|其他摘要||Two FADH2 dependent halogenases were cloned, heterologously expressed and characterized in vitro, screened out from the metagenomic library of microbiota enriched from the stem barks of Mallotus nudiflorus. Acyltransferase gene asm19 and halogenase gene asm12 proved to be involved in the post-PKS modifications of ansamitocin, were studied through conjugal transfer, heterologous expression and in vitro enzymatic characterization.
A stepwise PCR screening approach was used on 75000 clones of the metagenomic library and thus 13 positive clones were isolated. As high GC content or complex spatial structures of fomsid in metagenomic library, through subcloning technique and walking sequencing, only 2 halogenase genes named 63D9Hal II and 64E9Hal were obtained (GenBank accession number: FJ493227 and FJ493228).
With specific primers，63D9Hal II and 64E9Hal were cloned into 3 expression vectors respectively. Thus, six recombinant expression vectors were constructed, named pKIB001 (pRSET B/64E9Hal), pKIB002 (pRSET B/63D9Hal II), pKIB003 (pET28a(+)/64E9Hal), pKIB004 (pET28a(+)/63D9Hal II), pKIB007 (pET32a(+)/64E9Hal), pKIB008 (pET32a(+)/63D9Hal II). These recombinant vectors were transformed into BL21(DE3)pLysE and expressed, but the majority of expressed product were inclusion bodies. Though we try to optimized the temperature, IPTG concentration, medium and engineered strains, the result was not improved obviously.
L-tryptophan、MCD、proansamitocin and tryptamine were used as substrates to detect the biological activity in vitro, but no obvious activity was detected except for a low peak of possible 7-chloro-L-tryptophan through LC-MS. In addition, 63D9Hal II and 64E9Hal were also expressed in Streptomyces lividans T7 strain, but no obvious expressing protein and biological activity were detected either. As we know nothing about the origin, the fittest expressing strain and natural substrates of the two new haolgenase genes, it is disadvantage to their heterologous expression and biological activity detection in vitro.
Asm19 and Asm12 act as acyltransferase and halogenase in the post-PKS modifications of ansamitocin, which were proved to be very important to its biological activity. Through conjugal transfer, pHGF9071 containing asm19 was transformed into Actinosynnema pretiosum ssp. auranticum ATCC31565/△asm19. Analysis of antagonistic action to Filobasidium uniguttulatum and LC-MS detection of transformants approved their antibacterial activity and AP1-4 in their fermentation production, similar to wild type strain ATCC31565. Probably, the different inserted position of asm19 resulted in the difference of their antibacterial activity.
asm19 was heterologously expressed，and similarly，the induction temperature, time and IPTG concentration were optimized. As a result, Asm19 has extensive adaptability to acetyl-CoA except some artificial synthesized fatty acyl enzyme substrate. The stability and influencing factors to crude enzymatic reactions were also investigated preliminarily. With affinity chromatography, the recombinant protein was purified, and the Wash buffer and Elution buffer were optimized. According to SDS-PAGE and analysis of Lab Works 4.6 software, the content of Asm19 in purified recombinant protein was about 45-48%.
Besides, an artificial synthesized substrate named HGsy14 from alanine was fed to ATCC31565 and ATCC31565 AP-2. According to LC-MS analysis of product fed with different HGsy14 concentrations, anticipated main product H5 (Maytansinol-HGsy14) was observed, but the peak was very low. The final submission is to be confirmed by means of the product structure determination next. This is valuable for understanding of Asm19’s function and the origin of maytansinoids in plants.
With pET32a(+) vector，asm12 (belonging to FADH2 dependent halogenase genes) was cloned and heterologously expressed. At the induction temperature of 37 ℃, Asm12 were expressed in the form of inclusion bodies, but more soluble protein was produced at 16 ℃. The recombinant protein was purified with affinity chromatography, and over 90% soluble pure protein was obtained. Analyzed through LC-MS, crude protein of Asm12 from E. coli BL21(DE3)pLysE possibly can convert proansamitocin to 19-chloroproansamitocin (low peak of 500 in LC-MS map) but with no activity on L-tryptophan (no peak of 261 in LC-MS map). As a result, Asm12 has no obvious biological activity to proansamitocin and L-tryptophan.
Besides, with different fixation solution, endophytes in Mallotus nudiflorus were enriched and their DNA was extracted. Then, the 16S rDNA analysis was used to check whether this method could produce more endophytes pellet. It is meaningful to explore the method of enriching endophytes from plant tissues. In addition, 10 compounds were isolated from twigs and leaves of Trichilia connaroides, which were collected from Xishuangbanna ,Yunnan, China. Among them, TrichilinA、Trichilin B and Trichilin C are new compounds. Among these, Trichilin B is a limonoid having an unprecedented highly rearranged ring system.|