Succinic acid production from softwood with genome‐edited Corynebacterium glutamicum using the CRISPR‐Cpf1 system

Abstract

Corynebacterium glutamicum is a useful microbe that can be used for producing succinic acid under anaerobic conditions. In this study, we generated a knock‐out mutant of the lactate dehydrogenase 1 gene (ΔldhA‐6) and co‐expressed the succinic acid transporter (Psod:SucE‐ ΔldhA) using the CRISPR‐Cpf1 genome editing system. The highly efficient HPAC (hydrogen peroxide and acetic acid) pretreatment method was employed for the enzymatic hydrolysis of softwood (Pinus densiflora) and subsequently utilized for production of succinic acid.Upon evaluating a 1%–5% hydrolysate concentration range, optimal succinic acid production with the ΔldhA mutant was achieved at a 4% hydrolysate concentration. This resulted in 14.82 g L−1 succinic acid production over 6 h. No production of acetic acid and lactic acid was detected during the fermentation. The co‐expression transformant, [Psod:SucE‐ΔldhA] produced 17.70 g L−1 succinic acid in 6 h. In the fed‐batch system, 39.67 g L−1 succinic acid was produced over 48 h. During the fermentation, the strain consumed 100% and 73% of glucose and xylose, respectively. The yield of succinic acid from the sugars consumed was approximately 0.77 g succinic acid/g sugars. These results indicate that the production of succinic acid from softwood holds potential applications in alternative biochemical processes.

Recent advances in bio-based production of top platform chemical, succinic acid: an alternative to conventional chemistry

“…Most recently, CRISPR–Cpf1 system was used for editing C. glutamicum ATCC 13032 so that it can consume fermentable sugars from enzymatic hydrolysate of H 2 O 2 –acetic acid (HPAC) pretreated Pinus densiflora [ 94 ]. CRISPR–Cpf1 was preferred over CRISPR–Cas9 as the latter could get deactivated due to secretion of various toxic metabolites while the former was more efficient.…”

“…Gene encoding for lactate dehydrogenase enzyme was knocked out whereas SA transporter was overexpressed. When fed-batch cultivation was performed with the engineered bacterium using 4% hydrolysate, a maximum of 39.47 g/L of SA was obtained in 48 h, with 100% and 73% glucose and xylose consumption, respectively [ 94 ].…”