Carbon Source In Minimal Medium Research Articles

Rapid combinatorial rewiring of metabolic networks for enhanced poly(3-hydroxybutyrate) production in Corynebacterium glutamicum

BackgroundThe disposal of plastic waste is a major environmental challenge. With recent advances in microbial genetic and metabolic engineering technologies, microbial polyhydroxyalkanoates (PHAs) are being used as next-generation biomaterials to replace petroleum-based synthetic plastics in a sustainable future. However, the relatively high production cost of bioprocesses hinders the production and application of microbial PHAs on an industrial scale.ResultsHere, we describe a rapid strategy to rewire metabolic networks in an industrial microorganism, Corynebacterium glutamicum, for the enhanced production of poly(3-hydroxybutyrate) (PHB). A three-gene PHB biosynthetic pathway in Rasltonia eutropha was refactored for high-level gene expression. A fluorescence-based quantification assay for cellular PHB content using BODIPY was devised for the rapid fluorescence-activated cell sorting (FACS)-based screening of a large combinatorial metabolic network library constructed in C. glutamicum. Rewiring metabolic networks across the central carbon metabolism enabled highly efficient production of PHB up to 29% of dry cell weight with the highest cellular PHB productivity ever reported in C. glutamicum using a sole carbon source.ConclusionsWe successfully constructed a heterologous PHB biosynthetic pathway and rapidly optimized metabolic networks across central metabolism in C. glutamicum for enhanced production of PHB using glucose or fructose as a sole carbon source in minimal media. We expect that this FACS-based metabolic rewiring framework will accelerate strain engineering processes for the production of diverse biochemicals and biopolymers.

Bioreactor Rhamnolipid Production Using Palm Oil Agricultural Refinery By-Products

Palm fatty acid distillate (PFAD) and fatty acid methyl ester (FAME) are used by P. aeruginosa PAO1 to produce rhamnolipid biosurfactant. The process of fermentation producing of biosurfactant was structured in a 2 L bioreactor using 2% of PFAD and FAME as carbon sources in minimal medium and with a nitrogen concentration of 1 g L−1. Mass spectrometry results show the crude biosurfactant produced was predominantly monorhamnolipid (Rha-C10-C10) and dirhamnolipid (Rha-Rha-C10-C10) at 503 and 649 m/z value for both substrates. Maximum production of crude rhamnolipid for PFAD was 1.06 g L−1 whereas for FAME it was 2.1 g L−1, with a reduction in surface tension of Tris-HCl pH 8.0 solution to 28 mN m−1 and a critical micelle concentration (CMC) of 26 mg L−1 measured for both products. Furthermore, the 24 h emulsification indexes in kerosene, hexadecane, sunflower oil, and rapeseed oil using 1 g L−1 of crude rhamnolipid were in the range 20–50%. Consequently, PFAD and FAME, by-products from the agricultural refining of palm oil, may result in a product that has a higher added-value, rhamnolipid biosurfactant, in the process of integrated biorefinery.

Characterization of an operon required for growth on cellobiose in Clostridioides difficile.

Cellobiose metabolism is linked to the virulence properties in numerous bacterial pathogens. Here, we characterized a putative cellobiose PTS operon of Clostridiodes difficile to investigate the role of cellobiose metabolism in C. difficile pathogenesis. Our gene knockout experiments demonstrated that the putative cellobiose operon enables uptake of cellobiose into C. difficile and allows growth when cellobiose is provided as the sole carbon source in minimal medium. Additionally, using reporter gene fusion assays and DNA pulldown experiments, we show that its transcription is regulated by CelR, a novel transcriptional repressor protein, which directly binds to the upstream region of the cellobiose operon to control its expression. We have also identified cellobiose metabolism to play a significant role in C. difficile physiology as observed by the reduction of sporulation efficiency when cellobiose uptake was compromised in the mutant strain. In corroboration to in vitro study findings, our in vivo hamster challenge experiment showed a significant reduction of pathogenicity by the cellobiose mutant strain in both the primary and the recurrent infection model - substantiating the role of cellobiose metabolism in C. difficile pathogenesis.

Carbon Source Influence on Extracellular pH Changes along Bacterial Cell-Growth.

The effect of initial pH on bacterial cell-growth and its change over time was studied under aerobic heterotrophic conditions by using three bacterial strains: Escherichia coli ATCC 25922, Pseudomonas putida KT2440, and Pseudomonas pseudoalcaligenes CECT 5344. In Luria-Bertani (LB) media, pH evolved by converging to a certain value that is specific for each bacterium. By contrast, in the buffered Minimal Medium (MM), pH was generally more stable along the growth curve. In MM with glucose as carbon source, a slight acidification of the medium was observed for all strains. In the case of E. coli, a sudden drop in pH was observed during exponential cell growth that was later recovered at initial pH 7 or 8, but was irreversible below pH 6, thus arresting further cell-growth. When using other carbon sources in MM at a fixed initial pH, pH changes depended mainly on the carbon source itself. While glucose, glycerol, or octanoate slightly decreased extracellular pH, more oxidized carbon sources, such as citrate, 2-furoate, 2-oxoglutarate, and fumarate, ended up with the alkalinization of the medium. These observations are in accordance with pH change predictions using genome-scale metabolic models for the three strains, thus revealing the metabolic reasons behind pH change. Therefore, we conclude that the composition of the medium, specifically the carbon source, determines pH change during bacterial growth to a great extent and unravel the main molecular mechanism behind this phenotype. These findings pave the way for predicting pH changes in a given bacterial culture and may anticipate the interspecies interactions and fitness of bacteria in their environment.

Valorisation of orange peel: supplement in fermentation media for ethanol production and source of limonene

The usage of the citrus wastes for strategic valorization is a concept for the production of various commercial products. Wastes from citrus fruits have a considerate amount of sugar and limonene. In the present work, partially removed limonene from hydrolysed orange peel was used for supplementation as carbon source in minimal media for the production of ethanol. A novel yeast strain, Saccharomyces cerevisiae strain BT1 (MK373758) was isolated from Toddy, obtained from Borassus flabellifer (toddy palm), Bhatan, Mumbai. This strain was used in the formulated fermentation media for the production of bioethanol. Optimization of critical parameters was done for pH, temperature and time of fermentation vs production of bioethanol. The Brix, pH, sugar content and ethanol content were measured during the fermentation process. The ethanol content was estimated by dichromate method and high performance liquid chromatography (HPLC) and the yield obtained was 0.95%. In addition, limonene was also extracted from hydrolysed orange peel and estimated by gas chromatography-flame ionization detection (GC-FID) and was found to be 0.4% (v/v). The two valuable products, bioethanol, and d-limonene, obtained from orange peel waste, promises its valorisation at a larger scale.

Mini Photobioreactors for in Vivo Real-Time Characterization and Evolutionary Tuning of Bacterial Optogenetic Circuit.

The current standard protocols for characterizing the optogenetic circuit of bacterial cells using flow cytometry in light tubes and light exposure of culture plates are tedious, labor-intensive, and cumbersome. In this work, we engineer a bioreactor with working volume of ∼10 mL for in vivo real-time optogenetic characterization of E. coli with a CcaS-CcaR light-sensing system. In the bioreactor, optical density measurements, reporter protein fluorescence detection, and light input stimuli are provided by four light-emitting diode sources and two photodetectors. Once calibrated, the device can cultivate microbial cells and record their growth and gene expression without human intervention. We measure gene expression during cell growth with different organic substrates (glucose, succinate, acetate, pyruvate) as carbon sources in minimal medium and demonstrate evolutionary tuning of the optogenetic circuit by serial dilution passages.

Isolation of Rhodococcus sp. CMGCZ Capable to Degrade High Concentration of Fluoranthene

A bacterial strain CMGCZ was isolated from an abandoned oil field soil sample and identified as Rhodococcus sp. by 16S rRNA sequencing. Rhodococcus sp. CMGCZ was investigated for the degradation of model polycyclic aromatic hydrocarbons (PAHs) and Iranian light crude oil (ILCO) as a sole carbon source in minimal medium. Biodegradation enhancement was attained by supplementing the minimal medium with yeast extract (YE). Rhodococcus sp. CMGCZ was capable to degrade 13.2 % naphthalene (Nap), 13.1 % phenanthrene (Phe), and 99.3 % fluoranthene (Fla) in 1 week and 11 % aliphatic fraction of ILCO in 2 weeks as a sole carbon and energy source. Effect of YE supplementation on degradation potential of Rhodococcus sp. CMGCZ depended upon the added hydrocarbon in the medium. YE completely inhibited Nap degradation, slightly enhanced degradation of Phe (14.8 %) and ILCO aliphatics (13.2 %), and promoted a more rapid degradation of Fla (100 %). YE addition promoted rapid degradation of Fla and eliminated delay of 24 h in Fla degradation that was observed in minimal medium. Rhodococcus sp. CMGCZ was capable to degrade high concentrations of Fla (1000 mg l−1). Rieske [Fe2-S2] center was amplified in Rhodococcus sp. CMGCZ that exhibited homology with Rieske [Fe2-S2] domain protein of Mycobacterium species and pahAC gene of uncultured bacterium clones.

Bioprospecting and evolving alternative xylose and arabinose pathway enzymes for use in Saccharomyces cerevisiae.

Bioprospecting is an effective way to find novel enzymes from strains with desirable phenotypes. Such bioprospecting has enabled organisms such as Saccharomyces cerevisiae to utilize nonnative pentose sugars. Yet, the efficiency of this pentose catabolism (especially for the case of arabinose) remains suboptimal. Thus, further pathway optimization or identification of novel, optimal pathways is needed. Previously, we identified a novel set of xylan catabolic pathway enzymes from a superior pentose-utilizing strain of Ustilago bevomyces. These enzymes were used to successfully engineer a xylan-utilizing S. cerevisiae through a blended approach of bioprospecting and evolutionary engineering. Here, we expanded this approach to xylose and arabinose catabolic pathway engineering and demonstrated that bioprospected xylose and arabinose catabolic pathways from U. bevomyces offer alternative choices for enabling efficient pentose catabolism in S. cerevisiae. By introducing a novel set of xylose catabolic genes from U. bevomyces, growth rates were improved up to 85 % over a set of traditional Scheffersomyces stipitis pathway genes. In addition, we suggested an alternative arabinose catabolic pathway which, after directed evolution and pathway engineering, enabled S. cerevisiae to grow on arabinose as a sole carbon source in minimal medium with growth rates upwards of 0.05 h(-1). This pathway represents the most efficient growth of yeast on pure arabinose minimal medium. These pathways provide great starting points for further strain development and demonstrate the utility of bioprospecting from U. bevomyces.

Xylan catabolism is improved by blending bioprospecting and metabolic pathway engineering in Saccharomyces cerevisiae.

Complete utilization of all available carbon sources in lignocellulosic biomass still remains a challenge in engineering Saccharomyces cerevisiae. Even with efficient heterologous xylose catabolic pathways, S. cerevisiae is unable to utilize xylose in lignocellulosic biomass unless xylan is depolymerized to xylose. Here we demonstrate that a blended bioprospecting approach along with pathway engineering and evolutionary engineering can be used to improve xylan catabolism in S. cerevisiae. Specifically, we perform whole genome sequencing-based bioprospecting of a strain with remarkable pentose catabolic potential that we isolated and named Ustilago bevomyces. The heterologous expression of xylan catabolic genes enabled S. cerevisiae to grow on xylan as a single carbon source in minimal medium. A combination of bioprospecting and metabolic pathway evolution demonstrated that the xylan catabolic pathway could be further improved. Ultimately, engineering efforts were able to achieve xylan conversion into ethanol of up to 0.22 g/L on minimal medium compositions with xylan. This pathway provides a novel starting point for improving lignocellulosic conversion by yeast.

Metagenomes of tropical soil-derived anaerobic switchgrass-adapted consortia with and without iron

Tropical forest soils decompose litter rapidly with frequent episodes of anoxia, making it likely that bacteria using alternate terminal electron acceptors (TEAs) such as iron play a large role in supporting decomposition under these conditions. The prevalence of many types of metabolism in litter deconstruction makes these soils useful templates for improving biofuel production. To investigate how iron availability affects decomposition, we cultivated feedstock-adapted consortia (FACs) derived from iron-rich tropical forest soils accustomed to experiencing frequent episodes of anaerobic conditions and frequently fluctuating redox. One consortium was propagated under fermenting conditions, with switchgrass as the sole carbon source in minimal media (SG only FACs), and the other consortium was treated the same way but received poorly crystalline iron as an additional terminal electron acceptor (SG + Fe FACs). We sequenced the metagenomes of both consortia to a depth of about 150 Mb each, resulting in a coverage of 26× for the more diverse SG + Fe FACs, and 81× for the relatively less diverse SG only FACs. Both consortia were able to quickly grow on switchgrass, and the iron-amended consortium exhibited significantly higher microbial diversity than the unamended consortium. We found evidence of higher stress in the unamended FACs and increased sugar transport and utilization in the iron-amended FACs. This work provides metagenomic evidence that supplementation of alternative TEAs may improve feedstock deconstruction in biofuel production.

Canthaxanthin production with modified Mucor circinelloides strains

Canthaxanthin is a natural diketo derivative of β-carotene primarily used by the food and feed industries. Mucor circinelloides is a β-carotene-accumulating zygomycete fungus and one of the model organisms to study the carotenoid biosynthesis in fungi. In this study, the β-carotene ketolase gene (crtW) of the marine bacterium Paracoccus sp. N81106 fused with fungal promoter and terminator regions was integrated into the M. circinelloides genome to construct stable canthaxanthin-producing strains. Different transformation methods including polyethylene glycol-mediated transformation with linear DNA fragments, restriction enzyme-mediated integration and Agrobacterium tumefaciens-mediated transformation were tested to integrate the crtW gene into the Mucor genome. Mitotic stability, site of integration and copy number of the transferred genes were analysed in the transformants, and several stable strains containing the crtW gene in high copy number were isolated. Carotenoid composition of selected transformants and effect of culturing conditions, such as temperature, carbon sources and application of certain additives in the culturing media, on their carotenoid content were analysed. Canthaxanthin-producing transformants were able to survive at higher growth temperature than the untransformed strain, maybe due to the effect of canthaxanthin on the membrane fluidity and integrity. With the application of glucose, trehalose, dihydroxyacetone and L-aspartic acid as sole carbon sources in minimal medium, the crtW-expressing M. circinelloides strain, MS12+pCA8lf/1, produced more than 200 μg/g (dry mass) of canthaxanthin.

BACTERIAL ISOLATES OF MARINE COAST AS COMMERCIAL PRODUCER OF PROTEASE

The objective of this work was to explore and exploit the extracellular protease secreting marine microbial biodiversity of the eastern coastal region of India. Culture dependent method was applied for isolation of microbes from the marine coast of West Bengal (Digha and Mandarmani) and Andhra Pradesh (Vizag) in India. Six protease secreting isolates were screened using casein hydrolysing property as well as azocasein assay and characterized on the basis of their morphological, biochemical, physiological and 16S rDNA based molecular properties. The enzymes were used for various commercial applications at a laboratory scale. Besides milk media and Luria Bertini broth, all the isolates grew in carbon minimal salt medium with jaggeri or tamarind as the carbon source (0.3% w/v). They showed intracellular metal accumulation when grown in presence of metal salts in the medium as evident from Energy Dispersive X-ray Fluorescense Analysis (EDXRF) data. Maximum accumulation of lead was found in case of Bacillus cereus SM2. It showed equal efficiency of metal removal from solid strip at zero valent state. The isolates were also capable of complete removal of silver from exposed X-ray film after 48 hrs of incubation except for Escherichia coli SD1. Bacillus cereus SM2, isolate SD2 (closest to Bacillus pumilus) and isolate SV1 (closest to Bacillus cibi) were able to enhance the cleaning efficiency of detergent when used as additive. Use of tamarind and jaggeri as carbon source in minimal medium would make the process cost effective during large scale application. The ability to grow in a wide range of temperature and pH and accumulation of heavy metals revealed that these isolates would be potential candidates for bioremediation. Thus the marine diversity for protease production is extremely rich with immense commercial applications.

English

In the present investigation, Planococcus halophilus was screened for hydrocarbon degradation and bioremediation of refinery effluent. The test organism, P.halophilus, showed the capability to utilize kerosene as carbon source in minimal medium. Biological treatment of the refinery effluent with P. halophilus reduced the oil and grease and sulphide content to about 91.2 and 28%, respectively, on the 4th day of the incubation. The present work defined that the test organism P. halophiluscan be exploited for bioremediation of sites contaminated with hydrocarbons and industrial effluents polluted with hydrocarbons even under adverse conditions.   Key words: Kerosene, Planococcus halophilus, bioremediation, parameters, hydrocarbon.

Acetate–glycerol cometabolism: Cultivating Schizosaccharomyces pombe on a non-fermentable carbon source in a defined minimal medium

The growth of the fission yeast Schizosaccharomyces pombe on glucose and glycerol was monitored on-line in shake flasks and microtiter plates. The Edinburgh Minimal Medium 2 was improved by doubling its concentrations, improving its buffer and increasing its sulphur and iron concentrations additionally. By growing S. pombe on mixed carbon sources, it was shown that glycerol and glucose complement one another. Several tests were performed to establish the cultivation of S. pombe with non-fermentable glycerol as the main carbon source in minimal medium. Interestingly, a synergistic effect of glycerol and acetate was discovered which can significantly improve the growth of the fission yeast on glycerol. S. pombe showed optimal respiration activity, growth, and product formation by co-utilizing 20g/L glycerol and 2.5g/L sodium acetate.

Biodegradability of bacterial surfactants

This work aimed at evaluating the biodegradability of different bacterial surfactants in liquid medium and in soil microcosms. The biodegradability of biosurfactants by pure and mixed bacterial cultures was evaluated through CO(2) evolution. Three bacterial strains, Acinetobacter baumanni LBBMA ES11, Acinetobacter haemolyticus LBBMA 53 and Pseudomonas sp. LBBMA 101B, used the biosurfactants produced by Bacillus sp. LBBMA 111A (mixed lipopeptide), Bacillus subtilis LBBMA 155 (lipopeptide), Flavobacterium sp. LBBMA 168 (mixture of flavolipids), Dietzia Maris LBBMA 191(glycolipid) and Arthrobacter oxydans LBBMA 201(lipopeptide) as carbon sources in minimal medium. The synthetic surfactant sodium dodecyl sulfate (SDS) was also mineralized by these microorganisms, but at a lower rate. CO(2) emitted by a mixed bacterial culture in soil microcosms with biosurfactants was higher than in the microcosm containing SDS. Biosurfactant mineralization in soil was confirmed by the increase in surface tension of the soil aqueous extracts after incubation with the mixed bacterial culture. It can be concluded that, in terms of biodegradability and environmental security, these compounds are more suitable for applications in remediation technologies in comparison to synthetic surfactants. However, more information is needed on structure of biosurfactants, their interaction with soil and contaminants and scale up and cost for biosurfactant production.

Optimization of mannanse production from Streptomyces sp. PG-08-03 in submerged fermentation

Streptomyces sp. PG-08-3 was isolated from the desert of Rajasthan (India). The organism produced mannanase (15 Umg-1 protein) in the presence of 0.5% guar gum as a sole carbon source in minimal media by submerged fermentation (SmF). Enzyme production was enhanced by 7.3-fold when 0.5% soyabean meal and 0.25% of leucine were added to the minimal media. Increasing the guar gum concentration in the media by 0.1-1.0% resulted in linearly enhanced the production of mannanase.

Properties of the Functioning of the Promoter of the Microcin C51 Operon under Different Conditions of Escherichia coli Cell Growth

The level of transcription from the promoter of the microcin C51 operon (P mcc ) depends on the growth phase of Escherichia colicells: transcription proceeds with low efficiency at the exponential phase of growth and with higher efficiency when growth of cells is delayed during entry into the stationary phase. The functioning of P mcc was studied in cells grown in different media by a single-copy construct, which contained the cloned promoter region of the microcin C51 operon and the promoterless lacoperon. A decrease in the rate of cell growth caused by changes in the sole carbon source in minimal medium correlated with an increase in the level of transcription from the P mcc promoter at the exponential phase of growth; the expression of P mcc –lacduring cell entry into the stationary phase was higher under less favorable medium conditions. The use of composite rich media impaired this feature. The addition of l-leucine (100 μg/ml) to the medium decreased the expression of P mcc –lacin wild-type cells and those carrying the Δlrpmutation. A further increase in leucine concentration and the presence of other amino acids in the medium enhanced transcription that started from P mcc during cell entry into the stationary growth phase. The capacity of the P mcc promoter and of the wild-type lacZgene promoter upon IPTG induction was virtually the same. A mutation in the ompRgene did not markedly influence transcription started from P mcc .

Cloning and characterization of sialidases with 2-6' and 2-3' sialyl lactose specificity from Pasteurella multocida.

Pasteurella multocida is a mucosal pathogen that colonizes the respiratory system of susceptible hosts. Most isolates of P. multocida produce sialidase activity, which may contribute to colonization of the respiratory tract or the production of lesions in an active infection. We have cloned and sequenced a sialidase gene, nanH, from a fowl cholera isolate of P. multocida. Sequence analysis of NanH revealed that it exhibited significant amino acid sequence homology with many microbial sialidases. Insertional inactivation of nanH resulted in a mutant strain that was not deficient in sialidase production. However, this mutant exhibited reduced enzyme activity and growth rate on 2-3' sialyl lactose compared to the wild type. Subsequently, we demonstrated the presence of two sialidases by cloning another sialidase gene that differed from nanH in DNA sequence and substrate specificity. NanB demonstrated activity on both 2-3' and 2-6' sialyl lactose, while NanH demonstrated activity only on 2-3' sialyl lactose. Neither enzyme liberated sialic acid from colominic acid (2-8' sialyl lactose). Recombinant E. coli containing the sialidase genes were able to utilize several sialoconjugants when they were provided as sole carbon sources in minimal medium. These data suggest that sialidases have a nutritional function and may contribute to the ability of P. multocida to colonize and persist on vertebrate mucosal surfaces.

L-lactate enzyme electrode obtained with immobilized respiratory chain From Escherichia coli and oxygen probe for specific determination of l-lactate in yogurt, wine and blood

An enzyme electrode for L-lactate measurements in various biological media was prepared with an immobilized bacterial respiratory chain fixed to Actark electrode. The enzymatic film, which was easy to prepare, contained bacteria immobilized in gelatin, tanned with glutaraldehyde. This electrode was sensitive to 0.1 mM L-lactate and could be utilized to 10 mM. The apparent Kso was 5 mM. Less than 8% of the respiration rate with l-lactate was measured with D-lactate and succinate. The competitive inhibitors d-lactate and pyruvate had a K 50 of 50 mM. They could be quantitatively measured by inhibition in a range between 5 and 50 mM. It was also possible to discriminate between l-lactate and various metabolites of the respiratory chain: l-malate, succinate, 3-glycero-phosphateorNAD(P)H. Growing E. coli on 1% d- l-lactate as the sole carbon source in minimal medium induced l-lactate respiration tenfold. All other respiratory activities remained below 10% of the activity with l-lactate. A computerized instrument allowed successive measurements every 3 min for more than 10 h with the same enzymatic film. Most of the measured samples required dilution but no clarification or purification. This enzyme electrode may have many applications in basic research (metabolism, enzymology) and applied research (blood, yogurt, juices, wine).

Isolation and some properties of cell envelope altered mutants of Escherichia coli.

Mutants of Escherichia coli which have a defect in their permeability barrier were selected. The technique used was to employ a strain of E. coli having a deletion in the gene for lactose permease and to select for mutants which can grow on lactose at 40 C. Twenty such mutants were isolated and six of these were found to be more sensitive to actinomycin D, sodium deoxycholate, and sodium dodecyl sulfate than was the parental strain. They were also more sensitive to the antibiotics vancomycin and bacitracin, which inhibit peptidoglycan biosynthesis. These mutants were no more sensitive to several different colicins or phages than was the wild-type strain. One of the mutants selected by this technique has an abnormal morphology when grown on certain carbon sources in minimal medium, and this mutant is more extensively studied in the accompanying paper.