Proteomic analysis on Aspergillus strains that are useful for industrial enzyme production.

A simple intracellular proteomic study was conducted to investigate the biological activities of Aspergillus niger during industrial enzyme production. A strain actively secreting a heterologous enzyme was compared to a reference strain. In total, 1824 spots on 2-D gels were analyzed using MALDI-TOF MS, yielding 343 proteins. The elevated levels of UPR components, BipA, PDI, and calnexin, and proteins related to ERAD and ROS reduction, were observed in the enzyme-producer. The results suggest the occurrence of these responses in the enzyme-producers. Major glycolytic enzymes, Fba1, EnoA, and GpdA, were abundant but at a reduced level relative to the reference, indicating a potential repression of the glycolytic pathway. Interestingly, it was observed that a portion of over-expressed heterologous enzyme accumulated inside the cells and digested during fermentation, suggesting the secretion capacity of the strain was not enough for completing secretion. Newly identified conserved-proteins, likely in signal transduction, and other proteins were also investigated. Abbreviations: 2-D: two-dimensional; UPR: unfolded protein response; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; PDI: protein disulfide-isomerase; ROS: reactive oxygen species; RESS: Repression under Secretion Stress; CSAP: Conserved Small Abundant Protein; TCTP: translationally controlled tumor protein.

Engineering the expression system for Komagataella phaffii (Pichia pastoris): an attempt to develop a methanol-free expression system.

The construction of a methanol-free expression system of Komagataella phaffii (Pichia pastoris) was attempted by engineering a strong methanol-inducible DAS1 promoter using Citrobacter braakii phytase production as a model case. Constitutive expression of KpTRM1, formerly PRM1-a positive transcription regulator for methanol-utilization (MUT) genes of K. phaffii,was demonstrated to produce phytase without addition of methanol, especially when a DAS1 promoter was used but not an AOX1 promoter. Another positive regulator, Mxr1p, did not have the same effect on the DAS1 promoter, while it was more effective than KpTrmp1 on the AOX1 promoter. Removing a potential upstream repression sequence (URS) and multiplying UAS1DAS1 in the DAS1 promoter significantly enhanced the yield of C. braakii phytase with methanol-feeding, which surpassed the native AOX1 promoter by 80%. However, multiplying UAS1DAS1 did not affect the yield of methanol-free expression by constitutive KpTrm1p. Another important region to enhance the effect of KpTrm1p under a methanol-free condition was identified in the DAS1 promoter, and was termed ESPDAS1. Nevertheless, methanol-free phytase production using an engineered DAS1 promoter outperformed phytase production with the GAP promoter by 25%. Difference in regulation by known transcription factors on the AOX1 promoter and the DAS1 promoter was also illustrated.

Identifying the inserted locus of randomly integrated expression plasmids by whole-genome sequencing of Aspergillus strains.

Whole-genome sequencing was conducted on two Aspergillus oryzae strains used for the manufacturing of food enzymes, Acrylaway® and Shearzyme®, with the aim of identifying the inserted locus of randomly integrated expression plasmid and obtaining flanking sequences for safety assessment. Illumina paired-end sequencing was employed, and the obtained reads were mapped to two references: the public genome sequence of Aspergillus oryzae RIB40 and the in-house sequence of the used expression plasmid. Introducing the concept of linking-reads, one locus for each was successfully identified as the integrated site. In the case of Acrylaway®, the obtained sequences suggested that the expression plasmid had been integrated as multiple copies in tandem form. In the case of Shearzyme®, however, information on one edge of the insert was missing, which required extra polymerase chain reaction (PCR) cloning for safety assessment. A 4-kb deletion was detected at the integrated site. There was also evidence of rearrangement occurring in Shearzyme® strain.

Characterization of two fungal lipoxygenases expressed in Aspergillus oryzae.

Two fungal lipoxygenase genes were cloned from a rice pathogen, Magnaporthe salvinii, and the take-all fungus, Gaeumannomyces graminis var. tritici, and successfully expressed in Aspergillus oryzae in secreted form. The lipoxygenases expressed, termed MLOX and GLOX, were purified and characterized to evaluate suitability for industrial applications. Both enzymes were active broadly at pH 4-11 and had optimum temperatures around 60 °C, but they were largely different in substrate specificity. Where MLOX was active broadly on arachidonic acid, EPA and DHA, and even on derivatives of fatty acids, such as methyl linoleate or linoleoyl alcohol, GLOX was more specific to linoleic acid and linolenic acid. The most remarkable difference between the two fungal LOXs was the positional and stereo-specificity of oxygenation reactions on polyunsaturated fatty acids. When using linoleic acid as the substrate, the product of MLOX was 9S-hydroperoxy-(E,Z)-octadecadienoic acid (9S(E,Z)-HPODE), on the other hand, the product of GLOX was 13R(E,Z)-HPODE. The enzymes were evaluated for a couple of potential applications and found to be effective on bleaching colored compounds such as carotenoids.

Codon optimization increases steady-state mRNA levels in Aspergillus oryzae heterologous gene expression.

We investigated the effect of codon optimization on the expression levels of heterologous proteins in Aspergillus oryzae, using the mite allergen Der f 7 as a model protein. A codon-optimized Der f 7 gene was synthesized according to the frequency of codon usage in A. oryzae by recursive PCR. Both native and optimized Der f 7 genes were expressed under the control of a high-level-expression promoter with their own signal peptides or in a fusion construct with A. oryzae glucoamylase (GlaA). Codon optimization markedly increased protein and mRNA production levels in both nonfused and GlaA-fused Der f 7 constructs. For constructs with native codons, analysis by 3' rapid amplification of cDNA ends revealed that poly(A) tracts tended to be added within the coding region, producing aberrant mRNAs that lack a termination codon. Insertion of a termination codon between the carrier GlaA and native Der f 7 proteins in the GlaA fusion construct resulted in increases in mRNA and secreted-carrier-GlaA levels. These results suggested that mRNAs without a termination codon as a result of premature polyadenylation are degraded, possibly through the nonstop mRNA decay pathway. We suggest that codon optimization in A. oryzae results in elimination of cryptic polyadenylation signals in native Der f 7, thereby circumventing the production of truncated transcripts and resulting in an increase in steady-state mRNA levels.

Effects of Choto-san and hooks and stems of Uncaria sinensis on antioxidant enzyme activities in the gerbil brain after transient forebrain ischemia.

Previously, we revealed that oral administrations of Choto-san, a Kampo formula, and the hooks and stems of Uncaria sinensis Haviland (Rubiaceae), a medicinal plant comprising Choto-san, enhanced superoxide anion and hydroxyl radical scavenging activities in the hippocampus, and prevented delayed neuronal death of pyramidal cells in the hippocampal CA1 region in a transient forebrain ischemia gerbil model. In the present study, for the purpose of clarifying whether the endogenous antioxidant enzymes contribute to these mechanisms, we investigated the effects of Choto-san extract (CSE) and Uncaria sinensis extract (USE) on superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities in the brain by using the same experimental model. 1.0% CSE or 3.0% USE were dissolved in water and provided to gerbils ad libitum from 7 days prior to ischemia/reperfusion (i/rp). Seven days of continuous administrations of CSE or USE without i/rp procedure enhanced CAT activity but not SOD and GSH-Px activities in both the hippocampus and cortex. CSE elevated CAT activity in the hippocampus at 7 days and in the cortex at 3h after i/rp. USE raised CAT activity in both the hippocampus and cortex at 3 h and 7 days after i/rp. These results suggest that one of the mechanisms of the protective effects of CSE and USE against transient brain ischemia-induced neuronal damage may be their enhancing effect on CAT activity in the brain.

Isolation of cathepsin B inhibitory peptides, Cabin-A1 and -A2, from a tryptic and chymotryptic hydrolysate of human serum albumin.

Two novel peptides that inhibit cathepsin B were isolated from a tryptic and chymotryptic hydrolysate of human serum albumin, and designated as Cabin-A1 and -A2. Cabin-A1 and -A2 were purified by reversed-phase HPLC and identified as Ser-Leu-His-Thr-Leu-Phe and Phe-Gln-Asn-Ala-Leu, respectively. These peptides correspond to f(65-70) and f(403-407) of human serum albumin. Human albutensin A (Ala-Phe-Lys-Ala-Trp-Ala-Val-Ala-Arg), which corresponds to f(210-218), was also isolated as a potent cathepsin B inhibitor. Synthetic Cabin-A1, -A2, and human albutensin A showed dose-dependent inhibition of cathepsin B, with K(i) values of 2.4, 290, and 3.8 microM, respectively.