3D visualization and quantification of bioplastic PHA in a living
bacterial cell

Date:
July 28, 2021
Source:
The Korea Advanced Institute of Science and Technology (KAIST)
Summary:
A research team has observed how bioplastic granule is being
accumulated in living bacteria cells through 3D holographic
microscopy. Their 3D imaging and quantitative analysis of the
bioplastic 'polyhydroxyalkanoate' (PHA) via optical diffraction
tomography provides insights into biosynthesizing sustainable
substitutes for petroleum-based plastics.



FULL STORY ==========================================================================
A research team at KAIST has observed how bioplastic granule is
being accumulated in living bacteria cells through 3D holographic
microscopy. Their 3D imaging and quantitative analysis of the bioplastic 'polyhydroxyalkanoate' (PHA) via optical diffraction tomography provides insights into biosynthesizing sustainable substitutes for petroleum-based plastics.


==========================================================================
The bio-degradable polyester polyhydroxyalkanoate (PHA) is being touted as
an eco-friendly bioplastic to replace existing synthetic plastics. While carrying similar properties to general-purpose plastics such as
polyethylene and polypropylene, PHA can be used in various industrial applications such as container packaging and disposable products.

PHA is synthesized by numerous bacteria as an energy and carbon storage material under unbalanced growth conditions in the presence of excess
carbon sources. PHA exists in the form of insoluble granules in the
cytoplasm.

Previous studies on investigating in vivo PHA granules have been performed
by using fluorescence microscopy, transmission electron microscopy (TEM),
and electron cryotomography.

These techniques have generally relied on the statistical analysis of
multiple 2D snapshots of fixed cells or the short-time monitoring
of the cells. For the TEM analysis, cells need to be fixed and
sectioned, and thus the investigation of living cells was not
possible. Fluorescence-based techniques require fluorescence labeling or
dye staining. Thus, indirect imaging with the use of reporter proteins
cannot show the native state of PHAs or cells, and invasive exogenous
dyes can affect the physiology and viability of the cells. Therefore,
it was difficult to fully understand the formation of PHA granules in
cells due to the technical limitations, and thus several mechanism models
based on the observations have been only proposed.

The team of metabolic engineering researchers led by Distinguished
Professor Sang Yup Lee and Physics Professor YongKeun Park, who
established the startup Tomocube with his 3D holographic microscopy,
reported the results of 3D quantitative label-free analysis of PHA
granules in individual live bacterial cells by measuring the refractive
index distributions using optical diffraction tomography. The formation
and growth of PHA granules in the cells of Cupriavidus necator, the most-studied native PHA (specifically, poly(3- hydroxybutyrate), also
known as PHB) producer, and recombinant Escherichia coli harboring
C. necatorPHB biosynthesis pathway were comparatively examined.

From the reconstructed 3D refractive index distribution of the cells, the
team succeeded in the 3D visualization and quantitative analysis of cells
and intracellular PHA granules at a single-cell level. In particular, the
team newly presented the concept of "in vivo PHA granule density." Through
the statistical analysis of hundreds of single cells accumulating
PHA granules, the distinctive differences of density and localization
of PHA granules in the two micro-organisms were found. Furthermore,
the team identified the key protein that plays a major role in making
the difference that enabled the characteristics of PHA granules in the recombinant E. coli to become similar to those of C. necator.

The research team also presented 3D time-lapse movies showing the actual processes of PHA granule formation combined with cell growth and division.

Movies showing the living cells synthesizing and accumulating PHA granules
in their native state had never been reported before.

Professor Lee said, "This study provides insights into the
morphological and physical characteristics of in vivo PHA as well
as the unique mechanisms of PHA granule formation that undergo
the phase transition from soluble monomers into the insoluble
polymer, followed by granule formation. Through this study, a deeper understanding of PHA granule formation within the bacterial cells
is now possible, which has great significance in that a convergence
study of biology and physics was achieved. This study will help
develop various bioplastics production processes in the future." ========================================================================== Story Source: Materials provided by The_Korea_Advanced_Institute_of_Science_and_Technology_ (KAIST). Note:
Content may be edited for style and length.


========================================================================== Journal Reference:
1. So Young Choi, Jeonghun Oh, JaeHwang Jung, YongKeun Park, Sang
Yup Lee.

Three-dimensional label-free visualization and quantification
of polyhydroxyalkanoates in individual bacterial cell in its
native state.

Proceedings of the National Academy of Sciences, 2021; 118 (31):
e2103956118 DOI: 10.1073/pnas.2103956118 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2021/07/210728105624.htm

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