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High cell density production of multimethyl-branched long-chain esters in Escherichia coli and determination of their physicochemical properties.

ABSTRACT: BACKGROUND:Microbial synthesis of oleochemicals derived from native fatty acid (FA) metabolism has presented significant advances in recent years. Even so, native FA biosynthetic pathways often provide a narrow variety of usually linear hydrocarbons, thus yielding end products with limited structural diversity. To overcome this limitation, we took advantage of a polyketide synthase-based system from Mycobacterium tuberculosis and developed an Escherichia coli platform with the capacity to synthesize multimethyl-branched long-chain esters (MBE) with novel chemical structures. RESULTS:With the aim to initiate the characterization of these novel waxy compounds, here, we describe the chassis optimization of the MBE producer E. coli strain for an up-scaled oil production. By carrying out systematic metabolic engineering, we improved the final titer to 138.1 ± 5.3 mg MBE L-1 in batch cultures. Fed-batch microbial fermentation process was also optimized achieving a maximum yield of 790.2 ± 6.9 mg MBE L-1 with a volumetric productivity of 15.8 ± 1.1 mg MBE (L h)-1. Purified MBE oil was subjected to various physicochemical analyses, including differential scanning calorimetry (DSC) and pressurized-differential scanning calorimetry (P-DSC) studies. CONCLUSIONS:The analysis of the pour point, DSC, and P-DSC data obtained showed that bacterial MBE possess improved cold flow properties than several plant oils and some chemically modified derivatives, while exhibiting high oxidation stability at elevated temperatures. These encouraging data indicate that the presence of multiple methyl branches in these novel esters, indeed, conferred favorable properties which are superior to those of linear esters.

SUBMITTER: Menendez-Bravo S

PROVIDER: S-EPMC5064953 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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High cell density production of multimethyl-branched long-chain esters in <i>Escherichia coli</i> and determination of their physicochemical properties.

Menendez-Bravo Simón S Roulet Julia J Sabatini Martín M Comba Santiago S Dunn Robert R Gramajo Hugo H Arabolaza Ana A

Biotechnology for biofuels 20161014

<h4>Background</h4>Microbial synthesis of oleochemicals derived from native fatty acid (FA) metabolism has presented significant advances in recent years. Even so, native FA biosynthetic pathways often provide a narrow variety of usually linear hydrocarbons, thus yielding end products with limited structural diversity. To overcome this limitation, we took advantage of a polyketide synthase-based system from <i>Mycobacterium tuberculosis</i> and developed an <i>Escherichia coli</i> platform with ...[more]

PMID: 27757170

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Project description:BackgroundA broad diversity of natural and non-natural esters have now been made in bacteria, and in other microorganisms, as a result of original metabolic engineering approaches. However, the fact that the properties of these molecules, and therefore their applications, are largely defined by the structural features of the fatty acid and alcohol moieties, has driven a persistent interest in generating novel structures of these chemicals.ResultsIn this research, we engineered Escherichia coli to synthesize de novo esters composed of multi-methyl-branched-chain fatty acids and short branched-chain alcohols (BCA), from glucose and propionate. A coculture engineering strategy was developed to avoid metabolic burden generated by the reconstitution of long heterologous biosynthetic pathways. The cocultures were composed of two independently optimized E. coli strains, one dedicated to efficiently achieve the biosynthesis and release of the BCA, and the other to synthesize the multi methyl-branched fatty acid and the corresponding multi-methyl-branched esters (MBE) as the final products. Response surface methodology, a cost-efficient multivariate statistical technique, was used to empirical model the BCA-derived MBE production landscape of the coculture and to optimize its productivity. Compared with the monoculture strategy, the utilization of the designed coculture improved the BCA-derived MBE production in 45%. Finally, the coculture was scaled up in a high-cell density fed-batch fermentation in a 2 L bioreactor by fine-tuning the inoculation ratio between the two engineered E. coli strains.ConclusionPrevious work revealed that esters containing multiple methyl branches in their molecule present favorable physicochemical properties which are superior to those of linear esters. Here, we have successfully engineered an E. coli strain to broaden the diversity of these molecules by incorporating methyl branches also in the alcohol moiety. The limited production of these esters by a monoculture was considerable improved by a design of a coculture system and its optimization using response surface methodology. The possibility to scale-up this process was confirmed in high-cell density fed-batch fermentations.

Project description:BACKGROUND:Quinolone resistant Escherichia coli (QREC) have been found in samples from Norwegian broiler chicken, despite quinolones not being administered to poultry in Norway. Biofilm production may be one factor contributing to the observed persistence in the broiler production chain. In the present study, 158 QREC strains from chicken caecal and retail meat samples were screened for biofilm production in microtiter plates, biofilm morphotype on Congo Red (CR) agar plates and phylotype by multiplex PCR. Furthermore, the dynamics in mixed biofilms with strains of different morphotypes were studied on glass slides and on CR agar plates. RESULTS:All strains but one produced biofilm in microtiter plates and/or on CR agar plates at room temperature. There were no differences between strains from chicken caecum and chicken retail meat in the mean amount of biofilm produced in microtiter plates. Furthermore, no differences in biofilm production were observed between phylotypes. However, significant differences in biofilm production were found between biofilm morphotypes. The morphotype RDAR (red dry and rough, which has both curli and cellulose in the matrix, was displayed by 70% of the strains. Mean biofilm production by these strains were significantly higher than by strains with the morphotypes PDAR (pink dry and rough) with only cellulose or BDAR (brown dry and rough) with only curli. Interestingly, the two latter morphotypes produced biofilms with the morphotype RDAR when grown together. None of the strains achieved significantly higher numbers of colony forming units (cfu) in mixed biofilms than in single strain biofilms on glass slides. CONCLUSIONS:The results indicate that QREC can form biofilm reservoirs on both inert and organic surfaces in production environments, as well as on meat. This may contribute to persistence and dissemination of the strains. Strains with both curli and cellulose in the biofilm matrix were significantly better biofilm formers than strains lacking one of these components. However, strains with only one of the components could compensate for this by producing mixed biofilms with strains having the other component, and thereby most likely enhance their probabilities of persistence in the production environment.

Project description:Plums (Prunus domestica); red currants (Ribes rubrum); black currants (Ribes nigrum); gooseberries (Ribes uva-crispa); sour cherries (Prunus cerasus); pumpkins (Cuccurbita spp.) are sources for valuable fruit- and berry-juice and cider production. This process leaves a large number of by-products (BP) in the form of pomace, which accounts for up to 80% of the raw material. This by-product represents a rich source of biologically active compounds, especially in the form of different pectic polysaccharides. The pectin extracted from commercial fruits such as citric fruits and apples has high medicinal properties, can be used as edible films and coatings, and is also useful in texture improvement and gel production in the food industry. However, many under-utilized fruits have received little attention regarding the extraction and characterization of their high/value pectin from their by-products. Moreover, the commercial extraction process involving strong acids and high temperature to obtain high-purity pectin leads to the loss of many bioactive components, and these lost components are often compensated for by the addition of synthetic antioxidants and colorants. The aim of the research is to extract pectin from juice production by-products with hot-water extraction using weak organic (0.1 N) citric acid, thus minimizing the impact on the environment. The yield of pectin (PY = 4.47-17.8% DM), galacturonic acid content (47.22-83.57 g 100-1), ash content (1.42-2.88 g 100 g-1), degree of esterification (DE = 45.16-64.06%), methoxyl content (ME = 4.27-8.13%), the total content of phenolic compounds (TPC = 2.076-4.668 µg mg-1, GAE) and the antiradical scavenging activity of the pectin samples (DPPH method (0.56-37.29%)) were determined. Free and total phenolic acids were quantified by saponification using high-pressure liquid chromatography (HPLC). The pectin contained phenolic acids-benzoic (0.25-0.92 µg mg-1), gallic (0.14-0.57 µg mg-1), coumaric (0.04 µg mg-1), and caffeic (0.03 µg mg-1). The pectin extracts from by-products showed glucose and galactose (3.89-21.72 g 100 g-1) as the main neutral sugar monosaccharides. Pectin analysis was performed using FT-IR, and the rheological properties of the pectin gels were determined. The quality of the obtained pectin from the fruit and berry by-products in terms of their high biological activity and high content of glucuronic acids indicated that the products have the potential to be used as natural ingredients in various food products and in pharmaceutical products.

Dataset Information

High cell density production of multimethyl-branched long-chain esters in Escherichia coli and determination of their physicochemical properties.

Publications

High cell density production of multimethyl-branched long-chain esters in <i>Escherichia coli</i> and determination of their physicochemical properties.

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