Wim Soetaert

Microbial metabolomics: past, present and future methodologies.

Microbial metabolomics has received much attention in recent years mainly because it supports and complements a wide range of microbial research areas from new drug discovery efforts to metabolic engineering. Broadly, the term metabolomics refers to the comprehensive (qualitative and quantitative) analysis of the complete set of all low molecular weight metabolites present in and around growing cells at a given time during their growth or production cycle. This review focuses on the past, current and future development of various experimental protocols in the rapid developing area of metabolomics in the ongoing quest to reliably quantify microbial metabolites formed under defined physiological conditions. These developments range from rapid sample collection, instant quenching of microbial metabolic activity, extraction of the relevant intracellular metabolites as well as quantification of these metabolites using enzyme based and or modern high tech hyphenated analytical protocols, mainly chromatographic techniques coupled to mass spectrometry (LC-MSn, GC-MSn, CE-MSn), where n indicates the number of tandem mass spectrometry, and nuclear magnetic resonance spectroscopy (NMR).

Construction and model-based analysis of a promoter library for E. coli: an indispensable tool for metabolic engineering

BackgroundNowadays, the focus in metabolic engineering research is shifting from massive overexpression and inactivation of genes towards the model-based fine tuning of gene expression. In this context, the construction of a library of synthetic promoters of Escherichia coli as a useful tool for fine tuning gene expression is discussed here.ResultsA degenerated oligonucleotide sequence that encodes consensus sequences for E. coli promoters separated by spacers of random sequences has been designed and synthesized. This 57 bp long sequence contains 24 conserved, 13 semi-conserved (W, R and D) and 20 random nucleotides. This mixture of DNA fragments was cloned into a promoter probing vector (pVIK165). The ligation mixtures were transformed into competent E. coli MA8 and the resulting clones were screened for GFP activity by measuring the relative fluorescence units; some clones produced high fluorescence intensity, others weak fluorescence intensity. The clones cover a range of promoter activities from 21.79 RFU/OD600 ml to 7606.83 RFU/OD600 ml. 57 promoters were sequenced and used for promoter analysis. The present results conclusively show that the postulates, which link promoter strength to anomalies in the -10 box and/or -35 box, and to the length of the spacer, are not generally valid. However, by applying Partial Least Squares regression, a model describing the promoter strength was built and validated.ConclusionFor Escherichia coli, the promoter strength can not been linked to anomalies in the -10 box and/or -35 box, and to the length of the spacer. Also a probabilistic approach to relate the promoter sequence to its strength has some drawbacks. However, by applying Partial Least Squares regression, a good correlation was found between promoter sequence and promoter strength. This PLS model can be a useful tool to rationally design a suitable promoter in order to fine tune gene expression.

Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase.

Significance In the transition from a fossil-based to a bio-based economy, bioethanol will be generated from the lignocellulosic biomass of second-generation biofuel crops, such as poplar. The lignin polymers in the plant cell walls represent the main factor determining the recalcitrance of biomass to enzymatic processing. We have grown genetically modified poplars, down-regulated for cinnamoyl-CoA reductase (CCR), an enzyme in the lignin biosynthetic pathway, in field trials in Belgium and France. We show that wood samples derived from the transgenic trees are more easily processed into ethanol. However, strong down-regulation also affected biomass yield. In conclusion, CCR down-regulation may become a successful strategy to improve biomass processing if the yield penalty can be overcome. Lignin is one of the main factors determining recalcitrance to enzymatic processing of lignocellulosic biomass. Poplars (Populus tremula x Populus alba) down-regulated for cinnamoyl-CoA reductase (CCR), the enzyme catalyzing the first step in the monolignol-specific branch of the lignin biosynthetic pathway, were grown in field trials in Belgium and France under short-rotation coppice culture. Wood samples were classified according to the intensity of the red xylem coloration typically associated with CCR down-regulation. Saccharification assays under different pretreatment conditions (none, two alkaline, and one acid pretreatment) and simultaneous saccharification and fermentation assays showed that wood from the most affected transgenic trees had up to 161% increased ethanol yield. Fermentations of combined material from the complete set of 20-mo-old CCR–down-regulated trees, including bark and less efficiently down-regulated trees, still yielded ∼20% more ethanol on a weight basis. However, strong down-regulation of CCR also affected biomass yield. We conclude that CCR down-regulation may become a successful strategy to improve biomass processing if the variability in down-regulation and the yield penalty can be overcome.

The genus Gluconobacter oxydans: comprehensive overview of biochemistry and biotechnological applications.

ABSTRACT The genus Gluconobacter comprises some of the most frequently used microorganisms when it comes to biotechnological applications. Not only has it been involved in “historical” production processes, such as vinegar production, but in the last decades many bioconversion routes for special and rare sugars involving Gluconobacter have been developed. Among the most recent are the biotransformations involved in the production of L-ribose and miglitol, both very promising pharmaceutical lead molecules. Most of these processes make use of Gluconobacters membrane-bound polyol dehydrogenases. However, recently other enzymes have also caught the eye of industrial biotechnology. Among them are dextran dextrinase, capable of transglucosylating substrate molecules, and intracellular NAD-dependent polyol dehydrogenases, of interest for co-enzyme regeneration. As such, Gluconobacter is an important industrial microbial strain, but it also finds use in other fields of biotechnology, such as biosensor-technology. This review aims to give an overview of the myriad of applications for Gluconobacter, with a special focus on some recent developments.

Enzymes for the biocatalytic production of rare sugars

Carbohydrates are much more than just a source of energy as they also mediate a variety of recognition processes that are central to human health. As such, saccharides can be applied in the food and pharmaceutical industries to stimulate our immune system (e.g., prebiotics), to control diabetes (e.g., low-calorie sweeteners), or as building blocks for anticancer and antiviral drugs (e.g., l-nucleosides). Unfortunately, only a small number of all possible monosaccharides are found in nature in sufficient amounts to allow their commercial exploitation. Consequently, so-called rare sugars have to be produced by (bio)chemical processes starting from cheap and widely available substrates. Three enzyme classes that can be used for rare sugar production are keto–aldol isomerases, epimerases, and oxidoreductases. In this review, the recent developments in rare sugar production with these biocatalysts are discussed.

Polyhydroxyalkanoates Production From Low-cost Sustainable Raw Materials

The unnerving price of petroleum will push a major change from a petroleum based economy to a natural feed- stock based economy. Production of polyhydroxyalkanoates (PHAs) using industrial and agriculture by-products can al- low the use of low-cost feedstock to produce materials with specific monomer composition and therefore, with the appro- priate physicochemical properties to be used in a broad range of applications. Depending on the monomer composition, PHAs properties can range from thermoplastic to elastomeric materials. Even though PHA has been described as useful polymers due to thier intrinsic biodegradability and biocompatibility, their high price has limited their application signifi- cantly. The raw material cost has been known to contribute significantly to the manufacturing cost of PHA. Therefore, much research has been carried out using renewable cheap raw materials to replace the expensive commercial medium, which should reduce the overall production cost. In this review, the production of PHAs using low-cost sustainable raw materials such as molasses, whey, lignocelluloses, fats and oils, glycerol and wastewater are described. Finally, the phys- icochemical properties of PHAs produced from various carbon sources are discussed.

The role of cytochrome P450 monooxygenases in microbial fatty acid metabolism

Cytochrome P450 monooxygenases (P450s) are a diverse collection of enzymes acting on various endogenous and xenobiotic molecules. Most of them catalyse hydroxylation reactions and one group of possible substrates are fatty acids and their related structures. In this minireview, the significance of P450s in microbial fatty acid conversion is described. Bacteria and yeasts possess various P450 systems involved in alkane and fatty acid degradation, and often several enzymes with different activities and specificities are retrieved in one organism. Furthermore, P450s take part in the formation of fatty acid‐based secondary metabolites. Finally, there are a substantial number of microbial P450s displaying activity towards fatty acids, but to which no biological role could be assigned despite the often quite intense research.

Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase

Disaccharide phosphorylases are interesting enzymes for the production of sugar phosphates from cheap starting materials and for the synthesis of novel glycosides. Cellobiose phosphorylase (CP) from Cellulomonas uda was subjected to directed evolution in order to create enzyme variants with significantly increased lactose phosphorylase (LP) activity, useful for the production of alpha-D-galactose 1-phosphate. In a first round, random mutagenesis was performed on part of the CP gene and the resultant library was selected on minimal lactose medium. One clone containing six amino acid mutations was found with increased LP activity compared with the wild-type CP enzyme. The negative and neutral mutations were eliminated by site-directed mutagenesis and the resultant enzyme variant containing two amino acid substitutions (T508A/N667T) showed more LP activity than the parent mutant. Saturation mutagenesis of the beneficial sites and screening for improved mutants allowed us to identify the T508I/N667A mutant which has 7.5 times higher specific activity on lactose than the wild-type. The kinetic parameters of the mutants were determined and showed that the increased LP activity was caused by a higher k(cat) value. This is the first report of an engineered CP with modified substrate specificity.

Effect of iclR and arcA knockouts on biomass formation and metabolic fluxes in Escherichia coli K12 and its implications on understanding the metabolism of Escherichia coli BL21 (DE3)

BackgroundGene expression is regulated through a complex interplay of different transcription factors (TFs) which can enhance or inhibit gene transcription. ArcA is a global regulator that regulates genes involved in different metabolic pathways, while IclR as a local regulator, controls the transcription of the glyoxylate pathway genes of the aceBAK operon. This study investigates the physiological and metabolic consequences of arcA and iclR deletions on E. coli K12 MG1655 under glucose abundant and limiting conditions and compares the results with the metabolic characteristics of E. coli BL21 (DE3).ResultsThe deletion of arcA and iclR results in an increase in the biomass yield both under glucose abundant and limiting conditions, approaching the maximum theoretical yield of 0.65 c-mole/c-mole glucose under glucose abundant conditions. This can be explained by the lower flux through several CO2 producing pathways in the E. coli K12 ΔarcAΔiclR double knockout strain. Due to iclR gene deletion, the glyoxylate pathway is activated resulting in a redirection of 30% of the isocitrate molecules directly to succinate and malate without CO2 production. Furthermore, a higher flux at the entrance of the TCA was noticed due to arcA gene deletion, resulting in a reduced production of acetate and less carbon loss. Under glucose limiting conditions the flux through the glyoxylate pathway is further increased in the ΔiclR knockout strain, but this effect was not observed in the double knockout strain. Also a striking correlation between the glyoxylate flux data and the isocitrate lyase activity was observed for almost all strains and under both growth conditions, illustrating the transcriptional control of this pathway. Finally, similar central metabolic fluxes were observed in E. coli K12 ΔarcA ΔiclR compared to the industrially relevant E. coli BL21 (DE3), especially with respect to the pentose pathway, the glyoxylate pathway, and the TCA fluxes. In addition, a comparison of the genome sequences of the two strains showed that BL21 possesses two mutations in the promoter region of iclR and rare codons are present in arcA implying a lower tRNA acceptance. Both phenomena presumably result in a reduced ArcA and IclR synthesis in BL21, which contributes to the similar physiology as observed in E. coli K12 ΔarcAΔiclR.ConclusionsThe deletion of arcA results in a decrease of repression on transcription of TCA cycle genes under glucose abundant conditions, without significantly affecting the glyoxylate pathway activity. IclR clearly represses transcription of glyoxylate pathway genes under glucose abundance, a condition in which Crp activation is absent. Under glucose limitation, Crp is responsible for the high glyoxylate flux, but IclR still represses transcription. Finally, in E. coli BL21 (DE3), ArcA and IclR are poorly expressed, explaining the similar fluxes observed compared to the ΔarcAΔiclR strain.

Towards a carbon-negative sustainable bio-based economy

The bio-based economy relies on sustainable, plant-derived resources for fuels, chemicals, materials, food and feed rather than on the evanescent usage of fossil resources. The cornerstone of this economy is the biorefinery, in which renewable resources are intelligently converted to a plethora of products, maximizing the valorization of the feedstocks. Innovation is a prerequisite to move a fossil-based economy toward sustainable alternatives, and the viability of the bio-based economy depends on the integration between plant (green) and industrial (white) biotechnology. Green biotechnology deals with primary production through the improvement of biomass crops, while white biotechnology deals with the conversion of biomass into products and energy. Waste streams are minimized during these processes or partly converted to biogas, which can be used to power the processing pipeline. The sustainability of this economy is guaranteed by a third technology pillar that uses thermochemical conversion to valorize waste streams and fix residual carbon as biochar in the soil, hence creating a carbon-negative cycle. These three different multidisciplinary pillars interact through the value chain of the bio-based economy.