Kwansei Gakuin University
Report of Research Outcome
2022/03/16

To President
Department： Science and Technology
Position： Postdoctoral fellow
Name： Hermanus Nawaly
I report the outcome of the research as follows.
□Sabbatical leave with grantName of theFund/Prog ra m

□KGU Joint Research□Sabbatical leave with no grant□Individual Special Research⊠Postdoctoral f ellow※ Please report by desi gnated f orm as f or "Inte rnational ResearchCollaboration".

ResearchThe meResearchSite/Venue

Research
periodCharacterized pyrenoid and CCM factor in diatom

Matsuda L aborat ory, Graduate School of Science and Technolog y2021/04/01

2022/03/31 （～12month）◆ Summary of the research outcome （ appr ox . 2 ,5 00 wo r ds）
Please write down the outcomes in detail regarding the research theme above.

In seawater, the concentration of CO 2 is very limited in the range of 10and 25 μM. In such constrained conditions, diatoms could successfully adapt
and become one of the ocean's primary producers, contributing to 20% ofglobal primary production. The mechanism to take up inorganic carbon, CO 2 ,and HCO 3 - from the environment and accumulate in the chloroplast, wellknown as CO 2 concentrating mechanism (CCM), is an evolutionary adaptationfrom aquatic photosynthesis organisms, including diatoms.
In the diatom CCM model, carbonic anhydrases (CAs) located invarious cellular compartments were hypothesized to have a crucial role. CA isan enzyme facilitating equilibrium between CO 2 and HCO 3 - . θ-type CA is thenewest type CA, which was recently identified in the marine diatom,1Phaeodactylum tricornutum. In Thalassiosira pseudonana, its orthologous
were identified, TpθCA1, TpθCA2, TpθCA3, and TpθCA4. Their localization
was confirmed except for TpθCA4. In this time, I focused on TpθCA2, which
localizedinthelumenof

thylakoid-penetrating

pyrenoid.

Usingtheknock-down approach by Cas13d, I could obtain four candidates mutants,
which showed lower cell density in low CO 2 conditions relative to high CO 2 .
Further characterization for these four mutants is still required. In addition,
localization of TpθCA4 was confirmed in the cytoplasm.
Pyrenoid is a large protein body that widely occurs in the chloroplast ofeukaryotic algae. Most ribulose-1,5-bisphosphate carboxylase/oxygenase(RubisCO) is sequestrated in the pyrenoid of diatom, strongly suggesting thefunction of this body as a hub for the diatom biophysical CO2-concentrating
mechanism (CCM) and photosynthesis. A proper arrangement of pyrenoidal
components is believed to be critical to achieving high [CO2] at the proximityof RubisCO. However, the molecular bases of the pyrenoid structure are notyet clear. Previous work in our lab identified a new pyrenoid factor. Thispyroid factor previously identified in P. tricornutum. Its orthologous wasidentified in T. pseudonana, and two out of six candidates showed highexpression levels in high and low CO 2 conditions. In the current work, weused the knock-out (KO) approach by Cas9 (D10A) to double KO the twotarget genes. Mutants KO was acquired, mutant 1 (M1) and mutant 2 (M2). M1
showed reducing of specific growth rate compared to wild type (W T) in low
and high CO 2 . This indicated that pyrenoid factor protein is an importantfactor for T. pseudonana. Further characterization for these mutants is stillrequired.

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Deadline： W ithin two months af ter f inishing the research pe riod.
Sabbatical leave with grant: Sub mit this report t o President with conf irmation by the dean ofschool you belong to.
※ Postdoctoral f ellow is required to sub mit this report with conf irmation by the de an of graduate

school bef ore the end of emplo yment pe riod.
W here to submit： Orga nization f or Research a nd Develop ment and Ou treach (N UC)◆ W e put this report on t he web of KGU. If there is any proble m abo ut it because of diff iculties on
your research, please le t us know.
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