Fungal statin pump protein improves monacolin J efflux and regulates its production in Komagataella phaffii

Abstract

Background: Monacolin J (MJ) is a key intermediate for the synthesis of cholesterol-lowering drug simvastatin. Current industrial production of MJ involves complicated chemical hydrolysis of microbial fermented lovastatin. Recently, heterologous production of MJ has been achieved in yeast and bacteria, but the resulting metabolic stress and excessive accumulation of the compound adversely affect cell activity. Results: Five genes, tapA, stapA, slovI, smokI and smlcE, coding for fungal statin pump proteins were expressed in an MJ producing yeast strain, Komagataella phaffii J#9. Overexpression of these genes facilitated MJ production. Among them, tapA from Aspergillus terreus highly improved MJ production and led to a titer increase of 108%. Exogenous MJ feeding study on an MJ non-producing strain GS-P GAP-TapA was then performed, and the results illustrated tough entry of MJ into cells and possible efflux action of TapA. Further, intracellular and extracellular MJ levels of J#9 and J#9-TapA were analyzed. The extracellular MJ level of J#9-TapA increased faster, but its intracellular MJ percentage kept lower as compared to J#9. The results proved that TapA effectively excreted MJ from cells. Then functions of TapA were evaluated in a high-production bioreactor fermentation. Differently, TapA expression caused a low MJ titer but high intracellular MJ accumulation in J#9-TapA compared with J#9. Conclusions: Statin pump proteins improved MJ production in K. phaffii in a shake flask. Exogenous MJ feeding and endogenous MJ producing experiments demonstrated the efflux function of TapA. TapA improved MJ production at low MJ levels in a shake flask, but decreased it at high MJ levels in a bioreactor. This finding is useful for statin pump improvement and metabolic engineering for statin bioproduction.

Strategies for the statins production in microbial cell factory：present and future

“…Liu et al ) 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.…”

Improving statins production: From non‐genetic strategies to genetic strategies

“…[ 61 ] Similar modulation of Trap proteins in P. pastoris GS115 has also been shown to increase monacolin J production. [ 78 ] Knocking the sterol regulatory element binding protein (SREBP) system out increases the lovastatin production by A. terreus ATCC 20542. Thus, knocking the SREBP system out should be considered significant for increasing the productivities of polyketides, such as HMG‐CoA reductase inhibitors, by filamentous fungi.…”