NADH Availability Research Articles

Rewiring the reductive TCA pathway and glyoxylate shunt of Escherichia coli for succinate production from corn stover hydrolysate using a two-phase fermentation strategy

Succinate was found extensive applications in the food additives, pharmaceutical, and biopolymers industries. However, the succinate biosynthesis in E. coli required IPTG, lacked NADH, and produced high yields only under anaerobic conditions, unsuitable for cell growth. To overcome these limitations, the glyoxylate shunt and reductive TCA pathway were simultaneously enhanced to produce succinate in both aerobic and anaerobic conditions and achieve a high cell growth meanwhile. On this basis, NADH availability and sugars uptake were increased. Furthermore, an oxygen-dependent promoter was used to dynamically regulate the expression level of key genes of reductive TCA pathway to avoid the usage of IPTG. The final strain E. coli Mgls7-32 could produce succinate from corn stover hydrolysate without an inducer, achieving a titer of 72.8 g/L in 5 L bioreactor (1.2 mol/mol of total sugars). Those findings will aid in the industrial production of succinate.

External redox couple enhanced anammox sludge activity at low temperature: Insight into intracellular resource synthesis

Anaerobic ammonium oxidation (anammox), an energy-efficient deamination biotechnology, faces operational challenges in low-temperature environments. Enhancing the metabolic activity of anammox bacteria (AnAOB) is pivotal for advancing its application in mainstream municipal wastewater treatment. Inspired by the metabolic adaptability of AnAOB and based on our previous findings, this work investigated the enhancement of intracellular ATP and NADH synthesis through the exogenous supply of reduced humic acid (HAred) and H2O2 redox couple, aiming to augment AnAOB activity under low-temperature conditions. Our experimental setup involved continuous dosing of 0.0067 μmol g-1 volatile suspended solid of H2O2 and 10 mg g-1 volatile suspended solid of HAred into a mainstream anammox reactor operated at 15 °C with an influent TN content of 60 mg/L. The results showed that HAred / H2O2 couple succeeded in maintaining the effluent TN at 10.72 ± 0.91 mg l-1. The specific anammox activity, ATP and NADH synthesis levels of sludge increased by 1.34, 2.33 and 6.50 folds, respectively, over the control setup devoid of the redox couple. High-throughput sequencing analysis revealed that the relative abundance of Candidatus Kuenenia after adding HAred / H2O2 couple reached 3.65 % at the end of operation, which was 5.14 folds higher than that of the control group. Further metabolomics analysis underscored an activation in the metabolism of amino acids, nucleotides, and phospholipids, which collectively enhanced the availability of ATP and NADH for the respiratory processes. These findings may provide guidance on strategy development for improving the electron transfer efficiency of AnAOB and underscore the potential of using redox couples to promote the mainstream application of anammox technology.

An optimized reverse β-oxidation pathway to produce selected medium-chain fatty acids in Saccharomyces cerevisiae

BackgroundMedium-chain fatty acids are molecules with applications in different industries and with growing demand. However, the current methods for their extraction are not environmentally sustainable. The reverse β-oxidation pathway is an energy-efficient pathway that produces medium-chain fatty acids in microorganisms, and its use in Saccharomyces cerevisiae, a broadly used industrial microorganism, is desired. However, the application of this pathway in this organism has so far either led to low titers or to the predominant production of short-chain fatty acids.ResultsWe genetically engineered Saccharomyces cerevisiae to produce the medium-chain fatty acids hexanoic and octanoic acid using novel variants of the reverse β-oxidation pathway. We first knocked out glycerolphosphate dehydrogenase GPD2 in an alcohol dehydrogenases knock-out strain (△adh1-5) to increase the NADH availability for the pathway, which significantly increased the production of butyric acid (78 mg/L) and hexanoic acid (2 mg/L) when the pathway was expressed from a plasmid with BktB as thiolase. Then, we tested different enzymes for the subsequent pathway reactions: the 3-hydroxyacyl-CoA dehydrogenase PaaH1 increased hexanoic acid production to 33 mg/L, and the expression of enoyl-CoA hydratases Crt2 or Ech was critical to producing octanoic acid, reaching titers of 40 mg/L in both cases. In all cases, Ter from Treponema denticola was the preferred trans-enoyl-CoA reductase. The titers of hexanoic acid and octanoic acid were further increased to almost 75 mg/L and 60 mg/L, respectively, when the pathway expression cassette was integrated into the genome and the fermentation was performed in a highly buffered YPD medium. We also co-expressed a butyryl-CoA pathway variant to increase the butyryl-CoA pool and support the chain extension. However, this mainly increased the titers of butyric acid and only slightly increased that of hexanoic acid. Finally, we also tested the deletion of two potential medium-chain acyl-CoA depleting reactions catalyzed by the thioesterase Tes1 and the medium-chain fatty acyl CoA synthase Faa2. However, their deletion did not affect the production titers.ConclusionsBy engineering the NADH metabolism and testing different reverse β-oxidation pathway variants, we extended the product spectrum and obtained the highest titers of octanoic acid and hexanoic acid reported in S. cerevisiae. Product toxicity and enzyme specificity must be addressed for the industrial application of the pathway in this organism.

Fluorescence lifetime imaging insight into the effects of Taxol on cell metabolism.

Mitochondria and microtubules are two of the main targets for many anticancer drugs, which has prompted us to investigate immediate effects of these classes of chemotherapeutic agents on the mitochondrial respiratory chain and enzyme-bound metabolic co-factors flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). Cancer cells depend on NADH availability for biogenesis and to protect them from reactive oxygen species (ROS) generated from cancer cell metabolism. Microtubule integrity, besides having key importance in cell division, also has a role in mitochondrial localization and regulation. Herein, we investigate the multiple effects of Taxol on the oxygenation level, optical redox ratio (oxidative phosphorylation vs. glycolytic) and ROS-induced carbonylation in the PC-3 human prostate cancer cell line. Inhibitors rotenone/antimycin and uncoupler 2,4-dinitrophenol (DNP) are also used to investigate the effects of oxygen consumption on the metabolic outcomes.

Microglial reprogramming by Hv1 antagonism protects neurons from inflammatory and glutamate toxicity.

Although the precise mechanisms determining the neurotoxic or neuroprotective activation phenotypes in microglia remain poorly characterized, metabolic changes in these cells appear critical for these processes. As cellular metabolism can be tightly regulated by changes in intracellular pH, we tested whether pharmacological targeting of the microglial voltage-gated proton channel 1 (Hv1), an important regulator of intracellular pH, is critical for activated microglial reprogramming. Using a mouse microglial cell line and mouse primary microglia cultures, either alone, or co-cultured with rat cerebrocortical neurons, we characterized in detail the microglial activation profile in the absence and presence of Hv1 inhibition. We observed that activated microglia neurotoxicity was mainly attributable to the release of tumor necrosis factor alpha, reactive oxygen species, and zinc. Strikingly, pharmacological inhibition of Hv1 largely abrogated inflammatory neurotoxicity not only by reducing the production of cytotoxic mediators but also by promoting neurotrophic molecule production and restraining excessive phagocytic activity. Importantly, the Hv1-sensitive change from a pro-inflammatory to a neuroprotective phenotype was associated with metabolic reprogramming, particularly via a boost in NADH availability and a reduction in lactate. Most critically, Hv1 antagonism not only reduced inflammatory neurotoxicity but also promoted microglia-dependent neuroprotection against a separate excitotoxic injury. Our results strongly suggest that Hv1 blockers may provide an important therapeutic tool against a wide range of inflammatory neurodegenerative disorders.

One-time ozone treatment improves the postharvest quality and antioxidant activity of Actinidia arguta fruit

The major aim of this study was to check the effect of one-time ozonation on selected quality parameters and antioxidant status of Actinidia arguta fruit. For this purpose, A. arguta fruit was ozonated with gas at a concentration of 10 and 100 ppm, which was carried out successively for 5, 15 and 30 min. Next, the selected quality attributes, antioxidants level as well as NADPH and mitochondrial energy metabolism in mini-kiwi fruit after ozonation were analysed. Our research has shown that ozonation reduced the level of yeast and mould without affecting the content of soluble solids or acidity. In turn, ozonation clearly influenced the antioxidant activity and the redox status of the fruit. The ozonated fruit was characterised by a lower level of ROS due to the higher level of low molecular weight antioxidants, as well as the higher activity of superoxide dismutase and catalase. In addition, improved quality and antioxidant activity of the fruit were indirectly due to improved energy metabolism and NADPH level. The ozonated fruit showed a higher level of ATP, due to both higher activity of succinate dehydrogenase and higher availability of NADH. Moreover, the increased level of NAD+ and the activity of NAD+ kinase and glucose-6-phosphate dehydrogenase contributed to higher levels of NADPH in the fruit.

The effect of pesticides on the NADH-supported mitochondrial respiration of permeabilized potato mitochondria

Pesticides can seriously affect the respiratory chain of the mitochondria of many crops, reducing the intensity of plant growth and its yield. Studying the effect of pesticides on the bioenergetic parameters of intact plant mitochondria is a promising approach for assessing their toxicity. In this study, we investigated the effect of some pesticides on isolated potato mitochondria, which used exogenous NADH as a substrate for respiration. We showed that succinate is the most preferred substrate for phosphorylating respiration of intact potato tubers mitochondria. Potato mitochondria poorly oxidize exogenous NADH, despite of the presence of external NADH dehydrogenases. Permeabilization of the mitochondrial membrane with alamethicin increased the availability of exogenous NADH to complex I. However, the pathway of electrons through complex I to complex IV makes intact potato mitochondria susceptible to a number of pesticides such as difenoconazole, fenazaquin, pyridaben and tolfenpyrad, which strongly inhibit the rate of mitochondrial respiration. However, these pesticides only slightly inhibited the rate of oxygen consumption during succinate-supported respiration. Dithianon, the inhibitor of Complex II, is the only pesticide which significantly increased the respiratory rate of NADH-supported respiration of permeabilized mitochondria of potato. Thus, it can be assumed that the alternative NADH dehydrogenases for electron flow represent a factor responsible for plant resistance to xenobiotics, such as mitochondria-targeted pesticides.

Arginine Deprivation and Autophagy Sustain Bioenergetics in Multiple Myeloma

Background and Objective:Multiple myeloma (MM) originates from a clone of transformed plasma cells (PCs) residing in the bone marrow (BM), which establish vicious interactions with the multicellular microenvironment. Autophagy is a highly conserved adaptive strategy that sustains metabolism under stress. In cancer, autophagy has been shown to mediate both onco-suppressive and pro-tumoral functions. Our group demonstrated that MM is addicted to autophagy for clonogenicity, survival, and drug resistance.Little being known on metabolic changes during MM progression, we deployed a comprehensive metabolic analysis at different stages of disease.Experimental design: First, we adopted ultra-high performance liquid and gas chromatography followed by mass spectrometry (UHPLC/GC-MS) on an independent series of 167 samples of bone marrow (BM) and peripheral plasma collected ad hoc from 125 individuals, comprising newly diagnosed MM (n=16), patients with relapsing or progressive disease (n=20), in clinical remission (n=13), with MGUS (n=30), smoldering MM (SMM) (n=17), and age-matched healthy volunteers (n=29). Upon exclusion of drug metabolites and xenobiotic compounds, 284 endogenous metabolites were processed by PCA (unsupervised) and OPLS-DA (supervised) multivariate analyses to identify myeloma-associated metabolites. Second, we explored the expression levels of key metabolic genes in a large series of highly purified BM PC samples from healthy donors (4N), 129 MM, 24 primary plasma cell leukemia (pPCL), 12 secondary PCL (sPCL) cases from a proprietary dataset (GSE66293).ResultsMetabolomics revealed the presence of robust metabolic differences in the progression from MGUS to MM, sustained by reduced amount of lysolipids and amino acids. These findings were validated by HPLC and ELISA in an independent cohort showing that arginine deprivation was associated to progression from MGUS through MM.Upon MM progression, enolase-1 (ENO-1), phosphoglycerate kinase 1 (PGK-1), and dihydrolipoamide dehydrogenase (DLD) were increased, suggesting that branched chain amino acids, alpha-ketoglutarate, and glycine cleavage products are used in MM to sustain NADH availability and energy production. Moreover, clustering analysis of expression profiles of genes involved in tricarboxylic acid (TCA) cycle grouped the majority of pPCL and (t 14;16) cases, showing an up-regulated TCA cycle signature.Using pharmacological inhibitors of glucose metabolism (2-deoxyglucose), lipolysis (atglistatin), and autophagy (bafilomycin-A1) we collected evidence that MM cells generally deploy TCA and oxidative phosphorylation, with glucose catabolism as a major source of ATP. Inhibition of glycolysis was partially compensated for by non-glycolytic oxidative phosphorylation; such compensation correlated with increased expression of transcripts encoding mediators of glutamine anaplerosis, lipid synthesis, lipid droplet biogenesis, lipolysis, and autophagy.Next, we sought to determine whether the adaptation to amino acid starvation through autophagy altered cellular glucose dependence in three MM cell lines chosen on the basis of their cytogenetic alterations: U266 (t 11;14); H929 (t 4; 14) and MM.1S (t 14; 16).Progressive arginine deprivation (range 1000-10 nM) did not affect proliferation in vitro, even in low-glucose media. However, long-term arginine deprivation, or short-term treatment with human recombinant arginase-1, altered the cellular dependence on mitochondrial ATP generation via oxidative phosphorylation and increased glutamine anaplerosis. Indeed, U266, MM.1S and H929 cell lines treated with oligomycin, an inhibitor of complex V in the electron transport chain, invariably showed reduced ATP and higher cell death.ConclusionOverall, our study reveals a substantial contribution of oxidative phosphorylation in MM bioenergetics, and point to decreased arginine availability in the microenvironment as the basis for the activation of metabolic compensatory pathways, offering new putative targets for synthetic lethality. DisclosuresCiceri:GSK: Other: B-thalassemia gene therapy was developed by Fondazione Telethon and Ospedale San Raffaele and has been inlicenced by GSK that provides funding for the clinical trial, Research Funding.

Engineering Clostridium cellulovorans for highly selective n-butanol production from cellulose in consolidated bioprocessing.

Cellulosic n-butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans, a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adhE2 from C. acetobutylicum for n-butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl-CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adhE2 would enhance n-butanol production. In this study, thiolase (thlACA ) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbdCT ) from C. tyrobutyricum were overexpressed in C. cellulovorans adhE2 to increase the flux from acetyl-CoA to butyryl-CoA. In addition, ferredoxin-NAD(P)+ oxidoreductase (fnr), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl-CoA, which resulted in increased production of butyrate and butanol. The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n-butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adhE2-fnrCA -thlACA -hbdCT produced n-butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n-butanol production from cellulosic biomass in consolidated bioprocessing.

Micro-aerobic production of isobutanol with engineered Pseudomonas putida.

Pseudomonas putida KT2440 is emerging as a promising microbial host for biotechnological industry due to its broad range of substrate affinity and resilience to physicochemical stresses. Its natural tolerance towards aromatics and solvents qualifies this versatile microbe as promising candidate to produce next generation biofuels such as isobutanol. In this study, we scaled‐up the production of isobutanol with P. putida from shake flask to fed‐batch cultivation in a 30 L bioreactor. The design of a two‐stage bioprocess with separated growth and production resulted in 3.35 gisobutanol L–1. Flux analysis revealed that the NADPH expensive formation of isobutanol exceeded the cellular catabolic supply of NADPH finally causing growth retardation. Concomitantly, the cell counteracted to the redox imbalance by increased formation of 2‐ketogluconic thereby providing electrons for the respiratory ATP generation. Thus, P. putida partially uncoupled ATP formation from the availability of NADH. The quantitative analysis of intracellular pyridine nucleotides NAD(P)+ and NAD(P)H revealed elevated catabolic and anabolic reducing power during aerobic production of isobutanol. Additionally, the installation of micro‐aerobic conditions during production doubled the integral glucose‐to‐isobutanol conversion yield to 60 mgisobutanol gglucose –1 while preventing undesired carbon loss as 2‐ketogluconic acid.

Inside Front Cover Image, Volume 118, Number 2, February 2021

The cover image is based on the Article Effects of benzyl viologen on increasing NADH availability, acetate assimilation, and butyric acid production by Clostridium tyrobutyricum by Hongxin Fu et al., https://doi.org/10.1002/bit.27602.

Modulating redox metabolism to improve isobutanol production in Shimwellia blattae

BackgroundIsobutanol is a candidate to replace gasoline from fossil resources. This higher alcohol can be produced from sugars using genetically modified microorganisms. Shimwellia blattae (p424IbPSO) is a robust strain resistant to high concentration of isobutanol that can achieve a high production rate of this alcohol. Nevertheless, this strain, like most strains developed for isobutanol production, has some limitations in its metabolic pathway. Isobutanol production under anaerobic conditions leads to a depletion of NADPH, which is necessary for two enzymes in the metabolic pathway. In this work, two independent approaches have been studied to mitigate the co-substrates imbalance: (i) using a NADH-dependent alcohol dehydrogenase to reduce the NADPH dependence of the pathway and (ii) using a transhydrogenase to increase NADPH level.ResultsThe addition of the NADH-dependent alcohol dehydrogenase from Lactococcus lactis (AdhA) to S. blattae (p424IbPSO) resulted in a 19.3% higher isobutanol production. The recombinant strain S. blattae (p424IbPSO, pIZpntAB) harboring the PntAB transhydrogenase produced 39.0% more isobutanol than the original strain, reaching 5.98 g L−1 of isobutanol. In both strains, we observed a significant decrease in the yields of by-products such as lactic acid or ethanol.ConclusionsThe isobutanol biosynthesis pathway in S. blattae (p424IbPSO) uses the endogenous NADPH-dependent alcohol dehydrogenase YqhD to complete the pathway. The addition of NADH-dependent AdhA leads to a reduction in the consumption of NADPH that is a bottleneck of the pathway. The higher consumption of NADH by AdhA reduces the availability of NADH required for the transformation of pyruvate into lactic acid and ethanol. On the other hand, the expression of PntAB from E. coli increases the availability of NADPH for IlvC and YqhD and at the same time reduces the availability of NADH and thus, the production of lactic acid and ethanol. In this work it is shown how the expression of AdhA and PntAB enzymes in Shimwellia blattae increases yield from 11.9% to 14.4% and 16.4%, respectively.

Effects of benzyl viologen on increasing NADH availability, acetate assimilation, and butyric acid production by Clostridium tyrobutyricum

Clostridium tyrobutyricum produces butyric and acetic acids from glucose. The butyric acid yield and selectivity in the fermentation depend on NADH available for acetate reassimilation to butyric acid. In this study, benzyl viologen (BV), an artificial electron carrier that inhibits hydrogen production, was used to increase NADH availability and butyric acid production while eliminating acetic acid accumulation by facilitating its reassimilation. To better understand the mechanism of and find the optimum condition for BV effect on enhancing acetate assimilation and butyric acid production, BV at various concentrations and addition times during the fermentation were studied. Compared with the control without BV, the addition of 1 μM BV increased butyric acid production from glucose by ∼50% in yield and ∼29% in productivity while acetate production was completely inhibited. Furthermore, BV also increased the coutilization of glucose and exogenous acetate for butyric acid production. At a concentration ratio of acetate (g/L) to BV (mM) of 4, both acetate assimilation and butyrate biosynthesis increased with increasing the concentrations of BV (0-6.25 μM) and exogenous acetate (0-25 g/L). In a fed-batch fermentation with glucose and ∼15 g/L acetate and 3.75 μM BV, butyrate production reached 55.9 g/L with productivity 0.93 g/L/h, yield 0.48 g/g, and 97.4% purity, which would facilitate product purification and reduce production cost. Manipulating metabolic flux and redox balance via BV and acetate addition provided a simple to implement metabolic process engineering approach for butyric acid production from sugars and biomass hydrolysates.

Improved production of 2,3-butanediol and isobutanol by engineering electron transport chain in Escherichia coli.

The electron transport chain (ETC) is one of the major energy generation pathways in microorganisms under aerobic condition. Higher yield of ATP can be achieved through oxidative phosphorylation with consumption of NADH than with substrate level phosphorylation. However, most value-added metabolites are in an electrochemically reduced state, which requires reducing equivalent NADH as a cofactor. Therefore, optimal production of value-added metabolites should be balanced with ETC in terms of energy production. In this study, we attempted to reduce the activity of ETC to secure availability of NADH. The ETC mutants exhibited poor growth rate and production of fermentative metabolites compared to parental strain. Introduction of heterologous pathways for synthesis of 2,3-butanediol and isobutanol to ETC mutants resulted in increased titres and yields of the metabolites. ETC mutants yielded higher NADH/NAD+ ratio but similar ATP content than that by the parental strain. Furthermore, ETC mutants operated fermentative metabolism pathways independent of oxygen supply in large-scale fermenter, resulting in increased yield and titre of 2,3-butanediol. Thus, engineering of ETC is a useful metabolic engineering approach for production of reduced metabolites.

The Expression of NOX From Synthetic Promoters Reveals an Important Role of the Redox Status in Regulating Secondary Metabolism of Saccharopolyspora erythraea.

Redox cofactors play a pivotal role in primary cellular metabolism, whereas the clear link between redox status and secondary metabolism is still vague. In this study we investigated effects of redox perturbation on the production of erythromycin in Saccharopolyspora erythraea by expressing the water-forming NADH oxidase (NOX) from Streptococcus pneumonia at different levels with synthetic promoters. The expression of NOX reduced the intracellular [NADH]/[NAD+] ratio significantly in S. erythraea which resulted in an increased production of erythromycin by 19∼29% and this increment rose to 60% as more oxygen was supplied. In contrast, the lower redox ratio resulted in a decreased production of another secondary metabolite, the reddish pigment 7-O-rahmnosyl flaviolin. The metabolic shifts of secondary metabolism results in a higher NADH availability which compensates for its oxidization via NOX. The expression of the erythromycin biosynthesis gene cluster (BGC) in the NOX-expression strains was upregulated as the activity of diguanylate cyclase was inhibited moderately by NADH. This study also suggested that lower intracellular [NADH]/[NAD+] ratio benefits the biosynthesis of erythromycin by potentially affecting the biosynthesis of the secondary messenger, bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP), which may stimulate the positive regulation of erythromycin BGC via BldD. The present work provides a basis for future cofactor manipulation in S. erythraea to improve the industrial production of erythromycin.

Osmolality as a lever to modulate the N-glycolylneuraminicacid (Neu5Gc) level of a recombinant glycoprotein produced in Chinese hamster ovary cells.

Glycoproteins could be highly sialylated, and controlling the sialic acid levels for some therapeutic proteins is critical to ensure product consistency and efficacy. N-acetylneuraminic acid (Neu5Ac, or NANA) and N-glycolylneuraminic acid (Neu5Gc, or NGNA) are the two most common forms of sialic acids produced in mammalian cells. As Neu5Gc is not produced in humans and can elicit immune responses, minimizing Neu5Gc formation is important in controlling this quality attribute for complex glycoproteins. In this study, a sialylated glycoprotein was used as the model molecule to study the effect of culture osmolality on Neu5Gc. A 14-day fed-batch process with osmolality maintained at physiological levels produced high levels of Neu5Gc. Increase of culture osmolality reduced the Neu5Gc level up to 70-80%, and the effect was proportional to the osmolality level. Through evaluating different osmolality conditions (300-450 mOsm/kg) under low or high pCO2 , we demonstrated that osmolality could be an effective process lever to modulate the Neu5Gc level. Potential mechanism of osmolality impact on Neu5Gc is discussed and is hypothesized to be cytosol NADH availability related. Compared with cell line engineering efforts, this simple process lever provides the opportunity to readily modulate the Neu5Gc level in a cell culture environment.

Development and characterization of Saccharomyces cerevisiae strains genetically modified to over-express the pentose phosphate pathway regulating transcription factor STB5 in the presence of xylose

Several enzymes in the pentose phosphate pathway of Saccharomyces cerevisiae have been identified as relating to the constraint of xylose consumption and conversion to ethanol. However, no strategy has been proposed for simultaneous regulation of all contributing enzymes. If multiple enzymes contribute to constraint, over expression of a native transcription factor controlling the entire constraining pathway may provide optimal pathway wide regulation. Further characterization of this strain on both pure sugars and lignocellulosic hydrolysates would provide an opportunity to identify additional bottlenecks not addressed by the modification of the pentose phosphate pathway expression pattern. A series of strains were developed expressing STB5 and PGI1 under the control of a novel xylose inducible promoter. Increased transcription of STB5 and its regulatory targets was verified via qRT-PCR. No statistically significant difference was found in terms of xylose consumption or ethanol yield in these strains versus control strains. Xylose consumption through both the fermentative and respiratory pathways appeared to be related to oxygen availability and culture density with high-density (low oxygen) cultures consuming xylose more slowly than low-density cultures. The maximum specific consumption rate for high-density cultures was 0.21 g xylose/gDCW/h versus 0.41 g xylose/gDCW/h in lower density cultures. Statistically similar ethanol yields at high and low density (approximately 0.25 g ethanol/ g xylose) suggest that the maximum rate of fermentation is linked to the rate of respiration in a stoichiometric fashion. This study did not find evidence supporting the pentose phosphate pathway constraint identified in other works. Instead, NAD + availability mediated by oxygen availability and citric acid cycle flux was suggested to limit fermentation. While increased aeration could provide increased conversion of NAD + to NADH (and a stoichiometric increase in fermentation flux), this increase would not be expected improve ethanol yield beyond 50% of the theoretical maximum. Based on these findings, future work in Saccharomyces cerevisiae development for fermentation of lignocellulosic hydrolysates should focus on balancing NAD + / NADH availability through non-respiratory pathways.

Higher NADH Availability of Lager Yeast Increases the Flavor Stability of Beer.

Flavor stability is a significant concern to brewers as the staling compounds impart unpleasant flavor to beer. Thus, yeasts with antistaling ability have been engineered to produce beer with improved flavor stability. Here, we proposed that increasing the NADH availability of yeast could improve the flavor stability of beer. By engineering endogenous pathways, we obtained an array of yeast strains with a higher reducing activity. Then, we carried out beer fermentation with these strains and found that the antistaling capacities of the beer samples were improved. For a better understanding of the underlying mechanism, we compared the flavor profiles of these strains. The production of staling components was significantly decreased, whereas the content of antistaling components, such as SO2, was increased, in line with the increased antistaling ability. The other aroma components were marginally changed, indicating that this concept was useful for improving the antistaling stability without changing the flavor of beer.

Metabolic engineering of Pseudomonas denitrificans for the 1,3-propanediol production from glycerol

Bio-production of 1,3-propanediol (1,3-PDO) from glycerol was studied using Pseudomonas denitrificans as host, which aerobically synthesizes coenzyme B12, an essential cofactor of glycerol dehydratase (GDHt). P. denitrificans was transformed with the 1,3-PDO synthesis pathway composed of GDHt and 1,3-PDO oxidoreductase (PDOR), and its putative 3-hydroxypropionaldehyde (3-HPA) dehydrogenase(s), leading to the production of 3-hydroxypropioninc acid form the intermediary 3-HPA, was identified and deleted. In addition, to improve the availability of NADH for PDOR, oxidation of NADH in the electron transport chain was disturbed by deletion of the nuo operon and/or ndh gene. Finally, acetate formation pathway was eliminated. One resulting strain could produce 68.95 mM 1,3-PDO with the yield of 0.92 mol 1,3-PDO/mol glycerol on flask scale and 440 mM with the yield of 0.89 mol 1,3-PDO/mol glycerol in a fed-batch bioreactor experiment. This study demonstrates that P. denitrificans is a promising recombinant host for the production of 1,3-PDO from glycerol.

PS1362 TRYPTOPHAN DEPRIVATION DUE TO INCREASED IDO‐1 TRIGGERS A METABOLIC ADAPTIVE RESPONSE AND DOWN‐REGULATES CD38 IN MULTIPLE MYELOMA

Background:There is an increasing interest about the role of amino acid degrading enzymes in cancer immunotherapy. Indoleamine 2,3 dioxygenase (IDO) is the rate‐limiting enzyme in the kynurenine pathway, catabolizing tryptophan (trp) to kynurenine (kyn), sustaining immune evasion and inhibiting effector T cells.Aims:Investigating the effects of trp deprivation in multiple myeloma (MM).Methods:By combining different assays (ELISA, RT‐PCR, immuofluorescence, flow cytometry and western blot) we evaluated the metabolic adaptive response to trp shortage in four human MM cell lines (HMCLs, MM1.s, H929, U266, OPM2), chosen for their cytogenetic alterations and previously characterized for differential expression of CD38 and c‐myc and primary BM samples of MGUS and MM. We validated in‐vitro results looking at the expression levels of key metabolic genes in a large series of highly purified BM PC samples from healthy donors (4N), 129 MM, 24 primary plasma cell leukemia (pPCL), 12 secondary PCL (sPCL) cases from a proprietary dataset (GSE66293). Then, we investigated the effects of IDO inhibition to delay MM engraftment in vivo.Results:In a series of 20 healthy, 25 MGUS, 30 newly diagnosed MM patient, 20 relapsed/refractory subjects IDO was progressively increased. As consequence, trp was reduced and Kyn increased. Trp deprivation induced cell cycle arrest, while progressive trp shortage (range 1000–10 nM) did not affect proliferation in vitro, as evaluated up to 72 hours. Long‐term trp deprivation altered the cellular dependence on mitochondrial ATP generation via oxidative phosphorylation and increased glutamine anaplerosis, while surface expression of CD38 and intra‐cellular amount of c‐myc was reduced. Metabolic re‐shape was associated to increased isocitrate dehydrogenase (IDH‐1), enolase‐1 (ENO‐1), phosphoglycerate kinase 1 (PGK‐1), and dihydrolipoamide dehydrogenase (DLD), suggesting that branched chain amino acids with tolerogenic meaning like trp are used in MM to sustain NADH availability and energy production.On the other hand, long‐term trp deprivation induced expression of CXCR4 and IRF4 in a time dependent manner, associated to increased p‐STAT3 without affecting other transcription factors relevant for MM cells like c‐myc or Helios. Counterintuitively, monoclonal componenent secretion was not affected, and both intra‐cellular and extra‐cellular lambda chain amount in U266 cells did not change after 72 hours of trp starvation.We confirmed these findings in short‐term culture of CD138+ primary plasma cells with autologous feeder cells in presence of trp‐deprived media: after 18 hours, IRF4 and CXCR4 expression was increased and CD38 reduced.In the GSE66293 MM, progression was associated to increased expression of ENO‐1, PGK‐1, and DLD, but were independent from CD38 expression, suggesting that changes in transcription of genes involved in bioenergetics could be clinically relevant, but not able to reflect the complex networking between malignant PCs and microenvironment.In zebrafishes IDO inhibitor epacadostat could not delay MM engraftment. Increased doses of epacadostat (range 500–1000 nM) were cytotoxic against both human T‐CD4+cells and T‐CD8+ cells and for this reason could not be further tested in pre‐clinical in‐vivo models.Summary/Conclusion:Trp shortage is associated to progression from MGUS through MM due to increased IDO, contributing to metabolic adaptation. However, other soluble factors are involved in co‐opting adaptive metabolic phenotype, since IDO inhibition is not sufficient to delay MM engraftment in vivo.