RESUMEN
Glycerol is considered as a promising substrate for biotechnological applications and the non-conventional yeast Yarrowia lipolytica has been used extensively for the valorization of this compound. Contrary to S. cerevisiae, Y. lipolytica seems to prefer glycerol over glucose and it has been reported previously that the presence of glycerol can suppress the consumption of glucose in co-substrate fermentations. Based on these observations, we hypothesized glycerol repression-like effects in Y. lipolytica, which are converse to well described carbon repression mechanisms ensuring the prioritized use of glucose (e.g., in S. cerevisiae). We therefore aimed to investigate this effect on the level of transcription. Strains varying in the degree of glucose suppression were chosen and characterized in high-resolution growth screenings, resulting in the detection of different growth phenotypes under glycerol-glucose mixed conditions. Two strains, IBT and W29, were selected and cultivated in chemostats using glucose, glycerol and glucose/glycerol as carbon sources, followed by an RNA-Seq-based transcriptome analysis. We could show that several transporters were significantly higher expressed in W29, which is potentially related to the observed physiological differences. However, most of the expression variation between the strains were regardless of the carbon source applied, and cross-comparisons revealed that the strain-specific carbon source responses underwent in the opposite direction. A deeper analysis of the substrate specific carbon source response led to the identification of several differentially expressed genes with orthologous functions related to signal transduction and transcriptional regulation. This study provides an initial investigation on potentially novel carbon source regulation mechanisms in yeasts.
Asunto(s)
Glucosa/metabolismo , Glicerol/farmacología , RNA-Seq , Transcriptoma/genética , Yarrowia/genética , Carbono/farmacología , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genes Fúngicos , Modelos Biológicos , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad de la Especie , Yarrowia/efectos de los fármacos , Yarrowia/crecimiento & desarrolloRESUMEN
Over the last 10 to 15 years, metabolic engineering of microbes has become a versatile tool for high-level de novo synthesis of terpenoids, with the sesquiterpenoids armopha-1,4-diene, farnesene and artemisinic acid as prime examples. However, almost all cell factory approaches towards terpenoids to date have been based on sugar as the raw material, which is mainly used as a food resource and subject to high price volatilities. In this study we present de novo synthesis of the sesquiterpenoid α-humulene from the abundantly available non-food carbon source methanol by metabolically engineered Methylobacterium extorquens AM1. Expression of α-humulene synthase from Zingiber zerumbet in combination with farnesyl pyrophosphate (FPP) synthase from Saccharomyces cerevisiae led to concentrations of up to 18 mg/L α-humulene. Introduction of a prokaryotic mevalonate pathway from Myxococcus xanthus in combination with ribosome binding site optimization of α-humulene and FPP synthases increased product concentration 3-fold. This value was additionally raised by 30% using a carotenoid synthesis deficient mutant strain. Final product concentrations of up to 1.65 g/L were obtained in methanol limited fed-batch cultivations, which is the highest titer of de novo synthesized α-humulene reported to date. This study demonstrates the potential of M. extorquens as a future platform strain for the production of high-value terpenoids from the alternative carbon source methanol.
Asunto(s)
Ingeniería Metabólica/métodos , Metanol/metabolismo , Methylobacterium extorquens/genética , Methylobacterium extorquens/metabolismo , Sesquiterpenos/metabolismo , Reactores Biológicos , Carotenoides/biosíntesis , Simulación por Computador , Medios de Cultivo , Fermentación , Redes y Vías Metabólicas/genética , Ácido Mevalónico/metabolismo , Sesquiterpenos Monocíclicos , PlásmidosRESUMEN
BACKGROUND: Production of monoterpenoids as valuable chemicals using recombinant microbes is a growing field of interest. Unfortunately, antimicrobial activity of most monoterpenoids hampers a wide application of microorganisms for their production. Strains of Pseudomonas putida, a fast growing and metabolically versatile bacterium, often show an outstanding high tolerance towards organic solvents and other toxic compounds. Therefore, Pseudomonas putida constitutes an attractive alternative host in comparison to conventionally used microorganisms. Here, metabolic engineering of solvent tolerant Pseudomonas putida as a novel microbial cell factory for de novo production of monoterpenoids is reported for the first time, exemplified by geranic acid production from glycerol as carbon source. The monoterpenoic acid is an attractive compound for application in the flavor, fragrance, cosmetics and agro industries. RESULTS: A comparison between Escherichia coli, Saccharomyces cerevisiae and Pseudomonas putida concerning the ability to grow in the presence of geranic acid revealed that the pseudomonad bears a superior resilience compared to the conventionally used microbes. Moreover, Pseudomonas putida DSM 12264 wildtype strain efficiently oxidized externally added geraniol to geranic acid with no further degradation. Omitting external dosage of geraniol but functionally expressing geraniol synthase (GES) from Ocimum basilicum, a first proof-of-concept for de novo biosynthesis of 1.35 mg/L geranic acid in P. putida DSM 12264 was achieved. Doubling the amount of glycerol resulted in twice the amount of product. Co-expression of the six genes of the mevalonate pathway from Myxococcus xanthus to establish flux from acetyl-CoA to the universal terpenoid precursor isopentenylpyrophosphate yielded 36 mg/L geranic acid in shake flask experiments. In the bioreactor, the recombinant strain produced 193 mg/L of geranic acid under fed-batch conditions within 48 h. CONCLUSION: Metabolic engineering turned Pseudomonas putida DSM 12264, a versatile monoterpenoid oxidation biocatalyst, into an efficient microbial cell factory for de novo geranic acid production. Improvements by metabolic and process engineering are expected to further increase the product concentration. To the best of the authors' knowledge, this is the first example of a de novo production of a monoterpenoid with Pseudomonas putida and of a microbial monoterpenoic acid synthesis in general.
Asunto(s)
Ingeniería Metabólica , Monoterpenos/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Terpenos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Novel chatechol/hydroxamate siderophores (named "fimsbactins") were identified in Acinetobacter baumannii ATCC 17978 and Acinetobacter baylyi ADP1. The major compound, fimsbactin A, was isolated from low-iron cultures of A. baylyi ADP1, and its chemical structure was elucidated by mass spectrometry, and detailed (1)H, (13)C and (15)N NMR spectroscopy. From inverse feeding experiments following HPLC-MS analysis, the structures of five additional derivatives were elucidated. The gene cluster encoding the fimsbactin synthetase (fbs) was identified in both genomes, and mutants in fbs genes in A. baylyi were analyzed, thus allowing prediction of the fimsbactin biosynthesis pathway.