title: Semirational Approach for Ultrahigh Poly(3-hydroxybutyrate) Accumulation in Escherichia coli by Combining One-Step Library Construction and High-Throughput Screening.
abstract: 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 × 106were 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.
authors.affiliation: MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University , Beijing 100084, China.
authors.forename: Teng
authors.initials: T
authors.lastname: Li
authors.affiliation: MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University , Beijing 100084, China.
authors.forename: Jianwen
authors.initials: J
authors.lastname: Ye
authors.affiliation: MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University , Beijing 100084, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Shen
authors.affiliation: Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, China.
authors.forename: Yeqing
authors.initials: Y
authors.lastname: Zong
authors.affiliation: Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, China.
authors.forename: Xuejin
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, China.
authors.forename: Chunbo
authors.initials: C
authors.lastname: Lou
authors.affiliation: MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University , Beijing 100084, China.|Center for Synthetic and Systems Biology, Tsinghua University , Beijing 100084, China.
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Chen
mesh_terms: D003001:Cloning, Molecular; D053938:DNA Fragmentation; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D006885:Hydroxybutyrates / Q000378:metabolism*; D008970:Molecular Weight; D009876:Operon; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011091:Polyesters / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
chemical_list: D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates
keywords: PHB; high-throughput screening; polyhydroxyalkanoates; ribosomal binding site calculator