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High-Throughput Part Characterization and Tool Development to Advance Chloroplast Synthetic Biology

ABSTRACT: Plant synthetic biology holds promise for developing climate-resilient crop varieties particularly through advancements in engineering the chloroplast genome. However, the field faces limitations due to the scarcity of genetic tools and the long generation times of photosynthetic eukaryotes. To address these challenges, we established Chlamydomonas reinhardtii as a prototyping chassis for chloroplast synthetic biology, by significantly advancing plastome engineering tools. We developed an automation workflow that enabled the generation, handling, and analysis of 3,156 transplastomic Chlamydomonas strains and expanded the repertoire of selection markers for chloroplast transformation in Chlamydomonas to five, alongside establishing six new reporter genes. Moreover, we characterized over 140 regulatory parts, including promoters, UTRs, and intercistronic expression elements, all integrated within the Phytobrick cloning framework, for which we demonstrated a broad range of gene expression strength. Additionally, we explored library-based approaches to overcome the throughput limitations of biolistic chloroplast transformation. Finally, we demonstrated the utility of all tools by introducing a chloroplast-based synthetic photorespiration pathway. The high conservation of the chloroplast genetic system suggests that our findings in Chlamydomonas could be translated to advance chloroplast engineering in land plants and provides a prototyping platform for future crop improvements to address the challenges posed by a changing global climate.

INSTRUMENT(S):

ORGANISM(S): Chlamydomonas Reinhardtii

TISSUE(S): Cell Culture

SUBMITTER: Timo Glatter

LAB HEAD: Timo Glatter

PROVIDER: PXD051642 | Pride | 2025-08-11

REPOSITORIES: Pride

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			Action	DRS
	Chlamy-8-DIA-32w-550-680-1.raw	Raw
	Chlamy-8-DIA-32w-550-680-2.raw	Raw
	Chlamy-8-DIA-32w-550-680-3.raw	Raw
	Chlamy-8-DIA-32w-550-680-4.raw	Raw
	Chlamy-8-DIA-32w-550-680-5.raw	Raw

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Project description:Metabolism is fundamental to life, all life activities are sustained through metabolism. Metabolic engineering, while modifying host endogenous metabolic network or introducing heterologous pathway for biotechnological applications, often results in unfitness and suboptimal characteristics, because of the lack of full knowledge on host metabolism. Adaptive laboratory evolution (ALE), harnessing the nature of life -- evolution, has become a potent approach in phenotype optimization, environmental adaptation, and cell biology study1–3. Through ALE, alternative pathways of β-alanine biosynthesis4, pyridoxal 5’-phosphate (PLP) biosynthesis5, isoleucine biosynthesis6, as well as ATP generation7 have been uncovered; E. coli aerobic citrate utilization8,9 and various synthetic C1-trophy10–14 have been realized. In this study, we describe E. coli recruits the same workforce, Sad, in evolution through various strategies, i.e., catalytic efficiency improvement, gene dose increase, and gene expression regulations, to overcome metabolic damages (Fig. 1). Sad, i.e. NAD(P)+-dependent succinate semialdehyde dehydrogenase (SSADH), is an aldehyde dehydrogenase (ALDH) family protein. Sad oxidizes succinate semialdehyde to succinate, preventing accumulation of the toxic aldehyde intermediate in nitrogen metabolism, such as putrescine, arginine and γ-aminobutyrate (GABA) degradations15–17. Some bacteria, for example E. coli, possess also a NADP+-dependent SSADH, GabD15,18. SSADH is ubiquitous in both prokaryotes and eukaryotes. In human, it involves in catabolism of GABA, a major neurotransmitter, its malfunction leads to a disorder SSADH deficiency19. In plants, SSADH involves in leaf development and morphogenesis20 as well as defense of abiotic stress21. And in cyanobacteria, SSADH is an essential enzyme of its uncanonical tricarboxylic acid cycle. SSADH was also found to be able to oxidize glutarate semialdehyde, participating in lysine catabolism in bacteria.

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High-Throughput Part Characterization and Tool Development to Advance Chloroplast Synthetic Biology

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