Limits...
Complete PHB mobilization in Escherichia coli enhances the stress tolerance: a potential biotechnological application.

Wang Q, Yu H, Xia Y, Kang Z, Qi Q - Microb. Cell Fact. (2009)

Bottom Line: Poly-beta-hydroxybutyrate (PHB) mobilization in bacteria has been proposed as a mechanism that can benefit their host for survival under stress conditions.Here we reported for the first time that a stress-induced system enabled E. coli, a non-PHB producer, to mobilize PHB in vivo by mimicking natural PHB accumulation bacteria.The successful expression of PHB biosynthesis and PHB depolymerase genes in E. coli was confirmed by PHB production and 3-hydroxybutyrate secretion.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Microbial Technology, National Glycoengineering Research Center, School of Life Science, Shandong University, Jinan, 250100 PR China. qiqingsheng@sdu.edu.cn.

ABSTRACT

Background: Poly-beta-hydroxybutyrate (PHB) mobilization in bacteria has been proposed as a mechanism that can benefit their host for survival under stress conditions. Here we reported for the first time that a stress-induced system enabled E. coli, a non-PHB producer, to mobilize PHB in vivo by mimicking natural PHB accumulation bacteria.

Results: The successful expression of PHB biosynthesis and PHB depolymerase genes in E. coli was confirmed by PHB production and 3-hydroxybutyrate secretion. Starvation experiment demonstrated that the complete PHB mobilization system in E. coli served as an intracellular energy and carbon storage system, which increased the survival rate of the host when carbon resources were limited. Stress tolerance experiment indicated that E. coli strains with PHB production and mobilization system exhibited an enhanced stress resistance capability.

Conclusion: This engineered E. coli with PHB mobilization has a potential biotechnological application as immobilized cell factories for biocatalysis and biotransformation.

No MeSH data available.


Related in: MedlinePlus

Supposed metabolic network between PHB biosynthesis and mobilization in recombinant E. coli DH5α (pQWQ2/pSCP-CAB). Dashed lines represent enzymatic steps engineered in recombinant E. coli. Solid lines represent enzymatic steps existed in wild type E. coli. A question mark denotes unidentified enzyme in E. coli. ① β-ketothiolase, PhaA ② NADPH-dependent acetoacetyl-CoA reductase, PhaB ③ PHB synthase, PhaC ④ PHB depolymerase, PhaZ ⑤ acyl-CoA synthetase or thioesterase ⑥ 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxybutyryl-CoA epimerase, FadB ⑦ acetoacetyl-CoA transferase, AtoA, AtoD ⑧ acetyl-CoA acetyltransferase, AtoB.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2746179&req=5

Figure 6: Supposed metabolic network between PHB biosynthesis and mobilization in recombinant E. coli DH5α (pQWQ2/pSCP-CAB). Dashed lines represent enzymatic steps engineered in recombinant E. coli. Solid lines represent enzymatic steps existed in wild type E. coli. A question mark denotes unidentified enzyme in E. coli. ① β-ketothiolase, PhaA ② NADPH-dependent acetoacetyl-CoA reductase, PhaB ③ PHB synthase, PhaC ④ PHB depolymerase, PhaZ ⑤ acyl-CoA synthetase or thioesterase ⑥ 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxybutyryl-CoA epimerase, FadB ⑦ acetoacetyl-CoA transferase, AtoA, AtoD ⑧ acetyl-CoA acetyltransferase, AtoB.

Mentions: The secretion of 3HB in E. coli has also been observed in some previous experiments [9,38,39]. In this study, we observed the consumption of 3HB as carbon and energy source in engineered E. coli. The confirmation that 3HB can be converted into 3HB-CoA in vivo indicated the existence of a complete PHB cycle in engineered E. coli, from acetyl-CoA to PHB and from PHB to acetyl-CoA. This complete PHB mobilization is the reason of enhanced starvation and stress tolerance of the host. It was reported that a broad-substrate-ranged thioesterase YciA from E. coli mediated CoA-activation of the corresponding carboxylic acid, which supports the possibility of 3HB-CoA formation from 3HB in E. coli [15]. The PHB depolymerization rate is low in recombinant E. coli, thus we could not easily observe the 3HB accumulation under normal conditions. This is the first description that heterologous expression PHB mobilization system may change the stress resistance of the engineered E. coli. All previous experiments that concerned PHB mobilization and indicated altered stress resistance capability were done by knocking out key enzyme involved in PHB production in natural PHB production strains [29,30]. Taking advantage of the knowledge acquired previously and our findings in this study, we developed a schematic pathway to gain insight into the possible PHB mobilization routes in engineered E. coli (Fig. 6). The proposed pathway established in recombinant E. coli in vivo can mimic PHB cycle of the natural PHB producers: 3-HB, which proved to be actived to a CoA-link form, will be reversibly catalyzed by acetoacetyl-CoA reductase and β-ketothiolase to acetyl-CoA or will serve as a linker between the PHB mobilization with β-oxidation pathway.


Complete PHB mobilization in Escherichia coli enhances the stress tolerance: a potential biotechnological application.

Wang Q, Yu H, Xia Y, Kang Z, Qi Q - Microb. Cell Fact. (2009)

Supposed metabolic network between PHB biosynthesis and mobilization in recombinant E. coli DH5α (pQWQ2/pSCP-CAB). Dashed lines represent enzymatic steps engineered in recombinant E. coli. Solid lines represent enzymatic steps existed in wild type E. coli. A question mark denotes unidentified enzyme in E. coli. ① β-ketothiolase, PhaA ② NADPH-dependent acetoacetyl-CoA reductase, PhaB ③ PHB synthase, PhaC ④ PHB depolymerase, PhaZ ⑤ acyl-CoA synthetase or thioesterase ⑥ 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxybutyryl-CoA epimerase, FadB ⑦ acetoacetyl-CoA transferase, AtoA, AtoD ⑧ acetyl-CoA acetyltransferase, AtoB.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2746179&req=5

Figure 6: Supposed metabolic network between PHB biosynthesis and mobilization in recombinant E. coli DH5α (pQWQ2/pSCP-CAB). Dashed lines represent enzymatic steps engineered in recombinant E. coli. Solid lines represent enzymatic steps existed in wild type E. coli. A question mark denotes unidentified enzyme in E. coli. ① β-ketothiolase, PhaA ② NADPH-dependent acetoacetyl-CoA reductase, PhaB ③ PHB synthase, PhaC ④ PHB depolymerase, PhaZ ⑤ acyl-CoA synthetase or thioesterase ⑥ 3-hydroxyacyl-CoA dehydrogenase, 3-hydroxybutyryl-CoA epimerase, FadB ⑦ acetoacetyl-CoA transferase, AtoA, AtoD ⑧ acetyl-CoA acetyltransferase, AtoB.
Mentions: The secretion of 3HB in E. coli has also been observed in some previous experiments [9,38,39]. In this study, we observed the consumption of 3HB as carbon and energy source in engineered E. coli. The confirmation that 3HB can be converted into 3HB-CoA in vivo indicated the existence of a complete PHB cycle in engineered E. coli, from acetyl-CoA to PHB and from PHB to acetyl-CoA. This complete PHB mobilization is the reason of enhanced starvation and stress tolerance of the host. It was reported that a broad-substrate-ranged thioesterase YciA from E. coli mediated CoA-activation of the corresponding carboxylic acid, which supports the possibility of 3HB-CoA formation from 3HB in E. coli [15]. The PHB depolymerization rate is low in recombinant E. coli, thus we could not easily observe the 3HB accumulation under normal conditions. This is the first description that heterologous expression PHB mobilization system may change the stress resistance of the engineered E. coli. All previous experiments that concerned PHB mobilization and indicated altered stress resistance capability were done by knocking out key enzyme involved in PHB production in natural PHB production strains [29,30]. Taking advantage of the knowledge acquired previously and our findings in this study, we developed a schematic pathway to gain insight into the possible PHB mobilization routes in engineered E. coli (Fig. 6). The proposed pathway established in recombinant E. coli in vivo can mimic PHB cycle of the natural PHB producers: 3-HB, which proved to be actived to a CoA-link form, will be reversibly catalyzed by acetoacetyl-CoA reductase and β-ketothiolase to acetyl-CoA or will serve as a linker between the PHB mobilization with β-oxidation pathway.

Bottom Line: Poly-beta-hydroxybutyrate (PHB) mobilization in bacteria has been proposed as a mechanism that can benefit their host for survival under stress conditions.Here we reported for the first time that a stress-induced system enabled E. coli, a non-PHB producer, to mobilize PHB in vivo by mimicking natural PHB accumulation bacteria.The successful expression of PHB biosynthesis and PHB depolymerase genes in E. coli was confirmed by PHB production and 3-hydroxybutyrate secretion.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Microbial Technology, National Glycoengineering Research Center, School of Life Science, Shandong University, Jinan, 250100 PR China. qiqingsheng@sdu.edu.cn.

ABSTRACT

Background: Poly-beta-hydroxybutyrate (PHB) mobilization in bacteria has been proposed as a mechanism that can benefit their host for survival under stress conditions. Here we reported for the first time that a stress-induced system enabled E. coli, a non-PHB producer, to mobilize PHB in vivo by mimicking natural PHB accumulation bacteria.

Results: The successful expression of PHB biosynthesis and PHB depolymerase genes in E. coli was confirmed by PHB production and 3-hydroxybutyrate secretion. Starvation experiment demonstrated that the complete PHB mobilization system in E. coli served as an intracellular energy and carbon storage system, which increased the survival rate of the host when carbon resources were limited. Stress tolerance experiment indicated that E. coli strains with PHB production and mobilization system exhibited an enhanced stress resistance capability.

Conclusion: This engineered E. coli with PHB mobilization has a potential biotechnological application as immobilized cell factories for biocatalysis and biotransformation.

No MeSH data available.


Related in: MedlinePlus