Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction

Zhou, Yu Min; Whitney, Spencer M.

doi:10.3390/ijms20205019

Cited by 56 publications

(54 citation statements)

References 58 publications

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“…Third, multiple efforts to improve the rate of rubisco carboxylation have made limited progress (Mueller-Cajar et al, 2007;Gomez-Fernandez et al, 2018;. Cases where some increase in the net carboxylation capacity of rubisco was achieved (via directed evolution) include only form-I and form-III isoforms (Wilson et al, 2016;Zhou & Whitney, 2019).…”

Section: Introductionmentioning

confidence: 99%

Highly active rubiscos discovered by systematic interrogation of natural sequence diversity

et al. 2020

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CO2 is converted into biomass almost solely by the enzyme rubisco. The poor carboxylation properties of plant rubiscos have led to efforts that made it the most kinetically characterized enzyme, yet these studies focused on < 5% of its natural diversity. Here, we searched for fast‐carboxylating variants by systematically mining genomic and metagenomic data. Approximately 33,000 unique rubisco sequences were identified and clustered into ≈ 1,000 similarity groups. We then synthesized, purified, and biochemically tested the carboxylation rates of 143 representatives, spanning all clusters of form‐II and form‐II/III rubiscos. Most variants (> 100) were active in vitro, with the fastest having a turnover number of 22 ± 1 s−1—sixfold faster than the median plant rubisco and nearly twofold faster than the fastest measured rubisco to date. Unlike rubiscos from plants and cyanobacteria, the fastest variants discovered here are homodimers and exhibit a much simpler folding and activation kinetics. Our pipeline can be utilized to explore the kinetic space of other enzymes of interest, allowing us to get a better view of the biosynthetic potential of the biosphere.

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Section: Introductionmentioning

confidence: 99%

Highly active rubiscos discovered by systematic interrogation of natural sequence diversity

et al. 2020

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“…Escherichia coli has long been the preferred host for the directed evolution of Rubisco for the sake of enhancing carboxylation activity toward CO 2 due to its high transformation efficiency and simple genetic tools ( Mueller-Cajar and Whitney, 2008a ). Several screening platforms for directed evolution of Rubisco have been developed by coupling cell growth to Rubisco activity ( Parikh et al, 2006 ; Mueller-Cajar et al, 2007 ; Mueller-Cajar and Whitney, 2008b ; Cai et al, 2014 ; Wilson et al, 2016 ; Zhou and Whitney, 2019 ). Unfortunately, affinity and catalytic efficiency toward CO 2 failed to synchronously increase ( Kubis and Bar-Even, 2019 ).…”

Section: Natural Co 2 Fixation Pathwaysmentioning

confidence: 99%

Recent Advances in Developing Artificial Autotrophic Microorganism for Reinforcing CO2 Fixation

Liang

Zhao²,

Jian-ming

2020

Front. Microbiol.

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With the goal of achieving carbon sequestration, emission reduction and cleaner production, biological methods have been employed to convert carbon dioxide (CO2) into fuels and chemicals. However, natural autotrophic organisms are not suitable cell factories due to their poor carbon fixation efficiency and poor growth rate. Heterotrophic microorganisms are promising candidates, since they have been proven to be efficient biofuel and chemical production chassis. This review first briefly summarizes six naturally occurring CO2 fixation pathways, and then focuses on recent advances in artificially designing efficient CO2 fixation pathways. Moreover, this review discusses the transformation of heterotrophic microorganisms into hemiautotrophic microorganisms and delves further into fully autotrophic microorganisms (artificial autotrophy) by use of synthetic biological tools and strategies. Rapid developments in artificial autotrophy have laid a solid foundation for the development of efficient carbon fixation cell factories. Finally, this review highlights future directions toward large-scale applications. Artificial autotrophic microbial cell factories need further improvements in terms of CO2 fixation pathways, reducing power supply, compartmentalization and host selection.

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“…Accordingly, it should be feasible in the current synthetic biology revolution to circumvent these evolutionary barriers to RuBisCO adaptation. Indeed, promising steps toward this goal have been already demonstrated using directed evolution of the enzyme to generate variants with improved catalytic traits in non-photosynthetic archaea (Wilson, Alonso and Whitney, 2016) and plant-like Form I hexadecameric RuBisCO from photosynthetic bacteria (Zhou and Whitney, 2019) and cyanobacteria (Wilson et al , 2018). Thus, these findings presented here provide optimism for engineering RuBisCO in food, fibre and fuel crops to have improved catalytic efficiency.…”

Section: Discussionmentioning

confidence: 99%

“…First, a recent analysis of the correlative nature of RuBisCO kinetics has demonstrated that associations between kinetic traits are weakened when a large number of species are considered (Flamholz et al , 2019; Iñiguez et al , 2020). Further, engineering efforts to alter RuBisCO kinetics have produced enzyme variants that deviate from proposed catalytic trade-offs between S C/O , k catC and K C (Wilson et al , 2018; Zhou and Whitney, 2019). Thus together, these results indicate that RuBisCO kinetic traits are perhaps not as inextricably linked as originally thought, and suggest that there is scope for increasing the catalytic efficiency of the enzyme as has happened in nature for RuBisCO in some red algae (Andersson and Backlund, 2008).…”

Section: Introductionmentioning

confidence: 99%

Rubisco adaptation is more limited by phylogenetic constraint than by catalytic trade-off

Emms

Rhodes

et al. 2020

Preprint

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RuBisCO assimilates CO2 to form the sugars that fuel life on earth. Correlations between RuBisCO kinetic traits across species have led to the proposition that RuBisCO adaptation is constrained by catalytic trade-offs. However, these correlations were founded on the assumption that kinetic measurements in different species are independent. Here, we show that this assumption is incorrect in angiosperms by evaluating the dependence of variation in RuBisCO kinetic traits on the phylogenetic tree that relates the enzymes. We show that there is significant phylogenetic signal in all carboxylase kinetic traits in angiosperms, and significant phylogenetic signal in the Michaelis constant for O2 in species that conduct C3 photosynthesis. When accounting for this non-independence, we show that the catalytic trade-off between carboxylase turnover and the Michaelis constant for CO2 is weak (∼30 % dependency) and that the correlations between all other RuBisCO kinetic traits are either not-significant or marginal (<9 % dependency). Finally, we demonstrate that phylogenetic constraints limit RuBisCO adaptation to a greater extent than catalytic trade-offs. Thus, the biochemical landscape of RuBisCO adaptation in angiosperms is predominantly limited by phylogenetic constraint and a partial trade-off between carboxylase turnover and the Michaelis constant for CO2.

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Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction

Cited by 56 publications

References 58 publications

Highly active rubiscos discovered by systematic interrogation of natural sequence diversity

Highly active rubiscos discovered by systematic interrogation of natural sequence diversity

Recent Advances in Developing Artificial Autotrophic Microorganism for Reinforcing CO2 Fixation

Rubisco adaptation is more limited by phylogenetic constraint than by catalytic trade-off

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