Combination Of Metabolic Engineering Research Articles

Petroleum Hydrocarbon Catabolic Pathways as Targets for Metabolic Engineering Strategies for Enhanced Bioremediation of Crude-Oil-Contaminated Environments

Anthropogenic activities and industrial effluents are the major sources of petroleum hydrocarbon contamination in different environments. Microbe-based remediation techniques are known to be effective, inexpensive, and environmentally safe. In this review, the metabolic-target-specific pathway engineering processes used for improving the bioremediation of hydrocarbon-contaminated environments have been described. The microbiomes are characterised using environmental genomics approaches that can provide a means to determine the unique structural, functional, and metabolic pathways used by the microbial community for the degradation of contaminants. The bacterial metabolism of aromatic hydrocarbons has been explained via peripheral pathways by the catabolic actions of enzymes, such as dehydrogenases, hydrolases, oxygenases, and isomerases. We proposed that by using microbiome engineering techniques, specific pathways in an environment can be detected and manipulated as targets. Using the combination of metabolic engineering with synthetic biology, systemic biology, and evolutionary engineering approaches, highly efficient microbial strains may be utilised to facilitate the target-dependent bioprocessing and degradation of petroleum hydrocarbons. Moreover, the use of CRISPR-cas and genetic engineering methods for editing metabolic genes and modifying degradation pathways leads to the selection of recombinants that have improved degradation abilities. The idea of growing metabolically engineered microbial communities, which play a crucial role in breaking down a range of pollutants, has also been explained. However, the limitations of the in-situ implementation of genetically modified organisms pose a challenge that needs to be addressed in future research.

Metabolic engineering and optimization of Escherichia coli co-culture for the de novo synthesis of genkwanin.

Genkwanin has various significant roles in nutrition, biomedicine, and pharmaceutical biology. Previously, this compound has been chiefly produced by plant-originated extraction or chemical synthesis. However, due to increasing concern and demand for safe food and environmental issues, the biotechnological production of genkwanin and other bioactive compounds based on safe, cheap, and renewable substrates has gained much interest. This paper described recombinant Escherichia coli-based co-culture engineering that was reconstructed for the de novo production of genkwanin from D-glucose. The artificial genkwanin biosynthetic chain was divided into two modules in which the upstream strain contained the genes for synthesizing p-coumaric acid from D-glucose. And, the downstream module contained a gene cluster that produced the precursor apigenin and the final product, genkwanin. The Box-Behnken design, a response surface methodology, was used to empirically model the production of genkwanin and optimize its productivity. As a result, the application of the designed co-culture improved the genkwanin production by 48.8±1.3mg/L or 1.7-fold compared to the mono-culture. In addition, the scale-up of genkwanin bioproduction by bioreactor resulted in 68.5±1.9mg/L at a 48h time point. The combination of metabolic engineering and fermentation technology was therefore a very efficient and applicable approach to enhance the production of other bioactive compounds.

High-yield ectoine production in engineered Corynebacterium glutamicum by fine metabolic regulation via plug-in repressor library

Ectoine is a high-value protective and stabilizing agent with different applications in biopharmaceuticals, biotechnology, and fine chemicals. Here, efficient production of ectoine in Corynebacterium glutamicum was achieved by combination of metabolic engineering and plug-in repressor library strategy. First, the ectBAC cluster from Pseudomonas stutzeri was introduced into strain K02, and the titer of the obtained strain was 2.12 g/L. Metabolic engineering was then performed for further optimization, including removal of competing pathways (pck and ldh knockout), deletion of glycolysis repressor (sugR knockout), and enhancement of precursor supply (overexpression of Ecasd and CglysCS301Y). Next, two repressor libraries were designed for targeted flux control to improve ectoine production. Finally, strain CB5L6 produced 45.52 g/L ectoine and had the highest yield in C. glutamicum. For the first time, plug-in repressor library was employed to engineer C. glutamicum for metabolites production, which will provide a guideline for the construction of microbial cell factories.

Stepwise metabolic engineering of Candida tropicalis for efficient xylitol production from xylose mother liquor

BackgroundCommercial xylose purification produces xylose mother liquor (XML) as a major byproduct, which has become an inexpensive and abundant carbon source. A portion of this XML has been used to produce low-value-added products such as caramel but the remainder often ends up as an organic pollutant. This has become an issue of industrial concern. In this study, a uracil-deficient Candida tropicalis strain was engineered to efficiently convert XML to the commercially useful product xylitol.ResultsThe xylitol dehydrogenase gene was deleted to block the conversion of xylitol to xylulose. Then, an NADPH regeneration system was added through heterologous expression of the Yarrowia lipolytica genes encoding 6-phosphate-gluconic acid dehydrogenase and 6-phosphate-glucose dehydrogenase. After process optimization, the engineered strain, C. tropicalis XZX-B4ZG, produced 97.10 g L− 1 xylitol in 120 h from 300 g L− 1 XML in a 5-L fermenter. The xylitol production rate was 0.82 g L− 1 h− 1 and the conversion rate was 92.40 %.ConclusionsIn conclusion, this study performed a combination of metabolic engineering and process optimizing in C. tropicalis to enhance xylitol production from XML. The use of C. tropicalis XZX-B4ZG, therefore, provided a convenient method to transform the industrial by-product XML into the useful material xylitol.

Biosynthesis of 2,5-dimethylpyrazine from L-threonine by whole-cell biocatalyst of recombinant Escherichia coli

2,5-dimethylpyrazine (2,5-DMP) is of important economic value in food industry and pharmaceutical industry, and is now commonly produced by chemical synthesis. In this study, a recombinant Escherichia coli high-efficiently converting L-threonine to 2,5-DMP was constructed by combination of metabolic engineering and cofactor engineering. To do this, the effect of different threonine dehydrogenase (TDH) on 2,5-DMP production was investigated, and the results indicate that overexpression of EcTDH in E. coli BL21(DE3) was beneficial to construct a 2,5-DMP producer with highest 2,5-DMP production. The recombinant strain E. coli pRSFDuet-tdh(Ec) produced (438.3±23.7) mg/L of 2,5-DMP. Furthermore, the expression mode of NADH oxidase (NoxE) from Lactococcus cremoris was optimized, and fusion expression of EcTDH and LcNoxE led to balance the intracellular NADH/NAD⁺ level and to maintain the high survival rate of cells, thus further increasing 2,5-DMP production. Finally, the accumulation of by-products was significantly decreased because of disruption of shunt metabolic pathway, thereby increasing 2,5-DMP production and the conversion ratio of L-threonine. Combination of these genetic modifications resulted in an engineered E. coli Δkbl ΔtynA ΔtdcB ΔilvA pRSFDuet-tdhEcnoxELc-PsstT (EcΔkΔAΔBΔA/TDH(Ec)NoxE(Lc)-PSstT) capable of producing (1 095.7±81.3) mg/L 2,5-DMP with conversion ratio of L-threonine of 76% and a yield of 2,5-DMP of 28.8% in 50 mL transformation system with 5 g/L L-threonine at 37 °C and 200 r/min for 24 h. Therefore, this study provides a recombinant E. coli with high-efficiently catalyzing L-threonine to biosynthesize 2,5-DMP, which can be potentially used in biosynthesis of 2,5-DMP in industry.

Efficient production of Pseudoionone with multipathway engineering in Escherichia coli.

This study aimed to achieve efficient production of pseudoionone in Escherichia coli. Firstly, a stable lycopene-producing strain was constructed by integrating lycopene biosynthetic pathway into the chromosome. Further introduction of the Cucumis melo carotenoid cleavage dioxygenase 1 (cmCCD1) gene resulted in the production of pseudoionone. Then, the tricarboxylic acid cycle (i.e. sdhABCD and sucAB), pentose phosphate pathway (i.e. talB), and methylerythritol 4-phosphate pathway (i.e. idi and dxs) genes were modulated to improve the production of lycopene and accordingly pseudoionone. Regulation of the expression of talB and sdhABCD for improving NADPH supplies led to a 2·81-fold increase of pseudoionone production. Further engineering of dxs and idi increased the production of pseudoionone by 8·34-fold. Followed by bioprocess optimization, 24mgl-1 pseudoionone with a specific production of 8·55mgg-1 in shake flask was achieved. The combination of metabolic engineering and bioprocess engineering increased the production of pseudoionone by more than 185-fold. The present study promises a green and sustainable route for pseudoionone production using a microbial cell factory.

Bio-orthogonal AIE Dots Based on Polyyne-Bridged Red-emissive AIEgen for Tumor Metabolic Labeling and Targeted Imaging.

In this work, we aim to develop cancer cell-targeting AIE dots based on a polyyne-bridged red-emissive AIEgen, 2TPE-4E, through the combination of metabolic engineering and bio-orthogonal reactions. Azide groups on a tumor were efficiently produced by intravenous injection of Ac4ManNAz and glycol-metabolic engineering. These bio-orthogonal azide groups could facilitate the specific targeting of DBCO-AIE dots to the tumor cells undergoing metal-free click reaction in vivo. The efficiency of this targeting strategy could be further improved with the development of new bio-orthogonal chemical groups with higher reactivity and a large amount of AIEgens could be delivered to the tumor for diagnosis.

Efficient production of 3-hydroxypropionate from fatty acids feedstock in Escherichia coli

The production of chemicals from renewable biomass resources is usually limited by factors including high-cost processes and low efficiency of biosynthetic pathways. Fatty acids (FAs) are an ideal alternative biomass. Their advantages include high-efficiently producing acetyl-CoA and reducing power, coupling chemical production with CO2 fixation, and the fact that they are readily obtained from inexpensive feedstocks. The important platform chemical 3-hydroxypropionate (3HP) can be produced from FAs as the feedstock with a theoretical yield of 2.49 g/g, much higher than the theoretical yield from other feedstocks. In this study, we first systematically analyzed the limiting factors in FA-utilization pathways in Escherichia coli. Then, we optimized FA utilization in Escherichia coli by using a combination of metabolic engineering and optimization of fermentation conditions. The 3HP biosynthesis module was introduced into a FA-utilizing strain, and the flux balance was finely optimized to maximize 3HP production. The resulting strain was able to produce 3HP from FAs with a yield of 1.56 g/g, and was able to produce 3HP to a concentration of 52 g/L from FAs in a 5-L fermentation process. The strain also could produce 3HP from various type of FAs feedstock including gutter oil. This is the first report of a technique for the efficient production of the platform chemical 3HP from FAs.

E. coli metabolic engineering for gram scale production of a plant-based anti-inflammatory agent

In this report, the heterologous production of salicylate (SA) is the basis for metabolic extension to salicylate 2-O-β-d-glucoside (SAG), a natural product implicated in plant-based defense mechanisms. Production was optimized through a combination of metabolic engineering, gene expression variation, and co-culture design. When combined, SA and SAG production titers reached ~0.9g/L and ~2.5g/L, respectively. The SAG compound was then tested for anti-inflammatory properties relative to SA and acetylsalicylate (aspirin). Results indicate comparable activity between SAG and aspirin in reducing nitric oxide (NO) and reactive oxygen species (ROS) from macrophage cells while no discernable negative effects on cellular viability were observed.

Evolutionary engineering of Geobacillus thermoglucosidasius for improved ethanol production

The ability to grow at high temperatures makes thermophiles attractive for many fermentation processes. In this work, we used evolutionary engineering to increase ethanol production in the thermophile Geobacillus thermoglucosidasius. This bacterium is a facultative anaerobe, grows at an optimal temperature of 60°C, and can ferment diverse carbohydrates. However, it natively performs mixed-acid fermentation. To improve ethanol productivity, we first eliminated lactate and formate production in two strains of G. thermoglucosidasius, 95A1 and C56-YS93. These deletion strains were generated by selection on spectinomycin, which represents, to the best of our knowledge, the first time this antibiotic has been shown to work with thermophiles. Both knockout strains, however, were unable to grow under microaerobic conditions. We were able to recover growth in G. thermoglucosidasius 95A1 by serial adaptation in the presence of acetic acid. The evolved 95A1 strain was able to efficiently produce ethanol during growth on glucose or cellobiose. Genome sequencing identified loss-of-function mutations in adenine phosphoribosyltransferase (aprt) and the stage III sporulation protein AA (spoIIIAA). Disruption of both genes improved ethanol production in the unadapted strains: however, the increase was significant only when aprt was deleted. In conclusion, we were able to engineer a strain of G. thermoglucosidasius to efficiently produce ethanol from glucose and cellobiose using a combination of metabolic engineering and evolutionary strategies. This work further establishes this thermophile as a platform organism for fuel and chemical production. Biotechnol. Bioeng. 2016;113: 2156-2167. © 2016 Wiley Periodicals, Inc.

Engineering the biosynthesis of novel rhamnolipids inEscherichia colifor enhanced oil recovery

The interfacial tension of rhamnolipids and their applications in enhanced oil recovery are dependent on their chemical structures and compositions. To improve their performances of interfacial tension and enhanced oil recovery, the engineered strategies were applied to produce novel rhamnolipids with different chemical structures and compositions. By introducing different key genes for rhamnolipid biosynthesis, Escherichia coli was firstly constructed to produce rhamnolipids that showed different performances in interfacial tension from those from Pseudomonas aeruginosa due to the different fatty acyl compositions. Then, the mutant RhlBs were created by directed evolution and subsequent site-directed mutagenesis and resulted in the production of the novel rhamnolipids with the different performances in interfacial tension as well as enhanced oil recovery. Lastly, computational modelling elucidates that the single amino acid mutation at the position 168 in RhlB would change the volume of binding pocket for substrate and thus affect the selectivity of rhamnolipid formation in E. coli. The novel rhamnolipids that showed the improved performances of interfacial tension and the potential different applications in enhanced oil recovery were successfully produced by engineered E. coli. This study proved that the combination of metabolic engineering and protein engineering is an important engineered strategy to produce many novel metabolites in micro-organisms.

Engineering cells to improve protein expression

Cellular engineering of bacteria, fungi, insect cells and mammalian cells is a promising methodology to improve recombinant protein production for structural, biochemical, and commercial applications. Increased understanding of the host organism biology has suggested engineering strategies targeting bottlenecks in transcription, translation, protein processing and secretory pathways, as well as cell growth and survival. A combination of metabolic engineering and synthetic biology has been used to improve the properties of cells for protein production, which has resulted in enhanced yields of multiple protein classes.

The combination of glycerol metabolic engineering and drug resistance marker-aided genome shuffling to improve very-high-gravity fermentation performances of industrial Saccharomyces cerevisiae

A challenge associated with the ethanol productivity under very-high-gravity (VHG) conditions, optimizing multi-traits (i.e. byproduct formation and stress tolerance) of industrial yeast strains, is overcome by a combination of metabolic engineering and genome shuffling. First, industrial strain Y12 was deleted with a glycerol exporter Fps1p and hetero-expressed with glyceraldehydes-3-phosphate dehydrogenase, resulting in the modified strain YFG12 with lower glycerol yield. Second, YFG12 was subjected to three rounds of drug resistance marker-aided genome shuffling to increase its ethanol tolerance, and the best shuffled strain TS5 was obtained. Compared with wild strain Y12, shuffled strain TS5 not only decreased glycerol formation by 14.8%, but also increased fermentation rate and ethanol yield by 3.7% and 7.6%, respectively. Moreover, the system of genetic modification and Cre/loxP in aid of three different drug-resistance markers presented in the study significantly improved breeding efficiency and will facilitate the application of breeding technologies in prototrophic industrial microorganisms.

Novel chemobiosynthetic approach for exclusive production of FK506

FK506, a widely used immunosuppressant, is produced by industrial fermentation processes using various Streptomyces species. Independently of the strain, structurally related compound FK520 is co-produced, resulting in complex and costly isolation procedures. In this paper, we report a chemobiosynthetic approach for exclusive biosynthesis of FK506. This approach is based on the Streptomyces tsukubaensis strain with inactivated allR gene, a homologue of crotonyl-CoA carboxylase/reductase, encoded in the FK506 biosynthetic cluster. This strain produces neither FK506 nor FK520; however, if allylmalonyl-S-N-acetylcysteamine precursor is added to cultivation broth, the production of FK506 is reestablished without FK506-related by-products. Using a combination of metabolic engineering and chemobiosynthetic approach, we achieved exclusive production of FK506, representing a significant step towards development of an advanced industrial bioprocess.