Engineering the synthetic ß-alanine pathway in Komagataella phaffii for conversion of methanol into 3-hydroxypropionic acid.

BACKGROUND: Methanol is increasingly gaining attraction as renewable carbon source to produce specialty and commodity chemicals, as it can be generated from renewable sources such as carbon dioxide (CO2). In this context, native methylotrophs such as the yeast Komagataella phaffii (syn Pichia pastoris) are potentially attractive cell factories to produce a wide range of products from this highly reduced substrate. However, studies addressing the potential of this yeast to produce bulk chemicals from methanol are still scarce. 3-Hydroxypropionic acid (3-HP) is a platform chemical which can be converted into acrylic acid and other commodity chemicals and biopolymers. 3-HP can be naturally produced by several bacteria through different metabolic pathways. RESULTS: In this study, production of 3-HP via the synthetic ß-alanine pathway has been established in K. phaffii for the first time by expressing three heterologous genes, namely panD from Tribolium castaneum, yhxA from Bacillus cereus, and ydfG from Escherichia coli K-12. The expression of these key enzymes allowed a production of 1.0 g l-1 of 3-HP in small-scale cultivations using methanol as substrate. The addition of a second copy of the panD gene and selection of a weak promoter to drive expression of the ydfG gene in the PpCß21 strain resulted in an additional increase in the final 3-HP titer (1.2 g l-1). The 3-HP-producing strains were further tested in fed-batch cultures. The best strain (PpCß21) achieved a final 3-HP concentration of 21.4 g l-1 after 39 h of methanol feeding, a product yield of 0.15 g g-1, and a volumetric productivity of 0.48 g l-1 h-1. Further engineering of this strain aiming at increasing NADPH availability led to a 16% increase in the methanol consumption rate and 10% higher specific productivity compared to the reference strain PpCß21. CONCLUSIONS: Our results show the potential of K. phaffii as platform cell factory to produce organic acids such as 3-HP from renewable one-carbon feedstocks, achieving the highest volumetric productivities reported so far for a 3-HP production process through the ß-alanine pathway.

Animal models for assessing impact of C-section delivery on biological systems.

There has been a significant increase in Caesarean section (C-section) births worldwide over the past two decades and although it can be a life-saving procedure, the enduring effects on host physiology are now undergoing further scrutiny. Indeed, epidemiological data have linked C-section birth with multiple immune, metabolic and neuropsychiatric diseases. Birth by C-section is known to alter the colonisation of the neonatal gut microbiota (with C-section delivered infants lacking vaginal microbiota associated with passing along the birth canal), which in turn can impact the development and maintenance of many important biological systems. Appropriate animal models are key to disentangling the role of missing microbes in brain health and disease in C-section births. In this review of preclinical studies, we interrogate the effects of C-section birth on the development (and maintenance) of several biological systems and we discuss the involvement of the gut microbiome on C-section-related alterations.

Anti-inflammatory cytokine-eluting collagen hydrogel reduces the host immune response to dopaminergic cell transplants in a rat model of Parkinson's disease.

In cell replacement approaches for Parkinson's disease, the intracerebral implantation of dopamine neuron-rich grafts generates a neuroinflammatory response to the grafted cells that contributes to its varied outcome. Thus, the aim of the present study was to fabricate an anti-inflammatory cytokine-eluting collagen hydrogel capable of delivering interleukin (IL)-10 to the brain for reduction of the neuroinflammatory response to intracerebral cellular grafts. In vitro assessment revealed that cross-linker concentration affected the microstructure and gelation kinetics of the hydrogels and their IL-10 elution kinetics, but not their cytocompatibility or the functionality of the eluted IL-10. In vivo evaluation revealed that the hydrogels were capable of delivering and retaining IL-10 in the rat striatum, and reducing the neuroinflammatory (microglial) response to hydrogel-encapsulated grafts. In conclusion, IL-10-eluting collagen hydrogels may have beneficial anti-inflammatory effects in the context of cellular brain repair therapies for Parkinson's disease and should be investigated further.

The potential of biomaterials for central nervous system cellular repair.

The central nervous system (CNS) can be injured or damaged through a variety of insults including traumatic injury, stroke, and neurodegenerative or demyelinating diseases, including Alzheimer's disease, Parkinson's disease and multiple sclerosis. Existing pharmacological and other therapeutics strategies are limited in their ability to repair or regenerate damaged CNS tissue meaning there are significant unmet clinical needs facing patients suffering CNS damage and/or degeneration. Through a variety of mechanisms including neuronal replacement, secretion of therapeutic factors, and stimulation of host brain plasticity, cell-based repair offers a potential mechanism to repair and heal the damaged CNS. However, over the decades of its evolution as a therapeutic strategy, cell-based CNS repair has faced significant hurdles that have prevented its translation to widespread clinical practice. In recent years, advances in cell technologies combined with advances in biomaterial-based regenerative medicine and tissue engineering have meant there is very real potential for many of these hurdles to be overcome. This review will provide an overview of the main CNS conditions that lend themselves to cellular repair and will then outline the potential of biomaterial-based approaches for improving the outcome of cellular repair in these conditions.

Proteomic profiling of striatal tissue of a rat model of Parkinson's disease after implantation of collagen-encapsulated human umbilical cord mesenchymal stem cells.

Parkinson's disease (PD) is the most common neurodegenerative disorder of movement worldwide. To date, only symptomatic treatments are available. Implantation of collagen-encapsulated human umbilical cord mesenchymal stem cells (hUC-MSCs) is being developed as a novel therapeutic approach to potentially modify PD progression. However, implanted collagen scaffolds may induce a host tissue response. To gain insight into such response, hUC-MSCs were encapsulated into collagen hydrogels and implanted into the striatum of hemi-Parkinsonian male Sprague-Dawley rats. One or 14 days after implantation, the area of interest was dissected using a cryostat. Total protein extracts were subjected to tryptic digestion and subsequent LC-MS/MS analyses for protein expression profiling. Univariate and multivariate analyses were performed to identify differentially expressed protein profiles with subsequent gene ontology and pathway analysis for biological interpretation of the data; 2,219 proteins were identified by MaxQuant at 1% false discovery rate. A high correlation of label-free quantification (LFQ) protein values between biological replicates (r = .95) was observed. No significant differences were observed between brains treated with encapsulated hUC-MSCs compared to appropriate controls. Proteomic data were highly robust and reproducible, indicating the suitability of this approach to map differential protein expression caused by the implants. The lack of differences between conditions suggests that the effects of implantation may be minimal. Alternatively, effects may only have been focal and/or could have been masked by nonrelevant high-abundant proteins. For follow-up assessment of local changes, a more accurate dissection technique, such as laser micro dissection, and analysis method are recommended.

Viral mimetic priming enhances α-synuclein-induced degeneration: Implications for Parkinson's disease.

Evidence is accumulating to suggest that viral infections and consequent viral-mediated neuroinflammation may contribute to the etiology of idiopathic Parkinson's disease. Moreover, viruses have been shown to influence α-synuclein oligomerization as well as the autophagic clearance of abnormal intra-cellular proteins aggregations, both of which are key neuropathological events in Parkinson's disease pathogenesis. To further investigate the interaction between viral-mediated neuroinflammation and α-synuclein aggregation in the context of Parkinson's disease, this study sought to determine the impact of viral neuroinflammatory priming on α-synuclein aggregate-induced neuroinflammation and neurotoxicity in the rat nigrostriatal pathway. To do so, male Sprague-Dawley rats were intra-nigrally injected with a synthetic mimetic of viral dsRNA (poly I:C) followed two weeks later by a peptidomimetic small molecule which accelerates α-synuclein fibril formation (FN075). The impact of the viral priming on α-synuclein aggregation-induced neuroinflammation, neurodegeneration and motor dysfunction was assessed. We found that prior administration of the viral mimetic poly I:C significantly exacerbated or precipitated the α-synuclein aggregate induced neuropathological and behavioral effects. Specifically, sequential exposure to the two challenges caused a significant increase in nigral microgliosis (p < 0.001) and astrocytosis (p < 0.01); precipitated a significant degeneration of the nigrostriatal cell bodies (p < 0.05); and precipitated a significant impairment in forelimb kinesis (p < 0.01) and sensorimotor integration (p < 0.01). The enhanced sensitivity of the nigrostriatal neurons to pathological α-synuclein aggregation after viral neuroinflammatory priming further suggests that viral infections may contribute to the etiology and pathogenesis of Parkinson's disease.

Encapsulation of young donor age dopaminergic grafts in a GDNF-loaded collagen hydrogel further increases their survival, reinnervation, and functional efficacy after intrastriatal transplantation in hemi-Parkinsonian rats.

Biomaterials have been shown to significantly improve the outcome of cellular reparative approaches for Parkinson's disease in experimental studies because of their ability to provide transplanted cells with a supportive microenvironment and shielding from the host immune system. However, given that the margin for improvement in such reparative therapies is considerable, further studies are required to fully investigate and harness the potential of biomaterials in this context. Given that several recent studies have demonstrated improved brain repair in Parkinsonian models when using dopaminergic grafts derived from younger foetal donors, we hypothesized that encapsulating these cells in a supportive biomaterial would further improve their reparative efficacy. Thus, this study aimed to determine the impact of a GDNF-loaded collagen hydrogel on the survival, reinnervation, and functional efficacy of dopaminergic neurons derived from young donors. To do so, hemi-Parkinsonian (6-hydroxydopamine-lesioned) rats received intrastriatal transplants of embryonic day 12 cells extracted from the rat ventral mesencephalon either alone, in a collagen hydrogel, with GDNF, or in a GDNF-loaded collagen hydrogel. Methamphetamine-induced rotational behaviour was assessed at three weekly intervals for a total of 12 weeks, after which rats were sacrificed for postmortem assessment of graft survival. We found that, following intrastriatal transplantation to the lesioned striatum, the GDNF-loaded collagen hydrogel significantly increased the survival (4-fold), reinnervation (5.4-fold), and functional efficacy of the embryonic day 12 dopaminergic neurons. In conclusion, this study further demonstrates the significant potential of biomaterial hydrogel scaffolds for cellular brain repair approaches in neurodegenerative diseases such as Parkinson's disease.