Date Published: May 2, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Ruijie Gao, Huaxiang Deng, Zhengbing Guan, Xiangru Liao, Yujie Cai.
Shiraia bambusicola is an important and valuable macrofungus and hypocrellins are its main secondary metabolites which have been widely applied in many medical fields. However, during SSF process of this filamentous fungus, use ratio of corn substrate and dissolved oxygen supply are two main limiting factors, which influence production cost, yield and product quality. To solve these problems, overexpressions of amy365-1 and vgb in S. bambusicola were investigated and three overexpression transformants were constructed. Results demonstrated that expressions and coexpression of AMY365-1 and VHb not only increased the productions of biomass, amylase, hypocrellin, but also up-regulated relative expression levels of four central carbon metabolism genes (pdc, ald, acs, acc) and seven hypocrellin biosynthesis genes (fad, mono, zftf, omef, msf, pks, mco). Furthermore, expression of VHb decreased SSF period. When amy365-1 and vgb were coexpressed, relative expression levels of zftf and pks reached their highest levels at 72 h under liquid fermentation, hypocrellin production reached the highest level 75.85 mg/gds which was 2.99-fold compared with wild type strain within 11 days under SSF, and residual starch of solid substrates was decreased from 35.47 to 14.57%.
Shiraia bambusicola is an important and valuable macrofungus in the medical and food industries. Hypocrellins are the main secondary metabolites of S. bambusicola, including hypocrellin A, hypocrellin B, hypocrellin C and hypocrellin D (Fang et al. 2006), a type of perylenequinone. Under illumination, hypocrellins react with oxygen and generate reactive oxygen species (ROS), including singlet oxygen (1O2) and the superoxide radical. Abundant ROS cause cellular oxidative stress and kill cells by damaging cellular macromolecules, including lipids, DNA and proteins (Trachootham et al. 2008). Based on this characteristic, hypocrellins have been widely applied in many medical fields, such as photodynamic tumor therapy and antivirus treatments (Gao et al. 2012; Jin et al. 2013). They also have been used to treat skin diseases, gastric diseases and some vascular diseases. In addition to benefiting the pharmaceutical industry, hypocrellins also have extensive potential applications in the agricultural, cosmetic, food, and feed industries (Shen et al. 2012; Su et al. 2010, 2011). So far, yield of hypocrellin from natural extraction was too low to meet it current medical demand. Therefore, improvement of hypocrellin production was needed to meet its demand.
In order to increase the use ratio of corn substrate and improve the dissolved oxygen supply, α-amylase gene amy365-1 and Vitreoscilla hemoglobin gene vgb were overexpressed, and three plasmids which included Phygamy365-1, Phygvgb and PhygPgpdAamy365-1-vgb were constructed and three transformants were obtained.
Shiraia bambusicola has demonstrated recently its potential for hypocrellin production in fermentation systems and particularly in SSF (Liang et al. 2009). Furthermore, corn was found to be the best substrate after evaluating eight kinds of agro-industrial crops and residues (Cai et al. 2010). Starch is the main constituent in corn, and it also is the main carbon source when hypocrellin was produced under SSF with S. bambusicola. In addition, it is generally assumed that there is a limitation in the oxygen supply to the cells that are in close contact with the substrate in solid-state cultures with filamentous fungi (Oostra et al. 2001; Rahardjo et al. 2005). The use ratio of corn substrate and oxygen supply were two key aspects which influenced the yield of hypocrellin under SSF. In order to produce hypocrellin at an industrial scale in the future, specific α-amylase AMY365-1 and VHb were selected and overexpressed in Shiraia sp. SUPER-H168. Three transformants included Phygamy365-1 strain, Phygvgb strain and PhygPgpdAamy365-1-vgb strain were constructed.