The amino acid L-lysine was produced from homoserine auxotrophic and S-(2-aminoethyl)-L-cysteineresistant mutants of a newly isolated gram-positive methylotrophic bacterium, capable of growth on methanol at 60°C. The temperature optimum for growth was between 50 and 53°C. These aerobic, gram-positive, endospore-forming, rod-shaped bacteria required biotin and vitamin B12 for growth. Extracts of the bacteria grown on methanol lacked hydroxypyruvate reductase and contained hexulose 6-phosphate synthase activity. Therefore, these bacteria were considered to be type I methylotrophic bacteria of the genus Bacillus. Fed-batch fermentations resulted in cell densities of 50 g of cell dry weight per liter. Biomass yields on carbon, nitrogen, phosphate, and sulfate were determined. Generation of homoserine auxotrophic and amino acid analogresistant mutants resulted in L-lysine concentrations of nearly 20 g/liter in fed-batch fermentations.

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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...

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