利用基因工程大腸桿菌生產菸鹼醯胺單核苷酸

Abstract

β-菸鹼醯胺單核苷酸 (NMN) 為煙醯胺腺嘌呤二核苷酸 (NAD+) 的前驅物，且先前研究顯示其可增強NAD+生合成並改善糖尿病和血管功能障礙。NMN因此成為一種廣受歡迎的營養保健品。然而，NMN生產的限制包含：(1) 使用化學或酵素合成方式生產NMN會需要昂貴的基質和催化劑；(2) 以微生物發酵方法生產 NMN 的效率和實用性低。因此，開發有效率且經濟的NMN生產方式是重要的。本研究以菸鹼酸 (NA) 為基質，大腸桿菌JM109為宿主生產NMN。本研究首先將大腸桿菌的pncC基因剔除，以防止其水解NMN上的醯胺基。 此pncC缺失的JM109菌株便為後續異源基因表現的宿主。為了生合成NMN，本研究也異源表現了來自土拉弗朗西斯菌 (Francisella tularensis) 的NMN合成酶ftNadE。ftNadE 催化菸鹼酸D-核糖核苷酸 (NaMN) 的醯胺化形成NMN。因此，當大腸桿菌吸收NA，其菸鹼酸磷酸核糖基轉移酶PncB將催化NaMN的合成。接著ftNadE便將合成的NaMN醯胺化，合成NMN。結果顯示，ftNadE 在異丙基-β-D-硫代半乳糖苷 (IPTG) 誘導後成功表現，且 pncC 也成功在大腸桿菌的基因組中剔除。經過搖瓶培養並使用高效液相層析法 (HPLC) 分析 NMN 的產量後，NMN 很可能在表現 ftNadE 的野生型菌株 (34.79 mg/L)、在表現ftNadE的pncC剔除菌株 (44.96 mg/L)、和在表現His-ftNadE的pncC剔除菌株中產生 (46.83 mg/L) 。未來將會需使用液相層析質譜儀確認NMN分子存在，並在生物反應器中培養，以進一步提高NMN 產量。

As a key precursor of nicotinamide adenine dinucleotide (NAD+), β-Nicotinamide mononucleotide (NMN) has been shown to enhance NAD+ synthesis and improve diabetes and vascular dysfunction. NMN has subsequently become a popular nutraceutical. Despite its popularity, NMN production has limitations which include: (a) the use of expensive substrates and catalysts in chemical or enzymatic synthesis and (b) low productivity and practicality in microbial fermentative methods. A cost-effective NMN production method is therefore welcome and worth exploring. This study aims to produce NMN using nicotinic acid (NA) as substrate and Escherichia coli JM109 (E. coli JM109) as host. E. coli’s pncC gene has been deleted to prevent it from hydrolyzing NMN’s amide group. The pncC-deleted JM109 strain (E. coli JM109 ΔpncC) then served as the heterologous gene expression host. To synthesize NMN, ftNadE, an NMN synthetase from Francisella tularensis (F. tularensis) that amidates nicotinate D-ribonucleotide (NaMN) to form NMN, has been heterologously expressed. So once E. coli cells take up NA, its nicotinate phosphoribosyltransferase, PncB, catalyzes the synthesis of NaMN. NMN synthetase ftNadE then amidates the synthesized NaMN to yield NMN. Results showed that ftNadE was successfully expressed after Isopropyl β-D-1 thiogalactopyranoside (IPTG) induction and that pncC was deleted from the E. coli genome. After shake-flask culturing and analyzing NMN production using high performance liquid chromatography (HPLC), NMN was likely produced in wild-type E. coli strains expressing ftNadE (34.79 mg/L), in E. coliΔpncC strains expressing ftNadE (44.96 mg/L) and in E. coli ΔpncC strains expressing His-ftNadE (46.83 mg/L). Future work would involve verifying NMN production using liquid chromatography-mass spectrometry (LC-MS) and maximizing NMN production in bioreactors.