Karin Lemuth

Engineering of a plasmid-free Escherichia coli strain for improved in vivo biosynthesis of astaxanthin

BackgroundThe xanthophyll astaxanthin is a high-value compound with applications in the nutraceutical, cosmetic, food, and animal feed industries. Besides chemical synthesis and extraction from naturally producing organisms like Haematococcus pluvialis, heterologous biosynthesis in non-carotenogenic microorganisms like Escherichia coli, is a promising alternative for sustainable production of natural astaxanthin. Recent achievements in the metabolic engineering of E. coli strains have led to a significant increase in the productivity of carotenoids like lycopene or β-carotene by increasing the metabolic flux towards the isoprenoid precursors. For the heterologous biosynthesis of astaxanthin in E. coli, however, the conversion of β-carotene to astaxanthin is obviously the most critical step towards an efficient biosynthesis of astaxanthin.ResultsHere we report the construction of the first plasmid-free E. coli strain that produces astaxanthin as the sole carotenoid compound with a yield of 1.4 mg/g cdw (E. coli BW-ASTA). This engineered E. coli strain harbors xanthophyll biosynthetic genes from Pantoea ananatis and Nostoc punctiforme as individual expression cassettes on the chromosome and is based on a β-carotene-producing strain (E. coli BW-CARO) recently developed in our lab. E. coli BW-CARO has an enhanced biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) and produces β-carotene in a concentration of 6.2 mg/g cdw. The expression of crtEBIY along with the β-carotene-ketolase gene crtW148 (NpF4798) and the β-carotene-hydroxylase gene (crtZ) under controlled expression conditions in E. coli BW-ASTA directed the pathway exclusively towards the desired product astaxanthin (1.4 mg/g cdw).ConclusionsBy using the λ-Red recombineering technique, genes encoding for the astaxanthin biosynthesis pathway were stably integrated into the chromosome of E. coli. The expression levels of chromosomal integrated recombinant biosynthetic genes were varied and adjusted to improve the ratios of carotenoids produced by this E. coli strain. The strategy presented, which combines chromosomal integration of biosynthetic genes with the possibility of adjusting expression by using different promoters, might be useful as a general approach for the construction of stable heterologous production strains synthesizing natural products. This is the case especially for heterologous pathways where excessive protein overexpression is a hindrance.

Biosynthesis of the Vitamin E Compound δ‐Tocotrienol in Recombinant Escherichia coli Cells

The biosynthesis of natural products in a fast growing and easy to manipulate heterologous host system, such as Escherichia coli, is of increasing interest in biotechnology. This procedure allows the investigation of complex natural product biosynthesis and facilitates the engineering of pathways. Here we describe the cloning and the heterologous expression of tocochromanol (vitamin E) biosynthesis genes in E. coli. Tocochromanols are synthesized solely in photosynthetic organisms (cyanobacteria, algae, and higher green plants). For recombinant tocochromanol biosynthesis, the genes encoding hydroxyphenylpyruvate dioxygenase (hpd), geranylgeranylpyrophosphate synthase (crtE), geranylgeranylpyrophosphate reductase (ggh), homogentisate phytyltransferase (hpt), and tocopherol‐cyclase (cyc) were cloned in a stepwise fashion and expressed in E. coli. Recombinant E. coli cells were cultivated and analyzed for tocochromanol compounds and their biosynthesis precursors. The expression of only hpd from Pseudomonas putida or crtE from Pantoea ananatis resulted in the accumulation of 336 mgL−1 homogentisate and 84 μgL−1 geranylgeranylpyrophosphate in E. coli cultures. Simultaneous expression of hpd, crtE, and hpt from Synechocystis sp. under the control of single tac‐promoter resulted in the production of methyl‐6‐geranylgeranyl‐benzoquinol (67.9 μg g−1). Additional expression of the tocopherol cyclase gene vte1 from Arabidopsis thaliana resulted in the novel formation of a vitamin E compound—δ‐tocotrienol (15 μg g−1)—in E. coli.

Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains

In biotechnology, the heterologous biosynthesis of isoprenoid compounds in Escherichia coli is a field of great interest and growth. In order to achieve higher isoprenoid yields in heterologous E. coli strains, it is necessary to quantify the pathway intermediates and adjust gene expression. In this study, we developed a precise and sensitive nonradioactive method for the simultaneous quantification of the isoprenoid precursors farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) in recombinant and wild-type E. coli cells. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ extraction followed by separation and detection using gas chromatography–mass spectrometry. The integration of a geranylgeranyl diphosphate synthase gene into the E. coli chromosome leads to the accumulation of GGPP, generating quantities as high as those achieved with a multicopy expression vector.

Adaptation, adhesion and invasion during interaction of Candida albicans with the host – Focus on the function of cell wall proteins

Infectious diseases have long been regarded as losing their threat to mankind. However, in the recent decades infectious diseases have been regaining grounds and are back in the focus of research. This is also due to the fact that medical progress has enabled us to treat and cure a much higher fraction of severe diseases or trauma, resulting in a significant proportion of temporarily or constantly immune-suppressed patients. Infectious diseases result from the interplay between pathogenic microorganisms and the hosts they infect, especially their defense systems. Consequently, immune-suppressed patients are at high risk to succumb from opportunistic infections, like Candida infections. To study the balance between host and C. albicans with regard to the establishment of disease or asymptomatic, commensal colonisation, we developed host-pathogen interaction systems to study both the adaptation of C. albicans to different epithelia as well as to investigate the sensors of the innate immune system, the pattern recognition receptors. These host-pathogen interaction systems, as well as some of the results gained are described in this review.

DNA Microarray for Genotyping Antibiotic Resistance Determinants in Acinetobacter baumannii Clinical Isolates

ABSTRACT In recent decades, Acinetobacter baumannii has emerged as an organism of great concern due to its ability to accumulate antibiotic resistance. In order to improve the diagnosis of resistance determinants in A. baumannii in terms of lead time and accuracy, we developed a microarray that can be used to detect 91 target sequences associated with antibiotic resistance within 4 h from bacterial culture to result. The array was validated with 60 multidrug-resistant strains of A. baumannii in a blinded, prospective study. The results were compared to phenotype results determined by the automated susceptibility testing system VITEK2. Antibiotics considered were piperacillin-tazobactam, ceftazidime, imipenem, meropenem, trimethoprim-sulfamethoxazole, amikacin, gentamicin, tobramycin, ciprofloxacin, and tigecycline. The average positive predictive value, negative predictive value, sensitivity, and specificity were 98, 98, 99, and 94%, respectively. For carbapenemase genes, the array results were compared to singleplex PCR results provided by the German National Reference Center for Gram-Negative Pathogens, and results were in complete concordance. The presented array is able to detect all relevant resistance determinants of A. baumannii in parallel. The short handling time of 4 h from culture to result helps to provide fast results in order to initiate adequate anti-infective therapy for critically ill patients. Another application would be data acquisition for epidemiologic surveillance.

A family of secreted pathogenesis‐related proteins in Candida albicans

Analysing culture supernatants of yeast and hyphal cells of Candida albicans, we found two close homologues of pathogenesis‐related (PR‐) 1 proteins, Rbe1p and Rbt4p, in the secretome. Due to sequence homology, three additional, yet not characterized open reading frames, ORF19.6200, ORF19.2787 and ORF19.2336, together with RBE1 and RBT4 were assigned to a novel family of CaPRY proteins. In a Δrbe1/Δrbt4 deletion strain, genome‐wide transcriptional analysis revealed differential transcription of only a limited set of genes implicated in virulence and oxidative stress response. Single deletion of RBE1 or RBT4 in a clinical C. albicans isolate resulted in a moderate but significant attenuation in virulence in a mouse model for disseminated candidiasis. However, a synergistic effect was observed in a Δrbe1/Δrbt4 double deletion strain, where virulence was strongly affected. Remarkably, transcription of RBT4 and RBE1 was each upregulated in blastospores of Δrbe1 or hyphae of Δrbt4 deletion strains respectively, indicating functional complementation thereby compensating a potential virulence defect in the single deletion strains. Furthermore, the double deletion strain showed increased sensitivity to attack by polymorphonuclear leucocytes. Therefore, the crucial contribution of both C. albicans pathogenesis‐related proteins to virulence might be vested in protection against phagocyte attack.

An Antifungal Benzimidazole Derivative Inhibits Ergosterol Biosynthesis and Reveals Novel Sterols.

ABSTRACT Fungal infections are a leading cause of morbidity and death for hospitalized patients, mainly because they remain difficult to diagnose and to treat. Diseases range from widespread superficial infections such as vulvovaginal infections to life-threatening systemic candidiasis. For systemic mycoses, only a restricted arsenal of antifungal agents is available. Commonly used classes of antifungal compounds include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapies, significant side effects, and high costs for several antifungals, there is a need for new antifungals in the clinic. In order to expand the arsenal of compounds with antifungal activity, we previously screened a compound library using a cell-based screening assay. A set of novel benzimidazole derivatives, including (S)-2-(1-aminoisobutyl)-1-(3-chlorobenzyl)benzimidazole (EMC120B12), showed high antifungal activity against several species of pathogenic yeasts, including Candida glabrata and Candida krusei (species that are highly resistant to antifungals). In this study, comparative analysis of EMC120B12 versus fluconazole and nocodazole, using transcriptional profiling and sterol analysis, strongly suggested that EMC120B12 targets Erg11p in the ergosterol biosynthesis pathway and not microtubules, like other benzimidazoles. In addition to the marker sterol 14-methylergosta-8,24(28)-dien-3β,6α-diol, indicating Erg11p inhibition, related sterols that were hitherto unknown accumulated in the cells during EMC120B12 treatment. The novel sterols have a 3β,6α-diol structure. In addition to the identification of novel sterols, this is the first time that a benzimidazole structure has been shown to result in a block of the ergosterol pathway.

A Screening Assay Based on Host-Pathogen Interaction Models Identifies a Set of Novel Antifungal Benzimidazole Derivatives

ABSTRACT Fungal infections are a serious health problem in clinics, especially in the immune-compromised patient. Disease ranges from widespread superficial infections like vulvovaginal infections to life-threatening systemic candidiasis. Especially for systemic mycoses, only a limited arsenal of antifungals is available. The most commonly used classes of antifungal compounds used include azoles, polyenes, and echinocandins. Due to emerging resistance to standard therapy, significant side effects, and high costs for several antifungals, there is a medical need for new antifungals in the clinic and general practice. In order to expand the arsenal of compounds with antifungal activities, we screened a compound library including more than 35,000 individual compounds derived from organic synthesis as well as combinatorial compound collections representing mixtures of compounds for antimycotic activity. In total, more than 100,000 compounds were screened using a new type of activity-selectivity assay, analyzing both the antifungal activity and the compatibility with human cells at the same time. One promising hit, an (S)-2-aminoalkyl benzimidazole derivative, was developed among a series of lead compounds showing potent antifungal activity. (S)-2-(1-Aminoisobutyl)-1-(3-chlorobenzyl) benzimidazole showed the highest antifungal activity and the best compatibility with human cells in several cell culture models and against a number of clinical isolates of several species of pathogenic Candida yeasts. Transcriptional profiling indicates that the newly discovered compound is a potential inhibitor of the ergosterol pathway, in contrast to other benzimidazole derivatives, which target microtubules.

Simultaneous Detection of Different MicroRNA Types Using the ZIP-Code Array System

MicroRNAs (miRNAs) are important negative regulators of gene expression. Their implication in tumorigenesis is based on their dysregulation in many human cancer diseases. Interestingly, in tumor cells, an altered ratio of precursor and mature miRNA levels has been described. Consequently, differences in miRNA type levels have a high potential as biomarkers and comparative high-throughput-based detection might permit a more accurate characterization of subtypes, especially in the case of very heterogeneous tumor entities. Several molecular methods exist for the detection of mature and precursor miRNAs. DNA microarrays are predestinated as a high-throughput method for comprehensive miRNA detection in tumors. However, the simultaneous array-based detection of both these miRNA types is limited because the mature miRNA sequence is identically present in both forms. Here we present a ZIP-code DNA microarray-based system in combination with a novel labeling approach, which enables the simultaneous detection of precursor and mature miRNAs in one single experiment. Using synthetic miRNA templates, we demonstrate the specificity of the method for the different miRNA types, as well as the detection range up to four orders of magnitude. Moreover, mature and precursor miRNAs were detected and validated in human tumor cells.

Dynamic Modeling of the Central Metabolism of E. coli – Linking Metabolite and Regulatory Networks

Coupling complex regulatory and metabolic networks for the purpose of dynamic modeling requires knowledge of the quantitative kinetics of the participating reactions as well as the variation of parameters in the context of the physiological state of the system. This chapter aims at demonstrating the integration of the different networks for E. coli exposed to an increasing carbon limitation of a fed-batch process with constant feeding of the carbon and energy source glucose. Starting from a global observation of the response of the bacteria in terms of flux distribution and gene expression in the central metabolism, emphasis is given to the dynamic modeling of regulation phenomena in the catabolism. The cra regulon which is linked to the dynamic response of the metabolite fructose 1,6- bis(phosphate) serves as an example to introduce a new concept, in which the binding constants are estimated from DNA-binding site sequences of the regulatory proteins. By comparison of the nucleotide frequencies within the DNA-binding sites for the individual target genes of the regulon, it is possible to perform a reasonable estimation of the kinetic parameters. Results of these estimations are compared with experimentally observed transcript concentrations measured with the aid of quantitative PCR. In addition it is shown how these outputs of the regulatory networks can be linked to the maximal rates of the enzymes for the metabolic system of interest. The discussion of this issue is embedded within a critical assessment of different conceptual frameworks for modeling the metabolic network, which covers the spectrum of dynamic modeling at different levels of complexity, such as genome scale, modular approaches and reduced models.