Elsevier

Bioresource Technology

Volume 271, January 2019, Pages 368-374
Bioresource Technology

Targeted knockout of phospholipase A2 to increase lipid productivity in Chlamydomonas reinhardtii for biodiesel production

https://doi.org/10.1016/j.biortech.2018.09.121Get rights and content

Highlights

  • An efficient way of genome editing using CRISPR-Cas9 in microalgae.

  • Knockout of a key effector gene in Lands cycle have increased in TAG accumulation.

  • Increase in overall lipid productivity of the green alga, Chlamydomonas reinhardtii.

  • Upstream process for reducing the production cost of the microalgal biofuel.

Abstract

Biofuel derived from microalgae have several advantages over other oleaginous crops, however, still needs to be improved with its cost aspect and can be achieved by developing of a strain with improved lipid productivity. In this study, the CRISPR-Cas9 system was incorporated to carry out a target-specific knockout of the phospholipase A2 gene in Chlamydomonas reinhardtii. The targeted gene encodes a key enzyme in the Lands cycle. As a result, the mutants showed a characteristic of increased diacylglycerol pool, followed by a higher accumulation of triacylglycerol without being significantly compensated with the cell growth. As a result, the overall lipid productivities of phospholipase A2 knockout mutants have increased by up to 64.25% (to 80.92 g L−1 d−1). This study can provide crucial information for the biodiesel industry.

Introduction

As one of the major greenhouse gases, CO2 aggravates the setbacks from the climate change and the global warming by causing the imbalance of the natural carbon cycle from a high CO2 emission, 30 Gt/year (Moreira and Pires, 2016). The production of biofuel from microalgae, in particular, provides the benefit of CO2 mitigation by utilizing CO2 at the stage of amassing of the microalgal biomass (Choi et al., 2018, Kwak et al., 2016, Moreira and Pires, 2016). In the recent years, microalgae have renewed its potential for negative emission technology via the synergistic implementation at a bioenergy with carbon capture and storage (BECCS) technology (Beal et al., 2018). In the technoeconomic analysis and the life cycle assessment of 121 ha algae facility with a 2680 ha eucalyptus forest for BECCS conducted by Beal et al. (2018), the microalgae production integrated bioenergy CCS system generated 61.5 TJ of electricity while sequestering 29,600 ton of CO2 per year. This study provides the possible case for requiring the economic feasibility for the algal biofuel industry by synergistically integrating with other industry to supplement the disadvantage of stand-alone algal biofuel industry of its high cost (Beal et al., 2018, Beal et al., 2015, Chu, 2017, Wijffels and Barbosa, 2010). According to Council (2012), the five key areas of research for a sustainable development of algal biofuel production included the genetic engineering of microalgae for strain development for a strain with higher biomass productivity and improved lipid productivity than the wild type.

The single-celled green alga, Chlamydomonas reinhardtii, is a useful model organism for studying algal metabolism due to its fully sequenced genome, along with sizable collections of mutant libraries (Neupert et al., 2009); extensive studies on photosynthesis, carbon and lipid metabolism of this microalgae have been performed (Siaut et al., 2011). Due to the ease of cultivation and performing genetic modifications in this model alga, it is emerging as the next industrial biotechnology platform that can produce chemical compounds, metabolites and proteins with sustainability (Scaife et al., 2015). Microalgae are already commercially exploited for their natural by-products such as beta-carotene, astaxanthin, agar as dietary supplements. In addition, several research groups are attempting to produce bio-hydrogen, HIV antigen, bio-fuel by means of genetic manipulation of C. reinhardtii (Baltz et al., 2014, Barahimipour et al., 2016).

A large proportion of microalgal biofuel is derived from triacylglycerol (TAG). It is a stress-responsive storage lipid, in a form of metabolic energy. Its chemical similarity to petroleum and its high content (%w/w) of fatty acids have made it a promising feedstock for the biodiesel production (Breuer et al., 2012, Fan et al., 2011, Merchant et al., 2012). Microalgae accumulate TAG reliably and efficiently via nitrogen starvation (Shin et al., 2018, Yeh and Chang, 2011). However, according to the several economic models and life cycle analyses, the long-term commercialization of algal biofuel production is not yet viable (Dutta et al., 2016, Kenny and Flynn, 2017). The average global maximum microalgal biofuel productivity is estimated to be 8.4 TOE ha−1 y−1 at optimal cultivation condition. The maximum biofuel productivity can be improved by 33% by using a microalgal strain with high lipid content (Park and Lee, 2016). Therefore, it is critical to have a better understanding of TAG regulatory metabolic pathways from cradle- to grave: the initiation of TAG accumulation in response to stress, the total lipid synthesis and the degradation of TAG as a regulatory response in cell to realize the algal biofuel production (Merchant et al., 2012, Moellering and Benning, 2010).

As for genetic engineering approaches to increase lipid productivity, the competing pathway of carbohydrate synthesis was disturbed, or the biosynthesis of lipid was facilitated (Radakovits et al., 2010, Trentacoste et al., 2013). However, the resulting engineered strains often grow slowly (Li et al., 2010b, Wang et al., 2009). Specifically, the starchless sta6 mutant exhibited decreased photosynthetic activity under both mixo- and auto-trophic conditions compared to the wild type (Davey et al., 2014). In addition, unfavorable changes in the energy partitioning step of photosystem II (PSII), resulted in lowered maximal PSII quantum yield (F'v/F'm) and photochemical quenching (qP) (Zhao et al., 2017, Zijffers et al., 2010). These changes are related to lowered water oxidation and CO2 fixation in the sta6 than in the wild type, leaving sta6 relatively incompetent in terms of bioenergy (Li et al., 2010a). In order to bring the economic viability to algal biofuel, the securing of an algal strain with high lipid content with comparable growth rate to the wild type is imperative (Davis et al., 2011).

As a competing pathway with lipid biogenesis, lipid catabolism has been overlooked. Lipid catabolism provides the adaptability to different culture conditions by supplying acyl groups at the time of remodeling of the membrane and rearrangement of the photosynthetic apparatus (Solovchenko, 2012, Trentacoste et al., 2013). With the recent development of genetic engineering tool CRISPR-Cas9 and with several optimizations of the tool in microalgae, more efficient targeting of a gene is possible (Ng et al., 2017). In the present study, it was hypothesized that specific-knockout mutants of a phospholipase gene (Cre02.g095000) created by using CRISPR-Cas9 could have increased lipid productivity in C. reinhardtii. The focus was on the physiological changes in the lipid profile and in lipid productivity of three biologically similar phospholipase A2 mutants.

Section snippets

Culture conditions

Chlamydomonas reinhardtii strains CC-4349 cw15 mt (Baek et al., 2016, Jeong et al., 2017) and mutants at final OD750 ∼ 0.1 were transferred into 50 mL of Tris–acetate phosphate medium (TAP medium) with 125 rpm rotary shaking incubator maintained at 25 °C under continuous white light of 80 μmol photons m-2s-1. For the purpose of lipid analysis, the samples were centrifuged at 3000 rpm for 10 min and the supernatant growth media was changed into the nitrogen-deficient medium (TAP(-N), TAP medium

Characterization of Cre02.g095000

According to a BLAST search, Cre02.g095000 contains a catalytically active and a conserved domain that belongs to the phospholipase A2 (PLA2)-like superfamily. PLA2 is a key enzyme in the Lands cycle, which serves for remodeling of membrane lipids in the endoplasmic reticulum membrane for TAG biosynthesis and deposition into lipid droplets (Zulu et al., 2018). In mice, PLA2 liberates free fatty acids and lysophospholipids by hydrolyzing glycerophospholipids at the sn-2 position (Murakami et

Conclusion

TAG from microalgae has been recognized as a promising feedstock for biodiesel. This study presents a successful case of increased overall lipid productivity from microalgae via the usage of CRISPR-cas9. Using the targeted genome editing, phospholipase A2 gene was knocked-out. The selected mutants were confirmed at the genomic level. By targeting a lipase gene, it was expected to increase the lipid accumulation without hindering the growth rate. As a result, it was confirmed that all the tested

Acknowledgements

The authors would like to acknowledge the support of the Korea CCS R&D Center (Korea CCS 2020 Project) funded by the Korea government (Ministry of Science and ICT) in 2017 (grant number: KCRC-2014M1A8A1049278) and National Research Foundation of Korea (NRF) funded by the Korea government (grant number: NRF-2016R1A2A1A05005465) and Korea Institute of Energy Technology Evaluation and Planning (KETEP) grant funded by the Korea government (Ministry of Trade, Industry and Energy) (grant number:

References (49)

  • H.S. Kwak et al.

    Synergistic effect of multiple stress conditions for improving microalgal lipid production

    Algal Res.

    (2016)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method

    Methods

    (2001)
  • S.S. Merchant et al.

    TAG, you're it! Chlamydomonas as a reference organism for understanding algal triacylglycerol accumulation

    Curr. Opin. Biotechnol.

    (2012)
  • D. Moreira et al.

    Atmospheric CO2 capture by algae: Negative carbon dioxide emission path

    Bioresour. Technol.

    (2016)
  • M. Murakami et al.

    Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice

    Biochimie

    (2010)
  • Z.T. Wang et al.

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

    Eukaryot. Cell

    (2009)
  • N.N. Zulu et al.

    Current trends to comprehend lipid metabolism in diatoms

    Prog. Lipid Res.

    (2018)
  • S. Bae et al.

    Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases

    Bioinformatics

    (2014)
  • K. Baek et al.

    DNA-free two-gene knockout in Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins

    Sci. Rep.

    (2016)
  • A. Baltz et al.

    Plastidial Expression of Type II NAD(P)H Dehydrogenase Increases the Reducing State of Plastoquinones and Hydrogen Photoproduction Rate by the Indirect Pathway in Chlamydomonas reinhardtii

    Plant Physiol.

    (2014)
  • R. Barahimipour et al.

    Efficient expression of nuclear transgenes in the green alga Chlamydomonas: synthesis of an HIV antigen and development of a new selectable marker

    Plant Mol. Biol.

    (2016)
  • C.M. Beal et al.

    Integrating Algae with Bioenergy Carbon Capture and Storage (ABECCS) Increases Sustainability

    Earth's Future

    (2018)
  • E.G. Bligh et al.

    A rapid method of total lipid extraction and purification

    Can. J. Biochem. Physiol.

    (1959)
  • W.-L. Chu

    Strategies to enhance production of microalgal biomass and lipids for biofuel feedstock

    Eur. J. Phycol.

    (2017)
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