Future Foods 3 (2021) 100025

Contents lists available at ScienceDirect

Future Foods
journal homepage: www.elsevier.com/locate/fufo

Synthetic biology for future food: Research progress and future directions
Xueqin Lv a,b, Yaokang Wu a,b, Mengyue Gong d, Jieying Deng a,b, Yang Gu a,b, Yanfeng Liu a,b,
Jianghua Li a,b, Guocheng Du a,b, Rodrigo Ledesma-Amaro c, Long Liu a,b,∗, Jian Chen a,b,∗
a
Science Center for Future Foods, Jiangnan University, Wuxi 214122, China
b
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China
c
Department of Bioengineering, Imperial College London, London, SW72AZ, UK
d
School of Biotechnology, Jiangnan University, Wuxi 214122, China

a r t i c l e i n f o a b s t r a c t

Keywords: Food is essential to provide energy for human cellular metabolism, and is usually made from plants or animals.
Future food Beside plants and animals, other important food sources are made by microorganisms, typically products of
Food science fermentation (e.g bread, wine, beer, soy sauce, etc). Nowadays, because of the increasing environmental pollution,
Synthetic biology
climate change and population growth, is becoming challenging to keep the food supply safe, nutritious and
Traditional food industry
sustainable. Importantly, the development of the synthetic biology field enable the engineering of cells that can
Synthetic biotechnology
Synthetic food be used in food manufacturing. These engineered cells can transform renewable raw materials into important food
components, functional food additives and nutritional chemicals. This review discusses the major challenges in
food industry and how synthetic biology has the potential to revolutionize the future of the food. Finally, the
prospects and challenges of synthetic biology for a sustainable food manufacturing are discussed.

1. Introduction trition, but also an important method to overcome the unsustainability

Food is indispensable for human survival and its balance is essential
for people’s health and wellbeing. With the development of society and
technology, people’s concept of food consumption has changed dramat-
ically and the demand for food has switched from basic “guarantee sup-
ply” to “nutrition and health” Palou (2006). In addition, the increasing
environmental pollution and world population, novel processes are re-
quired to meet the higher demand while maintaining safety, nutritional
value and sustainability (Myers et al., 2017; Awange and Kiema, 2019).
In recent years, synthetic biology has emerged as a discipline that
merges biological research and engineering concepts (Shapira et al.,
2017). As a novel domain, synthetic biology is a discipline that gathers
different fields of life and social sciences, engineering and information
science with the goals of wiring biological circuitry to achieve cellu-
lar control, and also understanding design rules of biological systems
(Khalil and Collins, 2010; Cameron et al., 2014). Through synthetic
biology, researchers can design and build new biomolecular compo-
nents, pathways and networks, and use these constructs to reprogram
organisms to obtain the so-called engineered cell factories Khalil and
Collins (2010). At present, food industry is being transformed by the de-
velopments of synthetic biology (Katz et al., 2018). The effective com-
bination of food science and synthetic biology is not only an impor-
tant technology to solve the existing problems of food safety and nu-
problems associated to traditional food technology (Fig. 1) Roell and
Zurbriggen (2020). Synthetic biology allows the improvement of food
production by using programmed monoclonal cell factories, engineered
microbial consortia or cell free biosynthesis platforms. By applying syn-
thetic biology technology in future food may be possible to get rid of the
drawbacks of the traditional agriculture and husbandry while improving
resource conversion efficiency. Overall, synthetic biology driven food
industry has the potential to address the challenges of sustainable food
supply in the future. In this review, we discuss the current and future
food revolution guided by synthetic biology through three different sec-
tions. First, synthetic biology can improve the traditional food produc-
tion and manufacturing. Second, synthetic biology can improve food
nutrition or add new functionalities. Third, synthetic biology can trans-
form the traditional fermentation food production style by the use of
engineered microbial communities. In addition, the major synthetic bi-
ology based biotechnologies expected to be used in future food are also
introduced. Finally, the challenges and prospects of synthetic biology
for future food are discussed.
2. Technologies commonly used in synthetic biology
The design-build-test-learn cycle is commonly used to construct effi-
cient cell factories that have widespread applications including those in

∗
Corresponding author.
E-mail addresses: longliu@jiangnan.edu.cn (L. Liu), jchen@jiangnan.edu.cn (J. Chen).

https://doi.org/10.1016/j.fufo.2021.100025
Received 14 September 2020; Received in revised form 27 February 2021; Accepted 1 March 2021
2666-8335/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/)
X. Lv, Y. Wu, M. Gong et al.
Fig. 1. Schematic diagram showing the role of synthetic biology in the food
industry
Synthetic biology has the potential to improve the traditional food production
system, improve food nutrition, add new functions to food, and redesign tradi-
tional fermentation technologies. The development and application of synthetic
biology in the food industry can develop new resources to provide safer, more
nutritious, more delicious and more sustainable future food to meet people’s
better life.
the food industry (Fig. 2). Firstly, computational tools and databases can
guide the precise pathway engineering approach (Heirendt et al., 2019).
Besides, expression elements including promoters, Ribosome Binding
Sites (RBS), and protein degradation tags have been identified and de-
signed to fine-tune the expression level of target genes (Guiziou et al.,
2016). Furthermore, protein engineering strategies were developed to
improve the properties of the key enzymes in a pathway (Xu et al.,
2020a). To control the metabolic flux more accurately and efficiently,
methods such as synthetic scaffold (Lv et al., 2020), modular engineer-
ing (Liu et al., 2014a), and genetic circuit (Wu et al, 2020a) have been
also developed. Moreover, CRISPR systems also have wide applications
not only in the genome reconstruction but also in the metabolic network
reprogramming (Wu et al, 2020b). Recently, machine learning technol-
ogy has been introduced into the field for achieving predictive engineer-
ing and cell factories optimization (Zhang et al., 2020a; Lawson et al.,
2020). Undoubtedly, these technologies will continue to promote the
transformation and revolution of the food industry (Table 1), which is
the following sections.
3. Improving the food production and manufacturing
With the use of synthetic biology, the traditional agriculture and
husbandry dependent food production systems will be changed and re-
formed (Fig. 3), which will improve land use efficiency, save water re-
sources and avoid the use of pesticides and fertilizers (Stephens et al.,
2018). In addition, the synthetic biology based food manufacturing sys-
tem is less affected by uncontrollable environmental factors and is easier
to carry out according to high quality standards. By constructing food-
based cell factory, foods such as the meat analogs, animal-free bioengi-
neered milk and sugar substitutes can be produced from fully renewable
resources (Stephens et al., 2018). In addition, current, well-established
manufacturing process of fermented food may also be transformed by
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Future Foods 3 (2021) 100025
synthetic biology, for example, the beer industry can move away from
the dependence on the farmed hops by using the engineered hoppy yeast
(Denby et al., 2018).
3.1. Meat analog
Meat is an important source of protein and also a contributor to a
healthy diet. However, with the increasing production and consumption
of meat products at global scale, traditional husbandry is facing serious
environmental and social problems such as greenhouse gas emissions,
land and water misuse, waste disposal issues, animal welfare ethics and
public health problems. Hence, the meat analogs including those either
manufactured from plant proteins or cultured from animal cells have
been receiving increasing attention to build sustainable alternative meat
production systems van der Weele et al., 2019. Although plant-based
meat analogs have been marketed for several years, it is yet dependent
on traditional husbandry for obtaining the proteins from soy, wheat,
pea or other sources. In addition, complex post processing is necessary
to give it the taste and texture that resemble meat. Cultured meat, also
known as synthetic or in vitro meat, is produced from animal tissue and
cells grown in a bioreactor but not in a living organism. The muscle cells
derived from stem cells in embryos or muscle tissue can proliferate in
a bioreactor. Then scaffolds or microcarriers are used to obtain specific
muscle fibers and larger tissues (Ben-Arye et al., 2020). Although the
cultured meat is still at the lab stage, it is hardly dependent on stock
farming and will eventually achieve industrial production in the future
with the aid of synthetic biology (Zhang et al., 2020b). In addition, the
meat protein may also be replaced by other sustainable protein sources
(like algae and mycoprotein), which can be produced from biomass and
other feedstocks upon conversion by cell factories (Dekkers et al., 2018;
van der Weele et al., 2019).
The emergence of synthetic biology allows meat analogs to mimic
real meat in appearance and characteristics such as color, taste and fla-
vor, thus meeting the increasing demand of consumers for both food
quantity and quality. The meat color is generated by hemoglobin or
myoglobin Shleikin and Medvedev (2014). Hence, hemoglobin is often
added into the meat substitutes. Cell factories able to synthesize heme
or hemoglobin have been designed (Zhao et al., 2018), which provide
an alternative production method to extraction from plant tissue or ani-
mal blood. Hemoglobin is composed of four globin subunits (𝛼 2 𝛽 2 ) with
a prosthetic heme group included in each subunit. There are two path-
ways named C4 and C5 pathways for the biosynthesis of heme. Recently,
an engineered Escherichia coli cell factory that can achieve the secre-
tory production of free heme from glucose was constructed by using the
programmed C5 pathway and the downstream heme biosynthesis path-
way (Zhao et al., 2018). In addition, the hemoglobin producing Sac-
charomyces cerevisiae cell factory have also been built by combining the
optimization of 𝛼 and 𝛽 peptides expression with the metabolic engi-
neering of the heme biosynthetic pathway (Liu et al., 2014b).
Both meat taste and flavor are also associated to the types and
amount of fatty acid and aromatic molecules. The fatty acid biosynthe-
sis (FAB) and fatty acid degradation (FAD) pathways are both tightly
regulated to maintain a dynamic balance. Synthetic biology makes pos-
sible to overproduce a variety of fatty acids in cell factories (Fernandez-
Moya and Da Silva, 2017; Marella et al., 2018; Ledesma-Amaro et al.,
2014; Ledesma-Amaro et al., 2015; Ledesma-Amaro et al., 2018). For in-
stance, the pathways including PDH-bypass, carnitine shuttle, pyruvate-
formate lyase route, and pyruvate-formate lyase route were engineered
to increase supplement of the required precursor acetyl-CoA (Xu et al.,
2016). In addition, the fatty acid synthase systems can be regulated to
control chain length and to improve production (Zhu et al., 2017). Fur-
thermore, the redox metabolism was also managed by rewriting the re-
lated pathways to keep cofactor supply and balance (Qiao et al., 2017).
Related transcriptional regulators have also been engineered to per-
form dynamic pathway regulation or screening of the fatty acid pro-
ducing cell factories (Xu et al., 2014; Dabirian et al., 2019). The flavor
X. Lv, Y. Wu, M. Gong et al. Future Foods 3 (2021) 100025

Table 1
Reform and revolution of food industry by synthetic biology.

Product Method Benefit Reference

Meat analog The biosynthesis pathway and an exporter of haem Fed-batch fermentation of the engineered strain (Zhao et al., 2018)
were introduced into the engineered E. coli. The secrete 141.4 mg/L haem, which can be added
haem-degrading pathway and synthetic pathways into the meat substitutes to give it a better
of by-products acetate and lactate were blocked. appearance.
Meat analog The hemoglobin-producing S. cerevisiae cell factory Resulting in a significant enhancement of (Liu et al., 2014b)
was constructed by combining the optimization hemoglobin production in yeast.
globin protein expression with the metabolic
engineering of the heme biosynthetic pathway.
Meat analog The acetyl-CoA pathways of Y. lipolytica were The engineered strain achieved a lipid titer of (Xu et al., 2016)
engineered to increase precursor supplement for 66.4 g/L.
fatty acid synthesis.
Meat analog Protein engineering was used to tailor the The fatty acid product portfolio and production (Zhu et al., 2017)
Rhodosporidium toruloides type I fatty acid have been changed by expressing these
synthases (FASs). synthetic FASs in S. cerevisiae.
Meat analog The synthetic pathways converting glycolytic NADH The best engineered strain achieved a (Qiao et al., 2017)
into the lipid biosynthetic precursors NADPH or productivity of 1.2 g/L/h and a process yield of
acetyl-CoA were constructed in Y. lipolytica. 0.27 g–fatty acid methyl esters/g-glucose.
Meat analog A genetically encoded metabolic switch that enables Yielding 15.7-fold improvement in FA titer (Xu et al., 2014)
dynamic regulation of fatty acids (FA) biosynthesis compared with the wild-type strain.
in E. coli was built.
Animal-free Expression of milk casein proteins using the Casein proteins including 𝛼(s1)-casein, 𝛽-casein, (Kim et al., 1999;
bioengineered milk engineered E. coli or yeast. and 𝜅-casein can be produced in the bioreactor. Choi and Jiménez-
Flores, 2001;
Simons et al., 1993;
Kang and
Richardson, 1988)
Animal-free Expression of milk whey proteins using the Whey proteins such as 𝛼-lactalbumin, (Chaudhuri et al.,
bioengineered milk engineered E. coli or yeast. 𝛽-lactoglobulin, and lactoferrin can be produced 1999; Kim et al.,
in the bioreactor. 1997; Wang et al.,
2002)
Next-generation Protein engineering was employed in the A variant was obtained to increase the (Olsson et al., 2016).
sweeteners glucosyltransferase UGT76G1 from Stevia accumulation of rebaudioside D (Reb D) and
rebaudiana. rebaudioside M (Reb M), as well as for
decreased accumulation of by-products.
Lycopene Engineered S. cerevisiae through modular enzyme The lycopene titer reached 2300 mg/L, which is (Kang et al., 2019b)
assembly of the lycopene pathway. the highest titer to date.
Lycopene Lipid engineering and systematic metabolic The lycopene accumulation improved to 73.3 (Ma et al., 2019)
engineering was employed in S. cerevisiae. mg/g CDW in fed-batch fermentation.
Astaxanthin The multidimensional heuristic process (MHP) was This method increases astaxanthin production to (Zhang et al., 2018c)
developed to balance different modules of the 320 mg/L.
astaxanthin biosynthetic pathway in E. coli.
Astaxanthin The 𝛽-carotene ketolase was co-localized to the E. coli The astaxanthin increased from 12.90 mg/L to (Park et al., 2018)
membrane using the signal peptide of outer 432.82 mg/L.
membrane protein (OmpF).
Menaquinone-7 Modular engineering was conducted to optimized MK-7 titer reached 281.4±5.0 mg/L (i.e., 12.0 (Yang et al., 2020)
(MK-7) MK-7 production in the engineered B. subtilis. mg/g DCW) in a 5 L fermenter.
MK-7 The quorum sensing-based genetic circuits was built MK-7 titer was increased from 9 to 360 mg/L. (Cui et al., 2019)
for the dynamic regulation of MK-7 synthesis in B.
subtilis.
2’-fucosyllactose A bifunctional gene expression circuit based on The 2’-FL titers was increased from 24.7 to 674 (Deng et al., 2019)
(2’-FL) nucleic acid aptamers have been established and mg/L
applied in the synthesis of 2 ’- FL in B. subtilis.
2’-FL The transport pathway of substrate lactose and the The total 2′-FL concentration reached 15 g/L and (Hallands et al., 2019)
synthetic pathway of 2′-FL was engineered both in 24 g/L in the S. cerevisiae and Y. lipolytic
S. cerevisiae and Y. lipolytic. fermentation experiment.
Lacto-N-neotetraose Modular pathway engineering was conducted to The LNnT titer reached 4.52 g/L in a 3-L (Dong et al., 2019)
(LNnT) balance the supply of two key precursors the bioreactor.
UDP-GlcNAc and UDP-Gal pathways.
LNnT The CRISPRi system was used to downregulate the The LNnT titer reached 2.30 g/L in shake flask and (Dong et al., 2020)
competitive modules in B. subtilis. 5.41 g/L in 3 L bioreactor.
Soy sauce An isolated strain Bacillus amyloliquefaciens JY06 with The accumulation of ethyl carbamate (EC) was (Zhang et al., 2016)
high arginine consuming capacity was added in soy significantly reduced, and the sensory
sauce fermentation. evaluation gave a higher score of esters
fragrant.
Soy sauce Engineering the microbial communities of soy sauce The browning of the soy sauce was reduced. (Det-udom et al.,
fermentation using a recombinant B. subtilis that 2019)
can degrade melanoidin.
Chinese rice wine The semi-synthetic microbial communities were The EC was reduced by 87% and 15% compared to (Wu et al., 2016)
created using an engineered urea degradation S. the original microbial communities,
cerevisiae strain. respectively.
Chinese rice wine An engineered S. cerevisiae strain with relieve The concentrations of urea were reduced by 63% (Zhao et al., 2014)
nitrogen catabolite repression was constructed and and EC were reduced by 72% in a model rice
used for rice wine fermentation. wine system.

3
X. Lv, Y. Wu, M. Gong et al.
Fig. 2. Technologies commonly used in synthetic biology.
Various synthetic biology methods and tools have been developed to promote the design-build-test-learn cycle of cell factory construction, and these technologies
are reforming the future food industry.
of meat is also determined by aromatic molecules such as disulphides,
pyrazines, thiol and thiazoles that are produced from the Maillard reac-
tion between sugars and amino acids at high temperatures (Kang et al.,
2019a). In a recent study, up to 57 volatile flavor compounds related
to meaty flavor were generated from the mixture of the enzymatic hy-
drolysates of solid-state fermentation soy sauce residue and defatted soy-
bean Wang and Cha (2018). Commercial enzymes including alcalase,
protamex and flavourzyme used in that study were produced by the cell
factory. In the future, the main aromatic molecules may be manufac-
tured from the well-designed cell factories in a controlled manner.
3.2. Animal-free bioengineered milk
Milk is an important part of the daily diet and contains a variety of
bioactive proteins. It can also be used as a raw material for the manu-
facture of cheese, ice cream, and many other dairy products. The milk
proteins, casein and whey proteins, are the main components of milk
4
Future Foods 3 (2021) 100025
(Caroli et al., 2009). Casein proteins such as 𝛼(s1)-casein (Kim et al.,
1999), 𝛽-casein (Choi and Jiménez-Flores, 2001; Simons et al., 1993),
and 𝜅-casein (Kang and Richardson, 1988) and whey proteins such as 𝛼-
lactalbumin (Chaudhuri et al., 1999), 𝛽-lactoglobulin (Kim et al., 1997),
and lactoferrin (Wang et al., 2002) have been successfully expressed in
engineered E. coli or yeast cell factories using a simple and defined cul-
ture media. Then, the purified casein and whey proteins can be mixed
with fats, water and other essential components to make synthetic milk.
In addition, these other components (fats, oligosaccharides, vitamins)
can be also made by the engineered cell factories (Bych et al., 2019;
Cui et al., 2020). There are many advantages of the milk substitutes pro-
duced with the aid of synthetic biology compared with the traditional
process. First, the milk producing cell factory can grow in a bioreactor,
which could avoid the problems caused by the traditional husbandry
such as antibiotics and hormones contamination or land occupancy. The
culture period of the engineered cell is much shorter than that of the
dairy cow. Furthermore, the components of the artificial bioengineered
X. Lv, Y. Wu, M. Gong et al.
Fig. 3. Improving food production by synthetic biology.
With the employment of synthetic biology, the traditional agriculture and husbandry dependent food production manner have the potential to be changed and
improved. By constructing enhanced cell factories, foods such as artificial meat, animal-free bioengineered milk, and sugar substitutes can be produced from renewable
substrates in a bioreactor.
milk can be configured as needed, and that is more convenient than
previous attempts to build transgenic cows (Van Berkel et al., 2002;
Brophy et al., 2003). In addition, it can be made with a composition
closer to the human breast milk for infants in order to promote a healthy
development by changing the ratio of the proteins lactoferrin, 𝛽-casein
and 𝜅-casein. Other non-essential ingredients such as lactose and major
milk allergen 𝛽-lactoglobulin can be easily excluded. One can imagine
that If these substitutes take a large portion of the market share, the
production and processing of dairy will be reformed completely. The
milk can be brew by yeast, and its production is no longer dependent on
the dairy farming that possess low energy conversion and occupy much
land resource.
3.3. Low-calorie and zero-calorie sweeteners
Some health problems including obesity, diabetes and cancer corre-
lates with an excessive dietary sugars consumption, which has increased
the public attention towrds plant-derived natural low-calorie and zero-
calorie sweeteners (Philippe et al., 2014). Because the pathways and
genes required to make many natural sweeteners such as erythritol,
steviol glycosides, mogrosides, and glycyrrhizin are known, producing
these sweeteners using engineered cell factories by fermentation in a
bioreactor is a viable alternative to the traditional plant extraction pro-
duction method (Rzechonek et al., 2018; Seki et al., 2018). Erythritol
5
Future Foods 3 (2021) 100025
belongs to the family of sugar alcohols also known as polyols, which
are naturally occurring in fruits and vegetables like pears and mush-
rooms. Its intake does not change blood glucose and insulin levels be-
cause it cannot be metabolized by humans. Although the commercial
production of erythritol is already done by yeasts and yeast-like fungi,
synthetic biology methods are still being employed to build erythritol
producing cell factory with improved productivity as well as the ability
to use inexpensive and abundant substrates (Rzechonek et al., 2018).
Steviol glycosides were found in the leaves of Stevia rebaudiana, which
have been used as a natural sweetener by the South America indigenous
population for centuries (Yang et al., 2019). By using synthetic biology,
the glucosyltransferase UGT76G1 of S. rebaudiana, which was respon-
sible for the biosynthesis of Reb D and Reb M, was engineered by in
vivo site‑saturation mutagenesis screen to weak its catalyzed ability on
the formation of undesired side-products (1,3-bioside, Reb G, Reb Q and
Reb I) (Olsson et al., 2016).
3.4. Other flavors
Flavors commonly used in the food industry such as limonene,
sabinene, and vanillin can be produced using engineered cell facto-
ries to achieve their economic and sustainable supply. Limonene and
sabinene belong to monoterpenoids, which are synthesized from the
terpene precursor geranyl diphosphate (GPP). However, GPP is also
X. Lv, Y. Wu, M. Gong et al.
closely related to the biosynthesis of isoprenoid compounds that are
essential for cell growth. Accordingly, a synthetic orthogonal monoter-
penoid pathway was built by introducing an alternative noncanonical
precursor, neryl diphosphate (NPP). Furthermore, the terpene synthases
were engineered for the synthesis of limonene and sabinene from NPP
(Ignea et al., 2019). Recently, a programmed E. coli cell factory has been
constructed to produce vanillin, one of the most extensively used fla-
vors in food products. Because vanillin is toxic to the cell, a product
feedback-activated dynamic expression system was built to keep cell
growth and improve vanillin production by using an engineered tran-
scriptional regulator (Liang et al., 2020). Beyond the applications men-
tioned above, the flavors generated by cell factories may be directly ap-
plied to food production process. For example, an engineered brewer’s
yeast that possesses the biosynthesis pathway of linalool and geraniol,
which are the primary flavor components contributed to beer by hops,
have been constructed. Hence, this programmed yeast cell could be used
for hoppy beer production without hops addition, and the beer produced
by this manner was perceived as hoppier than traditionally hopped beers
(Denby et al., 2018).
4. Nutrition improvement and new functions of food
With the economic development and the improvement of living stan-
dard, people’s consumption trend has gradually turned to functional
food (Dias et al., 2017). Functional food does not only have the nu-
tritional and sensory functions of ordinary food, but also benefits for
human physiology McClements (2020). With more and more metabolic
pathways of functional food being elucidated, researchers can construct
metabolic pathways to regulate functional food in microbial cells with
the help of synthetic biology. Nowadays, synthetic biology has been
widely used in the biosynthesis of functional foods such as carotenoids
(like lycopene, beta-carotene and astaxanthin), Menaquinone-7 (vita-
min K2) and human milk oligosaccharides (HMOs).
4.1. Lycopene and astaxanthin
Significant progress has been achieved in understanding the
carotenoid biosynthesis and catabolism using biochemical and genetics
approaches (Liu et al., 2012). Lycopene is a bright red linear carotenoid
abundant in red fruits and vegetables. It is of particular nutritional in-
terest in promoting health and reducing the risk of various diseases
including cancer and cardiovascular disease (Zhang et al., 2018a).
Many kinds of microbes have been used in biosynthesis of lycopene in-
cluding Blakeslea trispora, Streptomyces chrestomyceticus, Candida utilis,
Phycomyces blakesleeanus, Yarrowia lipolytica, E. coli and S. cerevisiae
(Liu et al., 2012). The biosynthesis of lycopene in E. coli requires three
steps starting with farnesyl pyrophosphate (FPP) (Fig. 4A,B). After cat-
alyzing by geranylgeranyl synthase, phytoene synthase, phytoene desat-
urase, FPP was transformed into lycopene Albermann (2011). To date,
the highest titer of lycopene was achieved in engineered S. cerevisiae
through modular enzyme assembly, 2300 mg/L (Kang et al., 2019b).
Ma et al. (2019) adopted lipid engineering and systematic metabolic
engineering to establish an effective platform where lipid metabolism
and lycopene accumulation were improved, reaching 73.3 mg/g CDW
in fed-batch fermentation.
Astaxanthin is a red fat-soluble xanthophyll carotenoid pigment
which is found in various microorganisms and marine animals
(Ambati et al., 2014). Currently, astaxanthin is widely used for its com-
mercial application in various industries such as aquaculture, food,
cosmetics, nutraceutical and pharmaceutical (Davinelli et al., 2018).
Most astaxanthin products in today’s world are manufactured by Phaf-
fia yeast, Haematococcus and through chemical synthesis (Ambati et al.,
2014). Many efforts aimed to overproduce astaxanthin using geneti-
cally engineered plants, yeasts, microalgae and other microorganisms.
The biosynthesis pathway of astaxanthin is an extension of the ly-
copene synthesis pathway, and formed by catalyzing lycopene with 𝛽-
6
Future Foods 3 (2021) 100025
Table 2
Concentrations of major HMOs in human milk.
Categories of HMO (% total Oligosaccharide Mean concentration
weight) (range), g/L
Fucosylated (35%-50%) 2’-FL 2.7 (1.88–4.97)
3’-FL 0.5 (0.25–0.86)
LNFP I 0.122
LNFP II+III (0.106–0.145)
0.156
(0.120–0.161)
Sialyated (12-14%) 3’SL 0.2 (0.1–0.3)
6’SL 0.5 (0.2–1.22)
Non fucosylated neutral LNnT 0.3 (0.17–0.45)
(42-55%)
carotene hydroxylase and 𝛽-carotene ketolase (Fig. 4A,B). Corynebac-
terium glutamicum and E. coli have been used for biosynthesis of as-
taxanthin, and 385.0 mg/L astaxanthin (Park et al., 2018; Li et al.,
2020). Zhang et al. (2018a) developed a multidimensional heuristic
process in E. coli and optimized the metabolic pathways consisting of
large number of genes for increasing astaxanthin titer to 320 mg/L.
Park et al. (2018) successfully developed an astaxanthin producing E.
coli with high concentration and high productivity through membrane
fused expression of 𝛽-carotene hydroxylase in metabolically engineered
E. coli, increasing the titer from 12.90 mg/L to 432.82 mg/L. In addi-
tion, Tramontin et al. (2019) obtained an engineered Y. lipolytica strain
with astaxanthin titer of 285 ± 19 mg/L by optimizing the pathway
of 𝛽-carotene and introducing exogenous 𝛽-ketolase and 𝛽-hydroxylase
of bacterial and algal origins with different copy numbers. It is worth
noting that Y. lipolytica has great potential for efficient astaxanthin syn-
thesis, because the yield of 𝛽-carotene in engineered strain can reach
6.5 g/L (Larroude et al., 2018).
4.2. Menaquinone-7
As the most valuable member of the vitamin K family, Menaquinone-
7 (MK-7) has a significant effect on preventing osteoporosis and cardio-
vascular diseases (CVD) besides its positive effects on blood coagula-
tion (Mahdinia et al., 2017; Mahdinia et al., 2019). MK-7 was origi-
nally obtained from soybean by Bacillus natto by solid-state fermenta-
tion, and the content of MK-7 in natto is 8-9 𝜇g/g (Kamao et al., 2007).
Many kinds of substrates such as legumes, cereals, and raw wheat have
been used as substrate (Ren et al., 2020). Because of the difficulty in
controlling the fermentation parameters, the large area ang long time
required, the application of solid-state fermentation at large-scale has
been limited (Berenjian et al., 2015). Alternatively, microbial liquid-
state fermentation is widely used and require less space and shorter
times (Mahdinia et al., 2017). The synthesis pathway of MK-7 in mi-
croorganisms (Fig. 4A,C) can be generally divided into five modules or
pathways, the glycerol dissimilation pathway, the shikimate (SA) path-
way, the pyrimidine metabolism pathway, the methylerythritol phos-
phate (MEP) pathway and the MK-7 pathway (Yang et al., 2020). Re-
cently, the strain B. subtilis 168 was used as the chassis for MK-7 produc-
tion. The MK-7 titer of B. subtilis was increased from 9 to 360 mg/L in
shake flask by using quorum sensing system to dynamically regulate the
expression of key genes and by optimization of fermentation conditions
(Cui et al., 2019).
4.3. 2’-fucosyllactose and Lacto-N-neotetraose
As the third most abundant solid component in breast milk, human
milk oligosaccharides (HMOs) were first identified as the most impor-
tant bifidogenic factor in human milk in the 1930s (Vandenplas et al.,
2018). HMOs can be roughly divided into three categories, fucosy-
lated oligosaccharides (FOs), sialylated oligosaccharides (SOs) and other
kinds of oligosaccharides (Table 2). Recently, 2’-fucosyllactose (2’-FL)
X. Lv, Y. Wu, M. Gong et al. Future Foods 3 (2021) 100025

Fig. 4. The biosynthesis of nutritious supplements including human milk oligosaccharides (HMOs), vitamin K2, lycopene, and astaxanthin.
(A) Central carbon module: Glc-6-P, glucose-6-phosphate; Fru-6-P, fructose-6-phosphate; GlcN-6-P, glucosamine-6-phosphate; FBP, fructose-1,6-bisphosphate;
G3P, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; Acetyl-CoA, acetyl Coenzyme A; TCA cycle, tricarboxylic acid cycle. (B) Carotenoids synthe-
sis module: DXP, 1-deoxyxylulose-5-phosphate; MEP, methyl-erythritol-4-diphosphate; HMBPP, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate; IPP, isopentenyl
diphosphate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; GPP, geranyl diphosphate; GGPP, Geranylgeranyl diphosphate; MVA, Mevalonate;
MVAP, Mevalonate-5-phosphate; MVAPP, Mevalonate-5-pyrophosphate. (C) MK-7 synthesis module: DAHP, 3-deoxy-arabino-heptulonate 7-phosphate; DHQ,
3-dehydroquinate; DHS, 3-dehydroshikimate; SA, shikimate; S3P, shikimate 3-phosphate; EPSP, 5-O-(1-carboxyvinyl)-3-phosphoshikimate; CHA, Chorismite;
ICHA, isochorismate; SEPHCHC, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate; SHCHC, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1- carboxy-
late; OSB, 2-succinylbenzoate; OSB-CoA, 2-succinyl benzoyl-CoA; DHNA-CoA, 1,4-dihydroxy-2-naphthoyl-CoA; DHNA, 1,4-dihydroxy- 2-naphthoate; DMK, 2-
demethylmenaquinone; MK-7, menaquinone-7; HDP, heptaprenyl diphosphate; (D) Human milk oligosaccharides synthesis module: GlcN-1-P, glucosamine-1-
phosphate; GlcNAc-1P, N-acetylglucosamine-1-phosphate; UDP-GlcNAc, uridine diphosphate-N-acetyglucosamine; Glc-1-P, glucose-1-phosphate; UDP-Glc, UDP-
glucose; UDP-GlcNAc, UDP-N-acetylglucosamine; UDP-Gal, UDP-galactose; LNTII, Lacto-N-triose II; LNnT, Lacto-N-neotetraose; Man-6-P, mannose-6-phosphate;
Man-1-P, mannose-1-phosphate; GDP-L-Fuc, guanosine 5′-diphosphate-L-fucose; GDP-Man, GDP-mannose; GDP-4-dehydro-6-deoxy-Man, GDP-4-dehydro-6-deoxy-
mannose; 2’-FL, 2’-fucosyllactose.

and Lacto-N-neotetraose (LNnT) were approved as additives of infant
milk powder and new food additives by the Food and Drug Adminis-
tration (FDA) of USA and the European Food Safety Authority (EFSA)
(Vandenplas et al., 2018).
2′-FL is the most abundant HMO and constitutes nearly 30% of the to-
tal HMOs found in human breast milk (Castanys-Muñoz et al., 2013), but
it is absent in bovine milk (Tao et al., 2008). The health benefits of 2’-FL
studied in vitro and in clinical tests suggest that 2’-FL supports the growth
of probiotic bacteria as well as lowers the risk of pathogenic infection
in infants (Reverri et al., 2018). The synthesis of 2’-FL consists of two
steps: the generation of the intermediate 5’-diphosphate-L-fucose (GDP-
L-fucose) and the lactose fucosylation with the donor GDP-L-fucose
(Fig. 4A and 3D). In 2014, 2’-FL was produced through microbial trans-
formation in E. coli, and it was the first structure-identified HMO that
was produced artificially (Vandenplas et al., 2018). Biosynthesis of 2’-
FL has been achieved in several microbes including E. coli, Bacillus sub-
tilis, Saccharomyces cerevisiae and Yarrowia lipolytica (Huang et al., 2017;
Hollands et al., 2019; Deng et al., 2019; Yu et al., 2018). Through the
establishment of a bifunctional gene expression circuit based on nucleic
acid aptamers and its application in the synthesis of 2 ’- FL in B. sub-
tilis, the 2’-FL titer was increased from 24.7 to 674 mg/L (Deng et al.,
2019). To date, the highest 2’-FL titer was obtained in recombinant E.
coli, 47.0 g/L (Jung et al., 2019). In yeasts and B. subtilis, the highest
2’-FL titer achieved 24 g/L and 5.01 g/L, respectively (Hollands et al.,
2019; Deng et al., 2019).
LNnT is beneficial for breast-feeding infants since it enhances immu-
nity, regulates intestinal flora, and promots cell maturation (Chen et al.,
2015; Holscher et al., 2014). The biosynthetic pathway of LNnT con-

7
X. Lv, Y. Wu, M. Gong et al.
Fig. 5. Artificially creating semi-synthetic microbial communities for food production.
Many traditional fermentation foods, including soy sauce, wine, pickle, and so on, are produced by the complex synergistic interactions of microbial communities
with numerous raw materials. Synthetic biology could redesign the traditional fermentation food production by creating semi-synthetic microbial communities to
control the compositions and capacities of microbes, in order to produce novel products or avoid the synthesis of toxic byproduct.
tains two steps: 1) UDP-N-acetyglucosamine (UDP-GlcNAc) and lactose
are used by LgtA to produce Lacto-N-tri- ose II (LNTII); 2) the LNTII and
UDP-galactose (UDP-Gal) are converted into LNnT by LgtB (Priem et al.,
2002). The biosynthetic pathway can be constructed in E. coli and in
B. subtilis by overexpressing of 𝛽-(1,3)-N-acetylglucosaminyltransferase
(LgtA) and 𝛽-(1,4)-galactostltransferase (LgtB) from Nesseria meningi-
tides, resulting 6 and 4.52 g/L LNnT, respectively (Priem et al., 2002;
Dong et al., 2019). Dong et al. (2020) carried out modular pathway en-
gineering and CRISPRi-guided multiplexed fine-tuning of metabolic flux
for LNnT production, increasing the LNnT titer from 1.32 g/L to 2.30 g/L
in shake flask and finally reached a titer of 5.41 g/L in 3-L bioreactor.
5. Transforming the traditional fermentation process
Many traditional fermentation foods, including soy (Fang et al.,
2018), wine (Zhang et al., 2018b), pickle (Li and Hotamisligil, 2010),
and so on, are produced by the complex synergistic interactions of nu-
merous microbes. For example, sourdough bread is obtained by the com-
bined use of lactic acid bacteria and yeasts, such as Lactobacillus, Pe-
diococcus, Saccharomyces cerevisiae, and Candida humilis. Through the
fermentation process, the nutritional content and flavor of these foods
can be significantly improved (Det-udom et al., 2019). In general, the
fermentation process operations include tuning the conditions, such as
temperate, time, and substrates. However, these approaches cannot be
easily controlled owing to the properties of natural occurred microbial
communities (Det-udom et al., 2019). Combing with metabolic engi-
neering, synthetic biology could reconstruct the traditional fermenta-
tion food production by creating semi-synthetic microbial communities
(Fig. 5), which can be enhanced in their fermentation capacities for ex-
ample making the products tastier or healthier. For example, salami is
a kind of cured sausage, which is made from air-dried and fermented
meat (Blaiotta et al., 2018). To create different colours and flavors of
salami, microbial communities have been constructed by using Staphy-
8
Future Foods 3 (2021) 100025
lococcus xylosus, Micrococcus, Lactobacillus plantarum and Lactobacillus
curvatus (Aquilanti et al., 2016). Here, we will introduce two applica-
tions of synthetic biology to create semi-synthetic microbial communi-
ties in food production, soy sauce and Chinese rice wine.
5.1. Soy sauce
Soy sauce is a popular liquid condiment, which reached $926.2 mil-
lion USD retail sales in 2018 and is predicted with 6.20% of Compound
Annual Growth rate (CAGR) during 2019-2021 (Det-udom et al., 2019).
Soy sauce is made from a fermented paste of soybeans and wheat con-
sisting of five processes, including: 1) soaking and cooking; 2) solid-
stage koji culture; 3) submerged moromi fermentation; 4) pressing and
5) pasteurization. The fermentation processes in soy sauce production
include solid-stage koji culture and submerged moromi fermentation,
which is carried out by sequential growth of fungal (includes Aspergillus
oryzae, Aspergillus sojae, and Aspergillus sojae), yeast (includes Saccha-
romyces cerevisiae) and bacterial (includes Bacillus species and Lacto-
bacillus species) communities. In the solid-stage koji culture step, As-
pergillus genus firstly decompose soybean proteins, starch and other com-
plex biomolecules into simpler ones, such as small peptides, glucose,
xylose and other simple sugars. Subsequently, these nutrients are used
for the growth of halophilic bacteria and yeasts in the submerged mo-
romi fermentation step. As a result, the metabolites produced by the
microbes give soy sauce a special flavour. However, a common harmful
component in fermentation food, ethyl carbamate, has been detected in
soy sauce, which could cause liver cancer, and is in Group 2A in the
carcinogen classification presented by the International Agency for Re-
search on Cancer (LARC) (Baan et al., 2007; Zhang et al., 2018b). The
formation of ethyl carbamate is a spontaneous reaction, which can be
accomplished with ethanol and another precursor, such as urea, car-
bamyl phosphate, citrulline, and hydrocyanic acid Weber and Shary-
pov (2009). In soy sauce, citrulline is the major alternative precursor,
X. Lv, Y. Wu, M. Gong et al.
which is synthetized from arginine by the arginine deiminase (ADI)
pathway with three enzymatic steps, including arginine deiminase, or-
nithine transcarbamylase, and carbamate kinase (Zhang et al., 2014).
In order to understand the formation mechanism of ethyl carbamate
in soy sauce fermentation, Fang et al. investigated the composition of
microbial communities and identified that Pediococcus acidilactici and
Staphylococcus are mainly responsible for the conversion of arginine to
citrulline in solid-stage koji culture and submerged moromi fermenta-
tion, respectively (Fang et al., 2018; Zhang et al., 2014). However, in-
hibiting the growth of Pediococcus acidilactici and Staphylococcus could
influence the flavour. Thus, they screened and isolated a salt-tolerant
strain, Bacillus amyloliquefaciens JY06, with high arginine consuming
capacity from the moromi mash (Zhang et al., 2016). Adding Bacillus
amyloliquefaciens JY06 to the soy sauce fermentation, the ethyl carba-
mate levels were significantly reduced, while keeping a good sensory
evaluation.
On the other hand, consumers showed growing preferences for
lightly-coloured soy sauce products. It is generally known that the brown
colouration of soy sauce is primarily attributed to the non-enzymatic
Maillard reaction (Det-udom et al., 2019) between reducing sugars and
amino acids which generate a set of high molecular weight, brown pig-
mented heterogenous polymers, known as melanoidins. In soy sauce
production, the Maillard reaction occurs in the fermentation mash
(Satoh et al., 2011). To get lightly-coloured soy sauce products, some
possible solutions have been proposed in previous reports, like absorp-
tion and filtration (Miyagi et al., 2013; Terasawa et al., 2000). However,
these approaches would impair flavors and nutrition. Alternatively, esp-
cifically removing melanoidins could de-brown the soy sauce. Recently,
Det-udom et al. (2019) identified a candidate chassis organism Bacillus
subtilis suitable to growth under soy sauce fermentation conditions. B.
subtilis strains were genetically engineered to degrade xylose (the pre-
cursor of the Maillard reaction) or melanoidins, which enabled the de-
browning of soy-sauce like medium (Det-udom et al., 2019). As a result,
the browning of the soy sauce-like medium was significantly reduced
compared to that without the use of engineered recombinant B. subtilis
(Det-udom et al., 2019).
5.2. Chinese rice wine
Chinese rice wine is one of the oldest alcoholic beverages, which is
made from glutinous rice and has been consumed for more than 4000
years in China (Zhao et al., 2014). The fermentation process of Chi-
nese rice wine is carried out by fungi and yeast. Among them, Saccha-
romyces cerevisiae performs the leading role in the microbial community
(Zhao et al., 2014). However, S. cerevisiae could produce some harmful
byproducts, such as ethyl carbamate (Wu et al., 2016), whose content
is up to 515 ug/kg with an average of 160 ug/kg in Chinese rice wine
(Wu et al., 2011). Ethyl carbamate is mainly synthesized from ethanol
and urea during the fermentation process. Urea is a non-preferred nitro-
gen source for S. cerevisiae, which could accumulate. Several strategies
of engineering S. cerevisiae have been applied in Chinese rice wine pro-
duction to eliminate the generation of ethyl carbamate. One feasible ap-
proach is to increase the ability of utilize urea of S. cerevisiae. Through
creating a semi-synthetic microbial communities by overexpressing of
genes DUR1,2 (encoding urea amidolyase, which could degrade urea
into ammonia) and DUR3 (encoding urea permease) in S. cerevisiae,
the production of ethyl carbamate reduced 87% and 15% compared
to the original microbial communities, respectively (Wu et al., 2016).
Another solution is to relieve the nitrogen catabolite repression. The
nitrogen catabolite repression is an important regulatory mechanism
of prioritizing the use of the preferred nitrogen sources and repress-
ing the metabolism of nonpreferred nitrogen sources in the environ-
ment Hofman-Bang (1999). To relieve nitrogen catabolite repression,
Zhao et al. (2014) mutated some phosphorylation sites on the nuclear
localization signal of Gln3p (the most important activator for nonpre-
ferred nitrogen metabolism) and truncated the nuclear localization reg-
9
Future Foods 3 (2021) 100025
ulation signal. In addition, the authors disrupted URE2 (negative regula-
tor of nonpreferred nitrogen metabolism), which provided an additional
method to reduce urea accumulation. As a result, the concentrations of
urea were reduced by 63% and ethyl carbamate were reduced by 72% in
a model rice wine system with the novel semi-synthetic microbial com-
munities compared to the original microbial communities (Zhao et al.,
2014).
6. Challenges and perspectives
With the emergence and development of synthetic biology, more
and more food will be manufactured by engineered cellular synthesis
(van der Weele et al., 2019; Stephens et al., 2018), and will be the way
to generate current agricultural products without pesticide residues and
food allergens. For example, cell culture meat represents an important
direction of efficient and low-carbon development of agriculture in the
future (van der Weele et al., 2019). However, there are still many tech-
nical difficulties to be overcome for the application of synthetic biology
in future food.
First, although the development of synthetic biology and genetic
tools provides strategies for the transformation and enhanced of
microbial-based food, the metabolic network of microorganisms is com-
plex, and the construction cell factory faces various challenges. The
key to the efficient design of enhanced cell factories is to under-
stand the regulation and key functional gene (Almaas et al., 2004;
Liu et al., 2014a). For example, through modular pathway engineer-
ing, Liu et al. (2014a) significantly enhanced N-acetylglucosamine pro-
duction in B. subtilis. In recent years, different types of microbial chas-
sis cells with relatively simple genetic manipulation processes, rapid
growth rate, and sufficient key central metabolites have been developed
(Liu et al., 2020; Matsumoto et al., 2017; Xu et al., 2020), which could
be the basis to engineer efficient food producer strains.
Next, high-quality and low-cost synthesis of food raw materials and
key functional nutrition factors is the key to realize the large-scale appli-
cation of synthesis biology in future food. How to dynamically control
cell factories to achieve an efficiency synthesis of target products is a
key problem to be solved (Tan et al., 2016). The real-time detection
of cell physiology is the basis to understand the characteristics of cell
metabolism during the process (Wu et al., 2013). The key factors affect-
ing cell growth and product synthesis can be revealed by using real-time
multi parameter data and different levels of intracellular histochemical
data (Wu et al., 2013), which provides targeted guidance for the opti-
mization and control strategy of fermentation processes. Compared to
traditional biochemistry technologies, the recently developed fluores-
cence sensor can accurately monitor the concentration of intracellular
metabolites, which may fill the gap in metabolite detection in real time
(Zhang et al., 2020c).
The research and production of recombinant food have achieved
some preliminary achievements; however, there are still some bottle-
necks to be solved in relation to the manufacturing and safety eval-
uation. For example, cell culture meat faces challenges such as the
availability of insufficient stem cell sources, limited production scale,
large color difference with real meat, and the high production cost
Alfieri (2019). In order to solve these problems, it is necessary to com-
bine reactor design and artificial intelligence to obtain bioreactors suit-
able for large-scale cell production (Specht et al., 2018). At the same
time, the efficient production of flavor substances such as heme and the
synthesis of nutrients such as vitamins are also required (Nicolussi et al.,
2018). In terms of safety evaluation, there is still a lack of risk and safety
management standards for recombinant food, like meat analogs, that
needs to be addressed Alfieri (2019). Furthermore, it is necessary to sys-
tematically evaluate the safety of components (such as scaffolds, culture
media, oxygen carriers) and new production processes (like bioreactors)
that have not yet been used in the production of food.
Although synthetic biology for food could bring a variety of benefits
for society, possible natural and social risks should also be considered
X. Lv, Y. Wu, M. Gong et al.
(Epstein and Vermeire, 2016; Lai et al., 2019). In the future, more de-
tailed supervision and access regimes must be made to cope with the
fast-moving field of synthetic biology (Kitney et al., 2019; LeDuc and
Yuan, 2019; Trump, 2017). Simultaneously, at the technical level, bio-
containment systems should be introduced in the cell factory to avoid
its spread into the environment (Lee et al., 2018). Furthermore, related
labels should be marked on the foods produced by synthetic biology
rather than just using “plant leaf” or “natural” to mislead consumers
McFadden (2017), and more in-depth popularization of relevant knowl-
edge is necessary to enhance public acceptance of new food ingredients
and novel foods produced by synthetic biology (Jin et al., 2019). More-
over, the application of synthetic biology may generate global inequal-
ity (Lai et al., 2019). For example, although steviol glycoside, which
was a sweetener from the stevia plant traditionally used by the indige-
nous Guarani people of Brazil, has achieved commercial production by
synthetic biology, the Guarani people never benefit from the commer-
cial use of their traditional knowledge and may face challenge from the
competitive production (French, 2019). Hence related policies should
be made to grant sovereign rights of indigenous populations over their
genetic resources and promote the fair and equitable sharing of benefits
generated from the use of genetic resources.
Author contributions
J.C. and L.L. conceived the project. Y.F.L., J.H.L., G.C.D., and L.L. su-
pervised the work. X.Q.L., Y.K.W., M.Y.G., Y.G., J.Y.D. and R.L.A. wrote
and revised the paper. All authors contributed to the manuscript .
Declaration of Competing Interest
The authors declare no competing interests.
Acknowledgments
This work was financially supported bythe National Natural Sci-
ence Foundation of China (32021005, 31930085, 21808084), and the
key research and development program of China (2018YFA0900300,
2020YFA0908300).
References
Albermann, C., 2011. High versus low level expression of the lycopene biosynthesis genes
from Pantoea ananatis in Escherichia coli. Biotechnol. Lett. 33, 313–319.
Alfieri, F.Novel, 2019. Foods: artificial meat. Encycl. Food Security Sustain. 1, 280–284.
Almaas, E., Kovács, B., Vicsek, T., Oltvai, Z.N., Barabási, A.L., 2004. Global organization
of metabolic fluxes in the bacterium Escherichia coli. Nature 427, 839–843.
Ambati, R.R., Phang, S.M., Ravi, S., Aswathanarayana, R.G., 2014. Astaxanthin: sources,
extraction, stability, biological activities and its commercial applications - a review.
Marine Drugs 12, 128–152.
Aquilanti, L., Garofalo, C., Osimani, A., Clementi, F., et al., 2016. Ecology of lactic acid
bacteria and coagulase negative cocci in fermented dry sausages manufactured in Italy
and other Mediterranean countries: an overview.. Int. Food Res. J. 23, 429–455.
Awange, J., Kiema, J., 2019. Environmental pollution: extreme hydro-climatic
and food security challenges: exploiting the big data. Environ. Geoinform.
doi:10.1007/978-3-030-03017-9_32.
Baan, R., Straif, K., Grosse, Y., Secretan, B., El Ghissassi, F., Bouvard, V., et al., 2007.
Carcinogenicity of alcoholic beverages. Lancet Oncol 8, 292–293.
Ben-Arye, T., Shandalov, Y., Ben-Shaul, S., Landau, S., Zagury, Y., Lanovici, I., et al., 2020.
Textured soy protein scaffolds enable the generation of three-dimensional bovine
skeletal muscle tissue for cell-based meat. Nat. Food doi:10.1038/s43016-020-0046-5.
Berenjian, A., Mahanama, R., Kavanagh, J., Dehghani, F., 2015. Vitamin K series: current
status and future prospects. Crit. Rev. Biotechnol. 35, 199–208.
Blaiotta, G., Murru, N., Di Cerbo, A., Romano, Raffaele., Aponte, M., 2018. Production of
probiotic bovine salami using Lactobacillus plantarum 299v as adjunct. J. Sci. Food
Agric. 98, 2285–2294.
Brophy, B., Smolenski, G., Wheeler, T., Wells, D., L’Huilier, P., Laible, G., 2003. Cloned
transgenic cattle produce milk with higher levels of 𝛽-casein and 𝜅-casein. Nat.
Biotechnol. 21, 157–162.
Bych, K., Mikš, M.H., Johanson, T., Hederos, M.J., Vigsnæs, L.K., Becker, P., 2019. Produc-
tion of HMOs using microbial hosts-from cell engineering to large scale production.
Curr. Opin. Biotechnol. 56, 130–137.
Cameron, D.E., Bashor, C.J., Collins, J.J., 2014. A brief history of synthetic biology. Nat.
Rev. Microbiol. 12, 381–390.
10
Future Foods 3 (2021) 100025
Caroli, A.M., Chessa, S., Erhardt, G.J., 2009. Invited review: milk protein polymorphisms
in cattle: Effect on animal breeding and human nutrition. J. Dairy Sci. 92, 5335–5352.
Castanys-Muñoz, E., Martin, M.J., Prieto, P.A., 2013. 2’-fucosyllactose: an abundant, ge-
netically determined soluble glycan present in human milk. Nutr. Rev. 71, 773–789.
Chaudhuri, T.K., Horii, K., Yoda, T., Arai, M., Nagata, S., Terada, T.P., et al., 1999. Effect
of the extra N-terminal methionine residue on the stability and folding of recombinant
𝛼-Lactalbumin expressed in Escherichia coli. J. Mol. Biol. 285, 1179–1194.
Chen, C., Zhang, Y., Xue, M., Liu, X.W., Li, Y., Chen, X., et al., 2015. Sequential one-pot
multienzyme (OPME) synthesis of lacto-N-neotetraose and its sialyl and fucosyl
derivatives. Chem. Commun. 51, 7689–7692.
Choi, B.-K., Jiménez-Flores, R., 2001. Expression and purification of glycosylated bovine
𝛽-Casein (L70S/P71S) in Pichia pastoris. J. Agric. Food Chem. 49, 1761–1766.
Cui, S., Lv, X., Wu, Y., Li, J., Du, G., Ledesma-amaro, R., et al., 2019. Engineering a bifunc-
tional phr60-rap60-spo0a quorum-sensing molecular switch for dynamic fine-tuning
of menaquinone-7 synthesis in Bacillus subtilis. ACS Synth. Biol. 8, 1826–1837.
Cui, S., Xia, H., Chen, T., Gu, Y., Lv, X., Liu, Y., et al., 2020. Cell membrane and elec-
tron transfer engineering for improved synthesis of menaquinone-7 in Bacillus subtilis.
iScience 23, 100918.
Dabirian, Y., Gonçalves Teixeira, P., Nielsen, J., Siewers, V., David, F., 2019. FadR-based
biosensor-assisted screening for genes enhancing fatty acyl-CoA pools in Saccha-
romyces cerevisiae. ACS Synth. Biol. 8, 1788–1800.
Davinelli, S., Nielsen, M.E., Scapagnini, G., 2018. Astaxanthin in skin health, repair, and
disease: a comprehensive review. Nutrients 10, 1–12.
Denby, C.M., Li, R.A., Vu, V.T., Costello, Z., Lin, W., Chan, L.J.G., et al., 2018. Industrial
brewing yeast engineered for the production of primary flavor determinants in hopped
beer. Nat. Commun. 9, 1–10.
Deng, J., Gu, L., Chen, T., Huang, H., Yin, X., Lv, X., et al., 2019. Engineering the substrate
transport and cofactor regeneration systems for enhancing 2’-fucosyllactose synthesis
in Bacillus subtilis. ACS Synth. Biol. 8, 2418–2427.
Dekkers, B.L., Boom, R.M., van der Goot, A.J., 2018. Structuring processes for meat ana-
logues. Trends Food Sci. Tech. 81, 25–36.
Det-udom, R., Gilbert, C., Liu, L., Prakitchaiwattana, C., Ellis, T., Ledesma-Amaro, R.,
2019. Towards semi-synthetic microbial communities: enhancing soy sauce fermen-
tation properties in B. subtilis co-cultures. Microb. Cell Fact. 18, 101.
Dias, D.R., Botrel, D.A., Fernandes, R.V.D.B., Borges, S.V, 2017. Encapsulation as a tool
for bioprocessing of functional foods. Curr. Opin. Food Sci. 13, 31–37.
Dong, X., Li, N., Liu, Z., Lv, X., Li, J., Du, G., et al., 2019. Modular pathway engineering of
key precursor supply pathways for lacto-N-neotetraose production in Bacillus subtilis.
Biotechnol. Biofuels. 12, 212.
Dong, X., Li, N., Liu, Z., Lv, X., Shen, Y., Li, J., et al., 2020. CRISPRi-Guided multiplexed
fine-tuning of metabolic flux for enhanced lacto-N-neotetraose production in Bacillus
subtilis. J. Agric. Food Chem. 68, 2477–2484.
Epstein, M.M., Vermeire, T., 2016. Scientific opinion on risk assessment of synthetic biol-
ogy. Trends Biotechnol 34 (8), 601–603.
Fang, F., Zhang, J., Zhou, J., Zhou, Z., Li, T., Lu, L., et al., 2018. Accumulation of citrulline
by microbial arginine metabolism during alcoholic fermentation of soy sauce. J. Agric.
Food Chem. 66, 2108–2113.
Fernandez-Moya, R., Da Silva, N.A., 2017. Engineering Saccharomyces cerevisiae for high-
-level synthesis of fatty acids and derived products. FEMS Yeast Res 17, 1–15.
French, K.E., 2019. Harnessing synthetic biology for sustainable development. Nature Sus-
tainability 2, 250–252.
Guiziou, S., Sauveplane, V., Chang, H.-J., Clerté, C., Declerck, N., Jules, M., et al., 2016.
A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acids Res 44,
gkw624.
Heirendt, L., Arreckx, S., Pfau, T., Mendoza, S.N., Richelle, A., Heinken, A., et al., 2019.
Creation and analysis of biochemical constraint-based models using the COBRA Tool-
box v.3.0. Nature Protocols 14, 639–702.
Hofman-Bang, J., 1999. Nitrogen catabolite repression in Saccharomyces cerevisiae. Mol.
Biotechnol. 12, 35–71.
Hollands, K., Baron, C.M., Gibson, J., Kelly, K.J., Krasley, E.A., Laffend, L.A., et al., 2019.
Engineering two species of yeast as cell factories for 2’-fucosyllactose. Metab. Eng. 52,
232–242.
Holscher, H.D., Davis, S.R., Tappenden, K.A., 2014. Human milk oligosaccharides influ-
ence maturation of human intestinal Caco-2Bbe and HT-29 cell lines. J. Nutr. 144,
586–591.
Huang, D., Yang, K., Liu, J., Xu, Y., Wang, Y., Wang, R., et al., 2017. Metabolic engineering
of Escherichia coli for the production of 2′-fucosyllactose and 3-fucosyllactose through
modular pathway enhancement. Metab. Eng. 41, 23–38.
Ignea, C., Raadam, M.H., Motawia, M.S., Makris, A.M., Vickers, C.E., Kampranis, S.C.,
2019. Orthogonal monoterpenoid biosynthesis in yeast constructed on an isomeric
substrate. Nat. Commun. 10, 1–15.
Jin, S., Clark, B., Kuznesof, S., Lin, X., Frewer, L.J., 2019. Synthetic biology applied in
the agrifood sector: Public perceptions, attitudes and implications for future studies
2019. Trends Food Sci. Tech. 91, 454–466.
Jung, S., Chin, Y., Lee, Y., Seo, J., 2019. Enhanced production of 2′-fuco-
syllactose from fucose by elimination of rhamnose isomerase and arabinose
isomerase in engineeredEscherichia coli. Biotechnol Bioeng. 116 (9), 2412–
2417.
Kamao, K., Suhara, Y., Tsugawa, N., Uwano, M., Yamaguchi, N., Uenishi, K., et al., 2007.
Vitamin K content of foods and dietary vitamin K intake in japanese young women.
J. Nutr. Sci. Vitaminol. 53, 464–470.
Kang, L., Alim, A., Song, H., 2019a. Identification and characterization of flavor precur-
sor peptide from beef enzymatic hydrolysate by Maillard reaction. J. Chromatogr. B.
Analyt. Technol. Biomed. Life. Sci. 1104, 176–181.
Kang, W., Ma, T., Liu, M., Qu, J., Liu, Z., Zhang, H., et al., 2019b. Modular enzyme assem-
bly for enhanced cascade biocatalysis and metabolic flux. Nat. Commun. 10, 4248.
X. Lv, Y. Wu, M. Gong et al.
Kang, Y.C., Richardson, T., 1988. Molecular cloning and expression of bovine 𝜅-Casein in
Escherichia coli. J. Dairy Sci. 71, 29–40.
Katz, L., Chen, Y.Y., Gonzalez, R., Peterson, T.C., Zhao, H., Baltz, R.H., 2018. Synthetic
biology advances and applications in the biotechnology industry: a perspective. J. Ind.
Microbiol. Biot. 45 (7), 449–461.
Khalil, A.S., Collins, J.J., 2010. Synthetic biology: applications come of age. Nat. Rev.
Genet. 11 (5), 367–379.
Kim, T.R., Goto, Y., Hirota, N., Kuwata, K., Denton, H., Wu, S.Y., et al., 1997. High-level
expression of bovine beta-lactoglobulin in Pichia pastoris and characterization of its
physical properties. Protein Eng. Des. Sel. 10, 1339–1345.
Kim, Y.K., Yu, D.Y., Kang, H.A., Yoon, S., Chung, B.H., 1999. Secretory expression of
human 𝛼(s1)-casein in Saccharomyces cerevisiae. J. Microbiol. Biotechn. 9, 196–200.
Kitney, R., Adeogun, M., Fujishima, Y., Goñi-Moreno, Á., Johnson, R., Maxon, M., Steed-
man, S., Ward, S., Winickoff, D., Philp, J., 2019. Enabling the advanced bioecon-
omy through public policy supporting biofoundries and engineering biology. Trends
Biotechnol. 37 (9), 917–920.
Lai, H.-E., Canavan, C., Cameron, L., Moore, S., Danchenko, M., Kuiken, T., Sekeyová, Z.,
Freemont, P.S., 2019. Synthetic biology and the United Nations. Trends Biotechnol
37 (11), 1146–1151.
Larroude, M., Celinska, E., Back, A., Thomas, S., Nicaud, J.M., Ledesma-Amaro, R., 2018. A
synthetic biology approach to transform Yarrowia lipolytica into a competitive biotech-
nological producer of 𝛽-carotene. Biotechnol. Bioeng. 115 (2), 464–472.
Lawson, C., Martí, J.M., Radivojevic, T., Jonnalagadda, S.V.R., Gentz, R., Hillson, N.J.,
et al., 2020. Machine learning for metabolic engineering: a review. Metab. Eng.
doi:10.1016/j.ymben.2020.10.005.
Ledesma-Amaro, R., Lozano-Martínez, P., Jiménez, A., Revuelta, J.L., 2015. Engineering
Ashbya gossypii for efficient biolipid production. Bioengineered 6 (2), 119–123.
Ledesma-Amaro, R., Santos, M., Jiménez, A., Revuelta, J.L., 2014. Tuning single-cell oil
production in Ashbya gossypii by engineering the elongation and desaturation sys-
tems. Biotechnol Bioengin 111 (9), 1782–1791.
Ledesma-Amaro, R., Jiménez, A., Revuelta, J.L., 2018. Pathway grafting for polyunsat-
urated fatty acids production inAshbya gossypii through golden gate rapid assembly.
ACS synth. Biol. 7 (10), 2340–2347.
LeDuc, J.W., Yuan, Z., 2019. Safety and security in the age of synthetic biology. J. Biosaf.
Biosecur. 1 (2), 77–79.
Lee, J.W., Chan, C.T.Y., Slomovic, S., Collins, J.J., 2018. Next-generation biocontainment
systems for engineered organisms. Nat. Chem. Biol. 14 (6), 530–537.
Li, C., Swofford, C.A., Sinskey, A.J., 2020. Modular engineering for microbial production
of carotenoids. Metab. Eng. Commun. 10, e00118.
Li, P., Hotamisligil, G.S., 2010. Host and microbes in a pickle. Nature 464, 1287–1288.
Liang, C., Zhang, X., Wu, J., Mu, S., Wu, Z., Jin, J., et al., 2020. Dynamic control of
toxic natural product biosynthesis by an artificial regulatory circuit. Metab. Eng. 57,
239–246.
Liu, J., Wu, X., Yao, M., Xiao, W., Zha, J., 2020. Chassis engineering for microbial produc-
tion of chemicals: from natural microbes to synthetic organisms. Curr. Opin. Biotech-
nol. 66, 105–112.
Liu, L., Martínez, J.L., Liu, Z., Petranovic, D., Nielsen, J., 2014b. Balanced globin pro-
tein expression and heme biosynthesis improve production of human hemoglobin in
Saccharomyces cerevisiae. Metab. Eng. 21, 9–16.
Liu, X.J., Liu, R.S., Li, H.M., Tang, Y.J., 2012. Lycopene production from synthetic medium
by Blakeslea trispora NRRL 2895 (+) and 2896 (-) in a stirred-tank fermenter. Biopro-
cess Biosyst. Eng. 35, 739–749.
Liu, Y., Zhu, Y., Li, J., Shin, H.D., Chen, R.R., Du, G., et al., 2014a. Modular pathway
engineering of Bacillus subtilis for improved N-acetylglucosamine production. Metab.
Eng. 23, 42–52.
Lv, X., Cui, S., Gu, Y., Li, J., Du, G., Liu, L., 2020. Enzyme assembly for compartmentalized
metabolic flux control. Metabolites 10, 125.
Ma, T., Shi, B., Ye, Z., Li, X., Liu, M., Chen, Y., et al., 2019. Lipid engineering combined
with systematic metabolic engineering of Saccharomyces cerevisiae for high-yield pro-
duction of lycopene. Metab. Eng. 52, 134–142.
Mahdinia, E., Demirci, A., Berenjian, A., 2017. Production and application of
menaquinone-7 (vitamin K2): a new perspective. World. J. Microbiol. Biotechnol. 33,
1–7.
Mahdinia, E., Demirci, A., Berenjian, A., 2019. Biofilm reactors as a promising method
for vitamin K (menaquinone-7) production. Appl. Microbiol. Biotechnol. 103,
5583–5592.
Marella, E.R., Holkenbrink, C., Siewers, V., Borodina, I., 2018. Engineering microbial fatty
acid metabolism for biofuels and biochemicals. Curr. Opin. Biotechnol. 50, 39–46.
Matsumoto, T., Tanaka, T., Kondo, A., 2017. Engineering metabolic pathways in Es-
cherichia coli for constructing a "microbial chassis" for biochemical production. Biore-
sour. Technol. 245, 1362–1368.
McClements, D.J., 2020. Future foods: a manifesto for research priorities in structural
design of foods. Food. Funct. 11, 1933.
McFadden, B.R., 2017. The unknowns and possible implications of mandatory labeling.
Trends Biotechnol 35 (1), 1–3.
Miyagi, A., Nabetani, H., Nakajima, M., 2013. Decolorization of Japanese soy sauce
(shoyu) using adsorption. J. Food. Eng. 116, 749–757.
Myers, S.S., Smith, M.R., Guth, S., Golden, C.D., Vaitla, B., Mueller, N.D., et al., 2017.
Climate change and global food systems: potential impacts on food security and un-
dernutrition. Annu Rev Public Health 38, 259–277.
Nicolussi, A., Auer, M., Sevcnika, r B., et al., 2018. Posttranslational modification of
heme in peroxidases -impact on structure and catalysis. Arch. Biochem. Biophys. 643,
14–23.
Olsson, K., Carlsen, S., Semmler, A., Simón, E., Mikkelsen, M.D., Møller, B.L., 2016. Mi-
crobial production of next-generation stevia sweeteners. Microb. Cell Fact. 15, 1–14.
11
Future Foods 3 (2021) 100025
Palou, A., 2006. New challenges in basic and applied nutrition. Rev. Med. Univ. Navarra.
50, 62–70.
Park, S.Y., Binkley, R.M., Kim, W.J., Lee, M.H., Lee, S.Y., 2018. Metabolic engineering of
Escherichia coli for high-level astaxanthin production with high productivity. Metab.
Eng. 49, 105–115.
Philippe, R.N., De Mey, M., Anderson, J., Ajikumar, P.K., 2014. Biotechnological produc-
tion of natural zero-calorie sweeteners. Curr. Opin. Biotechnol. 26, 155–161.
Priem, B., Gilbert, M., Wakarchuk, W.W., Heyraud, A., Samain, E., 2002. A new fer-
mentation process allows large-scale production of human milk oligosaccharides by
metabolically engineered bacteria. Glycobiology 12, 235–240.
Qiao, K., Wasylenko, T.M., Zhou, K., Xu, P., Stephanopoulos, G., 2017. Lipid production
inYarrowia lipolytica is maximized by engineering cytosolic redox metabolism. Nat.
Biotechnol. 35, 173–177.
Ren, L., Peng, C., Hu, X., Han, Y., Huang, H., 2020. Microbial production of vitamin K2:
current status and future prospects. Biotechnol. Adv. 39, 107453.
Reverri, E., Devitt, A., Kajzer, J., Baggs, G., Borschel, M., 2018. Review of the clinical
experiences of feeding infants formula containing the human milk oligosaccharide
2′-fucosyllactose. Nutrients 10, E1346.
Roell, M.S., Zurbriggen, M.D., 2020. The impact of synthetic biology for future agriculture
and nutrition. Curr. Opin. Biotechnol. 61, 102–109.
Rzechonek, D.A., Dobrowolski, A., Rymowicz, W., Mirończuk, A.M., 2018. Recent ad-
vances in biological production of erythritol. Crit. Rev. Biotechnol. 38, 620–633.
Satoh, M., Nomi, Y., Yamada, S., Takenaka, M., Ono, H., Murata, M., 2011. Identifica-
tion of 2, 4-Dihydroxy-2, 5-dimethyl-3 (2H)-thiophenone as a low-molecular-weight
yellow pigment in soy sauce. Biosci. Biotechnol. Biochem. 75, 1240–1244.
Seki, H., Tamura, K., Muranaka, T., 2018. Plant-derived isoprenoid sweeteners: recent
progress in biosynthetic gene discovery and perspectives on microbial production.
Biosci. Biotechnol. Biochem. 82, 927–934.
Shapira, P., Kwon, S., Youtie, J., 2017. Tracking the emergence of synthetic biology. Sci-
entometrics 112, 1439–1469.
Shleikin, A.G., Medvedev, Y.V., 2014. Role of peroxidation and heme catalysis in col-
oration of raw meat. Acta. Sci. Pol. Technol. Aliment. 13, 123–127.
Simons, G., van den Heuvel, W., Reynen, T., Frijters, A., Rutten, G., Slangen, C.J., et al.,
1993. Overproduction of bovine 𝛽-casein in Escherichia coli and engineering of its main
chymosin cleavage site. Protein Eng. Des. Sel. 6, 763–770.
Specht, E.A., Welch, D.R., Rees Clayton, E.M, Lagally, C.D., 2018. Opportunities for ap-
plying biomedical production and manufacturing methods to the development of the
clean meat industry. Biochem. Eng. J. 132, 161–168.
Stephens, N., Di Silvio, L., Dunsford, I., Ellis, M., Glencross, A., Sexton, A., 2018. Bring-
ing cultured meat to market: technical, socio-political, and regulatory challenges in
cellular agriculture. Trends. Food. Sci. Technol. 78, 155–166.
Tan, J., Dai, W., Lu, M., Lv, H., Guo, L., Zhang, Y., et al., 2016. Study of the dynamic
changes in the non-volatile chemical constituents of black tea during fermentation
processing by a non-targeted metabolomics approach. Food Res. Int. 79, 106–113.
Tao, N., DePeters, E.J., Freeman, S., German, J.B., Grimm, R., Lebrilla, C.B., 2008. Bovine
milk glycome. J. Dairy. Sci. 91, 3768–3778.
Terasawa, N., Murata, M., Homma, S., 2000. Decolorization of brown pigments in foods
by immobilized mycelia of Coriolus versicolor IFO 30340 and Paecilomyces canadensis
NC-1. J. Food. Sci. 65, 870–875.
Tramontin, L.R.R., Kildegaard, K.R., Sudarsan, S., Borodina, I, 2019. Enhancement of as-
taxanthin biosynthesis in oleaginous yeast Yarrowia lipolytica via microalgal pathway.
Microorganisms 7 (10), 472.
Trump, B.D., 2017. Synthetic biology regulation and governance: lessons from TAPIC for
the United States, European Union, and Singapore. Health Policy, Governance for
intersectoral action 121 (11), 1139–1146.
Van Berkel, P.H.C., Welling, M.M., Geerts, M., van Veen, H.A., Ravensbergen, B., Salahed-
dine, M., et al., 2002. Large scale production of recombinant human lactoferrin in the
milk of transgenic cows. Nat. Biotechnol. 20, 484–487.
van der Weele, C., Feindt, P., Jan van der Goot, A., van Mierlo, B., van Boekel, M., 2019.
Meat alternatives: an integrative comparison. Trends Food Sci. Technol. 88, 505–512.
Vandenplas, Y., Berger, B., Carnielli, V.P., Ksiazyk, J., Lagström, H., Luna, M.S., et al.,
2018. Human milk oligosaccharides: 2’-fucosyllactose (2’-FL) and lacto-n-neotetraose
(LNnT) in infant formula. Nutrients 10, E1161.
Wang, S.H., Yang, T.S., Lin, S.M, Tsai, M.S, Wu, S.H., T Mao, S.J., et al., 2002. Expres-
sion, characterization, and purification of recombinant porcine lactoferrin in Pichia
pastoris. Protein. Expr. Purif. 25, 41–49.
Wang, W., Cha, Y.J., 2018. Volatile compounds in seasoning sauce produced from soy
sauce residue by reaction flavor technology. Prev. Nutr. Food Sci. 23, 356–363.
Weber, J.V., Sharypov, V.I., 2009. Ethyl carbamate in foods and beverages: a review.
Environ. Chem. Lett. 7, 233–247.
Wu, C., Zhou, J., Hu, N., Ha, D., Miao, X., Wang, P., 2013. Cellular impedance sens-
ing combined with LAPS as a new means for real-time monitoring cell growth and
metabolism. Sensor. Actuat. A Phys. 199, 136–142.
Wu, D., Li, X., Lu, J., Chen, J., Zhang, L., Xie, G., 2016. Constitutive expression of the
DUR1,2 gene in an industrial yeast strain to minimize ethyl carbamate production
during Chinese rice wine fermentation. FEMS. Microbiol. Lett. 363, fnv214.
Wu, P.G., Yang, D.J., Shen, X.H., Wang, L.Y., Pan, X.D., Zhang, J., et al., 2011. Sur-
vey and analysis of ethyl carbamate in commercial fermented foods in Hangzhou
in 2010. Zhonghua yu fang yi xue za zhi (Chinese J. Prevent. Med.) 45, 609–
611.
Wu, Y., Chen, T., Liu, Y., Tian, R., Lv, X., Li, J., et al., 2020a. Design of a programmable
biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of
metabolic flux in Bacillus subtilis. Nucleic Acids Res. 48, 996–1009.
Wu, Y., Liu, Y., Lv, X., Li, J., Du, G., Liu, L., 2020b. Applications of CRISPR in a microbial
cell factory: from genome reconstruction to metabolic network reprogramming. ACS
Synth. Biol. 9, 2228–2238.
X. Lv, Y. Wu, M. Gong et al.
Xu, N., Liu, Y., Jiang, H., Liu, J., Ma, Y., 2020a. Combining protein and metabolic en-
gineering to construct efficient microbial cell factories. Curr. Opin. Biotechnol. 66,
27–35.
Xu, P., Li, L., Zhang, F., Stephanopoulos, G., Koffas, M., 2014. Improving fatty acids pro-
duction by engineering dynamic pathway regulation and metabolic control. Proc.
Natl. Acad. Sci. USA 111, 11299–11304.
Xu, P., Qiao, K., Ahn, W.S., Stephanopoulos, G., 2016. Engineering Yarrowia lipolytica as
a platform for synthesis of drop-in transportation fuels and oleochemicals. Proc. Natl.
Acad. Sci. USA 113, 10848–10853.
Xu, X., Liu, Y., Du, G., Ledesma-Amaro, R., Liu, L., 2020. Microbial chassis development
for natural product biosynthesis. Trends Biotechnol 38, 779–796.
Yang, S., Wang, Y., Cai, Z., Zhang, G., Song, H., 2020. Metabolic engineering of Bacillus
subtilis for high-titer production of menaquinone-7. AIChE. J. 66, 1–11.
Yang, T., Zhang, J., Ke, D., Yang, W., Tang, M., Jiang, J., et al., 2019. Hydrophobic recog-
nition allows the glycosyltransferase UGT76G1 to catalyze its substrate in two orien-
tations. Nat. Commun. 10 (1), 3214.
Yu, S., Liu, J., Yun, E.J., Kwak, S., Kim, K.H., Jin, Y., 2018. Production of a human milk
oligosaccharide 2’-fucosyllactose by metabolically engineered Saccharomyces cere-
visiae. Microb. Cell. Fact. 17, 1–10.
Zhang, C., Seow, V.Y., Chen, X., Too, H.-P., 2018c. Multidimensional heuristic process for
high-yield production of astaxanthin and fragrance molecules in Escherichia coli. Nat.
Commun. 9, 1858.
Zhang, G.Q., Zhao, X.R., Li, X.L, Du, G.C., Chen, J., 2020b. Challenges and
possibilities for bio-manufacturing cultured meat. Trends Food Sci Tech
doi:10.1016/j.tifs.2020.01.026.
Zhang, J., Petersen, S.D., Radivojevic, T., Ramirez, A., Pérez-Manríquez, A., Abeliuk, E.,
et al., 2020a. Combining mechanistic and machine learning models for predictive
engineering and optimization of tryptophan metabolism. Nat. Commun. 11, 4880.
12
Future Foods 3 (2021) 100025
Zhang, J.R., Du, G.C., Chen, J., Fang, F., 2016. Characterization of a Bacillus amylolique-
faciens strain for reduction of citrulline accumulation during soy sauce fermentation.
Biotechnol. Lett. 38, 1723–1731.
Zhang, J.R., Fang, F., Chen, J., Du, G.C, 2014. The arginine deiminase pathway of koji
bacteria is involved in ethyl carbamate precursor production in soy sauce. FEMS.
Microbiol. Lett. 358, 91–97.
Zhang, W.P, Cheng, Y., Li, Y.D, Du, G.C, Xie, G.F, Zou, H.J, et al., 2018b. Adaptive evolu-
tion relieves nitrogen catabolite repression and decreases urea accumulation in cul-
tures of the chinese rice wine yeast strain Saccharomyces cerevisiae XZ-11. J. Agric.
Food. Chem. 66, 9061–9069.
Zhang, Y., Li, Z., Tu, Y., Cheng, W., Yang, Y., 2018a. Tomato (Solanum lycopersicum)
SlIPT4, encoding an isopentenyltransferase, is involved in leaf senescence and ly-
copene biosynthesis during fruit ripening. BMC. Plant. Biol. 18, 10–12.
Zhang, Z., Cheng, X., Zhao, Y., Yang, Y., 2020c. Lighting up live-cell andin vivo central
carbon metabolism with genetically encoded fluorescent sensors. Annu. Rev. Anal.
Chem. 13, 293–314.
Zhao, X.R, Zou, H.J, Fu, J.W, Zhou, J.W, Du, G.C, Chen, J., 2014. Metabolic engineering
of the regulators in nitrogen catabolite repression to reduce the production of ethyl
carbamate in a model rice wine system. Appl. Environ. Microbiol. 80, 392–398.
Zhao, X.R., Choi, K.R., Lee, S.Y., 2018. Metabolic engineering of Escherichia coli for se-
cretory production of free haem. Nat. Catal. 1, 720–728.
Zhu, Z., Zhou, Y.J., Krivoruchko, A., Grininger, M., Zhao, Z.K., Nielsen, J., 2017. Expand-
ing the product portfolio of fungal type I fatty acid synthases. Nat. Chem. Biol. 13,
360–362.