A Novel Cre/lox-Based Genetic Tool for Repeated, Targeted and Markerless Gene Integration in Yarrowia lipolytica

The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by the limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox site-specific recombination system. The developed tool required only a single selection marker and could completely excise the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of the protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed that the genetic tool was effective for the integration of other genes, loci, and strains. Thus, this study significantly promotes the application of the Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.

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A novel and effective Cre/lox-based genetic tool for repeated, targeted and markerless integration in Yarrowia lipolytica

10.1101/2020.12.30.424803 ◽

2020 ◽

Author(s):

Qinghua Zhou ◽

Liangcheng Jiao ◽

Wenjuan Li ◽

Zhiming Hu ◽

Yunchong Li ◽

...

Keyword(s):

Yarrowia Lipolytica ◽

Protein Production ◽

Genome Engineering ◽

Selection Marker ◽

The Novel ◽

Genetic Tool ◽

Recombination System ◽

Marker Free ◽

Free Gene ◽

Gene Integration

AbstractThe unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox sitespecific recombination system. The developed tool required only a single selection marker and could completely excise all of the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed the genetic tool was effective for integration of other genes, loci, and strains. Thus, this study significantly promotes the application of Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.

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Study of a upp-Based Counterselective Method for Large-Scale Deletion of Genome Fragments in Bacillus subtilis

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.634-638.1076 ◽

2013 ◽

Vol 634-638 ◽

pp. 1076-1080

Author(s):

Xue Yu Zhang ◽

Jing Fu ◽

Tao Chen

Keyword(s):

Bacillus Subtilis ◽

Large Scale ◽

Gene Knockout ◽

Engineering Strain ◽

Large Genome ◽

Study Research ◽

Genetic Tool ◽

Reduced Genome ◽

Marker Free ◽

Free Gene

The generation of an engineering strain with reduced genome relies on efficient methods for marker-free gene knockout. In this study, research on the efficiency of deletion of genome fragments with upp-based counterselective method in Bacillus subtilis was carried out. Using this method, the 3.053 Kb, 12.414 Kb and 34.148 Kb fragments of the skin element were marker-free deleted successfully, with the efficiency of 27%, 11.4% and 25%, respectively. Here we demonstrate that this method is an efﬁcient genetic tool for large genome fragments marker-free deletion.

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Marker-free genome engineering in Amycolatopsis using the pSAM2 site-specific recombination system

10.1101/2021.09.18.460463 ◽

2021 ◽

Author(s):

Luísa D. F. Santos ◽

Laëtitia Caraty-Philippe ◽

Emmanuelle Darbon ◽

Jean-Luc Pernodet

Keyword(s):

Genome Engineering ◽

High Efficiency ◽

Genetic Manipulation ◽

Marker Genes ◽

Biotechnological Applications ◽

Site Specific ◽

Site Specific Recombination ◽

Recombination System ◽

Marker Free ◽

Site Specific Recombination System

ABSTRACTActinobacteria belonging to the genus Amycolatopsis are important for antibiotic production and other valuable biotechnological applications such as biodegradation or bioconversion. Despite their industrial importance, tools and methods for the genetic manipulation of Amycolatopsis are less developed than in other actinobacteria such as Streptomyces. Moreover, most of the existing methods do not support convenient marker-free genome engineering. Here, we report the use of the pSAM2 site-specific recombination system for the efficient deletion of marker genes or large DNA regions in Amycolatopsis. For this purpose, we constructed a shuttle vector, replicating in Escherichia coli and Amycolatopsis, expressing the Xis and Int proteins from the Streptomyces integrative and conjugative element pSAM2. These proteins are sufficient for site-specific recombination between the attachment sites attL and attR. We also constructed two plasmids, replicative in E. coli but not in Amycolatopsis, for the integration of the recombination sites attL and attR on each side of a region targeted for deletion. We exemplified the use of these tools in Amycolatopsis mediterranei DSM 40773 by obtaining with high efficiency (>95%) a marker-free deletion of one single gene in the rifamycin biosynthetic gene cluster or of the entire 90-kb cluster.IMPORTANCEThe genus Amycolatopsis is regarded as an important source of diverse specialized metabolites. Members of this genus are used in industry for the production of valuable antibiotics such as rifamycins or vancomycin. Amycolatopsis spp. also present a great interest for biotechnological applications such as biodegradation or bioconversion. Despite their importance, their genetic manipulation was somehow hampered by the lack of efficient tools. Here we report the successful use of the pSAM2 site-specific recombination system to construct unmarked deletion mutants, allowing marker recycling, or to create large deletions in A. mediterranei DSM 40773. The high efficiency of this site-specific recombination system and it possible application to other Amycolatopsis species open new opportunities for marker-free genome engineering in this genus.

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Strategies for Vaccination: Conventional Vaccine Approaches Versus New-Generation Strategies in Combination with Adjuvants

Pharmaceutics ◽

10.3390/pharmaceutics13020140 ◽

2021 ◽

Vol 13 (2) ◽

pp. 140

Author(s):

Abdellatif Bouazzaoui ◽

Ahmed A. H. Abdellatif ◽

Faisal A. Al-Allaf ◽

Neda M. Bogari ◽

Saied Al-Dehlawi ◽

...

Keyword(s):

Viral Vectors ◽

Protein Production ◽

Vaccine Development ◽

Effective Vaccine ◽

The Novel ◽

Research Teams ◽

Advantages And Disadvantages ◽

Rapid Spread ◽

New Generation ◽

Conventional Vaccine

The current COVID-19 pandemic, caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), has raised significant economic, social, and psychological concerns. The rapid spread of the virus, coupled with the absence of vaccines and antiviral treatments for SARS-CoV-2, has galvanized a major global endeavor to develop effective vaccines. Within a matter of just a few months of the initial outbreak, research teams worldwide, adopting a range of different strategies, embarked on a quest to develop effective vaccine that could be effectively used to suppress this virulent pathogen. In this review, we describe conventional approaches to vaccine development, including strategies employing proteins, peptides, and attenuated or inactivated pathogens in combination with adjuvants (including genetic adjuvants). We also present details of the novel strategies that were adopted by different research groups to successfully transfer recombinantly expressed antigens while using viral vectors (adenoviral and retroviral) and non-viral delivery systems, and how recently developed methods have been applied in order to produce vaccines that are based on mRNA, self-amplifying RNA (saRNA), and trans-amplifying RNA (taRNA). Moreover, we discuss the methods that are being used to enhance mRNA stability and protein production, the advantages and disadvantages of different methods, and the challenges that are encountered during the development of effective vaccines.

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