scholarly journals Start-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering

2018 ◽  
Vol 47 (3) ◽  
pp. e17-e17 ◽  
Author(s):  
George M Taylor ◽  
Paweł M Mordaka ◽  
John T Heap
Keyword(s):  
Metabolic Engineering ◽  
Assembly System ◽  
Dna Assembly

MIDAS: A Modular DNA Assembly System for Synthetic Biology

2018 ◽  
Vol 7 (4) ◽  
pp. 1018-1029 ◽  
Author(s):  
Craig J. van Dolleweerd ◽  
Sarah A. Kessans ◽  
Kyle C. Van de Bittner ◽  
Leyla Y. Bustamante ◽  
Rudranuj Bundela ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Assembly System ◽  
Dna Assembly

scholarly journals Start-Stop Assembly: a functionally scarless DNA assembly system optimised for metabolic engineering

2018 ◽  
Author(s):  
George M. Taylor ◽  
Paweł M. Mordaka ◽  
John T. Heap
Keyword(s):  
Metabolic Engineering ◽  
Design Space ◽  
Combinatorial Libraries ◽  
Dna Assembly ◽  
Destination Vector ◽  
E Coli ◽  
Assembly Method ◽  
Wide Range ◽  
Combinatorial Assembly ◽  
Sequence Boundaries

ABSTRACTDNA assembly allows individual DNA constructs or designed mixtures to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable ‘scar’ sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimising the number of vectors. Only one destination vector is required per organism, reflecting our optimisation of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways inE. coliresulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.GRAPHICAL ABSTRACT

scholarly journals ZeBRα a universal, multi-fragment DNA-assembly-system with minimal hands-on time requirement

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
David Richter ◽  
Katharina Bayer ◽  
Thomas Toesko ◽  
Stefan Schuster
Keyword(s):  
Assembly System ◽  
Dna Assembly ◽  
Time Requirement ◽  
Hands On

One step DNA assembly for combinatorial metabolic engineering

2014 ◽  
Vol 23 ◽  
pp. 70-77 ◽  
Author(s):  
Pieter Coussement ◽  
Jo Maertens ◽  
Joeri Beauprez ◽  
Wouter Van Bellegem ◽  
Marjan De Mey
Keyword(s):  
Metabolic Engineering ◽  
Dna Assembly ◽  
One Step

Recombination-Based DNA Assembly and Mutagenesis Methods for Metabolic Engineering

2011 ◽  
pp. 93-109 ◽  
Author(s):  
Xiquan Liang ◽  
Lansha Peng ◽  
Billyana Tsvetanova ◽  
Ke Li ◽  
Jian-Ping Yang ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Dna Assembly

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

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