Synergistic co-utilization of biomass-derived sugars enhances aromatic amino acid production by engineered Escherichia coli.

Efficient co-utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose-xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co-utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose-to-glucose flux ratio. A mutant copy of the xylose-specific activator (XylR) was then introduced into the phenylalanine-overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose-xylose co-utilization. Carbon contribution analysis through 13 C-fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose-xylose mixture; a threefold increase over NST74. Then, using biomass-derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9-fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L-h). This study presents a novel strategy for enhancing phenylalanine production through the co-utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products.

Genetic transformation of Coniochaeta sp. 2T2.1, key fungal member of a lignocellulose-degrading microbial consortium.

Coniochaeta sp. strain 2T2.1 is a key member of a microbial consortium that degrades lignocellulosic biomass. Due to its ecological niche and ability to also grow in pure culture on wheat straw, protocols for transformation and antibiotic selection of the strain were established. Hygromycin was found to be a reliable selectable transformation marker, and the mammalian codon-optimized green fluorescent protein was expressed and used to visualize fluorescence in transformed cells of strain 2T2.1.

Use of green fluorescent protein to monitor fungal growth in biomass hydrolysate.

A reporter gene encoding green fluorescent protein (GFP) was introduced into the ascomycete Coniochaeta ligniaria NRRL30616, and fluorescence of cultures was monitored as a measure of cell growth. Fluorescence in the GFP-expressing strain was measured during growth of cells in defined and complex media as well as in the liquor derived from pretreatment of corn stover, an agricultural residue. Fluorescence mirrored growth of cultures, as measured by optical density and counts of colony forming units. Because traditional methods to monitor growth cannot be used in biomass liquors due to its fibrous, dark-colored nature, the speed and convenience of using GFP to monitor growth is advantageous. Fluorescence of cultures in biomass hydrolysate also correlated with the concentration of furfural in hydrolysate. Furfural and other compounds, present in hydrolysate due to physico-chemical pretreatment of biomass, are inhibitory to fermenting microbes. Therefore, measurement of fluorescence in GFP-expressing C. ligniaria is a proxy for measures of microbial growth and furfural consumption, and serves as a convenient indicator of metabolism of fermentation inhibitors in biomass hydrolysate.

Transcriptomic and anatomic parcellation of 5-HT3AR expressing cortical interneuron subtypes revealed by single-cell RNA sequencing.

Cortical GABAergic interneurons constitute a highly diverse population of inhibitory neurons that are key regulators of cortical microcircuit function. An important and heterogeneous group of cortical interneurons specifically expresses the serotonin receptor 3A (5-HT3AR) but how this diversity emerges during development is poorly understood. Here we use single-cell transcriptomics to identify gene expression patterns operating in Htr3a-GFP+ interneurons during early steps of cortical circuit assembly. We identify three main molecular types of Htr3a-GFP+ interneurons, each displaying distinct developmental dynamics of gene expression. The transcription factor Meis2 is specifically enriched in a type of Htr3a-GFP+ interneurons largely confined to the cortical white matter. These MEIS2-expressing interneurons appear to originate from a restricted region located at the embryonic pallial-subpallial boundary. Overall, this study identifies MEIS2 as a subclass-specific marker for 5-HT3AR-containing interstitial interneurons and demonstrates that the transcriptional and anatomical parcellation of cortical interneurons is developmentally coupled.

Maleic acid treatment of biologically detoxified corn stover liquor.

Elimination of microbial and enzyme inhibitors from pretreated lignocellulose is critical for effective cellulose conversion and yeast fermentation of liquid hot water (LHW) pretreated corn stover. In this study, xylan oligomers were hydrolyzed using either maleic acid or hemicellulases, and other soluble inhibitors were eliminated by biological detoxification. Corn stover at 20% (w/v) solids was LHW pretreated LHW (severity factor: 4.3). The 20% solids (w/v) pretreated corn stover derived liquor was recovered and biologically detoxified using the fungus Coniochaeta ligniaria NRRL30616. After maleic acid treatment, and using 5 filter paper units of cellulase/g glucan (8.3mg protein/g glucan), 73% higher cellulose conversion from corn stover was obtained for biodetoxified samples compared to undetoxified samples. This corresponded to 87% cellulose to glucose conversion. Ethanol production by yeast of pretreated corn stover solids hydrolysate was 1.4 times higher than undetoxified samples, with a reduction of 3h in the fermentation lag phase.

Bioabatement with hemicellulase supplementation to reduce enzymatic hydrolysis inhibitors.

A stepwise removal of inhibitory compounds by bioabatement combined with hemicellulase supplementation was conducted to enhance cellulose hydrolysis of liquid hot water-pretreated corn stover. Results showed that the fungus Coniochaeta ligniaria NRRL30616 eliminated most of the enzyme and fermentation inhibitors from liquid hot water-pretreated corn stover hydrolysates. Moreover, addition of hemicellulases after bioabatement and before enzymatic hydrolysis of cellulose achieved 20% higher glucose yields compared to non-treated samples. This work presents the mechanisms by which supplementation of the fungus with hemicellulase enzymes enables maximal conversion, and confirms the inhibitory effect of xylo-oligosaccharides in corn stover hydrolysates once the dominant inhibitory effect of phenolic compounds is removed.

Serotonin receptor 3A controls interneuron migration into the neocortex.

Neuronal excitability has been shown to control the migration and cortical integration of reelin-expressing cortical interneurons (INs) arising from the caudal ganglionic eminence (CGE), supporting the possibility that neurotransmitters could regulate this process. Here we show that the ionotropic serotonin receptor 3A (5-HT(3A)R) is specifically expressed in CGE-derived migrating interneurons and upregulated while they invade the developing cortex. Functional investigations using calcium imaging, electrophysiological recordings and migration assays indicate that CGE-derived INs increase their response to 5-HT(3A)R activation during the late phase of cortical plate invasion. Using genetic loss-of-function approaches and in vivo grafts, we further demonstrate that the 5-HT(3A)R is cell autonomously required for the migration and proper positioning of reelin-expressing CGE-derived INs in the neocortex. Our findings reveal a requirement for a serotonin receptor in controlling the migration and laminar positioning of a specific subtype of cortical IN.

Dopamine from the brain promotes spinal motor neuron generation during development and adult regeneration.

Coordinated development of brain stem and spinal target neurons is pivotal for the emergence of a precisely functioning locomotor system. Signals that match the development of these far-apart regions of the central nervous system may be redeployed during spinal cord regeneration. Here we show that descending dopaminergic projections from the brain promote motor neuron generation at the expense of V2 interneurons in the developing zebrafish spinal cord by activating the D4a receptor, which acts on the hedgehog pathway. Inhibiting this essential signal during early neurogenesis leads to a long-lasting reduction of motor neuron numbers and impaired motor responses of free-swimming larvae. Importantly, during successful spinal cord regeneration in adult zebrafish, endogenous dopamine promotes generation of spinal motor neurons, and dopamine agonists augment this process. Hence, we describe a supraspinal control mechanism for the development and regeneration of specific spinal cell types that uses dopamine as a signal.

Lesion-induced generation of interneuron cell types in specific dorsoventral domains in the spinal cord of adult zebrafish.

In contrast to mammals, adult zebrafish regenerate neurons in the lesioned spinal cord. For example, motor neurons are generated from an olig2-expressing population of pMN-like ependymoradial glial cells in a ventrolateral position at the central canal. However, the extent of neuronal regeneration is unclear. Here we show, using a transgenic fish in which V2 interneurons are labeled by green fluorescent protein (GFP) under the control of the vsx1 promoter, that after a complete spinal cord transection, large numbers of V2 interneurons are generated in the vicinity of the lesion site. Tg(vsx1:GFP)⁺ cells are not present in the unlesioned spinal cord and label with the proliferation marker bromodeoxyuridine (BrdU) after a lesion. Some mediolaterally elongated Tg(vsx1:GFP)⁺ cells contact the central canal in a medial position. These cells likely arise from a p2-like domain of ependymoradial glial progenitor cells, indicated by coexpression of Pax6 and Nkx6.1, but not DsRed driven by the olig2 promoter in these cells. We also present evidence that Pax2⁺ interneurons are newly generated after a spinal lesion, whereas the generation rate for a dorsal population of parvalbuminergic interneurons is comparatively low. Our results identify the regenerative potential of different interneuron types for the first time and support a model in which different progenitor cell domains in distinct dorsoventral positions around the central canal are activated by a lesion to give rise to diverse neuronal cell types in the adult zebrafish spinal cord.