Tag Archives: MetabolicEngineering

Metabolic engineering of Escherichia coli for the production of cinnamaldehyde

Metabolic engineering of Escherichia coli for the production of cinnamaldehyde

Background
Plant parasitic nematodes are harmful to agricultural crops and plants, and may cause severe yield losses. Cinnamaldehyde, a volatile, yellow liquid commonly used as a flavoring or food additive, is increasingly becoming a popular natural nematicide because of its high nematicidal activity and, there is a high demand for the development of a biological platform to produce cinnamaldehyde.

Results
We engineered Escherichia coli as an eco-friendly biological platform for the production of cinnamaldehyde. In E. coli, cinnamaldehyde can be synthesized from intracellular l-phenylalanine, which requires the activities of three enzymes: phenylalanine-ammonia lyase (PAL), 4-coumarate:CoA ligase (4CL), and cinnamoyl-CoA reductase (CCR). For the efficient production of cinnamaldehyde in E. coli, we first examined the activities of enzymes from different sources and a gene expression system for the selected enzymes was constructed. Next, the metabolic pathway for l-phenylalanine biosynthesis was engineered to increase the intracellular pool of l-phenylalanine, which is a main precursor of cinnamaldehyde. Finally, we tried to produce cinnamaldehyde with the engineered E. coli. According to this result, cinnamaldehyde production as high as 75 mg/L could be achieved, which was about 35-fold higher compared with that in the parental E. coli W3110 harboring a plasmid for cinnamaldehyde biosynthesis. We also confirmed that cinnamaldehyde produced by our engineered E. coli had a nematicidal activity similar to the activity of commercial cinnamaldehyde by nematicidal assays against Bursaphelenchus xylophilus.

Conclusion
As a potential natural pesticide, cinnamaldehyde was successfully produced in E. coli by construction of the biosynthesis pathway and, its production titer was also significantly increased by engineering the metabolic pathway of l-phenylalanine.

Read the full article here.

Mechanism of imidazolium ionic liquids toxicity in Saccharomyces cerevisiae and rational engineering of a tolerant, xylose-fermenting strain

Mechanism of imidazolium ionic liquids toxicity in Saccharomyces cerevisiae and rational engineering of a tolerant, xylose-fermenting strain

Background
Imidazolium ionic liquids (IILs) underpin promising technologies that generate fermentable sugars from lignocellulose for future biorefineries. However, residual IILs are toxic to fermentative microbes such as Saccharomyces cerevisiae, making IIL-tolerance a key property for strain engineering. To enable rational engineering, we used chemical genomic profiling to understand the effects of IILs on S. cerevisiae.

Results
We found that IILs likely target mitochondria as their chemical genomic profiles closely resembled that of the mitochondrial membrane disrupting agent valinomycin. Further, several deletions of genes encoding mitochondrial proteins exhibited increased sensitivity to IIL. High-throughput chemical proteomics confirmed effects of IILs on mitochondrial protein levels. IILs induced abnormal mitochondrial morphology, as well as altered polarization of mitochondrial membrane potential similar to valinomycin. Deletion of the putative serine/threonine kinase PTK2 thought to activate the plasma-membrane proton efflux pump Pma1p conferred a significant IIL-fitness advantage. Conversely, overexpression of PMA1 conferred sensitivity to IILs, suggesting that hydrogen ion efflux may be coupled to influx of the toxic imidazolium cation. PTK2 deletion conferred resistance to multiple IILs, including [EMIM]Cl, [BMIM]Cl, and [EMIM]Ac. An engineered, xylose-converting ptk2∆ S. cerevisiae (Y133-IIL) strain consumed glucose and xylose faster and produced more ethanol in the presence of 1 % [BMIM]Cl than the wild-type PTK2 strain. We propose a model of IIL toxicity and resistance.

Conclusions
This work demonstrates the utility of chemical genomics-guided biodesign for development of superior microbial biocatalysts for the ever-changing landscape of fermentation inhibitors.

Read the full article here.

Adaptive evolution and metabolic engineering of a cellobiose- and xylose- negative Corynebacterium glutamicum that co-utilizes cellobiose and xylose

Adaptive evolution and metabolic engineering of a cellobiose- and xylose- negative Corynebacterium glutamicum that co-utilizes cellobiose and xylose

An efficient microbial cell factory requires a microorganism that can utilize a broad range of substrates to economically produce value-added chemicals and fuels. The industrially important bacterium Corynebacterium glutamicum has been studied to broaden substrate utilizations for lignocellulose-derived sugars. However, C. glutamicum ATCC 13032 is incapable of PTS-dependent utilization of cellobiose because it has missing genes annotated to β-glucosidases (bG) and cellobiose-specific PTS permease.

Read the full article here.

Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose

Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose

Sustainable alternatives for the production of fuels and chemicals are needed to reduce our dependency on fossil resources and to avoid the negative impact of their excessive use on the global climate. Lignocellulosic feedstock from agricultural residues, energy crops and municipal solid waste provides an abundant and carbon-neutral alternative, but it is recalcitrant towards microbial degradation and must therefore undergo extensive pretreatment to release the monomeric sugar units used by biofuel-producing microbes. These pretreatment steps can be reduced by using microbes such asClostridium cellulolyticum that naturally digest lignocellulose, but this limits the range of biofuels that can be produced. We therefore developed a metabolic engineering approach in C. cellulolyticum to expand its natural product spectrum and to fine tune the engineered metabolic pathways.

Read the full article here.

Metabolic engineering of Escherichia coli for poly(3-hydroxybutyrate) production via threonine bypass

Metabolic engineering of Escherichia coli for poly(3-hydroxybutyrate) production via threonine bypass

Background
Poly(3-hydroxybutyrate) (PHB), have been considered to be good candidates for completely biodegradable polymers due to their similar mechanical properties to petroleum-derived polymers and complete biodegradability. Escherichia coli has been used to simulate the distribution of metabolic fluxes in recombinant E. coli producing poly(3-hydroxybutyrate) (PHB). Genome-scale metabolic network analysis can reveal unexpected metabolic engineering strategies to improve the production of biochemicals and biofuels.

Results
In this study, we reported the discovery of a new pathway called threonine bypass by flux balance analysis of the genome-scale metabolic model of E. coli. This pathway, mainly containing the reactions for threonine synthesis and degradation, can potentially increase the yield of PHB and other acetyl-CoA derived products by reutilizing the CO 2 released at the pyruvate dehydrogenase step. To implement the threonine bypass for PHB production in E. coli, we deregulated the threonine and serine degradation pathway and enhanced the threonine synthesis, resulting in 2.23-fold improvement of PHB titer. Then, we overexpressed glyA to enhance the conversion of glycine to serine and activated transhydrogenase to generate NADPH required in the threonine bypass.

Conclusions
The result strain TB17 (pBHR68) produced 6.82 g/L PHB with the yield of 0.36 g/g glucose in the shake flask fermentation and 35.92 g/L PHB with the yield of 0.23 g/g glucose in the fed-batch fermentation, which was almost 3.3-fold higher than the parent strain. The work outlined here shows that genome-scale metabolic network analysis can reveal novel metabolic engineering strategies for developing efficient microbial cell factories.

Read the full article here.