Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering

Zhao, Shujuan; Jones, J. Andrew; Lachance, Daniel M.; Bhan, Namita; Khalidi, Omar; Venkataraman, Sylesh K.; Wang, Zhengtao; Koffas, Mattheos

doi:10.1016/j.ymben.2014.12.002

Cited by 122 publications

(82 citation statements)

References 23 publications

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“…Lastly, by improving culture conditions through higher initial substrate concentration, substrate and glucose pulsing, and inducing at stationary phase, a C3G titer of 350 mg/liter was attained, representing a yield of 22.3 g of C3G per g of catechin. This optimized system, coupled with recent advances in producing high-yield, high-titer (ϩ)-catechin, offers a process that can lead to a commercially viable production of the vibrant natural colorant C3G (23,44). In addition, C3G can be semipurified after one-step solvent extraction, and its coloration is comparable to that of synthetic dye.…”

Section: Resultsmentioning

confidence: 99%

“…1B), we rap- idly optimized the culture condition and induction parameters, leading to a C3G titer of 350 mg/liter, which is about a 3.3-fold improvement over our previous efforts and is the highest production level reported to date. This work, coupled with the recently reported high-yield, high-titer production of catechin in E. coli, can lead to a commercially viable process for the sustainable production of anthocyanins (23).…”

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confidence: 87%

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Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Lim

Wong

Bhan

et al. 2015

Appl Environ Microbiol

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bAnthocyanins are water-soluble colored pigments found in terrestrial plants and are responsible for the red, blue, and purple coloration of many flowers and fruits. In addition to the plethora of health benefits associated with anthocyanins (cardioprotective, anti-inflammatory, antioxidant, and antiaging properties), these compounds have attracted widespread attention due to their promising potential as natural food colorants. Previously, we reported the biotransformation of anthocyanin, specifically cyanidin 3-O-glucoside (C3G), from the substrate (؉)-catechin in Escherichia coli. In the present work, we set out to systematically improve C3G titers by enhancing substrate and precursor availability, balancing gene expression level, and optimizing cultivation and induction parameters. We first identified E. coli transporter proteins that are responsible for the uptake of catechin and secretion of C3G. We then improved the expression of the heterologous pathway enzymes anthocyanidin synthase (ANS) and 3-O-glycosyltransferase (3GT) using a bicistronic expression cassette. Next, we augmented the intracellular availability of the critical precursor UDP-glucose, which has been known as the rate-limiting precursor to produce glucoside compounds. Further optimization of culture and induction conditions led to a final titer of 350 mg/liter of C3G. We also developed a convenient colorimetric assay for easy screening of C3G overproducers. The work reported here constitutes a promising foundation to develop a cost-effective process for large-scale production of plant-derived anthocyanin from recombinant microorganisms.

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 87%

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Lim

Wong

Bhan

et al. 2015

Appl Environ Microbiol

Self Cite

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“…Thus combinatorial metabolic engineering is required to explore the complex genetic spaces to search for the best phenotype (Ajikumar et al, 2010;Alper et al, 2005;Blazeck et al, 2014;Du et al, 2012;Santos and Stephanopoulos, 2008;Zhao et al, 2014). At the final stage of our metabolic engineering efforts, we performed combinatorial multiplexing of the selected positive targets and found unpredictable effect.…”

Section: Combinatorial Metabolic Engineeringmentioning

confidence: 99%

Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing

Lin

Huang

et al. 2015

Metabolic Engineering

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“…Using a Gibson assembly protocol optimized for high GC content, the pathway specific regulators of pristinamycin biosynthesis were mutated in a combinatorial fashion to increase production levels to over 1 g/L 101 . An improved three-gene pathway for catechin production was created via combinatorial assembly by drawing from eight homologous biosynthetic genes from different plant species 102 . Certain classes of NPs, for example indolocarbazoles, have been greatly expanded using combinatorial DNA assembly with more than fifty derivatives created to date 78,103 .…”

Section: High-throughput Genetic Optimization Of Multi-gene Systemsmentioning

confidence: 99%

Synthetic biology to access and expand nature's chemical diversity

et al. 2016

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Bacterial genomes encode the biosynthetic potential to produce hundreds of thousands of complex molecules with diverse applications, from medicine to agriculture and materials. Economically accessing the potential encoded within sequenced genomes promises to reinvigorate waning drug discovery pipelines and provide novel routes to intricate chemicals. This is a tremendous undertaking, as the pathways often comprise dozens of genes spanning as much as 100+ kiliobases of DNA, are controlled by complex regulatory networks, and the most interesting molecules are made by non-model organisms. Advances in synthetic biology address these issues, including DNA construction technologies, genetic parts for precision expression control, synthetic regulatory circuits, computer aided design, and multiplexed genome engineering. Collectively, these technologies are moving towards an era when chemicals can be accessed en mass based on sequence information alone. This will enable the harnessing of metagenomic data and massive strain banks for high-throughput molecular discovery and, ultimately, the ability to forward design pathways to complex chemicals not found in nature.

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Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering

Cited by 122 publications

References 23 publications

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Metabolic engineering of Escherichia coli using CRISPR–Cas9 meditated genome editing

Synthetic biology to access and expand nature's chemical diversity

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