Masana Hirai

Overproduction of Geranylgeraniol by Metabolically Engineered Saccharomyces cerevisiae

ABSTRACT (E, E, E)-Geranylgeraniol (GGOH) is a valuable starting material for perfumes and pharmaceutical products. In the yeast Saccharomyces cerevisiae, GGOH is synthesized from the end products of the mevalonate pathway through the sequential reactions of farnesyl diphosphate synthetase (encoded by the ERG20 gene), geranylgeranyl diphosphate synthase (the BTS1 gene), and some endogenous phosphatases. We demonstrated that overexpression of the diacylglycerol diphosphate phosphatase (DPP1) gene could promote GGOH production. We also found that overexpression of a BTS1-DPP1 fusion gene was more efficient for producing GGOH than coexpression of these genes separately. Overexpression of the hydroxymethylglutaryl-coenzyme A reductase (HMG1) gene, which encodes the major rate-limiting enzyme of the mevalonate pathway, resulted in overproduction of squalene (191.9 mg liter−1) rather than GGOH (0.2 mg liter−1) in test tube cultures. Coexpression of the BTS1-DPP1 fusion gene along with the HMG1 gene partially redirected the metabolic flux from squalene to GGOH. Additional expression of a BTS1-ERG20 fusion gene resulted in an almost complete shift of the flux to GGOH production (228.8 mg liter−1 GGOH and 6.5 mg liter−1 squalene). Finally, we constructed a diploid prototrophic strain coexpressing the HMG1, BTS1-DPP1, and BTS1-ERG20 genes from multicopy integration vectors. This strain attained 3.31 g liter−1 GGOH production in a 10-liter jar fermentor with gradual feeding of a mixed glucose and ethanol solution. The use of bifunctional fusion genes such as the BTS1-DPP1 and ERG20-BTS1 genes that code sequential enzymes in the metabolic pathway was an effective method for metabolic engineering.

Transgenic sweet potato expressing thionin from barley gives resistance to black rot disease caused by Ceratocystis fimbriata in leaves and storage roots

Black rot of sweet potato caused by pathogenic fungus Ceratocystis fimbriata severely deteriorates both growth of plants and post-harvest storage. Antimicrobial peptides from various organisms have broad range activities of killing bacteria, mycobacteria, and fungi. Plant thionin peptide exhibited anti-fungal activity against C. fimbriata. A gene for barley α-hordothionin (αHT) was placed downstream of a strong constitutive promoter of E12Ω or the promoter of a sweet potato gene for β-amylase of storage roots, and introduced into sweet potato commercial cultivar Kokei No. 14. Transgenic E12Ω:αHT plants showed high-level expression of αHT mRNA in both leaves and storage roots. Transgenic β-Amy:αHT plants showed sucrose-inducible expression of αHT mRNA in leaves, in addition to expression in storage roots. Leaves of E12Ω:αHT plants exhibited reduced yellowing upon infection by C. fimbriata compared to leaves of non-transgenic Kokei No. 14, although the level of resistance was weaker than resistance cultivar Tamayutaka. Storage roots of both E12Ω:αHT and β-Amy:αHT plants exhibited reduced lesion areas around the site inoculated with C. fimbriata spores compared to Kokei No. 14, and some of the transgenic lines showed resistance level similar to Tamayutaka. Growth of plants and production of storage roots of these transgenic plants were not significantly different from non-transgenic plants. These results highlight the usefulness of transgenic sweet potato expressing antimicrobial peptide to reduce damages of sweet potato from the black rot disease and to reduce the use of agricultural chemicals.

Efficient Production of Artificially Designed Gelatins with a Bacillus brevis System

ABSTRACT Artificially designed gelatins comprising tandemly repeated 30-amino-acid peptide units derived from human αI collagen were successfully produced with a Bacillus brevis system. The DNA encoding the peptide unit was synthesized by taking into consideration the codon usage of the host cells, but no clones having a tandemly repeated gene were obtained through the above-mentioned strategy. Minirepeat genes could be selected in vivo from a mixture of every possible sequence encoding an artificial gelatin by randomly ligating the mixed sequence unit and transforming it intoEscherichia coli. Larger repeat genes constructed by connecting minirepeat genes obtained by in vivo selection were also stable in the expression host cells. Gelatins derived from the eight-unit and six-unit repeat genes were extracellularly produced at the level of 0.5 g/liter and easily purified by ammonium sulfate fractionation and anion-exchange chromatography. The purified artificial gelatins had the predicted N-terminal sequences and amino acid compositions and a solgel property similar to that of the native gelatin. These results suggest that the selection of a repeat unit sequence stable in an expression host is a shortcut for the efficient production of repetitive proteins and that it can conveniently be achieved by the in vivo selection method. This study revealed the possible industrial application of artificially designed repetitive proteins.

Extracellular production of an intact and biologically active human growth hormone by the Bacillus brevis system

The characteristic features of the Bacillus brevis system are very high productivity of heterologous proteins and very low extracellular protease activity. However, degradation of some heterologous proteins, especially mammalian proteins, can be observed and resulted in a lowering of protein productivity. By using a mutant expressing low levels of proteases and the addition of EDTA to the medium, intact human growth hormone (hGH) was successfully produced with the B. brevis system. Signal peptide modification with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a twelve-fold increase in hGH production. The hGH yield was further elevated to 240 mg L−1 by optimization of culture conditions. Thus, biologically active and mature hGH can be efficiently produced directly in the medium with the B. brevis system.

THERMOPHILIC FUNGAL PROTEIN DISULFIDE ISOMERASE

Publisher Summary For the study the authors have isolated and characterized a thermostable protein disulfide isomerase (PDI) from a thermophilic fungus, Humicola insolens . The cDNA encoding the fungal PDI has been cloned and expressed in Bacillus brevis. PDIs from vertebrates and yeast are relatively heat labile. Even in the case of an algal enzyme that is most stable of the known PDIs, the stability against heat is not enough for industrial use of the enzyme. Hence, there is continuing interest in finding new, stable PDIs. Refolding activity using scrambled ribonuclease (RNase) as a substrate is discussed. The fungal PDI cDNA is expressed in a heterologous protein production system using B. brevis as a host. In the purification process, anion-exchange chromatography, lectin affinity chromatography, and high-performance liquid chromatography are discussed. The chapter also presents the data for purification of PDI from the fungus, Humicola insolens . To amplify the DNA fragment around the N-terminal consensus region of fungal PDI, two oligonucleotides corresponding to the amino acid sequence are synthesized and used as primers for a reverse transcriptase-mediated polymerase chain reaction (RT-PCR).

Isolation of a protease-deficient mutant of Bacillus brevis and efficient secretion of a fungal protein disulfide isomerase by the mutant

The efficient production of a thermostable protein disulfide isomerase (PDI) was successfully achieved using the newly isolated protease-deficient mutant, Bacillus brevis 31-OK. Extracellular protease (exoprotease) activity was about a quarter of that in the parent, and the mutant was deficient in at least one of the major exoproteases. The cDNA encoding the fungal PDI was inserted downstream of the signal peptide-encoding region in an expression-secretion vector for B. brevis. Efficient production of PDI was feasible using B. brevis 31-OK as a host and modified signal sequences composed of three leucine residues inserted in the hydrophobic region of the MWP (middle wall protein) signal sequence. The maximal secretion of PDI into the culture medium was 1.1 g/l, which is about twice that by the parent strain and fifty times greater than the amount of rat and murine PDIs produced by Escherichia coli. The enzymatic properties such as the specific activity and thermal stability of the recombinant PDI are similar to those of natural PDI derived from Humicola insolens mycelia. B. brevis 31-OK was able to maintain its exoprotease activity at a low level throughout the cultivation and is considered to be useful host for production of a protease-sensitive protein and for increase of protein productivity due to stable accumulation.

Effect of plasma polymerization conditions on gas permeability of plasma polymerized films.

複合膜の気体透過特性に及ぼすプラズマ重合条件の影響を把握するため, モノマーとしてヘキサメチルジシロキサン (M2) 及びオクタメチルシクロテトラシロキサン (D4) を用い, 重合位置, モノマー圧力, ラジオ波入力, 重合時間を変化させ, プラズマ重合膜を多孔質膜上に形成した. 重合条件とプラズマ重合膜の堆積速度及び複合膜の酸素-窒素透過特性との関係を調べた. 重合位置が電極に近いほど分離性を発現するのに必要な重合時間が短くて済むことが分かった. M2の重合速度は, 入力/圧力値の増大とともに増加した. D4にはその関係はみられず, 圧力が高い時には入力が律速であるものの, 低圧時には入力に無関係でありモノマー供給が律速となることが示唆された. 過剰なエネルギーが供給される条件では重合膜の堆積速度及び膜の分離性の低下がみられた. 重合速度の大きい条件で作製した膜は分離性が高いことが分かった.