Metabolic potentials of Liquorilactobacillus nagelii AGA58 isolated from Shalgam based on genomic and functional analysis

The aim of present study was to perform functional and genomic characterization of a novel Liquorilactobacillus nagelii AGA58 isolated from Shalgam to understand its metabolic potentials. AGA58 is gram-positive,catalase-negative and appears as short-rods under light-microscope. The AGA58 chromosome composed of a single linear chromosome of 2,294,535 bp that is predicted to carry 2151 coding sequences, including 45 tRNA genes, 4 rRNA operons. Genome has a GC content of 36.9% includes 45 pseudogenes, 32 transposases and one intact-prophage. AGA58 is micro-anaerobic owing to shorter doubling time and faster growth rate achieved compared microaerofilic condition. It carries flagellar biosynthesis protein-encoding genes predicting motile behavior. AGA58 is an obligatory homofermentative where hexose sugars such as galactose, glucose, fructose, sucrose, mannose, N-acetyl glucosamine, maltose, trehalose are fermented to lactate thru glycolysis and no acid production from pentose sugars achieved due to lack of key enzyme namely phosphoketolase in pentose phosphate pathway. Carbohydrate fermentation tests showed AGA58 cannot ferment pentoses which was also confirmed in silico. Putative pyruvate metabolism revealed formate, malate, oxaloacetate, acetate, acetaldehyde, acetoin and lactate forms from pyruvate. AGA58 predicted to carry bacteriocin genes for type A2 lantipeptide, Blp family class II bacteriocins showing antimicrobial potential of this bacterium which can be linked to antagonism tests that AGA58 can inhibit E. coli O157:H7, S. Typhimurium ATCC14028, and K. pneumonia ATCC13883. Moreoever, AGA58 is tolerant to acid and bile concentrations simulating the human gastrointestinal conditions. L. nagelii AGA58 depicting the probiotic potential of AGA58 as a first report in literature within same species.

A Combination of Structural, Genetic, Phenotypic and Enzymatic Analyses Reveals the Importance of a Predicted Fucosyltransferase to Protein O-Glycosylation in the Bacteroidetes

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species—Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked β1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.

Production of Omega-3 Fatty Acids from the Microalga Crypthecodinium cohnii by Utilizing Both Pentose and Hexose Sugars from Agricultural Residues

The core objective of this work was to take advantage of the unexploited wheat straw biomass, currently considered as a broadly available waste stream from the Greek agricultural sector, towards the integrated valorization of sugar streams for the microbial production of polyunsaturated omega-3 fatty acids (PUFAs). The OxiOrganosolv pretreatment process was applied using acetone and ethanol as organic solvents without any additional catalyst. The results proved that both cellulose-rich solid pulp and hemicellulosic oligosaccharides-rich aqueous liquid fraction after pretreatment can be efficiently hydrolyzed enzymatically, thus resulting in high yields of fermentable monosaccharides. The latter were supplied as carbon sources to the heterotrophic microalga Crypthecodinium cohnii for the production of PUFAs, more specifically docosahexaenoic acid (DHA). The solid fractions consisted mainly of hexose sugars and led to higher DHA productivity than their pentose-rich liquid counterparts, which can be attributed to the different carbon source and C/N ratio in the two streams. The best performance was obtained with the solid pulp pretreated with ethanol at 160 °C for 120 min and an O2 pressure of 16 bar. The total fatty acids content reached 70.3 wt% of dried cell biomass, of which 32.2% was DHA. The total DHA produced was 7.1 mg per g of untreated wheat straw biomass.

Overexpression of an oxidoreductase YghA confers tolerance against furfural in ethanologenic Escherichia coli strain SSK42

Furfural is a common furan inhibitor formed due to dehydration of pentose sugar like xylose and acts as an inhibitor of microbial metabolism. Overexpression of NADH specific FucO and deletion of NADPH specific YqhD had been a successful strategy in the past in conferring tolerance against furfural in E. coli , which highlight the importance of oxidoreductases in conferring tolerance against furfural. In a screen consisting of various oxidoreductases, dehydrogenases, and reductases, we identified yghA gene as an overexpression target to confer tolerance against furfural. YghA preferably used NADH as a cofactor and had apparent K m value of 0.03 mM against furfural. In presence of 1 g L −1 furfural and 10% xylose (wt/vol), yghA overexpression in an ethanologenic E. coli strain SSK42 resulted in a 5.3-fold increase in ethanol titers as compared to the control strain with an efficiency of ∼97%. YghA also exhibited activity against the lesser toxic inhibitor 5-hydroxymethyl furfural that is formed due to dehydration of hexose sugars and thus is a formidable target for overexpression in ethanologenic strain for fermentation of sugars in biomass hydrolysate. IMPORTANCE Lignocellulosic biomass represents an inexhaustible source of carbon for second-generation biofuels. Thermo-acidic pretreatment of biomass is performed to loosen the lignocellulosic fibers and make the carbon bioavailable for microbial metabolism. The pretreatment process also results in the formation of inhibitors that inhibit microbial metabolism and increase production costs. Furfural is a potent furan inhibitor that increases the toxicity of other inhibitors present in the hydrolysate. Thus it is desirable to engineer furfural tolerance in E. coli for efficient fermentation of hydrolysate sugars.

Utilization of Monosaccharides by Hungateiclostridium thermocellum ATCC 27405 through Adaptive Evolution

Hungateiclostridium thermocellum ATCC 27405 is a promising bacterium for consolidated bioprocessing with a robust ability to degrade lignocellulosic biomass through a multienzyme cellulosomal complex. The bacterium uses the released cellodextrins, glucose polymers of different lengths, as its primary carbon source and energy. In contrast, the bacterium exhibits poor growth on monosaccharides such as fructose and glucose. This phenomenon raises many important questions concerning its glycolytic pathways and sugar transport systems. Until now, the detailed mechanisms of H. thermocellum adaptation to growth on hexose sugars have been relatively poorly explored. In this study, adaptive laboratory evolution was applied to train the bacterium in hexose sugars-based media, and genome resequencing was used to detect the genes that got mutated during adaptation period. RNA-seq data of the first culture growing on either fructose or glucose revealed that several glycolytic genes in the Embden–Mayerhof–Parnas pathway were expressed at lower levels in these cells than in cellobiose-grown cells. After seven consecutive transfer events on fructose and glucose (~42 generations for fructose-adapted cells and ~40 generations for glucose-adapted cells), several genes in the EMP glycolysis of the evolved strains increased the levels of mRNA expression, accompanied by a faster growth, a greater biomass yield, a higher ethanol titer than those in their parent strains. Genomic screening also revealed several mutation events in the genomes of the evolved strains, especially in those responsible for sugar transport and central carbon metabolism. Consequently, these genes could be applied as potential targets for further metabolic engineering to improve this bacterium for bio-industrial usage.

Localization and phosphorylation in the Snf1 network is controlled by two independent pathways

AMPK/SNF1 is the master regulator of energy homeostasis in eukaryotic cells and has a key role in glucose de-repression. If glucose becomes depleted, Snf1 is phosphorylated and activated. Activation of Snf1 is required but is not sufficient for mediating glucose de-repression indicating a second glucose-regulated step that adjusts the Snf1 pathway. To elucidate this regulation, we further explore the spatial dynamics of Snf1 and Mig1 and how they are controlled by concentrations of hexose sugars. We utilize fluorescence recovery after photobleaching (FRAP) to study the movement of Snf1 and how it responds to external glucose concentrations. We show that the Snf1 pathway reacts both to the presence and to the absolute concentration of glucose. Furthermore, we identify a negative feedback loop regulating Snf1 activity. We also show that Mig1 localization correlates with the Snf1 phosphorylation pattern and not with the Mig1 phosphorylation pattern, suggesting that inactivation of Snf1 has a more pronounced effect on the localization of Mig1 than on the phosphorylation of Mig1. Our data offer insight into the true complexity of regulation of this central signaling pathway by one signal (glucose depletion) interpreted by the cell in different ways.

Construction of engineered RuBisCO Kluyveromyces marxianus for a dual microbial bioethanol production system

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) genes play important roles in CO2 fixation and redox balancing in photosynthetic bacteria. In the present study, the kefir yeast Kluyveromyces marxianus 4G5 was used as host for the transformation of form I and form II RubisCO genes derived from the nonsulfur purple bacterium Rhodopseudomonas palustris using the Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO) method. Hungateiclostridium thermocellum ATCC 27405, a well-known bacterium for its efficient solubilization of recalcitrant lignocellulosic biomass, was used to degrade Napier grass and rice straw to generate soluble fermentable sugars. The resultant Napier grass and rice straw broths were used as growth media for the engineered K. marxianus. In the dual microbial system, H. thermocellum degraded the biomass feedstock to produce both C5 and C6 sugars. As the bacterium only used hexose sugars, the remaining pentose sugars could be metabolized by K. marxianus to produce ethanol. The transformant RubisCO K. marxianus strains grew well in hydrolyzed Napier grass and rice straw broths and produced bioethanol more efficiently than the wild type. Therefore, these engineered K. marxianus strains could be used with H. thermocellum in a bacterium-yeast coculture system for ethanol production directly from biomass feedstocks.

Laboratory evolution and reverse engineering of Clostridium thermocellum for growth on glucose and fructose

The native ability of Clostridium thermocellum to efficiently solubilize cellulose makes it an interesting platform for sustainable biofuel production through consolidated bioprocessing. Together with other improvements, industrial implementation of C. thermocellum, as well as fundamental studies into its metabolism, would benefit from improved and reproducible consumption of hexose sugars. To investigate growth of C. thermocellum on glucose or fructose, as well as the underlying molecular mechanisms, laboratory evolution was performed in carbon-limited chemostats with increasing concentrations of glucose or fructose and decreasing cellobiose concentrations. Growth on both glucose and fructose was achieved with biomass yields of 0.09 ± 0.00 and 0.18 ± 0.00 gbiomass gsubstrate−1, respectively, compared to 0.15 ± 0.01 gbiomass gsubstrate−1 for wild type on cellobiose. Single-colony isolates had no or short lag times on the monosaccharides, whilst wild type showed 42 ± 4 hours on glucose and >80 hours on fructose. With good growth on glucose, fructose, and cellobiose, the fructose isolates were chosen for genome sequence-based reverse metabolic engineering. Deletion of a putative transcriptional regulator (Clo1313_1831), which upregulated fructokinase activity, reduced lag time on fructose to 12 hours with a growth rate of 0.11 ± 0.01 h−1 and resulted in immediate growth on glucose at 0.24 ± 0.01 h−1. Additional introduction of a cbpAG148V mutation resulted in immediate growth on fructose at 0.32 ± 0.03 h−1. These insights can guide engineering of strains for fundamental studies into transport and the upper glycolysis, as well as maximizing product yields in industrial settings. Importance. C. thermocellum is an important candidate for sustainable and cost-effective production of bioethanol through consolidated bioprocessing. In addition to unsurpassed cellulose deconstruction, industrial application and fundamental studies would benefit from improvement of glucose and fructose consumption. This study demonstrated that C. thermocellum can be evolved for reproducible constitutive growth on glucose or fructose. Subsequent genome sequencing, gene editing and physiological characterization identified two underlying mutations with a role in (regulation of) transport or metabolism of the hexose sugars. In light of these findings, such mutations have likely (and unknowingly) also occurred in previous studies with C. thermocellum using hexose-based media with possible broad regulatory consequences. By targeted modification of these genes, industrial and research strains of C. thermocellum can be engineered to i) reduce glucose accumulation, ii) study cellodextrin transport systems in vivo, iii) allow experiments at >120 g L−1 soluble substrate concentration, or iv) reduce costs for labelling studies.

Physiological Profiling of Embryos and Dormant Seeds in Two Arabidopsis Accessions Reveals a Metabolic Switch in Carbon Reserve Accumulation

In flowering plants, sugars act as carbon sources providing energy for developing embryos and seeds. Although most studies focus on carbon metabolism in whole seeds, knowledge about how particular sugars contribute to the developmental transitions during embryogenesis is scarce. To develop a quantitative understanding of how carbon composition changes during embryo development, and to determine how sugar status contributes to final seed or embryo size, we performed metabolic profiling of hand-dissected embryos at late torpedo and mature stages, and dormant seeds, in two Arabidopsis thaliana accessions with medium [Columbia-0 (Col-0)] and large [Burren-0 (Bur-0)] seed sizes, respectively. Our results show that, in both accessions, metabolite profiles of embryos largely differ from those of dormant seeds. We found that developmental transitions from torpedo to mature embryos, and further to dormant seeds, are associated with major metabolic switches in carbon reserve accumulation. While glucose, sucrose, and starch predominantly accumulated during seed dormancy, fructose levels were strongly elevated in mature embryos. Interestingly, Bur-0 seeds contain larger mature embryos than Col-0 seeds. Fructose and starch were accumulated to significantly higher levels in mature Bur-0 than Col-0 embryos, suggesting that they contribute to the enlarged mature Bur-0 embryos. Furthermore, we found that Bur-0 embryos accumulated a higher level of sucrose compared to hexose sugars and that changes in sucrose metabolism are mediated by sucrose synthase (SUS), with SUS genes acting non-redundantly, and in a tissue-specific manner to utilize sucrose during late embryogenesis.

Sugar transport for enhanced xylose utilization in Ashbya gossypii

The co-utilization of mixed (pentose/hexose) sugars constitutes a challenge for microbial fermentations. The fungus Ashbya gossypii, which is currently exploited for the industrial production of riboflavin, has been presented as an efficient biocatalyst for the production of biolipids using xylose-rich substrates. However, the utilization of xylose in A. gossypii is hindered by hexose sugars. Three A. gossypii homologs (AFL204C, AFL205C and AFL207C) of the yeast HXT genes that code for hexose transporters have been identified and characterized by gene-targeting approaches. Significant differences in the expression profile of the HXT homologs were found in response to different concentrations of sugars. More importantly, an amino acid replacement (N355V) in AFL205Cp, introduced by CRISPR/Cas9-mediated genomic edition, notably enhanced the utilization of xylose in the presence of glucose. Hence, the introduction of the afl205c-N355V allele in engineered strains of A. gossypii will further benefit the utilization of mixed sugars in this fungus.