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