Improving Process Yield in Succinic Acid Production by Cell Recycling of Recombinant Corynebacterium glutamicum

Toru Jojima,Ryoji Noburyu,Maysayuki Inui,Masako Suda,Hideaki Yukawa,Shohei Okino

doi:10.3390/fermentation2010005

Abstract

Aerobically cultivated cells of Corynebacterium glutamicum produce mixed organic acids, including succinic acid (SA), from glucose when the cells are transferred to oxygen-deprived conditions. Genetic modification, including inactivation of lactate dehydrogenase and overexpression of pyruvate carboxylase, allows this microbe to be an efficient SA producer under the conditions of oxygen deprivation. High productivity and high titers can be achieved in the production process by using the genetically engineered strain of C. glutamicum under the given conditions. However, glucose consumption for cell preparation decreases process yield (defined as the quantity of SA produced divided by the total quantity of glucose used in cell preparation and SA production). In this study, we investigated cell recycle fed-batch fermentation for SA production to improve the process yield by reducing the effect of glucose consumption for cell preparation on the process yield. A genetically stable and markerless strain, harboring nine genomic copies of the pyruvate carboxylase gene, was newly constructed and used for cell recycle fermentation. During 26 reaction cycles, only 0.7% decrease in specific productivity per reaction was observed. Overall, the process yield was improved by 79% compared to that in a single fed-batch reaction without cell recycling.

Highlights

Succinic acid (SA), a C4 dicarboxylic acid, is used as a building block for specialty chemicals such as surfactants and chelators and as an additive in the pharmaceutical and food industries [1]
A 3.8-kb nucleotide fragment, containing the native promoter and the coding sequences for the pyc gene was integrated into non-essential strain-specific islands (SSIs) of the C. glutamicum genome to construct a plasmid-free strain for SA production [30]
The resulting strain, named CRZ19, showed lower volumetric productivity than that of the plasmid-bearing CRZ1-pCRA717 strain (Table 2). These results suggest that more copies of the pyc gene were required to exhibit SA volumetric productivity similar to that of the CRZ1-pCRA717 strain, as a copy number of the pBL1-based plasmid pCRA717 is estimated to be 10–30 [34]

Summary

Introduction

Succinic acid (SA), a C4 dicarboxylic acid, is used as a building block for specialty chemicals such as surfactants and chelators and as an additive in the pharmaceutical and food industries [1]. Corynebacterium glutamicum is used for the industrial production of amino acids [4]. Gram-positive microbe has been utilized for the production of fuels and commodity chemicals including ethanol [5,6], isobutanol [7,8,9], D-lactic acid [10], cadaverine [11], putrescine [12], xylitol [13], 2,3-butanediol [14,15], and SA. SA-producing strains of C. glutamicum have been developed separately for aerobic [16,17,18,19] and anaerobic processes [20,21,22,23,24]. Anaerobic processes with C. glutamicum have been found to be superior to aerobic processes in terms of the productivity, titer, and yield of SA [20,21]

Methods

Results

Conclusion