Engineering topics

Flamholz, Avi I.

Publications and source records attributed to Flamholz, Avi I..

5 records

DOE OSTI2023

Carbon isotope fractionation by an ancestral rubisco suggests that biological proxies for CO 2 through geologic time should be reevaluated

The history of Earth’s carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO 2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. Therefore, we measured both biomass (ε p ) and enzymatic (ε Rubisco ) carbon isotope fractionations of a cyanobacterial strain ( Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to ≫1 Ga. This strain, nicknamed ANC, grows in ambient pCO 2 and displays larger ε p values than WT, despite having a much smaller ε Rubisco (17.23 ± 0.61‰ vs. 25.18 ± 0.31‰, respectively). Surprisingly, ANC ε p exceeded ANC ε Rubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. Such models can be rectified by introducing additional isotopic fractionation associated with powered inorganic carbon uptake mechanisms present in Cyanobacteria, but this amendment hinders the ability to accurately estimate historical pCO 2 from geological data. Understanding the evolution of rubisco and the CO 2 concentrating mechanism is therefore critical for interpreting the carbon isotope record, and fluctuations in the record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO 2 .

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2022

Trajectories for the evolution of bacterial CO 2 -concentrating mechanisms

Cyanobacteria rely on CO 2 -concentrating mechanisms (CCMs) to grow in today’s atmosphere (0.04% CO 2 ). These complex physiological adaptations require ≈15 genes to produce two types of protein complexes: inorganic carbon (Ci) transporters and 100+ nm carboxysome compartments that encapsulate rubisco with a carbonic anhydrase (CA) enzyme. Mutations disrupting any of these genes prohibit growth in ambient air. If any plausible ancestral form—i.e., lacking a single gene—cannot grow, how did the CCM evolve? Here, we test the hypothesis that evolution of the bacterial CCM was “catalyzed” by historically high CO 2 levels that decreased over geologic time. Using an E. coli reconstitution of a bacterial CCM, we constructed strains lacking one or more CCM components and evaluated their growth across CO 2 concentrations. We expected these experiments to demonstrate the importance of the carboxysome. Instead, we found that partial CCMs expressing CA or Ci uptake genes grew better than controls in intermediate CO 2 levels (≈1%) and observed similar phenotypes in two autotrophic bacteria, Halothiobacillus neapolitanus and Cupriavidus necator . To understand how CA and Ci uptake improve growth, we model autotrophy as colimited by CO 2 and HCO 3 − , as both are required to produce biomass. Our experiments and model delineated a viable trajectory for CCM evolution where decreasing atmospheric CO 2 induces an HCO 3 − deficiency that is alleviated by acquisition of CA or Ci uptake, thereby enabling the emergence of a modern CCM. This work underscores the importance of considering physiology and environmental context when studying the evolution of biological complexity.

Flamholz, Avi I.↗

DOE OSTI2022

Optical O 2 Sensors Also Respond to Redox Active Molecules Commonly Secreted by Bacteria

From a metabolic perspective, molecular oxygen (O 2 ) is arguably the most significant constituent of Earth’s atmosphere. Nearly every facet of microbial physiology is sensitive to the presence and concentration of O 2 , which is the most favorable terminal electron acceptor used by organisms and also a dangerously reactive oxidant. As O 2 has such sweeping implications for physiology, researchers have developed diverse approaches to measure O 2 concentrations in natural and laboratory settings. Recent improvements to phosphorescent O 2 sensors piqued our interest due to the promise of optical measurement of spatiotemporal O 2 dynamics. However, we found that our preferred bacterial model, Pseudomonas aeruginosa PA14, secretes more than one molecule that quenches such sen sors, complicating O 2 measurements in PA14 cultures and biofilms. Assaying supernatants from cultures of 9 bacterial species demonstrated that this phenotype is common: all super natants quenched a soluble O 2 probe substantially. Phosphorescent O 2 probes are often embedded in solid support for protection, but an embedded probe called O 2 NS was quenched by most supernatants as well. Measurements using pure compounds indicated that quenching is due to interactions with redox-active small molecules, including phena zines and flavins. Uncharged and weakly polar molecules like pyocyanin were especially potent quenchers of O 2 NS. These findings underscore that optical O 2 measurements made in the presence of bacteria should be carefully controlled to ensure that O 2 , and not bacterial secretions, is measured, and motivate the design of custom O 2 probes for specific organisms to circumvent sensitivity to redox-active metabolites.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Protein cost minimization promotes the emergence of coenzyme redundancy

Metabolism relies on a small class of molecules (coenzymes) that serve as universal donors and acceptors of key chemical groups and electrons. Although metabolic networks crucially depend on structurally redundant coenzymes [e.g., NAD(H) and NADP(H)] associated with different enzymes, the criteria that led to the emergence of this redundancy remain poorly understood. Our combination of modeling and structural and sequence analysis indicates that coenzyme redundancy may not be essential for metabolism but could rather constitute an evolved strategy promoting efficient usage of enzymes when biochemical reactions are near equilibrium. Our work suggests that early metabolism may have operated with fewer coenzymes and that adaptation for metabolic efficiency may have driven the rise of coenzyme diversity in living systems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

eQuilibrator 3.0: a database solution for thermodynamic constant estimation

Abstract eQuilibrator (equilibrator.weizmann.ac.il) is a database of biochemical equilibrium constants and Gibbs free energies, originally designed as a web-based interface. While the website now counts around 1,000 distinct monthly users, its design could not accommodate larger compound databases and it lacked a scalable Application Programming Interface (API) for integration into other tools developed by the systems biology community. Here, we report on the recent updates to the database as well as the addition of a new Python-based interface to eQuilibrator that adds many new features such as a 100-fold larger compound database, the ability to add novel compounds, improvements in speed and memory use, and correction for Mg2+ ion concentrations. Moreover, the new interface can compute the covariance matrix of the uncertainty between estimates, for which we show the advantages and describe the application in metabolic modelling. We foresee that these improvements will make thermodynamic modelling more accessible and facilitate the integration of eQuilibrator into other software platforms.

59 BASIC BIOLOGICAL SCIENCES↗