Semirational Approach for Ultrahigh Poly(3-hydroxybutyrate)
Accumulation in Escherichia coli by Combining One-Step
Library Construction and High-Throughput Screening
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Version 1 2016-05-10, 19:04Version 1 2016-05-10, 19:04
Posted on 2016-05-02 - 00:00
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%–92% PHB contents in cell dry weight
(CDW) were achieved. PHB with various weight-average molecular weights
(Mw) of 2.7–6.8
× 106 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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Li, Teng; Ye, Jianwen; Shen, Rui; Zong, Yeqing; Zhao, Xuejin; Lou, Chunbo; et al. (2016). Semirational Approach for Ultrahigh Poly(3-hydroxybutyrate)
Accumulation in Escherichia coli by Combining One-Step
Library Construction and High-Throughput Screening. ACS Publications. Collection. https://doi.org/10.1021/acssynbio.6b00083