Improved phloroglucinol production by metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Phloroglucinol is a valuable chemical which has been successfully produced by metabolically engineered Escherichia coli. However, the low productivity remains a bottleneck for large-scale application and cost-effective production. In the present work, we cloned the key biosynthetic gene, phlD (a type III polyketide synthase), into a bacterial expression vector to produce phloroglucinol in E. coli and developed different strategies to re-engineer the recombinant strain for robust synthesis of phloroglucinol. Overexpression of E. coli marA (multiple antibiotic resistance) gene enhanced phloroglucinol resistance and elevated phloroglucinol production to 0.27 g/g dry cell weight. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) level through coordinated expression of four acetyl-CoA carboxylase (ACCase) subunits increased phloroglucinol production to around 0.27 g/g dry cell weight. Furthermore, the coexpression of ACCase and marA caused another marked improvement in phloroglucinol production 0.45 g/g dry cell weight, that is, 3.3-fold to the original strain. Under fed-batch conditions, this finally engineered strain accumulated phloroglucinol up to 3.8 g/L in the culture 12 h after induction, corresponding to a volumetric productivity of 0.32 g/L/h. This result was the highest phloroglucinol production to date and showed promising to make the bioprocess economically feasible.     

Increasing fatty acid production in <Emphasis Type="Italic">E. coli</Emphasis> by simulating the lipid accumulation of oleaginous microorganisms

Unlike many oleaginous microorganisms, E. coli only maintains a small amount of natural lipids in cells, impeding its utility to overproduce fatty acids. In this study, acetyl-CoA carboxylase (ACC) from Acinetobacter calcoaceticus was expressed in E. coli to redirect the carbon flux to the generation of malonyl-CoA, which resulted in a threefold increase in intracellular lipids. Moreover, providing a high level of NADPH by overexpressing malic enzyme and adding malate to the culture medium resulted in a fourfold increase in intracellular lipids (about 197.74 mg/g). Co-expression of ACC and malic enzyme resulted in 284.56 mg/g intracellular lipids, a 5.6-fold increase compared to the wild-type strain. This study provides some attractive strategies for increasing lipid production in E. coli by simulating the lipid accumulation of oleaginous microorganisms, which could aid the development of a prokaryotic fatty acid producer.     

Metabolic Engineering of Escherichia coli for the Production of Xylonate

Xylonate is a valuable chemical for versatile applications. Although the chemical synthesis route and microbial conversion pathway were established decades ago, no commercial production of xylonate has been obtained so far. In this study, the industrially important microorganism Escherichia coli was engineered to produce xylonate from xylose. Through the coexpression of a xylose dehydrogenase (xdh) and a xylonolactonase (xylC) from Caulobacter crescentus, the recombinant strain could convert 1 g/L xylose to 0.84 g/L xylonate and 0.10 g/L xylonolactone after being induced for 12 h. Furthermore, the competitive pathway for xylose catabolism in E. coli was blocked by disrupting two genes (xylA and xylB) encoding xylose isomerase and xylulose kinase. Under fed-batch conditions, the finally engineered strain produced up to 27.3 g/L xylonate and 1.7 g/L xylonolactone from 30 g/L xylose, about 88% of the theoretical yield. These results suggest that the engineered E. coli strain has a promising perspective for large-scale production of xylonate.     

Physiological and subjective responses to low relative humidity in young and elderly men

In order to compare the physiological and the subjective responses to low relative humidity of elderly and young men, we measured saccharin clearance time (SCT), frequency of blinking, hydration state of the skin, transepidermal water loss (TEWL), sebum level recovery and skin temperatures as physiological responses. We asked subjects to evaluate thermal, dryness and comfort sensations as subjective responses using a rating scale. Eight non-smoking healthy male students (21.7+/-0.8 yr) and eight non-smoking healthy elderly men (71.1+/-4.1 yr) were selected. The pre-room conditions were maintained at an air temperature (Ta) of 25 degrees C and a relative humidity (RH) of 50%. The test-room conditions were adjusted to provide 25 degrees C Ta and RH levels of 10%, 30% and 50%. RH had no effect on the activity of the sebaceous gland or change of mean skin temperature. SCT of the elderly group under 10% RH was significantly longer than that of the young group. In particular, considering the SCT change, the nasal mucous membrane seems to be affected more in the elderly than in the young in low RH. Under 30% RH, the eyes and skin become dry, and under 10% RH the nasal mucous membrane becomes dry as well as the eyes and skin. These findings suggested that to avoid dryness of the eyes and skin, it is necessary to maintain greater than 30% RH, and to avoid dryness of the nasal mucous membrane, it is necessary to maintain greater than 10% RH. On the thermal sensation of the legs, at the lower humidity level, the elderly group felt cooler than the young group. On the dry sensation of the eyes and throat, the young group felt drier than the elderly group at the lower humidity levels. From the above results, the elderly group had difficulty in feeling dryness in the nasal mucous membrane despite being easily affected by low humidity. On the other hand, the young group felt the change of humidity sensitively despite not being severely affected by low humidity. Ocular mucosa and physiology of skin by dryness showed no difference by age. In the effect of longer exposure (180 min.) to low RH, only TEWL showed a slight decrease after 120 minutes in 30% RH, and all the measured results showed no noticeable differences compared with the result at 120 minutes.     

Determination of salivary dehydroepiandrosterone using liquid chromatography--tandem mass spectrometry combined with charged derivatization

A sensitive liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS-MS) method for the quantification of dehydroepiandrosterone (DHEA) in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a Strata-X cartridge, derivatized with the permanently charged reagent, 2-hydrazino-1-methylpyridine (HMP), and subjected to LC-MS-MS. The derivatization with HMP was very effective for increasing the detectability of DHEA in the positive-ESI-MS. Quantification was based on the selected reaction monitoring and androsterone was used as an internal standard. This method allowed the reproducible and accurate quantification of the salivary DHEA using a 200-microl sample and the limit of quantitation for DHEA was 25 pg/ml. No significant matrix effect or change in the measured value by freeze/thaw repetition was observed. The developed method was applied to clinical studies, and produced satisfactory results.     

Isl1 Directly Controls a Cholinergic Neuronal Identity in the Developing Forebrain and Spinal Cord by Forming Cell Type-Specific Complexes

The establishment of correct neurotransmitter characteristics is an essential step of neuronal fate specification in CNS development. However, very little is known about how a battery of genes involved in the determination of a specific type of chemical-driven neurotransmission is coordinately regulated during vertebrate development. Here, we investigated the gene regulatory networks that specify the cholinergic neuronal fates in the spinal cord and forebrain, specifically, spinal motor neurons (MNs) and forebrain cholinergic neurons (FCNs). Conditional inactivation of Isl1, a LIM homeodomain factor expressed in both differentiating MNs and FCNs, led to a drastic loss of cholinergic neurons in the developing spinal cord and forebrain. We found that Isl1 forms two related, but distinct types of complexes, the Isl1-Lhx3-hexamer in MNs and the Isl1-Lhx8-hexamer in FCNs. Interestingly, our genome-wide ChIP-seq analysis revealed that the Isl1-Lhx3-hexamer binds to a suite of cholinergic pathway genes encoding the core constituents of the cholinergic neurotransmission system, such as acetylcholine synthesizing enzymes and transporters. Consistently, the Isl1-Lhx3-hexamer directly coordinated upregulation of cholinergic pathways genes in embryonic spinal cord. Similarly, in the developing forebrain, the Isl1-Lhx8-hexamer was recruited to the cholinergic gene battery and promoted cholinergic gene expression. Furthermore, the expression of the Isl1-Lhx8-complex enabled the acquisition of cholinergic fate in embryonic stem cell-derived neurons. Together, our studies show a shared molecular mechanism that determines the cholinergic neuronal fate in the spinal cord and forebrain, and uncover an important gene regulatory mechanism that directs a specific neurotransmitter identity in vertebrate CNS development.