Scientists ID enzyme for making key industrial chemical in plants

Date:
August 5, 2021
Source:
DOE/Brookhaven National Laboratory
Summary:
Scientists studying the biochemistry of plant cell walls have
identified an enzyme that could turn woody poplar trees into a
source for producing a major industrial chemical. The research could
lead to a new sustainable pathway for making "p-hydroxybenzoic
acid," a chemical building block currently derived from fossil
fuels, in plant biomass.



FULL STORY ========================================================================== Scientists studying the biochemistry of plant cell walls have identified
an enzyme that could turn woody poplar trees into a source for producing a major industrial chemical. The research, just published in Nature Plants,
could lead to a new sustainable pathway for making "p-hydroxybenzoic
acid," a chemical building block currently derived from fossil fuels,
in plant biomass.


========================================================================== "P-hydroxybenzoic acid is a versatile chemical feedstock. It can serve
as a building block for making liquid crystals, a plasticizer of nylon
resin, a sensitizer for thermal paper, and a raw material for making
paraben, dyes, and pigments," said Chang-Jun Liu, a plant biochemist at
the U.S. Department of Energy's Brookhaven National Laboratory and lead
author on the paper.

The global market value of p-hydroxybenzoic acid stood at U.S. $59 million
in 2020 and is projected to reach $80 million by 2026. But the current
process for making this important chemical relies on petrochemicals. Its synthesis requires harsh reaction conditions (high temperature and high pressure) and has negative environmental impacts. Finding an economical
and sustainable way to make p- hydroxybenzoic acid in plants could help mitigate environmental impacts and contribute to an emerging bioeconomy.

"We've identified a key enzyme responsible for the synthesis and
accumulation of p-hydroxybenzoate (pBA) -- the conjugate base of p-hydroxybenzoic acid -- in lignin, one of three major polymers that make
up the structural support that surrounds plant cells," said Liu. "This discovery may enable us to engineer plants to accumulate more of this
chemical building block in their cell walls, thereby potentially adding
value to the biomass." Biofuels and bioproducts Cell walls are made of
a combination of chainlike polymers -- cellulose, hemicellulose, and
lignin -- which are the major source of plant biomass. Liu and other
scientists have been exploring the biochemical pathways that build up
these plant polymers. One goal has been to understand how changing the
mix of polymers could make it easier and more cost-effective to convert
biomass into biofuels.



========================================================================== Lignin, which gives plants structural integrity, mechanical strength, and waterproofing, is particularly hard to break down. But recent research
aimed at generating cellulosic ethanol has driven technical advances
and opportunities to increase the uses and therefore the value of lignin.

Scientists have known that the building blocks that make up lignin often
have various chemical groups, including pBA, attached as sidechains. The
exact function of these side groups was unknown. But Liu's team
was interested in exploring their influence on lignin structure and
properties. So, they set out to discover the enzyme responsible for
attaching pBA to lignin.

"If we could identify this enzyme, and then control the expression of
the gene that makes this enzyme, we could effectively control the level
of pBA in the biomass of bioenergy plants," Liu said.

Searching for the gene The scientists conducted their study on
poplar. This fast-growing tree species has rich woody biomass. It has
emerged as a promising renewable feedstock for biofuel and bio-based
chemical production. It also has pBA as the main sidechain "decoration"
on its lignin.



==========================================================================
To systematically identify and characterize the enzyme(s) involved in
attaching pBA or other chemical groups to lignin, Liu's team screened
a series of candidate genes identified through a related genomic study
of poplar.

"We cloned 20 candidate genes that are primarily expressed in woody
tissues and encode enzymes called acyltransferases. These are the enzymes
most likely involved in transferring chemical groups to the particular
accepter molecules," Liu said.

The scientists expressed the enzymes coded for by these genes and mixed
each one with various building blocks including one isotope-labeled
carbon compound.

Tracing the isotope label and a range of other test-tube based
biomolecular techniques allowed the scientists to monitor whether each candidate enzyme was involved in attaching sidechains such as pBA (or
the other chemical groups).

They were able to zero in on the most likely candidate for the reaction
of interest.

Firmly proving the enzyme's function in plants, however, was a formidable
task.

It took the scientists many years -- and required the emergence of new
advances in molecular biology.

One of those was a technique known as CRISPR/Cas9, a modern "genetic
scissor" that permits precise editing of genes in the genome of a target organism. The team used CRISPR/Cas9 to generate a poplar variant in
which the candidate enzyme-encoding gene had been deleted. Subsequent
analysis found almost no pBA on the lignin in stems of these plants.

They also tried another genetic test by over-expressing the gene that
produces the candidate enzyme. Those plants accumulated increased levels
of pBA.

"Together these data provide conclusive proof that the gene/enzyme we
have identified can attach pBA to the lignin building blocks," Liu said.

Ramping up plants' pBA content through genetic manipulation could be
one way to sustainably produce p-hydroxybenzoic acid.

The scientists also found that lignin from plants that were engineered to accumulate lower pBA were easier to dissolve in a solvent. This implies
that, in nature, pBA helps to strengthen lignin.

Therefore, another potential outcome of identifying the enzyme for adding
pBA to lignin could be genetic strategies for tailoring the chemical
properties of lignin.

Lowering pBA might improve the "delignification" of woody biomass for
processes such as pulping, paper making, and biofuel production.

Conversely, increasing pBA levels on lignin could potentially enhance
timber durability while also providing a pathway for long-term carbon sequestration by locking up more carbon in plant biomass -- another key
DOE goal.

"This work is a good example of basic scientific research leading to potentially valuable downstream applications," said John Shanklin,
Chair of the Brookhaven Lab Biology Department.

The research was performed in collaboration with Yuki Tobimatsu and
Pui-Ying Lam at Kyoto University in Japan. The work at Brookhaven was
funded by the DOE Office of Science (including through the Joint BioEnergy Institute, one of DOE's Bioenergy Research Centers) and by Brookhaven's Laboratory Directed Research and Development Program.

========================================================================== Story Source: Materials provided by
DOE/Brookhaven_National_Laboratory. Note: Content may be edited for
style and length.


========================================================================== Journal Reference:
1. Zhao, Y., Yu, X., Lam, PY. et al. Monolignol acyltransferase
for lignin
p-hydroxybenzoylation in Populus. Nat. Plants, 2021 DOI:
10.1038/s41477- 021-00975-1 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2021/08/210805115437.htm

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