A kinetic metabolic study of lipid production in  Chlorella protothecoides  under heterotrophic condition

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Abstract

Background

Microalgae have been proposed as potential platform to produce lipid-derived products, such as biofuels. Knowledge on the intracellular carbon flow distribution may identify key metabolic processes during lipid synthesis thus refining culture/genetic strategies to maximize cell lipid productivity. A kinetic metabolic model simulating cell metabolic behavior and lipid production was first applied in the microalgae platform Chlorella protothecoides under heterotrophic condition. It combines both physiology and flux information in a kinetic approach. Cell nutrition, growth, lipid production and almost 30 metabolic intermediates covering central carbon metabolism were included and simulated.

Results

Model simulations were shown to adequately agree with experimental data, which is suggesting that the proposed model copes with Chlorella protothecoides cells’ biology. The dynamic metabolic flux analysis using the model showed a reversible starch flux from accumulation to decomposing when glucose reached depletion, while net lipid flux shows a quasi-constant rate. The sensitive flux parameters on starch and lipid metabolism suggested that starch synthesis is the major competing pathway that affects lipid accumulation in C. protothecoides. Flux analysis also demonstrated that high lipid yield under heterotrophic condition is accompanied with high lipid flux and low TCA activity. Meanwhile, the dynamic flux distribution also suggests a relatively constant ratio of glucose distributed to biomass, lipid, starch, nucleotides as well as pentose phosphate pathway.

Conclusion

The model described not only experimental data, but also unraveled intracellular carbon flow distribution and identify key metabolic processes during lipid synthesis. Most of the metabolic kinetics also showed statistical significance for metabolic mechanism. Therefore, this study unravels the mechanisms of the glucose impact on the dynamic carbon flux distribution, thus improving our understanding of the links between carbon fluxes and lipid metabolism in C. protothecoides.

Electronic supplementary material

The online version of this article (10.1186/s12934-019-1163-4) contains supplementary material, which is available to authorized users.

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Most cited references 22

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Algal lipid bodies: stress induction, purification, and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii.

S-I Joo, Ursula Goodenough, Sabine Waffenschmidt … (2009)

When the unicellular green soil alga Chlamydomonas reinhardtii is deprived of nitrogen after entering stationary phase in liquid culture, the cells produce abundant cytoplasmic lipid bodies (LBs), as well as abundant starch, via a pathway that accompanies a regulated autophagy program. After 48 h of N starvation in the presence of acetate, the wild-type LB content has increased 15-fold. When starch biosynthesis is blocked in the sta6 mutant, the LB content increases 30-fold, demonstrating that genetic manipulation can enhance LB production. The use of cell wall-less strains permitted development of a rapid "popped-cell" microscopic assay to quantitate the LB content per cell and permitted gentle cell breakage and LB isolation. The highly purified LBs contain 90% triacylglycerol (TAG) and 10% free fatty acids (FFA). The fatty acids associated with the TAGs are approximately 50% saturated (C(16) and C(18)) fatty acids and approximately 50% unsaturated fatty acids, half of which are in the form of oleic acid (C(18:1)). The FFA are approximately 50% C(16) and approximately 50% C(18). The LB-derived TAG yield from a liter of sta6 cells at 10(7) cells/ml after starvation for 48 h is calculated to approach 400 mg. The LB fraction also contains low levels of charged glycerolipids, with the same profile as whole-cell charged glycerolipids, that presumably form LB membranes; chloroplast-specific neutral glycerolipids (galactolipids) are absent. Very low levels of protein are also present, but all matrix-assisted laser desorption ionization-identified species are apparent contaminants. Nitrogen stress-induced LB production in C. reinhardtii has the hallmarks of a discrete pathway that should be amenable to additional genetic and culture condition manipulation.

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Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii

Nanette R Boyle, John A. Morgan (2009)

Background Photosynthetic organisms convert atmospheric carbon dioxide into numerous metabolites along the pathways to make new biomass. Aquatic photosynthetic organisms, which fix almost half of global inorganic carbon, have great potential: as a carbon dioxide fixation method, for the economical production of chemicals, or as a source for lipids and starch which can then be converted to biofuels. To harness this potential through metabolic engineering and to maximize production, a more thorough understanding of photosynthetic metabolism must first be achieved. A model algal species, C. reinhardtii, was chosen and the metabolic network reconstructed. Intracellular fluxes were then calculated using flux balance analysis (FBA). Results The metabolic network of primary metabolism for a green alga, C. reinhardtii, was reconstructed using genomic and biochemical information. The reconstructed network accounts for the intracellular localization of enzymes to three compartments and includes 484 metabolic reactions and 458 intracellular metabolites. Based on BLAST searches, one newly annotated enzyme (fructose-1,6-bisphosphatase) was added to the Chlamydomonas reinhardtii database. FBA was used to predict metabolic fluxes under three growth conditions, autotrophic, heterotrophic and mixotrophic growth. Biomass yields ranged from 28.9 g per mole C for autotrophic growth to 15 g per mole C for heterotrophic growth. Conclusion The flux balance analysis model of central and intermediary metabolism in C. reinhardtii is the first such model for algae and the first model to include three metabolically active compartments. In addition to providing estimates of intracellular fluxes, metabolic reconstruction and modelling efforts also provide a comprehensive method for annotation of genome databases. As a result of our reconstruction, one new enzyme was annotated in the database and several others were found to be missing; implying new pathways or non-conserved enzymes. The use of FBA to estimate intracellular fluxes also provides flux values that can be used as a starting point for rational engineering of C. reinhardtii. From these initial estimates, it is clear that aerobic heterotrophic growth on acetate has a low yield on carbon, while mixotrophically and autotrophically grown cells are significantly more carbon efficient.

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Central carbon metabolism and electron transport in Chlamydomonas reinhardtii: metabolic constraints for carbon partitioning between oil and starch.

Xenie Johnson, Jean Alric (2013)

The metabolism of microalgae is so flexible that it is not an easy task to give a comprehensive description of the interplay between the various metabolic pathways. There are, however, constraints that govern central carbon metabolism in Chlamydomonas reinhardtii that are revealed by the compartmentalization and regulation of the pathways and their relation to key cellular processes such as cell motility, division, carbon uptake and partitioning, external and internal rhythms, and nutrient stress. Both photosynthetic and mitochondrial electron transfer provide energy for metabolic processes and how energy transfer impacts metabolism and vice versa is a means of exploring the regulation and function of these pathways. A key example is the specific chloroplast localization of glycolysis/gluconeogenesis and how it impacts the redox poise and ATP budget of the plastid in the dark. To compare starch and lipids as carbon reserves, their value can be calculated in terms of NAD(P)H and ATP. As microalgae are now considered a potential renewable feedstock, we examine current work on the subject and also explore the possibility of rerouting metabolism toward lipid production.

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Author and article information

Contributors

Mario Jolicoeur: 514 340 4711 , mario.jolicoeur@polymtl.ca

Journal

Journal ID (nlm-ta): Microb Cell Fact

Journal ID (iso-abbrev): Microb. Cell Fact

Title: Microbial Cell Factories

Publisher: BioMed Central (London )

ISSN (Electronic): 1475-2859

Publication date (Electronic): 28 June 2019

Publication date PMC-release: 28 June 2019

Publication date Collection: 2019

Volume: 18

Electronic Location Identifier: 113

Affiliations

[1 ]ISNI 0000 0004 1808 3414, GRID grid.412509.b, Colin Ratledge Center for Microbial Lipids, School of Agriculture Engineering and Food Science, , Shandong University of Technology, ; Zibo, China

[2 ]ISNI 0000 0004 0435 3292, GRID grid.183158.6, Research Laboratory in Applied Metabolic Engineering, Department of Chemical Engineering, , École Polytechnique de Montreal, Centre-ville Station, ; P.O. Box 6079, Montreal, H3C 3A7 QC Canada

[3 ]ISNI 0000 0001 2185 197X, GRID grid.265702.4, Université du Québec à Rimouski, ; 310 allée des Ursulines, Rimouski, QC G5L 3A1 Canada

[4 ]ISNI 0000 0001 2112 9282, GRID grid.4444.0, LRI, Université Paris-Sud, , CNRS, Université Paris-Saclay, ; 91405 Orsay, France

[5 ]ISNI 0000 0004 4910 6535, GRID grid.460789.4, MaIAGE, INRA, , Université Paris-Saclay, ; 78350 Jouy-en-Josas, France

Article

Publisher ID: 1163

DOI: 10.1186/s12934-019-1163-4

PMC ID: 6598345

PubMed ID: 31253148

SO-VID: 8e2955f4-24a7-44d7-a668-d76c42c664b4

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 9 April 2019

Date accepted : 19 June 2019

Funding

Funded by: Shandong Provincial Natural Science Foundation, China

Award ID: ZR2019BC099

Award Recipient : Xiaojie Ren

Funded by: FundRef http://dx.doi.org/10.13039/501100003150, Fonds Québécois de la Recherche sur la Nature et les Technologies;

Award ID: RS-171172

Award Recipient : Mario Jolicoeur

Funded by: FundRef http://dx.doi.org/10.13039/501100002790, Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada;

Award ID: 093865-RGPIN2014-04329

Award Recipient : Mario Jolicoeur

Funded by: China Postdoctoral Science Foundation

Award ID: 2019M650167

Award Recipient : Xiaojie Ren

Custom metadata

ScienceOpen disciplines: Biotechnology

Keywords: metabolic modelling,kinetic model,central carbon metabolism,microalgae,chlorella protothecoides,dynamic flux analysis

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ScienceOpen disciplines: Biotechnology

Keywords: metabolic modelling, kinetic model, central carbon metabolism, microalgae, chlorella protothecoides, dynamic flux analysis

A kinetic metabolic study of lipid production in Chlorella protothecoides under heterotrophic condition

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Abstract

Background

Results

Conclusion

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Genome Engineering using CRISPR

Most cited references 22

Algal lipid bodies: stress induction, purification, and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii.

Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii

Central carbon metabolism and electron transport in Chlamydomonas reinhardtii: metabolic constraints for carbon partitioning between oil and starch.

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