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\begin{document}

\title{Harnessing Pivotal Advances for Production and Structural Derivation of
the Promising Molecule Ursolic Acid}



\author[1]{Hao-ran Liu}%
\author[1]{Nadeem Ahmad}%
\author[1]{Bo Lv}%
\author[1]{Chun Li}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Ursolic acid (UA) is a ursane-type pentacyclic triterpenoid compound,
naturally produced in plants via specialized metabolism and exhibits
vast range of remarkable physiological activities and pharmacological
manifestations. Owing to significant safety and efficacy in different
medical conditions, UA may serve as a backbone to produce its
derivatives with novel therapeutic functions. This review systematically
provides an overview of the pharmacological activities, acquisition
methods and structural modification methods of UA. In addition, we
focused on the synthetic modifications of UA to yield its valuable
derivatives with enhanced therapeutic potential. Furthermore, harnessing
the essential advances for green synthesis of UA and its derivatives by
advent of metabolic engineering and synthetic biology are highlighted.
In combination with the advantages of UA biosynthesis and transformation
strategy, large-scale production and applications of UA is a promising
platform for further exploration.%
\end{abstract}%



\sloppy


\textbf{Harnessing Pivotal Advances for Production and Structural
Derivation of the Promising Molecule Ursolic Acid}

Hao-ran Liu\textsuperscript{a} Nadeem Ahmad\textsuperscript{a} Bo
Lv\textsuperscript{a}\emph{**} Chun Li\textsuperscript{abc}*

\begin{enumerate}
\tightlist
\item
  \emph{Institute of Biochemical Engineering, School of Chemistry and
  Chemical Engineering, Beijing Institute of Technology, Beijing 100081,
  P.R. China}
\item
  \emph{Key Lab for Industrial Biocatalysis, Ministry of Education,
  Department of Chemical Engineering, Tsinghua University, Beijing
  100084, PR China}
\item
  \emph{Center for Synthetic and Systems Biology, Tsinghua University,
  Beijing, 100084, China}
\end{enumerate}

Hao-ran Liu\textsuperscript{a}\emph{Liuhr\_bit@163.com}

Nadeem Ahmad\textsuperscript{a}\emph{dr.nadeem444@gmail.com}

Bo Lv\textsuperscript{a}\emph{**wavelvbo@163.com}

Chun Li\textsuperscript{abc}*\emph{lichun@tsinghua.edu.cn}

*Corresponding author. Institute of Biochemical Engineering, School of
Chemistry and Chemical Engineering, Beijing Institute of Technology,
Beijing 100081, P.R. China

** Corresponding author.

\emph{E-mail addresses: wavelvbo@163.com (B. Lv), lichun@tsinghua.edu.cn
(C. Li).}

\par\null

\textbf{Abstract:} Ursolic acid (UA) is a ursane-type pentacyclic
triterpenoid compound, naturally produced in plants via specialized
metabolism and exhibits vast range of remarkable physiological
activities and pharmacological manifestations. Owing to significant
safety and efficacy in different medical conditions, UA may serve as a
backbone to produce its derivatives with novel therapeutic functions.
This review systematically provides an overview of the pharmacological
activities, acquisition methods and structural modification methods of
UA. In addition, we focused on the synthetic modifications of UA to
yield its valuable derivatives with enhanced therapeutic potential.
Furthermore, harnessing the essential advances for green synthesis of UA
and its derivatives by advent of metabolic engineering and synthetic
biology are highlighted. In combination with the advantages of UA
biosynthesis and transformation strategy, large-scale production and
applications of UA is a promising platform for further exploration.

\textbf{Keywords:} Ursolic Acid; Ursolic Acid derivatives; Biosynthesis;
Metabolic engineering

Graphical abstract\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Introduction}

Ursolic acid (UA, 3\selectlanguage{greek}β-\selectlanguage{english}hydroxy-urs-12-en-28-oic acid) is a kind of
pentacyclic triterpenoids existing naturally in plants, which plays a
significant role as a hormone or regulatory factor in plant growth and
insect resistance\textsuperscript{1,2}. Structurally, it is a
ursane-type pentacyclic triterpenoid with C-3 hydroxy and C-28
carboxylic acid group (\emph{Fig. 1} ). Apart from its significance in
plants, UA and its derivatives also exhibit remarkable physiological and
pharmacological manifestations. Up to now, it has shown a wide range of
clinical manifestations, including anti-inflammatory
\textsuperscript{3}, anti-oxidation\textsuperscript{4}, diabetes
treatment \textsuperscript{5}, antibacterial \textsuperscript{6},
anti-tumor metastasis\textsuperscript{7}, anti-tumor activity
\textsuperscript{8}, anti-angiogenesis \textsuperscript{9}, skin
care\textsuperscript{10}, and superior liver protection
function\textsuperscript{11}.

\emph{\textbf{Fig. 1} Chemical structure of Ursolic acid (UA)}

The main way to obtain UA on a large scale currently relies on
extraction of plants rich in it. However, it encounters several
limitations, such as high energy consumption and low yield by plant
extraction, which make it difficult to meet commercial needs. In
addition, UA endures obstacles in therapeutic applications and drug
development due to fast metabolism, poor water solubility and low
bioavailability \textsuperscript{12,13}.

In view of the excellent potential of UA as therapeutic drug, its
structural modification is an attractive step to synthesize the
potentially active derivatives in order to expand the pharmacological
properties for further exploration \textsuperscript{14-16}. More
importantly, with the advent of synthetic biology and metabolic
engineering, transition towards the synthesis of natural product by
employing these approaches is of great concern. Cell factories such
as\emph{Saccharomyces cerevisiae} (\emph{S. cerevisiae} ) have been
genetically engineered for green production of terpenoids, such as
artemisinic acid \textsuperscript{17}, ginsenoside \textsuperscript{18},
oleanolic acid \textsuperscript{19} and lycopene \textsuperscript{20},
which provide new thoughts for industrial production of UA. In this
article, we systematically reviewed the significance of production and
modification of UA to explore more applications in pharmacy and
pharmaceutical chemistry. Particularly, we focused on the de novo
synthesis of UA and its derivatives in engineered microbial cell
factories in advancements for green production and green chemistry.

\textbf{Physiological and pharmacological profile of UA}

Studies have shown that UA has broad application prospects, which is
employed in the management of various chronic diseases by mediating and
regulating the pharmacological processes and related signaling pathways
\textsuperscript{21,22}.

UA plays an anti-inflammatory role in inflammatory cascade. It inhibits
the activation, proliferation and cytokine secretion of T cells, B cells
and macrophages of mouse lymphocytes. By inhibiting phosphorylation of
Extracellular Regulated protein Kinases (ERK) and c-Jun N-terminal
Kinase (JNK) induced by mitogen, it limits the activation of
immunomodulatory transcription factors NF-\selectlanguage{russian}кB, NF-AT and AP-1 in
lymphocytes \textsuperscript{23}.

In another study, UA has been revealed to exhibit hepatoprotective
properties as it significantly inhibits the HCV genotype 1b replicon and
HCV genotype 2aJFH1 virus \textsuperscript{24}. It is partly due to the
inhibition of HCV-NS5BRdRp activity as a noncompetitive inhibitor.
Researchers explored the expression of HPVE6/E7 by Reverse
Transcription-Polymerase Chain Reaction (RT-PCR) and found that UA
down-regulate it \textsuperscript{25}.

UA also shown the promising results in the management of hepatitis A and
B with the cure rate of 89.3\% as it reduces the liver weight, ALT/AST
level, hepatic steatosis, and palmitic acid induced fat accumulation in
L02 cells\textsuperscript{11,26}. It exerts hepatoprotective effects by
increasing \selectlanguage{greek}β-\selectlanguage{english}oxidation of fat and inhibiting endoplasmic reticulum
pressure.

In addition, UA exhibits significant therapeutic potential in cancer
treatment due to its high activity and low toxicity. Studies have shown
that UA decreases the activity of extracellular phosphorylated signal
regulated kinase and depolarizes the mitochondrial
membrane\textsuperscript{27}. It has also been found that UA promotes
the apoptosis of human bladder cancer cell line T24 by activating the
ASK1-JNK signaling pathway and induction of the endoplasmic reticulum
stress response\textsuperscript{28}. Bioactivities of UA and its
relevant mechanism of action have been summarized in \emph{Table 1} . On
the basis of understanding pharmacological and physiological activities,
UA will be better developed to meet more daily and medical needs.

\emph{Table 1. Bioactivities of UA and related mechanisms of action}\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Bioactivities} & \textbf{Mechanism of Action} &
\textbf{Reference}\tabularnewline
\midrule
\endhead
Antibacterial & effective on the biofilm of Gram-positive bacteria such
as \emph{Staphylococcus aureus} to prevent persistent infection. &
\textsuperscript{6}\tabularnewline
Anti-Angiogenesis & interference with Signal Transducer and Activator of
Transcription 3 (STAT3), Mitogen-Activated Protein Kinase (MAPK), NF-\selectlanguage{russian}кB
signaling pathway; inhibits vascular smooth muscle proliferation. &
\textsuperscript{9}\tabularnewline
Antihepatodamage & increase the \selectlanguage{greek}β-\selectlanguage{english}oxidation of fat and inhibiting
endoplasmic reticulum pressure. & \textsuperscript{11}\tabularnewline
Anti-Inflammatory & inhibits the phosphorylation of ERK and JNK, limits
the activation of NF-\selectlanguage{russian}кB, NF-AT and AP-1 in lymphocytes. &
\textsuperscript{23}\tabularnewline
Antiviral & down-regulate HPVE6/E7 &
\textsuperscript{24,25}\tabularnewline
Anti-Tumor & regulate transcription factors expression and protein
kinases related to tumorigenesis. & \textsuperscript{29}\tabularnewline
Anti-Oxidation & regulates the changes of reactive oxygen species levels
induced by ultraviolet B. & \textsuperscript{30}\tabularnewline
Diabetes Treatment & inhibits the activity of \selectlanguage{greek}α-\selectlanguage{english}amylase; reduces blood
glucose levels and body mass index. &
\textsuperscript{31,32}\tabularnewline
\bottomrule
\end{longtable}

\textbf{How to obtain UA and what's challenges for application}

UA is widely distributed as triterpene saponin aglycone or free acid in
various medicinal plants as well as several fruits, such
as\emph{Ligustrum lucidum} \textsuperscript{33}, \emph{Centella
asiatica}\textsuperscript{34}, \emph{Lysimachia
clethroides}\textsuperscript{35}, \emph{Crataegus pinnatifida}
\textsuperscript{36}and \emph{Malus domestica} \textsuperscript{37}.
However, its percentage varies from species to species due to related
biosynthetic enzymes and metabolism \textsuperscript{38}. Currently,
industrial production of UA mainly depends on extraction of natural
plants. A variety of extraction methods have been developed to extract
UA from different plants.

\emph{Table 2. Selected Methods for Isolation from Available Plant
Sources for UA}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{Method} & \textbf{Extraction and purification} & \textbf{Result}
& \textbf{Reference}\tabularnewline
\midrule
\endhead
Microwave Method & Weigh the plant powder and mix with the appropriate
amount of extractant. After soaking for 30 minutes, immerse the glass
tube and the sample in the water of the microwave device, set the
parameters and then start the extraction. & 5.8 mg/g ursolic acid
extracted from the fruit of \emph{Ligustrum lucidum} &
\textsuperscript{33}\tabularnewline
Rapid Solvent Extraction & The fast solvent method can be implemented
using systems introduced by related companies, such as Dionex ASE 100.
The samples were placed in the extraction tank and extracted in two
static cycles at 40  and 60  under 10MPa pressure. & 9.4 mg/g dry
weight ursolic acid extracted from leaves of \emph{Thymus mongolicus
Ronn}. & \textsuperscript{35}\tabularnewline
Impregnation & Wash the fruit and leaves with distilled water, then air
dry for a week to remove all water. At 258 , soak in anhydrous alcohol
for 48 hours, shake it irregularly and repeat twice. Among them, 20 ml/g
ethanol was used in leaves, 2 ml/g ethanol was used in fruits, and then
ethanol extract was obtained by filtration. & 0.46-1.08 and 1.99-4.54
mg/g dry weight ursolic acid extracted from the fruits and leaves of
Morocco nuts respectively & \textsuperscript{39}\tabularnewline
Soxhlet Extraction & Degrease 4 parts of 2.00g aliquots in Soxhlet
extraction unit for 4 hours. After drying, 80\% hot methanol extract
material was used, and ethanol extract and methanol extract were
collected and concentrated in a rotating evaporator at 60 . Add
deionized water and stir thoroughly, and introduce the extract into the
extraction column. Then the column is washed with butanol soaked with
water, and the filtrate is evaporated to dryness in 70  evaporator &
20.53 mg/g ursolic acid extracted from flowers of \emph{Calendula
officinalis L.} & \textsuperscript{40}\tabularnewline
Hot Reflux Method & The dried plant material was mixed with 100 mL of
acetone. Connect with cooling water and heat in a 60\selectlanguage{ngerman}°C water bath for 30
minutes, and repeat three times with new solvent. Finally, the extract
was collected, filtered and concentrated. & 92.6 mg/g ursolic acid
extracted from \emph{Lamium album} & \textsuperscript{41}\tabularnewline
Ultrasonic Method & First, the ground material and solvent are mixed
into the flask, and the flask is placed in a device that can provide
ultrasonic wave. The parameters are adjusted, and the mixing of solvent
and solid material is promoted by the diffusion of ultrasonic vibration
energy. & 9.11 mg/g ursolic acid extracted from flowers of \emph{Punica
granatum L.} & \textsuperscript{42}\tabularnewline
Supercritical Fluid Extraction & First, the liquid carbon dioxide is
pressurized to the required pressure in the coolant pump while being
heated and reaches the working temperature before reaching the
extractor. After flowing through the extractor pre-loaded with sample,
the cooled ethanol is used as a collector for the extraction liquid, and
the effluent of the extractor is depressurized by a heated back pressure
regulator. The solvent was then evaporated in a nitrogen stream on a
rotary evaporator, evaporated in a vacuum oven for 6 hours, and the
temperature was 60\selectlanguage{ngerman}°C. & 1.8 mg/g ursolic acid extracted from
\emph{Eucalyptus grandis} & \textsuperscript{43}\tabularnewline
\bottomrule
\end{longtable}

However, plant cultures occupy a large area of cultivated land and plant
growth may be affected by climate change and growth cycle, which
subsequently reduces the adequate supply of raw materials. The
challenges for UA's application also do include current production
methods which consume a lot of organic reagents, and eventually produce
waste liquids to cause serious ecological pressure to surrounding
environment. So the traditional extractions of UA make it difficult to
meet the commercial needs and environmental protection goals.

\textbf{Structure modification to improve the efficacy of UA}

Although UA exhibits remarkable physiological and pharmacological
activities with effective and safe therapeutic profile, its poor water
solubility, short plasma half-life and poor bioavailability limit
further clinical applications \textsuperscript{44}. In this regard,
structural modifications of UA to cope with these limitations have been
an attractive area of research to explore its valuable derivatives with
enhanced pharmacological profile. In recent years, structural
modification of UA is achieved by means of chemical transformation and
microbial transformation to yield valuable derivatives.

\textbf{4.1Chemical modification}

UA structure is characterized by a hydroxyl group and carboxylic group
which can be involved in the transfer of lone pair electrons to metal
atoms \textsuperscript{45}. According to the structural properties of
reported derivatives, UA usually undergoes structural modifications on
C-3/C-28 positions or the ring A of UA skeleton.

\textbf{4.1.1Modifications on C-3/C-28}

Researchers isolated UA and five triterpenoids from apple peel and
synthesized a series of UA analogs. Structural modification of UA at C-3
indicated significant anti-proliferative activity \textsuperscript{46}.
In another study, acetyl group at the C-3 and an alkylamino and/or
piperidine moiety at the C-28 enhanced the anticancer activity of UA
derivative \textsuperscript{46} (\emph{Fig. 2A} ). In addition,
structural modification of UA by addition of piperazine moieties may
enhance its anti-cancer properties. Addition of acyl piperazine motif at
C-28 position while C-3 retains the polar group significantly enhanced
the anticancer activity against breast cancer and gastric cancer cell
lines \textsuperscript{47} (\emph{Fig. 2B} ).

In order to clear on the mechanism of chemical modification at C-3 and
C-28 in detail, researchers synthesized a series of UA derivatives by
employing different electronic chemical modification at the two sites.
Derivatives showed stronger cytotoxicity due to presence of positive
charge (\emph{Fig. 2C} ), indicating that the increased lipophilicity
may enhance the therapeutic potential of gastric
carcinoma\textsuperscript{48}. These findings of UA derivatives led the
ways to structurally modify the UA skeleton to yield its valuable
derivatives with diverse pharmacological properties.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\emph{\textbf{Fig. 2} Modification on C-3/C-28}

\textbf{4.1.2 Modifications on ring A}

UA and Oleanolic acid (OA) are isomers, the difference between which is
only at the position of Me29(\emph{Fig. 3} ). Derivatives of OA such as
2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) exhibit significant
anti-inflammatory and anti-tumor activity due to the modification of the
ring A \textsuperscript{49,50}. Considering that both UA and OA are
characterized by similar triterpene templates, similar chemical
modifications have been applied to UA in recent years.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\emph{\textbf{Fig. 3} Oleanolic Acid and CDDO}

UA derivatives have been synthesized after oxidation, lactoseization and
ring-opening reaction with increased cytotoxicity to NTUB1 cells
compared to UA by 2-fold \textsuperscript{51} (\emph{Fig.4A} ),
providing new insights for the further development of its derivatives.
Moreover, UA derivatives containing N-acyl imidazole or N-alkyl
imidazole derivatives with the help of \selectlanguage{greek}α,β-\selectlanguage{english}unsaturated ketone have also
been characterized as significant anticancer activity
\textsuperscript{52}(\emph{Fig.4B} ). Introduction of N-alkyl
heterocyclic motifs bound to \selectlanguage{greek}α,β-\selectlanguage{english}unsaturated ketones in ring A can
provide better Michael receptors, which allows UA derivatives to
interact with certain target proteins interaction potentially.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\emph{\textbf{Fig. 4} UA derivatives modified on ring A}

\textbf{4.2Directed transformation of UA by microorganisms}

Biosynthesis of valuable products have advantages over chemical synthsis
in terms of milder reaction conditions and presence of abundant enzymes
along with high stereo-, regio- and chemo-
selectivities\textsuperscript{53}. Therefore, microbial transformation
is increasingly adopted as an alternative way to modify the structure of
natural and synthetic compounds \textsuperscript{54,55}. In particular,
it is easy to achieve the glycosylation of pentacyclic triterpenes that
is difficult during chemical synthesis \textsuperscript{56}. Generally,
UA biotransformation is carried out by dissolving the appropriate
quantity of UA in strain suspended in fresh sterile broth under sterile
conditions. After co-cultivation with strains, the transformed products
of UA are eventually separated through suction filtration and
extraction.

In recent years, various UA derivatives have been synthesized through
the transformation of numerous microbial strains. It is reported
that\emph{Nocardia sp.} NRRL 5646 converts UA into oleanolic acid methyl
ester (\emph{Fig.5A} ), in which two intermediates are formed by
participation in the ''reverse biosynthesis'' involving methyl migration
from C-19 to C-20 \textsuperscript{57}. A single component is produced
by this biotransformation followed by hydrolysis to yield the
corresponding triterpenoid acid. At the same time, UA has been found to
be transformed into different derivatives by the same strain (\emph{Fig.
5B} ). Researchers speculate that 3\selectlanguage{greek}β-\selectlanguage{english}hydroxysteroid dehydrogenase and
3-ketosteroid-\selectlanguage{greek}Δ\selectlanguage{english}1-dehydrogenase are involved in the transformation of
steroid \textsuperscript{58}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image10/image10}

\end{center}
\end{figure}

\emph{\textbf{Fig. 5} Transformation of UA by Nocardia sp. NRRL 5646}

Moreover, UA has been well modified by various
fungi\textsuperscript{59}. \emph{Aspergillus flavus} (ATCC 9170) was
able to convert UA into the new natural product 3-oxo-UA derivative with
C-3 hydroxylation (\emph{Fig. 6A} ) \textsuperscript{60}. In addition,
four UA derivatives have been produced by \emph{Syncephalastrum
racemosum}(Cohn) Schroter AS3.264 \textsuperscript{61}, indicating the
inhibitory effect against protein tyrosine phosphatase 1B (\emph{Fig.
6C} ). UA was also biotransformed into six glycosylated and hydroxylated
products by an endophytic fungi \emph{Alternaria longipes}
AS3.2875\textsuperscript{62}. One of the resultant new compounds,
28-o-\selectlanguage{greek}β-\selectlanguage{english}d-glucopyranosyl-3-oxo-UA, showed stronger antibacterial activity
against \emph{Staphylococcus aureus} and
methicillin-resistant\emph{Staphylococcus aureus} (\emph{Fig. 6B} ). Liu
\emph{et al.}obtained three new derivatives transformed by \emph{Mucor
spinosus}AS3.3450 \textsuperscript{63}(\emph{Fig.6D} ). Among them,
3\selectlanguage{greek}β,\selectlanguage{english}7\selectlanguage{greek}β,\selectlanguage{english}21\selectlanguage{greek}β-\selectlanguage{english}trihydroxy-UA showed stronger cytotoxicity against HeLa cells,
K562 cells and KB cells, enlightening its potential therapeutic role in
anticancer therapy.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image11/image11}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image12/image12}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image13/image13}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image14/image14}

\end{center}
\end{figure}

\emph{\textbf{Fig.6} UA derivatives biotransformed by fungi}

\textbf{Efficient synthesis of UA and its derivatives in engineered
microbial cell factories}

As mentioned above, traditional extraction methods are increasingly
unfavorable for large-scale production of UA. Furthermore, complex
structure and oxidation position, as well as the optical purity of the
final product all increase the difficulty for its chemical synthesis.
Green biosynthesis of valuable compounds by microbial cell factories may
reduce the limitations of traditional extraction methods from plants as
it is not restricted by the natural climate and is suitable for
large-scale industrial production under high density fermentation. More
importantly, some terpenoids are synthesized efficiently in
microorganisms with various regulatory strategies, such as excavation
and modification of key enzymes \textsuperscript{18,64}, optimization of
endogenous metabolic pathways \textsuperscript{65-67}, inhibition of
branching pathways \textsuperscript{68-70}. Therefore, microbial
engineering also has a wide application prospect in the green synthesis
of UA and its derivatives.

\textbf{5.1 The biosynthesis pathway of UA}

In recent years, synthetic pathway of UA has been well explored in
plants, which is roughly divided into three modules: synthesis of
isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate
(DMAPP), synthesis of \selectlanguage{greek}α-\selectlanguage{english}amyrin and oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin. In plant
organelles, IPP and DMAPP are synthesized by methyl
erythritol-4-phosphate (MEP) pathway in plastids and mevalonate (MVA)
pathway in cytoplasm. Afterwards, two IPP molecules are combined with
one molecule of DMAPP end-to-end, and condense to farnesyl diphosphate
(FPP). Two FPP molecules bind together to form the triterpenoid
precursor, squalene followed by oxidiation to form 2,3-oxidosqualene,
which subsequently undergoes cyclization into \selectlanguage{greek}α-\selectlanguage{english}amyrin, the direct
precursor of UA. Eventually, \selectlanguage{greek}α-\selectlanguage{english}amyrin is oxidized at C-28 to form UA
through a three-step oxidation reaction by employing the action of
Cytochrome P450 (CYP450) monooxygenase and its redox chaperone
Cytochrome P450 Reductase (CPR). In addition to the common rate-limiting
enzymes for triterpenoid, \selectlanguage{greek}α-\selectlanguage{english}amyrin synthase (\selectlanguage{greek}α\selectlanguage{english}AS) and CYP450 greatly
affect the synthesis of UA (\emph{Fig. 7A} ).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image15/image15}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image16/image16}

\end{center}
\end{figure}

\emph{\textbf{Fig. 7} Biosynthesis of UA (A) Biosynthesis pathway; (B)
The electron transfer between CYP450 and CPR}

It is generally believed that cyclization of 2,3-oxidosqualene is
carried out by the Oxidosqualene Cyclase (OSC) by employing protonation,
cascade cyclization of polyene addition, hydride and/or methyl
translocation, and unwinding \textsuperscript{71}. \selectlanguage{greek}α\selectlanguage{english}ASs are
multifunctional enzymes, which catalyze 2,3-oxidosqualene to several
pentacyclic triterpenoids like \selectlanguage{greek}α-\selectlanguage{english}amyrin, \selectlanguage{greek}β-\selectlanguage{english}amyrin and lupeol
etc\textsuperscript{72,73}. However, product isomerization limits their
catalytic ability and ultimately affects the downstream synthesis of UA.
CYP450s are most versatile proteases found in nature, the first one of
which was discovered in the rat liver microsomes in
1958\textsuperscript{74}. CYP450s contain the heme, which primarily
binds to the cytoplasmic surface of Endoplasmic Reticulum (ER) and
catalyze a series of oxidation reactions like oxidation, hydroxylation,
dealkylation and breakage of carbon-carbon bonds \textsuperscript{75}.
Mainly, CYP450s catalysis involves the introduction of oxygen into
inactive carbon-hydrogen bonds \textsuperscript{76}, while the
corresponding reduction process is assisted by CPR. CYP450s utilize two
electrons from NADH or NADPH transferred by CPR to the heme center of
iron to activate the oxygen molecules and further catalyze the
substrates to form functional groups.

\textbf{5.2 De novo synthesis of UA in} \textbf{engineered microbial
cell factories}

With strong resistance to low pH, osmotic stress and environmental
factors, engineered microbial cell factories such as \emph{S.
cerevisiae} is widely used to biosynthesize the valuable natural
products on industrial scale. What's more, \emph{S. cerevisiae} has
proven to be an enormously suitable candidate for synthesis of complex
terpenoids due to its endogenous MVA pathway, which provides sufficient
precursors to synthesize the terpenoids to meet subsequent
demand\textsuperscript{65}. Terpene skeleton is modified by CYP450 in a
better way due to presence of protein modification system, biofilm
system and redox system in \emph{S. cerevisiae} \textsuperscript{18}.
With clear genetic background and suitable biological safety, various
gene manipulation and genetic modification methods have been widely
employed in \emph{S. cerevisiae}for the green synthesis of terpenoids.

As early as 2012, \selectlanguage{greek}α-\selectlanguage{english}amyrin has been synthesized in microbial cell
factories \textsuperscript{77}, but the UA synthesis was inadequate. It
is generally accepted that UA biosynthesis in \emph{S. cerevisiae} is
limited due to the accumulation of \selectlanguage{greek}α-\selectlanguage{english}amyrin and the oxidation ability of
CYP450 \textsuperscript{78}. These limitations led researchers to adopt
metabolic engineering and synthetic biology approaches to synthesize UA
more efficiently in \emph{S. cerevisiae} . Dai \emph{et al.} introduced
CYP716A15 and CPR from \emph{Vitis vinifera} and \selectlanguage{greek}α\selectlanguage{english}AS
from\emph{Eriobotrya japonica} into \emph{S. cerevisiae} . With ERG1,
ERG 9 and ERG 20 overexpressed to enhance the precursor production, they
subsequently got a final UA yield of 1.76
mg/L/OD\textsubscript{600}\textsuperscript{75}. In another study, Lu
\emph{et al.} heterologously expressed CYP716A12 from \emph{Medicago
Sativa} , \selectlanguage{greek}α\selectlanguage{english}AS from\emph{Catharanthus roseus} , ERG1 from \emph{Candida
albicans} , and CPR from \emph{Arabidopsis thaliana} in
\emph{Saccharomyces cerevisiae} . By optimization of fermentation
conditions and overexpression of tHMG1, ERG9, ERG20 to strengthen MVA
pathway, the yield of UA in shake flask culture reached 25.85mg/L
\textsuperscript{79}.

\textbf{5.3 Optimization strategies to enhance the microbial synthesis
of UA}

Biosynthesis of UA in microbial cell factories provides an environment
friendly platform for synthesis of specialized terpenoids, while the
bioproduction of UA still needs to be improved as compared to its
counterpart triterpenes. Therefore, multiple optimization strategies are
still needed to further develop the production potential of \emph{S.
cerevisiae.} On the basis of UA's synthetic pathway, we propose three
strategies to enhance the green biosynthesis of UA at industrial scale.

\textbf{5.3.1Strengthening precursors supplementation}

Microbial biosynthesis of natural products is improved by adopting
effective metabolic engineering approaches to enhance the precursors
supply and reducing their unnecessary consumption. Common strategy
includes the overexpression of MVA-related (e.g. ERG9, ERG1and ERG20)
genes and tHMG1 (the truncated version of HMG1) gene. Lu \emph{et
al.}enhanced the supply of 2,3-oxidosqualene as mentioned above,
resulting in 25.99 mg/L \selectlanguage{greek}α-\selectlanguage{english}amyrin in shake flask culture
\textsuperscript{79}.

Enhancement of precursor supply is also achieved by adopting the
traditional metabolic engineering approaches to overexpress the key
genes, utilization of balanced pathways and downregulation of
competitive pathways \textsuperscript{80}. In another study, researchers
balanced the metabolic pathway and achieved transcriptional regulation
of aligned oleanane-type triterpenoids by overexpression of UPC2-1, a
global transcription factor for ergosterol synthesis in
yeast\textsuperscript{81}. In addition, they reconstructed the promoter
at the binding site of UPC2 and the galactose regulatory network to
promote gene expression, resulting in a 65 and 6.8-fold increase in
\selectlanguage{greek}β-\selectlanguage{english}amyrin and oleanolic acid, respectively \textsuperscript{82}. This
also provides a new idea for the synthesis and regulation of both
\selectlanguage{greek}α-\selectlanguage{english}amyrin and UA in yeast.

Furthermore, the catalytic potential of \selectlanguage{greek}α\selectlanguage{english}AS also plays a decisive role
in the synthesis of \selectlanguage{greek}α-\selectlanguage{english}amyrin and UA. A highly active \selectlanguage{greek}α\selectlanguage{english}AS, \emph{Md} OSC1
from \emph{Malus domestica} , have been identified through
bioinformatics screening and phylogenetic analysis \textsuperscript{37}.
\emph{Md} OSC1 expression combined with overexpression of genes related
to the MVA pathway in \emph{S. cerevisiae} yielded an \selectlanguage{greek}α-\selectlanguage{english}amyrin titer of
11.97\(\pm\)0.61 mg/L \textsuperscript{83}. Furthermore, the
yield of \selectlanguage{greek}α-\selectlanguage{english}amyrin was increased to 11-fold higher than that of the
control with the triple mutant \emph{Md}
OSC1\textsuperscript{N11T/P250H/P373A} by remodeling \emph{Md} OSC1
\textsuperscript{84}. Owing to these strategy, UA biosynthesis will be
guaranteed by enhancing the supply of its precursors.

\textbf{5.3.2Enhancing the coupling efficiency of CPR with CYP450}

Lower microbial production of UA as compared to \selectlanguage{greek}α-\selectlanguage{english}amyrin depicts the
inefficient oxidation process which in turn depends upon both the
catalytic efficiency of CYP450 as well as electron transfer between
CYP450 and CPR \textsuperscript{80}. In this regard, it is essential to
excavate and characterize CYP450 and CPR with high catalytic efficiency.
By means of gene linkage, genome sequencing and transcriptome
sequencing, many CYP450s with the ability to oxidize C-28 of \selectlanguage{greek}α-\selectlanguage{english}amyrin
have been identified from \emph{Arabidopsis thaliana} , \emph{Medicago
truncatula} , \emph{Barbarea vulgaris} and other plants. Combining
different sources of CYP with CPR and finding the best combination is a
common way to improve its oxidation ability (\emph{Table 3} ).

\selectlanguage{greek}\emph{Table 3. Oxidation of C-28 position of α-amyrin}\selectlanguage{english}\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
\textbf{CYP450} & \textbf{Source} & \textbf{Function} &
\textbf{Reference}\tabularnewline
\midrule
\endhead
CYP716A1 & \emph{Arabidopsis thaliana} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin and
\selectlanguage{greek}β-\selectlanguage{english}amyrin; C-28 hydroxylation of lupeol &
\textsuperscript{85}\tabularnewline
CYP716A2 & \emph{Arabidopsis thaliana} & C-28/C-22\selectlanguage{greek}α \selectlanguage{english}hydroxylation of
\selectlanguage{greek}α-\selectlanguage{english}amyrin; C-16/C-22\selectlanguage{greek}α/\selectlanguage{english}C-28 hydroxylation of \selectlanguage{greek}β-\selectlanguage{english}amyrin; C-28 hydroxylation
of lupeol & \textsuperscript{85}\tabularnewline
CYP716A12 & \emph{Medicago truncatula} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin and lupeol & \textsuperscript{86}\tabularnewline
CYP716A15 & \emph{Vitis vinifera.} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin and lupeol & \textsuperscript{86}\tabularnewline
CYP716A44 & \emph{Lycopersicum esculentum} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin & \textsuperscript{87}\tabularnewline
CYP716A46 & \emph{Lycopersicum esculentum} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin & \textsuperscript{88}\tabularnewline
CYP716A48 & \emph{Oleaeuropaea.} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin, \selectlanguage{greek}β-\selectlanguage{english}amyrin
and lupeol & \textsuperscript{89}\tabularnewline
CYP716A49 & \emph{Beta vulgaris.} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin, \selectlanguage{greek}β-\selectlanguage{english}amyrin
and lupeol & \textsuperscript{89}\tabularnewline
CYP716A50 & \emph{Coffea arabica.} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin and lupeol & \textsuperscript{89}\tabularnewline
CYP716A52V2 & \emph{Panax schinseng} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin and lupeol & \textsuperscript{90}\tabularnewline
CYP716A80 & \emph{Barbarea vulgaris} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin and lupeol & \textsuperscript{91}\tabularnewline
CYP716A81 & \emph{Barbarea vulgaris} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin and lupeol & \textsuperscript{91}\tabularnewline
CYP716A83 & \emph{Centella asiatica} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin & \textsuperscript{92}\tabularnewline
CYP716A175 & \emph{Malus domestica} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin, lupeol and germanicol & \textsuperscript{93}\tabularnewline
CYP716A179 & \emph{Glycyrrhiza uralensis} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin and lupeol & \textsuperscript{94}\tabularnewline
CYP716A252 & \emph{Ocimum basilicum} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin & \textsuperscript{95}\tabularnewline
CYP716A253 & \emph{Ocimum basilicum} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin & \textsuperscript{95}\tabularnewline
CYP716A265 & \emph{Lagerstroemia speciosa.} & C-28 oxidation of
\selectlanguage{greek}α-\selectlanguage{english}amyrin, \selectlanguage{greek}β-\selectlanguage{english}amyrin and lupeol & \textsuperscript{96}\tabularnewline
CYP716A266 & \emph{Lagerstroemia speciosa.} & C-28 oxidation of
\selectlanguage{greek}α-\selectlanguage{english}amyrin, \selectlanguage{greek}β-\selectlanguage{english}amyrin and lupeol & \textsuperscript{96}\tabularnewline
CYP716AL1 & \emph{Catharanthus roseus} & C-28 oxidation of \selectlanguage{greek}α-\selectlanguage{english}amyrin,
\selectlanguage{greek}β-\selectlanguage{english}amyrin and lupeol & \textsuperscript{97}\tabularnewline
\bottomrule
\end{longtable}

In addition, exact mechanism of action of CYP450 and CPR is not clear
yet and there is a need to explore the ultimate cause that affects the
electron transfer efficiency between them. Analysis of the protein
structure of CYP450 helps to elucidate the underlying causes of electron
transfer from CPR to CYP450. In addition, with the advent of protein
engineering along with high throughput screening, rational design and
modification of CYP450 and CPR have attracted much attention, which with
optimized structures have also become common means to solve the problem
of insufficient oxidation \textsuperscript{80}.

\textbf{5.3.3Optimization of subcellular structure}

\emph{S. cerevisiae} harbors diverse organelles with different
structures and specialized functions. Rational design and harnessing of
these organelles to produce valuable compounds is of great application
prospect in terpenoid synthesis. Heterologous FPP synthase and
sesquiterpene synthase to mitochondria by fusing the mitochondrial
targeting signal peptide of yeast COX4, resulting in a 20-fold increase
in sophoradiene production \textsuperscript{98}. ER can also be
subjected to morphological modifications such as increasing the membrane
area to harbor more enzymes to enhance the catalysis processes. As many
endogenous or exogenous proteins are located in ER membrane, like
CYP450, the area of ER membrane have been significantly increased by
knocking out the PAH1 by CRISPR/cas9 in \emph{S. cerevisiae} to enhance
the activity of enzymes related to terpene synthesis for efficient
biosynthesis of terpenoids \textsuperscript{99}. This engineering
strategy increased the yield of \selectlanguage{greek}β-\selectlanguage{english}amyrin, artemisinic acid, alfalfa acid
and its glycosylated derivatives by 8-fold, 2-fold, 6-fold and 16-fold
respectively, which showed great potential of microbial cell factories
to enhance the yield of terpenoids.

In addition, mass transfer efficiency can significantly boost the
synthetic capacity of microorganisms \textsuperscript{65}. The highest
yield of \selectlanguage{greek}α-\selectlanguage{english}amyrin was obtained in engineered \emph{S. cerevisiae} by
expanding the storage pool, where DGA1 (Diacylglycerol acyltransferase)
was overexpressed to enhance the intracellular storage
capacity\textsuperscript{84}. Draw lessons from it, UA biosynthesis can
also be improved by modifying chemical mass transfer such as the
translocation of its synthesis and the condition of transportation.

By combining all these strategies including the precursor
supplementation, enhancing the coupling efficiency of CPR with CYP450
and optimization of subcellular structure, de novo biosynthesis of UA
will be significantly enhanced in microbial cell factories. These
studies fully demonstrated the potential of utilizing \emph{S.
cerevisiae} cell factories to synthesize UA efficiently, providing an
effective means to replace traditional extraction and chemical
synthesis.

\textbf{5.4UA derivatives decorated by engineered microorganisms}

Biosynthesis of UA derivatives by construction of metabolic pathways in
engineered microbial cell factories have been promoted along with the
development of de novo microbial synthesis of UA. In recent years,
biosynthesis of UA derivatives mainly involved in hydroxylation on C-2\selectlanguage{greek}α
\selectlanguage{english}and glycosylation.

For C-2\selectlanguage{greek}α \selectlanguage{english}hydroxylation of UA, sequence analysis of RNA of
\emph{Avicennia marina} leaves revealed the functional CYP716C53, which
catalyzed the C-2\selectlanguage{greek}α \selectlanguage{english}hydroxylation of UA to yield Corosolic acid
(\emph{Fig. 8} ) \textsuperscript{100}. It's a triterpenoid compound
which has attracted commercial and research interest for unique
anti-diabetic properties \textsuperscript{101}. In addition, the
heterologous expression of \emph{Lagerstroemia speciose} -CYP716C55 in
\emph{S. cerevisiae} also led to C-2\selectlanguage{greek}α \selectlanguage{english}hydroxylation of UA
\textsuperscript{96}. Moreover, CYP716C49 was identified in
\emph{Crataegus pinnatifida} and its expression along with the \selectlanguage{greek}α\selectlanguage{english}AS from
\emph{E. japonica} as well as CYP716A15 and CPR from\emph{V. vinifera}
in microbial cell factories increased the production titer of Corosolic
acid to 141.0 mg/L \textsuperscript{75}.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image17/image17}

\end{center}
\end{figure}

\selectlanguage{greek}\emph{\textbf{Fig. 8} Structures of Corosolic Acid and Ursolic Acid
28-O-Β-D-Glucopyranoside}\selectlanguage{english}

Glycosylation involves the attachment of one or more sugar moieties to
the parent compound and contributes for diverse physicochemical
properties, such as water solubility, structural integrity and
pharmacological efficacy \textsuperscript{80}. To achieve the
glycosylated derivatives, heterologous expression of
UDP-glycosyltransferases (UGTs) is less frequent due to their lower in
vivo catalytic activity. In relation to glycyrrhetinic acid, structural
diversification of UA by glycosylation is still in infancy. In one
study, transcriptomic data analysis of \emph{Ilex Asprella} revealed
UGT74AG5 and its expression in UA-producing \emph{S. cerevisiae} strain
confirmed its glycosylation activity against UA to synthesize its
glycosylated derivative,
UA-28-O-\selectlanguage{greek}β-\selectlanguage{english}D-glucopyranoside\textsuperscript{102} (\emph{Fig. 8} ).
Although the synthesis of UA derivatives in cell factories is still in
its infancy, the de novo synthesis of UA in \emph{S. cerevisiae}
provides strong support for its development.

\textbf{Perspective}

UA exhibits significant therapeutic properties to be used in disease
management and drug development. UA derivatives with different
structural modifications have been designed and synthesized to explore
more effective therapeutic products with higher oral bioavailability and
efficacy. Particularly, many structurally modified UA derivatives by
chemical means seem to be effective against variety of cancer cell lines
in vitro. Due to the great value potential and pharmacological
manifestations of UA and its derivatives, there is a need to develop an
economical and feasible approach to produce the high yield of UA and its
varied derivatives.

Although considerable efforts are currently being made to develop
effective approaches for sequestering UA from various medicinal plants,
its biosynthesis in microbial cell factories is a more attractive
strategy. In this regard, \emph{S. cerevisiae} presents a potential
microbial host to synthesize terpenoids efficiently. However,
bioengineering strategies for bioproduction of either UA or its
derivatives have not been developed effectively. With the advent in
synthetic biology and metabolic engineering, improved metabolic pathways
and key enzyme-protein engineering lead to efficient biosynthesis of
valuable products, which provides strong support for better
utilizing\emph{S. cerevisiae} as a cell factory. Increased production of
terpene derivatives in \emph{S. cerevisiae} can be achieved by
optimization of endogenous pathways, employing CRISPR gene editing
technology, modification of key enzymes and utilization of synthetic
\emph{S. cerevisiae} chromosome system (SCRaMbLE). More importantly,
there are powerful tools such as computational biology, genomics and
transcriptomics accompanied by synthetic biology to identify and
manipulate novel terpenoid synthesis pathways in microbial cell
factories for their green production. Advances in synthetic biology
technology will promote to develop a fully automated robotic screening
platform for high-throughput screening of engineered strains.
Combination of different regulation strategies to design and improve the
metabolic pathways in \emph{S. cerevisiae} is expected to be an
efficient approach over the traditional methods to sequester UA and its
derivatives in future for further exploration. Although there is a long
way to go to develop an economically feasible and easy method for UA,
its rich pharmacological activity and great value potential are worthy
of extensive exploration in many scientific fields.

\textbf{List of abbreviations}

\emph{S. cerevisiae = Saccharomyces cerevisiae}

UA = Ursolic acid

IPP = Isopentenyl diphosphate

DMAPP = Dimethylallyl diphosphate

FPP = Farnesyl diphosphate

CYP450 = Cytochrome P450

UGTs = UDP-glycosyltransferases

\selectlanguage{greek}α\selectlanguage{english}AS = \selectlanguage{greek}α-\selectlanguage{english}amyrin synthase

OSCs = Oxidosqualene Cyclases

ER = Endoplasmic reticulum

\textbf{Declaration of Competing Interest}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

\textbf{Acknowledgements}

This work was supported by the National Key Research and Development
Program of China (2018YFA0901800, 2019YFA0905700) and the National
Natural Science Foundation of China (21736002, 21878021).

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