Role and neural regulation of clock genes in the rat pineal gland: Clock modulates amplitude of rhythmic expression of Aanat encoding the melatonin-producing enzyme.

Circadian clock gene expression in the suprachiasmatic nucleus (SCN) controls 24 h rhythms in body functions, but clock genes are also expressed in extra-hypothalamic tissues, including the melatonin-producing pineal gland. The nocturnal increase in pineal melatonin synthesis is a hallmark in circadian biology, but the role of local clock gene oscillations in the mammalian pineal gland is unknown. The aim of this work is to determine the role of clock genes in endocrine function of the pineal gland with focus on the Aanat transcript encoding the rhythm-generating enzyme of melatonin synthesis. Using the rat as a model, we here established 24 h expression patterns of clock genes in the pineal gland in vivo. Lesion studies showed that rhythmic clock gene expression in the pineal gland to a large extent depends on the SCN; further, clock gene rhythms could be re-established in cultured pineal cells synchronized by rhythmic stimulation with norepinephrine in 12 h pulses, suggesting that pineal cells house a slave oscillator controlled by adrenergic signaling in the gland. Histological analyses showed that clock genes are expressed in pinealocytes and colocalize with Aanat transcripts, thus potentially enabling clock gene products to control cellular melatonin production. To test this, cultured pineal cells were transfected using small interfering RNA to knock down clock gene expression. While successful knockdown of Per1 had a minor effect on Aanat, Clock knockdown produced a marked overexpression of Aanat in the pinealocytes. Our study suggests that SCN-dependent rhythmic Clock gene expression in the pinealocytes regulates the daily profile of Aanat expression.

The ISL LIM-homeobox 2 transcription factor is negatively regulated by circadian adrenergic signaling to repress the expression of Aanat in pinealocytes of the rat pineal gland.

Melatonin is synthesized in the pineal gland during nighttime in response to nocturnal increase in the activity of the enzyme aralkylamine N-acetyltransferase (AANAT), the transcription of which is modulated by several homeodomain transcription factors. Recent work suggests that the homeodomain transcription factor ISL LIM homeobox 2 (ISL2) is expressed in the pineal gland, but its role is currently unknown. With the purpose of identifying the mechanisms that control pineal expression of Isl2 and the possible function of Isl2 in circadian pineal biology, we report that Isl2 is specifically expressed in the pinealocytes of the rat pineal gland. Its expression exhibits a 24 h rhythm with high transcript and protein levels during the day and a trough in the second half of the night. This rhythm persists in darkness, and lesion studies reveal that it requires intact function of the suprachiasmatic nuclei, suggesting intrinsic circadian regulation. In vivo and in vitro experiments show that pineal Isl2 expression is repressed by adrenergic signaling acting via cyclic AMP; further, Isl2 is negatively regulated by the nocturnal transcription factor cone-rod homeobox. During development, pineal Isl2 expression is detectable from embryonic day 19, preceding Aanat by several days. In vitro knockdown of Isl2 is accompanied by an increase in Aanat transcript levels suggesting that ISL2 represses its daytime expression. Thus, rhythmic expression of ISL2 in pinealocytes is under the control of the suprachiasmatic nucleus acting via adrenergic signaling in the gland to repress nocturnal expression, while ISL2 itself negatively regulates daytime pineal expression of Aanat and thereby suggestively enhances the circadian rhythm in melatonin synthesis.

Metabolic engineering of Aspergillus niger via ribonucleoprotein-based CRISPR-Cas9 system for succinic acid production from renewable biomass.

BACKGROUND: Succinic acid has great potential to be a new bio-based building block for deriving a number of value-added chemicals in industry. Bio-based succinic acid production from renewable biomass can provide a feasible approach to partially alleviate the dependence of global manufacturing on petroleum refinery. To improve the economics of biological processes, we attempted to explore possible solutions with a fungal cell platform. In this study, Aspergillus niger, a well-known industrial production organism for bio-based organic acids, was exploited for its potential for succinic acid production. RESULTS: With a ribonucleoprotein (RNP)-based CRISPR-Cas9 system, consecutive genetic manipulations were realized in engineering of the citric acid-producing strain A. niger ATCC 1015. Two genes involved in production of two byproducts, gluconic acid and oxalic acid, were disrupted. In addition, an efficient C4-dicarboxylate transporter and a soluble NADH-dependent fumarate reductase were overexpressed. The resulting strain SAP-3 produced 17 g/L succinic acid while there was no succinic acid detected at a measurable level in the wild-type strain using a synthetic substrate. Furthermore, two cultivation parameters, temperature and pH, were investigated for their effects on succinic acid production. The highest amount of succinic acid was obtained at 35 °C after 3 days, and low culture pH had inhibitory effects on succinic acid production. Two types of renewable biomass were explored as substrates for succinic acid production. After 6 days, the SAP-3 strain was capable of producing 23 g/L and 9 g/L succinic acid from sugar beet molasses and wheat straw hydrolysate, respectively. CONCLUSIONS: In this study, we have successfully applied the RNP-based CRISPR-Cas9 system in genetic engineering of A. niger and significantly improved the succinic acid production in the engineered strain. The studies on cultivation parameters revealed the impacts of pH and temperature on succinic acid production and the future challenges in strain development. The feasibility of using renewable biomass for succinic acid production by A. niger has been demonstrated with molasses and wheat straw hydrolysate.