Fermentative production of enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis.

BACKGROUND: Linalool, an acyclic monoterpene alcohol, is extensively used in the flavor and fragrance industries and exists as two enantiomers, (S)- and (R)-linalool, which have different odors and biological properties. Linalool extraction from natural plant tissues suffers from low product yield. Although linalool can also be chemically synthesized, its enantioselective production is difficult. Microbial production of terpenes has recently emerged as a novel, environmental-friendly alternative. Stereoselective production can also be achieved using this approach via enzymatic reactions. We previously succeeded in producing enantiopure (S)-linalool using a metabolically engineered Pantoea ananatis, a member of the Enterobacteriaceae family of bacteria, via the heterologous mevalonate pathway with the highest linalool titer ever reported from engineered microbes. RESULTS: Here, we genetically modified a previously developed P. ananatis strain expressing the (S)-linalool synthase (AaLINS) from Actinidia arguta to further improve (S)-linalool production. AaLINS was mostly expressed as an insoluble form in P. ananatis; its soluble expression level was increased by N-terminal fusion of a halophilic ß-lactamase from Chromohalobacter sp. 560 with hexahistidine. Furthermore, in combination with elevation of the precursor supply via the mevalonate pathway, the (S)-linalool titer was increased approximately 1.4-fold (4.7 ± 0.3 g/L) in comparison with the original strain (3.4 ± 0.2 g/L) in test-tube cultivation with an aqueous-organic biphasic fermentation system using isopropyl myristate as the organic solvent for in situ extraction of cytotoxic and semi-volatile (S)-linalool. The most productive strain, IP04S/pBLAAaLINS-ispA*, produced 10.9 g/L of (S)-linalool in "dual-phase" fed-batch fermentation, which was divided into a growth-phase and a subsequent production-phase. Thus far, this is the highest reported titer in the production of not only linalool but also all monoterpenes using microbes. CONCLUSIONS: This study demonstrates the potential of our metabolically engineered P. ananatis strain as a platform for economically feasible (S)-linalool production and provides insights into the stereoselective production of terpenes with high efficiency. This system is an environmentally friendly and economically valuable (S)-linalool production alternative. Mass production of enantiopure (S)-linalool can also lead to accurate assessment of its biological properties by providing an enantiopure substrate for study.

Stereospecific linalool production utilizing two-phase cultivation system in Pantoea ananatis.

Linalool is a monoterpene alcohol, which imparts floral scents to a variety of plants and is extensively used in various kinds of products, such as processed foods and beverages for fragrances and flavors. However, linalool from natural resources is racemate, and production of linalool enantiomers is difficult. To produce stereospecific linalool, we evaluated linalool synthase genes (LINS) from plants, such as Actinidia arguta (AaLINS) and Coriandrum sativum (CsLINS) for (S)-specific LINS and a gram-positive bacterium Streptomyces clavuligerus (ScLINS) for (R)-specific LINS, with Pantoea ananatis strain as the host. Among the 16 LINS examined, AaLINS and ScLINS showed the best (S)-linalool production and (R)-linalool production, respectively, with 100 % enantio excess. Co-expression of the mutated farnesyl diphosphate synthase gene, ispA* (S80 F), from Escherichia coli along with the LINS genes also improved linalool production. In order to prevent volatilization and cell toxicity of linalool, two-phase cultivation with isopropyl myristate was done, which had positive effects on linalool production. The carbon flux to the MVA pathway from glucose was increased by inactivating a membrane-bound glucose dehydrogenase. Overall, 5.60 g/L (S)-linalool and 3.71 g/L (R)-linalool were produced from 60.0 g/L glucose by introduction of AaLINS-ispA* and ScLINS-ispA* in P. ananatis, respectively.

Production of glutamate and stereospecific flavors, (S)-linalool and (+)-valencene, by Synechocystis sp. PCC6803.

Cyanobacteria can grow photoautotrophically, producing a range of substances by absorbing sunlight and utilizing carbon dioxide, and can potentially be used as industrial microbes that have minimal sugar requirements. To evaluate this potential, we explored the possibility of l-glutamate production using the Synechocystis sp. PCC6803. The ybjL gene encoding the putative l-glutamate exporter from Escherichia coli was introduced, and l-glutamate production reached 2.3 g/L in 143 h (34°C, 100 µmol m-2 s-1). Then, we attempted to produce two flavor substances, (S)-linalool, a monoterpene alcohol, and the sesquiterpene (+)-valencene. The Synechocystis sp. PCC6803 strain in which the linalool synthase gene (LINS) from Actinidia arguta (AaLINS) was expressed under control of the tac promoter (GT0846K-Ptac-AaLINS) produced 11.4 mg/L (S)-linalool in 160 h (30°C, 50 µmol m-2 s-1). The strain in which AaLINS2 and the mutated farnesyl diphosphate synthase gene ispA∗ (S80F) from E. coli (GT0846K-PpsbA2-AaLINS-ispA∗) were expressed from the PpsbA2 promoter accumulated 11.6 mg/L (S)-linalool in 160 h. Genome analysis revealed that both strains had mutations in slr1270, suggesting that loss of Slr1270 function was necessary for high linalool accumulation. For sesquiterpene production, the valencene synthase gene from Callitropsis nootkatensis and the fernesyl diphosphate synthase (ispA) gene from E. coli were introduced, and the resultant strain produced 9.6 mg/L of (+)-valencene in 166 h (30°C, 50 µmol m-2 s-1). This study highlights the production efficiency of engineered cyanobacteria, providing insight into potential industrial applications.

Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin-like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins.

Directed evolution of a Cry1Aa toxin using phage display and biopanning was performed to generate an increased binding affinity to the Bombyx mori cadherin-like receptor (BtR175). Three mutant toxins (371 WGLA374 , 371 WPHH374 , 371 WRPQ374 25) with 16-, 16-, and 50-fold higher binding affinities, respectively, for BtR175 were selected from a phage library containing toxins with mutations in domain II loop 2. However, the observed toxicities of the three mutants against B. mori larvae and cultured cells expressing the BtR175 toxin-binding region did not increase, suggesting that increased binding affinity to cadherins does not contribute to the insecticidal activity. Affinity maturation of a Cry toxin to a receptor via directed evolution was relatively simple to achieve, and seems to have potential for generating a toxin with increased insecticidal activity.

Reduction of hydrogen peroxide stress derived from fatty acid beta-oxidation improves fatty acid utilization in Escherichia coli.

Fatty acids are a promising raw material for substance production because of their highly reduced and anhydrous nature, which can provide higher fermentation yields than sugars. However, they are insoluble in water and are poorly utilized by microbes in industrial fermentation production. We used fatty acids as raw materials for L-lysine fermentation by emulsification and improved the limited fatty acid-utilization ability of Escherichia coli. We obtained a fatty acid-utilizing mutant strain by laboratory evolution and demonstrated that it expressed lower levels of an oxidative-stress marker than wild type. The intracellular hydrogen peroxide (H2O2) concentration of a fatty acid-utilizing wild-type E. coli strain was higher than that of a glucose-utilizing wild-type E. coli strain. The novel mutation rpsA(D210Y) identified in our fatty acid-utilizing mutant strain enabled us to promote cell growth, fatty-acid utilization, and L-lysine production from fatty acid. Introduction of this rpsA(D210Y) mutation into a wild-type strain resulted in lower H2O2 concentrations. The overexpression of superoxide dismutase (sodA) increased intracellular H2O2 concentrations and inhibited E. coli fatty-acid utilization, whereas overexpression of an oxidative-stress regulator (oxyS) decreased intracellular H2O2 concentrations and promoted E. coli fatty acid utilization and L-lysine production. Addition of the reactive oxygen species (ROS) scavenger thiourea promoted L-lysine production from fatty acids and decreased intracellular H2O2 concentrations. Among the ROS generated by fatty-acid ß-oxidation, H2O2 critically affected E. coli growth and L-lysine production. This indicates that the regression of ROS stress promotes fatty acid utilization, which is beneficial for fatty acids used as raw materials in industrial production.

Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin-like receptor by directed evolution.

Improvement of the activity and insecticidal spectrum of cloned Cry toxins of Bacillus thuringiensis should allow for their wider application as biopesticides and a gene source for gene-modified crops. The insecticidal activity of Cry toxins depends on their binding to the receptor. Therefore, as a model, we aimed to generate improved binding affinity mutant toxins against Bombyx mori cadherin-like receptor (BtR175) using methods of directed evolution with the expectation of insecticidal activity improved mutants. Four serial amino acid residues of (439)QAAG(442) or (443)AVYT(446) of Cry1Aa were replaced with random amino acids and were displayed on the T7 phage for library construction. Through five cycles of panning of the phage libraries using BtR175, 11 mutant phage clones were concentrated, and mutant toxin sequences were confirmed. The binding affinities of the three mutants were 42-, 15-, and 13-fold higher than that of the wild type, indicating that mutants with improved binding affinity to cadherin can be easily selected from randomly replaced loop 3 mutant libraries using directed evolution. We discuss the development of a genetic engineering method based on directed evolution to improve the binding affinity of Cry toxin to receptors.

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops.

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.

A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis.

Theoretically, the activity of AB-type toxin molecules such as the insecticidal toxin (Cry toxin) from B. thuringiensis, which have one active site and two binding site, is improved in parallel with the binding affinity to its receptor. In this experiment, we tried to devise a method for the directed evolution of Cry toxins to increase the binding affinity to the insect receptor. Using a commercial T7 phage-display system, we expressed Cry1Aa toxin on the phage surface as fusions with the capsid protein 10B. These recombinant phages bound to a cadherin-like protein that is one of the Cry1Aa toxin receptors in the model target insect Bombyx mori. The apparent affinity of Cry1Aa-expressing phage for the receptor was higher than that of Cry1Ab-expressing phage. Phages expressing Cry1Aa were isolated from a mixed suspension of phages expressing Cry1Ab and concentrated by up to 130,000-fold. Finally, random mutations were made in amino acid residues 369-375 in domain 2 of Cry1Aa toxin, the mutant toxins were expressed on phages, and the resulting phage library was screened with cadherin-like protein-coated beads. As a result, phages expressing abnormal or low-affinity mutant toxins were excluded, and phages with high-affinity mutant toxins were selected. These results indicate that a method combining T7 phage display with selection using cadherin-like protein-coated magnetic beads can be used to increase the activity of easily obtained, low-activity Cry toxins from bacteria.

Safe introduction of in-hospital wireless LAN.

Insufficient research on electromagnetic interference (EMI) with medical electronic equipment by the signals of wireless LAN has been done. Therefore, electromagnetic compatibility between medical electronic equipment and wireless LAN data communications was done (IEEE802.11a, b, and g). First, to determine if medical electronic equipment is affected by EMI caused by radio waves, we irradiated radio waves to ten types of medical electronic equipment in an electromagnetic anechoic chamber. EMI were observed on three pieces of equipment. Next, to determine if the electromagnetic field emitted by the medical devices might interfere with wireless LAN communication, we measured the electric field intensity. Data analysis showed that the electromagnetic wave emitted by microwave ovens was at almost the same center frequency of the communication channel specified by the IEEE802.11b for wireless LAN. Although the number of medical electronic equipment investigated in this study was small, hospital administrators should consider electromagnetic wave testing of all medical electronic equipment to be used in areas of common wireless LAN use when planning the installation of wireless LAN.

Negligible electromagnetic interaction between medical electronic equipment and 2.4 GHz band wireless LAN.

Wireless LANs using radio waves have recently gained popularity for installation in hospitals. Because electromagnetic waves transmitted from mobile telephones have been shown to cause interference with medical electronic equipment, prudence would seem necessary when introducing radio wave communication devices into hospitals. Therefore, we tested the effect of wireless LAN communication on medical electronic equipment and the effect of electronic equipment on wireless LAN communication. We observed nine pieces of electronic equipment in the operating mode while transmitting radio waves from a wireless LAN. Even when the access point was put very close to the medical electronic equipment surface and data was transmitted, no malfunction of the equipment was observed. The medical electronic equipment caused little change in the effectiveness of the communication device, although radio waves emitted from electric knives and a remote patient monitor reduced the reception rate to about 60%. The communication speed of the wireless LAN was temporarily reduced only when a microwave oven was located close to and facing the access point. Because output in Japan is limited to a maximum of 10 mW wireless LAN following the IEEE802.11b standard should be able to be installed safely in Japanese hospitals. However, wireless LAN access points should not be installed near microwave ovens.