Journal
ACS SYNTHETIC BIOLOGY
Volume 5, Issue 11, Pages 1308-1317Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.6b00083
Keywords
polyhydroxyalkanoates; PHB; high-throughput screening; ribosomal binding site calculator
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Funding
- National Natural Science Foundation of China [31430003, 31270146]
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As a product of a multistep enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by overexpression of the PHB synthesis pathway from a native producer involving three genes phbC, phbA, and phbB. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product. Here, we reported a semirational approach for highly efficient PHB pathway optimization in E. coli based on a phbCAB operon cloned from the native producer Ralstonia entropha (R. entropha). Rationally designed ribosomal binding site (RBS) libraries with defined strengths for each of the three genes were constructed based on high or low copy number plasmids in a one-pot reaction by an oligo-linker mediated assembly (OLMA) method. Strains with desired properties were evaluated and selected by three different methodologies, including visual selection, high-throughput screening, and detailed in-depth analysis. Applying this approach, strains accumulating 0%similar to 92% PHB contents in cell dry weight (CDW) were achieved. PHB with various weight-average molecular weights (M-w) of 2.7-6.8 x 10(6) were also efficiently produced in relatively high contents. These results suggest that the semirational approach combining library design, construction, and proper screening is an efficient way to optimize PHB and other multienzyme pathways.
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