Co-cuture
In fact, co-culture technique has also been introduced to improve the yield of lovastatin. Panda et al.(Panda, Javed et al. 2010) co-culturedM. purpureus MTCC 369 and M. ruber MTCC 1880, which lead to maximum lovastatin production of 2.83 mg/g.

Improving statins production by engineering strains

Traditional fermentation culture production of statins usually poses some problems such as a long culture period, difficult manipulation, and multiple byproducts. With the rapid development of synthetic biology, the construction of engineering strains for the production of statins may be a major strategy for present and future statin production. At the same time, the improvement of metabolic engineering strategies should be rational pathway design and modification. All strains are modified to meet production requirements. We summarized the metabolic engineering strategies from the perspectives of heterologous expression of genes, modification of regulatory proteins, inhibiting by-product synthesis, respectively. In contrast to metabolic engineering strategies, evolution of strains is another alternative to improve the production. Engineering strains strategies for statins biosynthesis was summarized in Table 4.

3.1 Heterologous expression of genes

Heterologous expression of genes is a common strategy in synthetic biology. Heterologous expression of genes strategies to improve statins production are described in the box at the upper left (Figure 3 A-C).S. cerevisiae is very suitable for heterologous expression of genes. Bond et al.(Bond and Tang 2019) introduced six heterosynthetic genes into S. cerevisiaeBY4741 and adding the acyl-donor dimethylbutyryl-S-methyl mercaptopropionate (DMB-SMMP) into the culture medium. Regulating the copy number of lovA and introducing the gene npgA and in situ chemical lysis of cell wall, lead to 55 mg/L simvastatin. Liu et al.(Liu, Tu et al. 2018) introduced lovastatin synthesis gene intoP. pastoris GS115. Using dihydromonacolin L as a metabolic node, the synthetic pathway is divided into upstream and downstream modules. Finally, the optimal co-culture strategy was selected by bioreactor fermentation, lead to 250.8 mg/L lovastatin (Figure 3(A)).
Currently, industrial production of simvastatin acid (SVA) is a multistep process starting from the natural product lovastatin. Monacolin J can be obtained by alkaline hydrolysis of lovastatin. Chemical method for transformation of monacolin J to simvastatin was generally divided into three steps, including hydroxyl group protection, reesterification, and deprotection. The processes from lovastatin to simvastatin are complicated, laborious, and environmentally unfriendly(Askin, Verhoeven et al. 1991, Xie, Watanabe et al. 2006, Huang, Liang et al. 2017). Monacolin J biosynthetic gene cluster were integrated into the genome of A. niger CBS513.88(Zeng, Zheng et al. 2022) which processes strong promoters and suitable integration sites, lead to 92.90 mg/L monacolin J. Optimizing culture conditions and adding precursors, improved the titer to 142.61 mg/L. Liang et al.(Liang, Huang et al. 2018) achieved single-step in vivo production of monacolin J by using lovastatin hydrolase (PcEST) in A. terreus HZ01 (Figure 3(B)). After modification of PcEST, cell activity was increased by 18-dold, which greatly promoted hydrolysis of lovastatin to monacolin J, which also laid a foundation for industrial production of simvastatin.
Compactin synthetic gene cluster has not been fully characterized. The function of specific genes of compactin synthetic is unclear. However, there are still some reports of compactin production in engineered strains. Abe et al.(Abe, Suzuki et al. 2002) improved the synthesis of compactin by adding some gene clusters related to compactin synthesis in P. piltrinum No.41520. Baba et al.(Baba, Abe et al. 2009) improved the titer of compactin by adding complete gene clusters in P. piltrinum No.41520, lead to the titer of compactin increase by 50%. These results indicate that increasing gene copy numbers can promote high titer of compactin.
Pravastatin is mostly produced by microbial fermentation using compactin or compactin sodium as substrate. Screening strains with high conversion rate is the key to obtain high yield of pravastatin. Lin et al.(Lin, Tang et al. 2011) isolated a strain and further identified as P. carboxydivorans PAH4. In the medium of 1 mg/ml compactin sodium, the conversion rate of pravastatin reached 68%. The results of this study suggested P. carboxydivorans PAH4 could be considered a candidate for the production of pravastatin on an industrial scale. Ahmad et al.(Ahmad, Mujeeb et al. 2013) tested the bioconversion of compactin to pravastatin by three A. species, named A. livida MTCC 1382, A. macra MTCC 2559, and A. madurae MTCC 1220. Bioconversion by A. macra MTCC 2559 was highest (87 %) in the yeast extract-amended medium. Park et al.(Park, Lee et al. 2003) isolated Streptomyces sp. Y-110 from soil. In batch culture, the maximum titer was 340 mg/L. By adding compactin to the medium intermittently, the titer was increased to 1000 mg/L. McLean et al.(McLean, Hans et al. 2015) introduced the compactin pathway into the beta-lactam-negative P. chrysogenum DS50662, a new cytochrome P450 (P450 or CYP) was isolated to catalyze the final compactin hydroxylation. They evolved the CYP enzyme to reverse stereoselectivity, lead to more than 6 g/L pravastatin at a pilot production scale (Figure 3(C)).
3.2 Modification of regulatory proteins
Modifying the regulatory element proteins strategies to improve statins production are described in the box at the upper right (Figure 3 D, E). Liu et al.(Liu, Bai et al. 2018) overexpressed the statins pump protein TapA (a membrane protein that enables lovastatin to flow out of cells) in P. pastoris GS115, resulted in 419.0±9.5 mg/L lovastatin, 46% higher than overexpression of lovastatin gene and 520% higher than single-copy strain, respectively (Figure 3(D)). They similarly modulated Trap proteins in P. pastoris GS115, successfully increasing monacolin J production(Bai, Liu et al. 2020). Itoh et al.(Itoh, Miura et al. 2018) knocked out the sterol regulatory element binding protein (SREBP) system, increased the lovastatin production by A. terreusATCC 20542. Thus, knockout of the SREBP system should be considered significant for increasing the productivities of polyketides, such as HMG-CoA reductase inhibitors, by filamentous fungi. Lu et al.(Huang, Tang et al. 2019) overexpressed the lovastatin transcriptional regulatorlove , increased the synthetic yield of monacolin J by 52.5% (Figure 3(E)).
3.3 Inhibiting by-product synthesis
The by-product (+)-Geodin is produced when lovastatin is synthesized from A. terreus ATCC 20542 in glycerol culture. Hasan et al.(Hasan, Abd Rahim et al. 2019) inserted the antibiotic marker hygromycin B (hyg ) within the gedC gene that encodes emodin anthrone polyketide synthase (PKS), got an A. terreusmutant strain (gedCΔ ). Compared with the wild-type strain, the yield of lovastatin was increased by 80% to 113 mg/L. This study also provided a practical method for controlling carbon flux (Figure 3(F)).
3.4 Evolution of strains
Evolution of strains is a method to obtain high-yield strains. Chemical inducers and ultraviolet radiation are common methods of random mutation. Kaur et al.(Kaur, Kaur et al. 2009) induced A. terreusGD13 for three cycles to get high-yielding lovastatinA. terreus EM19, increased 7.5-fold to 1424 mg/L. Azeem et al.(Azeem, Arshad et al. 2020) induced A. terreus with ethidium bromide for a long time, which greatly improved the yield of lovastatin in solid-state fermentation. El-Bondkly et al.(El-Bondkly, El-Gendy et al. 2021) obtained 4.51 mg/gds lovastatin by solid-state fermentation of straw by Fusarium sp. Alaa-20. Enhancing mutagenesis induction and three successive gene recombination of Fusarium alternium, increased the titer to 52.1 mg/gds. Dzhavakhiya et al.(Dzhavakhiya, Voinova et al. 2015) obtained a strain S. xanthochromogenes S33-1 that is high tolerance of compactin by multi-step random UV mutagenesis of S. xanthochromogenes RIA 1098. After the fermentation medium improvement, the maximum bioconversion rate of this strain has reached 91% at the daily compactin dose equal to 1 g/L and still remained high (83%) even at the doubled dose (2 g/L) (Figure 3(G)).

4、Challenges and future prospects

From the perspective of statins production strategies, most of the research is based on solid-state fermentation and submerged fermentation. Most of them are optimized for the composition of carbon source, nitrogen source and inorganic salt in the medium. Some papers have also studied the fermentation parameters and the substances produced in the fermentation process that may affect the yield of statins. It can be concluded that simply optimizing the culture medium components and fermentation parameters will not lead to significant progress in statins production. At the same time, the lack of microbial growth and catalytic activity in industrial fermenters will lead to low product yield, weak cell growth and other problems. Global screening or random mutagenesis of existing strains to obtain more adaptable strains may solve this problem(Maltsev, Maltseva et al. 2020, Chekanov, Litvinov et al. 2021). Metabolic engineering strategies have also been used to increase statins production, but these have been relatively infrequently reported. This is partly because some of the statins synthesis gene clusters have not been fully characterized(Abe, Suzuki et al. 2002, Baba, Abe et al. 2009), limiting gene modification. Therefore, a complete analysis and characterization of the statins synthesis gene cluster will further promote statins synthesis.
S. cerevisiae is an ideal host for heterologous gene expression(Novo, Bigey et al. 2009, Vatanparast and Kim 2019, Davies, Tsyplenkov et al. 2021). The mature technologies of gene editing and expression, high cell-density culture and fermentation process control made S. cerevisiae to be a very promising microorganism for statins production. The successful synthesis of simvastatin(Bond and Tang 2019) has demonstrated thatSaccharomyces cerevisiae may be a promising microorganism for the production of statins. In particular, new statins can be synthesized by introducing exogenous synthetic genes into S. cerevisiae (Giugliano, Maiorino et al. 2019, Chioua and Marco-Contelles 2021). However, some challenges still exist to translate bio-statins into practical industrial applications.
In the future, major advances in statins production will depend on metabolic engineering strategies, which also need biotechnology innovation. Methods such as protein engineering, synthetic biology, metabolic engineering and fermentation engineering will be used to overcome challenges and solve biotechnology problems(Liu, Xue et al. 2022). Synthetic biology and systems biology tools help to explore and construct shorter alternatives to the classical statins synthesis pathway(2012, Nielsen and Pronk 2012). Protein engineering and structural biology tools are needed to improve transformation efficiency and mitigate the inhibition of key intermediates and end products. Adaptively directed evolution of enzymes has also benefited from advances in protein engineering. Therefore, through the further study of metabolic engineering strategies, the production of statins will make significant progress. Compared to review papers on statins production previously published(Manzoni and Rollini 2002, Barrios-González and Miranda 2010), We describe the strategies of statins synthesis in more detail. And we outlook the challenges and possible solutions of statins synthesis in more detail and comprehensively. Overall, statins biosynthesis is a worthy-studied theme, as statins still have high application and value.

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