Growth-coupled anaerobic production of isobutanol from glucose in minimal medium with Escherichia coli.

BACKGROUND: The microbial production of isobutanol holds promise to become a sustainable alternative to fossil-based synthesis routes for this important chemical. Escherichia coli has been considered as one production host, however, due to redox imbalance, growth-coupled anaerobic production of isobutanol from glucose in E. coli is only possible if complex media additives or small amounts of oxygen are provided. These strategies have a negative impact on product yield, productivity, reproducibility, and production costs. RESULTS: In this study, we propose a strategy based on acetate as co-substrate for resolving the redox imbalance. We constructed the E. coli background strain SB001 (ΔldhA ΔfrdA ΔpflB) with blocked pathways from glucose to alternative fermentation products but with an enabled pathway for acetate uptake and subsequent conversion to ethanol via acetyl-CoA. This strain, if equipped with the isobutanol production plasmid pIBA4, showed robust exponential growth (µ = 0.05 h-1) under anaerobic conditions in minimal glucose medium supplemented with small amounts of acetate. In small-scale batch cultivations, the strain reached a glucose uptake rate of 4.8 mmol gDW-1 h-1, a titer of 74 mM and 89% of the theoretical maximal isobutanol/glucose yield, while secreting only small amounts of ethanol synthesized from acetate. Furthermore, we show that the strain keeps a high metabolic activity also in a pulsed fed-batch bioreactor cultivation, even if cell growth is impaired by the accumulation of isobutanol in the medium. CONCLUSIONS: This study showcases the beneficial utilization of acetate as a co-substrate and redox sink to facilitate growth-coupled production of isobutanol under anaerobic conditions. This approach holds potential for other applications with different production hosts and/or substrate-product combinations.

Characterizing and utilizing oxygen-dependent promoters for efficient dynamic metabolic engineering.

Promoters adjust cellular gene expression in response to internal or external signals and are key elements for implementing dynamic metabolic engineering concepts in fermentation processes. One useful signal is the dissolved oxygen content of the culture medium, since production phases often proceed in anaerobic conditions. Although several oxygen-dependent promoters have been described, a comprehensive and comparative study is missing. The goal of this work is to systematically test and characterize 15 promoter candidates that have been previously reported to be induced upon oxygen depletion in Escherichia coli. For this purpose, we developed a microtiter plate-level screening using an algal oxygen-independent flavin-based fluorescent protein and additionally employed flow cytometry analysis for verification. Various expression levels and dynamic ranges could be observed, and six promoters (nar-strong, nar-medium, nar-weak, nirB-m, yfiD-m, and fnrF8) appear particularly suited for dynamic metabolic engineering applications. We demonstrate applicability of these candidates for dynamic induction of enforced ATP wasting, a metabolic engineering approach to increase productivity of microbial strains that requires a narrow level of ATPase expression for optimal function. The selected candidates exhibited sufficient tightness under aerobic conditions while, under complete anaerobiosis, driving expression of the cytosolic F1-subunit of the ATPase from E. coli to levels that resulted in unprecedented specific glucose uptake rates. We finally utilized the nirB-m promoter to demonstrate the optimization of a two-stage lactate production process by dynamically enforcing ATP wasting, which is automatically turned on in the anaerobic (growth-arrested) production phase to boost the volumetric productivity. Our results are valuable for implementing metabolic control and bioprocess design concepts that use oxygen as signal for regulation and induction.

Enabling anaerobic growth of Escherichia coli on glycerol in defined minimal medium using acetate as redox sink.

Glycerol has become an attractive substrate for bio-based production processes. However, Escherichia coli, an established production organism in the biotech industry, is not able to grow on glycerol under strictly anaerobic conditions in defined minimal medium due to redox imbalance. Despite extensive research efforts aiming to overcome these limitations, anaerobic growth of wild-type E. coli on glycerol always required either the addition of electron acceptors for anaerobic respiration (e.g. fumarate) or the supplementation with complex and relatively expensive additives (tryptone or yeast extract). In the present work, driven by model-based calculations, we propose and validate a novel and simple strategy to enable fermentative growth of E. coli on glycerol in defined minimal medium. We show that redox balance could be achieved by uptake of small amounts of acetate with subsequent reduction to ethanol via acetyl-CoA. Using a directed laboratory evolution approach, we were able to confirm this hypothesis and to generate an E. coli strain that shows, under anaerobic conditions with glycerol as the main substrate and acetate as co-substrate, robust growth (µ = 0.06 h-1), a high specific glycerol uptake rate (10.2 mmol/gDW/h) and an ethanol yield close to the theoretical maximum (0.92 mol per mol glycerol). Using further stoichiometric calculations, we also clarify why complex additives such as tryptone used in previous studies enable E. coli to achieve redox balance. Our results provide new biological insights regarding the fermentative metabolism of E. coli and offer new perspectives for sustainable production processes based on glycerol.

Cell-Free Multi-Enzyme Synthesis and Purification of Uridine Diphosphate Galactose.

High costs and low availability of UDP-galactose hampers the enzymatic synthesis of valuable oligosaccharides such as human milk oligosaccharides. Here, we report the development of a platform for the scalable, biocatalytic synthesis and purification of UDP-galactose. UDP-galactose was produced with a titer of 48 mM (27.2 g/L) in a small-scale batch process (200 µL) within 24 h using 0.02 genzyme /gproduct . Through in-situ ATP regeneration, the amount of ATP (0.6 mM) supplemented was around 240-fold lower than the stoichiometric equivalent required to achieve the final product yield. Chromatographic purification using porous graphic carbon adsorbent yielded UDP-galactose with a purity of 92 %. The synthesis was transferred to 1 L preparative scale production in a stirred tank bioreactor. To further reduce the synthesis costs here, the supernatant of cell lysates was used bypassing expensive purification of enzymes. Here, 23.4 g/L UDP-galactose were produced within 23 h with a synthesis yield of 71 % and a biocatalyst load of 0.05 gtotal_protein /gproduct . The costs for substrates per gram of UDP-galactose synthesized were around 0.26 €/g.

Deciphering the physiological response of Escherichia coli under high ATP demand.

One long-standing question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild-type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate-controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.

Increasing ATP turnover boosts productivity of 2,3-butanediol synthesis in Escherichia coli.

BACKGROUND: The alcohol 2,3-butanediol (2,3-BDO) is an important chemical and an Escherichia coli producer strain was recently engineered for bio-based production of 2,3-BDO. However, further improvements are required for realistic applications. RESULTS: Here we report that enforced ATP wasting, implemented by overexpressing the genes of the ATP-hydrolyzing F1-part of the ATPase, leads to significant increases of yield and especially of productivity of 2,3-BDO synthesis in an E. coli producer strain under various cultivation conditions. We studied aerobic and microaerobic conditions as well as growth-coupled and growth-decoupled production scenarios. In all these cases, the specific substrate uptake and 2,3-BDO synthesis rate (up to sixfold and tenfold higher, respectively) were markedly improved in the ATPase strain compared to a control strain. However, aerobic conditions generally enable higher productivities only with reduced 2,3-BDO yields while high product yields under microaerobic conditions are accompanied with low productivities. Based on these findings we finally designed and validated a three-stage process for optimal conversion of glucose to 2,3-BDO, which enables a high productivity in combination with relatively high yield. The ATPase strain showed again superior performance and finished the process twice as fast as the control strain and with higher 2,3-BDO yield. CONCLUSIONS: Our results demonstrate the high potential of enforced ATP wasting as a generic metabolic engineering strategy and we expect more applications to come in the future.

ATPase-based implementation of enforced ATP wasting in Saccharomyces cerevisiae for improved ethanol production.

BACKGROUND: Enforced ATP wasting has been recognized as a promising metabolic engineering strategy to enhance the microbial production of metabolites that are coupled to ATP generation. It also appears to be a suitable approach to improve production of ethanol by Saccharomyces cerevisiae. In the present study, we constructed different S. cerevisiae strains with heterologous expression of genes of the ATP-hydrolyzing F1-part of the ATPase enzyme to induce enforced ATP wasting and quantify the resulting effect on biomass and ethanol formation. RESULTS: In contrast to genomic integration, we found that episomal expression of the αßγ subunits of the F1-ATPase genes of Escherichia coli in S. cerevisiae resulted in significantly increased ATPase activity, while neither genomic integration nor episomal expression of the ß subunit from Trichoderma reesei could enhance ATPase activity. When grown in minimal medium under anaerobic growth-coupled conditions, the strains expressing E. coli's F1-ATPase genes showed significantly improved ethanol yield (increase of 10% compared to the control strain). However, elevated product formation reduces biomass formation and, therefore, volumetric productivity. We demonstrate that this negative effect can be overcome under growth-decoupled (nitrogen-starved) operation with high and constant biomass concentration. Under these conditions, which mimic the second (production) phase of a two-stage fermentation process, the ATPase-expressing strains showed significant improvement in volumetric productivity (up to 111%) compared to the control strain. CONCLUSIONS: Our study shows that expression of genes of the F1-portion of E. coli's ATPase induces ATPase activity in S. cerevisiae and can be a promising way to improve ethanol production. This ATP-wasting strategy can be easily applied to other metabolites of interest, whose formation is coupled to ATP generation.

Synthesis of lipid-linked oligosaccharides by a compartmentalized multi-enzyme cascade for the in vitro N-glycosylation of peptides.

A wide range of glycoproteins can be recombinantly expressed in aglycosylated forms in bacterial and cell-free production systems. To investigate the effect of glycosylation of these proteins on receptor binding, stability, efficacy as drugs, pharmacodynamics and pharmacokinetics, an efficient glycosylation platform is required. Here, we present a cell-free synthetic platform for the in vitro N-glycosylation of peptides mimicking the endoplasmic reticulum (ER) glycosylation machinery of eukaryotes. The one-pot, two compartment multi-enzyme cascade consisting of eight recombinant enzymes including the three Leloir glycosyltransferases, Alg1, Alg2 and Alg11, expressed in E. coli and S. cerevisiae, respectively, has been engineered to produce the core lipid-linked (LL) oligosaccharide mannopentaose-di-(N-acetylglucosamine) (LL-Man5). Pythanol (C20H42O), a readily available alcohol consisting of regular isoprenoid units, was utilized as the lipid anchor. As part of the cascade, GDP-mannose was de novo produced from the inexpensive substrates ADP, polyphosphate and mannose. To prevent enzyme inhibition, the nucleotide sugar cascade and the glycosyltransferase were segregated into two compartments by a cellulose ester membrane with 3.5 kDa cut-off allowing for the effective diffusion of GDP-mannose across compartments. Finally, as a proof-of-principle, pythanyl-linked Man5 and the single-subunit oligosaccharyltransferase Trypanosoma brucei STT3A expressed in Sf9 insect cells were used to in vitro N-glycosylate a synthetic peptide of ten amino acids bearing the eukaryotic consensus motif N-X-S/T.

Broadening the Scope of Enforced ATP Wasting as a Tool for Metabolic Engineering in Escherichia coli.

The targeted increase of cellular adenosine triphosphate (ATP) turnover (enforced ATP wasting) has recently been recognized as a promising tool for metabolic engineering when product synthesis is coupled with net ATP formation. The goal of the present study is to further examine and to further develop the concept of enforced ATP wasting and to broaden its scope for potential applications. In particular, considering the fermentation products synthesized by Escherichia coli under anaerobic conditions as a proxy for target chemical(s), i) a new genetic module for dynamic and gradual induction of the F1 -part of the ATPase is developed and it is found that ii) induction of the ATPase leads to higher metabolic activity and increased product formation in E. coli under anaerobic conditions, and that iii) ATP wasting significantly increases substrate uptake and productivity of growth-arrested cells, which is vital for its use in two-stage processes. To the best of the authors' knowledge, the glucose uptake rate of 6.49 mmol gCDW-1 h-1 achieved with enforced ATP wasting is the highest value reported for nongrowing E. coli cells. In summary, this study shows that enforced ATP wasting can be used to improve yield and titer (in growth-coupled processes) as well as volumetric productivity (in two-stage processes) depending on which of the performance measures is more crucial for the process and product of interest.

Correction to: Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides.

[This corrects the article DOI: 10.1186/s40694-018-0048-3.].

Aspergillus niger is a superior expression host for the production of bioactive fungal cyclodepsipeptides.

BACKGROUND: Fungal cyclodepsipeptides (CDPs) are non-ribosomally synthesized peptides produced by a variety of filamentous fungi and are of interest to the pharmaceutical industry due to their anticancer, antimicrobial and anthelmintic bioactivities. However, both chemical synthesis and isolation of CDPs from their natural producers are limited due to high costs and comparatively low yields. These challenges might be overcome by heterologous expression of the respective CDP-synthesizing genes in a suitable fungal host. The well-established industrial fungus Aspergillus niger was recently genetically reprogrammed to overproduce the cyclodepsipeptide enniatin B in g/L scale, suggesting that it can generally serve as a high production strain for natural products such as CDPs. In this study, we thus aimed to determine whether other CDPs such as beauvericin and bassianolide can be produced with high titres in A. niger, and whether the generated expression strains can be used to synthesize new-to-nature CDP derivatives. RESULTS: The beauvericin and bassianolide synthetases were expressed under control of the tuneable Tet-on promoter, and titres of about 350-600 mg/L for bassianolide and beauvericin were achieved when using optimized feeding conditions, respectively. These are the highest concentrations ever reported for both compounds, whether isolated from natural or heterologous expression systems. We also show that the newly established Tet-on based expression strains can be used to produce new-to-nature beauvericin derivatives by precursor directed biosynthesis, including the compounds 12-hydroxyvalerate-beauvericin and bromo-beauvericin. By feeding deuterated variants of one of the necessary precursors (d-hydroxyisovalerate), we were able to purify deuterated analogues of beauvericin and bassianolide from the respective A. niger expression strains. These deuterated compounds could potentially be used as internal standards in stable isotope dilution analyses to evaluate and quantify fungal spoilage of food and feed products. CONCLUSION: In this study, we show that the product portfolio of A. niger can be expanded from enniatin to other CDPs such as beauvericin and bassianolide, as well as derivatives thereof. This illustrates the capability of A. niger to produce a range of different peptide natural products in titres high enough to become industrially relevant.

The Natural Fungal Metabolite Beauvericin Exerts Anticancer Activity In Vivo: A Pre-Clinical Pilot Study.

Recently, in vitro anti-cancer properties of beauvericin, a fungal metabolite were shown in various cancer cell lines. In this study, we assessed the specificity of this effect by comparing beauvericin cytotoxicity in malignant versus non-malignant cells. Moreover, we tested in vivo anticancer effects of beauvericin by treating BALB/c and CB-17/SCID mice bearing murine CT-26 or human KB-3-1-grafted tumors, respectively. Tumor size and weight were measured and histological sections were evaluated by Ki-67 and H/E staining as well as TdT-mediated-dUTP-nick-end (TUNEL) labeling. Beauvericin levels were determined in various tissues and body fluids by LC-MS/MS. In addition to a more pronounced activity against malignant cells, we detected decreased tumor volumes and weights in beauvericin-treated mice compared to controls in both the allo- and the xenograft model without any adverse effects. No significant differences were detected concerning percentages of proliferating and mitotic cells in tumor sections from treated and untreated mice. However, a significant increase of necrotic areas within whole tumor sections of beauvericin-treated mice was found in both models corresponding to an enhanced number of TUNEL-positive, i.e., apoptotic, cells. Furthermore, moderate beauvericin accumulation was detected in tumor tissues. In conclusion, we suggest beauvericin as a promising novel natural compound for anticancer therapy.

Rational biosynthetic approaches for the production of new-to-nature compounds in fungi.

Filamentous fungi have the ability to produce a wide range of secondary metabolites some of which are potent toxins whereas others are exploited as food additives or drugs. Fungal natural products still play an important role in the discovery of new chemical entities for potential use as pharmaceuticals. However, in most cases they cannot be directly used as drugs due to toxic side effects or suboptimal pharmacokinetics. To improve drug-like properties, including bioactivity and stability or to produce better precursors for semi-synthetic routes, one needs to generate non-natural derivatives from known fungal secondary metabolites. In this minireview, we describe past and recent biosynthetic approaches for the diversification of fungal natural products, covering examples from precursor-directed biosynthesis, mutasynthesis, metabolic engineering and biocombinatorial synthesis. To illustrate the current state-of-the-art, challenges and pitfalls, we lay particular emphasis on the class of fungal cyclodepsipeptides which have been studied longtime for product diversification and which are of pharmaceutical relevance as drugs.

Tet-on, or Tet-off, that is the question: Advanced conditional gene expression in Aspergillus.

In Aspergillus, controlled gene expression is often achieved using the reverse tetracycline-controlled transactivator (rtTA) dependent Tet-on system, whereby transcription is activated in a titratable manner by addition of the tetracycline derivative doxycycline. The complementary Tet-off system utilises the tetracycline-controlled transactivator (tTA) component to quantitatively reduce gene expression. In this study, we utilised a synthetic biological approach to engineer highly optimised Tet-off conditional expression systems in Aspergillus niger and Aspergillus fumigatus. Steps for delivery of these tools include utilising codon optimised cassette components, testing several promoters for improved genetic stability and validating two modified luciferase reporters for highly accurate measurements of gene expression. The Tet-off cassettes developed in this study enable facile and quantitative functional analysis, as validated by Tet-off analysis of genes involved in chitin synthesis and cell wall polarity in A. niger, and para-aminobenzoic acid synthesis in A. fumigatus. We also used a racA(G18V) dominant allele to demonstrate that Tet-off in A. niger enables gene over-expression and downregulation in a single isolate. Additionally, we used the improved luciferase reporters to show that the Tet-off cassette in A. niger enables quantification of gene oscillations. In order to demonstrate that synthetic biological approaches developed here are broadly applicable to engineering transcriptional circuits in filamentous fungi, we used our strategy for improving cassette stability by promoter replacement in the A. niger Tet-on system, which resulted in a modified Tet-on cassette with higher stability in recipient genomes.

Reprogramming the Biosynthesis of Cyclodepsipeptide Synthetases to Obtain New Enniatins and Beauvericins.

Non-ribosomal peptide synthetases are complex multimodular biosynthetic machines that assemble various important and medically relevant peptide antibiotics. An interesting subgroup comprises the cyclodepsipeptide synthetases from fungi synthesizing cyclohexa- and cyclo-octadepsipeptides with antibacterial, anthelmintic, insecticidal, and anticancer properties; some are marketed drugs. We exploit the modularity of these highly homologous synthetases by fusing the hydroxy-acid-activating module of PF1022 synthetase with the amino-acid-activating modules of enniatin and beauvericin synthetase, thus yielding novel hybrid synthetases. The artificial synthetases expressed in Escherichia coli and the fungus Aspergillus niger yielded new cyclodepsipeptides, thus paving the way for the exploration of these derivatives for their bioactivity.

Engineering of Aspergillus niger for the production of secondary metabolites.

BACKGROUND: Filamentous fungi can each produce dozens of secondary metabolites which are attractive as therapeutics, drugs, antimicrobials, flavour compounds and other high-value chemicals. Furthermore, they can be used as an expression system for eukaryotic proteins. Application of most fungal secondary metabolites is, however, so far hampered by the lack of suitable fermentation protocols for the producing strain and/or by low product titers. To overcome these limitations, we report here the engineering of the industrial fungus Aspergillus niger to produce high titers (up to 4,500 mg • l-1) of secondary metabolites belonging to the class of nonribosomal peptides. RESULTS: For a proof-of-concept study, we heterologously expressed the 351 kDa nonribosomal peptide synthetase ESYN from Fusarium oxysporum in A. niger. ESYN catalyzes the formation of cyclic depsipeptides of the enniatin family, which exhibit antimicrobial, antiviral and anticancer activities. The encoding gene esyn1 was put under control of a tunable bacterial-fungal hybrid promoter (Tet-on) which was switched on during early-exponential growth phase of A. niger cultures. The enniatins were isolated and purified by means of reverse phase chromatography and their identity and purity proven by tandem MS, NMR spectroscopy and X-ray crystallography. The initial yields of 1 mg • l-1 of enniatin were increased about 950 fold by optimizing feeding conditions and the morphology of A. niger in liquid shake flask cultures. Further yield optimization (about 4.5 fold) was accomplished by cultivating A. niger in 5 l fed batch fermentations. Finally, an autonomous A. niger expression host was established, which was independent from feeding with the enniatin precursor d-2-hydroxyvaleric acid d-Hiv. This was achieved by constitutively expressing a fungal d-Hiv dehydrogenase in the esyn1-expressing A. niger strain, which used the intracellular α-ketovaleric acid pool to generate d-Hiv. CONCLUSIONS: This is the first report demonstrating that A. niger is a potent and promising expression host for nonribosomal peptides with titers high enough to become industrially attractive. Application of the Tet-on system in A. niger allows precise control on the timing of product formation, thereby ensuring high yields and purity of the peptides produced.