Mackinder Lab Vision

The Mackinder Lab focuses on systems and synthetic biology of algal (both eukaryotic and prokaryotic) carbon fixation. By using cutting edge approaches and high-throughput methodologies we aim to rapidly advance our molecular understanding of how algae account for approximately half of global carbon fixation.

Research Overview

Photosynthesis harnesses energy from sunlight to drive the fixation of CO₂ into the organic carbon building blocks of life. Eukaryotic algae and cyanobacteria play fundamental roles in global biogeochemical cycles, yet there are still substantial gaps in our knowledge of how they acquire their CO₂. The Mackinder Lab research focuses on how algae efficiently transport CO₂ from their surrounding environment and concentrate it in the proximity of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) - the principle carbon fixing enzyme. We use high-throughput, systems biology approaches to rapidly identify key components of this CO₂ concentrating mechanism (CCM). This data is used to guide synthetic biology experiments to reconstruct CCMs in heterologous systems. Ultimately, we aim to transfer components to higher plants to improve photosynthetic performance.

We currently use green algae, diatoms and cyanobacteria as model systems to study CCM function.

Protein-protein interaction network of the Chlamydomonas CCM.

​Research Areas

The spatial organization of CCMs

Ultimately we want to understand the location, interactors and function of every CCM component. To achieve this we have developed high-throughput fluorescence protein tagging and affinity purification followed by mass spectrometry methods in diverse photosynthetic microbes including green algae, diatoms and cyanobacteria.

At the centre of the eukaryotic algal CCM is an enigmatic organelle called the pyrenoid, where the cell releases CO₂ in the proximity of Rubisco. To determine the localization of putative CCM proteins and to identify novel pyrenoid components we performed a large-scale fluorescent protein tagging study to screen CCM candidates in the green algae Chlamydomonas reinhardtii. This approach has led to the discovery of 10’s of novel components of the pyrenoid and has shown that the pyrenoid has multiple spatially distinct regions. To further expand this dataset, we performed affinity purification mass spectrometry to create a spatially defined protein-protein interaction network. The lab has recently developed novel cloning methods to enable the expansion of this network and is adding further dimensions to understand changes in protein localization and interacting partners in response to abiotic changes.

To provide rapid advances in out understanding of how cyanobacteria adapt to changing CO₂ we have developed a pipeline to enable the fluorescence protein tagging of the complete proteome. This library will then be used to monitor protein abundance, protein localization, and protein-protein interaction changes in response to changing CO₂.

Quick-freeze deep-etched EM of the Chlamydomonas pyrenoid. Image courtesy of Ursula Goodenough.

Robotically propagated colonies of Chlamydomonas; each expressing a different fluorescently tagged CCM protein.

Diverse localization patterns of cyanobacterial proteins fluorescently tagged with mNeonGreen. Cartoon credit: Kelvinsong / CC BY-SA (https://creativecommons.org/licenses/by-sa/3.0)

Understanding the structure and function of the pyrenoid

We discovered that the pyrenoid is a liquid-liquid phase separated organelle that can rapidly dissolve and recondense during cell division and changes in CO₂ concentration (Freeman Rosenzweig et al., 2017 Cell). Enabling this is an intrinsically disordered repeat protein, called EPYC1 (Essential Pyrenoid Component 1; pronounced “epic one”), that links Rubisco together in the green algal pyrenoid (Mackinder et al., 2016 PNAS). Our recent fluorescence protein tagging and protein-protein interaction screen has further identified multiple additional pyrenoid and Rubisco interacting proteins (Mackinder et al. 2017 Cell). We are currently characterizing these proteins using cell physiology, biochemical and structural biology approaches.

Synthetic assembly of a CO₂ concentrating mechanism

One of the major challenges facing human civilization is how to sustainably feed a rapidly expanding population with limited resources. A currently pursued approach is to increase crop yields by enhancing photosynthesis through the introduction of a CCM. However, the engineering of complex pathways in plants is severely limited by low throughput and slow reproduction times. We aim to rapidly speed up the transition of a CCM to higher plants by developing a “stepping-stone” approach where thousands of component combinations can be tested in a heterologous system and screened for a functional CCM. This data can then be used to guide the engineering of higher plants.