17 research outputs found
Generation of structurally novel short carotenoids and study of their biological activity
Recent research interest in phytochemicals has consistently driven the efforts in the metabolic engineering field toward microbial production of various carotenoids. In spite of systematic studies, the possibility of using C(30) carotenoids as biologically functional compounds has not been explored thus far. Here, we generated 13 novel structures of C(30) carotenoids and one C(35) carotenoid, including acyclic, monocyclic, and bicyclic structures, through directed evolution and combinatorial biosynthesis, in Escherichia coli. Measurement of radical scavenging activity of various C(30) carotenoid structures revealed that acyclic C(30) carotenoids showed higher radical scavenging activity than did DL-α-tocopherol. We could assume high potential biological activity of the novel structures of C(30) carotenoids as well, based on the neuronal differentiation activity observed for the monocyclic C(30) carotenoid 4,4′-diapotorulene on rat bone marrow mesenchymal stem cells. Our results demonstrate that a series of structurally novel carotenoids possessing biologically beneficial properties can be synthesized in E. coli
Superlinkers – A versatile and automatable DNA backbone exchange standard for synthetic biology
Accurate DNA assembly and genome engineering with optimized uracil excision cloning
Simple
and reliable DNA editing by uracil excision (a.k.a. USER
cloning) has been described by several research groups, but the optimal
design of cohesive DNA ends for multigene assembly remains elusive.
Here, we use two model constructs based on expression of <i>gfp</i> and a four-gene pathway that produces β-carotene to optimize
assembly junctions and the uracil excision protocol. By combining
uracil excision cloning with a genomic integration technology, we
demonstrate that up to six DNA fragments can be assembled in a one-tube
reaction for direct genome integration with high accuracy, greatly
facilitating the advanced engineering of robust cell factories
Identification and Engineering of Transporters for Efficient Melatonin Production in Escherichia coli
Transporter discovery and engineering play an important role in cell factory development. Decreasing the intracellular concentration of the product reduces product inhibition and/or toxicity. Lowering intracellular concentrations is especially beneficial for achieving a robust strain at high titers. However, the identification of transporters for xenobiotic chemicals in the host strain is challenging. Here we present a high-throughput workflow to discover Escherichia coli transporters responsible for the efflux of the inhibitory xenobiotic compound melatonin. We took advantage of the Keio collection and screened about 400 transporter knockouts in the presence of a high concentration of melatonin. We found five transporters that when knocked out showed decreased tolerance to melatonin, indicating they are exporters of melatonin. We overexpressed these five genes individually in the production strain and found that one of them, yhjV, encoding a transporter with unknown substrates, resulted in a 27% titer increase in cultivation mimicking fed-batch fermentation. This study demonstrates how microbial cell factories can be improved through transporter identification and engineering. Further, these results lay the foundation for the scale-up of melatonin production in E. coli
Accurate Dna Assembly And Direct Genome Integration With Optimized Uracil Excision Cloning To Facilitate Engineering Of <i>Escherichia Coli</i> As A Cell Factory
SEVA Linkers: A Versatile and Automatable DNA Backbone Exchange Standard for Synthetic Biology
DNA vectors serve to maintain and
select recombinant DNA in cell
factories, and as design complexity increases, there is a greater
need for well-characterized parts and methods for their assembly.
Standards in synthetic biology are top priority, but standardizing
molecular cloning contrasts flexibility, and different researchers
prefer and master different molecular technologies. Here, we describe
a new, highly versatile and automatable standard “SEVA linkers”
for vector exchange. SEVA linkers enable backbone swapping with 20
combinations of classical enzymatic restriction/ligation, Gibson isothermal
assembly, uracil excision cloning, and a nicking enzyme-based methodology
we term SEVA cloning. SEVA cloning is a simplistic one-tube protocol
for backbone swapping directly from plasmid stock solutions. We demonstrate
the different performance of 30 plasmid backbones for small molecule
and protein production and obtain more than 10-fold improvement from
a four-gene biosynthetic pathway and 430-fold improvement with a difficult-to-express
membrane protein. The standardized linkers and protocols add to the
Standard European Vectors Architecture (SEVA) resource and are freely
available to the synthetic biology community
Heterologous Carotenoid-Biosynthetic Enzymes: Functional Complementation and Effects on Carotenoid Profiles in Escherichia coli
Redesign, Reconstruction, and Directed Extension of the Brevibacterium linens C40 Carotenoid Pathway in Escherichia coliâ–ż â€
In this study, the carotenoid biosynthetic pathways of Brevibacterium linens DSMZ 20426 were reconstructed, redesigned, and extended with additional carotenoid-modifying enzymes of other sources in a heterologous host Escherichia coli. The modular lycopene pathway synthesized an unexpected carotenoid structure, 3,4-didehydrolycopene, as well as lycopene. Extension of the novel 3,4-didehydrolycopene pathway with the mutant Pantoea lycopene cyclase CrtY2 and the Rhodobacter spheroidene monooxygenase CrtA generated monocyclic torulene and acyclic oxocarotenoids, respectively. The reconstructed β-carotene pathway synthesized an unexpected 7,8-dihydro-β-carotene in addition to β-carotene. Extension of the β-carotene pathway with the B. linens β-ring desaturase CrtU and Pantoea β-carotene hydroxylase CrtZ generated asymmetric carotenoid agelaxanthin A, which had one aromatic ring at the one end of carotene backbone and one hydroxyl group at the other end, as well as aromatic carotenoid isorenieratene and dihydroxy carotenoid zeaxanthin. These results demonstrate that reconstruction of the biosynthetic pathways and extension with promiscuous enzymes in a heterologous host holds promise as a rational strategy for generating structurally diverse compounds that are hardly accessible in nature