Enhanced Single-Step Bioproduction of the Simvastatin Precursor Monacolin J in an Industrial Strain ofAspergillus terreusby Employing the Evolved Lovastatin Hydrolase

Abstract

Biosynthesis of simvastatin, the active pharmaceutical ingredient of cholesterol-lowering drug Zocor, has drawn increasing global attention in recent years. Although single-step in vivo production of monacolin J, the intermediate biosynthetic precursor of simvastatin, has been realized by utilizing lovastatin hydrolase (PcEST) in our previous study, about 5% of residual lovastatin is still a problem for industrial production and quality control. In order to improve conversion efficiency and reduce lovastatin residues, modification of PcEST is carried out through directed evolution and a novel two-step high-throughput screening method. The mutant Q140L shows 18-fold improved whole-cell activity as compared to the wild-type, and one fold enhanced catalytic efficiency and 3 °C increased T over the wild-type are observed by characterizing the purified protein. Finally, the engineered A. terreus strain overexpressing Q140L mutant exhibited the increased conversion efficiency and the reduced lovastatin residues by comparing with A. terreus strain overexpressing the wild-type PcEST, where almost 100% of the produced lovastatin is hydrolyzed to monacolin J. Therefore, this improved microbial cell factory can realize single-step bioproduction of monacolin J in a more efficient way, providing an attractive and eco-friendly substitute over the existing chemical synthetic routes of monacolin J and promoting complete bioproduction of simvastatin at industrial scale.

Growing a circular economy with fungal biotechnology: a white paper

“…It, furthermore, serves as a starter molecule for manufacturing semisynthetic statins, such as simvastatin (trade name Zocor), which is the second leading statin on the market [23]. Current research is focused on re-routing lovastatin biosynthesis to one of its biosynthetic intermediates, monacolin J, which is the preferred precursor for Zocor synthesis [24]. Notably, a genetically engineered A. oryzae strain can reach monacolin J concentrations of about 5.5 g/L, which is considerably higher than has been achieved in heterologous hosts, such as S. cerevisiae (75 mg/L) or Pichia pastoris (renamed Komagataella phaffii; 600 mg/L) [25].…”

Structural insights into the catalytic mechanism of lovastatin hydrolase

“…These changes imply that the Tyr 127 -containing R2 region, together with loop 238 -245, is a latch for the entrance of the substrate-binding tunnel. Our previously reported Q140L mutant in the R2 helix region, causing a decrease in K m and an increase in k cat toward lovastatin (9), also indicates the importance of R2 in lovastatin hydrolysis. Further, the Phe 309 benzene ring at the bottom of the substrate-binding pocket swings away from the hydrophobic core and forms ainteraction with are separately involved in binding the two acid side chains of lovastatin (Fig.…”

“…Further, we achieved ϳ95% conversion by heterologously expressing PcEST in an industrial A. terreus strain (2). Recently, we further reduced the lovastatin residual using a directed evolutionary PcEST mutant, Q140L, which presents 2.2-fold improved solubility, 1-fold enhanced catalytic efficiency, and 3°C increased T 50 10 over the purified WT PcEST (9). All of these results clearly suggest the huge industrial application potential of PcEST.…”

“…Our previous work showed that the main problems with PcEST are its poor soluble expression and thermostability (9). The highly flexible regions on protein surfaces sometimes affect the solubility of the proteins and/or reduce their thermostability (17,18), because they might enhance the entropy during protein unfolding by increasing the numbers of unfolded conformations (19).…”

“…The analysis strategy for the soluble expression levels of PcEST and its mutants was described previously (9). The expression plasmids lacking fusion tags, pET22-D106A and pET22-D131A, were constructed using the QuikChange sitedirected mutagenesis method with pET22-PcEST as the template (9). Plasmids were transformed into BL21 (DE3) cells and induced with 0.2 mM isopropyl ␤-D-thiogalactopyranoside at 25°C for 16 h. The harvested cells were lysed by sonication.…”