Model-based high cell density cultivation of Rhodospirillum rubrum under respiratory dark conditions.

The potential of facultative photosynthetic bacteria as producers of photosynthetic pigments, vitamins, coenzymes and other valuable products has been recognized for decades. However, mass cultivation under photosynthetic conditions is generally inefficient due to the inevitable limitation of light supply when cell densities become very high. The previous development of a new cultivation process for maximal expression of photosynthetic genes under semi-aerobic dark conditions in common bioreactors offers a new perspective for utilizing the facultative photosynthetic bacterium Rhodospirillum rubrum for large-scale applications. Based on this cultivation system, the present study aimed in determining the maximal achievable cell density of R. rubrum in a bioreactor, thereby providing a major milestone on the way to industrial bioprocesses. As a starting point, we focus on aerobic growth due to higher growth rates and more facile process control under this condition, with the option to extend the process by an anaerobic production phase. Process design and optimization were supported by an unstructured computational process model, based on mixed-substrate kinetics. Key parameters for growth and process control were determined in shake-flask experiments or estimated by simulation studies. For fed-batch cultivation, a computer-controlled exponential feed algorithm in combination with a pH-stat element was implemented. As a result, a maximal cell density of 59 g cell dry weight (CDW) L(-1) was obtained, representing so far not attainable cell densities for photosynthetic bacteria. The applied exponential fed-batch methodology therefore enters a range which is commonly employed for industrial applications with microbial cells. The biochemical analysis of high cell density cultures revealed metabolic imbalances, such as the accumulation and excretion of tetrapyrrole intermediates of the bacteriochlorophyll biosynthetic pathway.

Nucleophosmin interacts with HEXIM1 and regulates RNA polymerase II transcription.

Hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) was identified earlier as an inhibitor of positive transcription elongation factor b (P-TEFb), which is a key transcriptional regulator of RNA polymerase II (Pol II). Studies show that more than half of P-TEFb in cells is associated with HEXIM1, which results in the inactivation of P-TEFb. Here, we identify a nucleolar protein, nucleophosmin (NPM), as a HEXIM1-binding protein. NPM binds to HEXIM1 in vitro and in vivo, and functions as a negative regulator of HEXIM1. Over-expression of NPM leads to proteasome-mediated degradation of HEXIM1, resulting in activation of P-TEFb-dependent transcription. In contrast, an increase in HEXIM1 protein levels and a decrease in transcription are detected when NPM is knocked down. We show that a cytoplasmic mutant of NPM, NPMc+, associates with and sequesters HEXIM1 in the cytoplasm resulting in higher RNA Pol II transcription. Correspondingly, cytoplasmic localization of endogenous HEXIM1 is detected in an acute myeloid leukemia (AML) cell line containing the NPMc+ mutation, suggesting the physiological importance of HEXIM1-NPMc+ interaction. Over-expression of NPM has been detected in tumors of various histological origins and our results may provide a possible molecular mechanism for the proto-oncogenic function of NPM. Furthermore, considering that 35% of AML patients are diagnosed with NPMc+ mutation, our findings suggest that in some cases of AML, RNA Pol II transcription may be disregulated by the malfunction of NPM and the mislocation of HEXIM1.