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

Bo Zhang,Shuang-Jiang Liu,Yi-Ming Liu,Nan Zhou,Chang Liu,Chun-Bo Lou,Cheng-Ying Jiang

doi:10.1186/s12934-015-0254-0

Abstract

BackgroundThe 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.ResultsIn 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 RES167ΔaroK carrying a genetic module produced 4.3 g/L of SA, which was 54 folds higher compared to that of strain RES167ΔaroK (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.ConclusionThese 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.

Highlights

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
As shown in (Fig. 4b), those modules (GHBLDLEL, GMBHDMEL, and GLBMDHEL) harbored low strengths of ribosome binding sites (RBS) exhibited low AroE activities and those modules (GHBHDHEH, GMBMDLEH, and GLBLDMEH) harbored higher strengths of RBS exhibited higher AroE activities. These results suggested that levels of gene translations in the 9 genetic modules were highly correlated to RBS strengths determined previously via EGFP fluorescence intensities
Plasmid pXMJ19-GBTDTE derivate, containing a terminator between aroG and aroB module. Several methods, such as overexpression of aro genes [31, 32] and the use of enzymes with improved properties [33], have been reported to enhance the metabolic flux into SA pathway, increase the production of aromatic amino acids or shikimic acid. This current study revealed a new synthetic biology strategy: Four aro genes were organized as continuous genetic modules and their transcriptions were coordinated by the same tac promoter, RiboJ and terminator

Summary

Introduction

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. Scientists have designed and constructed a series of parts libraries of promoters, ribosome binding sites (RBS) and terminators, which enabled the regulation of gene expression over wide dynamic ranges in Escherichia coli cells [14, 15]. Synthetic parts and modules are very limited for C. glutamicum, an important industry production workhorse that has been used for decades to produce amino acids, vitamins, nucleotides [17,18,19,20], and recently biofuels and chemicals [21,22,23,24]

Methods

Results

Discussion

Conclusion