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Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum.

ABSTRACT: The shikimic acid (SA) pathway is a fundamental route to synthesize aromatic building blocks for cell growth and metabolic processes, as well as for fermentative production of various aromatic compounds. Genes encoding enzymes of SA pathway are not continuous on genome and they are differently regulated.In this study, efforts were made to construct continuous genetic modules of SA pathway that are regulated by a same Ptac promoter. Firstly, aro genes [aroG (NCgl2098), aroB (NCgl1559), aroD (NCgl0408) and aroE (NCgl1567)] from Corynebacterium glutamicum and ribosome binding site (RBS) libraries that were tailored for the above genes were obtained, and the strength of each RBS in the 4 libraries was quantified. Secondly, 9 genetic modules were built up from the RBS libraries, a previously characterized ribozyme insulator (RiboJ) and transcriptional promoter (Ptac) and terminator, and aroG, aroB, aroD and aroE. The functionality and efficiency of the constructed genetic modules were evaluated in C. glutamicum by determination of SA synthesis. Results showed that C. glutamicum RES167?aroK carrying a genetic module produced 4.3 g/L of SA, which was 54 folds higher compared to that of strain RES167?aroK (80 mg/L, without the genetic module) during fermentation in 250-mL flasks. The same strain produced 7.4, and 11.3 g/L of SA during 5-L batch and fed-batch fermentations, respectively, which corresponding to SA molar yields of 0.39 and 0.24 per mole sucrose consumption.These results demonstrated that the constructed SA pathway modules are effective in increasing SA synthesis in C. glutamicum, and they might be useful for fermentative production of aromatic compounds derived from SA pathway.

SUBMITTER: Zhang B

PROVIDER: S-EPMC4453273 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum.

Zhang Bo B Zhou Nan N Liu Yi-Ming YM Liu Chang C Lou Chun-Bo CB Jiang Cheng-Ying CY Liu Shuang-Jiang SJ

Microbial cell factories 20150517

<h4>Background</h4>The shikimic acid (SA) pathway is a fundamental route to synthesize aromatic building blocks for cell growth and metabolic processes, as well as for fermentative production of various aromatic compounds. Genes encoding enzymes of SA pathway are not continuous on genome and they are differently regulated.<h4>Results</h4>In this study, efforts were made to construct continuous genetic modules of SA pathway that are regulated by a same Ptac promoter. Firstly, aro genes [aroG (NCg ...[more]

PMID: 25981633

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Project description:BackgroundLignocellulose provides the only practical carbohydrates feedstock for sustainable bioproduction of hydrocarbons as future alternative of fossil fuels. Production of hydrocarbons from lignocellulose is achieved by a biorefinery process chain including pretreatment to breakdown the crystalline structure for cellulase-catalyzed hydrolysis, detoxification of inhibitory compounds generated during pretreatment, enzymatic hydrolysis to fermentable monosaccharide sugars, and fermentation to hydrocarbon products. The major barriers on fermentative production of hydrocarbons from lignocellulose include two aspects: one is the inherent stress of pretreatment-derived inhibitors on microbial cells, the other is the toxicity of hydrocarbons to cell membranes. The microbial cell factory should be tolerant to both inhibitor stress and hydrocarbons toxicity.ResultsCorynebacterium glutamicum was selected as the starting strain of hydrocarbons synthesis since it is well adapted to lignocellulose hydrolysate environment. The dual hydrocarbon synthesis pathways were constructed in an industrial C. glutamicum S9114 strain. The first pathway was the regular one in microalgae composed of fatty acyl-acyl carrier protein (fatty acyl-ACP) reductase (AAR) and aldehyde deformylating oxygenase (ADO) with fatty acyl-ACP as precursor. The second pathway was the direct decarboxylation of free fatty acid by fatty acid decarboxylase (OleT) using the rich fatty acids from the disruption of the transcriptional regulator fasR gene. The transmembrane transportation of hydrocarbon products was avoided by secretively expressing the fatty acid decarboxylase (OleT) to the extracellular space. The hydrocarbons generation from glucose reached 29.2 mg/L, in which the direct decarboxylation pathway contributed more than 70% of the total hydrocarbons generation, and the AAR-ADO pathway contributed the rest 30%.ConclusionThe dual hydrocarbon synthesis pathways (OleT and AAR-ADO pathways) were constructed in the inhibitors tolerant C. glutamicum S9114 strain for hydrocarbon production using lignocellulose feedstock as the starting feedstock. When corn stover was used for hydrocarbons production after dry acid pretreatment and biodetoxification, the hydrocarbons generation reached 16.0 mg/L. This study provided a new strategy for hydrocarbons synthesis using microbial cell factory suitable for lignocellulose feedstock.

Dataset Information

Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum.

Publications

Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum.

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