Fermentation of dihydroxyacetone by engineered Escherichia coli and Klebsiella variicola to products.
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ABSTRACT:
SUBMITTER: Wang L
PROVIDER: S-EPMC5924927 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundBear bile powder is a valuable medicinal material characterized by high content of tauroursodeoxycholic acid (TUDCA) at a certain ratio to taurochenodeoxycholic acid (TCDCA). We had created an engineered E. coli harboring two-step bidirectional oxidative and reductive enzyme-catalyzing pathway that could rapidly convert TCDCA to TUDCA at a specific percentage in shake flasks.ResultsWe reported here the large-scale production of TUDCA containing products by balancing the bidirectional reactions through optimizing fermentation process of the engineered E. coli in fermenters. The fermentation medium was firstly optimized based on M9 medium using response surface methodology, leading to a glycerol and yeast extract modified M9-GY medium benefits for both cell growth and product conversion efficiency. Then isopropylthio-β-galactoside induction and fed-stock stage was successively optimized. Finally, a special deep-tank static process was developed to promote the conversion from TCDCA to TUDCA. Applying the optimal condition, fermentation was performed by separately supplementing 30 g refined chicken bile powder and 35 g crude chicken bile powder as substrates, resulting in 29.35 ± 2.83 g and 30.78 ± 3.04 g powder products containing 35.85 ± 3.85% and 27.14 ± 4.23% of TUDCA at a ratio of 1.49 ± 0.14 and 1.55 ± 0.19 to TCDCA, respectively, after purification and evaporation of the fermentation broth. The recovery yield was 92.84 ± 4.21% and 91.83 ± 2.56%, respectively.ConclusionThis study provided a practical and environment friendly industrialized process for producing artificial substitute of bear bile powder from cheap and readily available chicken bile powder using engineered E. coli microbial cell factory. It also put forward an interesting deep-tank static process to promote the enzyme-catalyzing reactions toward target compounds in synthetic biology-based fermentation.
| S-EPMC6368744 | biostudies-literature
Project description:Tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) found in diverse pharmaceutical compounds, is used as a starting material for the production of BIA. THP also has various neurobiological properties but is difficult to synthesize. Therefore, a simple method for THP production is desired. Recent studies have shown that microbes, especially bacteria, can serve as platforms for synthesizing these complex compounds; however, because bacteria lack organelles, the designed synthetic pathway cannot be compartmentalized. Thus, the metabolic flow is frequently inhibited or disrupted by undesirable reactions. Indeed, in the first attempt to synthesize THP using a single strain of engineered Escherichia coli, the yield was quite low (<5??M), mainly because of the oxidation of THP by tyrosinase, an essential enzyme in our production system. To circumvent these problems, we constructed a stepwise (R,S)-THP production system, in which the dopamine-producing step and the subsequent THP-producing step were separated. The yield of (R,S)-THP reached 1.0?mM (287?mg/L), the highest yielding BIA production method using a microbe reported to date. Furthermore, we demonstrated that (R,S)-THP produced by stepwise fermentation is useful for the production of reticuline, an important BIAs intermediate. Based on these observations, applying the stepwise fermentation method is discussed.
| S-EPMC4204043 | biostudies-literature
Project description:Opiates such as morphine and codeine are mainly obtained by extraction from opium poppies. Fermentative opiate production in microbes has also been investigated, and complete biosynthesis of opiates from a simple carbon source has recently been accomplished in yeast. Here we demonstrate that Escherichia coli serves as an efficient, robust and flexible platform for total opiate synthesis. Thebaine, the most important raw material in opioid preparations, is produced by stepwise culture of four engineered strains at yields of 2.1 mg l(-1) from glycerol, corresponding to a 300-fold increase from recently developed yeast systems. This improvement is presumably due to strong activity of enzymes related to thebaine synthesis from (R)-reticuline in E. coli. Furthermore, by adding two genes to the thebaine production system, we demonstrate the biosynthesis of hydrocodone, a clinically important opioid. Improvements in opiate production in this E. coli system represent a major step towards the development of alternative opiate production systems.
| S-EPMC4748248 | biostudies-literature
Project description:The fermentative metabolism of Escherichia coli was reengineered to efficiently convert glycerol to succinate under anaerobic conditions without the use of foreign genes. Formate and ethanol were the dominant fermentation products from glycerol in wild-type Escherichia coli ATCC 8739, followed by succinate and acetate. Inactivation of pyruvate formate-lyase (pflB) in the wild-type strain eliminated the production of formate and ethanol and reduced the production of acetate. However, this deletion slowed growth and decreased cell yields due to either insufficient energy production or insufficient levels of electron acceptors. Reversing the direction of the gluconeogenic phosphoenolpyruvate carboxykinase reaction offered an approach to solve both problems, conserving energy as an additional ATP and increasing the pool of electron acceptors (fumarate and malate). Recruiting this enzyme through a promoter mutation (pck*) to increase expression also increased the rate of growth, cell yield, and succinate production. Presumably, the high NADH/NAD(+) ratio served to establish the direction of carbon flow. Additional mutations were also beneficial. Glycerol dehydrogenase and the phosphotransferase-dependent dihydroxyacetone kinase are regarded as the primary route for glycerol metabolism under anaerobic conditions. However, this is not true for succinate production by engineered strains. Deletion of the ptsI gene or any other gene essential for the phosphotranferase system was found to increase succinate yield. Deletion of pflB in this background provided a further increase in the succinate yield. Together, these three core mutations (pck*, ptsI, and pflB) effectively redirected carbon flow from glycerol to succinate at 80% of the maximum theoretical yield during anaerobic fermentation in mineral salts medium.
| S-EPMC2849195 | biostudies-literature
Project description:BackgroundEngineered strains of Escherichia coli have been used to produce bioconjugate vaccines using Protein Glycan Coupling Technology (PGCT). Nanovaccines have also entered the vaccine development arena with advances in nanotechnology and have been significantly developed, but chassis cells for conjugate nanovaccines have not been reported.ResultsTo facilitate nanovaccine preparation, a generic recombinant protein (SpyCather4573) was used as the acceptor protein for O-linked glycosyltransferase PglL, and a glycol-engineered Escherichia coli strain with these two key components (SC4573 and PglL) integrated in its genome was developed in this study. The targeted glycoproteins with antigenic polysaccharides produced by our bacterial chassis can be spontaneously bound to proteinous nanocarriers with surface exposed SpyTag in vitro to form conjugate nanovaccines. To improve the yields of the targeted glycoprotein, a series of gene cluster deletion experiments was carried out, and the results showed that the deletion of the yfdGHI gene cluster increased the expression of glycoproteins. Using the updated system, to the best of our knowledge, we report for the first time the successful preparation of an effective Klebsiella pneumoniae O1 conjugate nanovaccine (KPO1-VLP), with antibody titers between 4 and 5 (Log10) after triple immunization and up to 100% protection against virulent strain challenge.ConclusionsOur results define a convenient and reliable framework for bacterial glycoprotein vaccine preparation that is flexible and versatile, and the genomic stability of the engineered chassis cells promises a wide range of applications for biosynthetic glycobiology research.
| S-EPMC10163571 | biostudies-literature
Project description:Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP-4-keto-6-deoxyglucose resulting in increased production of the heterologous sugars TDP-L-mycarose and TDP-D-desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6-deoxyerythronolide B (6-dEB) or 3-?-mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60- and 25-fold more erythromycin D (EryD) than the original strain K207-3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars.
| S-EPMC3815289 | biostudies-literature
Project description:Ammonia is used as a fertilizer for agriculture, chemical raw material, and carrier for transporting hydrogen, and with economic development, the demand for ammonia has increased. The Haber-Bosch process, which is the main method for producing ammonia, can produce ammonia with high efficiency. However, since it consumes a large amount of fossil energy, it is necessary to develop an alternative method for producing ammonia with less environmental impact. Ammonia production from food by-products is an appealing production process owing to unused resource usage, including waste, and mild reaction conditions. However, when food by-products and biomass are used as feedstocks, impurities often reduce productivity. Using metabolic profiling, glucose was identified as a potential inhibitor of ammonia production from impure food by-products. We constructed the recombinant Escherichia coli, in which glucose uptake was reduced by ptsG gene disruption and amino acid catabolism was promoted by glnA gene disruption. Ammonia production efficiency from okara, a food by-product, was improved in this strain; 35.4 mM ammonia was produced (47% yield). This study might provide a strategy for efficient ammonia production from food by-products.
| S-EPMC7434829 | biostudies-literature
Project description:Curcumin is a plant secondary metabolite with outstanding therapeutic effects. Therefore, there is a great interest in developing new strategies to produce this high-value compound in a cheaper and environmentally friendly way. Curcumin heterologous production in Escherichia coli using artificial biosynthetic pathways was previously demonstrated using synthetic biology approaches. However, the culturing conditions to produce this compound were not optimized and so far only a two-step fermentation process involving the exchange of culture medium allowed high concentrations of curcumin to be obtained, which limits its production at an industrial scale. In this study, the culturing conditions to produce curcumin were evaluated and optimized. In addition, it was concluded that E. coli BL21 allows higher concentrations of curcumin to be produced than E. coli K-12 strains. Different isopropyl β-d-thiogalactopyranoside concentrations, time of protein expression induction and substrate type and concentration were also evaluated. The highest curcumin production obtained was 959.3 µM (95.93% of per cent yield), which was 3.1-fold higher than the highest concentration previously reported. This concentration was obtained using a two-stage fermentation with lysogeny broth (LB) and M9. Moreover, terrific broth was also demonstrated to be a very interesting alternative medium to produce curcumin because it also led to high concentrations (817.7 µM). The use of this single fermentation medium represents an advantage at industrial scale and, although the final production is lower than that obtained with the LB-M9 combination, it leads to a significantly higher production of curcumin in the first 24 h of fermentation. This study allowed obtaining the highest concentrations of curcumin reported so far in a heterologous organism and is of interest for all of those working with the heterologous production of curcuminoids, other complex polyphenolic compounds or plant secondary metabolites.
| S-EPMC5582133 | biostudies-literature
Project description:The rapid detection and species-level differentiation of bacterial pathogens facilitates antibiotic stewardship and improves disease management. Here, we develop a rapid bacteriophage-based diagnostic assay to detect the most prevalent pathogens causing urinary tract infections: Escherichia coli, Enterococcus spp., and Klebsiella spp. For each uropathogen, two virulent phages were genetically engineered to express a nanoluciferase reporter gene upon host infection. Using 206 patient urine samples, reporter phage-induced bioluminescence was quantified to identify bacteriuria and the assay was benchmarked against conventional urinalysis. Overall, E. coli, Enterococcus spp., and Klebsiella spp. were each detected with high sensitivity (68%, 78%, 87%), specificity (99%, 99%, 99%), and accuracy (90%, 94%, 98%) at a resolution of ≥103 CFU/ml within 5 h. We further demonstrate how bioluminescence in urine can be used to predict phage antibacterial activity, demonstrating the future potential of reporter phages as companion diagnostics that guide patient-phage matching prior to therapeutic phage application.
| S-EPMC10359277 | biostudies-literature
Project description:Microbial fermentation for L-methionine (L-Met) production based on natural renewable resources is attractive and challenging. In this work, the effects of medium composition and fermentation conditions were investigated to improve L-Met production by genetically engineered Escherichia coli MET-3. Statistical optimization techniques including Plackett-Burman (PB) design and Box-Behnken design (BBD) were adopted first to optimize the culture medium. Results of PB-designed experiments indicated that the culture medium components including glucose, yeast extract, KH2PO4, and MgSO4.7H2O had significant effects on L-Met biosynthesis. With their best-predicted concentration established by BBD (glucose 37.43 g/L, yeast extract 0.95 g/L, KH2PO4 1.82 g/L, and MgSO4.7H2O 4.51 g/L), L-Met titer was increased to 3.04 g/L from less than 2.0 g/L. For further enhancement of L-Met biosynthesis, the fermentation conditions of batch cultivation carried out in a 5-L fermentor were optimized, and the optimum results were obtained at an agitation rate of 300 rpm, medium pH of 7.0, and induction temperature of 28 °C. Based on the optimization parameters, fed-batch fermentation with the modified medium was conducted. As a result, great improvement of L-Met titer (12.80 g/L) and yield (0.13 mol/mol) were achieved, with an increase of 38.53% and 30.0% compared with those of the basal medium, respectively. Furthermore, higher L-Met productivity of 0.261 g/L/h was obtained, representing 2.13-fold higher in comparison to the original medium. The results may provide a helpful reference for further study on strain improvement and fermentation control.
| S-EPMC6385071 | biostudies-literature