts citric acid and mannitol level and observed that they are reincorporated into cell metabolism at the late stage of fermentation. This work may serve as a baseline to harness Y. lipolytica as an oleaginous cell factory for production of squalene or terpene-based chemicals.

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