CRISPR interference-mediated metabolic engineering of Corynebacterium glutamicum for homo-butyrate production.

Combinatorial metabolic engineering enabled the development of efficient microbial cell factories for modulating gene expression to produce desired products. Here, we report the combinatorial metabolic engineering of Corynebacterium glutamicum to produce butyrate by introducing a synthetic butyrate pathway including phosphotransferase and butyrate kinase reactions and repressing the essential acn gene-encoding aconitase, which has been targeted for downregulation in a genome-scale model. An all-in-one clustered regularly interspaced short palindromic repeats interference system for C. glutamicum was used for tunable downregulation of acn in an engineered strain, where by-product-forming reactions were deleted and the synthetic butyrate pathway was inserted, resulting in butyrate production (0.52 ± 0.02 g/L). Subsequently, biotin limitation enabled the engineered strain to produce butyrate (0.58 ± 0.01 g/L) without acetate formation for the entire duration of the culture. These results demonstrate the potential homo-production of butyrate using engineered C. glutamicum. This method can also be applied to other industrial microorganisms.

Modular pathway engineering of Corynebacterium glutamicum to improve xylose utilization and succinate production.

Xylose-negative Corynebacterium glutamicum has been engineered to utilize xylose as the sole carbon source via either the xylose isomerase (XI) pathway or the Weimberg pathway. Heterologous expression of xylose isomerase and overexpression of a gene encoding for xylulose kinase enabled efficient xylose utilization. In this study, we show that two functionally-redundant transcriptional regulators (GntR1 and GntR2) present on xylose repress the pentose phosphate pathway genes. For efficient xylose utilization, pentose phosphate pathway genes and a phosphoketolase gene were overexpressed with the XI pathway in C. glutamicum. Overexpression of the genes encoding for transaldolase (Tal), 6-phosphogluconate dehydrogenase (Gnd), or phosphoketolase (XpkA) enhanced the growth and xylose consumption rates compared to the wild-type with the XI pathway alone. However, co-expression of these genes did not have a synergetic effect on xylose utilization. For the succinate production from xylose, overexpression of the tal gene with the XI pathway in a succinate-producing strain improved xylose utilization and increased the specific succinate production rate by 2.5-fold compared to wild-type with the XI pathway alone. Thus, overexpression of the tal, gnd, or xpkA gene could be helpful for engineering C. glutamicum toward production of value-added chemicals with efficient xylose utilization.