PMID- 27417934
OWN - NLM
STAT- MEDLINE
DCOM- 20170808
LR  - 20201209
IS  - 1557-7988 (Electronic)
IS  - 0076-6879 (Linking)
VI  - 575
DP  - 2016
TI  - Rapid Optimization of Engineered Metabolic Pathways with Serine Integrase 
      Recombinational Assembly (SIRA).
PG  - 285-317
LID - S0076-6879(16)00093-8 [pii]
LID - 10.1016/bs.mie.2016.02.009 [doi]
AB  - Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of 
      commodity compounds and fine chemicals offers a cheaper, greener, and more 
      reliable method of production than does chemical synthesis. However, engineering 
      metabolic pathways within a microbe is a complicated process: levels of gene 
      expression, protein stability, enzyme activity, and metabolic flux must be 
      balanced for high productivity without compromising host cell viability. A major 
      rate-limiting step in engineering microbes for optimum biosynthesis of a target 
      compound is DNA assembly, as current methods can be cumbersome and costly. Serine 
      integrase recombinational assembly (SIRA) is a rapid DNA assembly method that 
      utilizes serine integrases, and is particularly applicable to rapid optimization 
      of engineered metabolic pathways. Using six pairs of orthogonal attP and attB 
      sites with different central dinucleotide sequences that follow SIRA design 
      principles, we have demonstrated that PhiC31 integrase can be used to (1) insert a 
      single piece of DNA into a substrate plasmid; (2) assemble three, four, and five 
      DNA parts encoding the enzymes for functional metabolic pathways in a one-pot 
      reaction; (3) generate combinatorial libraries of metabolic pathway constructs 
      with varied ribosome binding site strengths or gene orders in a one-pot reaction; 
      and (4) replace and add DNA parts within a construct through targeted 
      postassembly modification. We explain the mechanism of SIRA and the principles 
      behind designing a SIRA reaction. We also provide protocols for making SIRA 
      reaction components and practical methods for applying SIRA to rapid optimization 
      of metabolic pathways.
CI  - (c) 2016 Elsevier Inc. All rights reserved.
FAU - Merrick, C A
AU  - Merrick CA
AD  - University of Edinburgh, Edinburgh, United Kingdom. Electronic address: 
      christine.merrick@ed.ac.uk.
FAU - Wardrope, C
AU  - Wardrope C
AD  - University of Edinburgh, Edinburgh, United Kingdom.
FAU - Paget, J E
AU  - Paget JE
AD  - University of Edinburgh, Edinburgh, United Kingdom.
FAU - Colloms, S D
AU  - Colloms SD
AD  - University of Glasgow, Glasgow, United Kingdom.
FAU - Rosser, S J
AU  - Rosser SJ
AD  - University of Edinburgh, Edinburgh, United Kingdom.
LA  - eng
PT  - Journal Article
DEP - 20160323
PL  - United States
TA  - Methods Enzymol
JT  - Methods in enzymology
JID - 0212271
RN  - 452VLY9402 (Serine)
RN  - 9007-49-2 (DNA)
RN  - EC 2.7.7.- (Integrases)
SB  - IM
MH  - Bacteriophages/*enzymology/genetics/metabolism
MH  - Base Sequence
MH  - DNA/genetics/metabolism
MH  - Escherichia coli/*genetics/metabolism
MH  - Gene Expression
MH  - Integrases/genetics/*metabolism
MH  - Metabolic Engineering/*methods
MH  - *Metabolic Networks and Pathways
MH  - Plasmids/genetics/metabolism
MH  - *Recombination, Genetic
MH  - Serine/metabolism
MH  - Synthetic Biology/methods
OTO - NOTNLM
OT  - DNA assembly
OT  - Metabolic pathway engineering
OT  - Serine integrase
EDAT- 2016/07/16 06:00
MHDA- 2017/08/09 06:00
CRDT- 2016/07/16 06:00
PHST- 2016/07/16 06:00 [entrez]
PHST- 2016/07/16 06:00 [pubmed]
PHST- 2017/08/09 06:00 [medline]
AID - S0076-6879(16)00093-8 [pii]
AID - 10.1016/bs.mie.2016.02.009 [doi]
PST - ppublish
SO  - Methods Enzymol. 2016;575:285-317. doi: 10.1016/bs.mie.2016.02.009. Epub 2016 Mar 
      23.