High cell density fed-batch production of insecticidal recombinant ribotoxin hirsutellin A from Pichia pastoris

Abstract

BackgroundThe fungal ribotoxin hirsutellin A (HtA) exhibits strong insecticidal activity; however, efficient systems for expressing recombinant HtA (rHtA) are lacking. Here, we established an efficient heterologous expression system to produce large amounts of rHtA.ResultsRecombinant Pichia pastoris transformants with high levels of secretory rHtA were screened, and in a fed-batch reactor, rHtA was secreted at levels up to 80 mg/l following methanol induction, which was more than sixfold higher than that in shake flasks. Approximately 7 mg of highly pure rHtA was obtained from 300 ml of fed-batch culture supernatant by Ni+-nitriloacetic acid affinity chromatography and CM Sepharose ion-exchange chromatography. Mass spectrometry results revealed rHtA as a native N-terminal non-glycosylated monomeric protein with a molecular weight of 15.3 kDa. Purified rHtA exhibited excellent thermal and protease stability and dose-dependent cytotoxicity to Sf9 insect cells and insecticidal activity against Galleria mellonella larvae.ConclusionsThis is the first report of rHtA expression in P. pastoris. The rHtA was expressed at a high level under high-cell-density fed-batch fermentation and was efficiently purified using a two-step purification method. Purified rHtA exhibited thermal and protease stability, as well as appropriate bioactivities. Our results indicate that fed-batch production by P. pastoris is an efficient method to produce functional rHtA.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0992-x) contains supplementary material, which is available to authorized users.

The Chemical Ecology Approach to Reveal Fungal Metabolites for Arthropod Pest Management

“…Methods for producing HtA using a recombinant strain of Pichia pastoris and fermentation in a bioreactor with a yield of 80 mg/L were developed. This allowed a better characterization to be achieved for this protein: its molecular weight (15.3 kDa), stability (high resistance to proteases and temperature), toxicity to Sf9 cells, and insecticidal activity against G. mellonella larvae were determined [ 230 ]. An analog of HtA, anisoplin, was found in M. anisopliae [ 231 ].…”

Trends and Applications of Omics Technologies to Functional Characterisation of Enzymes and Protein Metabolites Produced by Fungi

“…Olombrada et al [ 59 ] reviewed the potential biotechnological applications of these highly specific enzymes. Research completed by Li and Xia [ 60 ] showed the efficacy of ribotoxins as pest control agents either alone or conjugated with other compounds. This was used in experiments involving a recombinant form of the insecticidal ribotoxin hirsutellin A (HtA) produced from Pichia pastoris .…”

Promising approaches to the search for fungal metabolites for management of arthropod pests

“…Разработан способ получения ХтА с использованием рекомбинантного штамма Pichia pastoris и ферментацией в биореакторе с выходом на уровне 80 мг/л. Это позволило полнее охарактеризовать этот белок: определить его молекулярную массу (15.3 кДа), стабильность (показана высокая устойчивость к протеазам и температуре), токсичность для клеток линии Sf9 и инсектицидную активность в отношении личинок G. mellonella (Li, Xia, 2018). Аналог ХтА -анизоплин -был обнаружен у M. anisopliae (Olombrada et al, 2017).…”