Single-Domain Peptidyl-Prolyl cis / trans Isomerase FkpA from Corynebacterium glutamicum Improves the Biomass Yield at Increased Growth Temperatures

Abstract

Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the rate-limiting protein folding step at peptidyl bonds preceding proline residues and were found to be involved in several biological processes, including gene expression, signal transduction, and protein secretion. Representative enzymes were found in almost all sequenced genomes, including Corynebacterium glutamicum, a facultative anaerobic Gram-positive and industrial workhorse for the production of amino acids. In C. glutamicum, a predicted single-domain FK-506 (tacrolimus) binding protein (FKBP)-type PPIase (FkpA) is encoded directly downstream of gltA, which encodes citrate synthase (CS). This gene cluster is also present in other Actinobacteria. Here we carried out in vitro and in vivo experiments to study the function and influence of predicted FkpA in C. glutamicum. In vitro, FkpA indeed shows typical PPIase activity with artificial substrates and is inhibited by FK-506. Furthermore, FkpA delays the aggregation of CS, which is also inhibited by FK-506. Surprisingly, FkpA has a positive effect on the activity and temperature range of CS in vitro. Deletion of fkpA causes a 50% reduced biomass yield compared to that of the wild type when grown at 37°C, whereas there is only a 10% reduced biomass yield at the optimal growth temperature of 30°C accompanied by accumulation of 7 mM L-glutamate and 22 mM 2-oxoglutarate. Thus, FkpA may be exploited for improved product formation in biotechnical processes. Comparative transcriptome analysis revealed 69 genes which exhibit >2-fold mRNA level changes in C. glutamicum ⌬fkpA, giving insight into the transcriptional response upon mild heat stress when FkpA is absent.T he soil microorganism Corynebacterium glutamicum is widely used in large-scale industrial processes to produce the flavorenhancing amino acid L-glutamate and the feed additive L-lysine (2,900,000 and 1,950,000 tons/year; Ajinomoto). C. glutamicum, which grows on a variety of carbon sources, also has the potential for the production of several organic acids and other commercially interesting compounds (see, for example, references 1, 2, and 3 and references therein). Parameters such as hyperoptimal temperatures and osmotic stress play an important role in biotechnical processes (4, 5). The optimum growth temperature of C. glutamicum is about 30°C to 33°C, although it is able to grow at temperatures ranging from 15°C to 40°C (6). As a soil organism, C. glutamicum is able to adapt to fast-changing conditions, including pH (7), salinity (8), and also temperature. Adaptation mechanisms of C. glutamicum toward nonoptimal growth temperatures include the heat shock response induced at temperatures above 40°C (9-11) and the cold shock response induced below 20°C (12), both involving distinct sets of chaperones.Adaptation mechanisms of cells toward temperature, including the responses to heat shock and cold shock as well as protein chaperones, have been reviewed many times in several model systems (see, for example, references 13-15, and 16). Beside chapero...