| At present there are many microbial secondary metabolites having biological activity, nearly half are produced by filamentous fungi, Trichoderma, Aspergillus and Penicillium chrysogenum are typical representative among them. At the same time, Filamentous fungal cell factories as expression system of heterologous protein system has the incomparable advantage compared with prokaryotic and yeast expression. So as a heterologous expression system, filamentous fungi play an important role in the discovery of a variety of active compounds and so on, scientists at home and abroad are more and more concerned with them and gained significant achievements. Therefore, mining of fungal secondary metabolites has very important value in the theoretical basis and the industrial demand, which provides a natural treasure to search for more effective drugs against resistant-drug bacteria and is worth developing for researcher, post genomic era of filamentous fungi has opened, but it should be seen, due to the genetic manipulation of many filamentous fungi and the fermentation culture system is not perfect, or the resulting secondary metabolite’s production is very low, and much complexity in metabolic regulation and genetic background, which are hindering deeply the study of these potentially new active compounds. So scientists still need to try their best to overcome these limitations.Filamentous fungi are not only the major producers in the traditional fermentation industry, but also are the important cell factories in the process of the expression of heterologous proteins in the metabolic engineering. Heterologous expression coupled with developing DNA clone and transformation technology has become an efficient strategy that enables the deeply mining of natural active compounds yields, production of new structural analogues. The study of beauvericin is about the Fusarium proliferatum LF061 screening from the ocean. It is a cyclic hexadepsipeptide mycotoxins belonging to enniatin family, which has insecticidal, antibacterial, antiviral efficacy, also has the role of physiological toxicity. It is a potential agent for pesticides and medicines. Our laboratory in 2007 found that low dose of beauvericin and low dose of ketoconazole can play a very good interactive antifungal effect, and can significantly reduce both the dosage and toxic side effects, so beauvericin is expected to become the potentially novel antifungal compound. Due to the producing beauvericin in Fusarium proliferatum LF061 is not stable in solid or liquid culture conditions and the strain’s genome sequence and genetic operation is not very clearly. Therefore, we choose the A. nidulans RJMP1.59 as heterologous host to express beauvericin, expecting to achieve a higher yield. The specific research contents are as follows:Firstly, we construct the vector pYWL21 containing beauvericin synthetase and α-ketoisovalerate acid reductase. Then, the recombinant plasmid pYWL21 was transformed into A.nidulans RJMP1.59 by the method of PEG mediated protoplast’s transformation. The TYAHO transformant with pRG-AMAI vector, the TYAH1 transformant with pYWL21 plasmid and are fermented in 50 mL GMM+Yeast Extract medium shake flask (A.nidulans RJMP1.59 need to add extra uridine and uracil in medium). We finally found that beauvericin is successfully heterologouly expressed in A.nidulans RJMP1.59 by LC-MS and MS-MS detection, and achieved the yield up to 0.79 mg/L.As we all know, filamentous fungi can usually express large amounts of endogenous protein, however, the expression of exogenous protein is very low, the existing literatures reported that these endogenous gene may be driven by the strong promoters. Therefore, we need to refactor stronger promoters to enhance the efficiency of gene transcription, and high level expression of the target product. In order to further improve the beauvericin production, we use the constitutive 3-phosphate glyceraldehyde dehydrogenase promoter gpdAP and xylose inducible promoter xylP together to fulfil co-expression of beauvericin synthetase and α-ketoisovaleric acid reductase. Likewise, we detect the beauvericin in TYAH2 and TYAH3 transformants by LC-MS analysis. Compared to endogenous promoters, the yield of beauvericin increased more than 44 times by utilizing the strongly heterologous promoters to drive expression. The highest production reached 4.52 mg/L.In the process of filamentous fungi fermentation, the growth morphology of microorganisms has a great relationship with the species of the fermentation products and the production. At the same time, it is necessary to find out the more suitable components of the fermentation medium and the fermentation process for the higher production. At first, we want to optimize the production of beauvericin by adding inorganic particles (2% talcum) into the medium, which to a great extent can solve the problems such as the viscous fermentation liquid causing oxygen deficiency and low nutrient utilization. Then we continued to explore the optimal fermentation temperature, time and precursor supply by the orthogonal experiment, eventually get beauvericin yield reaching up to 157.2 mg/L in the optimized medium including 1% glucose,1% xylose,0.5% Yeast Extract,.25 mM valine, 1×Nitrate solution, 1×Trace element solution at 28℃,220 rpm for 7 days.This paper reported successfully that engineering of A. nidulans RJMP1.59 as heterologous expression of beauvericin through co-expressing synthetase anda-ketoisovalerate acid reductase. Then we optimize the production of beauvericin heterologously expressed in A. nidulans RJMP1.59 by replacing with strong promoters, exploring the most suitable fermentation conditions and process by the orthogonal experiment, and eventually obtained beauvericin at the highest yield of 157.2 mg/L. On the one hand, these methods and results provide the foundation for for us to keep trying to optimize beauvericin in A. nidulans RJMP1.59, on the other hand, it provides a new basis for mining more lipopeptide compounds, and has important significance with finding more novel active compounds. |
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