By Huimin Zhao

  • “The proposed ebook is completely curious about man made biology. It brings jointly the idea leaders during this rising box, giving professional bills and their reviews of distinctive issues in artificial biology…. this could be the 1st devoted, edited ebook for man made biology.”
    Dr. Qing Lin, nation collage of recent York at Buffalo, Buffalo, manhattan, united states

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18. Ong T, De Serres F. Mutagenicity of chemical carcinogens in Neurospora crassa. Cancer Res. 1972;32:1890À1893. 19. Myers RM, Lerman LS, Maniatis T. A general method for saturation mutagenesis of cloned DNA fragments. Science. 1985;229:242À247. 20. Lai YP, Huang J, Wang LF, Li J, Wu ZR. A new approach to random mutagenesis in vitro. Biotechnol Bioeng. 2004;86:622À627. 21. Muteeb G, Sen R. Random mutagenesis using a mutator strain. Methods Mol Biol. 2010;634:411À419. 22. Cirino PC, Mayer KM, Umeno D.

Cerevisiae-based research In vitro one-step assembly tool [24,25] Type II restriction enzyme digestion One-step pathway assembly in B. 1 (Continued) Tool Description Codon Optimization Express a heterologous enzyme, change the DNA sequence to complement host codon bias Tuning Intergenic Improve gene expression far from Regions promoter, improve stability of mRNA by introducing secondary structures to reduce mRNA degradation TIGR Library Avoid rationally designing the intergenic regions, creation of a library Promoter Engineering Engineer different promoters with different strengths and apply them to a pathway to overexpress rate-limiting enzymes Combinatorial Library approach to promoter Promoter Library engineering Dynamic Gene circuit designed to express Transcriptional certain genes to redirect flux Regulation towards desired product RBS Engineering Improve translation efficiency by optimizing the RBS site by mathematical models Advantages References Can be done by DNA synthesis [50À53] Control of gene expression in an operon structure [54] Library approach allows for nonintuitive mutations [55] Allows fine-tuning of gene expression for a single gene [57] Fine tuning of multiple gene expression simultaneously Express superfluous genes only when needed, reducing burden Reduce the library size required to screen [58] Proteins with higher activity do not need high expression [64] Reduce side reactions and redirect the flux towards the product Reduce the competition for cofactors between required metabolism reactions and the desired pathway [65] Can select proteins for pathway with best activity, most substrate specificity, and cofactor usage simultaneously Shuttle intermediates directly to the next reaction, reducing diffusion [75] [59,60] [63] Optimizing protein activity Increasing Protein Activity Improving Substrate Specificity Alter Cofactor Specificity Library of Homologous Enzymes Protein Scaffolds In vitro enzyme engineering to improve activity increases product titer In vitro enzyme engineering to alter specificity to desired substrate In vitro enzyme engineering to alter specificity to desired cofactor, can create internal regeneration mechanisms for cofactor In vivo library combination of different enzyme homologues with different activities Anchor the protein in a special manner to stimulate active site channeling DESIGN AND CONSTRUCTION OF PATHWAYS Pathway Design Tools In the design of metabolic pathways for synthesis of target compounds, traditional methods simply modify existing endogenous pathways or recruit partial/entire pathways into a heterologous host.

Anal Biochem. 1999;269:359À366. 10. Bornscheuer UT, Pohl M. Improved biocatalysts by directed evolution and rational protein design. Curr Opin Chem Biol. 2001;5:137À143. 11. Koide S. Generation of new protein functions by nonhomologous combinations and rearrangements of domains and modules. Curr Opin Biotechnol. 2009;20:398À404. 12. Yüksel D, Pamuk D, Ivanova Y, Kumar K. Protein engineering using noncanonical amino acids. In: Park SJ, Cochran JR, eds. Protein Engineering and Design. Boca Raton: CRC Press; 2009:206À218.

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