Lipid Accumulation Mechanisms in Autotrophic and Heterotrophic

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Lipid Accumulation Mechanisms in Autotrophic and Heterotrophic Microalgae Hao-Hong Chen, and Jian-Guo Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03495 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 25, 2017

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Journal of Agricultural and Food Chemistry

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Lipid Accumulation Mechanisms in Autotrophic and Heterotrophic

2

Microalgae

3 4

Hao-Hong Chen, Jian-Guo Jiang *

5 6

College of Food Science and Engineering, South China University of Technology, Guangzhou,

7

510640, China

8 9 10

*Author

(Jian-Guo

Jiang)

for

correspondence

(e-mail:

[email protected];

+86-20-87113849; fax: +86-20-87113849).

11

1

ACS Paragon Plus Environment

phone:


12

ABSTRACT

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Microalgae lipids have attracted great attention in the world due to their potential use for biodiesel

14

productions. Microalgae are cultivated in the photoautotrophic condition in most case, but several

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species are able to grow under the heterotrophic condition in which the microalgae are cultivated

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in the dark where the cell growth and reproduction are supported by organic carbons. This review

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is covering the related researches concerning the difference between heterotrophic and autotrophic

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cultivation of microalgae. The autotrophic and heterotrophic central carbon metabolic pathways in

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microalgae are described, and the catalyzing reactions of several key metabolic enzymes and their

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corresponding changes in protein level are summarized. Under adverse environmental conditions

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such as nutrient deprivation, microalgae have the ability to highly store energy by forming

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triacylglycerol (TAG), the reason of which is analyzed. In addition, the biosynthesis of fatty acids

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and triacylglycerols and their difference between autotrophic and heterotrophic conditions are

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compared at the molecular level. The positive regulatory enzymes such as the glucose transporter

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protein, fructose-1, 6-bisphosphate aldolase, glycerol-3-phosphate dehydrogenase, and the

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negative regulation enzymes such as triose phosphate isomerase, played a crucial role in the lipid

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accumulation autotrophic and heterotrophic conditions.

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KEYWORDS: microalgae, heterotrophy, autotrophy, lipid biosynthesis, metabolism

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INTRODUCTION

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Microalgae can not only regulate the circulation of substances in water and atmosphere, but also

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be regarded as the considerable resource in the near future.1 Many microalgaes have the ability to

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accumulate considerable amounts (20-50% DCW) of tricaylglycerols (TAG) under adverse

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environmental conditions, especially during nitrogen starvation.2 Oleaginous microalgae have

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been regarded as a promising alternative source for next-generation renewable fuels. The benefit

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of exploiting microalgae as biofuel feedstock are due to their short life cycle, less affected by

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geographical regions than higher plant, high lipid contents, low labor requirement, strong

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reproductive capacity.1, 2

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At present, the most popular procedure for microalgae cultivation is autotrophic growth. In

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illuminated environment, the microalgae cell harvest solar energy and utilize carbon dioxide (CO2)

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as carbon source.3 Most commercial microalgaes are cultivated in outdoor open ponds. However,

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these ponds are susceptible to bacteria, climate, light, nutrition and temperature, making their

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productivities very low.4 Some microalgaes have the ability to grow in dark using organic carbon

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sources.5 Moreover, hererotrophic cultivation can be well controlled so that a more productive

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yield of valuable products can be obtained. It has been reported that heterotrophic growth of

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Chlorella protothecoides accumulated higher lipid content in cells than autotrophic growth ones.6

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After cultivating in open ponds or reactors, microalgae cells can be harvested to extract the lipid

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which is turned into biodiesel via ester exchange reaction.7 A latest report showed that wet algae

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slurries could be converted into gravity separable biocrude product at relatively temperature

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(350°C) and pressurized environment (20Mpa) directly (Fig. 1).8 This may considerably improve

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the productivity and reduce the cost in industry.

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The heterotrophic growth approach has three major advantages, cost effectiveness and

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relative simplicity of operations and daily maintenance. But this culture also has several

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disadvantage: (1) Limited number of microalgae species that can grow heterotrophically; (2) Costs

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by adding an organic substrate; (3) Susceptible to bacterial infection in a nutrient rich medium; (4)

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Inability to produce light-induced metabolites. 6

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The decisive factor to utilize microalgaes for biodiesel is to improve their oil content of cells

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and cell density, and reduce the cost. In microalgae, the lipid biosynthetic pathway includes TAG 3



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synthesis and fatty acid synthesis. This review is covering the related researches about difference

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and coherence between autotrophic and heterotrophic lipid metabolism in microalgae, and focus

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on the carbon metabolism pathway and lipid biosynthesis. It is hoped that the analysis in this

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review will facilitate the development of targeted strategies to improve biomass production and

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lipid accumulation in microalgae.

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OLEAGINOUS MICROALGAE

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Oleaginous microalgaes are those whose lipid content in cells are more than 20% of the dry

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weight. Many microalgae species are capable of accumulating lipid contributing to a high oil

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yield.9 The average lipid content of oleaginous microalgaes are between 20 and 70%, while under

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certain cultural environment the lipid content of some species can reach 90% of dry weight.7, 10

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Most of oleaginous microalgaes fix CO2 for cellular metabolism and growth. Organic carbon

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sources, such as pyruvate, lactate, ethanol, saturated fatty acids, acetate, glycolate, glycerol,

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disaccharides (eg. cellobiose, lactose, and sucrose), C6 sugars (eg. glucose and fructose), C5

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monosaccharides (eg. xylose and arabinose) and amino acids, can be metabolized by several

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microalgaes to produce lipid.11

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As is shown in Table 1, the quality of biodiesel from heterotrophic cultured microalgae was

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guaranteed during the process improvement. Among them, Chaetoceros gracilis was the most

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productive oleaginous microalgae in autotrophic condition. As for heterotrophic culture, Chlorella

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protothecoides has the most lipid productivity cultured with glucose. These distinctions result

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from their species and culture methods. Even so, the proportions of main components in biodiesel

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(C19:1, C19:2, C17:0) are maintained to be relatively stable. Additionally, under autotrophic

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conditions, the production of polyunsaturated fatty acids (C16:3 and C18:3) is favored, while

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highly saturated fatty acids mainly produced under heterotrophic conditions.

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Scaling up for autotrophic microalgae is more complicated, open ponds have three main

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inherent disadvantages: (1) To minimize the cost, free sunlight is needed during the cultivation

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process, despite daily and seasonal variations in light levels.12 (2) Because of constant airborne

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contamination, mono-cultivation of the desired microalgae is susceptible to most microalgae

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species. (3) Environmental growth parameters of cultivation may not be controlled and make

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production seasonal. Heterotrophic cultures are easily controlled and cultivated in fermenters. 4


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However, they require organic carbon sources for lipid accumulation, which might limit the

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application for biodiesel production.13 Under heterotrophic conditions, the biomass yields of

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microalgae are reproducible and consistent reaching cells densities of 50 to 100 g/L of dry cell

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biomass, much higher than the maximum 30 g/L of dry biomass in autotrophic cultures.

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Heterotrophic microalgae containing as large as 100,000 L is able to produce useful biomass

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reaching hundreds of kilograms. These high productivity and large volumes of cultures make the

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heterotrophic strategy far cheaper than the autotrophic approach.15 For example, in Japan,

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heterotrophic cultures of Chlorella sp. was utilized to generate almost 500 ton of dry biomass,

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which was 50% of total Japanese production of this algae.17

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CARBON METABOLISM OF MICROALGAE

14-16

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The process of carbon absorption in heterotrophic growth and carbon sequestration in autotrophic

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have a significant influence on the central carbon metabolism of microalgae. This section

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describes the central carbon metabolic pathway of autotrophic and heterotrophic microalgae, and

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summarizes several of metabolic enzymes catalyzing these reactions. Furthermore, the central

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metabolic flux distribution and the physiological changes in those two different cultivating modes

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were provided.

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Light Capture and Carbon Fixation in Autotrophic Culture. It is well known

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that green algae and higher plants initially capture photons via light harvesting complexes (LHCs).

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Previous work indicated that the pigments relating to the LHCs in green algae account for about

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80% of the total chlorophyll content with the remaining 20% related to the proximal antenna and

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reaction center complexes where charge separation occurs.44 The carotenoids and chlorophyll of

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the LHCs are bound to thylakoid membrane proteins in close association with the reaction center

112

complexes. It was reported that energy transfer between these pigments of LHCs occurred on the

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femtosecond time scale.45 Eukaryotic photosynthetic cells absorb light energy from the P680 and

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P700, driving the flow of electrons from H2O to NADP+. The Z scheme thus describes the

115

complete route by which electrons flow H2O to NADP+ in noncyclic photosynthesis (Fig. 2).46

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NADPH is the source of energy produced by chloroplasts in light-dependent reactions, which is

117

used to generate ATP at the respiratory chain then goes on to provide a source of energetic

5



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electrons in other cellular reactions. Some reports show that electronic energy transfer between

119

pigments in the peripheral LHCs is about 100% efficiency under long-lived quantum coherence.47,

120

48

In summing up it may be stated that the pigments are the key factor in the carbon sequestration.

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It has been indicated that limitation in the rate of photosynthesis was determined by ribulose

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bisphosphate carboxylase oxygenase (EC 4.1.1.39 ) and cytochrome b6-f complex (Cytb6f;

123

plastoquinol-plastocyanin reductase; EC 1.10.99.1) in the thylakoid membrane. And the relatively

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slow rate of turnover of Mn-containing H2O-oxidation complex may also restrict the rate of

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photosynthesis. Above all, the initial fixation of CO2 through the Ribulose-1,5-bisphosphate

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(RuBP) carboxylase/oxygenase (Rubisco) is the overall rate-limiting step in photochemistry. This

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enzyme has very high concentrations of active sites. It is inactivated by loss of carbamylation and

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competitively inhibited by O2 and RuBP.49 This mechanism may help balance the carbon content

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and energy conversion of plants and algae.

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RuBisCo is only active under the light while the ribulose 1,5-bisphosphate is not regenerated

131

in the dark. Recent experiments suggested that Rubisco was regulated by several other enzymes

132

and factors in the Calvin cycle including Rubisco activase, ATP/ADP, stromal reduction/oxidation

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state, phosphate, CO2 and ions.50-52 In coping with the situation that the active sites of Rubisco

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became more and more restrictive by the competition between O2 and CO2, many microalgaes

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needed to utilize ATP and relative enzymes to elevate the CO2 concentrations in the near of

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Rubisco.53 In addition, bicarbonate cannot passively cross membranes. In C. reinhardtii,

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bicarbonate is actively transported across the plasmamembrane, there is an ABC-type transporter,

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HLA3, that is associated with HCO3− transport.54 In Nannochloropis gaditana, cytosolic carbonic

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anhydrase (CA) can form HCO3− either for transferring into the chloroplast by a bicarbonate

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transporter or for utilizing in C4 cycle-like carbon concentrating mechanisms. Chloroplastic CA

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formed CO2 in the near of Rubisco from actively transferred HCO3−. 55

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Additionally, CO2 assimilation of Calvin cycle was strikingly repressed under N-limiting

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conditions (substantial lipid accumulation situation), both CA-mediated carbon metabolism and C4

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cycle-like mechanism was upregulated.56 The investigation carried out above has revealed that

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these two intracellular CO2 concentrating mechanisms affect the oil produce.

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Carbon Absorption in Heterotrophic Culture. The nutrient and metabolites were 6


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transferred through certain vesicular transport, transmembrane and gated transports, which are

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regulated by the cytomembrane. In some cases, some microalgae cells can grow heterotrophically,

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as shown by recent reports.57, 58 Yet, not all microalgae species are able to survive in heterotrophic

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situation.59 Recently, it has been reported that the growth rate and cells density of microalgae

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were significantly improved after several genes was introduced into microalgae including

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Chlamydomonas, Chlorella, Diatoms and other algae.

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uptake protein (HUP1, from Chlorella), Glut1 (from human erythrocyte), and Hxt1, Hxt2, Hxt4

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(from Saccharomyces cerevisiae). First, Glut1, a member of glucose transporter protein, is located

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in cell membrane surface and is the main carrier of glucose transporter.61 Additionally, HUP1,

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HUP2 and HUP3 were the glucose transporter proteins in Chlorella kessleri, which demonstrated

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that certain microalgaes can uptake glucose naturally (Fig. 3).63 It also suggested that the presence

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of HUPs would drive other green algae to use glucose.

60-62

These genes were including hexose

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The absorption of glucose starts with the phosphorylation of hexose, producing glucose-6-

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phosphate. When glucose was the only carbon source in N. oleoabundans, it was absorbed by the

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proton-motive force, giving evidence that hexose was transported by symporter system.64 In C.

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vulgaris the minimum time required for inducing synthesis of the hexose/H+ symport system

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proteins via glucose is 15-18 mins.65, 66 The hexose/H+ symport system protein was coded by the

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HUP gene (hup1).67 The gene transcripts of hup1 appear within 5 min after glucose or sugar is

165

added.

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glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (gap1) are activated when

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autotrophically grown C. kessleri cells are switched to heterotrophic culture.68 The overexpression

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of gap1 in yeast resulted in 1.4~1.5-fold increase of lipid content.69, 70 GsSPT1, GsSPT2 and

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GsSPT4 were the plasma menmbrane transporter in the Galdieria sulphuraria. Among them, the

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GsSPT1 was a conserved type of sugar/H+ symporter with 12 predicted transmembrane-spanning

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domains, while GsSPT2 and GsSPT4 were typical for monosaccharide transporters, characterized

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by only 9 hydrophobic domains.71 Although growing with adequate glucose, the expression of

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hexose/ H+ symport is inhibited under illuminous culture in C. vulgaris. It was shown that the blue

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end of the visible spectrum plays an important role in inhibiting the uptake of hexoses via the

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blue-light photoreceptors flavoproteins cryptochromes 1/2 and NPH1, while the red end is slightly

Simultaneously,

the

mitochondrial

ATP/ADP

translocator

7


(AAT)

and

the


72

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effective.

In conclusion, heterotrophic microalgaes need the symporter system and ATP to

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uptake glucose or sugar, and their activity may affect the carbon assimilation even so the oil

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accumulation.

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Further, in comparison with autotrophic growth, Pentose phosphate pathway (PPP) is mainly

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metabolized in the darkness condition, nevertheless Embden-Meyerhof-Parnas (EMP) pathway is

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the main glycolytic pathway under light.25, 73, 74 This is the most significant difference between

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heterotrophic and autotrophic growth of microalgae. Of course, both aerobic glycolysis

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(breakdown of glucose) are implemented in the cytosol. The PPP may operate at a higher flux rate

184

than EMP, which depends on the presence of glucose and light.75 It has been demonstrated that in

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the heterotrophic culture of the Chlorella pyrenoidosa, there were about 90% of glucose metabolic

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flux distribution through PPP catalized glucose-6-phosphate dehydrogenase (EC: 1.1.1.49) but

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practically not through the EMP pathway catalized by glucose-6-phosphate isomerase (EC:

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5.3.1.9).73 This is different from the autotrophic flux distribution through the EMP.

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In addition, several enzymes found in the PPP were also carried out in the Calvin cycle,

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indicating that the photosynthetic CO2 fixation and the cytoplasmic carbon metabolism were

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associated with common cellular controls. The major source of NADPH in heterotrophic

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microalgae is from the PPP, and it is also the biosynthetic precursors such as ribose 5-phospate

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and erythrose 4-phosphate. In addition, the remaining NADPH is reoxidized to provide energy

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during respiration. Glucose-6-phosphate dehydrogenase is the rate-controlling enzyme of this

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pathway. And the activity of glucose-6-phosphate dehydrogenase was regulated by the ratio of

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NADP+/NADPH.76 Thus the light-modulated regulation of glucose-6-phosphate dehydrogenase

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may bring about the lower activity of PPP in autotrophic nutrition.73 Yet, it does not mean that the

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EMP pathway is completely shut down in heterotrophic condition. Glucose-6-phosphate isomerase

199

(EC:

200

6-phosphofructokinase (EC:2.7.1.11) are influenced, but the other reactions in the EMP pathway

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still carry out such as in autotrophic growth.73, 77 In heterotrophic growth, the high ratios of

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ATP/ADP would affect the mitochondrial electron transports. At the transcriptional level, the gnd

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(codes for 6-phosphogluconate dehydrogenase, EC: 1.1.1.44) was up-regulated about 60%, while

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the gap2 (glyceraldehyde-3-phosphate dehydrogenase-NADP; EC: 1.2.1.59) and rbcl (codes for

5.3.1.9),

fructose-1,

6-bisphosphate

aldolase

(ALDO;

8


EC:

4.1.2.13),

and

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ribulose bisophosphate carboxylase/oxygenase large subunit, EC: 4.1.1.39) were down-regulated

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about two-fold in the heterotrophic culture.78 Comparing the protein expression patterns of

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Synechocystis grown in mixotrophic and heterotrophic cultures, it was found that certain of

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cellular proteins were induced or repressed by light.78

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Under aerobic and dark conditions, eukaryotic cells can carry molecules with one carboxylate

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group (monocarboxylates, such as pyruvate, lactate and acetate) across biological membranes

211

using monocarboxylic/proton transporters protein (MCTs) (Fig. 3 ).79,

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microalgae cells, the assimilation of acetic acid starts with the usage of ATP for acetylation of

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coenzyme A by acetyl-CoA synthetase (EC 6.2.1.1) to form acetyl-CoA in a single-step catalyzed

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reaction.81, 82 There are two pathways for acetic acid oxidization. One is via the tricarboxylic acid

215

(TCA) to citrate in the mitochondria. The other is via the glyoxylate cycle to form malate in

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glyoxysomes, which is a variation of the TCA cycle.82, 83 The glyoxylate cycle is similar to TCA

217

cycle. The difference between these two pathways is that isocitrate is converted into glyoxylate

218

and succinate by isocitrate lyase (EC 4.1.3.1) in glyoxylate cycle instead of into α-ketoglutarate in

219

TCA cycle.84 In general, the absorption of acetic acid in microalgae cell must combine acetyl

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groups of acetyl-CoA to carbon skeletons. In the grown cells with acetate, the isocitrate lyase (EC

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4.1.3.1) and malate synthetase (EC 2.3.3.9) are induced.82, 83 When Scenedesmus obliquus grown

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on acetate in the dark for 24h, the activity of isocitrate lyase, was up-regulated about four fold in

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order to increasing the concentration of acetate.85 In C. reinhardtii, the oxidative PPP can also

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provide reducing power in the form of NADPH for cytosol in the presence of acetate.82 The

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assimilation of acetate in pH-auxostats (pH is maintained as a constant) is linked to succinic acid

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production which also inhibits microalgae growth. However, adding propionate to the reactor

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provides oxaloacaetate and promotes growth to the microalgae cells.75

80

In the cytoplasm of

228

Glycerol is another main substrate under heterotrophic culture, which enters the cell by

229

simple diffusion.86 In plant cells, GlpF-like intrinsic proteins, nodulin 26-like intrinsic proteins

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and plasmamembrane intrinsic proteins facilitate the movement of glycerol across the cytoplasmic

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membrane. In the view of Fig. 3, Glycerol was transformed to glyceraldehyde 3-phosphate (GAP)

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and glycerate via glycerol kinase (EC: 2.7.1.30), sn-glycerol-3-phosphate NAD+ oxidoreductase

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(EC: 1.1.1.8) and triose-phosphate (EC: 5.3.1.1). It was shown that glycerate and 9



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glyceraldehyde-3-phosphate are intermediate in the EMP pathway to form pyruvate which then

235

enters the TCA cycle.83 The activity of glucose-6-phosphate isomerase (EC: 5.3.1.9) was inhibited

236

by sn-glycerol-3- phosphate when fructose-6-phosphate serves as the substrate, which limits the

237

reversible glycolytic pathway, and the function of the plastidial and cytosolic pentose phosphate

238

pathways

239

3-phosphoglycerate, which is a crucial intermediate in the Calvin–Benson cycle.75

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LIPID BIOSYNTHESIS IN MICROALGAE

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Recently the genome sequence and transcriptome of several high oil accumulation microalgae

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species have been reported, such as Monoraphidium neglectum, Fistulifera solaris, N.

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oleoabundans, C. protothecoides, Nannochloropsis gaditana and Nannochloropsis oceanica

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IMET1. In C. protothecoides, compared with the autotrophic cells, 30.4% of the genes were

245

expressed differently in heterotrophic cells. In addition, as for proteomes, 205 proteins were

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upregulated while 293 proteins were downregulated in heterotrophic culture.88 Significantly, as for

247

transcriptomes, the expression of genes involved in photosynthesis and CO2 assimilation were

248

almost downregulated. This indicated that the lipid biosythesis mechanism in microalgae has been

249

changed by their alimentation mode.

250 251

are

arrested.87

Also,

Glyceraldehyde-3-phosphate

may

be

reduced

from

In the following parts, we illustrate the biosynthesis of fatty acids and triacylglycerols at the molecular level and the difference between these two cultures.

252

Biosynthesis of Fatty Acids. Under adverse environmental conditions such as nutrient

253

deprivation, microalgae inclined to store energy by forming triacylglycerol (TAG). Under

254

appropriate conditions, the microalgae cells will resume growth and division. The first step of

255

fatty acid biosynthesis is the transformation of acetyl-CoA into malonyl-CoA catalyzed by

256

AcetylCoA Carboxylase (ACCase). This reaction is the rate-limited step of the biosynthesis

257

pathway. However, the gene encoding ACCase, which predominantly locate in the cytosol where

258

lipid biosynthesis does not typically occur in transcription, was repressed under the N-depleted

259

(N-).56, 89 Interestingly, biotin carboxylase (BC), the biotin containing subunit of ACCase, was

260

significantly upregulated under the N-.89 BC presents in the plastid which is the primary cite of

261

lipid biosynthesis in microalgae and catalyzes the ATP-dependent carboxylation of the biotin

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subunit.90 This may be due to allosteric regulation of subunit of BC by α-ketoglutarate.

263

Coincidentally, it was found that gene expression of both the biotin carboxyl carrier subunit and

264

the biotin carboxylase subunit of the heteromeric ACCase were elevated in heterotrophic cells, as

265

suggested by Gao et al.(2014).88 Davis MS et al. showed that coexpression of ACCase (encoded

266

by accA, accB, accC, accD) and thioesterase I (encoded by the tesA gene) resulted in a 6-fold

267

increase in the rate of fatty acid synthesis. 91

268

In the view of Fig. 4, malonyl-CoA is transferred to an acyl-carrier protein (ACP) by

269

malonyl-CoA ACP transacylase (MAT; EC 2.3.1.39), one of the fatty acid synthase (FAS)

270

multi-enzymatic complex subunits, to form malonyl-acyl- carrier protein (molonyl-ACP). To

271

proceed with fatty acid biosynthesis, molony-ACP enters a cycle of condensation, reduction,

272

dehydration, and again reduction reactions to form 16- or 18-carbon fatty acid. As shown in Fig.

273

4, the fatty acid synthase of type II (FASII) was catalyzed by beta-ketocayl-ACP synthase (KAS),

274

beta-ketoacyl-ACP reductase (KAR; EC 1.1.1.100), beta-hydroxyacyl-ACP dehydrase (HAD; EC

275

4.2.1.-), and enoyl-ACP reductase (EAR; EC 1.3.1.9), respectively. In N. oleoabundans, the

276

transcriptional expression of the plastid type II fatty acid synthase system in chloroplast was

277

globally upregulated, while the KAR encoding gene was suppressed under N-limiting condition.89

278

By contrast, in N. oceanic the expression of genes coding for MAT, KAS, HAD, and EAR was

279

downregulated in response to N deprivation, whereas KAR was upregulate. The fact that the

280

absolute transcript levels of FA biosynthesis enzymes in the plastid were 3 to 4 times higher than

281

those responsible for TAG synthesis suggested that the machinery for de novo FA biosynthesis is

282

completely surplus in N. oceanic.56

283

In addition, with the appearance of acyl-ACP there were three different types of KAS to

284

catalyze the condensation of acetyl-CoA in vascular plants, including KAS I, KAS II and KAS III

285

(Fig. 4). Among them, KAS III catalyzed acetyl-CoA and malonyl-ACP in the first condensation

286

reaction; KAS I catalyzed the formation of C16:0-ACP in the subsequent condensation reactions;

287

KAS II catalyzed the formation of C18:0-ACP in the final condensation reactions.92 All these were

288

detected in N. oceanic and N. oleoabundans, as suggested by Tsuyoshi Tanaka et al.93 and Jing Li

289

et al.56 KAS III from Spinacia oleracea was overexpressed in Nicotiana tabacum and resulted in a

290

300-fold increase in activity above the wild type but 20% decrease of lipid.94 When the number of 11



291

carbon chain reached sixteen (C16: 0-ACP) or eighteen (C18: 0-ACP), the progress of these

292

synthesis would halt. In the end of condensation reactions, the gene expression of oleoyl- ACP

293

hydrolase (OAH; EC 3.1.2.14) and acyl-ACP thioesterase A (FatA; EC 3.1.2.-) were

294

overexpressed under N-limiting condition, as reported in N. oceanic and P. tricornutum (Fig. 4).89,

295

95

296

acyl ACPs may repress the fatty acid synthesis, and the overexpression of genes cleaved ACP

297

residues from the long chain fatty acyl ACPs will increase the production of fatty acids. The

298

overexpression of FatA gene from Diploknema butyracea, Ricinus communis, Jatropha curcas in

299

E. coli produced the quantity of free fatty acid (>0.2 g/L), confirming that the amount of free fatty

300

acid accumulated depends on the FatA in mircroalgae cells.96

These thioesterases cleaved off the ACP residues. Meanwhile, the buildup of long chain fatty

301

Conclusively, FA synthesis in the plastid is converted by either an acyl-ACP thioesterase or

302

a plastidic acyltansferases to generate glycerolipid.97 In the plastid envelopes of Arabidopsis, free

303

FA is subsequently converted to acyl-CoA by long-chain acyl-CoA synthetases (LC-FACS; EC

304

6.2.1.3), which plays a crucial role in the intermediary metabolism.98, 99 The resulting acyl-CoA

305

molecules was then transferred to the ER by the aid of cytosolic acyl-CoA binding proteins

306

(ACBP).100 During the nitrogen limitation, the genes encoding for the double bonds in fatty acids

307

were changed in N. oleoabundans. Among them, the gene expression of acyl-ACP desaturase

308

(AAD; EC 1.14.19.2) and delta-15 desturase (EC 1.4.19.-) were strikingly upregulated, while the

309

delta-12 desaturase was downregulated.89 Also, the transcription level of AAD (EC 1.14.19.2) was

310

significantly upregulated following N deprivation in Nannochloropsis oceanic.56 These enlighten

311

us that we can overexpress the AAD gene to increase the lipid and TAG content. Therefore, the

312

cellular content of C18: 1 drastically increased.101 The proportion of unsaturated of fatty acids

313

inclines to increase in the N-limiting condition. This may indicate its significantly physiological

314

role in the stress response, which releases the overreduced photosynthetic electron transport to

315

prevent the production of excess reactive oxygen species in photosynthesis process.

316

Moreover, because of the utilization of NADPH to power fatty acid biosynthesis, genes

317

encoding for the PPP were remarkablely upregulated under the nitrogen starvation condition, as

318

suggested by Rismani-Yazdi et al. (2012).89

319

Biosynthesis of Triacylglycerols. TAG is the major storage lipid in oleaginous 12


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Page 13 of 31


320

microalgae, and glycerolipid pathway focused on TAG formation via the Kennedy pathway. There

321

are two locations for glycerolipid biosynthesis, either in the ER or in the chloroplast. Biosynthesis

322

of TAG in both locations began with two consecutive acyl transfers from acyl-CoA to positions 1

323

and 2 of glycerol-3-phosphate to generate lysophosphatidic acid and phosphatidic acid (PA).

324

These reactions were catalyzed by enzymes glycerol-3-phosphate acyltransferase (GPAT, EC

325

2.3.1.15) and lysophosphatidic acid acyltransferases (LPAAT, EC 2.3.1.51) in ER and by ATS1

326

and ATS2 in the chloroplast membranes (Fig. 4).86

327

As shown in Fig. 4, PA is either transformed to phosphatidylglycerol (PG) by a chloroplast

328

phosphatidylglycerol phosphate synthase (PGP; EC 2.7.8.5) or dephosphorylated to generate 1,

329

2-diacylglycerol (DAG) by phosphatidate phosphatase (PP; EC 3.1.3.4). Then the DAG is

330

transformed to monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and

331

sulfoquinovosyldiacylglycerol (SQDG), catalyzed by monogalactosyldiacylglycerol synthase

332

(MGD; EC 2.4.1.46), digalactosyldiacylglycerol synthase (DGD; EC 2.4.1.241) and

333

sulfoquinovosyltransferase (SQD2; EC 2.4.1.-) enzymes, respectively.86, 102 On the other hand, in

334

the ER, PA can be transformed to PG, phosphoinositides (PI) and DAG. Recently, Tsuyoshi et al.

335

(2015) reported that DAG kinase (DGK) catalyzed reverse phosphorylation of DAG into PA. The

336

DAG is transformed to triacylglycerol (TAG) by the DAG acyltransferase (DGAT; EC 2.3.1.20),

337

following the canonical Kennedy pathway.93 It has been suggested that the overexpression of

338

Arabidopsis DGAT resulted in a 200~600-fold increase in activity above the wild type and 3~9

339

fold increase of TAG.100 Besides, It was shown that phospholipid diacylglycerol acyltransferase

340

(PDAT) catalyzed the synthesis of TAG from phosphatidylcholine (PC).103 Coincidently, the lipid

341

content decrease 40% when the PDAT gene (LRO1) was knocked out in yeast.101

342

Under the nitrogen starvation condition, in the N. oleoabundans, the expression of genes

343

encoding GPAT and AGPAT was up-regulated, whereas the expression of genes encoding PP and

344

DGAT remained relatively unchanged, as reported by Rismani-Yazdi et al.89 So the overexpression

345

of DGAT in algal may also increase the content of lipid.

346

Difference Mechanism between Autotrophic and Heterotrophic Condition.

347

Compared with autotrophic cells in C. protothecoides, the genes coding for glycolysis/

348

gluconeogenesis, the TCA cycle, pyruvate metabolism, oxidative phosphorylation, PPP and fatty 13



349

acid biosynthesis were strongly upregulated. On the other hand, photosynthesis, porphyrin and

350

chlorophyll metabolism, and carotenoid biosynthesis were dramatically downregulated.88 Because

351

glycolysis/ gluconeogenesis, the TCA cycle, pyruvate metabolism were able to provide ATP and

352

acetyl-CoA. Those are important for fatty acid synthesis in heterotrophic algae. Particularly, PPP

353

can provide NADPH to power fatty acid biosynthesis.88, 89

354

In addition, under heterotrophic and nitrogen starvation conditions, accumulation of lipids is

355

mainly produced from chloroplast membrane. And the chloroplast nitrogen was relocated by

356

Rubisco.98 This proposal was supported by the evidence that development of chloroplast was

357

associated with nitrogen. For example, under dark and nutrient shortage conditions, chloroplast

358

breakdown for the internal supply of nitrogen leads to cell survival and growth if an external

359

carbon source is not supplied. 99

360

Particularly, under heterotrophic addition, the protein level of ALDO were boosted, which

361

catalyzed the conversion of fructose-1,6-bisphosphate to dihydroxyacetone phosphate (DHAP)

362

and GAP. Secondly, the protein level of glycerol-3-phosphate dehydrogenase was upregulated too,

363

which produced glycerol-3-phosphate (G3P) from DHAP.104 To meet the cells consequent

364

requirement for DHAP, the triose phosphate isomerase (TPI; EC 5.3.1.1) was inactive in the

365

heterotrophic microalgae cells, which catalyzed the isomerization between DHAP and GAP,

366

directly linking TAG and glycolysis synthesis.1, 3 It was reported that deficiency of TPI elevated

367

the fatty acid or oil content in root cells of plants1 to ensure the supply of G3P in the heterotrophic

368

cells. Significantly, both protein and RNA levels of the major lipid droplet protein (MLDP) was

369

upregulated, which is attributed to the accumulation of lipid droplets.88

370

In conclusion, it is tempting to cultivated microglages in heterotrophic condition for the

371

production of economically useful metabolites. Heterotrophic cultivation is simple, cheap, and

372

usually used by fermentation industries for other production applications. Several obligate

373

photoautotrophs are transformed to heterotrophy via the intervention of glucose transporters,

374

suggesting that the absence of these proteins cause the inability of other green algae to use glucose.

375

Examples of single-genetic transformation of glucose transporter indicated the feasibility to

376

transform microalgae from photoautotrophic growth into heterotrophic growth. In comparison

377

with autotrophic growth, PPP is mainly metabolized in the darkness condition, while EMP is the 14


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Page 15 of 31


378

main glycolytic pathway under light. Under heterotrophic conditions, the expression of genes

379

involved in photosynthesis and CO2 are almost completely degraded. TCA cycle, pyruvate

380

metabolism, oxidative phosphorylation, PPP and fatty acid biosynthesis are strongly upregulated,

381

while the enzymes involved in fatty acid degradation are downregulated. Several enzymes play a

382

crucial role, such as glucose transporter protein, fructose-1, 6-bisphosphate aldolase,

383

glycerol-3-phosphate dehydrogenase and other positive regulatory enzymes, and negative

384

regulation enzyme such as triose phosphate isomerase.

385

ABBREVIATIONS USED

386

AAT, ATP/ADP translocator; Accase, acetyl-CoA carboxylase; ACP, acyl-carrier protein; ACS1,

387

acetyl-CoA synthase; ALDO, fructose-1, 6-bisphosphate aldolase; BC, biotin carboxylase; CA,

388

carbonic

389

acyltransferase; DGDG, digalactosyldiacylglycerol (DGDG); DGK, 1, 2-diacylglycerol kinase;

390

DGD,

391

Embden–Meyerhof–Parnas

392

fructose-1,6-bisphosphatase; GAP1, glyceraldehyde-3-phosphate dehydrogenase-NAD; GAP2,

393

glyceraldehyde-3-phosphate dehydrogenase-NADP dependent;

394

glyceraldehyde-3-phosphate; GLPK, glycerol kinase; GND, 6-phosphogluconate dehydrogenase;

395

GPAT,

396

oxidoreductase; G3P, glycerol-3-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; HAD,

397

beta-hydroxyacyl-ACP dehydrase; HUP, hexose/H+ symport systems; ICL, isocitrate lyase; LACS,

398

long-chain acyl-CoA synthetase; LHCs, light harvesting complexes; LPAAT, lysophosphatidate

399

acyltransferase; KAR, 3-oxoacyl-ACP reductase; KAS, 3-oxoacyl-ACP synthase II; MAS1,

400

malate

401

monocarboxylic/H+

402

monogalactosyldiacylglycerol; MLDP, major lipid droplet protein; NPQ, non-photochemical

403

quenching;

404

diacylglycerol acyltransferase; PGL, 6-phosphogluconolactonase; PP, phosphatidate phosphatase;

405

PPP,

406

ribose-5-phosphate

anhydrase;

Cytb6f,

cytochrome

digalactosyldiacylglycerol

synthase;

PA,

pentose

synthase;

pathway;

glycerol-3-phosphate

FAT,

acyl-carrier

transporter;

phosphatidic

MGD,

acid;

pathway;

isomerase;

complex;

DHAP, fatty

O-acyltransferase;

MCAT,

phosphate

b6-f

1,

dihydroxyacetone acyl-ACP

GPD1,

protein

DGAT,

(ACP)

RPE, RuBP,

phosphate;

thioesterase

GLK,

A;

glucokinase;

sn-glycerol-3-phosphate

S-malonyltransferase;

monogalactosyldiacylglycerol

PFK2

2-diacylglycerol

phosphofructokinase;

ribulose-5-phosphate

synthase;

PDAT,

15


FBP,

GIP2,

NAD+

MCT, MGDG,

phospholipid:

3-epimerase;

Ribulose-1,5-bisphosphate;

EMP,

RPIA, Rubisco,


407

Ribulose-1,5-bisphosphate carboxylase/ oxygenase; SQDG, sulfoquinovosyldiacylglycerol; SQD2,

408

sulfoquinovosyltransferase; TAL, transaldolase; TAG, tricaylglycerols; TKT, transketolase; TPIC,

409

Triose-phosphate isomerase;

410

Reference

411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448

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685 686

Table 1. Summary of microalgae strains studied under heterotrophic and autotrophic conditions

Microalgae species

Carbon source

Lipid content(% dry weight biomass)

Biomass Lipid Main fatty acids composition Reference productivity productivity (g/L/day) (mg/L/day)

glucose

50.3

7.31

3678.5

Jerusalem artichoke hydrolysate Cassava starch hydrolysate

44±3.4

4.1±0.2

1700±200

26.5

1.58

419

Molasses hydrolysate

40.6

3.58

1454

Sweet sorghum juice

52.5

1.2

586.8

Heterotrophic Chlorella protothecoides

Chlorella Vulgaris Acetate and crude 36 glycerol Neochloris glucose 52 oleoabundans

C17H34O2 (11.34%) C19H34O2 (19.48%) C19H36O2 (53.75%) C17H34O2 (14.28%) C19H34O2 (9.72%) C19H36O2 (71.57%) 16:0(21.53±0.042%) 16:1(D9) (11.02±0.67%) 18:1(D9) (24.65±1.28%) 18:2(D9,12) (31.37±1.35%) C17H34O2 (7.63%) C19H34O2 (14.17%) C19H36O2 (67.95%) 9-Octadecenoic acid methyl ester (66.80%) 9,12-Octadecadienoic acid methyl ester (15.12%) Hexadecenoic acid methyl ester (12.66%)

528.5

19, 20

21

22

23

24

0.076-0.082 25-33 0.3-0.47

18

C16H30O2 C18H34O2 C18H32O2

25

C16H32O2(20.6-26.0%) C16H30O2(6.60-10.80%) C18H34O2(11.40-20.8%) C18H32O2(27.2-33.4%) C16 FFA(20.24±1.4%) C18 FFA(46.37±2.5%)

26, 27

Autotrophic Chlorella sp.

CO2

32.6-66.1

0.077-0.338 51-124

Chlorella vulgaris CO2

20-42

0.21-0.346

44-147

Dunaliella sp. Haematococcus pluvialis

12-30.12 15.61-34.85

1.3-3.0

360-390

7-40

0.31-0.63

CO2 CO2

N. oleoabundans CO2

38-133

28, 29

30

C16H32O2(18.87-22.49%) C18H34O2(18.35-19.36%) C18H32O2(26.9-30.47%) C18H30O2(12.01-18.69%) C16H32O2(22.3-31.1%)

23


31

10, 32


C18H34O2(12.9-43.6%) C18H32O2(23.3-27.2%) C18H30O2(4.9-23.6%) 10.67-38.78 16:1w7(34-42%) 16:0(15-22%) 18:0(14-18%) 53-350

N. oleoabundans CO2 UTEX #1185

19-56

0.03-0.15

Pseudochlorococc CO2 um sp. Scenedesmus CO2 obliquus

24.6-52.1

0.234-0.76

21-58

0.070-0.094 19.0-43.3

Tetraselmis sp.

8.2-33.0

0.158-0.214 18.6-22.7

Chaetoceros CO2 calcitrans CS 178 Chaetoceros CO2 gracilis

39.8

0.04

17.6

15.5-60.28

3.4-3.7

530-2210

Chaetoceros muelleri

CO2

11.67-25.25

0.7-2.7

150-180

Nitzschia cf.pusilla YSR02 Phaeodactylum tricornutum

CO2

48±3.1

0.065

31.4

CO2

18.7

0.24

44.8

Isochrysis sp.

CO2

6.5-21.25

0.7-2.7

150-180

Isochrysis zhangjiangensis

CO2

29.8-40.9

0.667-3.1

66.2-140.9

Nannochloropsis CO2 oculata

22.75-23.0

2.4-3.4

550-790

Nannochloropsis CO2 sp.

21.3-37.8

0.021-0.064 4.59-20.0

CO2

18:1n9c(18-25%) 18:2n6c(14-18%) 18:3n3(20-33%) 16:0(25.3-38.6%) 16:1n-7(21.4-40.8%) 20:5n-3(EPA)(7.6-30.8%)

Page 24 of 31

33

34

35

36

37

14:0(13.01-20.32%) 16:0(24.03-40.35%) 18:1(D9)(17.42-31.05%) 16:0(17.04-30.55%) 16:1n-7(24.94-31.73%) 20:5n-3 (12.5-20.62%) 16:0 (31±1.3%) 16:1(57±2.4%) 16:0(16.1%) 16:1n-7(19.2%) 20:5n-3(EPA)(23.7%) 14:0(12.94-23.92%) 16:0(8.33-14.38%) 18:1n-9(10.07-12.32%) 18:4n-3(9.05-19.86%) 22:6n-3(11.23-21.58%) 14:0(21.0±0.8%) 16:0(26.1±1.5%) 18:1(28.3±1.2%) 22:6(DHA)(10.2±0.2%) 16:0(14.0-24.2%) 16:1n-7(24.8-25.8%) 20:5n-3(27.8-30.8%) 16:0(17.8-19.8%) 16:1n-7(11.0-14.2%) 20:5n-3(EPA)(29.4-32.1%)

687 688 689 24


30, 38

30, 39

35

37, 40

30, 39

41

30, 42

43

Page 25 of 31


690

691 692 693 694

Fig 1. The process of producing biocrude via microalgae in economic industry.

25



695 696 697 698 699 700

Fig 2. Photosynthetic electron and proton transfer mechanism of microalgaes. Enzymes and molecules include: PS I, Photosystem I; PS II, Photosystem II; Fd, Ferredoxin; PQ, Plastoquinone; PC, Plastocyanin.

26


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701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717

Fig 3. Overview of the carbon absorption metabolism in heterotrophic microalgae. The metabolisms contain glyoxylate cycle, glycolysis and pentose phosphate pathway. The glyoxylate cycle is the metabolic pathway that converts acetyl-CoA into succinate. Glycolysis centers on the conversion of glucose to pyruvate. The pentose phosphate pathway is a metabolic pathway parallel to glycolysis, generating NADPH and pentoses. ACS1, acetyl-CoA synthase; FBP, Fructose-1,6-bisphosphatase; GAP1, Glyceraldehyde-3-phosphate dehydrogenase-NAD; GAP2, Glyceraldehyde-3-phosphate dehydrogenase-NADP dependent; GLK, Glucokinase; GLPK, Glycerol kinase; GIP2, Glyceraldehyde-3-phosphate; GND, 6-phosphogluconate dehydrogenase; GPD1, sn-glycerol-3-phosphate NAD+ oxidoreductase; G6PDH, glucose-6-phosphate dehydrogenase; HUP, Hexose/H+ symport systems; ICL, isocitrate lyase; MAS1, malate synthase; MCT monocarboxylic/H+ transporter; PFK2 phosphofructokinase; PGL, 6-phosphogluconolactonase; RPE, ribulose-5-phosphate 3-epimerase; RPIA, ribose-5-phosphate isomerase; TAL, transaldolase; TKT, transketolase; TPIC, Triose-phosphate isomerase;

27



718 719 720 721 722 723 724 725 726 727

Fig 4. Fatty acid and TAG biosynthesis pathway in microalgae under autotrophic and heterotrophic growth conditions. The metabolisms contain Calvin Benson cycle, triacylglycerol biosynthesis and fatty acid biosynthesis. Calvin Benson cycle are chemical reactions that convert carbon dioxide and other compounds into glucose. Fatty acid biosynthesis is the creation of fatty acids from acetyl-CoA and NADPH. Triacylglycerol biosynthesis is the metabolic pathway that converts acetyl-CoA and G3P into TAG. Accase, acetyl-CoA carboxylase; CA, carbonic anhydrase; DGAT, 1, 2-diacylglycerol acyltransferase; DGD, digalactosyldiacylglycerol synthase; DGK, 1, 2-diacylglycerol kinase; FAT, fatty acyl-ACP thioesterase A; GPAT, glycerol-3-phosphate O-acyltransferase; HAD, beta-hydroxyacyl-ACP dehydrase; KAR, 3-oxoacyl-ACP reductase;

28


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Page 29 of 31


728 729 730 731 732 733 734

KAS, 3-oxoacyl-ACP synthase II; LPAAT, lysophosphatidate acyltransferase; LACS, long-chain acyl-CoA synthetase; MAT, malonyl-CoA ACP transacylase; MCAT, acyl-carrier protein (ACP) S-malonyltransferase; MGD, Monogalactosyldiacylglycerol synthase; PP, phosphatidate phosphatase; PDAT, phospholipid: diacylglycerol acyltransferase; Rubisco, Ribulose-1,5-bisphosphate carboxylase/ oxygenase; SQD2, sulfoquinovosyltransferase; TAG, Triacylglycerol.

29



735

TOC graphic

736 737

30


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Page 31 of 31


738

Figure captions

739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774

Fig 1. The process of producing biocrude via microalgae in economic industry. Fig 2. Photosynthetic electron and proton transfer mechanism of microalgaes. Enzymes and molecules include: PS I, Photosystem I; PS II, Photosystem II; Fd, Ferredoxin; PQ, Plastoquinone; PC, Plastocyanin. Fig 3. Overview of the carbon absorption metabolism in heterotrophic microalgae. The metabolisms contain glyoxylate cycle, glycolysis and pentose phosphate pathway. The glyoxylate cycle is the metabolic pathway that converts acetyl-CoA into succinate. Glycolysis centers on the conversion of glucose to pyruvate. The pentose phosphate pathway is a metabolic pathway parallel to glycolysis, generating NADPH and pentoses. ACS1, acetyl-CoA synthase; FBP, Fructose-1,6-bisphosphatase; GAP1, Glyceraldehyde-3-phosphate dehydrogenase-NAD; GAP2, Glyceraldehyde-3-phosphate dehydrogenase-NADP dependent; GLK, Glucokinase; GLPK, Glycerol kinase; GIP2, Glyceraldehyde-3-phosphate; GND, 6-phosphogluconate dehydrogenase; GPD1, sn-glycerol-3-phosphate NAD+ oxidoreductase; G6PDH, glucose-6-phosphate dehydrogenase; HUP, Hexose/H+ symport systems; ICL, isocitrate lyase; MAS1, malate synthase; MCT monocarboxylic/H+ transporter; PFK2 phosphofructokinase; PGL, 6-phosphogluconolactonase; RPE, ribulose-5-phosphate 3-epimerase; RPIA, ribose-5-phosphate isomerase; TAL, transaldolase; TKT, transketolase; TPIC, Triose-phosphate isomerase; Fig 4. Fatty acid and TAG biosynthesis pathway in microalgae under autotrophic and heterotrophic growth conditions. The metabolisms contain Calvin Benson cycle, triacylglycerol biosynthesis and fatty acid biosynthesis. Calvin Benson cycle are chemical reactions that convert carbon dioxide and other compounds into glucose. Fatty acid biosynthesis is the creation of fatty acids from acetyl-CoA and NADPH. Triacylglycerol biosynthesis is the metabolic pathway that converts acetyl-CoA and G3P into TAG. Accase, acetyl-CoA carboxylase; CA, carbonic anhydrase; DGAT, 1, 2-diacylglycerol acyltransferase; DGD, digalactosyldiacylglycerol synthase; DGK, 1, 2-diacylglycerol kinase; FAT, fatty acyl-ACP thioesterase A; GPAT, glycerol-3-phosphate O-acyltransferase; HAD, beta-hydroxyacyl-ACP dehydrase; KAR, 3-oxoacyl-ACP reductase; KAS, 3-oxoacyl-ACP synthase II; LPAAT, lysophosphatidate acyltransferase; LACS, long-chain acyl-CoA synthetase; MAT, malonyl-CoA ACP transacylase; MCAT, acyl-carrier protein (ACP) S-malonyltransferase; MGD, Monogalactosyldiacylglycerol synthase; PP, phosphatidate phosphatase; PDAT, phospholipid: diacylglycerol acyltransferase; Rubisco, Ribulose-1,5-bisphosphate carboxylase/ oxygenase; SQD2, sulfoquinovosyltransferase; TAG, Triacylglycerol.

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Lipid Accumulation Mechanisms in Autotrophic and Heterotrophic

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