Production of L-Lysine and Some Other Amino Acids by Mutants of B. methanolicus

Abstract

INTRODUCTION:Bacillus methanolicus is is a Gram-positive, endosporeforming, facultative methylotroph with an optimal growth temperature of 50-53 0 C. This bacterium employs the efficient ribulose-monophosphate pathway for formaldehyde assimilation and possesses a novel nicotinamide adenine nucleotide-dependent methanol dehydrogenase (Dijkhuisen et ai, 1988) rather than the cytochrome linked MDH found in gram-negative methylotrophs. Several isolates similar to those isolated by us (Schendel et ai, 1990) have been described and have been shown to be related to Bacillus frimus and B. aiotoJormans (Arfman, Dijhuizen, 1993) .. We have previously shown that mutants of B. methanolicus strains MGA3 and NOA2 which lacked homoserine dehydrogenase (methionine, threonine auxotrophs) and were also resistant to the lysine analogue, S-(2-aminoethyl)-L-cysteine (ABC) produced significant amounts of lysine when grown in a mineral salts medium under threonine limited conditions in fed-batch fermentations (Schendel et ai, 1990).Lysine is used to supplement feeds for poultry, swine and other livestock that contain cereal grains which contain limited quantities of this amino acid. Fermentation processes that employ strains of Corynebacterium glutamicum or Brevibacterium lactoJermentum with starch hydrolysate or molasses as feedstocks have been used as the major methods for L-Iysine production (Tosaka et ai, 1983) Methanol is available in pure form, it is highly soluble in water, methanol solutions are not explosive and residual methanol can be readily removed from products after fermentations are completed. Methanol is also relatively inexpensive, the price is stable and it is easily stored and transported. Therefore, methanol has potential as a good substrate for the production of fermentation products (Lentin, Neikus, 1987).Aspartokinase isoenzymes catalyze the first reaction required for the common pathway involved in the biosynthesis of lysine, threonine, methionine and isoleucine. (Figure 1) Previous studies have indicated that B. methanolicus MGA3 possesses three isoenzymes (Schendel, Flickinger, 1992) .. One is inhibited by lysine alone (aspartokinase II), another is subject to concerted feedback inhibition by threonine and lysine (aspartokinase ill) and a third is believed to be inhibited by diaminopimelic acid. The lysE gene which encodes aspartokinase II has been cloned and sequenced, and purified from E. coli clones that express the gene (Schendel, Flickinger, 1992). The gene product was shown to be similar in structure and sensitivity to lysine inhibition to the aspartokinase II from B. subtilis. Aspartokinase ill accounts for the major portion of the activity of aspartokinases (approximately 50%) while aspartokinase II accounts for about 40% of the total activity.The lys A gene which encodes diaminopimelate (DAP) decarboxylase, the enzyme that catalyzes the final reaction in lysine biosynthesis has also been cloned from B. methanolicus MGA3 (Mills, Flickinger, 1993). The gene was found to be 57% similar in nucleoti...

Analysis and Manipulation of Aspartate Pathway Genes for l -Lysine Overproduction from Methanol by Bacillus methanolicus

“…We therefore proceeded to analyze transcription levels for the aspartate pathway genes in this mutant by using RT-PCR as described above. To obtain comparable conditions for growth, cells were cultivated in methanol medium supplemented with 1 mM DL-methionine and 1.5 mM L-threonine (see Materials and Methods), as required for growth of NOA2#13A52-8A66 (19). Both MGA3 and M168-20 were included as control strains, and the results of these experiments are shown in Table 4.…”

Plasmid-Dependent Methylotrophy in Thermotolerant Bacillus methanolicus

“…B. methanolicus uses an NAD(P)-dependent methanol dehydrogenase (MDH) to oxidize methanol to formaldehyde, and in addition to entering the RuMP pathway, a linear branch for dissimilation of formaldehyde to CO 2 was recently demonstrated (29). It was previously shown that this bacterium can secrete 55 g of glutamate per liter at 50°C by using methanol as a carbon source in fed-batch fermentation (11), and a homoserine dehydrogenase mutant (13A52-8A66) that secreted up to 35 g of L-lysine per liter at 50°C was described (18). In a previous study (13) it was found that the B. methanolicus NOA2 mutant 13A52 possesses a plasmid with an estimated size of 17 kb, and a plasmid of a similar size was later identified (N. Tsujimoto, H. Yasueda, and S. Sugimoto, 24 October 2000, Japanese patent application JP2000295988) in B. methanolicus PB1 (ϭ NCIMB 13113).…”

Overexpression of Wild-Type Aspartokinase Increases l -Lysine Production in the Thermotolerant Methylotrophic Bacterium Bacillus methanolicus

“…The gram-positive and thermotolerant methylotroph Bacillus methanolicus has been studied as an alternative producer of L-glutamate and L-lysine using methanol as the raw material (for a review, see reference 5). We have previously demonstrated that wild-type B. methanolicus produces 58 g/liter of L-glutamate (6), while mutants generated by random chemical mutagenesis have been reported to produce up to 37 g/liter of L-lysine (16,23,34). Favorable properties of B. methanolicus, such as a lack of sporulation at high temperatures, utilization of methanol as an energy and carbon source, a high methanol conversion rate, a high theoretical yield of L-lysine, and an optimal growth temperature of 50°C, indicate that this organism may represent a possible future noncarbohydrate substrate alternative for large-scale production of L-lysine (5).…”