An atlas of rational genetic engineering strategies for improved xylose metabolism in Saccharomyces cerevisiae.

Xylose is the second most abundant carbohydrate in nature, mostly present in lignocellulosic material, and representing an appealing feedstock for molecule manufacturing through biotechnological routes. However, Saccharomyces cerevisiae-a microbial cell widely used industrially for ethanol production-is unable to assimilate this sugar. Hence, in a world with raising environmental awareness, the efficient fermentation of pentoses is a crucial bottleneck to producing biofuels from renewable biomass resources. In this context, advances in the genetic mapping of S. cerevisiae have contributed to noteworthy progress in the understanding of xylose metabolism in yeast, as well as the identification of gene targets that enable the development of tailored strains for cellulosic ethanol production. Accordingly, this review focuses on the main strategies employed to understand the network of genes that are directly or indirectly related to this phenotype, and their respective contributions to xylose consumption in S. cerevisiae, especially for ethanol production. Altogether, the information in this work summarizes the most recent and relevant results from scientific investigations that endowed S. cerevisiae with an outstanding capability for commercial ethanol production from xylose.

PKA regulatory subunit Bcy1 couples growth, lipid metabolism, and fermentation during anaerobic xylose growth in Saccharomyces cerevisiae.

Organisms have evolved elaborate physiological pathways that regulate growth, proliferation, metabolism, and stress response. These pathways must be properly coordinated to elicit the appropriate response to an ever-changing environment. While individual pathways have been well studied in a variety of model systems, there remains much to uncover about how pathways are integrated to produce systemic changes in a cell, especially in dynamic conditions. We previously showed that deletion of Protein Kinase A (PKA) regulatory subunit BCY1 can decouple growth and metabolism in Saccharomyces cerevisiae engineered for anaerobic xylose fermentation, allowing for robust fermentation in the absence of division. This provides an opportunity to understand how PKA signaling normally coordinates these processes. Here, we integrated transcriptomic, lipidomic, and phospho-proteomic responses upon a glucose to xylose shift across a series of strains with different genetic mutations promoting either coupled or decoupled xylose-dependent growth and metabolism. Together, results suggested that defects in lipid homeostasis limit growth in the bcy1Δ strain despite robust metabolism. To further understand this mechanism, we performed adaptive laboratory evolutions to re-evolve coupled growth and metabolism in the bcy1Δ parental strain. The evolved strain harbored mutations in PKA subunit TPK1 and lipid regulator OPI1, among other genes, and evolved changes in lipid profiles and gene expression. Deletion of the evolved opi1 gene partially reverted the strain's phenotype to the bcy1Δ parent, with reduced growth and robust xylose fermentation. We suggest several models for how cells coordinate growth, metabolism, and other responses in budding yeast and how restructuring these processes enables anaerobic xylose utilization.

Towards universal synthetic heterotrophy using a metabolic coordinator.

Engineering the utilization of non-native substrates, or synthetic heterotrophy, in proven industrial microbes such as Saccharomyces cerevisiae represents an opportunity to valorize plentiful and renewable sources of carbon and energy as inputs to bioprocesses. We previously demonstrated that activation of the galactose (GAL) regulon, a regulatory structure used by this yeast to coordinate substrate utilization with biomass formation during growth on galactose, during growth on the non-native substrate xylose results in a vastly altered gene expression profile and faster growth compared with constitutive overexpression of the same heterologous catabolic pathway. However, this effort involved the creation of a xylose-inducible variant of Gal3p (Gal3pSyn4.1), the sensor protein of the GAL regulon, preventing this semi-synthetic regulon approach from being easily adapted to additional non-native substrates. Here, we report the construction of a variant Gal3pMC (metabolic coordinator) that exhibits robust GAL regulon activation in the presence of structurally diverse substrates and recapitulates the dynamics of the native system. Multiple molecular modeling studies suggest that Gal3pMC occupies conformational states corresponding to galactose-bound Gal3p in an inducer-independent manner. Using Gal3pMC to test a regulon approach to the assimilation of the non-native lignocellulosic sugars xylose, arabinose, and cellobiose yields higher growth rates and final cell densities when compared with a constitutive overexpression of the same set of catabolic genes. The subsequent demonstration of rapid and complete co-utilization of all three non-native substrates suggests that Gal3pMC-mediated dynamic global gene expression changes by GAL regulon activation may be universally beneficial for engineering synthetic heterotrophy.

Codon Optimization Improves the Prediction of Xylose Metabolism from Gene Content in Budding Yeasts.

Xylose is the second most abundant monomeric sugar in plant biomass. Consequently, xylose catabolism is an ecologically important trait for saprotrophic organisms, as well as a fundamentally important trait for industries that hope to convert plant mass to renewable fuels and other bioproducts using microbial metabolism. Although common across fungi, xylose catabolism is rare within Saccharomycotina, the subphylum that contains most industrially relevant fermentative yeast species. The genomes of several yeasts unable to consume xylose have been previously reported to contain the full set of genes in the XYL pathway, suggesting the absence of a gene-trait correlation for xylose metabolism. Here, we measured growth on xylose and systematically identified XYL pathway orthologs across the genomes of 332 budding yeast species. Although the XYL pathway coevolved with xylose metabolism, we found that pathway presence only predicted xylose catabolism about half of the time, demonstrating that a complete XYL pathway is necessary, but not sufficient, for xylose catabolism. We also found that XYL1 copy number was positively correlated, after phylogenetic correction, with xylose utilization. We then quantified codon usage bias of XYL genes and found that XYL3 codon optimization was significantly higher, after phylogenetic correction, in species able to consume xylose. Finally, we showed that codon optimization of XYL2 was positively correlated, after phylogenetic correction, with growth rates in xylose medium. We conclude that gene content alone is a weak predictor of xylose metabolism and that using codon optimization enhances the prediction of xylose metabolism from yeast genome sequence data.

Enhanced glycolic acid yield through xylose and cellobiose utilization by metabolically engineered Escherichia coli.

Microbial biorefinery is a promising route toward sustainable production of glycolic acid (GA), a valuable raw material for various industries. However, inherent microbial GA production has limited substrate consumption using either D-xylose or D-glucose as carbon catabolite repression (CCR) averts their co-utilization. To bypass CCR, a GA-producing strain using D-xylose via Dahms pathway was engineered to allow cellobiose uptake. Unlike glucose, cellobiose was assimilated and intracellularly degraded without repressing D-xylose uptake. The final GA-producing E. coli strain (CLGA8) has an overexpressed cellobiose phosphorylase (cep94A) from Saccharophagus degradans 2-40 and an activated glyoxylate shunt pathway. Expression of cep94A improved GA production reaching the maximum theoretical yield (0.51 g GA g-1 xylose), whereas activation of glyoxylate shunt pathway enabled GA production from cellobiose, which further increased the GA titer (2.25 g GA L-1). To date, this is the highest reported GA yield from D-xylose through Dahms pathway in an engineered E. coli with cellobiose as co-substrate.

Identification of a xylose-inducible promoter and its application for improving vitamin B12 production in Sinorhizobium meliloti.

Sinorhizobium meliloti 320 is a vitamin B12 (VB12 ) high-producing strain that has been isolated and identified in our previous study. Because the regulatory toolbox for S. meliloti is limited, we searched for new genetic components and identified the two xylose-inducible promoters PA and PB based on a promoter-probe vector with a green fluorescent protein (GFP) as reporter. Compared with the ParaA promoter from S. meliloti, both promoters exhibited higher induced expression and lower basal expression. Subsequently, the influence of glucose or sucrose on the expression of GFP driven by these three promoters was assayed. Glucose repressed all three promoters, and the expression of ParaA was the lowest in the presence of glucose. Although sucrose repressed the expression of PA by 35% and improved the expression of ParaA by 16%, the expression level of PA was the highest and was 13% higher than that of ParaA . Lastly, we overexpressed the hemA gene in the C4 pathway using the PA promoter in S. meliloti 320, and the VB12 production of the engineered strain increased by 11%. The VB12 production was further increased by 11% by adding 0.1% sodium succinate to the culture medium.

Characterization of two sugar transporters responsible for efficient xylose uptake in an oleaginous yeast Candida tropicalis SY005.

Microbial conversion of lignocellulosic feedstock to the target bioproduct requires efficient assimilation of its constituent sugars, a large part of which comprises of glucose and xylose. This study aims to identify and characterize sugar transporters capable of xylose uptake in an oleaginous strain of the industrially relevant yeast Candida tropicalis. In silico database mining resulted in two sugar transporter proteins- CtStp1 and CtStp2, containing conserved amino acid residues and motifs that have been previously reported to be involved in xylose transport in other organisms. Several softwares predicted the likelihood of 10-12 transmembrane (TM) helices to be present in both the Stps, while molecular modelling showed 12 TM helices that were organized into a typical structure found in the major facilitator superfamily of transporters. Docking with different sugars also predicted favorable interactions. Heterologous expression in a Saccharomyces cerevisiae strain harboring functional xylose metabolic genes validated the broad substrate specificity of the two Stps. Each transporter supported prominent growth of recombinant S. cerevisiae strains on six sugars including xylose at various concentrations. Expression of CtSTP1 and CtSTP2 along with the xylose metabolic genes in yeast transformants grown in presence of xylose was confirmed by transcript detection. Growth curve and sugar consumption profiles revealed uptake of both glucose and xylose simultaneously by the recombinant yeast strains, though CtStp1 showed relatively less effect of glucose repression in mixed sugars and was a better transporter of xylose than CtStp2. Such glucose-xylose utilizing efficient transporters can be effective tools for developing co-fermenting yeasts through genetic engineering in future, with noteworthy applications in renewable biomass utilization.

Engineering transcription factor-based biosensors for repressive regulation through transcriptional deactivation design in Saccharomyces cerevisiae.

BACKGROUND: With the development of engineering the microbial cell factories, biosensors have been used widely for regulation of cellular metabolism and high-throughput screening. However, most of the biosensors constructed in Saccharomyces cerevisiae are designed for transcriptional activation. Very few studies have dedicated to the development of genetic circuit for repressive regulation, which is also indispensable for the dynamic control of metabolism. RESULTS: In this study, through transcriptional deactivation design, we developed transcription-factor-based biosensors to allow repressive regulation in response to ligand. Using a malonyl-CoA sensing system as an example, the biosensor was constructed and systematically engineered to optimize the dynamic range by comparing transcriptional activity of the activators, evaluating the positions and numbers of the operators in the promoter and comparing the effects of different promoters. A biosensor with 82% repression ratio was obtained. Based on this design principle, another two biosensors, which sense acyl-CoA or xylose and downregulate gene expression, were also successfully constructed. CONCLUSIONS: Our work systematically optimized the biosensors for repressive regulation in yeast for the first time. It provided useful framework to construct similar biosensors. Combining the widely reported biosensors for transcriptional activation with the biosensors developed here, it is now possible to construct biosensors with opposing transcriptional activities in yeast.

Exploiting the NADPH pool for xylitol production using recombinant Saccharomyces cerevisiae.

Xylitol is a five-carbon sugar alcohol that has a variety of uses in the food and pharmaceutical industries. In xylose assimilating yeasts, NAD(P)H-dependent xylose reductase (XR) catalyzes the reduction of xylose to xylitol. In the present study, XR with varying cofactor specificities was overexpressed in Saccharomyces cerevisiae to screen for efficient xylitol production. Xylose consumption and xylitol yields were higher when NADPH-dependent enzymes (Candida tropicalis XR and S. cerevisiae Gre3p aldose reductase) were expressed, indicating that heterologous enzymes can utilize the intracellular NADPH pool more efficiently than the NADH pool, where they may face competition from native enzymes. This was confirmed by overexpression of a NADH-preferring C. tropicalis XR mutant, which led to decreased xylose consumption and lower xylitol yield. To increase intracellular NADPH availability for xylitol production, the promoter of the ZWF1 gene, coding for the first enzyme of the NADPH-generating pentose phosphate pathway, was replaced with the constitutive GPD promoter in a strain expressing C. tropicalis XR. This change led to a ~12% increase in xylitol yield. Deletion of XYL2 and SOR1, whose gene products can use xylitol as substrate, did not further increase xylitol yield. Using wheat stalk hydrolysate as source of xylose, the constructed strain efficiently produced xylitol, demonstrating practical relevance of this approach.

Alternative metabolic routes in channeling xylose to cordycepin production of Cordyceps militaris identified by comparative transcriptome analysis.

The responsive mechanism of C. militaris TBRC7358 on xylose utilization was investigated by comparative analysis of transcriptomes, growth kinetics and cordycepin productions. The result showed that the culture grown on xylose exhibited high production yield of cordycepin on dry biomass. Comparing xylose to other carbon sources, a set of significantly up-regulated genes in xylose were enriched in pentose and glucuronate interconversion, and cordycepin biosynthesis. After validating up-regulated genes using quantitative real-time PCR, interestingly, putative alternative 3'-AMP-associated metabolic route on cordycepin biosynthesis was identified. Through reporter metabolites analysis of C. militaris, significant metabolites (e.g., AMP, glycine and L-glutamate) were identified guiding involvement of growth and cordycepin production. These findings suggested that there was a cooperative mechanism in transcriptional control of the supplying precursors pool directed towards the cordycepin biosynthesis through main and putative alternative metabolic routes for leverage of cell growth and cordycepin production on xylose of C. militaris strain TBRC7358.

Transcriptional Changes in the Xylose Operon in Bacillus licheniformis and Their Use in Fermentation Optimization.

The xylose operon is an efficient biological element used for the regulation of gene expression in Bacillus licheniformis. Although the mechanism underlying the xylose-mediated regulation of this operon has been elucidated, the transcriptional changes that occur under various fermentation conditions remain unclear. In this study, the effects of different conditions on xylose operon expression were investigated. Significant upregulation was observed during the transition from the logarithmic phase to the stationary phase (2.5-fold, n = 3, p < 0.01). Glucose suppressed transcription over 168-fold (n = 3, p < 0.01). Meanwhile, the inhibitory effect of glucose hardly strengthened at concentrations from 20 to 180 g/L. Furthermore, the transcription of the xylose operon increased at elevated temperatures (25-42 °C) and was optimal at a neutral pH (pH 6.5-7.0). Based on these findings, relevant fermentation strategies (delaying the induction time, using dextrin as a carbon source, increasing the fermentation temperature, and maintaining a neutral pH) were proposed. Subsequently, these strategies were validated through the use of maltogenic amylase as a reporter protein, as an 8-fold (n = 3, p < 0.01) increase in recombinant enzyme activity compared to that under unoptimized conditions was observed. This work contributes to the development of fermentation optimization and furthers the use of the xylose operon as an efficient expression element.

Identification of modifications procuring growth on xylose in recombinant Saccharomyces cerevisiae strains carrying the Weimberg pathway.

The most prevalent xylose-assimilating pathways in recombinant Saccharomyces cerevisiae, i.e. the xylose isomerase (XI) and the xylose reductase/xylitol dehydrogenase (XR/XDH) pathways, channel the carbon flux through the pentose phosphate pathway and further into glycolysis. In contrast, the oxidative and non-phosphorylative bacterial Weimberg pathway channels the xylose carbon through five steps into the metabolic node α-ketoglutarate (αKG) that can be utilized for growth or diverted into production of various metabolites. In the present study, steps preventing the establishment of a functional Weimberg pathway in S. cerevisiae were identified. Using an original design where a S. cerevisiae strain was expressing the essential four genes of the Caulobacter crescentus pathway (xylB, xylD, xylX, xylA) together with a deletion of FRA2 gene to upregulate the iron-sulfur metabolism, it was shown that the C. crescentus αKG semialdehyde dehydrogenase, XylA was not functional in S. cerevisiae. When replaced by the recently described analog from Corynebacterium glutamicum, KsaD, significantly higher in vitro activity was observed but the strain did not grow on xylose. Adaptive laboratory evolution (ALE) on a xylose/glucose medium on this strain led to a loss of XylB, the first step of the Weimberg pathway, suggesting that ALE favored minimizing the inhibiting xylonate accumulation by restricting the upper part of the pathway. Therefore three additional gene copies of the lower Weimberg pathway (XylD, XylX and KsaD) were introduced. The resulting S. cerevisiae strain (ΔΔfra2, xylB, 4x (xylD-xylX-ksaD)) was able to generate biomass from xylose and Weimberg pathway intermediates were detected. To our knowledge this is the first report of a functional complete Weimberg pathway expressed in fungi. When optimized this pathway has the potential to channel xylose towards value-added specialty chemicals such as dicarboxylic acids and diols.

Design and development of a "Y-shaped" microbial consortium capable of simultaneously utilizing biomass sugars for efficient production of butanol.

Microbial production of chemicals from lignocellulosic biomass is usually hampered by the low efficiency of the simultaneous utilization of C5 and C6 sugars. In nature, this is not a problem because different C5- and C6-utilizing microorganisms cooperate. Nevertheless, the diverse metabolism of microorganisms in nature makes it difficult to synchronize the utilization of biomass sugars toward a specific goal. To address this problem, we sought to develop a novel microbial consortium that can mimic nature's ability of efficiently use biomass sugars, while synchronizing this capability toward a useful goal to maximize the power of nature and engineering. Starting from a completely chromosomally engineered butanol hyper-producing Escherichia coli strain that we developed previously, we developed a consortium comprising two E. coli strains with nearly identical genomic backgrounds, thus creating a "Y-shaped" consortium with two different "heads" (using xylose or glucose) but the same "body" (from glycolysis to butanol production). This "Y-shaped" chimeric consortium achieved the most efficient butanol production from mixed sugars reported to date, by equally efficient and orthogonal consumption of C5 and C6 sugars. Furthermore, we show that the consortium structure is not only adaptive to environmental perturbations, but can be arbitrarily changed to simultaneously utilize C5/C6 sugars in different ratio. The design and development of such a "Y-shaped" chimeric consortium provides a novel approach to address the need for simultaneous efficient utilization of different biomass sugars for the production of useful chemicals.

Towards semi-synthetic microbial communities: enhancing soy sauce fermentation properties in B. subtilis co-cultures.

BACKGROUND: Many fermented foods and beverages are produced through the action of complex microbial communities. Synthetic biology approaches offer the ability to genetically engineer these communities to improve the properties of these fermented foods. Soy sauce is a fermented condiment with a vast global market. Engineering members of the microbial communities responsible for soy sauce fermentation may therefore lead to the development of improved products. One important property is the colour of soy sauce, with recent evidence pointing to a consumer preference for more lightly-coloured soy sauce products for particular dishes. RESULTS: Here we show that a bacterial member of the natural soy sauce fermentation microbial community, Bacillus, can be engineered to reduce the 'browning' reaction during soy sauce production. We show that two approaches result in 'de-browning': engineered consumption of xylose, an important precursor in the browning reaction, and engineered degradation of melanoidins, the major brown pigments in soy sauce. Lastly, we show that these two strategies work synergistically using co-cultures to result in enhanced de-browning. CONCLUSIONS: Our results demonstrate the potential of using synthetic biology and metabolic engineering methods for fine-tuning the process of soy sauce fermentation and indeed for many other natural food and beverage fermentations for improved products.

Parallel experimental evolution reveals a novel repressive control of GalP on xylose fermentation in Escherichia coli.

Efficient xylose utilization will facilitate microbial conversion of lignocellulosic sugar mixtures into valuable products. In Escherichia coli, xylose catabolism is controlled by carbon catabolite repression (CCR). However, in E. coli such as the succinate-producing strain KJ122 with disrupted CCR, xylose utilization is still inhibited under fermentative conditions. To probe the underlying genetic mechanisms inhibiting xylose utilization, we evolved KJ122 to enhance its xylose fermentation abilities in parallel and characterized the potential convergent genetic changes shared by multiple independently evolved strains. Whole-genome sequencing revealed that convergent mutations occurred in the galactose regulon during adaptive laboratory evolution potentially decreasing the transcriptional level or the activity of GalP, a galactose permease. We showed that deletion of galP increased xylose utilization in both KJ122 and wild-type E. coli, demonstrating a common repressive role of GalP for xylose fermentation. Concomitantly, induced expression of galP from a plasmid repressed xylose fermentation. Transcriptome analysis using RNA sequencing indicates that galP inactivation increases transcription levels of many catabolic genes for secondary sugars including xylose and arabinose. The repressive role of GalP for fermenting secondary sugars in E. coli suggests that utilization of GalP as a substitute glucose transporter is undesirable for conversion of lignocellulosic sugar mixtures.

Exploring the xylose paradox in Saccharomyces cerevisiae through in vivo sugar signalomics of targeted deletants.

BACKGROUND: There have been many successful strategies to implement xylose metabolism in Saccharomyces cerevisiae, but no effort has so far enabled xylose utilization at rates comparable to that of glucose (the preferred sugar of this yeast). Many studies have pointed towards the engineered yeast not sensing that xylose is a fermentable carbon source despite growing and fermenting on it, which is paradoxical. We have previously used fluorescent biosensor strains to in vivo monitor the sugar signalome in yeast engineered with xylose reductase and xylitol dehydrogenase (XR/XDH) and have established that S. cerevisiae senses high concentrations of xylose with the same signal as low concentration of glucose, which may explain the poor utilization. RESULTS: In the present study, we evaluated the effects of three deletions (ira2∆, isu1∆ and hog1∆) that have recently been shown to display epistatic effects on a xylose isomerase (XI) strain. Through aerobic and anaerobic characterization, we showed that the proposed effects in XI strains were for the most part also applicable in the XR/XDH background. The ira2∆isu1∆ double deletion led to strains with the highest specific xylose consumption- and ethanol production rates but also the lowest biomass titre. The signalling response revealed that ira2∆isu1∆ changed the low glucose-signal in the background strain to a simultaneous signalling of high and low glucose, suggesting that engineering of the signalome can improve xylose utilization. CONCLUSIONS: The study was able to correlate the previously proposed beneficial effects of ira2∆, isu1∆ and hog1∆ on S. cerevisiae xylose uptake, with a change in the sugar signalome. This is in line with our previous hypothesis that the key to resolve the xylose paradox lies in the sugar sensing and signalling networks. These results indicate that the future engineering targets for improved xylose utilization should probably be sought not in the metabolic networks, but in the signalling ones.

Atmospheric and room temperature plasma (ARTP) mutagenesis enables xylitol over-production with yeast Candida tropicalis.

Xylitol is a sugar alcohol that is used as a sweetener in food and confections. Industrially, xylitol is manufactured by chemical hydrogenation of d-xylose, which requires expensive separation and purification steps as well as high pressure and temperature. The microbial production of xylitol has been examined as an alternative to the chemical process. In this study, a xylitol over-producing strain is breeded by mutagenesis of a newly isolated yeast Candida tropicalis with a new mutation breeding system named atmospheric and room temperature plasma. The highest yield strain T31 was screened among more than 200 mutants with a xylitol yield of 0.61 g/g, which represents a yield increase of 22%. Furthermore, a two-stage dissolved oxygen supply strategy was used in a fermentation process resulting the maximum xylitol yield 0.79 g/g, which makes it a promising candidate for xylitol production. Further biochemical analysis indicating the relative gene expression and the enzyme activity of xylose reductase were higher in mutants than those in the original strain, which partly explained the high yield of xylitol. Thus, this study provides a new strategy to breed the over-producing strains for the xylitol industry.

GXYLT2 accelerates cell growth and migration by regulating the Notch pathway in human cancer cells.

Glucoside xylosyltransferase2 (GXYLT2), a member of the human α-1,3-D-xylosyltransferases, functions to modify the first xylose to the O-Glucose residue on epidermal growth factor (EGF) repeats of Notch receptors. It is well-established that the Notch signaling pathway plays a critical role in proper development and homeostasis. However, the regulatory role of EGF xylosylation in Notch signaling and different cell activities in human cells remains unknown. In this study, we showed that knockdown of GXYLT2 suppressed human cell proliferation and induced G1/S phase cell cycle arrest. GXYLT2 downregulation also inhibited cell migration and invasion, whereas the overexpression of GXYLT2 had the opposite effects. Additionally, GXYLT2 activated Notch signaling and promoted the phosphorylation of MAPKs but not PI3K and Akt. Taken together, our findings indicated that GXYLT2 plays an important role in cell activities via regulation of the Notch signaling.

Enhancing the Secretion of a Glyco-Engineered Anti-CD20 scFv-Fc Antibody in Hairy Root Cultures.

Hairy root (HR) cultures represent an attractive platform for the production of heterologous proteins, due to the possibility of secreting the molecule of interest in the culture medium. The main limitation is the low accumulation yields of heterologous proteins. The aim of this study is to enhance the accumulation of a tumor-targeting antibody with a human-compatible glycosylation profile in HR culture medium. To this aim, the authors produce Nicotiana benthamiana HR cultures expressing the red fluorescent protein (RFP) to easily screen for different auxins able to induce heterologous protein secretion in the medium. The hormone 2,4-dichlorophenoxyacetic acid (2,4-D) is found to induce high accumulation levels (334 mg L-1 ) of RFP in the culture medium. The same protocol is used to improve the secretion of the tumor-targeting, CD20-specific 2B8-FcΔXF recombinant antibody from glyco-engineered ΔXTFT N. benthamiana HR cultures. The addition of 2,4-D determine a 28-fold increase of the accumulation of fully functional 2B8-FcΔXF in the culture medium, at levels of ≈16 mg L-1 . Antibody N-glycosylation profiling reveal the prominent occurrence of GnGn structures and low levels of xylose- and fucose-containing counterparts. This result is the first example of the expression of an engineered anti-CD20 antibody with a scFv-Fc format at high levels in HR.

l-Arabinose induces d-galactose catabolism via the Leloir pathway in Aspergillus nidulans.

l-Arabinose and d-galactose are the principal constituents of l-arabinogalactan, and also co-occur in other hemicelluloses and pectins. In this work we hypothesized that similar to the induction of relevant glycoside hydrolases by monomers liberated from these plant heteropolymers, their respective catabolisms in saprophytic and phytopathogenic fungi may respond to the presence of the other sugar to promote synergistic use of the complex growth substrate. We showed that these two sugars are indeed consumed simultaneously by Aspergillus nidulans, while l-arabinose is utilised faster in the presence than in the absence of d-galactose. Furthermore, the first two genes of the Leloir pathway for d-galactose catabolism - encoding d-galactose 1-epimerase and galactokinase - are induced more rapidly by l-arabinose than by d-galactose eventhough deletion mutants thereof grow as well as a wild type strain on the pentose. d-Galactose 1-epimerase is hyperinduced by l-arabinose, d-xylose and l-arabitol but not by xylitol. The results suggest that in A. nidulans, l-arabinose and d-xylose - both requiring NADPH for their catabolisation - actively promote the enzyme infrastructure necessary to convert ß-d-galactopyranose via the Leloir pathway with its α-anomer specific enzymes, into ß-d-glucose-6-phosphate (the starting substrate of the oxidative part of the pentose phosphate pathway) even in the absence of d-galactose.