Monoterpenoid production and monoterpenoid resistance mechanisms in Pseudomonas putida

Monoterpenes and their monoterpenoid derivatives form a subclass of terpene(oid)s. They are widely used in medicines/pharmaceuticals, as flavor and fragrance compounds, or in agriculture and are also considered as future biofuels. However, for many of these substances, the extraction from natural sources poses challenges such as occurring at low concentrations in their raw material or because the natural sources are diminishing. Furthermore, many of the structurally more complex terpenoids cannot be chemically synthesized in an economic way. Therefore, microbial production provides an attractive alternative, taking advantage of the often distinct regio- and stereoselectivity of enzymatic reactions. However, monoterpenes and monoterpenoids are challenging products for industrial biotechnology processes due to their pronounced cytotoxicity, which complicates the production in microorganisms compared to longer-chain terpenes (sesquiterpenes, diterpenes, etc.). The aim of this thesis was to generate a biotechnological complement to fossil-resources-based chemical processes for industrial monoterpenoid production. Therefore, a starting point for the further development of a microbial cell factory based on the microbe Pseudomonas putida KT2440 was aimed to be created. This production organism should be able to conduct a whole- cell biocatalysis to selectively oxyfunctionalize monoterpene hydrocarbons using renewable industrial by-products and waste streams as raw material for monoterpenoid production (Figure 1). As a model substance, the production of (-)-menthol should be addressed due to its industrial significance. (-)-Menthol is one of the world’s most widely-used flavor and fragrance compounds by volume as well as a medical component, having an annual production volume of over 30,000 tons. An approach for (-)-menthol production from renewable resources could be a biotechnological(-chemical) two-step conversion (Figure 1), starting from (+)-limonene, a by-product of the citrus fruit processing industry. The thesis project was divided into three parts. In the first part, enzymes (limonene-3- hydroxylases) were to be identified that can convert (+)-limonene into the precursor of (-)-menthol, (+)-trans-isopiperitenol. To counteract product toxicity, in the second part, the tolerance of the intended production organism P. putida KT2440 towards monoterpenes and their monoterpenoid derivatives should be increased. Finally, in the third part, the identified hydroxylase enzymes would be expressed in the improved P. putida KT2440 strain to create a whole-cell biocatalyst for the first reaction step of a two-step (-)-menthol production, starting from (+)-limonene. To achieve these objectives, different genetic/molecular biology and analytical methods were applied. In this way, two cytochrome P450 monooxygenase enzymes from the fungi Aureobasidium pullulans and Hormonema carpetanum could be identified and functionally expressed in Pichia pastoris, which can catalyze the intended hydroxylation reaction on (+) limonene with high stereo- and regioselectivity. A further characterization of the enzyme from A. pullulans showed that apart from (+) limonene the protein can also hydroxylate ( ) limonene, - and -pinene, as well as 3-carene. Furthermore, within this thesis, mechanisms of microbial monoterpenoid resistance of P. putida could be identified. It was shown that the different monoterpenes and monoterpenoids tested have very different toxicity levels and that mainly the Ttg efflux pumps of P. putida GS1 are responsible for the tolerance to many of these compounds. Based on these results, a P. putida KT2440 strain with increased resistance to various monoterpenoids, including isopiperitenol, could then be generated, which can be used as a host organism for the further development of monoterpenoid-producing cell factories. While within the scope of this work the heterologous expression of the fungal gene in prokaryotic cells in a functional form could not be realized despite different approaches, the identified enzymes, the monoterpenoid-tolerant P. putida strain and a plasmid developed for heterologous gene expression in P. putida provide a starting point for the further design of a microbial cell factory for biotechnological monoterpenoid production.Monoterpene und ihre Monoterpenoid-Derivate bilden eine Unterklasse der Terpen(oid)e und finden eine breite Anwendung als Duft- und Aromastoffe, Arzneimittelbestandteile, in Kosmetika und als Agrochemikalien. Seit einigen Jahren wird aber auch ein möglicher Einsatz als zukünftige Biokraftstoffe diskutiert. Für viele dieser Substanzen birgt die Extraktion aus natürlichen Quellen, wie etwa Pflanzenteilen, jedoch einige Herausforderungen, wie z. B. eine sehr geringe Konzentration im Ausgangsmaterial oder die Tatsache, dass die benötigten Pflanzen immer weniger verfügbar sind. Während für einige Monoterpene und Monoterpenoide inzwischen chemische Herstellungsverfahren entwickelt werden konnten, ist es besonders für strukturell komplexere Moleküle immer noch schwierig oder sogar unmöglich, diese wirtschaftlich chemisch zu synthetisieren. Deshalb bietet die biotechnologische Herstellung mit Mikroorganismen eine attraktive Alternative. Eine der großen Herausforderung bei der Biosynthese einiger Monoterpenoide ist jedoch ihre ausgeprägte Zytotoxizität, welche die Herstellung von Monoterpenen und Monoterpenoiden in Mikroorganismen erschwert. Das Ziel der vorliegenden Arbeit war es, eine biotechnologische Ergänzung zu den auf fossilen Rohstoffen basierenden, chemischen Prozessen für die industrielle Monoterpenoid-Produktion zu schaffen. Dafür sollte die Grundlage für die weitere Entwicklung einer mikrobiellen Zellfabrik auf Basis des Bakteriums Pseudomonas putida KT2440 generiert werden. Der Produktionsorganismus sollte selektiv Monoterpenkohlenwasserstoffe oxygenieren und dafür erneuerbare industrielle Abfallströme als Ausgangsstoffe für die Monoterpenoid-Produktion nutzen können (Abbildung 1). Als Modellsubstanz sollte die Herstellung von (-) Menthol realisiert werden, das wegen seiner industriellen Bedeutung als einer der weltweit meist genutzten Duft- und Aromastoff sowie seines Einsatzes als medizinischer Bestandteil ausgewählt wurde. Ein Ansatz für ein ( ) Menthol-Produktionsverfahren aus erneuerbaren Rohstoffen könnte eine biotechnologische( chemische), zweistufige Umwandlung ausgehend vom Nebenprodukt der Zitrussaft-Industrie (+) Limonen sein (Abbildung 1). Das Projekt gliederte sich in drei Teile. Im ersten Teil sollten Enzyme (Limonen-3-Hydroxylasen) identifiziert werden, die (+) Limonen in die Vorstufe von (-) Menthol, (+) trans-Isopiperitenol, umwandeln können. Um dem Problem der Produkttoxizität entgegenzuwirken, sollte im zweiten Teil die Toleranz des gewählten Produktionsorganismus P. putida KT2440 gegenüber Monoterpenen und deren Monoterpenoid-Derivaten gesteigert werden. Im dritten Teil sollten die identifizierten Hydroxylase-Enzyme im verbesserten P. putida KT2440-Stamm exprimiert werden, um einen Ganzzell-Biokatalysator für den ersten Schritt der zweistufigen ( ) Menthol-Produktion ausgehend von (+) Limonen zu schaffen. Zur Erreichung dieser Ziele wurden verschiedene genetische/molekularbiologische und analytische Methoden angewendet. Auf diese Weise konnten zwei Cytochrom P450 Monooxygenase-Enzym aus den Pilzen Aureobasidium pullulans und Hormonema carpetanum identifiziert und funktionell in Pichia pastoris exprimiert werden, die die gewünschte Hydroxlierungsreaktion an (+) Limonen mit hoher Stereo- und Regioselektivität katalysieren können. Eine weitere Charakterisierung des Enzyms aus A. pullulans zeigte, dass das Protein neben (+) Limonen auch (-)-Limonen, - und -Pinen sowie 3 Caren hydroxylieren kann. Des Weiteren konnten im Rahmen dieser Arbeit Mechanismen der mikrobiellen Monoterpenoid-Resistenz von P. putida identifiziert werden. Dabei zeigte sich, dass die verschiedenen getesteten Monoterpene und Monoterpenoide sehr unterschiedlichen Toxizitätsniveaus aufweisen und dass hauptsächlich die Ttg-Efflux-Pumpen von P. putida GS1 für die Toleranz gegenüber vielen der getesteten Verbindungen verantwortlich sind. Auf Grundlage dieser Ergebnisse konnte dann ein P. putida KT2440-Stamm mit erhöhter Resistenz gegenüber verschiedenen Monoterpenoiden, einschließlich Isopiperitenol, generiert werden, der als Wirtsorganismus für die weitere Entwicklung von Monoterpenoid-produzierenden Zellfabriken genutzt werden kann. Während im Rahmen dieser Arbeit die heterologe Expression des pilzlichen Limonen-3-Hydroxylase-Gens in einer funktionalen Form in prokaryotischen Zellen trotz verschiedener Ansatzpunkte nicht mehr realisiert werden konnte, bilden die identifizierten Enzyme, der monoterpenoid-tolerante P. putida-Stamm und ein für die heterologe Gen-Expression in P. putida entwickeltes Plasmid einen Ausgangspunkt für die weitere Entwicklung einer mikrobiellen Zellfabrik für die biotechnologische Monoterpenoid-Produktion

A novel and widespread class of ketosynthase is responsible for the head-to-head condensation of two acyl moieties in bacterial pyrone biosynthesis

The biosynthesis of photopyrones, novel quorum sensing signals in Photorhabdus, has been studied by heterologous expression of the photopyrone synthase PpyS catalyzing the head-to-head condensation of two acyl moieties. The biochemical mechanism of pyrone formation has been investigated by amino acid exchange and bioinformatic analysis. Additionally, the evolutionary origin of PpyS has been studied by phylogenetic analyses also revealing homologous enzymes in Pseudomonas sp. GM30 responsible for the biosynthesis of pseudopyronines including a novel derivative. Moreover this novel class of ketosynthases is only distantly related to other pyrone-forming enzymes identified in the biosynthesis of the potent antibiotics myxopyronin and corallopyronin

Antibacterial marinopyrroles and pseudilins act as protonophores

Elucidating the mechanism of action (MoA) of antibacterial natural products is crucial to evaluating their potential as novel antibiotics. The marinopyrroles, pentachloropseudilin, and pentabromopseudilin are densely halogenated, hybrid pyrrole-phenol natural products with potent activity against Gram-positive bacterial pathogens like Staphylococcus aureus. However, the exact way in which they exert this antibacterial activity has not been established. In this study, we explore their structure-activity relationship, determine their spatial location in bacterial cells, and investigate their MoA. We show that the natural products share a common MoA based on membrane depolarization and dissipation of the proton motive force (PMF) that is essential for cell viability. The compounds show potent protonophore activity, but do not appear to destroy the integrity of the cytoplasmic membrane via the formation of larger pores or interfere with the stability of the peptidoglycan sacculus. Thus, our current model for the antibacterial MoA of marinopyrrole, pentachloropseudilin, and pentabromopseudilin stipulates that the acidic compounds insert into the membrane and transport protons inside the cell. This MoA may explain many of the deleterious biological effects in mammalian cells, plants, phytoplankton, viruses, and protozoans that have been reported for these compounds

Mutations in <i>CERS3</i> Cause Autosomal Recessive Congenital Ichthyosis in Humans

<div><p>Autosomal recessive congenital ichthyosis (ARCI) is a rare genetic disorder of the skin characterized by abnormal desquamation over the whole body. In this study we report four patients from three consanguineous Tunisian families with skin, eye, heart, and skeletal anomalies, who harbor a homozygous contiguous gene deletion syndrome on chromosome 15q26.3. Genome-wide SNP-genotyping revealed a homozygous region in all affected individuals, including the same microdeletion that partially affects two coding genes (<i>ADAMTS17</i>, <i>CERS3</i>) and abolishes a sequence for a long non-coding RNA (<i>FLJ42289</i>). Whereas mutations in <i>ADAMTS17</i> have recently been identified in autosomal recessive Weill-Marchesani-like syndrome in humans and dogs presenting with ophthalmologic, cardiac, and skeletal abnormalities, no disease associations have been described for <i>CERS3</i> (ceramide synthase 3) and <i>FLJ42289</i> so far. However, analysis of additional patients with non-syndromic ARCI revealed a splice site mutation in <i>CERS3</i> indicating that a defect in ceramide synthesis is causative for the present skin phenotype of our patients. Functional analysis of patient skin and <i>in vitro</i> differentiated keratinocytes demonstrated that mutations in <i>CERS3</i> lead to a disturbed sphingolipid profile with reduced levels of epidermis-specific very long-chain ceramides that interferes with epidermal differentiation. Taken together, these data present a novel pathway involved in ARCI development and, moreover, provide the first evidence that CERS3 plays an essential role in human sphingolipid metabolism for the maintenance of epidermal lipid homeostasis.</p></div

Histological analysis and CERS3 protein expression in skin biopsies and cultured keratinocytes from healthy controls and patient H.

<p>(<b>A</b>) Hematoxylin and eosin staining (H&E) shows acanthosis with a thickening of the granular layer and psoriasiform epidermal hyperplasia in the patient compared to a healthy control. The inset (same scale) shows the detached stratum corneum of the patient. Scale bars, 50 µm (<b>B</b>) Immunofluorescence staining using a specific antibody for CERS3 (red) and DAPI (blue) as nuclear counterstaining. CERS3 staining is present at the interface between the stratum granulosum and the stratum corneum in control skin but not detectable in the patient skin. The thin dashed lines indicate the interface between the stratum granulosum and the stratum corneum as well as the upper edge of the stratum corneum. Scale bars, 50 µm (<b>C</b>) Western blot analysis of CERS3 in control and patient keratinocytes before differentiation (0 d) and at day 4, 7, and 14 after induction of differentiation. CERS3 was detected at ∼37 kDa (indicated by an asterisk) using an antibody targeting the C-terminus of the protein. An antibody that recognizes actin was used as a loading control.</p

<i>In situ</i> ceramide localization and sphingolipid profile of cultured keratinocytes.

<p>(<b>A</b>) Diagram depicting the <i>de novo</i> synthesis pathway of (glucosyl)ceramides. Loss-of-function of ceramide synthase 1–6 results in an impaired <i>N</i>-acylation of dihydrosphingosine as indicated by a cross. (<b>B</b>) Confocal microscopy images of healthy control and patient H skin biopsies immunostained with an antibody targeting ceramides (red) with DAPI (blue) as nuclear counterstaining. Ceramides localize to the stratum granulosum and the stratum corneum in healthy control skin. Note the loss of ceramide staining in patient's skin. We observed immunostaining of ceramides in the uppermost layer of the mutant stratum corneum, which results from unspecific binding of the secondary antibody. The thin dashed lines indicate the interface between the stratum granulosum and the stratum corneum as well as the upper edge of the stratum corneum. Scale bars, 50 µm (<b>C</b>) Upper panel: TLC of lipid extracts from healthy control and patient H keratinocytes 14 days after induction of differentiation. Lipids corresponding to 400 µg of cellular protein were extracted from cultures, separated twice by TLC using the solvent system chloroform/methanol/glacial acetic acid (190/9/1 v/v/v), and quantified after carbonization. An epidermal lipid extract of a healthy control individual was used as a reference. Lower panel: Data are presented as mean values +S.D. of triplicate samples and are representative for three independent experiments. Statistical significance was determined by unpaired two-tailed Student's <i>t</i>-test (* <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001). Ceramide species are classified according to the sphingoid base (S, sphingosine or P, phytosphingosine). Abbreviations: AcylCer, acylceramides; DAG, diacylglycerols; GlcAcylCer, glucosylacylceramides; GlcCer, glucosylceramides; MAG, monoacylglycerols; M/LC-Cer, middle and long-chain ceramides; NEFA, non-esterified fatty acids; TAG, triacylglycerols; VLC-Cer, very long-chain ceramides.</p

Deletion and <i>CERS3</i> mutation scheme.

<p>(<b>A</b>) Homozygous regions on chromosome 15q26.3 in patients. The smallest common interval is defined by rs2684811 in patient C and rs11247226 in patient S. The genomic deletion characterized by breakpoint spanning PCR (in blue) encompasses 106,960 bp with the borders 100,856,031 to 100,962,985 on chromosome 15 (UCSC hg19, February 2009) in patient D1, D2, C, and S. The splice site mutation in patient H is marked by an asterisk. The diagram shows the deletion and the limiting SNPs of the homozygous regions in patient H, D1, D2, C, and S (not to scale). (<b>B</b>) The scale illustration of the deleted region shows the missing SNPs and genes. The internal limit of the deletion is between SNP rs1080429 and rs7179355. (<b>C</b>) FISH signal pattern in a healthy individual shows the control signal on 15q21.2 in red (digoxigenin-labeled BAC RP11-562A8) and the signal corresponding to 15q26.3 in green (arrow). In patients D1 and D2 the green FISH signal is missing confirming the microdeletion in 15q26.3. (<b>D</b>) Diagram depicting the structure of the human <i>CERS3</i> gene and the site of mutation in exon 9 (c.609+1G>T) of patient H indicated by an asterisk. Below is illustrated the predicted structure of human CERS3 protein including transmembrane domains (TMD), homeobox, polyglutamic acid region (G), and the TLC (<u>T</u>RAM, <u>L</u>AG1 and <u>C</u>LN8 homology) domain.</p

Epidermal differentiation in healthy control and patient H.

<p>(<b>A</b>) Light microscopy images of skin biopsies from control and patient H immunolabeled with antibodies specific for keratin 14 (K14) and Ki-67 with hematoxylin as nuclear counterstaining reveal an abnormal differentiation process in patient skin. (<b>B</b>) Confocal microscopy images of the same control and patient skin biopsies immunostained with antibodies specific for filaggrin, involucrin, and loricrin with DAPI (blue) as nuclear counterstaining. The patient skin biopsy shows a thickening of the stratum granulosum compared to the healthy control. Arrows indicate hyperplastic basal cells in the patient skin. The thin dashed lines indicate the interface between the stratum granulosum and the stratum corneum as well as the upper edge of the stratum corneum. Scale bars, 50 µm.</p