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ScienceDirect
Recent advances in biotechnology for marine enzymes
and molecules
Jingyu Zhang1,3, Lan Jiang1,3, Xiangyin Chen1,3, Kangjie Lv1,
Mostafa Basiony1, Guoliang Zhu1, Loganathan Karthik2,
Liming Ouyang1, Lixin Zhang1 and Xueting Liu1
The marine environment is the most biologically and chemically
diverse habitat on Earth, and provides numerous marine-
derived products, including enzymes and molecules, for
industrial and pharmaceutical applications. Marine
biotechnology provides important biological resources from
marine habitat conservation to applied science. In recent years,
advances in techniques in interdisciplinary research fields,
including metabolic engineering and synthetic biology have
significantly improved the production of marine-derived
commodities. In this review, we outline the recent progress in
the use or marine enzymes and molecules in biotechnology,
including newly discovered products, function optimization of
enzymes, and production improvement of small molecules.
Addresses
1
State Key Laboratory of Bioreactor Engineering, East China University
of Science and Technology, Shanghai 200237, China
2
R and D Center, Salem Microbes Private Limited, Salem, Tamil Nadu
636301, India
Corresponding author: Liu, Xueting (liuxueting@ecust.edu.cn)
3
These authors contributed to this work equally.
Current Opinion in Biotechnology 2021, 69:308–315
This review comes from a themed issue on Chemical biotechnology
Edited by Ajikumar Parayil and Christine Santos
For a complete overview see the Issue and the Editorial
Available online 8th June 2021
https://doi.org/10.1016/j.copbio.2021.05.009
0958-1669/ã 2021 Elsevier Ltd. All rights reserved.
Introduction
Marine organisms are a recent research focus due to their
ability to provide a huge source of products, including
enzymes and small molecules, for applications in the
medical, agricultural, and fine chemical industries [1

].
Many marine enzymes possess remarkable properties,
including hyperthermostability, salt tolerance, barophili-
city, cold adaptability, chemoselectivity, regioselectivity,
and stereoselectivity. These new chemical and stereo-
chemical characteristics enable novel industrial uses [2
].
In the last decade, biocatalytic processes catalyzed by
marine enzymes, including oxidoreductases, hydrolases,
Current Opinion in Biotechnology 2021, 69:308–315
transferases, isomerases, ligases, and lyases, have become
of great interest due to the usefulness of these products in
biotechnological applications [1

,3–6]. Marine organisms
also produce numerous bioactive compounds, such as
antibiotics, probiotics, melanins, and anticancer sub-
stances, for pharmaceutical applications [7,8]. Owing to
their limited availability, bulk production of marine
products with biotechnology and synthetic biology
approaches has become extremely important for their
further applications.
In this review, we summarize the recent progress on
marine enzymes and molecules with a particular focus
on enzymes used for production of high-value added
lipids and biofuels, and enzymes used for healthcare
and bioremediation. Additionally, we briefly outline the
production of several marine-derived small molecules
used for pharmaceutical applications by systems biology
and metabolic engineering.
Marine enzymes as the catalyst for lipid and
biofuel production in biotechnological
processes
Lipids are a group of compounds including fats, oils,
hormones, and components of membranes. Among the
marine-derived lipids, which are produced by cyanobac-
teria [9] and microalgae [10
], triglycerides (TAGs) and
poly-unsaturated fatty acids (PUFAs) have been the most
studied for biotechnological applications (Figure 1).
TAGs can be used to produce biodiesels and nutraceu-
ticalsc, while PUFA products are widely used for human
health, especially V-3 fatty acids such as docosahexaenoic
acid (DHA) and eicosapentaenoic acid (EPA) [11]. TAGs
have shown as alternative fuels. However, it is unsatisfied
and impractical for a direct use of oil blends/vegetable oils
because of gum formation and polymerization, high vis-
cosity, and acid composition of those oils [12]. A number
of efforts have gone into developing a renewably, non-
toxic, biodegradable fuel. Traditionally, plants and fish
are the primary sources of PUFA. With the increasing
concerns about food security and environmental contami-
nants in fish oil, alternative resources with more safer,
sustainable, and economical characteristics have been
considered for the production of PUFAs. Obviously,
enzymatic method has proven to be a promising, low-
energy, and environmentally friendly strategy to boost
production of TAG and PUFA.
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Figure 1
Representative marine-derived lipids and their applications.
In biosynthesis of TAG, several enzymes have been
recently assessed for their ability to increase high-value
added lipid production. TAG was converted from acyl
coenzyme A (acyl-CoA) and diacylglycerol (DAG) cata-
lyzed by diacylglycerol acyltransferases (DGATs), which
is the most studied enzyme involved in lipid synthesis
and participates in the final and committed step [13].
Microalgae is an ideal TAG producer, they usually harbor
larger numbers of DGAT2s (type-2 DGAT gene) than
most higher plants or animals, and can accumulate tria-
cylglycerols (TAGs) when subjected to adverse environ-
mental conditions such as high light and nutrient starva-
tion [14]. Chlamydomonas reinhardtii and Nannochloropsis
oceanica have been used to produce TAG and study the
key enzyme in its biosynthesis pathway, such as the gene
dgd1 encoding digalactosyldiacylglycerol synthase, an
enzyme involved in the biosynthesis of digalactosyldia-
cylglycerol (DGDG), was characterized from a mutant of
the photosynthetic unicellular microalgae Nannochloropsis
gaditana proved to be responsible for a nearly 80%
increase in lipid productivity [15]. Additionally, new
technologies provide efficient tools for metabolism engi-
neering. Shin et al. developed a CRISPR-Cas9 system to
carry out the targeted knockout of phospholipase A2, a
key enzyme in the Lands cycle that remodels membrane
lipids in TAG biosynthesis and deposition into lipid
droplets [16] in the green alga C. reinhardtii. This proce-
dure resulted in higher accumulation of triacylglycerol
and the overall lipid production reaching up to 64.25%
(80.92 g/L/d), without hindering growth rate [17].
Marine enzymes due to habitat-related characteristics can
be desirable features recognized from a general biotech-
nological perspective. Haslam et al. conducted co-over-
expression of endogenous DGAT in the marine diatom
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Marine enzymes and molecules Zhang et al. 309
Current Opinion in Biotechnology
strain Phaeodactylum tricornutum and the heterologous
D5-elongase from the picoalga Ostreococcus tauri, which
resulted in a significant increase in DHA levels (3.8–8.5%
compared to 1.8% in the wildtype strain) without nutrient
stress [18]. G6PDH is the rate-limiting enzyme in the
oxidative pentose phosphate pathway (OPPP). Both lipid
content and the total proportions of saturated and unsat-
urated fatty acids are highly increased in high-CO2 culti-
vated G6PDH overexpressing strains [19]. Chen et al.
carried out overexpression of ACCa, the first rate-limiting
step of fatty acids (FAs) biosynthesis, in the model algae
C. reinhardtii and succeeded in increasing content of
unsaturated FAs, and increased the content of lipids
1.16-fold greater than the control [20].
Marine enzymes used for pharmaceutical
applications
Therapeutic enzymes have been well-documented for
their function and clinical implications, including onco-
lytics, thrombolytics, anticoagulants, and replacements
for metabolic disorders [21

]. Because of clinical usage
requirements, enzymes used for therapeutic applications
are required in relatively smaller amounts, but with a
higher degree of purity and specificity, compared to
industrial applications [22]. These requirements increase
the demand for refinement through biotechnology and for
the discovery of new enzymes from unique environments,
including marine habitats. In this section, we focus on
recent progress in investigations of the marine-derived L-
asparaginase and L-glutaminase widely used in cancer
therapy.
L-asparaginaseplays an important role in the treatment of
cancer and has been used in the treatment of acute
lymphoblastic leukemia for half a century [23]. Taking
Current Opinion in Biotechnology 2021, 69:308–315
310 Chemical biotechnology
advantage of leukemic cells and other cancer cells have
little or no asparagine synthetase, L-asparaginase could
depletion of the circulating pool of L-asparagine which
cause the inhibition of the protein synthesis and ultimately
apoptosis in susceptible leukemic cells [24,25]. Although
several microorganisms could produce L-asparaginase, only
Escherichia coli and Dickeya chrysanthemi are used as producer
on an industrial scale for pharmaceutical use [26], and only
the type II L-asparaginase has been used for clinical appli-
cation due to a higher affinity to L-asparagine (Km = 10–
15 mM) [27]. High substrate specificity is crucial for thera-
peutic enzymes, as this is usually the cause of the side
effects. Common side effects when used L-asparaginase,
mainly attributes to the dual activity of L-asparaginase as it
can also hydrolysis L-glutamine to glutamic acid and ammo-
nia. However, commercial L-asparaginase are not
‘glutaminase-free’, hydrolyzes L-glutamine up to 9% of
total hydrolysis activity [26]. Marine organisms have
become an intriguing source for L-asparaginase, due to
their novel structures, lower molecular weights, and high
substrate specificity [28]. They have high affinity for
L-asparagine but a lesser extent conversion of glutaminase
(L-asparagine Km = 1.8 mM, L-glutamine Km = 6.1 mM)
[29]. A marine-derived fungi Aspergillus niger with a high-
yield L-asparaginase could led to an increase of 108.62% in
yield after optimizing the fermentation conditions (from
7.5642 U/mL to 15.7807 U/mL) [30]. A new L-asparaginase
discovered in the marine strain Bacillus velezensis
showed high anti-breast cancer activity with an IC50 of
17.3  2.8 mg/mL, with no glutaminase activity (which
causes side effects such as liver and kidney diseases) [31].
Unlike L-asparaginase which is reported to cause allergic
reactions, L-glutaminase is primarily used to catalyze the
hydrolysis of the amino bond of L-glutamine and can be
used in cancer therapy [32]. For industry production, the
important concern is that most of microorganisms have
low L-asparaginases production. Screening and selection
of new glutaminase producing organisms for the
production of L-glutaminase are particularly desired.
Marine bacteria have increased production of glutamin-
ase than terrestrial microorganisms [33]. Kiruthika et al.
reported an increased production of L-glutaminase from
236.67 U/mL to 672.28 U/mL in marine Bacillus subtilis
JK-79 under solid state fermentation [34]. Further stud-
ies characterized a new extracellular L-glutaminase from
marine B. subtilis JK-79 and B. subtilis OHEM11 with
strong anticancer activity [35,36].
Marine enzymes as catalysts for
bioremediation
Marine is a tremendous trove for novel biocatalysts. Those
biocatalysts derived from marine usually show appealing
properties due to their long term evolution in unfriendly
habitats. Compared with terrestrial biocatalysts, some of the
marine enzyme catalysts perform novel chemical or stereo-
chemical properties [37], including substrate specificity and
Current Opinion in Biotechnology 2021, 69:308–315
enantioselectivity, which shows potential applications in the
industry.
Marine organisms produce many important hydrolytic
enzymes, such as cellulase, xylanase, protease, and chit-
inase, which have been widely used for degrading various
biopolymers to produce biofuels and production of many
biochemicals of interest from renewable biomass [38
].
Cellulase (b-1,4 glucan hydrolase) is an enzyme complex
that possesses several activities like exoglucanase, endo-
glucanase, and b-glucosidase [39]. It is used in the produc-
tion of biofuels by microbial fermentation through sacchar-
ification of lignocellulosic materials, which is the most
abundant biopolymer in nature, and is a renewable and
inexpensive feedstock for biorefinery [40]. Akurathi et al.
used the marine bacterium Streptomyces fungicidicus strain
RPBS-A4 as the production strain and agricultural proces-
sing waste as the medium for the production of cellulase
with an activity of 13.18 U/mL [41]. Ratnakomala et al.
optimized the fermentation medium for the production of
carboxymethyl cellulase (CMCase) by Streptomyces sp. Bse
7–9 and obtained a maximum activity of 4.496 U/mL [42].
Xylanase is one of the most important enzymes that
degrades hemicellulose into fermentable sugars, and is
produced by a wide diversity of marine organisms, such
as algae, bacteria, fungi, protozoa, gastropods, and arthro-
pods [43]. Xylan, one kind of hemicellulose, due to it is the
variety and structural complexity, understanding of the
synergistic effects of xylanolytic enzymes and new candi-
dates are needed for full xylan hydrolysis [44]. Yu et al.
identified a first example of multi-domain high-molecular-
weight xylanase XylM18 from the marine halophilic bacte-
ria Marinimicrobium sp. LS-A18 with potential applications
for xylan biodegradation under high-salt and alkali condi-
tions [45].
Chitinase is the enzyme degrading chitin, a widely dis-
tributed biopolymer composed of b-1,4-linked units of N-
acetyl-D-glucosamine (GlcNAc), to generate GlcNAc and
chitooligosaccharides [46]. Chemical methods are most
frequently applied for GlcNAc industrial production. In
the traditional chemical approach, using high tempreture
(175℃) and sulfuric acid, adding co-solvents an 80%
GlcN yield can be produced in 1 hour, pure GlcNAc
can be obtained after a series purification of GlcN [47].
Current production of GlcNAc requires chemical hydro-
lysis of chitin with low yield and high cost as well as
environmentally unfriendly [48], significant attention has
been focused on the enzymatic hydrolysis of chitin.
Cardozo et al. described the bioconversion of a-chitin
into GlcNAc using the crude enzyme extracts of the
marine bacterium Aeromonas caviae which resulted in a
yield of 93% [49]. That study presented the potential
applications of a relatively inexpensive method for
GlcNAc production from colloidal a-chitin. Two more
highly similar chitinases (Chi1557 and Chi4668) were
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identified from the marine bacteria Vibrio rotiferianus and
Vibrio harveyi, respectively, and showed different catalytic
activities, degradation products, and responses to envi-
ronmental conditions. These two enzymes add to the
existing roster of marine Vibrio-derived chitinases, indi-
cating that Vibrio strains may play important roles in the
marine chitin cycle [50].
Recent advancements in marine enzyme conversion have
shown a high proficiency for many fields. However, to
reach the commercial-scale needs a lot more research. It
will be facilitated by the combination of progress in
synthetic biology, system biology, metabolic engineering,
and computational modeling and simulation.
Marine molecules used for pharmaceutical
applications
Many marine organisms inhabit extreme conditions, and
their diverse adaptations to these environments constitute
a significant potential source for active substances used in
functional foods and drugs [51]. However, the application
of these many highly active substances found in marine
organisms was hampered by the minute concentrations at
which they are naturally produced. Systems biology and
metabolic engineering can augment production of these
pure substances and facilitate further pharmaceutical
research and applications. Detailed information about
Figure 2
Structure of small marine molecules outlined in this review.
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Marine enzymes and molecules Zhang et al. 311
representative marine-derived molecules and their produc-
tion during the period 2018–2020 is outlined in Figure 2 and
Table 1.
Carotenoids are a group of the tetraterpenes family (C40-
based isoprenoid) of molecules, and show a broad spectrum
of biological activities, including antioxidant activity,
immune enhancement, regression of malignant lesions,
and inhibition of mutagenesis [65]. Astaxanthin, zeaxan-
thin, and fucoxanthin have been targeted for production in
recent years. Astaxanthin is a reddish carotenoid, one of the
most valuable natural antioxidants, and a promising sub-
stance with anticancer, immune response enhancing, and
anti-inflammatory properties [66]. Systems for scaling up
astaxanthin production by metabolic strategies in native
organisms and heterologous hosts have been reviewed by
Mao et al. [67]. Recent progress on astaxanthin production
has been made in different host cells, including E. coli,
Saccharomyces cerevisiae, Yarrowia lipolytica, and Haemato-
coccus pluvialis (Table 1).
Marine bacterial Paracoccus sp. strain N-81106 and Bre-
vundimonas sp. SD212 are the natural producers of astax-
anthin, the genes in astaxanthin biosynthetic pathway are
usually used as heterologous modules to be expressed in
different chassis [68]. The b-carotene hydroxylase and
ketolase from the marine bacterium Brevundimonas sp.
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Current Opinion in Biotechnology 2021, 69:308–315
312 Chemical biotechnology
Table 1
Production and detailed information about key marine-derived molecules reported from 2018–2020
Compound Source Host strain
Astaxanthin (1) Escherichia coli
E. coli
Shrimp wastes;
Haematococcus E. coli
pluvialis; Saccharomyces cerevisiae
Xanthophyllomyces S. cerevisiae
dendrorhous Yarrowia lipolytica
Haematococcus pluvialis
Zeaxanthin (2) Cyanobacteria; Chlorella zofingiensis CZ-bkt1
Microalgae; Higher Sphingobium DIZ
plants
Fucoxanthin (3) Diatoms; Seaweeds Cylindrotheca closterium
Squalene (4) S. cerevisiae Y2805
Shark liver oils E. coli
Shinorine (5) Cyanobacteria; Synechocystis sp. PCC6803
Macroalgae
were transferred into Synechococcus sp. PCC7002, which
made astaxanthin yield to be 3 mg/g of DCW (3.35 mg/
L/d) [69]. Heterologous expression of b-carotene ketolase
(crtW) from Paracoccus sp. N81106 and hydroxylase (crtZ)
from Pantoea ananatis in the oleaginous yeast Y. lipolytica,
after modulating the copy numbers of crtW and crtZ resulted
in astaxanthin content of 6 mg/g DCW (285 mg/L) in a
bioreactor [57

].
Astaxanthin content can be increased with several differ-
ent approaches. Park et al. achieved astaxanthin content
of 7.12 mg/g DCW from E. coli in a 5 L bioreactor fed-
batch fermentation [52]. Increased astaxanthin content
(11.92 mg/g DCW) was obtained from E. coli ASTA-4 by
complementarily expressing the morphology-related
/membrane-related genes and the oxidative stress-related
genes [53]. Yet higher content (15.1 mg/g DCW) of 100%
pure enantiomer 3S,30 S-astaxanthin was reported in
engineered E. coli by regulating the transcription and
translation of the biosynthetic enzymes through a multi-
dimensional heuristic process [54

]. By integration of
heterologous genes (b-carotene ketolase, crtW; b-caro-
tene hydroxylase, crtZ) of the astaxanthin biosynthetic
pathway and random mutagenesis of the host, astaxanthin
yield was successively raised to 13.8 mg/g DCW in
S. cerevisiae without any addition of inducers [55]. An
efficient astaxanthin-producing S. cerevisiae strain was con-
structed by combining protein engineering and dynamic
metabolic regulation and reached a 3S,30 S-astaxanthin titer
of 235 mg/L in a bioreactor, a new strategy to increase the
production of astaxanthin in yeast [56]. A biorefinery
approach was also employed with H. pluvialis to increase
astaxanthin production (45.8 mg/Kg of raw biomass) [58].
Zeaxanthin is one of the carotenoids produced by
cyanobacteria, microalgae, and some higher plants, with
important pharmaceutical applications, including strong
antioxidant activity, and prevention of cancer and
Current Opinion in Biotechnology 2021, 69:308–315
Content or titer Reference
7.12 mg/g DCW (432.82 mg/L) (bioreactor) [52]
11.92 mg/g DCW (shake-flasks) [53]
15.1 mg/g DCW (shake-flasks)
320 mg/L (bioreactor) [54

]
13.8 mg/g DCW (217.9 mg/L) (bioreactor) [55]
235 mg/L (bioreactor) [56]
6 mg/g DCW (285  19 mg/L) (bioreactor) [57

]
45.8 mg/Kg of raw biomass [58]
36.79  2.23 mg/L (bioreactor) [59]
479.5 mg/L (bioreactor) [60]
25.5 mg/g DCW (bioreactor) [61]
2011  75 mg/L (bioreactor) [62]
28.5 mg/g DCW (52.1 mg/L) (bioreactor) [63]
2.37  0.21 mg/g DCW (shake-flasks) [64]
cardiovascular disease [70,71]. Many efforts have been
made to improve zeaxanthin production in several organ-
isms. A Chlorella zofingiensis mutant (CZ-bkt1) accumu-
lated zeaxanthin up to 36.79  2.23 mg/L after feeding
with glucose [59]. A high-level producing zeaxanthin
Sphingobium DIZ strain was obtained by a combination
of chemical mutagenesis and co-overexpression of three
rate-limiting enzymes: DXS and IDI from E. coli, and
CrtZ from Brevundimonas sp. The DIZ strain produced
479.5 mg/L zeaxanthin with optimization of culture con-
ditions [60]. Fucoxanthin is a natural light-harvesting
carotenoid in diatoms and seaweeds. It provides signifi-
cant health benefits in light protection for humans [72
].
Optimization of light conditions for Cylindrotheca closter-
ium increased fucoxanthin content up to 25.5 mg/g [61].
Squalene is a triterpene natural product traditionally
derived from deep-sea shark liver oils and has attracted
attention due to its various biological activities [73]. Many
attempts have been directed towards producing squalene
through biotechnology approaches. S. cerevisiae strains are
commonly used for accumulating squalene. Co-overex-
pression of ERG10 and tHMG1 in the HX4HE strain
resulted in squalene concentration up to 150 mg/L on
xylose. Subsequently, through a fed-batch fermentation
with xylose, squalene production was elevated to 532 mg/L
[74]. By homologous recombination, a high level of squa-
lene yield was obtained in 5-L fed-batch fermentations
with terbinafine supplementation (2011  75 mg/L) [62].
There have been attempts to produce squalene in other
model strains. The engineered Synechococcus elongatus PCC
7942 overexpressing the gene dosage for CpcB1-SQS pro-
duced 79.2 mg/g DCW squalene in a photobioreactor with
light optimization [75]. Additionally, novel strategies using
systematic metabolic engineering to improve squalene
yield include increasing the NADPH/NADP+ ratio, modi-
fying the isoprenoid pathway module, and choking the
menaquinone pathway in E. coli (28.5 mg/g DCW) [63].
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The production of several other types of marine-derived
molecules have been reported. Mycosporine-like amino
acids (MAAs) are a series of natural products derived from
cyanobacteria and macroalgae. They can be used as
biological sunscreens owing to their strong ultraviolet
(UV) absorption properties [76,77]. One MAA, shinorine,
is an active ingredient in two sunscreen products cur-
rently on the market. A combination of heterologous
expression of a shinorine gene cluster from cyanobacte-
rium Fischerella sp. PCC9339 in Synechocystis sp. PCC6803
and multiple promoters insertions led to a yield of
2.37  0.21 mg/g DCW, 10-fold higher than its native
producer [64].
Conclusions and outlook
Biotechnology has been widely applied in the production
of high-profit marine enzymes and small molecules.
Growing commercial demand requires the discovery of
new and more efficient enzymes and optimization of their
yield. Additionally, current research focuses on renewable
and environmentally sustainable sources for the produc-
tion of these enzymes. Marine biotechnology performs a
crucial role in the delivery of important biological
resources from the marine habitat to applications. Syn-
thetic biology creates new biological parts, devices, and
systems and utilizes genetic circuits in order to carry out
new functions in living cells [78
,79]. Emerging synthetic
biology approaches have been applied in engineering for
the production of enzymes [80] and active compounds
[81]. Further work in our lab will build on our established
success with a well-developed 5 M strategy (Mine,
Model, Manipulate, Measure, Manufacture) and inte-
grated multi-scale data-driven engineering that have
been used for successful overproduction of avermectins
[82] and other natural products in previous work [83]. We
will continue to use these strategies and others in the
biotechnology of marine products.
Conflict of interest statement
Nothing declared.
Acknowledgements
We gratefully acknowledge the financial support from the National Key
Research and Development Program of China (2019YFA0906200,
2020YFA090032, and 2020YFA0907200), the National Natural Science
Foundation of China (21877038, 21907031, 21977029, 31720103901, and
81903529), Shanghai Rising-Star Program (20QA1402800), the Open Project
Funding of the State Key Laboratory of Bioreactor Engineering, the
111 Project (B18022), Shanghai Science and Technology Commission
(18JC1411900).
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