Engineering of Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex to improve poly(3-hydroxybutyrate) production in Escherichia coli.

Yan Zhang,Qiaojie Liu,Tao Chen,Zhiwen Wang,Yifan Li,Zhenquan Lin,Hongwu Ma,Xueming Zhao

doi:10.1186/s12934-014-0172-6

Abstract

BackgroundPoly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs). PHB is synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses. Bio-based production of PHB from renewable feedstock is a promising and sustainable alternative to the petroleum-based chemical synthesis of plastics. In this study, a novel strategy was applied to improve the PHB biosynthesis from different carbon sources.ResultsIn this research, we have constructed E. coli strains to produce PHB by engineering the Serine-Deamination (SD) pathway, the Entner-Doudoroff (ED) pathway, and the pyruvate dehydrogenase (PDH) complex. Firstly, co-overexpression of sdaA (encodes L-serine deaminase), L-serine biosynthesis genes and pgk (encodes phosphoglycerate kinase) activated the SD Pathway, and the resulting strain SD02 (pBHR68), harboring the PHB biosynthesis genes from Ralstonia eutropha, produced 4.86 g/L PHB using glucose as the sole carbon source, representing a 2.34-fold increase compared to the reference strain. In addition, activating the ED pathway together with overexpressing the PDH complex further increased the PHB production to 5.54 g/L with content of 81.1% CDW. The intracellular acetyl-CoA concentration and the [NADPH]/[NADP+] ratio were enhanced after the modification of SD pathway, ED pathway and the PDH complex. Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source.ConclusionsSignificant levels of PHB biosynthesis from different kinds of carbon sources can be achieved by engineering the Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex in E. coli JM109 harboring the PHB biosynthesis genes from Ralstonia eutropha. This work demonstrates a novel strategy for improving PHB production in E. coli. The strategy reported here should be useful for the bio-based production of PHB from renewable resources.

Highlights

Poly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs)
Polyhydroxyalkanoates (PHAs) are diverse polyesters synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses [1]
Overexpressing L-serine deaminase for improved PHB production Under aerobic or anaerobic conditions, acetyl-CoA was derived mostly from the decarboxylation of pyruvate which respectively catalyzed by the pyruvate dehydrogenase (PDH) complex or pyruvate-formate lyase in E.coli [15,16]

Summary

Introduction

Poly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs). PHB is synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses. Polyhydroxyalkanoates (PHAs) are diverse polyesters synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses [1]. Recombinant E. coli harboring the exogenous PHB synthetic pathway was one of the most frequently used hosts for biopolymer production because of its advantages such as having a wide range of utilizable carbon sources, accumulating of large amounts of polymers with a high level of productivity, high cell density fermentation, and lacking PHA degradation system. By overexpressing the fructosebisphosphate aldolase (encoded by fbaA) or/and triosephosphate isomerase (encoded by tpi) [4], the recombinant E. coli accumulated more PHB than the reference strains due to the increase in acetyl-CoA concentration. To increase product yields, NADPH levels have been manipulated in the past by overexpressing the NADP+-dependent D-glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans [9]

Methods

Results

Conclusion