Reconstruction of tricarboxylic acid cycle in Corynebacterium glutamicum with a genome‐scale metabolic network model for trans‐4‐hydroxyproline production. Issue 1 (27th October 2018)

• Record Type: Journal Article
• Authors: Zhang, Yu; Zhang, Yun; Shang, Xiuling; Wang, Bo; Hu, Qitiao; Liu, Shuwen; Wen, Tingyi
• Is Part Of: Biotechnology and bioengineering. Volume 116:Issue 1(2019)
• Page Start: 99
• Page End: 109
• Publication Date: 2018-10-27
• Subjects: iCW773 -- in silico model‐based simulation -- ribosome binding site optimization -- reconstruction of tricarboxylic acid cycle -- trans‐4‐hydroxy‐ l‐proline; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.26818
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: trans ‐4‐Hydroxy‐l ‐proline (Hyp) is an abundant component of mammalian collagen and functions as a chiral synthon for the syntheses of anti‐inflammatory drugs in the pharmaceutical industry. Proline 4‐hydroxylase (P4H) can catalyze the conversion ofl ‐proline to Hyp; however, it is still challenging for the fermentative production of Hyp from glucose using P4H due to the low yield and productivity. Here, we report the metabolic engineering of Corynebacterium glutamicum for the fermentative production of Hyp by reconstructing tricarboxylic acid (TCA) cycle together with heterologously expressing the p4h gene from Dactylosporangium sp . strain RH1. In silico model‐based simulation showed that α‐ketoglutarate was redirected from the TCA cycle toward Hyp synthetic pathway driven by P4H when the carbon flux from succinyl‐CoA to succinate descended to zero. The interruption of the TCA cycle by the deletion of sucCD ‐encoding the succinyl‐CoA synthetase (SUCOAS) led to a 60% increase in Hyp production and had no obvious impact on the growth rate. Fine‐tuning of plasmid‐borne ProB* and P4H abundances led to a significant increase in the yield of Hyp on glucose. The final engineered Hyp‐7 strain produced up to 21.72 g/L Hyp with a yield of 0.27 mol/mol (Hyp/glucose) and a volumetric productivity of 0.36 g·L −1 ·hr −1 in the shake flask fermentation. To our knowledge, this is the highest yield and productivity achieved by microbial fermentation in a glucose‐minimal medium for Hyp production. This strategy provides new insights into engineering C. glutamicum by flux coupling for the fermentative production of Hyp and related products. Abstract : The TCA cycle in Corynebacterium glutamicum was reconstructed by inactivating succinyl‐CoA synthetase to redirect α‐ketoglutarate toward proline hydroxylation. P4H bridged the broken TCA cycle through the conversion of cosubstrate α‐ketoglutarate to succinate and led to a significant increase in Hyp production. Fine‐tuning of ProB* and P4H abundances gave rise to the improvement in the yield of Hyp on glucose.