Effective pH pretreatment and cell disruption method for real-time intracellular enzyme activity assay of a marine fungus covered with pigments

<p>Filamentous fungi are capable producers of many bioactive compounds, and real-time intracellular enzyme activity assay is an essential guidance for their bioprocess developments. However, there are many difficulties in preparing homogenate for enzyme activity assay, such as disrupting fungal cell with complicated cellular structure and solid cell wall, removing abundant extracellular metabolites accumulating on mycelia, and so on. <i>Halorosellinia</i> sp. (No. 1403) was a marine-derived filamentous fungus producing a potential antitumor compound 1403C, and the deep red pigments (with main component of 1403C) covering on its mycelia showed strong absorption in a wide range, which critically affected the measurement of many enzyme activities. In this study, we developed an effective pH pretreatment and cell disruption method to prepare homogenate for enzyme activity assay. When mycelia were washed by the solution with pH 5.0 for 3 min, most pigments could be removed without severe loss on enzyme activities. Afterward, grinding with mini bead for 15 min with alternating cooling could effectively disrupt both cell wall and mitochondrial membrane. These methods have been successfully applied on real-time intracellular enzyme activity assay of <i>Halorosellinia</i> sp. (No. 1403) and can offer enlightenment for other filamentous fungi with similar problems.</p

Bioprocess exploration for thermostable <i>α</i>-amylase production of a deep-sea thermophile <i>Geobacillus</i> sp. in high-temperature bioreactor

<p><i>Geobacillus</i> sp. 4j, a deep-sea high-salt thermophile, was found to produce thermostable α-amylase. In this work, culture medium and conditions were first optimized to enhance the production of thermostable α-amylase by statistical methodologies. The resulting extracellular production was increased by five times and reached 6.40 U/ml. Then, a high-temperature batch culture of the thermophile in a 15 l in-house-designed bioreactor was studied. The results showed that a relatively high dissolved oxygen (600 rpm and 15 l/min) and culture temperature of 60°C facilitated both cell growth and α-amylase production. Thus, an efficient fermentation process was established with initial medium of pH 6.0, culture temperature of 60°C, and dissolved oxygen above 20%. It gave an α-amylase production of 79 U/ml and productivity of 19804 U/l·hr, which were 10.8 and 208 times higher than those in shake flask, respectively. This work is useful for deep-sea high-salt thermophile culture, where efforts are lacking presently.</p

MOESM1 of Improved methanol-derived lovastatin production through enhancement of the biosynthetic pathway and intracellular lovastatin efflux in methylotrophic yeast

Additional file 1. Additional figures and tables

Peroxisome-targeted and tandem repeat multimer expressions of human antimicrobial peptide LL37 in <i>Pichia pastoris</i>

<p>Although the human antimicrobial peptide LL37 has a broad spectrum of antimicrobial activities, it easily damages host cells following heterologous expressions. This study attempted two strategies to alleviate its damage to host cells when expressed in <i>Pichia pastoris</i> using the <i>AOX1</i> promoter. Tandem repeat multimers of LL37 were first designed, and secretion expression strains GS115-9K-(DPLL37DP)_<i>n</i> (<i>n</i> = 2, 4, 6 and 8) containing different copies of the LL37 gene were constructed. However, LL37 tandems still killed the cells after 96 hr of induction. Subsequently, peroxisome-targeted expression was performed by adding a peroxisomal targeting signal 1 (SKL) at the C-terminus of LL37. The LL37 expression strain GS115-3.5K-LL37-SKL showed no significant inhibition in the cells after induction. Antibacterial activity assays showed that the recombinant LL37 expressed in peroxisomes had good antimicrobial activities. Then, a strain GS115-3.5K-LL37-GFP-SKL producing LL37, green fluorescent protein, and SKL fusion proteins was constructed, and the fusion protein was confirmed to be targeting the peroxisomes. However, protein extraction analysis indicated that most of the fusion proteins were still located in the cell debris after cell disruption, and further studies are required to extract more proteins from the peroxisome membrane.</p

Kinase Screening in <i>Pichia pastoris</i> Identified Promising Targets Involved in Cell Growth and <i>Alcohol Oxidase 1</i> Promoter (P<i>_AOX1</i>) Regulation

<div><p>As one of the most commonly used eukaryotic recombinant protein expression systems, <i>P</i>. <i>pastoris</i> relies heavily on the <i>AOX1</i> promoter (P_<i>AOX1</i>), which is strongly induced by methanol but strictly repressed by glycerol and glucose. However, the complicated signaling pathways involved in P_<i>AOX1</i> regulation when supplemented with different carbon sources are poorly understood. Here we constructed a kinase deletion library in <i>P</i>. <i>pastoris</i> and identified 27 mutants which showed peculiar phenotypes in cell growth or P_<i>AOX1</i> regulation. We analyzed both annotations and possible functions of these 27 targets, and then focused on the MAP kinase Hog1. In order to locate its potential downstream components, we performed the phosphoproteome analysis on glycerol cultured WT and Δ<i>hog1</i> strains and identified 157 differentially phosphorylated proteins. Our results identified important kinases involved in <i>P</i>. <i>pastoris</i> cell growth and P_<i>AOX1</i> regulation, which could serve as valuable targets for further mechanistic studies.</p></div

Kinase Screening in <i>Pichia pastoris</i> Identified Promising Targets Involved in Cell Growth and <i>Alcohol Oxidase 1</i> Promoter (P<i>_AOX1</i>) Regulation - Fig 2

<p>GFP reporter assay showing the AOX1 promoter activity of several kinase mutants in methanol (A) or glycerol (B). A WT strain is used as control here.</p

Summary of kinase genes specifically involved in methanol supported cell growth.

<p>Summary of kinase genes specifically involved in methanol supported cell growth.</p

A cluster of orthologous groups of proteins analysis of the phosphoproteins in H-G, W-G and W-M.

<p>H-G: glycerol cultured Δ<i>hog1</i> strain; W-G: glycerol cultured WT strain; W-M: methanol cultured WT strain.</p

The growth rates (shown by spotting assay) and Aox activities (shown by colorimetrical assay) of the 27 knockouts.

<p>D: glucose; G: glycerol; M: methanol.</p

The characteristics of the identified phosphoproteins.

<p>The characteristics of the identified phosphoproteins.</p