Improving diacetyl production in Corynebacterium glutamicum via modifying respiratory chain

Abstract

To explore the suitability of Corynebacterium glutamicum as a chassis for diacetyl production from glucose, diacetyl metabolic pathway and the respiratory chain were linked to achieve redox balance. The carbon flux was redirected from pyruvate to diacetyl by overexpressing the α-acetolactate synthase, in combination with disruption the biosynthetic pathways of lactate, acetoin, 2,3-butanediol and acetate in C. glutamicum ATCC 13032. These modifications resulted in a sharp increase of the NADH/NAD⁺ ratio from 0.53 to 1.10, and produced 0.58 g/L diacetyl under aerobic conditions, representing a 58-fold increase over the wild type. Although the modification of the by-product pathways is an effective strategy, these disruption led to intracellular cofactor imbalance. NADH re-oxidization was further successfully solved by overexpressing of cytochrome bd oxidase. We constructed an efficient respiration-dependent cell factory by modification of the respiratory chain, improving diacetyl titer to 1.29 g/L in CGC11, decreased NADH/NAD⁺ ratio to 0.45, increased the ATP concentration from 8.51 to 10.64 μM/gDCW. To our best knowledge, this is the first report of diacetyl synthesis in C. glutamicum. Intracellular cofactor imbalance can be reduced by modification of the respiratory chain for production of diacetyl as well as other bio-based products with cofactor imbalance in C. glutamicum.

Keywords: Cofactor; Corynebacterium glutamicum; Diacetyl; Respiratory chain.