Protein production in Pichia pastoris is often based on the methanol‐inducible P AOX1 promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose‐regulated P GTH1 promoter which we reported recently. To further increase its potential, we investigated its regulation in more details by the screening of promoter variants harboring deletions and mutations. Thereby we could identify the main regulatory region and important putative transcription factor binding sites of P GTH1. Concluding from that, yeast metabolic regulators, monomeric Gal4‐class motifs, carbon source‐responsive elements, and yeast GC‐box proteins likely contribute to the regulation of the promoter. We engineered a P GTH1 variant with greatly enhanced induction properties compared with that of the wild‐type promoter. Based on that, a model‐based bioprocess design for high volumetric productivity in a limited time was developed for the P GTH1 variant, to employ a glucose fed‐batch strategy that clearly outperformed a classical methanol fed‐batch of a P AOX1 strain in terms of titer and process performance.

Integrated operation of biotransformation and simulated moving bed (SMB) separation is an attractive option for high-yield manufacturing of commercially relevant compounds such as rare sugars and sialic acids from equilibrium-limited isomerase- or aldolase-catalyzed reactions. Here, we present the first lab-scale implementation of such a process using the production of d-psicose, which is currently under consideration as low calorie sweetener, by d-tagatose epimerase-catalyzed epimerization from d-fructose as a model system. While a typical batchwise eprimerization of d-fructose would stop at 25%, a yield of 97% was obtained when operating the fully integrated process consisting of SMB, enzyme membrane reactor (EMR) and nanofiltration (NF) for a number of days with absolute product purities. Next to the proof of principle, important process characteristics such as startup time, stability and robustness were investigated. By pre-equilibrating the NF unit to the projected conditions, startup times could be reduced to the contributions from EMR and SMB (in this case below 5 h) which was perfectly in line with the projected range of operation time of a few days. Robustness was probed by introduction of a perturbation, specifically a 2-fold increase in process feed concentration, which did not compromise any of the set specifications. Next, long-term operation of the respective units indicated a potential process time of at least 5 days, which could be easily extended in the future by engineering a more stable enzyme variant and implementing a cleaning-in-place approach for SMB column regeneration. In summary, the principle feasibility of such process integration for fine chemical synthesis could be successfully demonstrated.

ABSTRACT:The aim of the present study was to investigate corticospinal and intracortical excitability in patients with congenital stroke. In adults, stroke sequelae reduce corticospinal excitability, as indicated by an elevated threshold for motor evoked potentials (MEP), and increase intracortical excitability, as indicated by reduced intracortical inhibition. Ten patients with pre-or perinatally acquired, unilateral cortico-subcortical infarctions in the middle cerebral artery territory were studied with single pulse transcranial magnetic stimulation (TMS) to measure motor threshold (MT) and with paired pulse TMS to study short interval intracortical inhibition (SICI) and intracortical facilitation (ICF). Eight healthy, age-matched subjects served as controls. MT over the affected hemisphere of patients compared with the dominant hemisphere of controls was significantly elevated, reflecting reduced corticospinal excitability, and SICI was significantly reduced, reflecting increased intracortical excitability. No such differences were found for ICF. Findings in patients with congenital stroke were comparable with adulthood stroke. Thus, similar assumptions can be made: reduced corticospinal excitability is probably a consequence of neuronal damage. Reduced intracortical inhibition might represent deficient inhibitory cortical properties or might reflect a compensational mechanism, dispositioning for use-dependent plasticity. T he human brain is capable of reorganization (1-3). In patients with congenital stroke, reorganization is determined by the maturational state of the brain at the time of the insult (4). Early studies using transcranial magnetic stimulation (TMS) focused on corticospinal connectivity and revealed in some patients preserved ipsilateral motor pathways from the undamaged motor cortex to the hemiplegic hand (5-9). Subsequent studies combining TMS with structural and functional magnetic resonance imaging (MRI) revealed that the type of corticospinal reorganization as well as the compensatory recruitment of areas within the contralesional hemisphere depend on the extent of the underlying brain lesion (10). Furthermore, location and timing period affect the type and efficacy of reorganization, with significant impact on clinical outcome (11).Until now, studies investigating intracortical excitability by transcranial magnetic paired pulse stimulation have been conducted only in patients with adulthood stroke (12-15). This technique allows us to study changes in the excitability of both inhibitory and excitatory intracortical circuits, a mechanism credited to be involved in reorganization of the brain (16 -19). In adulthood, cortical stroke intracortical excitability is increased, as indicated by a reduced intracortical inhibition with a heightened amplitude of the conditioned motor evoked potential (MEP) (12-15). There is evidence from developmental studies that corticospinal excitability as well as intracortical excitability are age dependent and are not fully established up to several years post...

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