Enhancing the Glucose Flux of an Engineered EP-Bifido Pathway for High Poly(Hydroxybutyrate) Yield Production

Ying Li; Zhijie Sun; Ya Xu; Yaqi Luan; Jiasheng Xu; Quanfeng Liang; Qingsheng Qi; Qian Wang

doi:10.3389/fbioe.2020.517336

Enhancing the Glucose Flux of an Engineered EP-Bifido Pathway for High Poly(Hydroxybutyrate) Yield Production

Front Bioeng Biotechnol. 2020 Aug 27:8:517336. doi: 10.3389/fbioe.2020.517336. eCollection 2020.

Authors

Ying Li¹, Zhijie Sun², Ya Xu¹, Yaqi Luan¹, Jiasheng Xu¹, Quanfeng Liang¹, Qingsheng Qi^{1

3}, Qian Wang¹

Affiliations

¹ National Glycoengineering Research Center, State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China.
² Marine Biology Institute, Shantou University, Shantou, China.
³ CAS Key Lab of Biobased Materials, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.

Abstract

Background: As the greenhouse effect becomes more serious and carbon dioxide emissions continue rise, the application prospects of carbon sequestration or carbon-saving pathways increase. Previously, we constructed an EP-bifido pathway in Escherichia coli by combining Embden-Meyerhof-Parnas pathway, pentose phosphate pathway and "bifid shunt" for high acetyl-CoA production. There is much room for improvement in the EP-bifido pathway, including in production of target compounds such as poly(hydroxybutyrate) (PHB).

Result: To optimize the EP-bifido pathway and obtain higher PHB yields, we knocked out the specific phosphoenolpyruvate phosphate transferase system (PTS) component II Cglc, encoded by ptsG. This severely inhibited the growth and sugar consumption of the bacterial cells. Subsequently, we used multiple automated genome engineering (MAGE) to optimize the ribosome binding site (RBS) sequences of galP (galactose: H (+) symporter) and glk (glucokinase gene bank: NC_017262.1), encoding galactose permease and glucokinase, respectively. Growth and glucose uptake were partially restored in the bacteria. Finally, we introduced the glf (UDP-galactopyranose) from Zymomonas mobilis mutase sugar transport vector into the host strain genome.

Conclusion: After optimizing RBS of galP, the resulting strain L-6 obtained a PHB yield of 71.9% (mol/mol) and a 76 wt% PHB content using glucose as the carbon source. Then when glf was integrated into the genome strain L-6, the resulting strain M-6 reached a 5.81 g/L PHB titer and 85.1 wt% PHB content.

Keywords: EP-bifido pathway; Escherichia coli; MAGE; glucose flux; poly(hydroxybutyrate) yield.