Abstract
Statins as a lipid-lowering drug can selectively inhibit
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and decrease
cholesterol synthesis effectively. With the improvement of nutritional
conditions, the demand for statins is increasing in global market. Due
to the rapid development of modern biotechnologies, the biosynthesis of
stains by microbial cell factory appears great advantages. It has the
advantages of simple operation and easy separation of products. This
review summarized the strategies on
statins production via microbial cell factory, including both
traditional fermentation culture and modern synthetic biology
manufacture. Firstly, the complex fermentation parameters and process
control technology on submerged fermentation (SmF) and solid-state
fermentation (SSF) were introduced in detail. Especially, the
possibility of recoverable agricultural wastes/(Biomass) as fermentation
substrate on solid-state fermentation to produce statins was emphasized.
Besides, metabolic engineering strategies to construct robust
engineering strains and strains evolution were also discussed. The
review highlights the potential and challenge of microbial cell factory
to yield the statins. Thus, it will facilitate the production of statins
in more green production mode.
KEYWORDS : Statins; strategies; microorganism; fermentation;
engineering strains
Introduction
Hypercholesterolemia is one of the leading causes of death from
cardiovascular disease in humans. Only one-third of the total body
cholesterol is diet derived, two-thirds is synthesized directly from
intracellular precursors 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)
(Alberts, Chen et al. 1980, Breedlove and Hedrick 1999). Statins drugs
can selectively inhibit HMG-CoA reductase thus reducing lipids synthesis
significantly as well as giving play to multiple biological effects such
as inhibiting atherosclerosis, thrombosis and alleviating rejection
reaction, treating osteoporosis, anti-tumor, etc. (Figure 1) (Cummings
and Bauer 2000, A. Massy and Guijarro 2001, Barrios-González and Miranda
2010, Osmak 2012). Statins block an early step, the conversion of
HMG-CoA to mevalonate, which reducing cholesterol synthesis precursors,
thus directly affecting the synthesis of cholesterol (such as the reduce
of LDL and the increase of HDL) (A. Massy and Guijarro 2001, Adhyaru and
Jacobson 2018). And the reduction of farnesyl pyrophosphate
(farnesyl-PP) and geranylgeranyl pyrophosphate (geranylgeranyl-PP)
interferes with protein isoprene (the binding of lipid isoprene to
proteins), thereby affecting the normal function of small glutamyl
transpeptidases (GTPases) (such as Ras, Rho, Rac and Rab) in the
osteoclasts, which may lead to osteoporosis, senile dementia and so
on(Rikitake and Liao 2005, Binnington, Nguyen et al. 2015, Petek,
Villa-Lopez et al. 2018, Healy, Berus et al. 2020). From January 1,
2002, to December 31, 2018, an average of 21.35 million statins were
purchased annually, with an average total annual cost of $24.5 billion
in the US (Lin, Baumann et al. 2021).
Statins can be produced through microbial synthesis and chemical
synthesis. There are totally four statins which can be produced by
microorganism cells, lovastatin (Alberts, Chen et al. 1980), compactin
(Endo, Kuroda et al. 1976), pravastatin, simvastatin. Meanwhile,
lovastatin and simvastatin are first-generation statins. Lovastatin fromAspergillus and compactin from Penicillium are two pure
natural statins till now. Simvastatin can be synthesized by the
precursor monacolin J, a hydrolysate of lovastatin. Pravastatin can be
obtained by stereoselective hydroxylation in the fermentation of
microorganism using compactin as precursor. Fluvastatin, atorvastatin,
rosuvastatin and pitavastatin are fully synthetic statins (Jahnke 2007).
Pravastatin and fluvastatin belong to the second-generation of statins.
Atorvastatin, rosuvastatin and pitavastatin are the third-generation
statins. Their chemical structures are quite different from natural
statins (Table 1) and couldn’t be produced by microbiology technology so
far (Zhou, Curtis et al. 2019). Although there are many types of statins
on the market, the first generation of statins produced by microbial
cells still maintain a good trend in the world market. Although there is
a strong commercial demand for statins, their production is usually at
low levels in native producers from rare natural sources. The small
quantities and poor purities limit the scale-up of stains production
through chemical synthesis (Tartaggia, Fogal et al. 2016). Today,
statins are mainly produced through microbial submerged fermentation
(SmF) and solid-state fermentation (SSF) (Pawlak and Bizukojc 2013,
Gonciarz, Kowalska et al. 2016). However, fermentation using nativeA. terreus usually poses some problems such as a long culture
period, difficult manipulation, and multiple byproducts. Synthetic
biology has many advantages compared to chemical synthesis, such as
carbon neutral, sustainable, low cost, etc. With the development of
synthetic biology, reconstruction of biosynthetic pathways in chassis
organisms has been proved to be a possible solution to these
problems(Ro, Paradise et al. 2006, Galanie, Thodey et al. 2015).
Therefore, a growing number of researchers are looking into using
microbial cell factories to yield statins (Ro, Paradise et al. 2006).
Here, we reviewed the strategies of microbial cell fermentation to
produce statins in recent 20 years, mainly include submerged
fermentation (SmF) and solid-state fermentation (SSF). As a vital
strategy to improve statins synthesis at the cellular level, we have
also reviewed findings that provide guidance on strains construction by
metabolic engineering strategies and evolution. We also illustrated the
great potential and challenges of producing statins through microbial
cell factories.
Microbial cell fermentation to produce
statins
Now, statins are produced mainly through microbial cell fermentation,
mainly divided into submerged fermentation (SmF) and solid-state
fermentation (SSF). According to the reports, three most aspects
including medium, dissolved oxygen and other effects, respectively,
affecting the submerged fermentation (SmF). The medium and other effects
are two most aspects that affecting solid-state fermentation (SSF)
(Figure 2).
2.1 Statins produced in
SmF
Submerged fermentation (SmF) technology has the advantages of short
cycle, low cost and high yield, and the purification of products is
easier. Medium components especially carbon, nitrogen sources and
inorganic salt are most influential aspects for statins production. The
dissolved oxygen in the medium also has a great influence on the
synthesis of statins. There are also many other effects such as
Antibiotics, surfactants, the age of selected spores and fed-batch
fermentation, affecting the production of statins. Submerged
fermentation for statins biosynthesis was summarized in Table 2.
2.1.1 Medium