Synthetic biology platform of CoryneBrick vectors for gene expression in Corynebacterium glutamicum and its application to xylose utilization

Abstract

Currently, the majority of tools in synthetic biology have been designed and constructed for model organisms such as Escherichia coli and Saccharomyces cerevisiae. In order to broaden the spectrum of organisms accessible to such tools, we established a synthetic biological platform, called CoryneBrick, for gene expression in Corynebacterium glutamicum as a set of E. coli-C. glutamicum shuttle vectors whose elements are interchangeable with BglBrick standard parts. C. glutamicum is an established industrial microorganism for the production of amino acids, proteins, and commercially promising chemicals. Using the CoryneBrick vectors, we showed various time-dependent expression profiles of a red fluorescent protein. This CoryneBrick platform was also applicable for two-plasmid expression systems with a conventional C. glutamicum expression vector. In order to demonstrate the practical application of the CoryneBrick vectors, we successfully reconstructed the xylose utilization pathway in the xylose-negative C. glutamicum wild type by fast BglBrick cloning methods using multiple genes encoding for xylose isomerase and xylulose kinase, resulting in a growth rate of 0.11 ± 0.004 h(-1) and a xylose uptake rate of 3.35 mmol/gDW/h when 1 % xylose was used as sole carbon source. Thus, CoryneBrick vectors were shown to be useful engineering tools in order to exploit Corynebacterium as a synthetic platform for the production of chemicals by controllable expression of the genes of interest.

Engineering of Corynebacterium glutamicum for minimized carbon loss during utilization of d-xylose containing substrates

“…Similarly, C. glutamicum has been also engineered for growth on d-xylose (Kawaguchi et al, 2006;Meiswinkel et al, 2013;Kang et al, 2014). In this case, heterologous expression of a xylose isomerase XLI (encoded by xylA) from Escherichia coli is sufficient to convert d-xylose to d-xylulose, which is then phosphorylated by an endogenous xylulokinase XLK (encoded by xylB) to yield xylulose-5-phosphate as intermediate of the pentose phosphate pathway (Fig.…”

Succinate production from CO2-grown microalgal biomass as carbon source using engineered Corynebacterium glutamicum through consolidated bioprocessing

“…Additional sugar transporters and hydrolytic enzymes could be necessary to uptake unused carbohydrates in the total sugars because 10% of total sugars in microalgal biomass were not fermentable. In addition to extensive studies on sugar metabolism27, pentose-sugar fermentations of engineered C. glutamicum have been well investigated2829. Current synthetic platform (CoryneBrick29) for the gene expression in this study can be easily expanded for additional gene expression of targets.…”

“…In addition to extensive studies on sugar metabolism27, pentose-sugar fermentations of engineered C. glutamicum have been well investigated2829. Current synthetic platform (CoryneBrick29) for the gene expression in this study can be easily expanded for additional gene expression of targets. Also, application of cell display system20 (i.e.…”

“…Each target gene was synthesized (Genscript, USA) with codon-optimization for C. glutamicum (represented as cg.co). Each gene was assembled using a standard BglBrick cloning method, where the target gene is inserted at the Eco RI and Xho I sites of the CoryneBrick plasmid pBbEB1c29. The Tat-specific TorA signal peptide sequence from C. glutamicum 33 was added to the coding sequence of the target gene where the native signal sequences was already deleted.…”

Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

“…The successful expression of the xylooligosaccharide ABC transporter gene cluster in a heterologous host in this study demonstrates the central role of the cluster in uptake of arabino-xylooligosaccharides. C. glutamicum has been engineered and improved to be an effective platform for pentose fermentation (31)(32)(33), producing ethanol (34), succinic acid (35), 1,5-diaminopentane (36), and amino acids (37,38) from pentose sugars. The findings described here could offer faster, broader, and more efficient pentose sugar utilization to such potential industrial strains, and could contribute significantly to advancement of microbial lignocellulosic biomass exploitation.…”