The fcl gene encodes GDP-fucose synthase, which catalyzes two-step differential isomerase and reductase reactions in the synthesis of GDP-L-fucose from GDP-D-mannose. It also participates in the biosynthesis of amino sugar and ribose sugar, and is one of the key enzymes to regulate the metabolism of sugar and nucleotides in organisms. The presence of fcl gene in Saccharopolyspora pogona was found through sequencing result of genome. The mutant S. pogona-fcl and S. pogona-Δfcl were constructed by gene engineering technology. The results showed that the gene had an effects on growth and development, protein expression and transcriptional level, insecticidal activity, and biosynthesis of butenyl-spinosyn of Saccharopolyspora pogona. The results of HPLC analysis showed that the yield of butenyl-spinosyn in S. pogona-Δfcl was 130% compared with that in S. pogona, which reduced by 25% in S. pogona-fcl. The results of determination of insecticidal activity showed that S. pogona-Δfcl had a stronger insecticidal activity against Helicoverpa armigera than that of S. pogona, while the S. pogona-fcl had a lower insecticidal activity against Helicoverpa armigera compared with S. pogona. Scanning electron microscopy (SEM) was used to observe the morphology of the mycelia. It was found that the surface of the S. pogona-Δfcl was wrinkled, and the mycelium showed a short rod shape. There was no significant difference in mycelial morphology between S. pogona-fcl and S. pogona. Aboved all showed that deletion of fcl gene in S. pogona hindered the growth and development of mycelia, but was beneficial to increase the biosynthesis of butenyl-spinosyn and improve insecticidal activity. Whereas the fcl gene over-expression was not conducive to the biosynthesis of butenyl-spinosyn and reduced their insecticidal activity. SDS-PAGE results showed that the difference of protein expression among the three strains was most obvious at 96 hours, which was identified by real-time fluorescence quantitative polymerase chain reaction, the results showed that there were significant differences of related genes in transcriptional levels among the three strains. Based on the results of the study, a network metabolic control map was constructed to analyze the effect of fcl gene on growth and the regulation pathway of butenyl-spinosyn biosynthesis, which provided an experimental basis for revealing the regulation mechanism of butenyl-spinosyn biosynthesis and related follow-up studies.
fcl 基因编码的GDP-岩藻糖合成酶 (GDP fucose synthetase，GFS)，能催化由GDP-D-甘露糖合成GDP-L-岩藻糖过程中的两步差向异构酶和还原酶反应；还参与氨基糖和核糖的生物合成，是调控生物体糖代谢、核苷酸代谢的关键酶之一。通过前期基因组测序表明须糖多孢菌Saccharopolyspora pogona 中存在fcl 基因。利用基因工程技术构建了fcl 基因的过表达菌株S. pogona-fcl 和敲除菌株S. pogona-Δfcl。结果表明该基因对菌株生长发育、蛋白表达及其转录水平、杀虫活性、丁烯基多杀菌素的生物合成均存在影响。经HPLC 分析显示，S. pogona-Δfcl的丁烯基多杀菌素产量增加为野生型菌株的130%，S. pogona-fcl 的丁烯基多杀菌素产量降低了25%。生测结果显示，与野生型菌株相比S. pogona-Δfcl 对棉铃虫的杀虫活性明显增强，而S. pogona-fcl 的杀虫活性降低。利用扫描电镜观察发现，S. pogona-Δfcl 菌丝体表面出现褶皱，呈现短棒状，S. pogona-fcl 菌丝形态与野生型菌株一致。以上结果表明，fcl 基因的敲除影响菌丝体的生长发育，能促进丁烯基多杀菌素的生物合成和增强杀虫活性，该基因的过表达抑制了丁烯基多杀菌素的生物合成和降低了杀虫活性。SDS-PAGE 结果表明，三株菌株在96 h 时蛋白表达差异最为明显。对差异蛋白通过实时荧光定量聚合酶链式反应结果显示，三菌株蛋白的转录水平存在显著表达差异。通过研究结果构建了网络代谢调控图，分析fcl 基因对须糖多孢菌生长发育及丁烯基多杀菌素生物合成代谢调控网络途径的影响，初步构建了fcl 基因调控的代谢途径，为揭示丁烯基多杀菌素生物合成的调控机制及相关后续研究提供了实验依据。.
Keywords: Saccharopolyspora pogona; biosynthesis; butenyl-spinosyn; fcl gene; growth and development.