Feb 9, 2017

Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening

PloS One
Daniela QuagliaJoelle N Pelletier

Abstract

The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A), the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random). Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid production of a series of diverse libraries, thus facilitating directed evolution. Screening to improve discrimination of short-chain versus long-chain fatty acid substrates was aided by development of a general, automated method for visual discrimination of the hydrolysis of varied substrates by whole cells.

  • References32
  • Citations5

References

Mentioned in this Paper

Directed Evolution
Molecular Dynamics
Transverse Tubule of Muscle Cell
Fatty Acids, Volatile
Olive oil
Carboxy-Terminal Amino Acid
Genes
Enzymes, antithrombotic
Agar
Structure-Activity Relationship

Related Feeds

Aminoglycosides (ASM)

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside. Discover the latest research on aminoglycoside here.

Biomaterial Engineering

Advances in biomaterial engineering have permitted the development of sophisticated drug-releasing materials with a biomimetic 3D support that allow a better control of the microenvironment of transplanted cells. Here is the latest research.

Aminoglycosides

Aminoglycoside is a medicinal and bacteriologic category of traditional Gram-negative antibacterial medications that inhibit protein synthesis and contain as a portion of the molecule an amino-modified glycoside. Discover the latest research on aminoglycoside here.