Isotopically Nonstationary MFA (INST-MFA) of Autotrophic Metabolism

Jazmin, Lara J.; O'Grady, John P.; Ma, Feifei; Allen, Doug K.; Morgan, John A.; Young, Jamey D.

doi:10.1007/978-1-62703-688-7_12

Cited by 36 publications

(26 citation statements)

References 43 publications

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“…All isotopic measurements used for flux determination are listed in Table 2, and a list of the reactions included in the biochemical reaction network is provided in the Supplementary Materials. INST-MFA was used to estimate intracellular metabolic fluxes (Jazmin et al, 2014). Least-squares parameter regression and statistical and sensitivity analysis of the optimal solution were performed by using the publicly available software package INCA (Young, 2014), which runs within MATLAB™.…”

Section: Methodsmentioning

confidence: 99%

“…PCC 6803 (Schwarz et al, 2013; Yang et al, 2002a; Yang et al, 2002b; Yang et al, 2002c). In addition, we have previously developed novel experimental approaches (Jazmin et al, 2014) and software packages (Young, 2014) that enable flux estimation in photoautotrophic cyanobacteria cultures using isotopically nonstationary 13 C MFA (INST-MFA). We applied these technologies to precisely quantify the rates of Calvin-Benson-Bassham (CBB) cycle and TCA pathway reactions in Synechocystis cultures, and we identified several “wasteful” side reactions that contributed to suboptimal photoautotrophic growth (Young et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

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Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production

Jazmin

Cheah

et al. 2017

Metabolic Engineering

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We applied isotopically nonstationary 13C metabolic flux analysis (INST-MFA) to compare the pathway fluxes of wild-type (WT) Synechococcus elongatus PCC 7942 to an engineered strain (SA590) that produces isobutyraldehyde (IBA). The flux maps revealed a potential bottleneck at the pyruvate kinase (PK) reaction step that was associated with diversion of flux into a three-step PK bypass pathway involving the enzymes PEP carboxylase (PEPC), malate dehydrogenase (MDH), and malic enzyme (ME). Overexpression of pk in SA590 led to a significant improvement in IBA specific productivity. Single-gene overexpression of the three enzymes in the proposed PK bypass pathway also led to improvements in IBA production, although to a lesser extent than pk overexpression. Combinatorial overexpression of two of the three genes in the proposed PK bypass pathway (mdh and me) led to improvements in specific productivity that were similar to those achieved by single-gene pk overexpression. Our work demonstrates how 13C flux analysis can be used to identify potential metabolic bottlenecks and novel metabolic routes, and how these findings can guide rational metabolic engineering of cyanobacteria for increased production of desired molecules.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production

Jazmin

Cheah

et al. 2017

Metabolic Engineering

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“…4B, [65]). Thus the study of leaves and other autotrophic cells may be best suited to transient or ''nonstationary'' isotopic analysis [66][67][68][69][70]. Pathways that have limited carbon bond rearrangements or network branching such as specialized metabolite production or pathways based on a single precursor like fatty acid biosynthesis that utilizes acetyl-CoA units have typically been investigated utilizing the kinetic incorporation of radioisotopes over time (i.e., pulse labeling at metabolic steady state as described below).…”

Section: Steady State and Transient Labelingmentioning

confidence: 99%

Tracking the metabolic pulse of plant lipid production with isotopic labeling and flux analyses: Past, present and future

Allen

Bates

Tjellström

2015

Progress in Lipid Research

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100

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Metabolism is comprised of networks of chemical transformations, organized into integrated biochemical pathways that are the basis of cellular operation, and function to sustain life. Metabolism, and thus life, is not static. The rate of metabolites transitioning through biochemical pathways (i.e., flux) determines cellular phenotypes, and is constantly changing in response to genetic or environmental perturbations. Each change evokes a response in metabolic pathway flow, and the quantification of fluxes under varied conditions helps to elucidate major and minor routes, and regulatory aspects of metabolism. To measure fluxes requires experimental methods that assess the movements and transformations of metabolites without creating artifacts. Isotopic labeling fills this role and is a long-standing experimental approach to identify pathways and quantify their metabolic relevance in different tissues or under different conditions. The application of labeling techniques to plant science is however far from reaching it potential. In light of advances in genetics and molecular biology that provide a means to alter metabolism, and given recent improvements in instrumentation, computational tools and available isotopes, the use of isotopic labeling to probe metabolism is becoming more and more powerful. We review the principal analytical methods for isotopic labeling with a focus on seminal studies of pathways and fluxes in lipid metabolism and carbon partitioning through central metabolism. Central carbon metabolic steps are directly linked to lipid production by serving to generate the precursors for fatty acid biosynthesis and lipid assembly. Additionally some of the ideas for labeling techniques that may be most applicable for lipid metabolism in the future were originally developed to investigate other aspects of central metabolism. We conclude by describing recent advances that will play an important future role in quantifying flux and metabolic operation in plant tissues.

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“…For this reason, isotopomer measurements obtained from proteinogenic amino acids are not useful for INST-MFA due to their slower rate of labeling. Instead, rapid sampling and cold-quenching of cells is required to fix the in vivo labeling state of central carbon metabolites, which can be measured in cell extract samples using a variety of mass spectrometry approaches (Jazmin and Young 2013;Jazmin et al 2014). Berla et al (2013) have recently reviewed applications of stoichiometric MFA to cyanobacteria, and a number of other recent articles have reviewed applications of 13 C MFA to examine general aspects of microbial metabolism (Matsuoka and Shimizu 2010;Tang et al 2009).…”

Section: Introductionmentioning

confidence: 99%

13C flux analysis of cyanobacterial metabolism

2014

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(13)C metabolic flux analysis (MFA) has made important contributions to our understanding of the physiology of model strains of E. coli and yeast, and it has been widely used to guide metabolic engineering efforts in these microorganisms. Recent advancements in (13)C MFA methodology combined with publicly available software tools are creating new opportunities to extend this approach to examine less characterized microbes. In particular, growing interest in the use of cyanobacteria as industrial hosts for photosynthetic production of biofuels and biochemicals has led to a critical need to better understand how cyanobacterial metabolic fluxes are regulated in response to changes in growth conditions or introduction of heterologous pathways. In this contribution, we review several prior studies that have applied isotopic steady-state (13)C MFA to examine heterotrophic or mixotrophic growth of cyanobacteria, as well as recent studies that have pioneered the use of isotopically nonstationary MFA (INST-MFA) to study autotrophic cultures. We also provide recommendations for the design and analysis of INST-MFA experiments in cyanobacteria, based on our previous experience and a series of simulation studies used to assess the selection of measurements and sample time points. We anticipate that this emerging knowledgebase of prior (13)C MFA studies, optimized experimental protocols, and public software tools will catalyze increasing use of (13)C MFA techniques by the cyanobacteria research community.

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Isotopically Nonstationary MFA (INST-MFA) of Autotrophic Metabolism

Cited by 36 publications

References 43 publications

Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production

Isotopically nonstationary 13C flux analysis of cyanobacterial isobutyraldehyde production

Tracking the metabolic pulse of plant lipid production with isotopic labeling and flux analyses: Past, present and future

13C flux analysis of cyanobacterial metabolism

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