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Microbiology is an extraordinary field which has been, and will continue to be, central to the advancement of human cultures. In fact, human cultures and even our planet would not be as they are today if microorganisms and microbiology would not exist. Basic, but at the same time, complex microbiological processes play key roles in biodegradation and nutrient (re)cycling, climate change, disease and biotechnology. Due to its importance, a brief history about microbiology is provided in Chapter 1 and the related (old or new) biotechnological applications are described. The importance of the microorganism-workhorses is highlighted and a “start-at-the-end” approach is recommended for choosing the appropriate microbe for the production of the final product. A brief introduction to clostridia species and their potential for the sustainable production of ester compounds is provided. Lastly, the importance of the revolutionary CRISPR-Cas genome engineering tools is highlighted.Chapter 2 reviews the state-of-the-art technologies used for the microbial production of short and medium chain esters. Several enzymatic processes are described with main focus on the AAT enzymes. In addition, several metabolic engineering processes are reviewed, along with the physical properties of the final ester product. Lastly, an outlook is provided on other products (e.g. alcohols, carboxylic acids and diols) that can be derived from (microbially produced) esters.In Chapter 3, the unique Eat1 enzyme was further characterized using in vitro and in vivo approaches. Two new activities (alcoholysis and thiolysis) were added to the existing activities (AAT, esterase and thioesterase) of Eat1. Alcoholysis appears to be the main activity of the enzyme as it highly prefers to exchange the alcohol moiety of the ester molecule with another free alcohol. This discovery exposes the limitations for using Eat1 as an AAT but also provides insights into the portfolio of ester compounds produced by many yeast

Clostridium beijerinckii has been an organism of great interest for industrial biotechnology for about a century because it can degrade a wide range of sugars to produce acetone, butanol, ethanol, and isopropanol, chemicals used in consumer goods (paints, cosmetics, food ingredients) and as alternatives to transport fuel. Still, C. beijerinckii has other less studied physiological traits, such as the formation of intracellular compartments, spores and microcompartments. Therefore, we decided to study sporulation and microcompartment formation in C. beijerinckii using functional genomics.Sporulation is a cell differentiation process that occurs in all Firmicutes. It is triggered when the cells are exposed to an unfavorable environmental condition (presence of oxygen, high pH variation), and yields spores, metabolically inactive cells resistant to harsh conditions (UV, chemicals, heat, oxygen). In Chapter 1, the research that has been done in the last 30 years on sporulation in solvent-producing clostridia, including the latest updates on the molecular regulation of sporulation in solventogenic clostridia, is presented. Common triggers and regulation mechanisms of sporulation in several solventogenic species are identified, underlining the differences and similarities between species. Potential links in the regulation of sporulation and solvent production are pinpointed. This chapter highlights the need for more studies on signal transduction pathways, transcriptional and post-translational regulation of sporulation, and solvent production. A better understanding of the connections between both physiological phenomena would provide us with relevant targets for strain engineering of solventogenic clostridia.Metabolic engineering of Clostridium used to be tedious and hampered the studies on strain physiology. An efficient and fast engineering tool was highly desired. Chapter 2 describes the adaptation of a CRISPR-Cas9 method for use in Clostridium beijerinckii. The tool

In order to reduce CO2 emissions and the use of fossil resources, microbial production platforms offer a vast range of possibilities. Many different compounds and/or precursors can be produced by micro-organisms from renewable resources and their production can be enhanced by means of metabolic engineering.Ethyl acetate is a popular solvent used in various applications but production mainly relies on petrochemical resources. Whereas yeast can naturally produce ethyl acetate, metabolism does not allow for a complete anaerobic process. Especially the production of inexpensive bulk chemicals, like ethyl acetate, need to proceed at high yields and rates to be economically viable. Therefore, anaerobic processes are desired as more carbons and electrons are directed to formation instead of biomass. With the identification of the bulk ethyl acetate producing enzyme ethanol acetyltransferase (Eat1) the path was finally cleared for targeted engineering approaches and the use of alternative production hosts for the bio-based production of ethyl acetate.This research explored different metabolic engineering approaches in Escherichia coli to improve the key parameters titer, rate, and yield (TRY). Three anaerobic scenarios were followed up on that differed in their use of electrons and carbons for the desired product. An integrated strategy on in situ product removal via gas stripping was included to benefit downstream processing and to prevent process limitations by product toxicity. The resulting bioprocess with optimizations on expression, protein and process level increased product yields to above 70 % of the pathway yield, and productivities by a factor 1.4. The thesis reflects on the results and discusses the next steps towards efficient bio-based ethyl acetate production

This PhD thesis describes the relation of the structure of RNA with its effect on protein expression and the efficacy of CRISPR-Cas12a. Direct control of protein expression by RNA is done via so-called riboswitches, which change their structure depending on the presence of a ligand or external factors like temperature. A theophylline-responsive riboswitch based on the phage T4 td group I intron is described in detail and a method is proposed to obtain riboswitches based on this intron. This method was used to generate a riboswitch library for 3-methylxanthine, a compound closely related to theophylline, and the unrelated citrulline. The second part focuses on the effect of coding and non-coding mRNA on gene translation. Coding regions that are in close proximity of other coding regions have their translation coupled to each other, and the order of the genes appears to be of surprisingly little influence. The next part investigates the effect of the 3’-UTR and the intergenic region. These non-coding elements may have a lot of influence on the translation rate, by a mechanism that is not yet understood. The last part shines a light on the influence of the RNA structure of the CRISPR-Cas12a crRNA. The pre-crRNA forms a characteristic pseudoknot structure that is required for the recognition by Cas12a, which then turns it into crRNA. The low GC content of the pseudoknot allows this structure to be outcompeted by other potential structures, which renders the pre-crRNA unrecognisable. A design strategy was proposed to improve the efficacy of CRISPR-Cas12a.

Enzymes are proteins that catalyse chemical reactions. They accelerate the reaction by lowering the activation energy, thereby allowing the equilibrium to be reached more quickly. The molecules upon which enzymes may act are called substrates and the enzyme converts the substrates into molecules known as products. Nearly all metabolic processes in a living cell need enzyme catalysis in order to proceed at rates fast enough to sustain life. In Chapter 1, the relevance of enzymes for society is described. This dates back to more than 8000 years ago, when people unknowingly already made use of enzymatic conversions via fermentation by whole-cell microorganisms to make early forms of bread and beer. In the last two centuries, our knowledge on how enzymes are functioning increased tremendously. They have remarkable features that make them interesting for industrial applications, such as a high specificity and selectivity, and they can make processes ‘greener’ by replacing often polluting or toxic chemical reactions. This resulted in the large scale industrial production and application of enzymes in diverse areas ranging from food industry and detergents to pharma and DNA technology. Currently, there is both the need and the room to increase the number of enzyme applications. A more extensive implementation of enzymes in industry is very important because, sooner or later, we have to make the shift from a fossil fuel based economy to a biobased economy. At this moment, only 5% of all chemical products are produced biologically. For the latter, only 150-170 of the 3000 different types of known enzymes are being applied. It is estimated that only 1% of enzymes is known and, therefore, there is much room to extend the number of industrial enzyme applications. But how to find novel enzymes for these applications? Enzymes can be obtained from nature (natural evolution, screening of metagenomics libraries) or by enzyme engineering via laboratory evolution (screening of random

Ester production by microorganisms is of great importance to biotechnological processes. These volatile compounds impart pleasant, fruity aromas to beer, wine, and other fermented products. Yeast in particular are well known for their ability to produce volatile esters. The genetic and enzymatic intricacies of ester production have been the focus of many studies, mainly in S. cerevisiae. Despite this, ester synthesis in yeast is not fully understood. This holds particularly true for yeasts that produce high amounts of ethyl acetate from sugars, such as Kluyveromyces marxianus and Wickerhamomyces anomalus. As was introduced in Chapter 1, this ability has been described more than a century ago, but the enzymatic mechanisms behind the synthesis were unclear. Circumstantial evidence suggested that an alcohol acetyltransferase (AAT) was responsible for the ethyl acetate formation. Ethyl acetate is a versatile commodity chemical that is currently produced in unsustainable processes. Understanding the enzymes responsible for bulk ethyl acetate synthesis in yeast could enable rational design of novel production strains. This, in turn could facilitate the development of biobased ethyl acetate production processes. This thesis has made several important breakthroughs in the field of ester production in yeast, particularly ethyl acetate. The most significant of these was the discovery of the elusive enzyme responsible for bulk ethyl acetate synthesis in yeast.The identification of the ethyl acetate-producing enzyme in W. anomalus is described in Chapter 2. The purified enzyme showed AAT activity with ethanol and acetyl-CoA and was therefore named Ethanol acetyltransferase 1 (Eat1). Production of Eat1 in Escherichia coli enabled efficient ethyl acetate production in E. coli. The enzyme could thus potentially be used to develop new biobased ethyl acetate production processes. However, Eat1 was also able to function as a thioesterase and esterase active against acetyl-CoA and eth

Esters are essential for the flavor and aroma of fermented products, and are mainly produced by alcohol acyl transferases (AATs). A recently discovered AAT family named Eat (Ethanol acetyltransferase) contributes to ethyl acetate synthesis in yeast. However, its effect on the synthesis of other esters is unknown. In this study, the role of the Eat family in ester synthesis was compared to that of other Saccharomyces cerevisiae AATs (Atf1p, Atf2p, Eht1p, and Eeb1p) in silico and in vivo. A genomic study in a collection of industrial S. cerevisiae strains showed that variation of the primary sequence of the AATs did not correlate with ester production. Fifteen members of the EAT family from nine yeast species were overexpressed in S. cerevisiae CEN.PK2-1D and were able to increase the production of acetate and propanoate esters. The role of Eat1p was then studied in more detail in S. cerevisiae CEN.PK2-1D by deleting EAT1 in various combinations with other known S. cerevisiae AATs. Between 6 and 11 esters were produced under three cultivation conditions. Contrary to our expectations, a strain where all known AATs were disrupted could still produce, e.g., ethyl acetate and isoamyl acetate. This study has expanded our understanding of ester synthesis in yeast but also showed that some unknown ester-producing mechanisms still exist.

The euryarchaeon Thermococcus kodakarensis is a well-characterized anaerobic hyperthermophilic heterotroph and due to the availability of genetic engineering systems it has become one of the model organisms for studying Archaea. Despite this prominent role among the Euryarchaeota, no data about the ploidy level of this species is available. While polyploidy has been shown to exist in various Euryarchaeota, especially Halobacteria, the chromosome copy number of species belonging to one of the major orders within that phylum, i.e., the Thermococcales (including Thermococcus spp. and Pyrococcus spp.), has never been determined. This prompted us to investigate the chromosome copy number of T. kodakarensis. In this study, we demonstrate that T. kodakarensis is polyploid with a chromosome copy number that varies between 7 and 19 copies, depending on the growth phase. An apparent correlation between the presence of histones and polyploidy in Archaea is observed

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is an essential electron donor in all organisms. It provides the reducing power that drives numerous anabolic reactions, including those responsible for the biosynthesis of all major cell components and many products in biotechnology. The efficient synthesis of many of these products, however, is limited by the rate of NADPH regeneration. Hence, a thorough understanding of the reactions involved in the generation of NADPH is required to increase its turnover through rational strain improvement. Traditionally, the main engineering targets for increasing NADPH availability have included the dehydrogenase reactions of the oxidative pentose phosphate pathway and the isocitrate dehydrogenase step of the tricarboxylic acid (TCA) cycle. However, the importance of alternative NADPH-generating reactions has recently become evident. In the current review, the major canonical and non-canonical reactions involved in the production and regeneration of NADPH in prokaryotes are described, and their key enzymes are discussed. In addition, an overview of how different enzymes have been applied to increase NADPH availability and thereby enhance productivity is provided

Caldicellulosiruptor saccharolyticus is one of the most thermophilic cellulolytic organisms known to date. This Gram-positive anaerobic bacterium ferments a broad spectrum of mono-, di- and polysaccharides to mainly acetate, CO2 and hydrogen. With hydrogen yields approaching the theoretical limit for dark fermentation of 4 mol hydrogen per mol hexose, this organism has proven itself to be an excellent candidate for biological hydrogen production. This review provides an overview of the research on C. saccharolyticus with respect to the hydrolytic capability, sugar metabolism, hydrogen formation, mechanisms involved in hydrogen inhibition, and the regulation of the redox and carbon metabolism. Analysis of currently available fermentation data reveal decreased hydrogen yields under non-ideal cultivation conditions, which are mainly associated with the accumulation of hydrogen in the liquid phase. Thermodynamic considerations concerning the reactions involved in hydrogen formation are discussed with respect to the dissolved hydrogen concentration. Novel cultivation data demonstrate the sensitivity of C. saccharolyticus to increased hydrogen levels regarding substrate load and nitrogen limitation. In addition, special attention is given to the rhamnose metabolism, which represents an unusual type of redox balancing. Finally, several approaches are suggested to improve biohydrogen production by C. saccharolyticus

10.1016/j.egypro.2012.09.036, Given the highly reduced state of carbon in glycerol and its availability as a substantial byproduct of biodiesel production, glycerol is of special interest for sustainable biofuel production. Glycerol was used as a substrate for biohydrogen production using the hyperthermophilic bacterium, Thermotoga maritima and Thermotoga neapolitana. Both species metabolized glycerol to mainly acetate and hydrogen. At glycerol concentrations of 2.5 g/L, hydrogen was produced with a yield of 2.75 and 2.65 mol H2/mol glycerol consumed by T. maritima and T. neapolitana respectively. Additionally, the effect of initial pH (ranging between pH 5.0-8.5) and yeast extract concentrations (0.5, 1, 2, 4 g/L) on glycerol fermentation by T. neapolitana was investigated in batch systems. An initial pH value of around 7 was optimal for hydrogen production by T. neapolitana. Lower concentration of yeast extract resulted in a lower H2 production, however increasing the concentration from 2 to 4 g/L did not affect H2 production.

Acetoin reductase (ACR) catalyzes the conversion of acetoin to 2,3-butanediol. Under certain conditions Clostridium acetobutylicum ATCC 824 (and derived strains) generates both D- and L-stereoisomers of acetoin, but due to the lack of an ACR enzyme, does not produce 2,3-butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 was functionally expressed in C. acetobutylicum under control of two strong promoters, the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains were grown in batch cultures, during which 89-90% of the natively produced acetoin was converted to 20-22 mM D-2,3-butanediol. Addition of a racemic mixture of acetoin did lead to the production of both, D-2,3-butanediol and meso-2,3-butanediol. A metabolic network is proposed that is in agreement with the experimental data. Native 2,3-butanediol production is a first step towards a potential homo-fermentative 2-butanol producing strain of C. acetobutylicum.

Glycosaminoglycans (GAGs) are polysaccharides that are typically present in a wide diversity of animal tissue. Most common GAGs are well-characterized and pharmaceutical applications exist for many of these compounds, e.g. heparin and hyaluronan. In addition, also bacterial glycosaminoglycan-like structures exist. Some of these bacterial GAGs have been characterized, but until now no bacterial GAG has been found that possesses the modifications that are characteristic for many of the animal GAGs such as sulfation and C5-epimerization. Nevertheless, the latter conversion may also occur in bacterial and archaeal GAGs, as some prokaryotic polysaccharides have been demonstrated to contain L-iduronic acid. However, experimental evidence for the enzymatic synthesis of L-iduronic acid in prokaryotes is as yet lacking. We therefore performed an in silico screen for D-glucuronyl C5-epimerases in prokaryotes. Multiple candidate C5-epimerases were found, suggesting that many more microorganisms are likely to exist possessing an L-iduronic acid residue as constituent of their cell wall polysaccharides

Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase (SacKdgA) displays optimal activity at 95 degrees C and is studied as a model enzyme for aldol condensation reactions. For application of SacKdgA at lower temperatures, a library of randomly generated mutants was screened for improved synthesis of 2-keto-3-deoxygluconate from pyruvate and glyceraldehyde at the suboptimal temperature of 50 degrees C. The single mutant SacKdgA-V193A displayed a threefold increase in activity compared with wild type SacKdgA. The increased specific activity at 40-60 degrees C of this mutant was observed, not only for the condensation of pyruvate with glyceraldehyde, but also for several unnatural acceptor aldehydes. The optimal temperature for activity of SacKdgA-V193A was lower than for the wild type enzyme, but enzymatic stability of the mutant was similar to that of the wild type, indicating that activity and stability were uncoupled. Valine193 has Van der Waals interactions with Lysine153, which covalently binds the substrate during catalysis. The mutation V193A introduced space close to this essential residue, and the increased activity of the mutant presumably resulted from increased flexibility of Lysine153. The increased activity of SacKdgA-V193A with unaffected stability demonstrates the potential for optimizing extremely thermostable aldolases for synthesis reactions at moderate temperatures

Co-utilization of hexoses and pentoses derived from lignocellulose is an attractive trait in microorganisms considered for consolidated biomass processing to biofuels. This issue was examined for the H2-producing, extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus growing on individual monosaccharides (arabinose, fructose, galactose, glucose, mannose and xylose), mixtures of these sugars, as well as on xylan and xyloglucooligosacchrides. C. saccharolyticus grew at approximately the same rate (td approximately 95 min) and to the same final cell density (1-3 x 10(8) cells/ml) on all sugars and sugar mixtures tested. In the monosaccharide mixture, while simultaneous consumption of all monosaccharides was observed, not all were utilized to the same extent (fructose > xylose/arabinose > mannose/glucose/galactose). Transcriptome contrasts for monosaccharide growth revealed minimal changes in some cases (e.g., 31 ORFs changed >/= 2-fold for glucose vs. galactose) while substantial changes occurred for cases involving mannose (e.g., 363 ORFs >/= 2-fold for glucose vs. mannose). Evidence for catabolite repression was noted neither for growth on multi-sugar mixtures nor in the corresponding transcriptomes. Based on the whole-genome transcriptional response analysis and comparative genomics, carbohydrate specificities for transport systems could be proposed for most of the 24 putative carbohydrate ATP-binding cassette (ABC) transporters and single phosphotransferase system (PTS) identified in C. saccharolyticus. While most transporter genes responded to individual monosacchrides and polysaccharides, Csac_0692-0694 was up-regulated only in the monosaccharide mixture. The results here affirm the broad growth substrate preferences of C. saccharolyticus on carbohydrates representative of lignocellulosic biomass and suggest that this bacterium holds promise for biofuels applications

Carboxylic ester hydrolyzing enzymes constitute a large group of enzymes that are able to catalyze the hydrolysis, synthesis or transesterification of an ester bond. They can be found in all three domains of life, including the group of hyperthermophilic bacteria and archaea. Esterases from the latter group often exhibit a high intrinsic stability, which makes them of interest them for various biotechnological applications. In this review, we aim to give an overview of all characterized carboxylic ester hydrolases from hyperthermophilic microorganisms and provide details on their substrate specificity, kinetics, optimal catalytic conditions, and stability. Approaches for the discovery of new carboxylic ester hydrolases are described. Special attention is given to the currently characterized hyperthermophilic enzymes with respect to their biochemical properties, 3D structure, and classification

Caldicellulosiruptor saccharolyticus is an extremely thermophilic, gram-positive anaerobe which ferments cellulose-, hemicellulose- and pectin-containing biomass to acetate, CO(2), and hydrogen. Its broad substrate range, high hydrogen-producing capacity, and ability to coutilize glucose and xylose make this bacterium an attractive candidate for microbial bioenergy production. Here, the complete genome sequence of C. saccharolyticus, consisting of a 2,970,275-bp circular chromosome encoding 2,679 predicted proteins, is described. Analysis of the genome revealed that C. saccharolyticus has an extensive polysaccharide-hydrolyzing capacity for cellulose, hemicellulose, pectin, and starch, coupled to a large number of ABC transporters for monomeric and oligomeric sugar uptake. The components of the Embden-Meyerhof and nonoxidative pentose phosphate pathways are all present; however, there is no evidence that an Entner-Doudoroff pathway is present. Catabolic pathways for a range of sugars, including rhamnose, fucose, arabinose, glucuronate, fructose, and galactose, were identified. These pathways lead to the production of NADH and reduced ferredoxin. NADH and reduced ferredoxin are subsequently used by two distinct hydrogenases to generate hydrogen. Whole-genome transcriptome analysis revealed that there is significant upregulation of the glycolytic pathway and an ABC-type sugar transporter during growth on glucose and xylose, indicating that C. saccharolyticus coferments these sugars unimpeded by glucose-based catabolite repression. The capacity to simultaneously process and utilize a range of carbohydrates associated with biomass feedstocks is a highly desirable feature of this lignocellulose-utilizing, biofuel-producing bacterium