Explore the vast range of titles available.

Glycolic acid (GA) and ethylene glycol (EG) are versatile two-carbon organic chemicals used in multiple daily applications. GA and EG are currently produced by chemical synthesis, but their biotechnological production from renewable resources has received a substantial interest. Several different metabolic pathways by using genetically modified microorganisms, such as Escherichia coli , Corynebacterium glutamicum and yeast have been established for their production. As a result, the yield of GA and EG produced from sugars has been significantly improved. Here, we describe the recent advancement in metabolic engineering efforts focusing on metabolic pathways and engineering strategies used for GA and EG production.

Background Anthranilate is a platform chemical used by the industry in the synthesis of a broad range of high-value products, such as dyes, perfumes and pharmaceutical compounds. Currently anthranilate is produced via chemical synthesis from non-renewable resources. Biological synthesis would allow the use of renewable carbon sources and avoid accumulation of toxic by-products. Microorganisms produce anthranilate as an intermediate in the tryptophan biosynthetic pathway. Several prokaryotic microorganisms have been engineered to overproduce anthranilate but attempts to engineer eukaryotic microorganisms for anthranilate production are scarce. Results We subjected Saccharomyces cerevisiae , a widely used eukaryotic production host organism, to metabolic engineering for anthranilate production. A single gene knockout was sufficient to trigger anthranilate accumulation both in minimal and SCD media and the titer could be further improved by subsequent genomic alterations. The effects of the modifications on anthranilate production depended heavily on the growth medium used. By growing an engineered strain in SCD medium an anthranilate titer of 567.9 mg l −1 was obtained, which is the highest reported with an eukaryotic microorganism. Furthermore, the anthranilate biosynthetic pathway was extended by expression of anthranilic acid methyltransferase 1 from Medicago truncatula . When cultivated in YPD medium, this pathway extension enabled production of the grape flavor compound methyl anthranilate in S. cerevisiae at 414 mg l −1 . Conclusions In this study we have engineered metabolism of S. cerevisiae for improved anthranilate production. The resulting strains may serve as a basis for development of efficient production host organisms for anthranilate-derived compounds. In order to demonstrate suitability of the engineered S. cerevisiae strains for production of such compounds, we successfully extended the anthranilate biosynthesis pathway to synthesis of methyl anthranilate.

The important platform chemicals ethylene glycol and glycolic acid were produced via the oxidative D-xylose pathway in the yeast Saccharomyces cerevisiae . The expression of genes encoding D-xylose dehydrogenase (XylB) and D-xylonate dehydratase (XylD) from Caulobacter crescentus and YagE or YjhH aldolase and aldehyde dehydrogenase AldA from Escherichia coli enabled glycolic acid production from D-xylose up to 150 mg/L. In strains expressing only xylB and xylD , 29 mg/L 2-keto-3-deoxyxylonic acid [( S )-4,5-dihydroxy-2-oxopentanoic acid] (2K3DXA) was produced and D-xylonic acid accumulated to ca. 9 g/L. A significant amount of D-xylonic acid (ca. 14%) was converted to 3-deoxypentonic acid (3DPA), and also, 3,4-dihydroxybutyric acid was formed. 2K3DXA was further converted to glycolaldehyde when genes encoding by either YagE or YjhH aldolase from E. coli were expressed. Reduction of glycolaldehyde to ethylene glycol by an endogenous aldo-keto reductase activity resulted further in accumulation of ethylene glycol of 14 mg/L. The possibility of simultaneous production of lactic and glycolic acids was evaluated by expression of gene encoding lactate dehydrogenase ldhL from Lactobacillus helveticus together with aldA . Interestingly, this increased the accumulation of glycolic acid to 1 g/L. The D-xylonate dehydratase activity in yeast was notably low, possibly due to inefficient Fe–S cluster synthesis in the yeast cytosol, and leading to D-xylonic acid accumulation. The dehydratase activity was significantly improved by targeting its expression to mitochondria or by altering the Fe–S cluster metabolism of the cells with FRA2 deletion.

d-Xylonic acid is a versatile platform chemical with reported applications as complexing agent or chelator, in dispersal of concrete, and as a precursor for compounds such as co-polyamides, polyesters, hydrogels and 1,2,4-butanetriol. With increasing glucose prices, d-xylonic acid may provide a cheap, non-food derived alternative for gluconic acid, which is widely used (about 80 kton/year) in pharmaceuticals, food products, solvents, adhesives, dyes, paints and polishes. Large-scale production has not been developed, reflecting the current limited market for d-xylonate. d-Xylonic acid occurs naturally, being formed in the first step of oxidative metabolism of d-xylose by some archaea and bacteria via the action of d-xylose or d-glucose dehydrogenases. High extracellular concentrations of d-xylonate have been reported for various bacteria, in particular Gluconobacter oxydans and Pseudomonas putida. High yields of d-xylonate from d-xylose make G. oxydans an attractive choice for biotechnical production. G. oxydans is able to produce d-xylonate directly from plant biomass hydrolysates, but rates and yields are reduced because of sensitivity to hydrolysate inhibitors. Recently, d-xylonate has been produced by the genetically modified bacterium Escherichia coli and yeast Saccharomyces cerevisiae and Kluyveromyces lactis. Expression of NAD^sup +^-dependent d-xylose dehydrogenase of Caulobacter crescentus in either E. coli or in a robust, hydrolysate-tolerant, industrial Saccharomyces cerevisiae strain has resulted in d-xylonate titres, which are comparable to those seen with G. oxydans, at a volumetric rate approximately 30 % of that observed with G. oxydans. With further development, genetically modified microbes may soon provide an alternative for production of d-xylonate at industrial scale.[PUBLICATION ABSTRACT]

Background Crude glycerol coming from biodiesel production is an attractive carbon source for biological production of chemicals. The major impurity in preparations of crude glycerol is methanol, which is toxic for most microbes. Development of microbes, which would not only tolerate the methanol, but also use it as co-substrate, would increase the feasibility of bioprocesses using crude glycerol as substrate. Results To prevent methanol conversion to CO 2 via formaldehyde and formate, the formaldehyde dehydrogenase (FLD) gene was identified in and deleted from Yarrowia lipolytica. The deletion strain was able to convert methanol to formaldehyde without expression of heterologous methanol dehydrogenases. Further, it was shown that expression of heterologous formaldehyde assimilating enzymes could complement the deletion of FLD. The expression of either 3-hexulose-6-phosphate synthase (HPS) enzyme of ribulose monosphosphate pathway or dihydroxyacetone synthase (DHAS) enzyme of xylulose monosphosphate pathway restored the formaldehyde tolerance of the formaldehyde sensitive Δ fld1 strain. Conclusions In silico, the expression of heterologous formaldehyde assimilation pathways enable Y. lipolytica to use methanol as substrate for growth and metabolite production. In vivo, methanol was shown to be converted to formaldehyde and the enzymes of formaldehyde assimilation were actively expressed in this yeast. However, further development is required to enable Y. lipolytica to efficiently use methanol as co-substrate with glycerol.

Hydrogen oxidizing autotrophic bacteria are promising hosts for conversion of CO 2 into chemicals. In this work, we engineered the metabolically versatile lithoautotrophic bacterium R. opacus strain DSM 43205 for synthesis of polymer precursors. Aspartate decarboxylase (panD) or lactate dehydrogenase (ldh) were expressed for beta-alanine or L-lactic acid production, respectively. The heterotrophic cultivations on glucose produced 25 mg L −1 beta-alanine and 742 mg L −1 L-lactic acid, while autotrophic cultivations with CO 2 , H 2 , and O 2 resulted in the production of 1.8 mg L −1 beta-alanine and 146 mg L −1 L-lactic acid. Beta-alanine was also produced at 345 μg L −1 from CO 2 in electrobioreactors, where H 2 and O 2 were provided by water electrolysis. This work demonstrates that R. opacus DSM 43205 can be engineered to produce chemicals from CO 2 and provides a base for its further metabolic engineering.

Fungal secondary metabolites are a rich source of medically useful compounds due to their pharmaceutical and toxic properties. Sequencing of fungal genomes has revealed numerous secondary metabolite gene clusters, yet products of many of these biosynthetic pathways are unknown since the expression of the clustered genes usually remains silent in normal laboratory conditions. Therefore, to discover new metabolites, it is important to find ways to induce the expression of genes in these otherwise silent biosynthetic clusters. We discovered a novel secondary metabolite in Aspergillus nidulans by predicting a biosynthetic gene cluster with genomic mining. A Zn(II)(2)Cys(6)-type transcription factor, PbcR, was identified, and its role as a pathway-specific activator for the predicted gene cluster was demonstrated. Overexpression of pbcR upregulated the transcription of seven genes in the identified cluster and led to the production of a diterpene compound, which was characterized with GC/MS as ent-pimara-8(14),15-diene. A change in morphology was also observed in the strains overexpressing pbcR. The activation of a cryptic gene cluster by overexpression of its putative Zn(II)(2)Cys(6)-type transcription factor led to discovery of a novel secondary metabolite in Aspergillus nidulans. Quantitative real-time PCR and DNA array analysis allowed us to predict the borders of the biosynthetic gene cluster. Furthermore, we identified a novel fungal pimaradiene cyclase gene as well as genes encoding 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase and a geranylgeranyl pyrophosphate (GGPP) synthase. None of these genes have been previously implicated in the biosynthesis of terpenes in Aspergillus nidulans. These results identify the first Aspergillus nidulans diterpene gene cluster and suggest a biosynthetic pathway for ent-pimara-8(14),15-diene.

Background Anthraquinones in the emodin family are produced by bacteria, fungi, and plants. They display various biological activities exploited, e.g., for crop protection, and may also be utilized as sustainable, bio-based colorants for the textile, paints, electronics, and cosmetic industries. Anthraquinone pigments from Cortinarius mushrooms have been used for artisan dyeing because they are stable, colorfast, and compatible with various dyeing methods. However, their chemical synthesis is complex and uneconomical, and harvesting wild mushrooms from forests in commercial quantities is not feasible. Results Here, we use genomics, transcriptomics, and synthetic biology to uncover the biosynthesis of the anthraquinone scaffold compounds emodin and endocrocin, and their methylation to the yellow pigments physcion and dermolutein in Cortinarius semisanguineus and C. sp. KIS-3. Both the nonreducing polyketide synthases (nrPKSs), and the regiospecific, fastidious O- methyltransferases (OMTs) are non-orthologous to their Ascomycete counterparts, suggesting a parallel evolutionary origin for the pathway in Basidiomycetes. The genes for the nrPKS and the OMTs are not all clustered in Cortinarius , revealing metabolic crosstalk among paralogous nrPKS biosynthetic gene clusters. Conclusions Heterologous biosynthesis of physcion and dermolutein in Saccharomyces cerevisiae opens the way to produce specific Cortinarius anthraquinones, and to modify these scaffolds to tune their chemistry towards their various applications.

Saccharomyces cerevisiae was engineered to produce D-xylonate by introducing the Trichoderma reesei xyd1 gene, encoding a D-xylose dehydrogenase. D-xylonate was not toxic to S. cerevisiae, and the cells were able to export D-xylonate produced in the cytoplasm to the supernatant. Up to 3.8 g of D-xylonate per litre, at rates of 25-36 mg of D-xylonate per litre per hour, was produced. Up to 4.8 g of xylitol per litre was also produced. The yield of D-xylonate from D-xylose was approximately 0.4 g of D-xylonate per gramme of D-xylose consumed. Deletion of the aldose reductase encoding gene GRE3 in S. cerevisiae strains expressing xyd1 reduced xylitol production by 67%, increasing the yield of D-xylonate from D-xylose. However, D-xylose uptake was reduced compared to strains containing GRE3, and the total amount of D-xylonate produced was reduced. To determine whether the co-factor NADP⁺ was limiting for D-xylonate production the Escherichia coli transhydrogenase encoded by udhA, the Bacillus subtilis glyceraldehyde 3-phosphate dehydrogenase encoded by gapB or the S. cerevisiae glutamate dehydrogenase encoded by GDH2 was co-expressed with xyd1 in the parent and GRE3 deficient strains. Although each of these enzymes enhanced NADPH consumption on D-glucose, they did not enhance D-xylonate production, suggesting that NADP⁺ was not the main limitation in the current D-xylonate producing strains.

Background The industrially important yeast Saccharomyces cerevisiae is able to grow both in the presence and absence of oxygen. However, the regulation of its metabolism in conditions of intermediate oxygen availability is not well characterised. We assessed the effect of oxygen provision on the transcriptome and proteome of S. cerevisiae in glucose-limited chemostat cultivations in anaerobic and aerobic conditions, and with three intermediate (0.5, 1.0 and 2.8% oxygen) levels of oxygen in the feed gas. Results The main differences in the transcriptome were observed in the comparison of fully aerobic, intermediate oxygen and anaerobic conditions, while the transcriptome was generally unchanged in conditions receiving different intermediate levels (0.5, 1.0 or 2.8% O 2 ) of oxygen in the feed gas. Comparison of the transcriptome and proteome data suggested post-transcriptional regulation was important, especially in 0.5% oxygen. In the conditions of intermediate oxygen, the genes encoding enzymes of the respiratory pathway were more highly expressed than in either aerobic or anaerobic conditions. A similar trend was also seen in the proteome and in enzyme activities of the TCA cycle. Further, genes encoding proteins of the mitochondrial translation machinery were present at higher levels in all oxygen-limited and anaerobic conditions, compared to fully aerobic conditions. Conclusion Global upregulation of genes encoding components of the respiratory pathway under conditions of intermediate oxygen suggested a regulatory mechanism to control these genes as a response to the need of more efficient energy production. Further, cells grown in three different intermediate oxygen levels were highly similar at the level of transcription, while they differed at the proteome level, suggesting post-transcriptional mechanisms leading to distinct physiological modes of respiro-fermentative metabolism.