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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