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► Biosynthesis Pathway Design &
► Biocatalysis & Bioconversion
► Enzyme Screening & Characterization
► Modification of Microbial Metabolic
► Development of Engineered Strains
► Protein Engineering
► Microbial Genome Editing
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Extending shikimate pathway for the production of muconic acid and salicylic acid in Escherichia coli
Metabolic Engineering (2013)
cis,cis-Muconic acid (MA) and salicylic acid (SA) are naturally-occurring organic acids having great commercial value. MA is a potential platform chemical for the manufacture of several widely-used consumer plastics; while SA is mainly used for producing pharmaceuticals (for example, aspirin and lamivudine) and skincare and haircare products. At present, MA and SA are commercially produced by organic chemical synthesis using petro-derived aromatic chemicals, such as benzene, as starting materials, which is not environmentally friendly. Here, we report a novel approach for efficient microbial production of MA via extending shikimate pathway by introducing the hybrid of an SA biosynthetic pathway with its partial degradation pathway. First, we engineered a well-developed phenylalanine producing Escherichia coli strain into an SA overproducer by introducing isochorismate synthase and isochorismate pyruvate lyase. The engineered strain is able to produce 1.2 g/L of SA from simple carbon sources, which is the highest titer reported so far. Further, the partial SA degradation pathway involving salicylate 1-monoxygenase and catechol 1,2-dioxygenase is established to achieve the conversion of SA to MA. Finally, a de novo MA biosynthetic pathway is assembled by integrating the established SA biosynthesis and degradation modules. Modular optimization enables the production of up to 1.5 g/L MA within 48 h in shake flasks. This study not only establishes an efficient microbial platform for the production of SA and MA, but also demonstrates a generalizable pathway design strategy for the de novobiosynthesis of valuable degradation metabolites.
The experts and research team of BiotecEra have rich and successful experiences in performing the work related to microbiology, metabolic engineering, synthetic biology, biocatalysis and enzymology. The strengths of BiotecEra include the design, validation and optimization of natural or artifial biosynthetic pathway, which are followed by scaled process development towards industrial production of target chemicals. These works have been published in many top-rated journals, including Nature Communications, Metabolic Engineering, ChemSusChem, ACS Synthetic Biology, Biotechnology and Bioengineering, exhibiting great potential for commercialization. Below are some of the representative works conducted by BiotecEra's researchers.
Microbial biosynthesis of the anticoagulant precursor 4-hydroxycoumarin
Nature Communications (2013)
4-Hydroxycoumarin (4HC) type anticoagulants (for example, warfarin) are known to have a significant role in the treatment of thromboembolic diseases—a leading cause of patient morbidity and mortality worldwide. 4HC serves as an immediate precursor of these synthetic anticoagulants. Although 4HC was initially identified as a naturally occurring product, its biosynthesis has not been fully elucidated. Here we present the design, validation, in vitro diagnosis and optimization of an artificial biosynthetic mechanism leading to the microbial biosynthesis of 4HC. Remarkably, function-based enzyme bioprospecting leads to the identification of a characteristic FabH-like quinolone synthase from Pseudomonas aeruginosa with high efficiency on the 4HC-forming reaction, which promotes the high-level de novo biosynthesis of 4HC in Escherichia coli (B500mg l1 in shake flasks) and further in situ semisynthesis of warfarin. This work has the potential to be scaled-up for microbial production of 4HC and opens up the possibility of biosynthesizing diverse coumarin molecules with pharmaceutical importance.
Combinatorial biosynthesis of plant-specific coumarins in bacteria
Metabolic Engineering (2013)
Coumarins are plant secondary metabolites that have demonstrated a variety of important therapeutic properties, such as antibacterial, anti-inflammatory, and anti-coagulant effects, as well as anti-cancer and anti-AIDS activities. However, knowledge regarding their biosynthesis is relatively limited even for the simplest coumarin molecule, which serves as the gateway molecule to many pharmaceutically important coumarin derivatives. Here we reported the design and validation of artificial pathways leading to the biosynthesis of plant-specific simple coumarins in bacteria. First, Escherichia colistrains were engineered to convert inexpensive phenylpropanoid acid precursors, 4-coumarate and ferulate to simple coumarins, umbelliferone (4.3 mg/L) and scopoletin (27.8 mg/L), respectively. Furthermore, we assembled the complete artificial pathways inE. coli and achieved de novo biosynthesis of umbelliferone and scopoletin without addition of precursors. This study lays the foundation for microbial production of more diverse coumarin compounds.
Caffeic acid production enhancement by engineering a phenylalanine over-producing Escherichia coli strain
Biotechnology and Bioengineering (2013)
Caffeic acid is a plant-specific phenylpropanoic acid with multiple health-improving effects reported, and its therapeutic derivatives have also been studied throughout the last decade. To meet its market need and achieve high-level production, microbial production of caffeic acid approaches have been developed in metabolically engineered Escherichia coli. In our previous work, we have established the first artificial pathway that realized de novo production of caffeic acid using E. coli endogenous 4-hydroxyphenylacetate 3-hydroxylase (4HP3H). In this work, we exploited the catalytic potential of 4HPA3H in the whole-cell bioconversion study and produced 3.82 g/L (461.12 mg/L/OD) caffeic acid from p-coumaric acid, a direct precursor. We further engineered a phenylalanine over-producer into a tyrosine over-producer and then introduced the artificial pathway. After adjusting the expression strategy and optimizing the inoculants timing, de novo production of caffeic acid reached 766.68 mg/L. Both results from the direct precursor and simple carbon sources represent the highest titers of caffeic acid from microbial production so far.
Biotechnological production of plant-specific hydroxylated phenylpropanoids
Biotechnology and Bioengineering (2014)
Hydroxylated phenylpropanoid compounds (e.g., esculetin, piceatannol, and eriodictyol) have been proved to possess important biological activities and pharmacological properties. These compounds exist at low abundance in nature, which hampers their cost-effective isolation, and broad application. Meanwhile, regiospecific hydroxylation of complex aromatic compounds is still quite challenging for chemical synthesis. In past decades, biocatalytic hydroxylation of plant phenylpropanoids was achieved due to the identification and engineering of some cytochrome P450 hydroxylases; however, the conversion efficiency was still too low for scale-up production use. In this work, we identify a non-P450 monooxygenase (HpaBC) from Escherichia coli, which is able to catalyze the efficient ortho-hydroxylation towards plant phenylpropanoids umbelliferone and resveratrol; meanwhile it also exhibits activity towards naringenin. On this basis, whole-cell biocatalysis enables the production of esculetin and piceatannol at high titers (2.7 and 1.2 g/L, respectively, in shake flasks) and high yields (close to 100%). To our knowledge, this work reports the highest titers and yields for biotechnological production of esculetin and piceatannol, representing a promising hydroxylation platform.
Microbial synthesis of human neurotransmitter precursor 5-hydroxytryptophan
ACS Synthetic Biology (2014)
5-Hydroxytryptophan (5-HTP) is a drug that is clinically effective against depression, insomnia, obesity,chronic headaches, etc. It is only commercially produced by the extraction from the seeds of Griffonia simplicifolia because of a lack of synthetic methods. Here, we report the efficient microbial production of 5-HTP via combinatorial protein and metabolic engineering approaches. First, we reconstituted and screened prokaryotic phenylalanine 4-hydroxylase activity in Escherichia coli. Then, sequence- and structure-based protein engineering dramatically shifted its substrate preference,allowing for efficient conversion of tryptophan to 5-HTP. Importantly, E. coli endogenous tetrahydromonapterin (MH4) could be utilized as the coenzyme, when a foreign MH4 recycling mechanism was introduced. Whole-cell bioconversion allowed the high-level production of 5-HTP (1.1−1.2 g/L) from tryptophan in shake flasks. On this basis, metabolic engineering efforts were further made to achieve the de novo 5-HTP biosynthesis from glucose. This work not only holds great scale-up potential but also demonstrates a strategy for expanding the native metabolism of microorganisms.