title: Establishing the Chlamydomonas reinhardtii chloroplast as a platform for photosynthesis engineering
title: Establishing the Chlamydomonas reinhardtii chloroplast as a platform for photosynthesis engineering
titleType: AlternativeTitle
Creators: name: Chotel, Tanguy
Publisher: name: Philipps-Universität Marburg
Year: 2024
Subjects: Synthetische Biologie
Life sciences
Biowissenschaften, Biologie
Fotosynthese

description: The detrimental effects of human activities on the environment have been clearly documented, leading
to a global effort to curb industrial pollution and decrease energy consumption. Human-generated
pollutants, especially carbon dioxide (CO2), play a significant role in atmospheric degradation. The
dramatic increase in industrial CO2 emissions over the centuries has resulted in a concerning accumulation
in the atmosphere, adversely affecting human health and ecosystem stability. Despite this, CO2
is vital for phototrophic organisms like plants, algae, and cyanobacteria, which utilize CO2 and use
the carbon to create organic molecules through photosynthesis. With its unique capacity to convert
light energy into inorganic carbon assimilation, it has been a central subject of scientific research, offering
substantial potential for fields such as agronomy and energy production, alongside fundamental
studies. Enhancing photosynthesis is a complex challenge that demands a deep understanding of its
mechanisms and the creation of advanced tools for practical use. This thesis seeks to dive into into
the complexities of improving photosynthetic efficiency, employing innovative methodologies from synthetic biology to enhance our comprehension and application of this essential biological process. The
aim is to establish Chlamydomonas reinhardtii as a chassis for carbon fixation engineering, through
the development of new tools to engineer the plastome. This should be implemented using a proof of
concept photorespiratory bypass in the chloroplast of C. reinhardtii and its characterization, and by
relocating the small subunit of Rubisco rbcS from the nuclear genome to the chloroplast genome. This
should contribute to fortifying the chloroplast genome of C. reinhardtii as a suitable testing ground for
complex endeavors, while remaining translatable to higher plants through the conservation of chloroplasts
throughout the tree of life. In the first part of the thesis we show the construction of a MoClo
toolkit for the chloroplast genome of C. reinhardtii, through the establishment of high-throughput part
characterization pipeline. This allowed us to screen for more than 300 parts, including untranslated
regions (UTRs) and promoters. By testing unique combination of regulatory elements, we identified
two transcritpion units suited for the implementation of the South pathway as a photorespiratory bypass.
The pathway is constituted of two genes, a glycolate dehydrogenase and a malate synthase, that
were assembled in the characterized transcription units. These transgenes were transformed in the
chloroplast of a characterized mutant strain displaying increased photorespiration. Through genomic,
proteomic, metabolomic and phenotypic characterization in photorespiratory conditions, we show the
successful implementation of the pathway. Transplastomic strains displayed reduced doubling time and
final cell density compared to the mutant strains, explained by lower intermediates of natural photorespiration
suggesting an increased flux in the bypass. Finally, we set out to re-localize the small subunit
of Rubisco from the nuclear genome to the chloroplast genome, by transforming the endogenous Crrbcs1 gene in the chloroplast of mutant strain for rbcS and the pyrenoid. Though the knock-out strain
does not behave as expected and maintains growth in minimal phototrophic conditions, the presence
of the transgene was confirmed and a drastic impact on growth and cell density was exhibited in the
transplastomic strain, reducing its doubling time by three compared to the mutant and reaching cell
densities comparable to wild-type levels. Overall in this thesis, I have establish the basic tools and
proofs of concept to use the chloroplast of C. reinhardtii as a testbed for photosynthesis engineering.
By showing that we can engineer a photorespiratory bypass and re-localize the small subunit of Rubisco
in the chloroplast genome, we now open the possibility of testing multiple pathways, cycles and
variants in a high-throughput manner, using the established pipeline and characterized mutants.