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Accumulation of Poly(3-hydroxybutyrate) Helps Bacterial Cells to Survive Freezing.

ABSTRACT: Accumulation of polyhydroxybutyrate (PHB) seems to be a common metabolic strategy adopted by many bacteria to cope with cold environments. This work aimed at evaluating and understanding the cryoprotective effect of PHB. At first a monomer of PHB, 3-hydroxybutyrate, was identified as a potent cryoprotectant capable of protecting model enzyme (lipase), yeast (Saccharomyces cerevisiae) and bacterial cells (Cupriavidus necator) against the adverse effects of freezing-thawing cycles. Further, the viability of the frozen-thawed PHB accumulating strain of C. necator was compared to that of the PHB non-accumulating mutant. The presence of PHB granules in cells was revealed to be a significant advantage during freezing. This might be attributed to the higher intracellular level of 3-hydroxybutyrate in PHB accumulating cells (due to the action of parallel PHB synthesis and degradation, the so-called PHB cycle), but the cryoprotective effect of PHB granules seems to be more complex. Since intracellular PHB granules retain highly flexible properties even at extremely low temperatures (observed by cryo-SEM), it can be expected that PHB granules protect cells against injury from extracellular ice. Finally, thermal analysis indicates that PHB-containing cells exhibit a higher rate of transmembrane water transport, which protects cells against the formation of intracellular ice which usually has fatal consequences.

SUBMITTER: Obruca S

PROVIDER: S-EPMC4912086 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Accumulation of Poly(3-hydroxybutyrate) Helps Bacterial Cells to Survive Freezing.

Obruca Stanislav S Sedlacek Petr P Krzyzanek Vladislav V Mravec Filip F Hrubanova Kamila K Samek Ota O Kucera Dan D Benesova Pavla P Marova Ivana I

PloS one 20160617 6

Accumulation of polyhydroxybutyrate (PHB) seems to be a common metabolic strategy adopted by many bacteria to cope with cold environments. This work aimed at evaluating and understanding the cryoprotective effect of PHB. At first a monomer of PHB, 3-hydroxybutyrate, was identified as a potent cryoprotectant capable of protecting model enzyme (lipase), yeast (Saccharomyces cerevisiae) and bacterial cells (Cupriavidus necator) against the adverse effects of freezing-thawing cycles. Further, the vi ...[more]

PMID: 27315285

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Project description:Poly-3-hydroxybutyrate (PHB) is an environmentally friendly polymer and can be produced in Escherichia coli cells after overexpression of the heterologous gene cluster phaCAB. The biosynthesis of the outer membrane (OM) consumes many nutrients and influences cell morphology. Here, we engineered the OM by disrupting all gene clusters relevant to the polysaccharide portion of lipopolysaccharide (LPS), colanic acid (CA), flagella, and/or fimbria in E. coli W3110. All these disruptions benefited PHB production. Especially, disrupting all these OM components increased the PHB content to 83.0 wt% (PHB content percentage of dry cell weight), while the wild-type control produced only 1.5 wt% PHB. The increase was mainly due to the LPS truncation to Kdo2 (3-deoxy-d-manno-octulosonic acid)-lipid A, which resulted in 82.0 wt% PHB with a 25-fold larger cell volume, and disrupting CA resulted in 57.8 wt% PHB. In addition, disrupting LPS facilitated advantageous fermentation features, including 69.1% less acetate, a 550% higher percentage of autoaggregated cells among the total culture cells, 69.1% less biofilm, and a higher broken cell ratio. Further detailed mechanism investigations showed that disrupting LPS caused global changes in envelope and cellular metabolism: (i) a sharp decrease in flagella, fimbria, and secretions; (ii) more elastic cells; (iii) much greater carbon flux toward acetyl coenzyme A (acetyl-CoA) and supply of cofactors, including NADP, NAD, and ATP; and (iv) a decrease in by-product acids but increase in γ-aminobutyric acid by activating σE factor. Disrupting CA, flagella, and fimbria also improved the levels of acetyl-CoA and cofactors. The results indicate that engineering the OM is an effective strategy to enhance PHB production and highlight the applicability of OM engineering to increase microbial cell factory performance. IMPORTANCE Understanding the detailed influence of the OM on the cell envelope and cellular metabolism is important for optimizing the E. coli cell factory and many other microorganisms. This study revealed the applicability of remodeling the OM to enhance PHB accumulation as representative inclusion bodies. The results generated in this study give essential information for producing other inclusion bodies or chemicals which need more acetyl-CoA and cofactors but less by-product acids. This study is promising to provide new ideas for the improvement of microbial cell factories.

Dataset Information

Accumulation of Poly(3-hydroxybutyrate) Helps Bacterial Cells to Survive Freezing.

Publications

Accumulation of Poly(3-hydroxybutyrate) Helps Bacterial Cells to Survive Freezing.

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