Construction of RNA silencing system of Penicillium brevicompactum and genetic manipulation of the regulator pbpcz in mycophenolic acid production.

Penicillium brevicompactum is a critical industrial strain for the production of mycophenolic acid (MPA). However, the genetic background of Penicillium brevicompactum is unclear, and there are few tools available for genetic manipulation. To investigate its gene function, we first verified the feasibility of a pair of citrate synthase promoter (Pcit) and terminator (Tcit) from P. brevicompactum by constructing a fluorescent expression cassette. Based on this, an RNAi vector was designed and constructed with reverse promoters. This study focused on the functional investigation of the pbpcz gene in P. brevicompactum, a regulator belonging to the Zn(II)2Cys6 family. RNAi was used to silence the pbpcz gene, providing a valuable tool for genetic studies in P. brevicompactum. After seven days, we observed differences in the number of spores between different phenotypes strains of pbpcz gene. Compared to the wild-type strain (WT), the spore yield of the pbpcz gene silencing mutant (M2) was only 51.4 %, while that of the pbpcz gene overexpressed mutant (SE4) was increased by 50 %. Expression levels of the three genes (brlA, abaA, and wetA) comprising conidia's central regulatory pathway were significantly reduced in the pbpcz gene silencing mutant, while fluorescence localization showed that PbPCZ protein was mainly distributed in spores. The results indicated that the pbpcz gene is critical for conidia and asexual development of P. brevicompactum. In addition, overexpressing the pbpcz gene resulted in a 30.3 % increase in MPA production compared to the wild type, with a final yield of 3.57 g/L. These results provide evidence that PbPCZ acts as a positive regulator in P. brevicompactum, controlling MPA production and regulating conidia and asexual development.

High-level expression of a ß-mannanase (manB) in Pichia pastoris GS115 for mannose production with Penicillium brevicompactum fermentation pretreatment of soybean meal.

An endo-1,4-ß-mannanase gene (manB) from a Bacillus pumilus Nsic-2 grown in a stinky tofu emulsion was cloned and expressed in Pichia pastoris GS115. After characterized, the endo-1,4-ß-mannanase (manB) show maximum activity at pH 6.0 and 50 °C with LBG as substrate and perform high stability at a range of pH 6-8. After applying for a shake flask fermentation, the specific activity of manB reached 3462 U/mg. To produce mannose, the soybean meal (SBM) was pretreated by biological fermentation for 11 days with Penicillium brevicompactum, and then hydrolyzed by manB. As a result, mannose yield reached 3.58 g per 1 kg SBM which indicated that 0.358% SBM was converted into mannose after hydrolyzation, and mean a total 20% mannan of SBM converting into mannose, while the control group demonstrated only 1.78% conversion. An effective ß-mannanase for the bioconversion of mannan-rich biomasses and an efficient method to produce mannose with soybean meal were introduced.

Mn2+ modulates the production of mycophenolic acid in Penicillium brevicompactum NRRL864 via reactive oxygen species signaling and the investigation of pb-pho.

Although Penicillium brevicompactum is a widely used commercial strain for the manufacture of mycophenolic acid, there are few findings of Ca2+, Mn2+, and reactive oxygen species (ROS) interaction. Metal ions play a crucial role in physiological metabolism. Calcium, as the important second messenger, influences fungus growth, virulence, and stress responses. The concentration of cytosolic Ca2+ was influenced by the Mn2+, which demonstrated the crosstalk between calcium and manganese. In the previous study, the crosstalk between calcium and ROS has been discovered and verified, which modified the secondary metabolism and enhanced the yield of MPA (Mycophenolic Acid). A higher concentration of Mn2+ in the fermentation broth causes an increase in cytoplasmic Ca2+ and ROS (Reactive Oxygen Species), enhancing the yield of MPA by about 20 % higher and disclosing the cascade regulation with the Mn2+, Ca2+, and ROS. To be more specific, the intracellular concentration of ROS at 6 mM Mn2+ is about 1.5 times higher than that at 0.6 mM. Furthermore, we identify an Mn2+ transport protein, designated as Pb-PHO, which shows 71.2 % identity to the inorganic phosphate transporter PHO84 (Q0CBJ6) from Aspergillus terreus. At the same time, the △Pb-pho exhibits damage to the cell wall integrity, while the OE-pho displays a more normal phenotype at high osmotic stress. The high-affinity Ca2+ channel, Pb-CCH, is examined via knockdown to demonstrate the crosstalk between Mn2+ and Ca2+. The results show that the addition of Mn2+ remits the negative influence of pb-cch knockdown and the addition of Ca2+ remits the negative influence of pb-pho knockdown, demonstrating the relationship between cytoplasmic Mn2+ and Ca2+. Taken together, our results demonstrate the mechanism of a manganese-induced cascade of manganese-calcium-ROS and reveal a signal pathway-relative method to illustrate the manganese-induced increase of MPA production in Penicillium brevicompactum. Furthermore, we discover and identify an Mn2+ transport protein, Pb-PHO, which is subcellular localized at the plasma membrane and proved to affect the cell wall integrity.

Synergistic Regulation of Metabolism by Ca2+/Reactive Oxygen Species in Penicillium brevicompactum Improves Production of Mycophenolic Acid and Investigation of the Ca2+ Channel.

Although Penicillium brevicompactum is a very important industrial strain for mycophenolic acid production, there are no reports on Ca2+/reactive oxygen species (ROS) synergistic regulation and calcium channels, Cch-pb. This study initially intensified the concentration of the intracellular Ca2+ in the high yielding mycophenolic acid producing strain NRRL864 to explore the physiological role of intracellular redox state in metabolic regulation by Penicillium brevicompactum. The addition of Ca2+ in the media caused an increase of intracellular Ca2+, which was accompanied by a strong increase, 1.5 times, in the higher intracellular ROS concentration. In addition, the more intensive ROS sparked the production of an unreported pigment and increase in mycophenolic acid production. Furthermore, the Ca2+ channel, the homologous gene of Cch1, Cch-pb, was investigated to verify the relationship between Ca2+ and the intracellular ROS. The Vitreoscilla hemoglobin was overexpressed, which was bacterial hemoglobin from Vitreoscilla, reducing the intracellular ROS concentration to verify the relationship between the redox state and the yield of mycophenolic acid. The strain pb-VGB expressed the Vitreoscilla hemoglobin exhibited a lower intracellular ROS concentration, 30% lower, and decreased the yield of mycophenolic acid as 10% lower at the same time. Subsequently, with the NRRL864 fermented under 1.7 and 28 mM Ca2+, the [NADH]/[NAD+] ratios were detected and the higher [NADH]/[NAD+] ratios (4 times higher with 28 mM) meant a more robust primary metabolism which provided more precursors to produce the pigment and the mycophenolic acid. Finally, the 10 times higher calcium addition in the media resulted in 25% enhanced mycophenolic acid production to 6.7 g/L and induced pigment synthesis in NRRL864.