Biotechnology Advances 48 (2021) 107725

Contents lists available at ScienceDirect

Biotechnology Advances

journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Biotechnological production of lipid and terpenoid from thraustochytrids

Fei Du a,1, Yu-Zhou Wang a,1, Ying-Shuang Xu a, Tian-Qiong Shi a, Wen-Zheng Liu a, Xiao-Man Sun a,*, He
Huang a,b
a
School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, 2 Xuelin Road, Qixia District, Nanjing, People’s Republic of China b College
of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing, People’s Republic of China

ARTICLEINFO ABSTRACT
Keywords: As fungus-like protists, thraustochytrids have been increasingly studied for their faster growth rates and high lipid content. In
Thraustochytrids the 1990s, thraustochytrids were used as docosahexaenoic acid (DHA) producers for the first time. Thraustochytrids genera,
Lipid such as Thraustochytrium, Schizochytrium, and Aurantiochytrium have been developed and patented as industrial strains for
Metabolic engineering DHA production. The high DHA yield is attributed to its unique and efficient polyketide-like synthase (PKS) pathway.
Downstream processing Moreover, thraustochytrids possess a completed mevalonate (MVA) pathway, so it can be used as host for terpenoid
production. In order to improve strain performance, the metabolic engineering strategies have been applied to promote or
disrupt intracellular metabolic pathways, such as genetic engineering and addition of chemical activators. However, it is
difficult to realize industrialization only by improving strain performance. Various operation strategies were developed to
enlarge the production quantities from the laboratory-scale, including two-stage cultivation strategies, scale-up technologies
and bioreactor design. Moreover, an economical and effective downstream process is also an important consideration for the
industrial application of thraustochytrids. Downstream costs accounts for 20–60% of the overall process costs, which
represents an attractive target for increasing the cost-competitiveness of thraustochytrids, including how to improve the
efficiency of lipid extraction and the further application of biomass residues. This review aims to overview the whole lipid
biotechnology of thraustochytrids to provide the background information for researchers.

1. Introduction Li et al., 2015). The main fatty acids of most thraustochytrids

Thraustochytrids are marine unicellular heterotrophic
microorganisms. Previously, thraustochytrids were classified as
microalgae, but taxonomists rarely used this classification
because they have no plastids and are incapable of
photosynthesis (Leyland et al., 2017). Nine genera of
thraustochytrids have been recognized to date, including
Thraustochytrium, Schizochytrium (Goldstein and Belsky, 1964),
Aurantiochytrium (Yokoyama and Honda, 2007), Japonochytrium,
Ulkenia, Monorhizochytrium (Doi and Honda, 2017),
include C14:0, C16:0, docosapentaenoic acid (DPA) and DHA, and
the total content of C16:0 and DHA occupy more than 65% of
total fatty acids (Jiang et al., 2004; Meesapyodsuk and Qiu, 2016).
Therefore, the lipid profiles was introduced into the classification
of thraustochytrids as biochemical characteristics. Based on this,
thraustochytrids can be separated into 5 major groups, including
DHA/docosapentaenoic acid (DPA), DHA/DPA/eicosapentaenoic
acid (EPA), DHA/EPA, DHA/DPA/ EPA/arachidonic acid (ARA), and
DHA/DPA/EPA/ARA/docosate-
traenoic acid. And this new classification is consistent with the

* Corresponding author at: School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, 2 Xuelin Road, Qixia District, Nanjing, People’s Republic of China.
E-mail address: xiaomansun@njnu.edu.cn (X.-M. Sun).
1
These two authors contributed equally to this work. https://doi.org/10.1016/j.biotechadv.2021.107725

Received 3 November 2020; Received in revised form 15 January 2021; Accepted 25 February 2021 Available online 13
March 2021
0734-9750/© 2021 Elsevier Inc. All rights reserved.

Sicyoidochytrium, Parietichytrium, and Botryochytrium
(Yokoyama and Honda, 2007). Thraustochytrids attracted
biotechnological interest because the lipid content of some
thraustochytrid strains can reach more than 55% of dry cell
weight (DCW) under optimized conditions (Jakobsen et al., 2008;
result of 18S rRNA gene classification, which provide great
models for studying the mechanism PUFA biosynthesis by the
genetic approach (Huang et al., 2003).
DHA has attracted great attention as essential for the
development of eyes and neuron of infants (Innis, 2008), and the
F. Du et al.
infant formulae is the fastest-growing segment for DHA
consumption, with a 14% of growth
rate from 2015 to 2025 (Ana et al., 2016). DHA is traditionally
obtained from fish oil, but mass fishing has become
unsustainable and unable to meet the demand for DHA in recent
years, suggesting the need to find new sustainable resources.
Currently, the largest commercial production of microbial DHA is
produced by Schizochytrium species, due to it has been approved
by FDA (Ratledge, 2012). DSM is the market leader for microbial
DHA production from thraustochytrids, including DHAGold and
Life’sDHA. In 2018, DSM and Evonik industries established a joint
venture to produce DHA and EPA from Schizochytrium, aimed to
meet about 15% of the global farmed salmon demand for
omega-3 algal oil (https://cen.acs.org/articles/95/i11/DSM-
Evonik-form-omega-3-join t-venture.html). Moreover, many
thraustochytrids species with higher saturated fatty acids (SFA)-
producing capacity were isolated. The 54% and 44% content of
SFAs was found in thraustochytrids that screened from the
environment in Australia and UK, respectively (Lee Chang et al.,
2013; Marchan et al., 2017). Recent, thraustochytrid strains with
high SFA production were isolated from the coastal waters of
Shenzhen, China, with SFA content of 62% (Wang et al., 2018b).
The quality of biodiesel from thraustochytrids was reconginzed
by international standards such as ASTM and EN14214 (Marchan
et al., 2017).
In addition to fatty acids, many thraustochytrids species with
high content of squalene or carotenoids have been isolated.
Among the few squalene-producing thraustochytrid strains
isolated to date, Aurantiochytrium can produce especially high
titers, reaching more than 30% of DCW (Li et al., 2009). Although
no impressive carotenoid yields was obtained in thraustochytrids,
experimental evidence shows that they have the potential to
produce a greater variety of carotenoids including β-carotene,
astaxanthin and canthaxanthin (Aki et al., 2003). The
thraustochytrid strain AS4-A1 has the highest carotenoid content
reported to date, reaching up to 40 mg/g astaxanthin, which
corresponds to 4% of DCW (Benita et al., 2010). Notably, this
thraustochytrid has a far higher carotenoid yield than the 6–7
mg/g obtained by the commercial production strain Phaffia
rhodozyma and Haematococcus pluvialis (Schmidt et al., 2011).
These production levels are only based on the optimization of
Fig. 1. The fermentation process and function of lipid in thraustochytrids.
Biotechnology Advances 48 (2021) 107725
fermentation conditions, so thraustochytrids are highly
interesting chassis cells for further systematic metabolic
engineering.
There are many other oleaginous microorganisms with an
inherent capacity to produce excess amounts of fatty acids and
store them inside the cell in the form of triacylglycerols (TAGs).
By engineering the intracellular redox metabolism, the highest
reported lipid titer of Yarrowia lipolytica reached to 98.9 g/L
(Qiao et al., 2017). Moreover, oleaginous microalgae have been
frequently employed for photosynthetic lipid production from
CO2, but a major drawback of this approach is the low
photosynthetic grow rate of the commonly used organisms such
as Chlorella vulgaris and Synechocystis sp. (Chu et al., 2020, Liang
and Lindblad, 2017). By contrast, thraustochytrids can produce
high biomass yields of 165 g/L, along with lipid titers of 113 g/L
without any genetic modification (Guo et al., 2017). In this
review, technologies applied in thraustochytrids to produce lipid
and terpenoid are summarized and discussed, including low-cost
cultivation, molecular approaches, engineering operation
strategies and downstream processing technologies (Fig. 1).
2. The biosynthetic pathways of lipid and terpenoid
Thraustochytrids are a promising future source for lipid
compound production.
In order to obtain good strains, it is indispensable to disturb
or rewrite metabolic pathways. Fulfilling these targets needs a
thorough understanding of lipid and terpenoid biosynthesis in
thraustochytrids. Microbial lipid synthesis routes can be divided
into fatty acid and TAG biosynthesis (Fig. 3). The PUFA
biosynthesis pathway of thraustochytrids is different to that of
most oleaginous microorganism, which is the most critical reason
for it to efficiently produce DHA. Moreover, the terpenoid
biosynthetic mechanisms in thraustochytrids also has been
characterized. The following section provide an overview of the
key biosynthetic pathways and metabolic engineering strategies.
2.1. Biosynthesis of SFAs
In general, shorter fatty acids (< C18) with high content of SFA
and monounsaturated fatty acid (MUFA) are preferable for
biodiesel production because they increase the thermal and
F. Du et al.
oxidative stability of the resulting biodiesel (Gupta et al., 2015).
Thraustochytrids can accumulate high amounts of SFA and MUFA
up to 76% of total fatty acids. It has proved that biodiesel derived
from thraustochytrids is comparable to fossil diesel in terms of
energy usage and greenhouse gas emissions (Chang et al.,
2015b). SFAs are produced by the type I fatty acid synthetase
(FAS) pathway in thraustochytrids. In the first step, acetyl-CoA is
converted into malonyl-CoA by acetyl-CoA carboxylase (ACCase)
(Fig. 3). A recent study found that the activity of ACCase was
inhibited in Schizochytrium, but overexpression of an elongase
enzyme for converting C16 into C18 fatty acids relieved the
inhibition effect, resulting in elevated conversion of C15:0 into
C17:0 (Wang et al., 2019). Subsequently, malonyl-CoA is ligated
with ACP by MAT, after which a sequence encompassing
condensation by KS, reduction by KR, dehydration by DH, and
repeated reduction by ER is performed by a multi- subunit
enzyme to yield C16 or C18 products (Parsons and Rock, 2013)
(Fig. 4). Importantly, fatty acid chain extension is controlled by
acyltransferase and acyl-ACP thioesterase (TE). Notably, TE
hydrolyzes acyl-ACP to release fatty acids, which is significant for
the production of desired fatty acids. For example, when C12 and
C14-specific TEs were overexpressed in Dunaliella tertiolecta, the
content of C12:0 and C14:0 was increased 7- and 4-fold,
respectively (Lin and Lee, 2017). There are two kinds of TEs,
which have different preferences for fatty acids chain lengths
(Moreno-Perez et al., 2011´ ), suggesting that they can be used to
improve lipid profile. 2.2. Biosynthesis of PUFAs
Two biosynthesis pathways of PUFAs are known to exist in
nature, the elongase-desaturase pathway and the polyketide-like
synthase (PKS) pathway. Starting from SFAs, various desaturases
and elongases are required to produce a targeted PUFA,
proceeding via different intermediate PUFAs (Fig. 4). By contrast,
the PKS pathway catalyzes the de novo synthesis of PUFAs, in
which several cycles of reduction, dehydration, reduction and
condensation add two carbons in each cycle (Fig. 5A). There is a
controversy about the biosynthesis pathway of PUFAs in
thraustochytrids. The Δ4 desaturase and Δ5 elongase were
detected in Thraustochytrium and Schizochytrium. Further fatty
acid analysis of two species showed that they had related
precursor fatty acids for DHA synthesis in the elongase-
desaturase pathway, which suggested that Thraustochytrium and
Schizochytrium could synthesize DHA via the elongase-desaturase
pathway (Nagano et al., 2011). When the Δ12 desaturase gene of
Thraustochytrium aureum was disrupted, the conversion of C18:1
into C18:2 decreased. However, the proportion of DHA increased,
which suggested that DHA biosynthesis is independent of the
elongase-desaturase pathway (Sakaguchi et al., 2012). Hoang et
al. (2016) supported that PKS and the elongase-desaturase
pathway co-exist in Schizochytrium mangrovei PQ6 for PUFA
biosynthesis. Altogether, different species of thraustochytrids
possessed different pathway of PUFAs biosynthesis: some via the
elongase-desaturase pathway, some via PKS pathway, and some
possess both pathways.
In fact, biosynthesis of PUFAs via PKS is more efficient
because it requires less NADPH (Figs. 4 and 5A). Using the
elongase-desaturase pathway requires 26 NADPH to synthesize
DHA from acetyl-CoA, while the PKS pathway only needs 14
NADPH molecules. Moreover, the PKS pathway produces fewer
intermediate fatty acids due to fewer catalytic steps compared to
the elongase-desaturase pathway. The PKS complex is composed
of different subunits, and the number, location, and order of
domains determine the finally produced fatty acids. For example,
the ARA-producing PKS possesses four subunits (Hayashi et al.,
2019), while the EPA-producing PKS has three subunits and a
number of ACPs varying from four to three. Exchange of domains
Biotechnology Advances 48 (2021) 107725
in PKS enzymes successfully changed the chain lengths of the
resulting fatty acids (Fisch et al., 2011; Liu et al., 2014a). There
are three PKS subunits in thraustochytrids, encoded by ORFA,
ORFB, and ORFC. The ORFA protein contains one KS, one
acyltransferase (AT), a variable number of ACP, and one KR
domain. The number of ACP domains present in different
thraustochytrids is different. For example, Aurantiochytrium sp.,
Thraustochytrium sp., Schizochytrium sp. and Ulkenia sp. possess
13, 8, 9, and 10 ACPs, respectively (Fig. 5B). It has been reported
that the number of ACPs is associated with the overall
productivity (Lippmeier et al., 2009). When the number of ACPs
in Schizochytrium sp. was increased from 9 to 11, the DHA
productivity was enhanced by 80% (Hayashi et al., 2016). The
ORFB protein contains one KS, one chain length factor (CLF), one
AT, and one ER domain. The ORFC protein contains two DH and
one ER domain. Xie et al. (2017) advocated that the KS domain in
ORFB is more related to PUFA biosynthesis, but the ORFA is more
effective to improve SFA biosynthesis in Thraustochytrium sp.
Recently, the control mechanism regulating carbon chain length
in EPA and DHA biosynthesis was analyzed, and the results
showed that the KS domain of ORFA controls the condensation
from C18 to C20, while that of ORFC catalyze the condensation
from C20 to C22. Based on this discovery, microalgal DHA
synthase was successfully converted to an EPA synthase (Dairi et
al., 2019). Subsequently, the mechanism of double-bond
formation in ARA and EPA biosynthesis was also clarified, in
which the DH domains of ORFB and ORFC play key roles in the
introduction of cis double bonds depending on the carbon chain
length (Hayashi et al., 2019). When the DH domain of ORFC was
disrupted, the PUFA yield of Schizochytrium sp. decreased by
more than half, highlighting its importance (Li et al., 2018a).
Taken together, research on the detailed biosynthetic pathways
and mechanisms controlling the specific PUFA profiles in
thraustochytrids is still in the preliminary stage, and further
exploration is needed in the future.
2.3. Biosynthesis of TAGs
The oil of thraustochytrids contains neutral lipids (NLs),
phospholipids (PLs), glycolipids (GLs) and unsaponifiable matter
(UM) (Ren et al., 2014), with notable differences of the relative
proportions in different species. For example, NLs and PLs
respectively account for 95% and 5% in Schizochytrium
mangrovei (Fan et al., 2007), compared to 75% and 20% in
Aurantiochytrium sp. (Liu et al., 2014b). The lipid fractions can be
regulated by changing the cultivation conditions (Papanikolaou et
al., 2010). Triacylglycerols (TAGs) are the major neutral storage
lipids in thraustochytrids, while PLs are essential components of
the cell membrane. TAGs are synthesized via the Kennedy
pathway (Fig. 3). As the first step, glycerol-3-phosphate (G3P) is
converted into lysophosphatidate (LPA) by the rate-limiting
enzyme glycerol-sn-3-phosphate acyl-transferase (GPAT).
Strategies to increase lipid accumulation by enhancing GPAT
expression have been successfully applied in a variety of
microalgal species. Subsequently, lysophosphatidate acyl-
transferase (LPAAT) transfers an acyl moiety from CoA onto the
sn-2 position of LPA, resulting the phosphatidate (PA). Notably,
LAPPT enzymes from different sources possess different substrate
preferences, and an appropriate choice is a key for engineering
strains with desired fatty acids (Balamurugan et al., 2017; Kim
and Huang, 2004). Finally, an acyl group is transferred to
diacylglycerol from CoA under the catalysis of diacylglycerol
acyltransferase (DGAT). There are type-I and type-II DGAT,
whereby the latter more effective for TAG biosynthesis.
Modification of DGAT genes has already been attempted and is
considered to be the best strategy for manipulating TAG
3
F. Du et al.
accumulation in other microorganisms (Hsin-Ju et al., 2012;
Irshad et al., 2015). However, this approach has not been applied
in thraustochytrids to date.
2.4. Biosynthesis of terpenoids
In addition to lipids, thraustochytrids can also be used as cell
factories for the production of terpenoids such as squalene and
carotenoids.
There are two common pathways for terpenoid biosynthesis in
microorganisms - the mevalonate (MVA) pathway and the 2-C-
methyl-D- erythritol-4-phosphate (MEP) pathway (Varela et al.,
2015). It has been reported that terpenoid accumulation in
thraustochytrids proceeds via the MVA pathway (Fig. 3).
Terpenoid biosynthesis shares the common carbon precursors
acetyl-CoA with lipid biosynthesis. Three molecules acetyl-CoA
are condensed into HMG-CoA by acetyl-CoA transferase (ERG10)
and HMG-CoA synthase (ERG13), which release two molecules of
CoA-SH. Next, HMG-CoA is reduced to mevalonate (MVA) by
HMG- CoA reductase (HMGR), and the reaction of which relies on
NADPH. Since this step is irreversible, HMGR is considered to be a
key rate- limiting enzyme in the MVA pathway. Phospho-
mevalonate kinase (ERG12) and mevanolate-5-P kinase (ERG8)
are responsible for transferring a pyrophosphate group from ATP
to MVA-PP, accompanied by the release of ADP. Subsequently,
mevanolate-5-PP decarboxylase (ERG19) and isopentenyl-PP
isomerase (IDI) produce IPP and DMAPP, which are common
precursors for terpenoid synthesis. Next, farnesyl-PP synthase
(ERG20) condenses DMAPP and two molecules of IPP to generate
FPP, two molecules of which are utilized to form squalene by
squalene synthase (Fig. 3). However, there are no reports on
improving squalene production by direct genetic regulation of
the MVA pathway in thraustochytrids. Starting from FPP, two and
four steps are respectively needed to produce β-carotene and
Biotechnology Advances 48 (2021) 107725
astaxanthin. Hong et al. (2013a) identified the squalene synthase
of Aurantiochytrium sp. KRS101, which was the first evidence of
the squalene biosynthesis pathway in thraustochytrids. Recently,
a top squalene producer named Aurantiochytrium sp. TWZ-97
was isolated from coastal waters of China, with a squalene yield
of 188.6 mg/L. Seven key genes of the MVA pathway were
revealed by transcriptome analysis (Zhang et al., 2019). From the
metabolic engineering point of view, squalene biosynthesis
shares the common precursor FPP with β-carotene, so
insufficient GGPP supply may be the reason for the lower
carotenoid content. When a homologous enzyme from
Archaeoglobus with high capacity for GGPP synthesis was further
overexpressed in Aurantiochytrium sp. SK4, the resulting
engineered strain produced 78% more total carotenoids and
5.02-fold more astaxanthin than the wild type (Ye et al., 2019). As
oleaginous microorganisms, thraustochytrids distribute the vast
majority of acetyl-CoA into the lipid biosynthesis pathway, thus
strategies for weakening lipid production as well as enhancing
the MVA pathway could be used in the future. 3. Low-cost
culture medium
Typical laboratory scale cultivation media for thraustochytrids
is based on artificial seawater, containing glucose as carbon
sources. However, in industrial fermentation of thraustochytrids,
the price of culture medium represents a significant proportion
of the operating costs. For example, in Schizochytrium sp.
fermentation for 120 h in 25L bioreactor, the cost of glucose
accounts for 73% of the total substrate cost (Fig. 2). Therefore,
low-cost culture medium such as crude glycerol, lignocellulosic
hydrolysate, and wastewater were tested in fermentation of
thraustochytrids (Table 1). However, these low-cost feedstocks
need further safety validation when seeking commercialization in
the field of food additives, for which it must be confirmed that
the product is safe for human consumption.
4
F. Du et al. Biotechnology Advances 48 (2021) 107725

Fig. 2. Substrate cost of Schizochytrium sp. fermentation in 25L bioreactor.

Table 1
Biomass accumulation and lipid content for thraustochytrids strain growth on low-cost carbon sources.
Strain Carbon source Biomass (g/L) Lipid content (% biomass) Fermentation (h) Reference

Aurantiochytrium sp. TC 20 glycerol 71 52% 69 Lee Chang et al., 2013

Thraustochytrium sp. BM2 glycerol 4.83 79% 48 Chen et al., 2020
Aurantiochytrium sp. glycerol 59.72 75.38% 168 Dilip et al., 2016
Schizochytrium sp. glycerol 54.15 72.38 168 Dilip et al., 2016
Aurantiochytrium limacinum SR21 Glucose and glycerol 88.32 83.84 96 Chang et al., 2015a
Aurantiochytrium sp. T66 glycerol 100 52 167 Jakobsen et al., 2008
Schizochytrium sp. S31 Glycerol 151.4 50.3 96 Chang et al., 2013b
Aurantiochytrium sp. FN21 sugarcane bagasse hydrolysate 33 35.9 120 Qi et al., 2017
Aurantiochytrium sp. T66 forest biomass hydrolysates 11.24 52.49 72 Patel et al., 2019
Schizochytrium limacinum SR21 sweet sorghum juice 9.4 73.4 96 Liang et al., 2010
Aurantiochytrium sp. KRS101 spent yeast 47.1 53.9 72 Ryu et al., 2013

5
F. Du et al. Biotechnology Advances 48 (2021) 107725

Fig. 3. Overview of lipid and terpenoid synthesis metabolism in thraustochytrids.

3.1. Glycerol acid cycle were significantly downregulated (Chen et al., 2016b).
Due to the generally poor regulation of glycerol assimilation, the
It has been reported that eukaryotic microorganisms conversion rate of glycerol into microbial oil is around 0.10 ± 0.02
dissimilate glycerol via phosphorylative pathway and g/g (Papanikolaou and Aggelis, 2011), lower than that of glucose.
dihydroxyacetone pathway (Wang et al., 2001). In Schizochytrium Similarly, the glycerol-to-oil conversion rate of thraustochytrids is
sp., glycerol is dissimilated via the phosphorylative pathway about 0.15 g/g (Chi et al., 2007; Pyle et al., 2008), indicating that
(Chang et al., 2013a). In this pathway, glycerol is converted into new strategies are needed to further improve the conversion rate
glycerol-3-phosphate (G-3-P) by glycerol kinase (GK), followed by in the future.
oxidation into dihydroxyacetone phosphate (DHAP) by FAD+-
dependent glycerol-3-phosphate dehydrogenase (FAD+-G-3- PDH)
(Makri et al., 2010). Previous studies had shown that lipid
accumulation by thraustochytrids grown on glycerol was
comparable with those grown on glucose (Chi et al., 2007).
Jakobsen et al., (2008) produced 90–100 g/L DCW containing 54–
63% of lipids by culturing Aurantiochytrium sp. on glycerol.
Combined with the highest kLa, the DCW and lipid concentration
of Schizochytrium sp. S31 can reach up to 151.4 g/L and 79.7 g/L,
respectively (Chang et al., 2013a). When 10 g/L glycerol was used
as carbon source and 12.5 g/L corn steep liquor as nitrogen
source, the maximal biomass of Thraustochytrium sp. BM2
reached 4.83 g/L, containing 79% of lipids, suggesting that this
strain could serve as a low-cost heterotrophic lipid producer
(Chen et al., 2020). However, the activity of the key enzymes GK
and G-3-PDH involved in the glycerol assimilation pathway may
be inhibited due to decreased oxygen supply at the later
fermentation stage. Therefore, mixed carbon sources with
glucose and glycerol were used to culture Thraustochytrids,
resulting maximal growth and DHA productivity (Li et al., 2015, Ye
et al., 2020). Moreover, the addition of calcium and magnesium
can also address these problems, because calcium can enhance
the activity of G-3-PDH and magnesium promote the activity of
malic enzyme in thraustochytrids (Dilip et al., 2016). Interestingly,
although the amounts of biomass and lipids of thraustochytrids
were similar in the cultivation with glucose or glycerol, the DHA
content was enhanced when glycerol was used as the carbon
source. Subsequent transcriptomic analysis revealed that acetyl-
CoA production was overall increased because the genes involved
in glycolysis and amino acid metabolism were remarkably
upregulated, while the pentose phosphate pathway and citric

6
F. Du et al.
Fig. 4. The fatty acid synthetase (FAS) pathway and the elongase-
desaturase pathway. 3.2. Lignocellulosic hydrolysate
The use of lignocellulosic biomass is expected to increase
significantly due to which is the most abundant renewable
organic materials. After pretreatment, sugars including glucose,
xylose, and arabinose can be obtained and then be utilized as
carbon sources by microorganisms. There are two xylose-
utilization pathways in microorganisms, the eukaryotic xylose
reductase/xylitol dehydrogenase pathway and prokaryotic xylose
isomerase pathway (Schellenberg et al., 1984; Xin et al., 2014).
Compared to the xylose isomerase pathway, the xylose
reductase/xylitol dehydrogenase pathway has more catalytic
steps, and the formation of xylitol affects the conversion rate of
xylose. In addition, heterologous expression of the xylose
reductase/xylitol dehydrogenase pathway can disturb the
intracellular cofactor balance. It should be noted that different
species of thraustochytrids vary greatly in their xylose utilization
capacity. Schizochytrium sp. can consume xylose from sugarcane
bagasse hydrolysate (Hoang et al., 2018). By using synthetic
biology techniques, the ability of microorganisms to metabolize
xylose can be enhanced. Thraustochytrids produce xylitol from
both glucose and xylose, but cannot grow in the presence of
xylose alone due to an incomplete xylose reductase/xylitol
dehydrogenase pathway. When introduced endogenous xylose
isomerase and heterologous xylulose kinase into thraustochytrid,
the resulting strain can utilize xylose ( (Merkx et al., 2018)). When
Aurantiochytrium sp. was cultivated using birch hydrolysate
pretreated with organic solvents, it resulted in 703.3 mg/L/ day
of DHA productivity and 1.0 g/L of squalene, which suggests
economic production of lipid compounds is technically feasible
(Patel et al., 2019). Furthermore, in order to endow the strain
Biotechnology Advances 48 (2021) 107725
with the ability to grow on carboxymethylcellulose, a β-
glucosidase from Aspergillus aculeatus was expressed in
Aurantiochytrium sp., and the resulting strain with cellobiose as
the sole carbon source exhibited increased growth and enzyme
activity.
The process of lignocellulose pretreatment inevitably yields
many toxic by-products, which can inhibit cell growth. Therefore,
an Aurantiochytrium sp. strain tolerant to lignocellulosic
hydrolysate was isolated and commercially applied for DHA
production (Huang et al., 2008). Furthermore, high-throughput
RNA sequencing was applied to clarify the tolerance mechanism,
and results showed that genes involved in the TCA cycle and
amino acid biosynthesis were highly upregulated, while genes
involved in the degradation of aromatic compounds were
downregulated (Qi et al., 2017). It should be noted that different
species of thraustochytrids vary greatly in their xylose utilization
capacity, and the mechanisms of hydrolysate tolerance should be
studied further.
3.3. Wastewater
Microbial fermentation often results in large amounts of
fermentation wastewater. For instance, the fermentation of
Aurantiochytrium generates 100,000 tons of wastewater every
year in China (Chen et al., 2016a). Although wastewater
treatment is not expensive, it requires high-power processing
equipment (Ruiz-Rosa et al., 2016). The fermentation wastewater
contains inorganic salts or growth-promoting factors, which can
potentially be valuable for the fermentation of thraustochytrids.
For example, the supernatant of distillery waste contains 2.5% of
proteins as well as 0.2% of amino acids, and has been used as the
sole nitrogen source for lipid and astaxanthin production using
Schizochytrium sp. (Yamasaki et al., 2006). The fermentation
wastewater from Mortierella alpina was also used to replace
pure water for Aurantiochytrium fermentation, resulting in a DHA
yield of 30.4 g/L, with no significant changes compared to the
use of pure water (Song et al., 2017). In addition, it has been
demonstrated that saline wastewater from cheesemaking
(Humhal et al., 2017), industrial wastewaters from pig
slaughtering plants (Villarroel Hipp and Silva Rodriguez, 2018),
K2HPO4-waste feeds (Leong et al., 2019), and recycled spent
media (Bagul and Annapure, 2020) can be used to lower the
production cost of thraustochytrid biomass and lipids. In general,
wastewater cannot be used directly because it contains cell
growth inhibitors, and it would be highly desirable to optimize
the handling methods and enhance the productivity of
wastewater fermentations in the future. Overall, using
wastewater for the production of biomass and value-added
products is in high demand.
4. Molecular approaches for enhancing lipid and terpenoid
production
The genome sequences of thraustochytrids such as
Schizochytrium sp., Aurantiochytrium limacinum SR21 and
Schizochytrium aggregatum have been published. In recent
years, a transformation method for genome editing has been
successfully established in thraustochytrids. Although the
efficiency is still low, metabolic engineering of thraustochytrids to
improve the accumulation of target products is gradually
becoming more feasible (Table 2).
7
F. Du et al. Biotechnology Advances 48 (2021) 107725
4.1. Genetic engineering

8
F. Du et al. Biotechnology Advances 48 (2021) 107725
Selection markers are genes that are introduced into a cell to
allow its survival under predetermined selection conditions. In
thraustochytrids, selectable markers such as resistance genes for
zeocin, G418, hygromycin, cycloheximide, and paromomycin can
be used (Sakaguchi et al., 2012; Suen et al., 2014; Hong et al.,
2013a; Bayne et al., 2013). In most studies, electroporation with
linear DNA is the most common strategy for transformation (Li et
al., 2018b; Wang et al., 2019). In addition, particle bombardment
and Agrobacterium-mediated transformation can also be used in
thraustochytrids (Cheng et al., 2012). In order to make the

Fig. 5. A: Catalytic steps of the PKS pathway in thraustochytrids. B: Structure of PKS pathway in different thraustochytrids species.
transformants suitable for industrial application, the cre/loxP
system was established to generate markerless mutations in A.
limacinum (Sun et al., 2015). Homologous recombination has
been shown to be effective for integrating a foreign gene into the
genome, but the required length of the homology arms is
generally 1000-2000 bp. Recently, a 2A peptide-based method
was developed in Aurantiochytrium for multiple genes expression
(Wang et al., 2020).

9
F. Du et al.
At present, there is little research improved lipid production
by modifying the FAS pathway. Recently, the record lipid yield of
110.5 g/L was obtained by overexpressing MAT in
Schizochytrium, leading to 39.6% higher production than in the
wild-type strain (Li et al., 2018b). When ACCase was
overexpressed in Schizochytrium using the strong constitutive
ccg1p promoter, the lipid content and DHA yield respectively
increased by 30.5 and 41.9% (Han et al., 2020). Several metabolic
engineering studies focused on the PUFA biosynthesis pathway.
Such approaches can be used to increase the content of naturally
occurring fatty acids that are present at low levels in the wild
type strain. For example, when a Δ5 desaturase from
Thraustochytrium aureum was overexpressed in A. limacinum,
the levels of EPA and ARA were respectively increased 4.6- and
13.6- fold in the transgenic strain (Hauvermale et al., 2006).
Disruption of the Δ12 desaturase of Thraustochytrium aureum
ATCC 34304 resulted in the accumulation of oleic acid (C18:1)
and a decrease of linoleic acid (C18:2) (Takanori et al., 2012). In
our previous study, the heterogeneous ω-3 desaturase of
Saprolegnia diclina was expressed in Schizochytrium sp., the
result showed that more DPA was transformed into DHA (Re et
al., 2015). While ω-3 desaturases possess an extensive substrate
specificity, the ω-3 desaturase from Saprolegnia diclina was
shown to be more specific for C20:4 (Pereira et al., 2004). Thus,
protein engineering of the ω-3 desaturase may be a feasible
strategy to improve its DPA specificity.
In theory, rewriting the PKS pathway is the most direct
method to regulate PUFA biosynthesis. ORFA from
Thraustochytrium contains 8
ACPs, while that of Schizochytrium possesses 9. The main role
of ACP domains is to covalently bind the acyl substrates. It has
been reported that the amount of ACPs influences PUFA
productivity. In order to
Table 2
Genetic engineering strategies to improve lipid accumulation in thraustochytrids.
Strain Transformation
Biotechnology Advances 48 (2021) 107725
modifying the PKS pathway, which may mainly due to the lack of
efficient genetic tools, together with the complexity and unclear
function of PKS enzymes.
In addition, many studies aimed to enhance the performance
of thraustochytrids by regulating the bypass pathway. It is
generally accepted that lipid biosynthesis requires large amounts
of acetyl-CoA and NADPH as the main precursor and source of
reducing power, respectively. In Aurantiochytrium sp., the acetyl-
CoA synthetase gene from Escherichia coli was introduced to
increase the pool of acetyl-CoA, which resulted in a maximal fatty
acid content of 46.8% (Yan et al., 2012). More significantly,
overexpression of acetyl-CoA synthetase in Schizochytrium not
only improved carbon source utilization, but also decreased the
production of the major byproduct acetate (Yan et al., 2012).
Based on β-galactosidase reporter system, a strong promoter of
ccg1p was screened, then ATP-citrate lyase and ACCase under
ccg1p were overexpressed in Schizochytrium sp. ATCC 20888,
resulting in the highest lipid content of 73% (Han et al., 2020).
The NADPH needed for fatty acid synthesis is mainly produced by
the transhydrogenase cycle and the PPP pathway, in which the
ME and the G6PDH is the key enzymes involved in two pathways,
respectively. Cui et al. (2016) overexpressed the G6PDH for
increasing NADPH supply via the pentose phosphate pathway,
and the PUFAs content was increased by 10.6%. Similarly, the
NADPH generation system of Aurantiochytrium sp. was
strengthen by overexpressing the gene encoding malic enzyme,
and the recombinant strain exhibited a high SFA yield of 8.65 g/L
(Cui et al., 2019).
4.2. Chemical activators
Many small-molecule drugs can be used to stimulate or
Strategy Performance Reference

Schizochytrium sp. TIO1101 Electroporation transformation (2100 Overexpression of ACS gene from The biomass and fatty acid proportion of transformants Yan et al., 2012
V, 4.5 ms) Escherichia coli was increased by 29.9 and 11.3%, respectively.

Thraustochytrium aureum
ATCC 34304
Schizochytrium
Thraustochytrium aureum Electroporation transformation
ATCC 34304
Schizochytrium sp
Schizochytrium sp
Aurantiochytrium sp. SK4
Schizochytrium sp
Schizochytrium sp. ATCC
20888
Aurantiochytrium
Electroporation transformation
(50 μF, 50 Ω, 7.5 kV/cm, 2 times)
Electroporation transformation
Electroporation transformation
Electroporation transformation (0.75
kV, 200 Ω, and 50 μF)
Electroporation transformation (1.8
kV, 300 Ω, and 50 μF)
Electroporation transformation (0.75
kV, 200 Ω, and 50 μF)
Electroporation transformation (0.75
kV, 200 Ω, and 50 μF)
Electroporation transformation
Disruption of △5 desaturase gene The content of dihomo-γ-linolenic acid (C20:3) and Sakaguchi et
eicosatetraenoic acid (C20:4) was increased. al., 2012
Overexpression of malonyl-CoA: ACP The DHA and EPA yield was enhanced by 81.5 and Li et al., 2018b
transacylase (MAT) 172.5% than that of wild type, respectively.
Disruption of △12 desaturase gene The oleic acid content was increased and linoleic acid Takanori et al.,
was decreased. 2012
Overexpression of G6PDH The PUFA was increased by 10.6% Cui et al., 2016
Introduction of ω-3 desaturase gene from The 3% (DPA) was converted to (DHA) Re et al., 2015
Saprolegnia diclina
Expression of vitreoscilla hemoglobin The fatty acid and astaxanthin content of Suen et al., 2014
transformants was 44% and 9-fold higher than that of
the wild type
Replacement of the native AT with AT The EPA content was increased by 3.7 times. Ren et al., 2018
domain from Shewanella sp.
Overexpression of ATP-citrate lyase The DHA yield was increased by 48.8%. Han et al., 2020
(ACL) and acetyl-CoA carboxylase
(ACCase)
Expression of the EPA biosynthetic gene The EPA yield was increased by 5-folds. Wang et al.,
cluster from Shewanella japonica 2020

examine their function, the number of ACPs in Schizochytrium
was increased from 9 to 10 and 11. The results showed that the
PUFA yield of the strain with more ACP domains is 50 to 100%
higher than that of the native group (Hayashi et al., 2016). The AT
domain of Schizochytrium sp. was replaced with the AT domain
from Shewanella sp., which resulted in a 3.7 times increase of
EPA production compared to the wild type (Ren et al., 2018).
Furthermore, in a recent study, the whole EPA biosynthesis gene
cluster from Shewanella japonica was expressed in
Aurantiochytrium, resulting in 5-fold increase of EPA yield (Wang
et al., 2020). To date, no major progress has been made in
inhibit intracellular metabolic pathways, which are equivalent to
effects of overexpression and suppression approaches. Compared
to genetic engineering, the application of chemicals is potentially
an easy and practical approach to improve lipid production in
thraustochytrids, which has become one of research hotspots.
Phytohormones and their derivatives are often used to improve
lipid accumulation by adjusting the internal biochemical
pathways. For example, lipid accumulation of Schizochytrium was
enhanced to 16.79% by adding 20 mg/L jasmonic acid and 2
mg/L naphthoxyacetic acid (Wang et al., 2018a). Similarly, the
addition of 6-benzylaminopurine increased the lipid yield of
10
F. Du et al.
Aurantiochytrium sp. by 25.9% (Yu et al., 2016). In a recent study,
the addition of 120 mg/L inositol improved the production of
lipids and DHA in Schizochytrium sp. by 13.9 and 20.8%,
respectively (Liu et al., 2019). Moreover, short-chain alcohols can
partition into membranes to increase their fluidity and interfere
with normal cell functions, which was also used to regulate the
accumulation of terpenoids. A remarkable 2000-fold increase of
astaxanthin was obtained in Schizochytrium limacinum by adding
5.6% methanol (Du et al., 2019). Interestingly, addition of 6 g/L
butanol in the same species decreased the DHA content by
nearly 40%, but the squalene content was increased 31 times
(Zhang et al., 2017).
Sterol biosynthesis shares acetyl-CoA as a common precursor
with the fatty acid and terpenoid biosynthesis pathways, so that
inhibiting the sterol biosynthesis pathway could be an effective
strategy to improve the production of the former two types of
products. Li et al. (2019a) used fluconazole to block the sterol
pathway by inhibiting lanosterol 14α- demethylase activity, which
resulted in a 16 and 45% increases of the lipid and squalene
content in Schizochytrium, respectively. In addition, Shirasaka et
al. (2005) found that benzoic acid enhanced EPA accumulation in
Schizochytrium sp. by suppressing the methyl-directed
desaturation from the Δ13 position. Based on this, four benzoic
acid derivatives were tested in Schizochytrium limacinum SR21,
and the addition of 200 mg/L of p-aminobenzoic acid increased
the lipid yield by 56.84% (Li et al., 2019b). In order to alleviate
lipid peroxidation, which is especially important for PUFAs,
antioxidants can been used to scavenge intracellular reactive
oxygen species. When 9 g/L of ascorbic acid was added to
cultures of Schizochytrium sp., the DCW and DHA yield
respectively increased by 16.16 and 30.44%, which was
accompanied by lower ROS levels and higher antioxidant capacity
(Ren et al., 2017). In addition, supplementing butylated
hydroxytoluene or propyl gallate also improved the lipid yield and
cell growth of thraustochytrids (Singh et al., 2015). Overall, the
addition of chemicals is a sustainable and economical strategy
that can be easily applied in large-scale production.
5. Engineering operation strategies for enhancing lipid and
terpenoid production
The lipid production in thraustochytrids is often influenced by
the culture environment, which exhibit two opposing
characteristics, including high biomass with low lipid content, or
low biomass with high lipid content. Thus, many operation
strategies were developed to balance the lipid accumulation and
cell growth (Table 3). In addition, it is difficult to enlarge the
production quantities from the laboratory-scale while keeping
the titers similar, thus scale-up technologies and bioreactor
design are key to industrialization.
5.1. Two-stage cultivation strategies
Thraustochytrids cells accumulate lipid as an energy storage
battery when exposed to a stress condition, but which could
generate growth limitation. Two-stage strategy is an effective
cultivation system due to which separate the lipid accumulation
phase from the biomass growth phase. Regulating the oxygen
Table 3
Operation strategies for high-cell-density fermentation.
Strain Strategies Biomass Lipid DHA
(g/L) yield yield
(g/L) (g/L)
Biotechnology Advances 48 (2021) 107725
supply is a classic and effective control strategy for
thraustochytrid fermentations, since DHA is produced by the
oxygen-independent PKS pathway. For example, stepwise
dissolved oxygen control was applied to thraustochytrids, with a
high DO stage for cell growth and low DO stage for lipid
accumulation. Moreover, when the DO level was maintained at
50% in the whole fermentation process, Aurantiochytrium
limacinum SR21 produced DCW at 8.82 g/L/day and DHA
productivity at 2.9 g/L/day (Huang et al., 2012). However, it has
been shown that DO does not fully reflect the oxygen supply
capacity, especially under DO-limited conditions (Chang et al.,
2013b). Consequently, the oxygen uptake rate (OUR) was
introduced as an online parameter to better reflect the oxygen
supply in fermentations of Schizochytrium sp. (Chang et al.,
2014). In addition, Guo et al. (2016) combined the OUR and
respiratory quotient (RQ) as the real-time monitoring
parameters, results showed that this method achieved the
highest DHA content (45% in total fatty acids), suggesting that it
is more effective than DO in Schizochytrium sp. fermentation.
Nutrient limitation is another effective strategy for lipid
production in thraustochytrids. For example, lipid production of
Aurantiochytrium sp. T66 was significantly boosted when the
cells were subjected to nitrogen limitation (Jakobsen et al., 2008).
Moreover, nitrogen limitation is conducive to neutral lipids
accumulation but nor polar lipids in Schizochytrium sp. (Sun et
al., 2014). However, temperature is more effective for regulating
fatty acid composition. At lower temperatures, more PUFAs need
to be synthesized to maintain the fluidity of the cell membrane
(An et al., 2013). Accordingly, when the temperature was
decreased to 12 ◦C from 30 ◦C, the DHA content of
Aurantiochytrium mangrovei Sk-02 was increased from 29 to 42%
in the late lipid accumulation phase (Chodchoey and Verduyn,
2012). However, this strategy could less feasible in industrial
production due to the increased cost of lower temperature
control. To overcome this bottlenecks, Sun et al. (2018) applied
adaptive evolution under low temperature to enhance the PUFA
content of Schizochytrium sp., which avoid the high cost and
production instability. In a recent study, ammonia and citric acid
was applied as pH regulators, then a two-stage pH control
strategy was developed for Schizochytrium sp. fermentation,
resulting a maximum DHA yield of 272.92 mg/L/h (Yin et al.,
2018b). Altogether, many bioprocess engineering strategies have
been developed to improve lipid accumulation of
thraustochytrids, with varying degrees of success.
5.2. Bioprocess scale-up strategies
The aim of scale-up is to enlarge the production quantities
while keeping the titers similar to those obtained on the
laboratory-scale. The oxygen supply conditions obviously affect
cell growth and lipid production, so the volumetric oxygen-
transfer coefficient (KLa) is often chosen as the critical factor for
scale-up. Using this scale-up strategy, the fermentation
performance of Schizochytrium sp. in a 7000L stirred-tank
bioreactor (DHA content of 19.72 g/L) was similar to that of the
experimental-scale process (DHA content of 13.84 g/L), but it
resulted in
References
11
F. Du et al. Biotechnology Advances 48 (2021) 107725
Schizochytrium sp. The Volumetric 151.40 76.15 28.93 Chang
S31 mass transfer et al.,
coefficient (kLa) 2013b
was maintained
at
1802 ± 105 h
−1
Schizochytrium sp. Two-phase pH 120.6 62.6 32.8 Yin et al.,
control with 2018b
ammonia and
citric acid
Schizochytrium sp. The fluid dynamics 128.3 74.8 39.2 Guo et al.,
was analyzed for 2018
improve the
efficiency of scale-
up, and and
impeller
combinations was
optimized.

Schizochytrium sp. Combined porous- 151.0 103.6 44.3 Guo et al.,

membrane- 2017
impeller
bioreactor and
three-stage DO
control strategy

Aurantiochytrium sp. Limitation of 100 58 11.84 Jakobsen et

T66 nitrogen and al.,
oxyen 2008
a 150-fold quantitative scale-up (Qu et al., 2013). Furthermore, Guo et al. (2018) analyzed the fluid dynamics of different scale
bioreactors and then developed the efficient and economical scale-up strategy for DHA production using Schizochytrium sp., which
resulted in 39.2 g/L DHA yield in 7000L bioreactor. In addition, feeding strategy is also an important factor in scale-up production. Using
continuous feeding strategy with a glucose concentration of 20–25 g/L achieved a maximum DHA productivity of 320.17 mg/L/ ⋅h in
500-L pilot-scale bioreactor, which represent increase of 37.68% over that of the intermittent feeding method (Guo et al., 2020).

5.3. Bioreactor design

In the fermentation process of thraustochytrids, new reactor designs need to provide an adequate oxygen supply but avoid high
shear forces, so air-lift bioreactors is a better choice. When an air-lift fermenter was used to culture Aurantiochytrium sp., the DHA
content was increased from 39.5 to 52.3% (Hong et al., 2013b). The highest DCW of 27.57 g/L and DHA productivity of 610 mg/L/day
were achieved in an air-lift fermenter, due to the low shear stress (Chen and Yang, 2018). Furthermore, the impeller configuration was
optimized to improve the lipid production of Schizochytrium sp. The combination of two upper 6- SBDTs and one bottom 6-ABDT was
proven to be highly beneficial for DHA synthesis (Zhao et al., 2016). Guo et al. (2018) applied an impeller configuration with two wide-
blade hydrofoil impellers pumping downward (located at the top and in the middle), and a six-parabolic-blade disk turbine (located at
the bottom) in a 7000L bioreactor. This configuration resulted in a DHA concentration of 326.67 mg/L/h, representing an increase of
133.3% over the original impeller. In addition, membrane materials are also widely used in new reactor design. For example, a hollow-
fiber polypropylene membrane was mounted in a
shake flask to transform it into a membrane reactor, which led to to the specificity and complexity of cell walls, this method may
a 60% relative increase of DHA productivity in Schizochytrium sp. require screening of wall- breaking enzymes for each species.
compared to single shake mode (Zhang et al., 2013). An Here, we mainly discuss lipid extraction technologies and the
innovative porous- membrane-impeller bioreactor was recently reuse of algal residues.
designed, and it was found to significantly facilitate cell
proliferation and DHA accumulation in Schizochytrium sp. when 6.1. Lipid extraction
combined with a multi-stage control strategy (Guo et al., 2017).
Lipid extraction is a crucial stage in industrial lipid production,
6. New developments in downstream processing aiming to facilitate the release of lipids from cells. Organic
solvent extraction is the most commonly used method in
The key stages of downstream processing include cell thraustochytrids, and the suitability of the organic solvent
harvesting and disruption, lipid extraction, and conversion of determines the extraction efficiency. Byreddy et al. (2015) tested
biomass into products (Fig. 1). Centrifugation has been suggested nine organic solvents for lipid extraction from thraustochytrids,
as the most effective cell harvesting method for thraustochytrids, and the results showed that hexane generated the highest lipid
due to advantages such as speed, efficiency, low cost, no toxic content of 12.5%. Furthermore, two or more organic solvents can
residues, and not affecting the quality of the biomass. Common be used simultaneously in polar/non-polar combinations to
cell disruption techniques for thraustochytrids include osmotic increase the lipid yield. The maximum lipid extraction yield from
shock, grinding, high-pressure homogenizers and other thraustochytrids was 22% using a chloroform: methanol ratio of
mechanical approaches. In contrast, enzymatic treatment for 2:1 (Byreddy et al., 2015). Organic solvent extraction is simple in
lipid release is a low-energy and environmentally friendly operation and has a high efficiency, but it leaves an unavoidable
process, which mainly includes the disruption of cell wall and solvent residue, which is unacceptable for many product
internal organelles for free lipid bodies releasing. However, due applications. In addition, organic solvent extraction can cause

12
F. Du et al.
environmental pollution and affect the health of operators
(Harris et al., 2018). Ionic liquids have been shown to improve
the efficiency of organic solvent extraction of lipids under mild
conditions. The two ionic liquids imidazolium 1-ethyl- 3-
methylimidazolium ethylsulfate [C2mim] [EtSO4] and
tetrabutylphosphonium propanoate [P4444] [Prop] were
evaluated in lipid extraction from Thraustochytrium sp., and the
results indicated that both ionic liquids facilitated the lipid
extraction (Zhang et al., 2018). Furthermore, supercritical fluids
can be used to replace organic solvents in extraction processes.
For instance, a lipid yield of 33.9% with a DHA content of 27.5%
was obtained from Schizochytrium limacinum using supercritical
CO2 (Tang et al., 2011). In a recent study, a novel method for lipid
extraction from thraustochytrids was developed. The biomass
was first combined with potassium hydroxide, after which
reaction mixture was added to an anti-solvent to generate solid
precipitates, and the final PUFA recovery rate reached 72–74%
(Pour et al., 2020). It is worth noting that thraustochytrid lipids
contain a series of fatty acids, mainly including C16:0, C22:5 and
C22:6. Since the lipid bodies are large and insoluble in water, it is
possible to extract lipids by simple centrifugation. Three-phase
centrifuges can separate the lipids, water and solids, and this
simple technology is used in industrial lipid extraction from
Schizochytrium sp.
6.2. Residue application
Microalgal biomass residues are major by-products of biodiesel
production, and can potentially be utilized as nutrient sources
for microbial cultivation due to their content of proteins and
carbohydrates. For instance, fermentation residues were
hydrolyzed into amino acids and sugars for Chlorella vulgaris
cultivation, which yielded 3.28 g/L of biomass with a lipid
content of 35% (Zheng et al., 2012). It has been reported that
Schizochytrium sp. contains 3.45% of total nitrogen (Yin et al.,
2018a), and its oil-extraction residues can potentially be
developed into a novel nitrogen source (Bryant et al., 2012). In
order to increase the economic and environmental sustainability
of Schizochytrium sp. fermentation, its residues were used to
replace yeast extract for its own fermentation (Yin et al., 2018a).
A 27.1 g/L of DHA yield was obtained when 80% of yeast extract
nitrogen was replaced with the residues, which was 20.07%
higher than that of the control.
In addition, residues of thraustochytrid fermentation are rich
in PUFAs and proteins that are beneficial to animal growth, so
which are widely used as an ingredient of livestock feed (Bruneel
et al., 2013). For example, the EPA and DHA content of eggs was
significantly increased by feeding chickens with Aurantiochytrium
biomass residues, thus improving the nutritional and economic
value of eggs (Moran et al., 2020). Moreover, numerous studies
indicate that enhancing the DHA content in feed can improve the
survival rate of aquatic animals (Ganuza et al., 2008; Gładkowski
et al., 2014). Fermentation residues of thraustochytrids have
been used as feed additives for a variety of aquatic animals,
including atlantic salmon parr and pacific white shrimp (Miller et
al., 2007). When Schizochytrium biomass residue was added to
pig feed, the serum triglycerides of the pigs significantly
decreased, and the DHA content in subcutaneous fat increased
13 times (Jon Meadus et al., 2011). These studies indicate that
feeding the fermentation residues of thraustochytrids can have
unique positive effects on the physiological function of livestock,
improving animal growth and product quality.
Biotechnology Advances 48 (2021) 107725
7. Conclusions
Thraustochytrids are a promising and competitive source of
lipid production compared to other microorganisms. Its high
growth rate, broad substrate utilization, efficient product
pathways, and mature operation technologies, in conjunction
with compact downstream processing, all point to great promise
for the future development of thraustochytrids. On one hand, the
fatty acid composition of thraustochytrids is simple, dominated
by C16:0 and DHA. Whereas DHA are used in the field of human
health, C16:0 is suitable for biodiesel production. Based on
precursor of acetyl-CoA, C16:0 and DHA are produced by two
different enzymatic systems, therefore it is possible to favor one
pathway over another by genetic engineering. In addition, the
PKS also generates about 10% of C22:5, so it is meaningful to
convert DPA into DHA via introducing w-3 desaturase. On the
other hand, the efficient PKS pathway is the key value of
thraustochytrids, so how to excavate and use this pathway needs
to be considered in the future. In fact, the FAS pathways has been
engineered to achieve desired fatty acids by in vitro and in vivo
assays, which encourage that similar approaches could be taken
to modify the PKS pathway for producing other high-value fatty
acids such as EPA. There is also considerable potential for the
production of more high-valued products using thraustochytrids.
In other words, it would be interesting to convert
thraustochytrids from DHA producer to other chemical producer
in the future, in which, squalene and carotenoids are surely good
examples. However, resources and tools used for genome editing
remains limited the development of thraustochytrids. A primary
challenge is the lack of the key enzyme information involved in
terpenoids biosynthesis. Thus, developing efficient genetic
editing tools and discovering molecular targets is therefore of
much significance.
Declaration of Competing Interest
The authors declare that they have no competing interests.
Acknowledgement
This work was supported by the Nature Science Foundation of
Jiangsu Province (No. BK20190706, No. BK20170988), the
National Natural Science Foundation of China (Number:
21908112, No. 22038007). References
Aki, T., Hachida, K., Yoshinaga, M., Katai, Y., Yamasaki, T., Kawamoto, S.,
Kakizono, T., Maoka, T., Shigeta, S., Suzuki, O., Ono, K., 2003.
Thraustochytrid as a potential source of carotenoids. J. Am. Oil Chem. Soc.
https://doi.org/10.1007/s11746-003- 0773-2.
An, M.L., Mou, S.L., Zhang, X.W., Ye, N.H., Zheng, Z., Cao, S.N., Xu, D., Fan, X.,
Wang, Y. T., Miao, J.L., 2013. Temperature regulates fatty acid desaturases
at a transcriptional level and modulates the fatty acid profile in the
Antarctic microalga Chlamydomonas sp. ICE-L. Bioresour. Technol.
https://doi.org/10.1016/j.biortech.2013.01.142.
Ana, M.D.O.F., Luis, D.G.M., Júlio, C.D.C., Gilberto, V,D,M,P., Vanete, T,S., Carlos,
R,S., 2016. Technological trends and market perspectives for production of
microbial oils rich in omega-3. Crit. Rev. Biotechnol.
https://doi.org/10.1080/ 07388551.2016.1213221.
Bagul, V.P., Annapure, U.S., 2020. Effect of sequential recycling of spent media
wastewater on docosahexaenoic acid production by newly isolated strain
Aurantiochytrium sp. ICTFD5. Bioresour. Technol. https://doi.org/10.1016/j.
biortech.2020.123153.
Balamurugan, S., Wang, X., Wang, H.L., An, C.J., Li, H., Li, D.W., Yang, W.D., Liu,
J.S., Li, H.Y., 2017. Occurrence of plastidial triacylglycerol synthesis and the
potential regulatory role of AGPAT in the model diatom Phaeodactylum
tricornutum.
Biotechnol. Biofuels. https://doi.org/10.1186/s13068-017-0786-0.
Bayne, A.C.V., Boltz, D., Owen, C., Betz, Y., Maia, G., Azadi, P., Archer, H.S., Zirkle,
R., Lippmeier, J.C., 2013. Vaccination against influenza with recombinant
hemagglutinin expressed by Schizochytrium sp. confers protective
immunity. PLoS One. https://doi.org/10.1371/journal.pone.0061790.
13
F. Du et al.
Benita, Q., Ivonne, H., Emilio, H. Andr´es, 2010. Docosahexaenoic acid (C22: 6n−
3, DHA) and astaxanthin production by Thraustochytriidae sp. AS4-A1 a
native strain with high similitude to Ulkenia sp.: evaluation of liquid
residues from food industry as nutrient sources. Enzyme. Microb. Tech.
https://doi.org/10.1016/j.
enzmictec.2010.04.002.
Bruneel, C., Lemahieu, C., Fraeye, I., Ryckebosch, E., Muylaert, K., Buyse, J.,
Foubert, I., 2013. Impact of microalgal feed supplementation on omega-3
fatty acid enrichment of hen eggs. J. Funct. Foods.
https://doi.org/10.1016/j.jff.2013.01.039.
Bryant, H.L., Gogichaishvili, I., Anderson, D., Richardson, J.W., Sawyer, J.,
Wickersham, T., Drewery, M.L., 2012. The value of post-extracted algae
residue.
Algal Res. https://doi.org/10.1016/j.algal.2012.06.001.
Byreddy, A.R., Gupta, A., Barrow, C.J., Puri, M., 2015. Comparison of cell
disruption methods for improving lipid extraction from Thraustochytrid
strains. Marine Drugs. https://doi.org/10.3390/md13085111.
Chang, G., Luo, Z., Gu, S., Wu, Q., Chang, M., Wang, X., 2013a. Fatty acid shifts
and metabolic activity changes of Schizochytrium sp. S31 cultured on
glycerol. Bioresour.
Technol. https://doi.org/10.1016/j.biortech.2013.05.030.
Chang, G.F., Gao, N.S., Tian, G.W., Wu, Q.H., Chang, M., Wang, X.G., 2013b.
Improvement of docosahexaenoic acid production on glycerol by
Schizochytrium sp. S31 with constantly high oxygen transfer coefficient.
Bioresour. Technol. https:// doi.org/10.1016/j.biortech.2013.04.107.
Chang, G.F., Wu, J., Jiang, C.H., Tian, G.W., Wu, Q.H., Chang, M., Wang, X.G.,
2014. The relationship of oxygen uptake rate and kLa with rheological
properties in high cell density cultivation of docosahexaenoic acid by
Schizochytrium sp. S31. Bioresour.
Technol. https://doi.org/10.1016/j.biortech.2013.11.002.
Chang, K.J.L., Rye, L., Dunstan, G.A., Grant, T., Koutoulis, A., Nichols, P.D.,
Blackburn, S. I., 2015b. Life cycle assessment: heterotrophic cultivation of
thraustochytrids for biodiesel production. J. Appl. Phycol.
https://doi.org/10.1007/s10811-014-0364-9.
Chang, M., Jing, G., Wang, X., Xiang, Y., Liu, Y., Rui, J., 2015a. A strategy for the
highly efficient production of docosahexaenoic acid by Aurantiochytrium
limacinum SR21 using glucose and glycerol as the mixed carbon sources.
Bioresour. Technol. https:// doi.org/10.1016/j.biortech.2014.11.046.
Chen, C.Y., Yang, Y.T., 2018. Combining engineering strategies and fermentation
technology to enhance docosahexaenoic acid (DHA) production from an
indigenous Thraustochytrium sp. BM2 strain. Biochem. Eng. J.
https://doi.org/10.1016/j. bej.2018.02.010.
Chen, C.Y., Lee, M.H., Dong, C.D., Leong, Y.K., Chang, J.S., 2020. Enhanced
production of microalgal lipids using a heterotrophic marine microalga
Thraustochytrium sp.
BM2. Biochem. Eng. J. https://doi.org/10.1016/j.bej.2019.107429.
Chen, W., Zhou, P., Zhu, Y., Xie, C., Ma, L., Wang, X., Bao, Z., Yu, L., 2016a.
Improvement in the docosahexaenoic acid production of Schizochytrium
sp. S056 by replacement of sea salt. Bioprocess Biosyst. Eng.
https://doi.org/10.1007/s00449- 015-1517-1.
Chen, W., Zhou, P.P., Zhang, M., Zhu, Y.M., Wang, X.P., Luo, X.A., Bao, Z.D., Yu,
L.J., 2016b. Transcriptome analysis reveals that up-regulation of the fatty acid
synthase gene promotes the accumulation of docosahexaenoic acid in
Schizochytrium sp. S056 when glycerol is used. Algal Res.
https://doi.org/10.1016/j.algal.2016.02.007. Cheng, R., Ma, R., Li, K., Rong, H.,
Lin, X., Wang, Z., Yang, S., Ma, Y., 2012.
Agrobacterium tumefaciens mediated transformation of marine microalgae
Schizochytrium. Microbiol. Res.
https://doi.org/10.1016/j.micres.2011.05.003.
Chi, Z., Pyle, D., Wen, Z., Frear, C., Chen, S., 2007. A laboratory study of
producing docosahexaenoic acid from biodiesel-waste glycerol by
microalgal fermentation.
Process Biochem. https://doi.org/10.1016/j.procbio.2007.08.008.
Chodchoey, K., Verduyn, C., 2012. Growth, fatty acid profile in major lipid classes
and lipid fluidity of Aurantiochytrium Mangrovei Sk-02 as a function of
growth temperature. Braz. J. Microbiol. https://doi.org/10.1590/S1517-
838220120001000020.
Chu, F.F., Cheng, J., Li, K., Wang, Y.G., Li, X., Yang, W.J., 2020. Enhanced lipid
accumulation through a regulated metabolic pathway of phosphorus luxury
uptake in the microalga Chlorella vulgaris under nitrogen starvation and
phosphorus repletion. ACS Sustain. Chem. Eng. https://doi.org/10.1021/
acssuschemeng.9b07447.
Cui, G., Wang, Z., Hong, W., Liu, Y.J., Chen, Z., Cui, Q., Song, X., 2019. Enhancing
tricarboxylate transportation-related NADPH generation to improve
biodiesel production by Aurantiochytrium. Algal Res.
https://doi.org/10.1016/j.
algal.2019.101505.
Cui, G.Z., Ma, Z., Liu, Y.J., Feng, Y., Sun, Z., Cheng, Y., Song, X., Cui, Q., 2016.
Overexpression of glucose-6-phosphate dehydrogenase enhanced the
polyunsaturated fatty acid composition of Aurantiochytrium sp. SD116.
Algal Res. https://doi.org/10.1016/j.algal.2016.08.005.
Dairi, T., Hayashi, S., Naka, M., Ikeuchi, K., Ohtsuka, M., Kobayashi, K., Satoh, Y.,
Ogasawara, Y., Maruyama, C., Hamano, Y., 2019. Control mechanism for
Biotechnology Advances 48 (2021) 107725
carbon- chain length in polyunsaturated fatty-acid synthases. Angew.
Chem. Int. Edit.
https://doi.org/10.1002/anie.201900771.
Dilip, Singh, Barrow, Colin J., Munish, Puri, Tuli, Deepak K., Mathur, Anshu S.,
2016. Combination of calcium and magnesium ions prevents substrate
inhibition and promotes biomass and lipid production in thraustochytrids
under higher glycerol concentration. Algal Res.
https://doi.org/10.1016/j.algal.2016.02.024.
Doi, K., Honda, D., 2017. Proposal of Monorhizochytrium globosum gen. nov.,
comb. nov. (Stramenopiles, Labyrinthulomycetes) for former
Thraustochytrium globosum based on morphological features and
phylogenetic relationships. Phycol. Res. https://doi.org/
10.1111/pre.12175.
Du, H., Liao, X., Gao, Z., Li, Y., Lei, Y., Chen, W., Chen, L., Fan, X., Zhang, K., Chen,
S., 2019. Effects of methanol on biomass, and fatty acid and carotenoid
biosynthesis in Schizochytrium limacinum B4D1. Appl. Environ. Microbiol.
https://doi.org/10.1128/ AEM.01243-19.
Fan, K.W., Jiang, Y., Faan, Y.W., Chen, F., 2007. Lipid characterization of mangrove
thraustochytrid-Schizochytrium mangrovei. J. Agric. Food Chem.
https://doi.org/ 10.1021/jf070058y.
Fisch, K.M., Bakeer, W., Yakasai, A.A., Song, Z., Pedrick, J., Wasil, Z., Bailey, A.M.,
Lazarus, C.M., Simpson, T.J., Cox, R.J., 2011. Rational domain swaps decipher
programming in fungal highly reducing polyketide synthases and resurrect an
extinct metabolite. J. Am. Chem. Soc. https://doi.org/10.1021/ja206914q.
Ganuza, E., Benítez, S.T., Atalah, Vega, O.O., Ganga, R., Izquierdo, M.S., 2008.
Crypthecodinium cohnii and Schizochytrium sp. as potential substitutes to
fisheries- derived oils from seabream (Sparus aurata) microdiets. Aquaculture.
https://doi. org/10.1016/j.aquaculture.2008.02.005.
Gładkowski, W., Kiełbowicz, G., Chojnacka, A., Bobak, Ł., Spychaj, R., Dobrzanski,
Z., ´ Trziszka, T., Wawrzenczyk, C., 2014. The effect of feed supplementation
with dietary ´ sources of n-3 polyunsaturated fatty acids, flaxseed and algae
Schizochytrium sp., on their incorporation into lipid fractions of Japanese
quail eggs. Int. J. Food Sci.
Technol. https://doi.org/10.1111/ijfs.12497.
Goldstein, S., Belsky, M., 1964. Avenic culture studies of a new marine
Phycomycete possessing an unusual type of asexual reproduction. Am. J.
Bot. https://doi.org/ 10.1002/j.1537-2197.1964.tb06602.x.
Guo, D.S., Ren, L.J., Yin, F.W., Huang, H., Li, G.L., 2016. Development of a real-
time bioprocess monitoring method for docosahexaenoic acid production
by Schizochytrium sp. Bioresour. Technol. https://doi.org/10.1016/j.
biortech.2016.05.044.
Guo, D.S., Ji, X.J., Ren, L.J., Li, G.L., Huang, H., 2017. Improving docosahexaenoic
acid production by Schizochytrium sp. using a newly designed high-oxygen-
supply bioreactor. AICHE J. https://doi.org/10.1002/aic.15783.
Guo, D.S., Ji, X.J., Ren, L.J., Li, G.L., Sun, X.M., Chen, K.Q., Gao, S., Huang, H.,
2018. Development of a scale-up strategy for fermentative production of
docosahexaenoic acid by Schizochytrium sp. Chem. Eng. Sci.
https://doi.org/10.1016/j.
ces.2017.11.021.
Guo, D.S., Tong, L.L., Ji, X.J., Ren, L.J., Ding, Q.Q., 2020. Development of a
strategy to improve the stability of culture environment for
docosahexaenoic acid fermentation by Schizochytrium sp. Appl. Biochem.
Biotechnol. https://doi.org/10.1007/s12010- 020-03298-7.
Gupta, A., Abraham, R.E., Barrow, C.J., Puri, M., 2015. Omega-3 fatty acid
production from enzyme saccharified hemp hydrolysate using a novel
marine Thraustochytrid strain. Bioresour. Technol.
https://doi.org/10.1016/j.biortech.2014.11.031.
Han, X., Zhao, Z.N., Wen, Y., Chen, Z., 2020. Enhancement of docosahexaenoic
acid production by overexpression of ATP-citrate lyase and acetyl-CoA
carboxylase in Schizochytrium sp. Biotechnol. Biofuels.
https://doi.org/10.1186/s13068-020- 01767-z.
Harris, J., Viner, K., Champagne, P., Jessop, P.G., 2018. Advances in microalgal
lipid extraction for biofuel production: a review: microalgal lipid extraction
for biofuel production. Biofuels Bioprod. Biorefin.
https://doi.org/10.1002/bbb.1923.
Hauvermale, A., Kuner, J., Rosenzweig, B., Guerra, D., Diltz, S., Metz, J.G., 2006.
Fatty acid production in Schizochytrium sp.: involvement of a
polyunsaturated fatty acid synthase and a type I fatty acid synthase. Lipids.
https://doi.org/10.1007/s11745- 006-5025-6.
Hayashi, S., Satoh, Y., Ujihara, T., Takata, Y., Dairi, T., 2016. Enhanced production
of polyunsaturated fatty acids by enzyme engineering of tandem acyl
carrier proteins. Sci. Rep-UK. https://doi.org/10.1038/srep35441.
Hayashi, S., Satoh, Y., Ogasawara, Y., Maruyama, C., Hamano, Y., Ujihara, T., Dairi,
T., 2019. Control mechanism for cis double-bond formation by
polyunsaturated fatty- acid synthases. Angew. Chem. Int. Ed.
https://doi.org/10.1002/anie.201812623.
Hoang, C., Nguyen, S., Chia, H., Yu, Y.K., Dinh, T., 2018. Sugarcane bagasse as a
novel carbon source for heterotrophic cultivation of oleaginous microalga
Schizochytrium sp. Ind. Crop. Prod.
https://doi.org/10.1016/j.indcrop.2018.05.005.
Hoang, M.H., Nguyen, C., Pham, H.Q., Van, N.L., Hoang, D.L., Van, S.L., Hai, T.N.,
Ha, C. H., Nhan, L.D., Anh, H.T.L., 2016. Transcriptome sequencing and
comparative analysis of Schizochytrium mangrovei PQ6 at different
14
F. Du et al.
cultivation times. Biotechnol. Lett. https://doi.org/10.1007/s10529-016-
2165-5.
Hong, W.K., Heo, S.Y., Park, H.M., Kim, C.H., Sohn, J.H., Kondo, A., Seo, J.W.,
2013a.
Characterization of a squalene synthase from the thraustochytrid microalga
Aurantiochytrium sp. KRS101. J. Microbiol. Biotechnol.
https://doi.org/10.4014/ jmb.1212.12023.
Hong, W.K., Yu, A., Oh, B.R., Park, J.M., Kim, C.H., Sohn, J.H., Kondo, A., Seo, J.W.,
2013b. Large-scale production of microalgal lipids containing high levels of
docosahexaenoic acid upon fermentation of Aurantiochytrium sp. KRS101.
Food Nutr.
Res. https://doi.org/10.4236/fns.2013.49A1001.
Hsin-Ju, Hsieh, Su, Chia-Hung, Chien, Liang-Jung, 2012. Accumulation of lipid
production in Chlorella minutissima by triacylglycerol biosynthesis-related
genes cloned from Saccharomyces cerevisiae and Yarrowia lipolytica. J.
Microbiol. https:// doi.org/10.1007/s12275-012-2041-5.
Huang, J., Aki, T., Yokochi, T., Nakahara, T., Honda, D., Kawamoto, S., Shigeta, S.,
Ono, K., Suzuki, O., 2003. Grouping newly isolated docosahexaenoic acid-
producing thraustochytrids based on their polyunsaturated fatty acid
profiles and comparative analysis of 18S rRNA genes. Mar. Biotechnol. 5,
450–457. https://doi.org/10.1007/ s10126-002-0110-1.
Huang, J., Jiang, X., Zhang, X., Chen, W., Hu, S., 2008. Expressed sequence tag
analysis of marine fungus Schizochytrium producing docosahexaenoic acid.
J. Biotechnol. https://doi.org/10.1016/j.jbiotec.2008.07.1994.
Huang, T.Y., Lu, W.C., Chu, I.M., 2012. A fermentation strategy for producing
docosahexaenoic acid in Aurantiochytrium limacinum SR21 and increasing
C22:6 proportions in total fatty acid. Bioresour. Technol.
https://doi.org/10.1016/j.
biortech.2012.07.068.
Humhal, T., Kastanek, P., Jezkova, Z., Cadkova, A., Kohoutkova, J., Branyik, T.,
2017.
Use of saline waste water from demineralization of cheese whey for
cultivation of Schizochytrium limacinum PA-968 and Japonochytrium
marinum AN-4. Bioprocess Biosyst. Eng. https://doi.org/10.1007/s00449-
016-1707-5.
Innis, S.M., 2008. Dietary omega 3 fatty acids and the developing brain. Brain
Res. https://doi.org/10.1016/j.brainres.2008.08.078.
Irshad, A., Sharma, A., Daniell, H., Shashi, K., 2015. Altered lipid composition
and enhanced lipid production in green microalga by introduction of
brassica diacylglycerol acyltransferase 2. Plant Biotechnol. J.
https://doi.org/10.1111/ pbi.12278.
Jakobsen, A.N., Aasen, I.M., Josefsen, K.D., Strøm, A.R., 2008. Accumulation of
docosahexaenoic acid-rich lipid in thraustochytrid Aurantiochytrium sp.
strain T66: effects of N and P starvation and O-2 limitation. Appl. Microbiol.
Biotechnol. https:// doi.org/10.1007/s00253-008-1537-8.
Jiang, Y., Fan, K.W., Wong, R.D.Y., Chen, F., 2004. Fatty acid composition and
squalene content of the marine microalga Schizochytrium mangrovei. J.
Agric. Food Chem. https://doi.org/10.1021/jf035004c.
Jon Meadus, W., Duff, P., Rolland, D., Lynn Aalhus, J., Uttaro, B., Russell Dugan,
M.E., 2011. Feeding docosahexaenoic acid to pigs reduces blood
triglycerides and induces gene expression for fat oxidation. Can. J. Chem.
https://doi.org/10.4141/cjas2011- 055.
Kim, H.U., Huang, A.H.C., 2004. Plastid lysophosphatidyl acyltransferase is
essential for embryo development in arabidopsis. Plant Physiol.
https://doi.org/10.1104/ pp.103.035832.
Lee Chang, K.J., Dumsday, G., Nichols, P.D., Dunstan, G.A., Blackburn, S.I.,
Koutoulis, A., 2013. High cell density cultivation of a novel
Aurantiochytrium sp. strain TC 20 in a fed-batch system using glycerol to
produce feedstock for biodiesel and omega-3 oils.
Appl. Microbiol. Biotechnol. https://doi.org/10.1007/s00253-013-4965-z.
Leong, H.Y., Su, C.A., Lee, B.S., Lan, C.W., Law, C.L., Chang, J.S., Show, P.L., 2019.
Development of Aurantiochytrium limacinum SR21 cultivation using salt-
rich waste feedstock for docosahexaenoic acid production and application
of natural colourant in food product. Bioresour. Technol.
https://doi.org/10.1016/j.
biortech.2018.09.093.
Leyland, Ben, Leu, Stefan, Boussiba, Sammy, 2017. Are Thraustochytrids algae?
Fungal. Biol. Rev. https://doi.org/10.1016/j.funbio.2017.07.006.
Li, J., Liu, R., Chang, G., Li, X., Chang, M., Liu, Y., Jin, Q., Wang, X., 2015. A
strategy for the highly efficient production of docosahexaenoic acid by
Aurantiochytrium limacinum SR21 using glucose and glycerol as the mixed
carbon sources. Bioresour. Technol.
https://doi.org/10.1016/j.biortech.2014.11.046.
Li, J., Zhou, H., Pan, X., Li, Z., Ling, X., 2019a. The role of fluconazole in the
regulation of fatty acid and unsaponifiable matter biosynthesis in
Schizochytrium sp. MYA 1381.
BMC Microbiol. https://doi.org/10.1186/s12866-019-1622-4.
Li, Q., Chen, G.Q., Fan, K.W., Lu, F.P., Aki, T., Jiang, Y., 2009. Screening and
characterization of squalene-producing Thraustochytrids from Hong Kong
mangroves. J. Agric. Food Chem. https://doi.org/10.1021/jf9003972.
Li, Z., Chen, X., Li, J., Meng, T., Wang, L., Chen, Z., Shi, Y., Ling, X., Luo, W., Liang,
D., Lu, Y., Li, Q., He, N., 2018a. Functions of PKS genes in lipid synthesis of
Schizochytrium sp. by gene disruption and metabolomics analysis. Mar.
Biotechnol. https://doi.org/10.1007/s10126-018-9849-x.
Biotechnology Advances 48 (2021) 107725
Li, Z., Meng, T., Ling, X.P., Li, J., He, N., 2018b. Overexpression of malonyl-CoA:
Acp transacylase in Schizochytrium sp. to improve polyunsaturated fatty
acids production. J. Agric. Food Chem.
https://doi.org/10.1021/acs.jafc.8b01026.
Li, Z., Ling, X., Zhou, H., Meng, T., Zeng, J., Hang, W., Shi, Y., He, N., 2019b.
Screening chemical modulators of benzoic acid derivatives to improve lipid
accumulation in Schizochytrium limacinum SR21 with metabolomics
analysis. Biotechnol. Biofuels. https://doi.org/10.1186/s13068-019-1552-2.
Lin, H.X., Lee, Y.K., 2017. Genetic engineering of medium-chain-length fatty acid
synthesis in Dunaliella tertiolecta for improved biodiesel production. J.
Appl.
Phycol. https://doi.org/10.1007/s10811-017-1210-7.
Liang, F., Lindblad, P., 2017. Synechocystis PCC 6803 overexpressing RuBisCO
grow faster with increased photosynthesis. Metab. Eng. Commun.
https://doi.org/ 10.1016/j.meteno.2017.02.002.
Liang, Y.N., Sarkany, N., Cui, Y., Yesuf, J., Trushenski, J., Blackburn, J.W., 2010. Use
of sweet sorghum juice for lipid production by Schizochytrium limacinum
SR21.
Bioresour. Technol. https://doi.org/10.1016/j.biortech.2009.12.087.
Lippmeier, J.C., Crawford, K.S., Owen, C.B., Rivas, A.A., Metz, J.G., Apt, K.E.,
2009. Characterization of both polyunsaturated fatty acid biosynthetic
pathways in Schizochytrium sp. Lipids. https://doi.org/10.1007/s11745-009-
3311-9.
Liu, T., Sanchez, J.F., Chiang, Y.M., Oakley, B.R., Wang, C.C.C., 2014a. Rational
domain swaps reveal insights about chain length control by ketosynthase
domains in fungal nonreducing polyketide synthases. Org. Lett.
https://doi.org/10.1021/ol5003384.
Liu, Y., Tang, J., Li, J., Daroch, M., Cheng, J.J., 2014b. Efficient production of
triacylglycerols rich in docosahexaenoic acid (DHA) by osmo-heterotrophic
marine protists. Appl. Microbiol. Biotechnol.
https://doi.org/10.1007/s00253-014-6032-9.
Liu, Z.X., You, S., Tang, B.P., Wang, B., Sheng, S., Wu, F.A., Wang, J., 2019. Inositol
as a new enhancer for improving lipid production and accumulation in
Schizochytrium sp. SR21. Environ. Sci. Pollut. Res.
https://doi.org/10.1007/s11356-019-06056-3.
Makri, A., Fakas, S., Aggelis, G., 2010. Metabolic activities of biotechnological
interest in Yarrowia lipolytica grown on glycerol in repeated batch cultures.
Bioresour. Technol. https://doi.org/10.1016/j.biortech.2009.11.024.
Marchan, L.F., Lee Chang, K.J., Nichols, P.D., Polglase, J.L., Mitchell, W.J.,
Gutierrez, T., 2017. Screening of new British Thraustochytrids isolates for
docosahexaenoic acid (DHA) production. J. Appl. Phycol.
https://doi.org/10.1007/s10811-017-1149-8.
Meesapyodsuk, D., Qiu, X., 2016. Biosynthetic mechanism of very long chain
polyunsaturated fatty acids in Thraustochytrium sp. 26185. J. Lipid Res.
https://doi. org/10.1194/jlr.M070136.
Merkx, J.A., Rasmussen, H., Muise, D.M., Benjamin, J.J.R., Kottwitz, H., Tanner,
K., Milway, M.T., Purdue, L.M., Scaife, M.A., Armenta, R.E., Woodhall, D.L.,
2018. Engineering xylose metabolism in thraustochytrid T18. Biotechnol.
Biofuels. https:// doi.org/10.1186/s13068-018-1246-1.
Miller, M.R., Nichols, P.D., Carter, C.G., 2007. Replacement of fish oil with
thraustochytrid Schizochytrium sp. L oil in Atlantic salmon parr (Salmo salar
L) diets. Comp. Biochem. Phys. Part A.
https://doi.org/10.1016/j.cbpa.2007.05.018.
Moran, C.A., Morlacchini, M., Keegan, J.D., Rutz, F., Fusconi, G., 2020.
Docosahexaenoic acid enrichment of layer hen tissues and eggs through
dietary supplementation with heterotrophically grown Aurantiochytrium
limacinum. J. Appl. Poult. Res. https://doi.
org/10.1016/j.japr.2019.10.002.
Moreno-P´erez, A.J., Sanchez-García, A., Salas, J.J., Garc´ ´es, R., Martínez-Force,
E., 2011. Acyl-ACP thioesterases from macadamia (Macadamia tetraphylla)
nuts: cloning, characterization and their impact on oil composition. Plant
Physiol. Biochem.
https://doi.org/10.1016/j.plaphy.2010.10.002.
Nagano, N., Sakaguchi, K., Taoka, Y., Okita, Y., Honda, D., Ito, M., Hayashi, M.,
2011. Detection of genes involved in fatty acid elongation and desaturation
in Thraustochytrid Marine Eukaryotes. J. Oleo Sci.
https://doi.org/10.5650/jos.60.475.
Papanikolaou, S., Aggelis, G., 2011. Lipids of oleaginous yeasts. Part I:
Biochemistry of single cell oil production. Eur. J. Lipid Sci. Technol. 113 (8),
1031–1051. https://doi. org/10.1002/ejlt.201100014.
Papanikolaou, S., Sarantou, S., Komaitis, M., Aggelis, G., 2010. Repression of
reserve lipid turnover in Cunninghamella echinulata and Mortierella
isabellina cultivated in multiple-limited media. J. Appl. Microbiol.
https://doi.org/10.1111/j.1365- 2672.2004.02376.x.
Parsons, J.B., Rock, C., 2013. Bacterial lipids: metabolism and membrane
homeostasis.
Prog. Lipid Res. https://doi.org/10.1016/j.plipres.2013.02.002.
Patel, A., Rova, U., Christakopoulos, P., Matsakas, L., 2019. Simultaneous
production of DHA and squalene from Aurantiochytrium sp. grown on
forest biomass hydrolysates.
Biotechnol. Biofuels. https://doi.org/10.1186/s13068-019-1593-6.
Pereira, S.L., Huang, Y.S., Bobik, E.G., Kinney, A.J., Stecca, K.L., Packer, J.C.L.,
Mukerji, P., 2004. A novel omega 3-fatty acid desaturase involved in the
15
F. Du et al.
biosynthesis of eicosapentaenoic acid. Biochem. J.
https://doi.org/10.1042/BJ20031319.
Pour, A.K., Mirmehrabi, M., Brar, S.K., 2020. A novel process for isolation and
purification of polyunsaturated fatty acids from a thraustochytrid. Algal Res.
https:// doi.org/10.1016/j.algal.2020.101806.
Pyle, D.J., Garcia, R.A., Wen, Z., 2008. Producing docosahexaenoic acid (DHA)-
rich algae from biodiesel-derived crude glycerol: effects of impurities on
DHA production and algal biomass composition. J. Agric. Food Chem.
https://doi.org/10.1021/ jf800602s.
Qi, F., Zhang, M., Chen, Y., Jiang, X., Lin, J., Cao, X., Huang, J., 2017. A
lignocellulosic hydrolysate-tolerant Aurantiochytrium sp. mutant strain for
docosahexaenoic acid production. Bioresour. Technol.
https://doi.org/10.1016/j.biortech.2016.12.011.
Qiao, K., Wasylenko, T.M., Zhou, K., Xu, P., Stephanopoulos, G., 2017. Lipid
production in Yarrowia lipolytica is maximized by engineering cytosolic
redox metabolism. Nat. Biotechnol. https://doi.org/10.1038/nbt.3763.
Qu, L., Ren, L.J., Huang, H., 2013. Scale-up of docosahexaenoic acid production
in fed- batch fermentation by Schizochytrium sp. based on volumetric
oxygen-transfer coefficient. Biochem. Eng. J.
https://doi.org/10.1016/j.bej.2013.05.011.
Ratledge, C., 2012. Omega-3 biotechnology: errors and omissions. Biotechnol.
Adv. https://doi.org/10.1016/j.biotechadv.2012.04.002.
Ren, L.J., Zhuang, X.Y., Chen, S.L., Ji, X.J., Huang, H., 2015. Introduction of ω-3
desaturase obviously changed the fatty acid profile and sterol content of
Schizochytrium sp. J. Agric. Food Chem.
https://doi.org/10.1021/acs.jafc.5b04238.
Ren, L.J., Sun, G.N., Ji, X.J., Hu, X.C., Huang, H., 2014. Compositional shift in lipid
fractions during lipid accumulation and turnover in Schizochytrium sp.
Bioresour. Technol. https://doi.org/10.1016/j.biortech.2014.01.078.
Ren, L.J., Sun, X.M., Ji, X.J., Chen, S.L., Guo, D.S., Huang, H., 2017. Enhancement
of docosahexaenoic acid synthesis by manipulation of antioxidant capacity
and prevention of oxidative damage in Schizochytrium sp. Bioresour.
Technol. https:// doi.org/10.1016/j.biortech.2016.10.040.
Ren, L.J., Chen, S.L., Geng, L.J., Ji, X.J., Xu, X., Song, P., Gao, S., Huang, H., 2018.
Exploring the function of acyltransferase and domain replacement in order
to change the polyunsaturated fatty acid profile of Schizochytrium sp. Algal
Res. https://doi. org/10.1016/j.algal.2017.11.021.
Ruiz-Rosa, I., Garcia-Rodriguez, F.J., Mendoza-Jimenez, J., 2016. Development
and application of a cost management model for wastewater treatment
and reuse processes. J. Clean. Prod.
https://doi.org/10.1016/j.jclepro.2015.12.044.
Ryu, B.G., Kim, K., Kim, J., Han, J.I., Yang, J.W., 2013. Use of organic waste from
the brewery industry for high-density cultivation of the docosahexaenoic
acid-rich microalga, Aurantiochytrium sp. KRS101. Bioresour. Technol.
https://doi.org/ 10.1016/j.biortech.2012.11.049.
Sakaguchi, K., Matsuda, T., Kobayashi, T., Ohara, J., Hamaguchi, R., Abe, E.,
Nagano, N., Hayashi, M., Ueda, M., Honda, D., Okita, Y., Taoka, Y., Sugimoto,
S., Okino, N., Ito, M., 2012. Versatile transformation system that is
applicable to both multiple transgene expression and gene targeting for
Thraustochytrids. Appl. Environ.
Microbiol. https://doi.org/10.1128/AEM.07129-11.
Schellenberg, G.D., Sarthy, A., Larson, A.E., Backer, M.P., Furlong, C.E., 1984.
Xylose isomerase from Escherichia coli. Characterization of the protein and
the structural gene. J. Biol. Chem. https://doi.org/10.1007/978-3-540-
85387-9_1.
Schmidt, I., Schewe, H., Gassel, S., Jin, C., Buckingham, J., Hümbelin, M.,
Sandmann, G., Schrader, J., 2011. Biotechnological production of
astaxanthin with Phaffia rhodozyma/Xanthophyllomyces dendrorhous.
Appl. Microbiol. Biotechnol. https://doi. org/10.1007/s00253-010-2976-6.
Shirasaka, N., Hirai, Y., Nakabayashi, H., Yoshizumi, H., 2005. Effect of
cyanocobalamin and p-toluic acid on the fatty acid composition of
Schizochytrium limacinum (Thraustochytriaceae, Labyrinthulomycota).
Mycoscience. https://doi.org/10.1007/ s10267-005-0259-3.
Singh, D., Mathur, A.S., Tuli, D.K., Puri, M., Barrow, C.J., 2015. Propyl gallate and
butylated hydroxytoluene influence the accumulation of saturated fatty
acids, omega-3 fatty acid and carotenoids in Thraustochytrids. J. Funct.
Foods. https://doi. org/10.1016/j.jff.2015.03.022.
Song, X., Ma, Z., Tan, Y., Zhang, H., Cui, Q., 2017. Wastewater recycling
technology for fermentation in polyunsaturated fatty acid production.
Bioresour. Technol. https:// doi.org/10.1016/j.biortech.2017.03.034.
Suen, Y.L., Tang, H., Huang, J., Chen, F., 2014. Enhanced production of fatty acids
and astaxanthin in Aurantiochytrium sp. by the expression of Vitreoscilla
hemoglobin. J. Agric. Food Chem. https://doi.org/10.1021/jf5048578.
Sun, H., Chen, H., Zang, X., Hou, P., Zhou, B., Liu, Y., Wu, F., Cao, X., Zhang, X.,
2015. Application of the Cre/loxP site-specific recombination system for
gene transformation in Aurantiochytrium limacinum. Molecules.
https://doi.org/10.3390/ molecules200610110.
Sun, L.N., Ren, L.J., Zhuang, X.Y., Ji, X.J., Yan, J.C., Huang, H., 2014. Differential
effects of nutrient limitations on biochemical constituents and
docosahexaenoic acid production of Schizochytrium sp. Bioresour. Technol.
https://doi.org/10.1016/j.
biortech.2014.02.106.
Sun, X.M., Ren, L.J., Bi, Z.Q., Ji, X.J., Zhao, Q.Y., Jiang, L., Huang, H., 2018.
Development of a cooperative two-factor adaptive-evolution method to
Biotechnology Advances 48 (2021) 107725
enhance lipid production and prevent lipid peroxidation in Schizochytrium
sp. Biotechnol.
Biofuels. https://doi.org/10.1186/s13068-018-1065-4.
Takanori, M., Keishi, S., Rie, H., Takumi, K., Eriko, A., Yoichiro, H., Masahiro, H.,
Daiske, H., Yuji, O., Shinichi, S., Nozomu, O., Makoto, Ito, 2012. Analysis of
12-fatty acid desaturase function revealed that two distinct pathways are
active for the synthesis of PUFAs in T. aureum ATCC 34304. J. Lipid Res.
https://doi.org/10.1016/ j.jpainsymman.2014.08.012.
Tang, S.K., Qin, C.R., Wang, H.G., Li, S.F., Tian, S.J., 2011. Study on supercritical
extraction of lipids and enrichment of DHA from oil-rich microalgae. J.
Supercrit.
Fluids. https://doi.org/10.1016/j.supflu.2011.01.010.
Varela, J., Pereira, H., Vila, M., Leon, R., 2015. Production of carotenoids by
microalgae: ´ achievements and challenges. Photosynth. Res.
https://doi.org/10.1007/s11120- 015-0183-0.
Villarroel Hipp, M.P., Silva Rodriguez, D., 2018. Bioremediation of piggery
slaughterhouse wastewater using the marine protist, Thraustochytrium
kinney VAL- B1. J. Adv. Res. https://doi.org/10.1016/j.jare.2018.01.010.
Wang, F., Bi, Y., Diao, J., Lv, M., Zhang, W., 2019. Metabolic engineering to
enhance biosynthesis of both docosahexaenoic acid and odd-chain fatty
acids in
Schizochytrium sp. S31. Biotechnol. Biofuels.
https://doi.org/10.1186/s13068-019- 1484-x.
Wang, K., Sun, T., Cui, J., Liu, L., Bi, Y., Pei, G., Chen, L., Zhang, W., 2018a.
Screening of chemical modulators for lipid accumulation in Schizochytrium
sp S31. Bioresour.
Technol. https://doi.org/10.1016/j.biortech.2018.03.104.
Wang, Q., Sen, B., Liu, X., He, Y., Xie, Y., Wang, G., 2018b. Enhanced saturated
fatty acids accumulation in cultures of newly-isolated strains of
Schizochytrium sp. and Thraustochytriidae sp. for large-scale biodiesel
production. Sci. Total Environ.
https://doi.org/10.1016/j.scitotenv.2018.03.078.
Wang, S., Lan, C., Wang, Z., Wan, W., Song, X., 2020. Optimizing
eicosapentaenoic acid production by grafting a heterologous polyketide
synthase pathway in the Thraustochytrid Aurantiochytrium. J. Agric. Food
Chem. https://doi.org/10.1021/acs. jafc.0c04299.
Wang, Z.X., Jian, Z.G., Fang, H., Prior, B.A., 2001. Glycerol production by
microbial fermentation: a review. Biotechnol. Adv.
https://doi.org/10.1016/S0734-9750(01) 00060-X.
Xie, X., Meesapyodsuk, D., Qiu, X., 2017. Ketoacylsynthase domains of a PUFA
synthase in Thraustochytrium can effectively function as stand-alone
enzymes in Escherichia coli. Appl. Environ. Microbiol.
https://doi.org/10.1128/AEM.03133-16.
Xin, F., Wu, Y.R., He, J., 2014. Simultaneous fermentation of glucose and xylose
to butanol by Clostridium sp. strain BOH3. Appl. Environ. Microbiol.
https://doi.org/ 10.1128/AEM.00337-14.
Yamasaki, T., Aki, T., Shinozaki, M., Taguchi, M., Kawamoto, S., Ono, K., 2006.
Utilization of Shochu distillery wastewater for production of
polyunsaturated fatty acids and xanthophylls using Thraustochytrid. J.
Biosci. Bioeng. https://doi.org/ 10.1016/j.enzmictec.2017.11.009.
Yan, J., Cheng, R., Lin, X., You, S., Ma, Y., 2012. Overexpression of acetyl-CoA
synthetase increased the biomass and fatty acid proportion in microalga
Schizochytrium. Appl. Microbiol. Biotechnol. 97 (5), 1933–1939.
https://doi.org/10.1007/s00253-012- 4481-6.
Ye, J.R., Liu, M.M., He, M.X., Ye, Y., Huang, J.C., 2019. Illustrating and enhancing
the biosynthesis of astaxanthin and docosahexaenoic acid in
Aurantiochytrium sp. SK4.
Mar. Drugs. https://doi.org/10.3390/md17010045.
Ye, H.K., He, Y.D., Xie, Y.X., Sen, B., Wang, G.Y., 2020. Fed-batch fermentation of
mixed carbon source significantly enhances the production of
docosahexaenoic acid in Thraustochytriidae sp PKU#Mn16 by differentially
regulating fatty acids biosynthetic pathways. Bioresour. Technol.
https://doi.org/10.1016/j.
biortech.2019.122402.
Yin, F.W., Guo, D.S., Ren, L.J., Ji, X.J., Huang, H., 2018a. Development of a
method for the valorization of fermentation wastewater and algal-residue
extract in docosahexaenoic acid production by Schizochytrium sp.
Bioresour. Technol. https:// doi.org/10.1016/j.biortech.2018.06.109.
Yin, F.W., Zhang, Y.T., Jiang, J.Y., Guo, D.S., Gao, S., Gao, Z., 2018b. Efficient
docosahexaenoic acid production by Schizochytrium sp. via a two-phase pH
control strategy using ammonia and citric acid as pH regulators. Process
Biochem. https:// doi.org/10.1016/j.procbio.2018.11.013.
Yokoyama, R., Honda, D., 2007. Taxonomic rearrangement of the genus
Schizochytrium sensu lato based on morphology, chemotaxonomic
characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae,
Labyrinthulomycetes): emendation for Schizochytrium and erection of
Aurantiochytrium and Oblongichytrium gen. nov.
Mycoscience. https://doi.org/10.1007/s10267-006-0362-0.
Yu, X.J., Sun, J., Zheng, J.Y., Sun, Y.Q., Wang, Z., 2016. Metabolomics analysis
reveals 6- benzylaminopurine as a stimulator for improving lipid and DHA
accumulation of Aurantiochytrium sp. J. Chem. Technol. Biotechnol.
https://doi.org/10.1002/ jctb.4869.
Zhang, A.Q., Xie, Y.X., He, Y.D., Wang, W.J., Sen, B., Wang, G.Y., 2019. Bio-based
squalene production by Aurantiochytrium sp. through optimization of
16
F. Du et al. Biotechnology Advances 48 (2021) 107725
culture conditions, and elucidation of the putative biosynthetic pathway
genes. Bioresour.
Technol. https://doi.org/10.1016/j.biortech.2019.121415.
Zhang, K., Chen, L., Liu, J., Gao, F., He, R., Chen, W., Guo, W., Chen, S., Li, D.,
2017. Effects of butanol on high value product production in
Schizochytrium limacinum B4D1. Enzym. Microb. Technol.
https://doi.org/10.1016/j.enzmictec.2017.03.007.
Zhang, L., Zhao, H., Lai, Y., Wu, J., Chen, H., 2013. Improving docosahexaenoic
acid productivity of Schizochytrium sp by a two-stage AEMR/shake mixed
culture mode.
Bioresour. Technol. https://doi.org/10.1016/j.biortech.2013.05.072.
Zhang, Y., Ward, V., Dennis, D., Plechkova, N.V., Armenta, R., Rehmann, L., 2018.
Efficient extraction of a docosahexaenoic acid (DHA)-rich lipid fraction from
Thraustochytrium sp. using lonic liquids. Mater. https://doi.org/10.3390/
ma11101986.
Zhao, X., Ren, L., Guo, D., Wu, W., Ji, X., Huang, H., 2016. CFD investigation of
Schizochytrium sp impeller configurations on cell growth and
docosahexaenoic acid synthesis. Bioprocess Biosyst. Eng.
https://doi.org/10.1007/s00449-016-1608-7.
Zheng, H.L., Gao, Z., Yin, F.W., Ji, X.J., Huang, H., 2012. Lipid production of
Chlorella vulgaris from lipid-extracted microalgal biomass residues through
two-step enzymatic hydrolysis. Bioresour. Technol.
https://doi.org/10.1016/j. biortech.2012.04.007.

17