异戊烯基转移酶N端截短强化异戊烯基柚皮素合成

N-terminal truncation of prenyltransferase enhances the biosynthesis of prenylnaringenin

摘要:

8-戊烯基柚皮素(8-prenylnaringenin,8-PN)是一种强有效的雌激素,具有很高的药用价值,同样也是多种异戊烯基黄酮的前体。微生物合成8-PN主要面临异戊烯基转移酶(prenyltransferases,PTs)催化活性较低以及前体供给不足等问题,严重阻碍了8-PN在微生物体内的高效合成。文中以苦参(Sophora flavescens)来源的SfN8DT-1为对象,研究如何实现更高效的柚皮素异戊烯基化反应。结构预测显示,SfN8DT-1主要由9个α-螺旋和8个环的主体,以及一个近120个氨基酸的长侧链组成。将侧链不同位点序列截断的SfN8DT-1在酿酒酵母中表达,发现截短至K62位点时8-PN产量最高,将该蛋白命名为SfND8T-1-t62。SfN8DT-1-t62与柚皮素和二甲基丙烯基二磷酸(dimethylallyl diphosphate,DMAPP)进行分子对接,发现K185是一个潜在的关键位点。对底物0.5 nm范围内的氨基酸进行丙氨酸扫描突变,突变体K185A对底物的亲和力下降,表明K185是一个潜在的关键催化位点。模拟饱和突变表明,突变体K185W对配体的亲和力增强,而K185饱和突变后8-PN产量显著下降,说明K185对SfN8DT-1的功能至关重要。另外,结合甲羟戊酸(mevalonate,MVA)途径关键基因的强化,8-PN产量达到31.31 mg/L,说明DMAPP的供给也是8-PN合成的关键因素。最后,将产量最高的菌株在5 L发酵罐中进行发酵,发酵120 h后8-PN产量达到44.92 mg/L,是目前报道的最高水平。

关键词:酿酒酵母;8-异戊烯基柚皮素;分子对接;丙氨酸扫描;饱和突变

Abstract:

8-prenylnaringenin (8-PN) is a potent estrogen with high medicinal values.It also serves as an important precursor for many prenylated flavonoids.Microbial synthesis of 8-PN is mainly hindered by the low catalytic activity of prenyltransferases (PTS) and insufficient supply of precursors.In this work,a SfN8DT-1 from Sophora flavescens was used to improve the efficiency of (2S)-naringenin prenylation.The predicted structure of SfN8DT-1 showed that its main body is comprised of 9 α-helices and 8 loops,along with a long side chain formed by nearly 120 amino acids.SfN8DT-1 mutants with different side-chain truncated were tested in Saccharomyces cerevisiae.A mutant expressing the truncated enzyme at K62 site,designated as SfND8T-1-t62,produced the highest 8-PN titer.Molecular docking of SfN8DT-1-t62 with (2S)-naringenin and dimethylallyl diphosphate (DMAPP) showed that K185 was a potentially crucial residue.Alanine scanning within a range of 0.5 nm around these two substrates showed that the mutant K185A may decrease its affinity to substrates,which also indicated K185 was a potentially critical residue.Besides,the mutant K185W enhanced the affinity to ligands implied by the simulated saturation mutation,while the saturated mutation of K185 showed a great decrease in 8-PN production,indicating K185 is vital for the activity of SfN8DT-1.Subsequently,overexpressing the key genes of Mevalonate (MVA) pathway further improved the titer of 8-PN to 31.31 mg/L,which indicated that DMAPP supply is also a limiting factor for 8-PN synthesis.Finally,44.92 mg/L of 8-PN was produced in a 5 L bioreactor after 120 h,which is the highest 8-PN titer reported to date.

Key words:Saccharomyces cerevisiae;8-prenylnaringenin;molecular docking;alanine scanning;saturated mutation