Applied Microbiology and Biotechnology

https://doi.org/10.1007/s00253-019-10157-x

MINI-REVIEW

Metabolic engineering for the production of fat-soluble vitamins:

advances and perspectives
Panhong Yuan 1,2 & Shixiu Cui 1,2 & Yanfeng Liu 1,2 & Jianghua Li 1,2 & Guocheng Du 1,2 & Long Liu 1,2

Received: 28 July 2019 / Revised: 19 September 2019 / Accepted: 24 September 2019

# Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Fat-soluble vitamins are vitamins that are insoluble in water, soluble in fat, and organic solvents; they are found in minute amount
in various foods. Fat-soluble vitamins, including vitamins A, D, E, and K, have been widely used in food, cosmetics, health care
products, and pharmaceutical industries. Fat-soluble vitamins are currently produced via biological and chemical synthesis. In
recent years, fat-soluble vitamin production by biotechnological routes has been regarded as a very promising approach. Based
on biosynthetic pathways, considerable advances of α-tocopherol and β-carotenes have been achieved in transgenic plants and
microalgae. Microbial fermentation, as an alternative method for the production of vitamin K and β-carotenes, is attracting
considerable attention because it is an environment friendly process. In this review, we address the function and applications of
fat-soluble vitamins, and an overview of current developments in the production of fat-soluble vitamins in transgenic plants,
microalgae, and microorganisms. We focus on the metabolic and process engineering strategies for improving production of fat-
soluble vitamins, and we hope this review can be useful for the people who are interested in the production of fat-soluble vitamins
by biotechnological routes.

Keywords Fat-soluble vitamins . Transgenic plant production . Microalgae biosynthetic product . Microbial production . Systems
metabolic engineering

Introduction are necessary for human body because of different physio-

Vitamins are organic compounds that are required in very
small quantities for growth and health of human (Pilz
et al. 2019), and they are grouped under an alphabetized
vitamin generic descriptor title (Zou et al. 2012). Each
kind of vitamin includes a group of compounds with spe-
cial biological and chemical activities (Perez-Hernandez
et al. 2016). Among them, fat-soluble vitamins include
vitamin A, D, E, and K.
The body needs vitamins in order to function properly and
stay healthy (Zittermann et al. 2014). Fat-soluble vitamins
* Guocheng Du
gcdu@jiangnan.edu.cn
* Long Liu
longliu@jiangnan.edu.cn
1
Key Laboratory of Carbohydrate Chemistry and Biotechnology,
Ministry of Education, Jiangnan University, Wuxi 214122, China
2
Key Laboratory of Industrial Biotechnology, Ministry of Education,
Jiangnan University, Wuxi 214122, China
logical function as shown in Fig. 1a–d. Vitamin A is used for
any compound possessing the biological activity of retinol
(Fig. 1e). Carotenoids have principal functions as antioxi-
dants in plants and animals. Vitamin D can be available as
either vitamin D2 or D3 in many nutritional supplements and
fortified foods (Fig. 1f). Vitamin D2 is formed from ergos-
terol by the UV irradiation in yeast, and vitamin D3 is formed
from cholesterol by the chemical conversion or lanolin by
the irradiation of 7-dehydrocholesterol. Furthermore, vita-
min E refers to a group of compounds, including four to-
copherols and four tocotrienols (DellaPenna and Last
2006) (Fig. 1g). For vitamin E, α-tocopherol has the highest
activity, and other tocochromanols have an activity range
from 0 to 50% of that of α-tocopherol. Vitamin K occurs in
its natural forms as vitamin K1 (phylloquinone), vitamin K2
(menaquinone) (Shearer and Newman 2008), and vitamin
K3 (menadione) (Fig. 1h). All vitamin K are fat-soluble
compounds, which share a corporate 2-methyl-1,4-
napthoquinone nucleus and the polyisoprenoid side chain
with different length and saturation degrees at the 3-
position (Schurgers and Vermeer 2000).
Appl Microbiol Biotechnol
 Appl Microbiol Biotechnol
ƒFig. 1 a Physiological function of vitamin A. b Physiological function of
vitamin D. c Physiological function of vitamin E. d Physiological
function of vitamin K. e Structure of vitamin A. f Structure of vitamin
D2 and vitamin D3. g Vitamin E structural diversity of aliphatic side-
chain. Tocopherol, δ-tocopherol, R1 = H, R2 = H; γ-tocopherol, R1 =
H, R2 = CH3; β-tocopherol, R1 = CH3, R2 = H; α-tocopherol, R1 = CH3,
R2 = CH3. Tocotrienol, δ-tocotrienol, R1 = H, R2 = H; γ-tocotrienol, R1
= H, R2 = CH3; β-tocotrienol, R1 = CH3, R2 = H; α-tocotrienol, R1 =
CH3, R2 = CH3. h Vitamin K structural diversity of aliphatic side-chain.
K1, 2-methyl-3-phytyl-1,4-naphthoquinone (phylloquinone); K2,
menaquinone-n (MKn, n = 1–14); K3, 2-methyl-1,4-naphthoquinone
(menadione)
Vitamin A is ingested in the form of carotenoids or retinyl
esters and metabolized to active compounds such as all-trans-
retinoic acid, which is the main medium of biological actions
of vitamin A, and 11-cis-retinal, which is important for vision
(Conaway et al. 2013). Although vitamin A can be extracted
from animal tissues, most commercial vitamin A is chemically
synthesized with the distributed resources, cumbersome steps,
and high cost involved in the extraction method. Furthermore,
vitamin D2 and vitamin D3 can be synthesized by photochem-
ical strategy. A low-pressure mercury lamp, a new nitrogen
agitated photoreactor, a new preparative liquid chromatogra-
phy, and computer control system were established. It was
successfully applied to the separation and purification of vita-
min D2 by preparative liquid chromatography purification
(Ramawat and Mérillon 2013). However, it is unfavorable
for scale production and cost reduction. At present, α-
tocopherol can be synthesized by a chemical process or ex-
tracted from vegetable oils. Extraction from oils is not highly
efficient, because of low yield of α-tocopherol. However,
synthetic α-tocopherols is a mixture of eight stereoisomers,
which consists of four 2R-isomers and four 2S-isomers
(Ajjawi and David 2004). The chemical synthesis of vitamin
K1 involves the condensation of menadione with isophytol;
this process has several drawbacks. First, the solvents and
catalysts are hazardous to both the environment and humans
(Bonrath and Netscher 2005), and second, menadione con-
denses with phytol to form 10–20% of the inactive Z-isomer
of vitamin K1 (Daines et al. 2003).
Thus far, vitamins are mainly produced by chemical syn-
thesis. However, chemical synthesis for vitamins production
could become limited because of variable raw material supply
as the increasing demand of vitamins (Xu 2008). Moreover,
vitamins obtained from chemical synthesis may not be envi-
ronment friendly. As the biosynthetic pathways of fat-soluble
vitamins are becoming clear, significant advances have been
achieved in transgenic plant production and microbial produc-
tion, and thus, it is necessary to summarize the progress to
further stimulate the research interest in this field.
In this review, we provide an overview of the function and
applications of fat-soluble vitamins. Then, the metabolic reg-
ulation of fat-soluble vitamins in transgenic plants,
microalgae, and microorganisms is summarized. Finally, we
emphasize on the metabolic engineering strategies and discuss
the perspective of research for fat-soluble vitamins
production.
Function and applications of fat-soluble
vitamins
Vitamins are trace amounts of organic substances that animals
and humans must obtain from food to maintain normal phys-
iological functions (Zhou et al. 2012). Vitamins are classified
as fat-soluble and water-soluble depending on their solubility
in polar and non-polar solvents (Zou et al. 2013). The human
body needs to absorb vitamins through various foods and
supplements as it cannot synthesize vitamins on its own
(Beveridge et al. 2018). Vitamins are related with correspond-
ing vitamin deficiency diseases (Chen et al. 2016). As report-
ed, a vitamin D deficiency can lead to bone diseases
(Savastano et al. 2017); the defect can cause symptoms chang-
ing from night blindness to keratomalacia and xerophthalmia,
which can lead to complete blindness (Leal et al. 2006).
Vitamin E deficiency, which is very rare, causes the nerve
damage; a vitamin K deficiency causes spontaneous bleeding.
Vitamins are widely used as dietary supplements, and their use
in beverages and functional food has also increased dramati-
cally (Tarento et al. 2018).
Vitamin A contains retinaldehyde, which combines with
protein to produce rhodopsin, which helps maintain visual
function in the dark. Vitamin A can also promote the differ-
entiation of various tissues and structures of the eyes to ensure
normal visual function. In addition to retinaldehyde, retinol,
retinic acid, and other derivatives of vitamin A are involved in
corneal protein synthesis and energy metabolism. Vitamin A
induces an enhanced expression of the epidermal growth fac-
tor receptor of an endothelial cell; it promotes corneal wound
healing and maintains normal vision and immune system in-
tegrity (Ruhl et al. 2004). The lack of vitamin A causes night
blindness because of slow and incomplete regeneration of
rhodopsin. Vitamin A plays a significant role in animal immu-
nity, especially in non-specific immunity (Jason et al. 2002).
Vitamin A can effectively maintain epidermal integrity, and it
is the barrier of the respiratory tract, digestive tract, and uri-
nary system and can effectively prevent pathogen invasion.
Vitamin A deficiency affects the animal’s lymphoid tissue,
causing some immune organs to atrophy; it in turn affects
the cellular immunity of the animal. Further, vitamin A defi-
ciency can also affect the humoral immunity of animals by
reducing the response of antigens and antibodies. Vitamin A is
an essential substance to maintain the normal epithelial tissue.
It participates in the synthesis of mucosal cells through the
formation of retinol-phosphomannose, and it then affects the
normal differentiation of the respiratory tract, digestive tract,
cornea, and reproductive urinary system. A vitamin A

deficiency can cause the keratinization of epithelial tissue,
which can cause diarrhea, colds, pneumonia, eye diseases,
and animal performance, and vitamin A has become an indis-
pensable material in feed (Knutson and Wessling-Resnick
2003).
The primary function of vitamin D is to promote the bone
calcification, absorption of phosphorus and calcium in the
intestine, and differentiation of intestinal mucosal cells
(Cutolo et al. 2007). Vitamin D can regulate animal immunity
by regulating the proliferation and differentiation of immune
cells and the secretion of cytokines (Matheu et al. 2003).
Vitamin D is produced by the skin or supplied by the diet
and is converted to 25-hydroxyvitamin D in the liver (Jones
2007). Vitamin D deficiency causes rickets and reduces im-
munity in animals (Wittke et al. 2004). Furthermore, vitamin
D can affect the production performance of animals, including
laying rate, meat quality, and blood biochemical indexes (Lu
et al. 2014).
The most important function of vitamin E is its biological
antioxidant effect (Kelly et al. 2004). Vitamin E also partici-
pates in phosphorylation reactions, synthesis of vitamin C,
metabolism of vitamin B12, and regulation of cell DNA syn-
thesis in animals. Also, vitamin E reduces the toxicity of some
heavy metals and toxic elements, improving animal immunity
and disease resistance (Albers et al. 2003). Moreover, vitamin
E can improve animal production performance, immunity, and
antioxidant capacity (Hanson et al. 2007).
Primarily, phylloquinone occurs in a singular form,
whereas menaquinones (MK) contain 14 compounds with
side chains as short as 1 isoprene unit MK-1, and up to 14
isoprene units MK-14; however, the most common ones
are MK-7 and MK-4 (Bentley and Meganathan 1982).
Menaquinone is the most effective cofactor in the treat-
ment and guard against calcified arterial, plaque athero-
sclerosis, and bone metabolism. MK-7 could be chemical-
ly synthesized or produced by microbial fermentation; and
its purity also varies relying on method and procedure
used to produce it (Orlando et al. 2019). Vitamin K par-
ticipates in clotting activities and activates clot-related en-
zyme activity in animals. The lack of vitamin K can pro-
long clotting time, and it is related to the formation of
calcium-binding protein.
Fat-soluble vitamins have been extensively applied in the
industrial production of animal feed additives, nutraceuticals,
cosmetics, and pharmaceuticals. Currently, most fat-soluble
vitamins are produced by chemical synthesis. In recent years,
owing to the increasing public concern about food safety, the
usage of chemically synthesized fat-soluble vitamin as func-
tional cosmetic agents or food additives has been strictly con-
trolled (Montano Velazquez et al. 2006). This has fired up the
enthusiasm for searching natural fat-soluble vitamins for sus-
tainable large-scale production using transgenic plants,
microalgae, and microorganisms.
Appl Microbiol Biotechnol
Metabolic production of vitamin A
Biosynthetic provitamin A in transgenic plant
Carotenoids are diverse classes of secondary metabolites syn-
thesized by bacteria, algae, fungi, and plants. They are exten-
sively applied in poultry, food, cosmetics, nutraceuticals, and
pharmaceuticals, on account of their antioxidant properties
and vitamin A activities (Saini and Keum 2017). Provitamin
A is synthesized in transgenic plants by enhancing the meta-
bolic flux upstream of a target product to achieve product
accumulation (Fig. 2a). The overexpression of one or more
biosynthetic enzymes is a significant strategy to improve the
metabolic flux upstream of a target product (Table 1). For
instance, Welsch et al. overexpressed a phytoene synthase
transgene in Cassava plants, producing yellow-fleshed, high-
carotenoid roots. The result suggested that through breeding
and genetic modification, the content of provitamin A in cas-
sava can be improved in the roots by a phytoene synthase
(PSY) gene expression (Welsch et al. 2010). In the same
way, Paul et al. expressed the Fe’i banana-derived phytoene
synthase 2a (MtPsy2a) gene in bananas, resulting in the pro-
duction of provitamin A 55 μg/g (Paul et al. 2017).
Heterologous gene expression is an effective approach to
increase provitamin A content. Wang et al. expressed carotene
desaturase gene (CrtI) and the bacterial phytoene synthase
gene (CrtB) in common wheat. Both genes were expressed,
resulting in a darker grain phenotype with an approximately 8-
fold increase in the content of carotenoid; a β-carotene in-
creases to 65-fold of wheat grain dry weight, and a provitamin
A content increases to 76-fold of wheat grain dry weight
(Wang et al. 2014). As tissues do not accumulate carotenoids
originally, Bai et al. studied the effect of precursor product
balance on the pathway of carotenoid by expressing the bac-
terial phytoene desaturase gene (PaCRTI), the maize phytoene
synthase gene (ZmPSY1), and Arabidopsis thaliana (A.
thaliana)1-deoxy-D-xylulose 5-phosphate synthase (AtDXS)
in transgenic plants. The result suggested that isoprenoid pre-
cursors from the α-C-methyl-D-erythritol-4-phosphate (MEP)
pathway is one of the key factors (Bai et al. 2016).
In another study, Che et al. demonstrated that the co-
expression of carotenoid biosynthesis genes stacked with
homogentisate geranylgeranyl transferase (HGGT) could alle-
viate oxidative degradation of β-carotene and thus enhance β-
carotene stability and accumulation. Meanwhile, a β-carotene
degradation kinetic study suggested that HGGT co-expression
increased the half-life of β-carotene to 10 weeks on average
(Che et al. 2016). Consequently, promoting the precursor-
product balance on the metabolic pathway is key to accumu-
lating higher quantities of the product.
Another strategy for accumulating the product is by de-
creasing the metabolic flux downstream of a target compound
or blocking a competing metabolic branch. Pons et al. blocked
Appl Microbiol Biotechnol
Fig. 2 a Biosynthesis pathway of provitamin A in transgenic plant. PSY,
phytoene synthase; PDS, phytoene desaturase; ZDS, carotene desaturase;
βLCY, lycopene β-cyclase; εLCY, lycopene ε-cyclase; βHYD, carotene
β-hydroxylase; εHYD, cytochrome P450 ε-hydroxylase. b Microbial
biosynthesis pathway of provitamin A. dxs, 1-deoxyxylulose-5-
phosphate synthase; dxr, 1-deoxy-D-xylulose-5-phosphate
reductoisomerase; ispD, 4-diphosphocytidyl-2-C-methyl-D-erythritol
synthase; ispE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase;
ispF, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; ispG, (E)-
4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; ispH, 4-hydroxy-
3-methylbut-2-enyl diphosphate reductase; crtY, phytoene synthase;
crtI, phytoene desaturase; crtB, phytoene synthase; crtE, geranylgeranyl
diphosphate synthase; ispA, farnesyl diphosphate synthase; idi,
isopentenyl diphosphate isomerase; hmgs, 3-hydroxy-3-methylglutaryl
CoA synthase; hmgr, 3-hydroxy-3-methylglutaryl CoA reductase; mk,
mevalonate kinase; pmk, phosphomevalonate kinase; pmd, mevalonate
diphosphate decarboxylase
 Appl Microbiol Biotechnol

Table 1 Overview of development of transgenic biosynthesis of provitamin A and vitamin E in recent years

Species Origin Enzyme/gene Products References

Provitamin A
Cassava A. thaliana Phytoene synthase Provitamin A (Welsch et al. 2010)
Potato Orange Cauliflower Orange gene (Or) Carotenoid (Li et al. 2012)
Orange Orange β-carotene hydroxylase (RNA interference) β-carotene (Pons et al. 2014)
Wheat Bacterial Phytoene synthase and carotene desaturase Provitamin A (Wang et al. 2014)
Wheat Wheat CrtB and silencing carotenoid hydroxylase Provitamin A (Zeng et al. 2015)
(TaHYD)
Rice Maize, bacteria, Maize phytoene synthase, bacterial phytoene β-carotene (Bai et al. 2016)
A. thaliana desaturase,1-deoxy-D-xylulose-5-phosphate
synthase
Sorghum Plant Homogentisate geranylgeranyl transferase β-carotene (Che et al. 2016)
Banana Banana Derived phytoene synthase 2a Provitamin A (Paul et al. 2017)
Vitamin E
Rice Rice Tocopherol cyclase Tocopherol (Ouyang et al. 2011)
Lettuce A. thaliana Homogentisate phytyltransferase (HPT) and Tocopherol (Li et al. 2011)
γ-tocopherol methyltransferase (γ-TMT)
Rice A. thaliana ρ-hydroxyphenylpyruvate dioxygenase α-Tocopherol (Farré et al. 2012)
Tobacco A. thaliana and P47/P80 and homogentisate phytyltransferase α-Tocopherol (Espinoza et al.
olanum chilense 2013)
Wheat A. thaliana TaHydroxyphenylpyruvate dioxygenase and Tocochromanol (Chaudhary and
Tag-Tocopherolmethyltransferase (TaHPPD Khurana 2013)
and TaγTMT)
Tobacco A. thaliana γ-Tocopherol methyltransferase α-Tocopherol (Jin and Daniell
2014)
Rice Rice Silence the homogentisate phytyltransferase (HPt) Tocopherol (Chen et al. 2014)
and tocopherol cyclase (tc) defcient
Tobacco Rice Homogentisate geranylgeranyl transferase Tocotrienol (Tanaka et al. 2015)
Soybean Soybean γ-Tocopherol methyl transferase α-Tocopherol (Vinutha et al. 2015)
Rice Rice γ-Tocopherol methyltransferase α-Tocopherol (Wang et al. 2015)
Catharanthus roseus Medicinal Plants N-methyltransferase Tocopherol (Levac et al. 2016)
Arabidopsis seeds, Medicago sativa γ-Tocopherol methyltransferase α-Tocopherol (Jiang et al. 2016)
Alfalfa leaves

endogenous β-carotene hydroxylase gene (Csb-CHX) in or-
ange through RNA interference to enhance the β-carotene
content accumulation (Pons et al. 2014). In addition, through
a combination of method silencing of the carotenoid hydrox-
ylase gene (TaHYD) and overexpression of phytoene synthase
(CrtB) in wheat metabolic pathway (Fig. 2a), the accumula-
tion of β-carotene in wheat was increased significantly (Zeng
et al. 2015).
Exploring the basic mechanism of carotenoid accumulation
and stability has certain significance for research. It has a
significant impact on increasing post-harvest carotenoid sta-
bility. Li et al. expressed the cauliflower Orange (Or) gene in
transgenic potato, which resulted in promoting the retention of
the β-carotene level, and they also clarified the structure of
sequestering lipoprotein-carotenoid and the abundance of key
enzyme-phytoene synthase (Li et al. 2012). These studies are
very interesting with respect to manipulating the levels of
provitamin A in different plant species. On the other hand,
these investigations suggest that design and fine tuning of
the synthetic pathway of carotenoid remains a challenge.
Microalgae biosynthesis of provitamin A
The microalgae species are considered major producers of
commercially important carotenoids (Christaki et al. 2013).
β-carotene is a worthy pigment in abundant microalgal spe-
cies; however, the major obstacle is the low productivity of β-
carotene in microalgae. Genetic engineering approaches are
chosen to increase the productivity of β-carotene in algae.
Chen et al. designed and synthesized phytoene synthase
(psy) gene based on the highest amino acids consensus of
homologous genes from Mariella zofingiensis, Dunaliella
salina, and Chlamydomonas reinhardtii; expression in the
microalgal host results in β-carotene content higher than ma-
jor algae-based production in Scenedesmus sp. CPC2 (Chen
et al. 2017). In another study, through devitalizing carotenoid
Appl Microbiol Biotechnol

ketolase by chemical mutagen, Huang et al. screened the
Chlorella zofingiensis mutant (CZ-bkt1) accumulating high
amounts of β-carotene, zeaxanthin, and lutein, which are es-
sential for human health (Huang et al. 2018).
Microbial biosynthesis of provitamin A
In nature, in addition to plants and algae, microorganisms can
also produce large amounts of β-carotene. Filamentous fungi
such as Blakeslea trispora, and yeast such as Phaffia
rhodozyma and Torulopsis candida, are used to produce β-
carotene. Currently, the microorganisms used to produce β-
carotene include Saccharomyces cerevisiae (S. cerevisiae) and
Escherichia coli (E. coli). The synthesis pathway of β-
carotene usually includes three relatively independent mod-
ules: glycolysis, precursor synthesis, and β-carotene synthe-
sis, as shown in Fig. 2b. Over the past decade, researchers
have conducted an in-depth study on the metabolic synthesis
of β-carotene (Table 2). The MEP pathway provides the pre-
cursor substance to synthesize β-carotene in E. coli. To im-
prove the isoprenoid flux, the chromosomal native promoters
of the rate-controlling steps (ispD, ispF, idi, and dxs) was
replaced by a strong bacteriophage T5 promoter (PT5) in the
isoprenoid synthetic pathway. The resulting high isoprenoid
flux can be used for β-carotene production in E. coli (Suh
2012).
Eukaryotic organisms, plant mitochondria, etc. use
mevalonate (MVA) pathways to provide precursors. The sup-
ply of DMAPP and IPP can be significantly increased by
introducing the foreign MVA pathway of Streptococcus
pneumonia and Enterococcus faecalis into E. coli. Under the
conditions of harboring β-carotene synthesis genes and the
entire MVA pathway, the recombinant E. coli DH5α increased
β-carotene production to 465 mg/L at a 2% (w/v) glycerol
concentration (Yoon et al. 2009). Yan et al. overexpressed 3-
hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)

Table 2 Overview of development of microbial biosynthesis of provitamin A and vitamin K2 in recent years

Strain Carbon sources Temp (°C) Fermentation time Fermentation Output References
(day) state

Provitamin A
E. coli Glycerol 29 2 L 465 mg/L (Yoon et al. 2009)
R. glutinis Chicken feathers 30 5 L 92 mg/L (Taskin et al. 2011)
R. glutinis Glycerol 30 3 L 135.2 mg/L (Saenge et al. 2011)
S. cerevisiae Glucose 30 0.75 L 6.29 mg/g (Yan et al. 2012)
E. coli Glucose 37 2–3 L ND (Suh 2012)
E. coli Glycerol 37 4 L 2.1 g/L (Zhao et al. 2013)
S. cerevisiae Glucose 30 3 L 7.41 mg/g (Xie et al. 2014)
S. cerevisiae Glucose 30 3 L 20.79 mg/g (Xie et al. 2015)
S. cerevisiae Fatty acid 30 1 L 2.83 mg/g (Sun et al. 2016)
E. coli Glycerol 30 2 L 44.2 mg/g (Wu et al. 2017)
Y. lipolytica Glucose 30 11 L 4 g/L (Gao et al. 2017)
Y. lipolytica Glucose 28 4 L 6.5 g/L (Larroude et al. 2018)
Vitamin K
B. subtilis natto Glycerol 37 6 L 50 mg/L (Benedetti et al. 2009)
B. subtilis natto Black soybeans 35, 40, 45, 50 0.75 S 8 ug/g (Wu and Chou 2009)
B. subtilis Glycerol 43 1.5 L 12.47 ug/g (Wu and Ahn 2011)
amyloliquefaciens
B. subtilis natto Glycerol 40 6 L 62.32 mg/L (Berenjian et al. 2011)
B. subtilis natto Cooked 40 6 S 67.01 mg/kg (Mahanama et al.
soybeans 2011)
B. subtilis natto glycerol 40 6 L 86.48 mg/L (Berenjian et al. 2012)
B. subtilis natto Glycerol 37 1 S 39 ug/g (Singh et al. 2015)
B. subtilis natto Glycerol 37 6 L 69.5 ± 2.8 (Yang et al. 2018)
mg/L
B. subtilis natto Glycerol 40 5 L 50 mg/L (Ma et al. 2019)
B. subtilis 168 Glycerol, 40 6 L 360 mg/L (Cui et al. 2019)
glucose

reductase by coupling with ergosterol biosynthesis inhibitors,
which resulted in added farnesyl diphosphate (FPP) fluxing
into the carotenoid pathway. Recombinant S. cerevisiae pro-
duce β-carotene up to 6.29 mg/g (Yan et al. 2012).
Furthermore, galactose regulatory network is one of the most
characterized transcriptional induction systems whose repres-
sion and induction are tightly regulated by glucose and galac-
tose. GAL1 and GAL10 can be induced in the galactose reg-
ulatory network at least 1000-fold in S. cerevisiae. By apply-
ing GAL10-GAL1 bidirectional promoters in rate-limiting
step on the MVA pathway from FPP to geranylgeranyl pyro-
phosphate (GGPP), 7.41 mg/g dry cell weight (DCW) of β-
carotene was achieved in S. cerevisiae (Xie et al. 2014).
Moreover, a repressor/inducer-free dynamic control strategy,
where all the repression and induction of pathways were
switched automatically in response to the glucose concentra-
tion in media, was used to increase the production of β-
carotene in the growth environment. Through the sequential
control of competitive, upstream, and downstream pathways
of FPP in S. cerevisiae, Xie et al. achieved carotenoid produc-
tion of 20.79 mg/g by high-cell density fermentation (Xie
et al. 2015).
CrtEYIB gene clusters were found in Erwinia uredovora
and Pantoea agglomerans and were integrated into the chro-
mosomes of model organisms to synthesize β-carotene (Yoon
et al. 2007). In recent years, the β-carotene synthesis module
and its optimization have also achieved progress. Kato et al.
identified and analyzed carotenoid biosynthesis in Euglena
gracilis and found EgcrtE expression was not affected by
the light-stress treatments (Kato et al. 2016). In another study,
by using carotenoid genes (crtE, crtB, and crtI) of Pantoea
ananatis and Pantoea agglomerans in lycopene synthetic
pathway, the crt genes of P. agglomerans turned out to be
more useful for lycopene production in E. coli than that of
P. ananatis (Yoon et al. 2007). Furthermore, the high yield
of the target product is often limited by its metabolic pathways
enzymes, including enzyme activity, stability, and specificity,
which leads to a low conversion rate and accumulation of by-
products; protein engineering can provide a method of in-
crease the specific properties and substrate specificity of the
enzyme (Damborsky and Brezovsky 2014), including direc-
tional degeneration (Zanghellini 2014) and rational design
(Jeschek et al. 2016). An efficient pathway was constructed
to produce β-carotene. Eleven synthetic genes with strong
promoters were investigated in β-carotene biosynthetic path-
way, which produced 4 g/L β-carotene in Yarrowia lipolytica
accompanied by sequential multiple-copy integration (Gao
et al. 2017). Y. lipolytica can synthesize β-carotene through
overexpression of three genes, including phytoene dehydro-
genase (carB), pytoene synthase/lycopene cyclase (carPR),
and geranylgeranyl diphosphate synthase (GGS1).
Simultaneously, a combinatorial synthetic biology approach
was applied for screening the optimum promoter-gene pairs
Appl Microbiol Biotechnol
and 6.5 g/L of β-carotene production was achieved in the fed-
batch fermentation (Larroude et al. 2018).
The regulation of energy supply plays an important role in
β-carotene biosynthesis. NADPH and ATP are two significant
cofactors for the terpenoids compound production.
Engineering central metabolic modules of E. coli can improve
the production of β-carotene. Zhao et al. engineered central
metabolic modules to improve NADPH and ATP supplies for
increasing production of β-carotene, yielding 2.1 g/L β-
carotene by fed-batch culture (Zhao et al. 2013).
To enhance its storage capacity for β-carotene in E. coli,
morphological and biosynthetic aspects of membrane were
investigated. The titer of β-carotene was increased to 19.6
mg/g by overexpressing membrane-bending proteins.
Simultaneously, this membrane engineering strategy was used
to engineer the β-carotene hyperproducing strain CAR025,
and the titer of β-carotene was improved to 44.2 mg/g DCW
(Wu et al. 2017).
In addition, controlled nutrition and physiological stress
can be used to increase the production of carotenoids. The
enormous commercial demand for natural production of ca-
rotenoids has attracted attention on the development of appro-
priate biotechnology strategies, including the use of liquid
waste substrates as nitrogen and carbon sources. Yeast ex-
hibits important growth capabilities and was used to produce
high levels of carotenoids with several agro-industrial wastes
(Mata-Gómez et al. 2014). Rhodotorula glutinis uses waste
chicken feathers effectively as a novel substrate to produce
carotenoid; the carotenoid concentration of 92 mg/L was
achieved (Taskin et al. 2011). The optimum condition for the
production of carotenoids was a C/N ratio of 85 and a glycerol
concentration of 9.5%. Carotenoids titer was increased to
135.25 mg/L in fed-batch fermentation (Saenge et al. 2011).
β-carotene production levels were significantly promoted to
2.83 mg/g by continuous expression of carotenogenic genes,
including diphosphomevalonate decarboxylase (ERG19),
mevalonate kinase (ERG12), and FPP synthase (ERG20), si-
multaneously with the supply of palmitoleic acid and oleic
acid in recombinant S. cerevisiae (Sun et al. 2016).
Metabolic production of vitamin D
Microbial production of vitamin D2
Vitamin D2, also known as anti-rickets vitamin, and therefore,
ergocalcification sterol, is a kind of fat-soluble vitamin derived
from sterol (Yu et al. 2015). Ergosterol is converted to the vita-
min D2 precursor under ultraviolet radiation, which is obtained
by heating isomerization and losing two hydrogen atoms.
Vitamin D2 is widely used in the medicine, food, and feed
industry, and it has a good market prospect. Ergosterol mainly
exists in the plasma membrane of plants and some fungi, among
Appl Microbiol Biotechnol

which yeast has the highest content, which is usually 0.03–4.6%
of the dry weight of cells. However, in a few species such as
S. cerevisiae, the content can be up to 7–10%. Therefore, yeast
is generally used as the production strain of ergosterol. The
selection and breeding of high yield ergosterol yeast species is
the first and most important step in vitamin D2 production.
The synthesis of pre-vitamin D2 by Ergosterol’s light-
singlet ring-opening reaction is the most successful example
of using photochemical technology. Due to the occurrence of
secondary photochemical reactions, the reaction products are
very complex, which still attracts the research interest of photo
chemists even now. In the metabolic pathway of ergosterol
synthesis (Fig. 3), HMG-CoA reductase is a speed-limiting
enzyme that controls the synthesis of ergosterol. If the feed-
back inhibition route can be interrupted, the inhibition effect
of ergosterol synthesis can be relieved, and a high yield of
ergosterol can be obtained. Transcriptional profiling study of
Penicillium digitatum about the cause of citrus green mold,
reveals that an inhibited ergosterol biosynthesis pathway ex-
ists in reaction to citral (OuYang et al. 2016). However, so far,
there are no relevant reports about screening ergosterol high-
yield strains by metabolic regulation.
To convert the accumulated ergosterol to ergosta-5,7-dien-
3β-ol in the workflow of the metabolic engineering of
S. cerevisiae, Ma et al. deleted the gene erg5 and
overexpressed the transcription factor upc2-1 and the rate-
limiting enzymes Erg1p, Hmg1p, and Erg11p for the co-
induction of the post-squalene pathway (Ma et al. 2018). In
the other case, the total production of ergosterol by the
S. cerevisiae fermentation depends on the biomass of yeast
and the content of ergosterol in the cells. Two preferred met-
abolic conditions, oxidative-fermentative growth on glucose
followed by oxidative growth on ethanol and glucose, were
studied simultaneously, which achieved ergosterol production
total efficiency value of 103.84 × 10−6 g/L/h and the final
content and purity of 2.43% and 86%. These results make
the product achieved by the control strategy conducive to
downstream processing (Nahlik et al. 2017).
Microbial production of activated vitamin D3
Activated vitamin D3 is an essential fat-soluble vitamin in the
human body (Xu et al. 2017), which possesses the important
physiological function of regulating the metabolism of

Fig. 3 Fungal ergosterol

biosynthetic pathway. ERG1,
squalene epoxidase; ERG7,
lanosterol synthase; ERG11,
sterol C14-demethylae; ERG24,
C-14 sterol reductase; ERG2, C-8
sterol isomerase; ERG25, C-4
sterol methyloxidase; ERG3, C-5
sterol desaturase; ERG5, C-22
sterol desaturase; ERG4, C-24
sterol reductase; ERG26, C-4
sterol decarboxylase; ERG6,
C-24 sterol methyl-transferase;
ERG27, C-3 sterol Keto reductase

calcium and phosphorus (Liu et al. 2007) and controlling the
growth and differentiation of skeletal cells (Li et al. 2019). At
present, the main method of preparation of active vitamin D3
is divided into two kinds: the first is the thermalization re-
search method, and the second is carried out through P450
enzymes in microbial reforming process. Microbial reforming
process has advantages of low production cost, highly specific
reaction, and extensive application prospect in the future
(Wang et al. 2018). Simultaneously, the restriction factors of
catalytic characteristics of the individual enzyme and micro-
bial reforming process exist in a recombinant strain for the
purpose of enhancing vitamin D3 production (Wang et al.
2019).
At present, researchers focus on the following aspects to
solve the restriction factors of bio-preparation of active vita-
min D3: recombinant engineering bacteria was constructed
with the expression of the vitamin D3 hydroxylase gene to
solve the problem of instability and low expression activity.
Successful applications of P450 reactions are the bioconver-
sion of vitamin D 3 to 1α,25-dihydroxyvitamin D 3 in
Streptomyces griseolus CYP105A1 and Pseudonocardia
autotrophica in CYP107 family. Through site-directed muta-
genesis of CYP105A1 based on its crystal structure and di-
rected evolution of CYP107, vitamin D3 hydroxylase activity
was dramatically increased (Sakaki et al. 2011). Furthermore,
the actinomycete Pseudonocardia auto-trophica is capable of
converting vitamin D3 into its physiologically active forms
1α,25-dihydroxyvitamin D3. Fujii et al isolated and mutated
vitamin D 3 hydroxylase (Vdh) from Pseudonocardia
autotrophica NBRC 12743, which can hydroxylate vitamin
D3 through actinobacterium Rhodococcus erythropolis with
Vdh coexpression (Fujii et al. 2009).
It is significant to select an optimal set of electron transport
chains to co-express with hydroxylase, simultaneously solve
the problem of coenzyme NADH (NADPH) electron transport
by constructing fusion proteins. Imoto et al. used nisin to form
nisin-lipid II pore complexes in the cell membrane, which
increase the chance of VD3-PMCD complexes entering the
Rhodococcus interior (Imoto et al. 2011).
Moreover, the cell catalytic system co-expresses the coen-
zyme regeneration system, which is an efficient biocatalytic
synthesis of 25(OH)VD3, a promising intermediate for the
synthesis of a variety of hydroxylated vitamin D3 derivatives,
and solves the problem of coenzyme supply in the reaction
system. Yasutake et al. used nisin-treated Rhodococcus
erythropolis cells containing VdhT107A. The gene of Bacillus
megaterium glucose dehydrogenase-IV was inserted into the
chromosome of R. erythropolis for expression, so as to avoid
the exhaustion of cytoplasmic NADH in the process of bio-
logical transformation (Yasutake et al. 2013).
In addition, the high hydrophobicity of substrate vitamin
D3 can be overcome by improving the membrane permeability
o f s u b s t r a t e v i t a m i n D 3 . Ya s u t a k e e t a l . u s e d
Appl Microbiol Biotechnol
lactostreptococcus peptide to treat the membrane of
R. erythrococcus, effectively improving the permeability of
substrate vitamin D3 (Yasutake et al. 2013).
Metabolic production of vitamin E
Biosynthetic vitamin E in transgenic plants
Plants can synthesize bioactive vitamin E in metabolism path-
ways (Cahoon et al. 2003). In addition to its anti-oxidant
function and its resistance to abiotic stresses, vitamin E also
affects important physiological processes such as seed germi-
nation, transport of photosynthetic assimilation products,
plant growth, and leaf senescence (Falk and Munne-Bosch
2010). The tocochromanol head group was synthesized by
cytosolic aromatic amino acid metabolism, and the hydropho-
bic tail was synthesized by the MEP pathway (Fig. 4). The
first step in synthesis of tocochromanol, homogentisic acid
(HGA) is achieved from ρ-hydroxyphenylpyruvic acid
(HPP) through hydroxyphenylpyruvic acid dioxygenase
(HPPD) (Janke and Krupinska 1997). Recently, in model pho-
tosynthetic organisms, genetic and genomics-based ap-
proaches have allowed the isolation of the genes and proteins
that synthesize tocochromanol. Progress has been made in
genetic and molecular biology of the tocochromanol synthetic
pathway in the organisms Synechocystis sp. PCC6803 and
A. thaliana (Herbers 2003).
Significant genes have been expressed and silenced collec-
tively or individually in diverse plants to explore metabolic
processes and improve vitamin E content and composition
(Table 1). Farré et al. overexpressed A. thaliana HPPD in
transgenic rice plants. The levels of total tocochromanols in
the transgenic grains were higher than those in the controls,
with a slightly increase in absolute synthesis of tocotrienol,
without changing tocopherol level (Farré et al. 2012).
Tocopherol is necessary in photoprotection. Chen et al. si-
lenced homogentisate phytyltransferase (HPT) and tocopherol
cyclase (TC) in transgenic rice, which exhibited different phe-
notypic and physiological characteristics (Chen et al. 2014). A
picrinine N-methyltransferase produces biologically active
monoterpenoid indole alkaloids through the bioinformatic
screening of transcriptomes from Rauvolfia serpentina,
Vinca minor, and Catharanthus roseus (Levac et al. 2016).
A genome-wide association study (GWAS) indicated nucleo-
tide variations in the coding region of tocopherol methyltrans-
ferase (OsγTMT) in rice. OsγTMT was significantly associ-
ated with the α-tocopherol content variations; furthermore,
nucleotide polymorphisms can partially prove the correlation
with α-tocopherol content changes in the promoter region of
OsγTMT in rice (Wang et al. 2015). As α-tocopherol has
antioxidant properties and is nutraceutical, it is of great signif-
icance to modulate the γ-tocopherol methyl transferase (γ-
Appl Microbiol Biotechnol
Fig. 4 Biosynthesis pathway of vitamin E in transgenic plant. GGR,
geranylgeranyl diphosphate reductase; HPPD, ρ-
hydroxyphenylpyruvate dioxygenase; MPBQMT, 2-methyl-6-
TMT) gene expression. Temporal differences exist in the γ-
TMT3 gene expression in seeds, α-tocopherol accumulation,
and the distinct roles of γ-TMT genes in soybean (Vinutha
et al. 2015). Overexpression of Medicago sativa L.’s γ-TMT
enhanced the concentrations of tocopherol 10–15-fold and
also had an effect on delayed leaf senescence phenotype
(Jiang et al. 2016).
With the development of molecular biology and genetic
engineering technology, research reports on the regulation of
vitamin E biosynthesis-related enzymes are increasing.
Among the various components of vitamin E, tocopherol
has the highest activity; however, the main component of vi-
tamin E is tocopherol in plant seeds. The overexpression of
genes can improve vitamin E composition, the overexpression
of homogentisate phytyltransferase (hpt) can increase the total
tocopherol content in lettuce, and the overexpression of γ-
tocopherol methyltransferase (γ-tmt) can alter the tocopherol
composition and enhance the tocopherol content (Li et al.
2011). Heterologous and homologous transgenic manipula-
tion can alter tocochromanol composition and content.
Triticum aestivum L. plants and transgenic wheat-
overexpressing TaHydroxyphenylpyruvate dioxygenase
displayed 2.4-fold increase in content of tocochromanol
phytylbenzoquinone methyltransferase; HPT, homogentisate
phytyltransferase; γ-TMT, γ-tocopherol methyltransferase; TC,
tocopherol cyclase
(Chaudhary and Khurana 2013). Through introducing rice
homogentisate geranylgeranyl transferase (OsHGGT) cDNA
into a nuclear genome of transgenic tobacco plants, the yield
of tocotrienol was enhanced in tobacco plants (Tanaka et al.
2015).
Simultaneously, researchers focus on the following aspects
to increase tolerance against environmental stresses. The
homogentisate phytyltransferase VTE2.1 from Solanum
chilense and stress inducible promoters isolated from
Arabidopsis can be used to increase the levels of vitamin E
in plants and decrease the environmental negative stress
(Espinoza et al. 2013). Transgenic tobacco plants showed
the content of γ-tocopherol increased by expression of
ScVTE2.1 in drought conditions. Jin and Daniell studied the
function of γ-tocopherol methyltransferase (γ-TMT) within
chloroplasts in regulation of abiotic stress. γ-TMT was
overexpressed in tobacco chloroplast genome, with leaf pro-
tein increased by 7.7%. Most of γ-tocopherol was converted
to γ-tocopherol in transplastomic seeds by a high-level ex-
pression of γ-TMT under abiotic stress (Jin and Daniell
2014). Interestingly, rice increased tolerance against salt and
simultaneously enhanced tocopherol content by transgenically
expressing the VTE1 gene encoding a rice tocopherol cyclase

(Ouyang et al. 2011). Therefore, through transgenic technol-
ogy methods, vitamin E biofortified crops are tolerant against
environmental stresses.
Microalgae biosynthesis of vitamin E
Microalgae are a novel source of sustainable natural antioxi-
dants (Goiris et al. 2015) and they produce a variety of natural
products such as vitamins, pigments, fatty acids, and enzymes
that are beneficial to humans (Harun et al. 2010). Tocopherols
are antioxidants that guard against many kinds of diseases
resulted from oxidative stress. Nannochloropsis oculata has
the potential as an additive, which can improve the shelf life
and nutritional value of the cooking oil during high-
temperature cooking (Lee et al. 2013).
Green microalgae stands for a renewable natural source of
vitamin E. A great deal of work has been carried out to im-
prove the tocopherols composition and contents by genetic
and metabolic engineering in higher plants, yet, little work
has been done to genetically modify microalgae gene to in-
crease the accumulation of tocopherol. Euglena gracilis has
relatively high α-tocopherol contents and growth rate. Thus
far, cultivation systems have been studied to improve the pro-
ductivity of tocopherol in Euglena gracilis (Ogbonna 2009).
In phytoplankton, the synthesis of the antioxidant α-
tocopherol could rely on changes in abiotic factors, including
photon flux density, temperature, and salinity fluctuations
(Haubner et al. 2014). Furthermore, Stichococcus bacillaris
strain siva2011 is a new green microalga which biosynthesizes
significant amounts of RRR-α-tocopherol in a 20 L balloon
bioreactor, achieving approximately 0.6 mg/g dry weight
(DW) of RRR-α-tocopherol (Sivakumar et al. 2014).
Metabolic production of vitamin K
Microbial biosynthesis of vitamin K
Vitamin K exists two major forms, including vitamin K1 and
K2. Vitamin K1, also known as phylloquinone, is naturally
produced in plants and found in the highest concentration in
green leafy vegetables (Widhalm et al. 2012). Vitamin K2,
called menaquinones, is synthesized in animal and bacterial
cells (Mahdinia and Demirci 2019). Contrast to other vitamin
K, MK-7 can be produced by many species of bacterial
(Geleijnse et al. 2004). How to enhance the MK-7 productiv-
ity has been a major issue. The MK-7 fermentation includes
solid-state fermentation (SSF) (Ebrahiminezhad et al. 2016)
and liquid-state fermentations (LSF); SSF has a water content
from 12 to 80% (Berenjian et al. 2013). Moreover, MK-7
production is a costly process, owning to its low productivities
(Berenjian et al. 2015).
Appl Microbiol Biotechnol
During the last decade, researchers have investigated
bioprocess engineering technologies for the industrial produc-
tion of MK-7 (Mahdinia and Demirci 2018). Table 2 shows an
overview of the development of MK-7 production in recent
decades. The formation of pellicle played a significant role in
MK-7 production in Bacillus subtilis natto (B. subtilis natto)
(Berenjian et al. 2013). Meanwhile, high stirring and aeration
rates enhance the MK7 production dramatically contrast to the
static state (Berenjian et al. 2014). Also, agitation can solve
the heat transfer problem in industrial MK-7 production
(Berenjian et al. 2015). The optimization of the initial fermen-
tation medium and the fed-batch with added nutrients have led
to significant improvements in menaquinone production.
Substrate pre-treatment with α-amylase increases the MK-7
yield to 67.01 mg/kg in B. subtilis natto (Mahanama et al.
2011). Singh et al. used several medium components to inves-
tigate the concentrations of MK-7 and obtained the highest
yield of 39 ug/g (Singh et al. 2015).
Meanwhile, the biosynthetic pathway of menaquinone
comprises three modules: isoprene side chain module,
menaquinone skeleton module, and MK7 synthesis module
(Fig. 5). The modular metabolic engineering was designed
to promote the biosynthesis of MK-7. Yang et al. constructed
the final B. subtilis strain MK3-MEP123-Gly2-ΔdhbB
through deletion of dhbB and excessive expression of dxs,
menA, dxr, glpD, and yacM-yacN, which yields MK-7 of
69.5 ± 2.8 mg/L after 6 days of fermentation (Yang et al.
2018). Overexpression of different combinations of the rate-
limiting enzymes dxs, dxr, idi, and menA can enhance pro-
duction of MK-7 11-fold to 50 mg/L (Ma et al. 2019).
Dynamic pathway regulation system has already been exten-
sively applied contrast to static regulation, to balance the prod-
ucts synthesis and cell growth. Quorum sensing (QS)-based
dynamic regulation system has been extensively applied to
fine-tune gene expression in the process of cell density chang-
es. A Phr60-Rap60-Spo0A QS regulation system was used to
increase MK-7 production to 360 mg/L in shake flasks (Cui
et al. 2019).
Conclusions and prospects
Compared to chemical synthesis, the production in a natural
environment may have the following advantages: vitamins
produced from plants, microalgae, and microorganism are
considered to be natural; therefore, they may be positively
recognized by the public. Relevant bioelectron stereoisomers
are produced specifically, and plant-based and microorganism
production systems may be cheaper. Thus far, only biosyn-
thetic pathways of plants have already been elucidated; how-
ever, their mechanisms remain unclear. Plant cultivation to
produce vitamins has not yet competed with existing produc-
tion systems. However, plant systems have the greatest
Appl Microbiol Biotechnol
Fig. 5 Biosynthesis pathway of MK-7 in B. subtilis. menD, 2-succinyl-5-
enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase; ispE, 4-
diphosphocytidyl-2-C-methylerythritol kinase; ytxM, 2-succinyl-6-
hydroxy-2,4-cyclohexadiene-1-carboxylate synthase; ispH, 4-hydroxy-
3-methylbut-2-enyl diphosphate reductase; menC, o-succinylbenzoate
synthase; ispD, 2-C-methylerythritol 4-phosphate cytidylyltransferase;
menE, o-succinylbenzoic acid-CoA ligase; ispA, farnesyl diphosphate
synthase; menB, 1,4-dihydroxy-2-naphthoyl-CoA synthase; dxs, 1-
deoxyxylulose-5-phosphate synthase; hepT/hepS, heptaprenyl
potential as vitamin factories contrast to others. The current
findings suggest that plants can be customized as functional
food with the increase of vitamin E and provitamin A levels.
The microbial production of vitamins is attracting increasing
attention due to its advantages, and considerable progresses
have been made to date. So far, biosynthesis of vitamin K2 in
bacteria has been industrialized and commercialized. Yet, a
biosynthetic pathway for phylloquinone has not been con-
firmed. This may attribute to the reality that phylloquinone
is considered to be the weakest steroid in terpenoid quinones
or higher focus on other photosynthetic organisms.
Microalgae are potential for the biosynthesis of phylloquinone
because of their industrially useful properties.
diphosphate synthase component II/I; yuxO, 1,4-dihydroxy-2-
naphthoyl-CoA hydrolase; menA, 1,4-dihydroxy-2-naphthoate
heptapren yltransferase; d xr,1-deoxyx ylulose-5-phosphate
reductoisomerase; ispF, 2-C-methylerythritol 2,4-cyclodiphosphate
synthase; ispG, 4-hydroxy-3-methylbut-2-enyl diphosphate synthase;
MenF, isochorismate synthase; menH, demethylmenaquinone
methyltransferase; DhbB, bifunctional isochorismate lyase/aryl carrier
protein
In the past decade, high yields of vitamin A and
menaquinone have been accomplished through protein and
metabolic engineering. In the future research, metabolic path-
ways of some fat-soluble vitamins will be constructed to
achieve the industrial production, based on well-reported bio-
synthesis pathways. At present, synthetic biology focuses on
the comprehensive analysis of all the interacting elements of
molecules, as well as how these generated dynamic and com-
plex biological systems behave, providing more biological
functions of proteins and technology to construct cell factories
in microorganism. It is feasible to develop microbial strains
which can produce vitamins by using the knowledge of higher
plants. After all, many strategies are available to improve the

productivity of secondary metabolites, including selecting
high yield organism, metabolic engineering, and optimizing
environmental conditions. Combined with modern engineer-
ing techniques, vitamins can be expected to be produced more
economically and sustainably.
Funding information This work was financially supported by the
National Natural Science Foundation of China (31871784, 31870069,
21676119, and 31671845), Postgraduate Research & Practice
Innovation Program of Jiangsu Provence (KYCX18_1786), and The
Fundamental Research Funds for the Central Universities
(JUSRP51713B).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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