Disruption of vacuolar protein sorting receptor gene Poxvps10 improves cellulolytic enzyme production by Penicillium oxalicum.

Penicillium oxalicum can secrete numerous of plant biomass-degrading enzymes, but limited information is available regarding the mechanisms associated with their secretion. In the Golgi-to-vacuole pathway, the type I transmembrane receptor Vps10p is involved in the sorting of the soluble vacuolar proteins and can also target recombinant and aberrant proteins from the Golgi to the vacuole for degradation. Here, we used the combination of phenotypic characterization and comparative secretome analysis to explore the effect of disruption of the vps10 gene in P. oxalicum (Poxvps10) on endogenous cellulolytic enzyme secretion. The study found that PoxVps10p is required for the targeting and delivery of vacuolar PoxCpyA to the vacuole in P. oxalicum. Poxvps10p deletion enhances extracellular protein and cellulase production by P. oxalicum when the cells are grown on a cellulosic substrate (wheat bran and Avicel). Furthermore, secretome analysis revealed higher relative amount of cellulases, lytic polysaccharide monooxygenase and post-translational modification-related proteins in the ΔPoxvps10 mutant than in the wild-type (WT) strain, which may explain the higher cellulase production by the ΔPoxvps10 than the WT strain. This study thus provides a new target for manipulating the secretory pathway to enhance the cellulolytic enzyme production.

Engineering Pichia pastoris to improve S-adenosyl- l-methionine production using systems metabolic strategies.

S-adenosyl-l-methionine (SAM) is a highly valued chemical that can be used as a dietary supplement and has been used to treat depression, osteoarthritis, and liver problems as well. We adopted systems metabolic engineering strategies to improve SAM production in a high-producing strain (GS115/DS56). First, the cystathionine ß-synthase gene CYS4 was downregulated using a weak promoter PG12 to reduce the removal of homocysteine from SAM cycle, thus leading to a 48.8% increase in the SAM titer (1.68 g/L) from the strain G12-CBS, while preventing cysteine auxotrophy induced by deletion of this essential gene. Subsequently, the SAM titer of G12-CBS was improved to 13.01 g/L in 15-L fed-batch fermentation using the optimal l-methionine feeding strategy. Finally, based on comparative transcriptomics, five genes were chosen and overexpressed for further enhancement of SAM production. Among them, GDH2 and ACS2 exhibited positive effects, and the additional overexpression of GDH2 led to a 52.3% increase of titer (2.71 g/L) in shake flask culture. Therefore, the engineered Pichia pastoris strains can be utilized in industrial production of SAM using a simple and cost-effective process, and these approaches could be employed for improving the production of other chemicals by P. pastoris.

Deletion of ligD significantly improves gene targeting frequency in the lignocellulolytic filamentous fungus Penicillium oxalicum.

To improve the gene targeting frequency (GTF) in the lignocellulolytic filamentous fungus Penicillium oxalicum HP7-1, the non-homologous end-joining (NHEJ) gene ligD was deleted. The obtained PoligD deletion mutant ΔPoligD showed no apparent defect in cellulase production, growth rate, and sensitivity towards osmotic stress and mutagen ethyl methanesulphonate (EMS), while increased sensitivity to high concentrations of methyl methanesulfonate (MMS). Deletion of PoligD gene resulted in significantly increased GTFs at three different loci in P. oxalicum, which are even higher than those in Poku70 deletion mutant. The GTF in ΔPoligD at PoargB (reached 97 %) and PoagaA (reached 90 %) loci increased 5.1- and 1.2-fold compared with that in wild-type strain (WT), while at the Podpp4 locus GTF was up to 27 % in ΔPoligD but close to 0 % in WT, with 0.5 kb homologous flanking regions. Furthermore, the argB and agaA nutritional selection in P. oxalicum was demonstrated and the PoargB and PoagaA genes could be used as selective markers in this fungus. Thus, the PoligD deletion mutant can be an important tool for the functional analysis of genes in P. oxalicum.

Expression, purification, and characterization of a recombinant methionine adenosyltransferase pDS16 in Pichia pastoris.

Methionine adenosyltransferase (MAT, EC2.5.1.6) catalyzes the synthesis of S-adenosylmethionine (SAM) using L-methionine and adenosine triphosphate (ATP) as substrates. The mutant MAT pDS16 was obtained through DNA shuffling previously in our lab. Overexpression of pDS16 in Pichia pastoris led to about 65 % increase of MAT activity and SAM accumulation, compared with the strain overexpressing Saccharomyces cerevisiae MAT gene SAM2. Different strategies were tested to facilitate the expression and purification of pDS16. However, addition of the hexahistidine tag to pDS16 was shown to decrease the enzyme activity, and the yeast α-factor signal sequence could not effectivley direct the secretion of pDS16. The intracellular pDS16 was purified by a simple two-step procedure combining an ion exchange and hydrophobic interaction chromatography. Protein purity was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 93%, with the specific activity of 1.828 U/mg. Two-dimensional electrophoresis revealed pI of ∼5.5. The purified enzyme followed Michaelis kinetics with a Km of 1.72 and 0.85 mM, and Vmax of 1.54 and 1.15 µmol/min/mg for ATP and L-methionine, respectively. pDS16 exhibited optimal activity at pH 8.5 and 45 °C with the requirement of divalent cation Mg(2+) and was slightly stimulated by the monovalent cation K(+). It showed an improved thermostability, about 50% of the enzyme activity was retained even after preincubation at 50 °C for 2 h.

GAP promoter library for fine-tuning of gene expression in Pichia pastoris.

A library of engineered promoters of various strengths is a useful genetic tool that enables the fine-tuning and precise control of gene expression across a continuum of broad expression levels. The methylotrophic yeast Pichia pastoris is a well-established expression host with a large academic and industrial user base. To facilitate manipulation of gene expression spanning a wide dynamic range in P. pastoris, we created a functional promoter library through mutagenesis of the constitutive GAP promoter. Using yeast-enhanced green fluorescent protein (yEGFP) as the reporter, 33 mutants were chosen to form the functional promoter library. The 33 mutants spanned an activity range between ∼0.6% and 19.6-fold of the wild-type promoter activity with an almost linear fluorescence intensity distribution. After an extensive characterization of the library, the broader applicability of the results obtained with the yEGFP reporter was confirmed using two additional reporters (ß-galactosidase and methionine adenosyltransferase [MAT]) at the transcription and enzyme activity levels. Furthermore, the utility of the promoter library was tested by investigating the influence of heterologous MAT gene expression levels on cell growth and S-adenosylmethionine (SAM) production. The extensive characterization of the promoter strength enabled identification of the optimal MAT activity (around 1.05 U/mg of protein) to obtain maximal volumetric SAM production. The promoter library permits precise control of gene expression and quantitative assessment that correlates gene expression level with physiologic parameters. Thus, it is a useful toolbox for both basic and applied research in P. pastoris.

Cloning and characterization of Kluyveromyces marxianus Hog1 gene.

The mitogen-activated protein kinase Hog1 gene (Kmhog1) was isolated from Kluyveromyces marxianus strain NBRC 1777 by degenerate PCR and genome walking, and then disrupted to construct a mutant strain hog1∆. The mutant was now more sensitive to acetic acid and its growth was nearly completely inhibited by 0.5 M NaCl (97%) and 10 mM H(2)O(2) (93%) as compared with the wild-type cells. However, neither strain grew at 47°C. Kmhog1 may thus be required for adaptation to acetic acid, osmotic, and oxidative stress but is not involved in thermotolerance.