scholarly journals In vitro and in vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Dan-Dan Qi ◽  
Jin Jin ◽  
Duo Liu ◽  
Bin Jia ◽  
Ying-Jin Yuan
Keyword(s):  
In Vivo Recombination

scholarly journals The Escherichia coli Fis Protein Stimulates Bacteriophage λ Integrative Recombination In Vitro

2003 ◽  
Vol 185 (10) ◽  
pp. 3076-3080 ◽  
Author(s):  
Dominic Esposito ◽  
Gary F. Gerard
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Essential Role ◽  
Bacteriophage Λ ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
In Vivo Recombination

ABSTRACT The Escherichia coli nucleoid-associated protein Fis was previously shown to be involved in bacteriophage lambda site-specific recombination in vivo, enhancing the levels of both integrative recombination and excisive recombination. While purified Fis protein was shown to stimulate in vitro excision, Fis appeared to have no effect on in vitro integration reactions even though a 15-fold drop in lysogenization frequency had previously been observed in fis mutants. We demonstrate here that E. coli Fis protein does stimulate integrative lambda recombination in vitro but only under specific conditions which likely mimic natural in vivo recombination more closely than the standard conditions used in vitro. In the presence of suboptimal concentrations of Int protein, Fis stimulates the rate of integrative recombination significantly. In addition, Fis enhances the recombination of substrates with nonstandard topologies which may be more relevant to the process of in vivo phage lambda recombination. These data support the hypothesis that Fis may play an essential role in lambda recombination in the host cell.

scholarly journals In vitro and In vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast

2020 ◽  
Author(s):  
Dan-Dan Qi ◽  
Jin Jin ◽  
Duo Liu ◽  
Bin Jia ◽  
Ying-Jin Yuan
Keyword(s):  
High Throughput Screening ◽  
Optimal Combination ◽  
In Vitro Recombination ◽  
In Vivo Recombination ◽  
Yeast Strains ◽  
Engineered Yeast ◽  
Heterologous Pathway ◽  
Β Carotene

Abstract Background : Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. Concerning the safety and economy issue the biosynthesis of astaxanthin has greater potential than chemical synthesis and extraction from natural producers. However, the production of astaxanthin in microorganisms is still limited by the poor efficiency of the heterologous pathway. Results: To address the bottleneck of astaxanthin yield in microbes, we developed the in vitro and in vivo recombination methods to optimize the combination of heterologous modules of β-carotene ketolase ( crtW ) and hydroxylase ( crtZ ) from different species in engineered yeast strains. Finally, the astaxanthin yield of in vitro recombination and in vivo recombination were enhanced 2.11- to 8.51-fold and 3.05- to 9.71-fold compared to the parent strains, respectively. The highest astaxanthin producing yeast yQDD022 was obtained by the in vivo recombination with 6.05 mg/g DCW of the astaxanthin yield. Moreover, it is demonstrated that the astaxanthin producing yeast of the in vivo recombination has higher efficiency and stability than that of the in vitro recombination. Conclusions: Recombination of heterologous modules by in vitro and in vivo provides a simple and efficient way to improve the astaxanthin yield in yeast. Both the in vitro and in vivo recombination methods enable high-throughput screening of heterologous pathways by combining crtW and crtZ from different species. And the heterologous pathway constructed by the in vivo recombination is more stable than that of the in vitro recombination. This study not only found the underlying optimal combination of crtZ and crtW , but also provided a reference to greatly enhance desired compounds accumulation by evolving heterologous pathways.

scholarly journals In vitro and In vivo recombination of heterologous modules for improving biosynthesis of astaxanthin in yeast

2020 ◽  
Author(s):  
Dan-Dan Qi ◽  
Jin Jin ◽  
Duo Liu ◽  
Bin Jia ◽  
Ying-Jin Yuan
Keyword(s):  
High Throughput Screening ◽  
Optimal Combination ◽  
In Vivo Recombination ◽  
Environmental Friendly ◽  
Yeast Strains ◽  
Engineered Yeast ◽  
The Cost ◽  
Heterologous Pathway

Abstract Background: Astaxanthin is a kind of tetraterpene and has strong antioxygenic property. The biosynthesis of astaxanthin in engineered microbial chassis has greater potential than its chemical synthesis and extraction from natural producers in an environmental-friendly way. However, the cost-offsetting production of astaxanthin in engineered microbes is still constrained by the poor efficiency of astaxanthin synthesis pathway as a heterologous pathway.Results: To address the bottleneck of limited production of astaxanthin in microbes, we developed in vitro and in vivo recombination methods respectively in engineered yeast chassis to optimize the combination of heterologousβ-carotene ketolase (crtW) and hydroxylase (crtZ) modules that were selected from different species. As a result, the in vitro and in vivo recombination methods enhanced the astaxanthin yield respectively to 2.11~8.51 folds and 3.0~9.71 folds compared to the initial astaxanthin pathway, according to the different combination of particular genes. The highest astaxanthin producing strain yQDD022 was constructed by in vivo method and produced 6.05 mg/g DCW of astaxanthin. Moreover, it was proved that the in vivo recombination method showed higher DNA-assembling efficiency than the in vitro method and contributed to higher stability to the engineered yeast strains.Conclusions: The in vitro and in vivo recombination methods of heterologous modules provide simple and efficient ways to improve the astaxanthin yield in yeast. Both the two methods enable high-throughput screening of heterologous pathways through recombination of certain crtW and crtZ derived from different species. This study not only exploited the underlying optimal combination of crtZ and crtW for astaxanthin synthesis, but also provided a general approach to evolve a heterologous pathway for the enhanced accumulation of desired biochemical products.

scholarly journals Determination of the curvature of epithelial cell mass by mesenchyme in branching morphogenesis of mouse salivary gland

Development ◽  
1983 ◽  
Vol 73 (1) ◽  
pp. 221-232
Author(s):  
Hiroyuki Nogawa
Keyword(s):  
Salivary Gland ◽  
Epithelial Cell ◽  
Cell Mass ◽  
Branching Morphogenesis ◽  
Epithelial Surface ◽  
In Vivo Recombination ◽  
Recombination Experiments

Mouse submaxillary epithelium undergoes branching morphogenesis with increase in the curvature of its surface in vivo. Recombination experiments in vitro of the epithelium and mesenchyme between 13- and 14-day rudiments showed (1) that the 14-day mesenchyme more actively induced the epithelium to branch than the 13-day mesenchyme, (2) that the 14-day mesenchyme could produce clefts on smaller epithelial lobes than the 13-day mesenchyme, (3) that the 14-day mesenchyme produced lobes with similar diameter to lobes of the 14-day intact rudiment, and (4) that the lobular morphology of assembled 14-day lobes became obscure in recombinates with the 13-day mesenchyme while it was well maintained in recombinates with the 14-day mesenchyme. From these results it is concluded that the mesenchyme determines the curvature of epithelial surface, and that clefts are formed on the epithelial surface as a result of increase in the epithelial curvature.

In vitro and In vivo recombination of heterologous modular for improving biosynthesis of astaxanthin in yeast

2020 ◽  
Author(s):  
Dan-Dan Qi ◽  
Jin Jin ◽  
Duo Liu ◽  
Bin Jia ◽  
Ying-Jin Yuan
Keyword(s):  
Chemical Synthesis ◽  
High Throughput Screening ◽  
Optimal Combination ◽  
In Vivo Recombination ◽  
The Poor ◽  
Yeast Strains ◽  
Engineered Yeast ◽  
Heterologous Pathway

Abstract Bacground: Astaxanthin is a kind of tetraterpene with strong antioxygenic property. Concerning the safety and economy issue the biosynthesis of astaxanthin has greater potential than chemical synthesis and extraction from natural producers. However, the production of astaxanthin in microorganism is still limited by the poor efficiency of heterologous pathway.Results: To address the bottleneck of astaxanthin yield in microbe, we developed the in vitro and in vivo recombination methods to optimize the combination of heterologous modular ofβ-carotene ketolase (crtW) and hydroxylation (crtZ) from different species in engineered yeast strains. Finally, the astaxanthin yield of in vitro recombination and in vivo recombination were enhanced 2.11- to 8.51-fold and 3.05- to 9.71-fold compared to the parent strains, respectively. The highest astaxanthin producing yeast yQDD022 was obtained by the in vivo recombination with 6.05mg/g DCW of the astaxanthin yield. Moreover, it is demonstrated that the astaxanthin producing yeast of the in vivo recombination has higher efficiency and stability than that of the in vitro recombination.Conclusions: Recombination of heterologous modular by in vitro and in vivo provides a simple and efficient way to improve the astaxanthin yield in yeast. Both the in vitro and in vivo recombination methods enable high throughput screening of heterologous pathway by combining crtW and crtZ from different species. And the heterologous pathway constructed by the in vivo recombination is more stable than that of the in vitro recombination. This study not only found the underlying optimal combination of crtZ and crtW, but also provided a reference to greatly enhance desired compounds accumulation by evolving heterologous pathway.

Producing Chimeric Genes by CLERY In Vitro and In Vivo Recombination

2003 ◽  
pp. 165-174 ◽  
Author(s):  
Valérie Abécassis ◽  
Denis Pompon ◽  
Gilles Truan
Keyword(s):  
Chimeric Genes ◽  
In Vivo Recombination

scholarly journals Point Mutations in the Integron Integrase IntI1 That Affect Recombination and/or Substrate Recognition

1998 ◽  
Vol 180 (20) ◽  
pp. 5437-5442 ◽  
Author(s):  
Annie Gravel ◽  
Nancy Messier ◽  
Paul H. Roy
Keyword(s):  
Dna Binding ◽  
Antibiotic Resistance Gene ◽  
Point Mutations ◽  
Wild Type ◽  
Conserved Residues ◽  
Site Specific ◽  
Gene Cassettes ◽  
In Vivo Recombination

ABSTRACT The site-specific recombinase IntI1 found in class 1 integrons catalyzes the excision and integration of mobile gene cassettes, especially antibiotic resistance gene cassettes, with a site-specific recombination system. The integron integrase belongs to the tyrosine recombinase (phage integrase) family. The members of this family, exemplified by the lambda integrase, do not share extensive amino acid identities, but three invariant residues are found within two regions, designated box I and box II. Two conserved residues are arginines, one located in box I and one in box II, while the other conserved residue is a tyrosine located at the C terminus of box II. We have analyzed the properties of IntI1 variants carrying point mutations at the three conserved residues of the family in in vivo recombination and in vitro substrate binding. We have made four proteins with mutations of the conserved box I arginine (R146) and three mutants with changes of the box II arginine (R280); of these, MBP-IntI1(R146K) and MBP-IntI1(R280K) bind to the attI1 site in vitro, but only MBP-IntI1(R280K) is able to excise cassettes in vivo. However, the efficiency of recombination and DNA binding for MBP-IntI1(R280K) is lower than that obtained with the wild-type MBP-IntI1. We have also made two proteins with mutations of the tyrosine residue (Y312), and both mutant proteins are similar to the wild-type fusion protein in their DNA-binding capacity but are unable to catalyze in vivo recombination.

Induction and Differentiation of Dental Epithelium in Vivo and in Vitro

1982 ◽  
Vol 40 ◽  
pp. 142-145
Author(s):  
E. J. Kollar
Keyword(s):  
Connective Tissue ◽  
Oral Mucosa ◽  
Basal Lamina ◽  
Functional Derivatives ◽  
Specific Source ◽  
Dental Epithelium ◽  
Connective Tissue Stroma

The differentiation and maintenance of many specialized epithelial structures are dependent on the underlying connective tissue stroma and on an intact basal lamina. These requirements are especially stringent in the development and maintenance of the skin and oral mucosa. The keratinization patterns of thin or thick cornified layers as well as the appearance of specialized functional derivatives such as hair and teeth can be correlated with the specific source of stroma which supports these differentiated expressions.

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