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Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit.

ABSTRACT: Metabolic engineering of microorganisms to produce desirable products on an industrial scale can result in unbalanced cellular metabolic networks that reduce productivity and yield. Metabolic fluxes can be rebalanced using dynamic pathway regulation, but few broadly applicable tools are available to achieve this. We present a pathway-independent genetic control module that can be used to dynamically regulate the expression of target genes. We apply our module to identify the optimal point to redirect glycolytic flux into heterologous engineered pathways in Escherichia coli, resulting in titers of myo-inositol increased 5.5-fold and titers of glucaric acid increased from unmeasurable to >0.8 g/L, compared to the parent strains lacking dynamic flux control. Scaled-up production of these strains in benchtop bioreactors resulted in almost ten- and fivefold increases in specific titers of myo-inositol and glucaric acid, respectively. We also used our module to control flux into aromatic amino acid biosynthesis to increase titers of shikimate in E. coli from unmeasurable to >100 mg/L.

SUBMITTER: Gupta A

PROVIDER: S-EPMC5340623 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit.

Gupta Apoorv A Reizman Irene M Brockman IM Reisch Christopher R CR Prather Kristala L J KL

Nature biotechnology 20170213 3

Metabolic engineering of microorganisms to produce desirable products on an industrial scale can result in unbalanced cellular metabolic networks that reduce productivity and yield. Metabolic fluxes can be rebalanced using dynamic pathway regulation, but few broadly applicable tools are available to achieve this. We present a pathway-independent genetic control module that can be used to dynamically regulate the expression of target genes. We apply our module to identify the optimal point to red ...[more]

PMID: 28191902

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Project description:Background:4-Hydroxyphenylacetic acid (4HPAA) is an important building block for synthesizing drugs, agrochemicals, biochemicals, etc. 4HPAA is currently produced exclusively via petrochemical processes and the process is environmentally unfriendly and unsustainable. Microbial cell factory would be an attractive approach for 4HPAA production. Results:In the present study, we established a microbial biosynthetic system for the de novo production of 4HPAA from glucose in Escherichia coli. First, we compared different biosynthetic pathways for the production of 4HPAA. The yeast Ehrlich pathway produced the highest level of 4HPAA among these pathways that were evaluated. To increase the pathway efficiency, the yeast Ehrlich pathway enzymes were directedly evolved via error-prone PCR. Two phenylpyruvate decarboxylase ARO10 and phenylacetaldehyde dehydrogenase FeaB variants that outperformed the wild-type enzymes were obtained. These mutations increased the in vitro and in vivo catalytic efficiency for converting 4-hydroxyphenylpyruvate to 4HPAA. A tunable intergenic region (TIGR) sequence was inserted into the two evolved genes to balance their expression. Regulation of TIGR for the evolved pathway enzymes further improved the production of 4HPAA, resulting in a 1.13-fold increase in titer compared with the fusion wild-type pathway. To prevent the toxicity of a heterologous pathway to the cell, an Esa quorum-sensing (QS) circuit with both activating and repressing functions was developed for inducer-free productions of metabolites. The Esa-PesaR activation QS system was used to dynamically control the biosynthetic pathway of 4HPAA in E. coli, which achieved 17.39?±?0.26 g/L with a molar yield of 23.2% without addition of external inducers, resulting in a 46.4% improvement of the titer compared to the statically controlled pathway. Conclusion:We have constructed an E. coli for 4HPAA production with the highest titer to date. This study also demonstrates that the combination of directed evolution of pathway enzymes and dynamic pathway regulation using a QS circuit is a powerful strategy of metabolic engineering for the productions of metabolites.

Dataset Information

Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit.

Publications

Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit.

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