Efficient 40�C fermentation of l-lysine by a new Corynebacterium glutamicum mutant developed by genome breeding

Abstract

We have recently developed a new L-lysine-producing mutant of Corynebacterium glutamicum by "genome breeding" consisting of characterization and reconstitution of a mutation set essential for high-level production. The strain AHP-3 was examined for L-lysine fermentation on glucose at temperatures above 35 degrees C, at which no examples of efficient L-lysine production have been reported for this organism. We found that the strain had inherited the thermotolerance that the original coryneform bacteria was endowed with, and thereby grew and produced L-lysine efficiently up to 41 degrees C. A final titer of 85 g/l after only 28 h was achieved at temperatures around 40 degrees C, indicating the superior performance of the strain developed by genome breeding. When compared with the traditional 30 degrees C fermentation, the 40 degrees C fermentation allowed an increase in yield of about 20% with a concomitant decrease in final growth level, suggesting a significant transition of carbon flux distribution in glucose metabolism. DNA array analysis of metabolic changes between the 30 degrees C and 40 degrees C fermentations identified several differentially expressed genes in central carbon metabolism although we could not find stringent control-like global induction of amino-acid-biosynthetic genes in the 40 degrees C fermentation. Among these changes, two candidates were picked out as the potential causes of the increased production at 40 degrees C; decreased expression of the citrate synthase gene gltA and increased expression of malE, the product of which involves regeneration of pyruvate and NADPH.

A genome-based approach to create a minimally mutated Corynebacterium glutamicum strain for efficient l-lysine production

“…A second is improved stress-tolerance, which has allowed fermentation at higher temperatures than is traditionally practiced. The new strain showed efficient L-lysine production at 40ºC, whereas classical strains could not function above 35ºC [14]. This feature not only allows reduction of cooling costs, but also enables cost-effective manufacture in tropical regions that have easy access to low-cost carbon sources such as molasses and tapioca.…”

“…Reconstructing an L-lysine producer according to genome information Reconstruction of an L-lysine producer is carried out in the following way [8,9,[14][15][16].…”

Comparisons of Potentials forL-Lysine Production among DifferentCorynebacterium glutamicumStrains

“…The L-lysine production by the six isogenic mutants were investigated using 5-liter jar fermentors both under traditional 30 C conditions and, in addition, under suboptimal 40 C conditions which could allow reduction of cooling costs in industrial processes. 23,24) Figure 1 shows the profiles obtained in fed-batch fermentor cultivation with LPG1 medium 19,20) to which glucose was added at an initial concentration of 5%, with a subsequent increase to 25%. The six lysC mutants all produced large amounts of L-lysine at both temperatures, but it was found that final titers and productivities were significantly different among the six isogenic mutants at either temperature (Table 2).…”

A new insertion sequence, IS14999, from Corynebacterium glutamicum

“…To date, numerous mutants that can produce amino acids at high efficiency have been created. The worldwide demand for amino acids such as L-glutamate, L-lysine and L-threonine for use as animal feed and food additives has continued to increase (Hermann, 2003;Ohnishi et al, 2003). Therefore, it is desirable to develop C. glutamicum strains with improved productivity.…”

“…One approach to developing strains of higher productivity using genome information has been reported (Ohnishi et al, 2003). But it does not yet deviate from the range of a single gene manipulation, and there still remain many unknown functions of genes in C. glutamicum.…”