Metabolic Engineering of Escherichia coli for Poly(3-hydroxybutyrate) Production under Microaerobic Condition.

Xiao-Xing Wei,Jian Liang,Zheng-Jun Li,Xue Hou,Wei-Tao Zheng

doi:10.1155/2015/789315

Xiao-Xing Wei, Jian Liang + Show 3 more

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https://doi.org/10.1155/2015/789315

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Abstract

The alcohol dehydrogenase promoter PadhE and mixed acid fermentation pathway deficient mutants of Escherichia coli were employed to produce poly(3-hydroxybutyrate) (P3HB) under microaerobic condition. The E. coli mutant with ackA-pta, poxB, ldhA, and adhE deletions accumulated 0.67 g/L P3HB, up to 78.84% of cell dry weight in tube cultivation. The deletion of pyruvate formate-lyase gene pflB drastically decreased P3HB production and P3HB content to 0.09 g/L and 24.44%, respectively. Overexpressing pflB via the plasmid in its knocked out mutant restored cell growth and P3HB accumulation, indicating the importance of the pyruvate formate-lyase in microaerobic carbon metabolism. The engineered E. coli BWapld (pWYC09) produced 5.00 g/L P3HB from 16.50 g/L glucose in 24 h batch fermentation, and P3HB production yield from glucose was 0.30 g/g, which reached up to 63% of maximal theoretical yield.

Highlights

Poly(3-hydroxybutyrate) (P3HB) is a polyester of 3-hydroxybutyrate that is synthesized by a variety of microorganisms as intracellular carbon and energy storages under unbalanced nutrients conditions [1]
The P3HB content obtained under fed-batch microaerobic cultures was 51%, lower than that produced by aerobic fermentation [9]
To investigate the possibility of the synergistic effect, plasmid pWYC09 carrying PadhE controlled phaCAB was transformed into the wild type E. coli and a series of mixed acid fermentation mutants BWa, BWap, BWapl, BWapld, and BWapldf (Table 1)

Summary

Introduction

Poly(3-hydroxybutyrate) (P3HB) is a polyester of 3-hydroxybutyrate that is synthesized by a variety of microorganisms as intracellular carbon and energy storages under unbalanced nutrients conditions [1]. P3HB was mainly produced from renewable carbon sources through aerobic fermentation [4, 5]. The P3HB content obtained under fed-batch microaerobic cultures was 51%, lower than that produced by aerobic fermentation [9]. E. coli undertakes mixed acid fermentation pathway, producing lactate, succinate, acetate, formate, and ethanol under oxygen limited conditions, which decreases the carbon flux into P3HB accumulation. E. coli mutant with deletions of ackA-pta, poxB, ldhA, and adhE was constructed and doubled the cell dry weight and improved P3HB production by 3.5-folds compared to the control [11]. We managed to further increase the microaerobic P3HB production by employing the PadhE promoter to drive the expression of phaCAB operon in E. coli mutants with defected mixed acid fermentation pathway. The pyruvate formate-lyase, which catalyzes the coenzyme A-dependent, nonoxidative cleavage of pyruvate to acetyl-CoA and formate under anaerobic or microaerobic conditions, was overexpressed to investigate its effect on cell growth and P3HB accumulation (Figure 1)

Materials and Methods

Results and Discussion

Conclusion