The unusual metabolic properties of aerobic, restrictive, thermotolerant methylotrophic bacteria make them useful for the production of recombinant proteins, vitamins, amino acids, coenzymes, and cytochromes (12). Bacillus methanolicus MGA3 (ATCC 53907) is a gram-positive bacterium with a growth optimum of 50°C to 53°C. Cell extracts of this bacterium grown on methanol contain an NAD-dependent methanol dehydrogenase (3, 13), have 3-hexulose-6-phosphate synthase, fructose bisphosphate aldolase, and transaldolase activity indicative of the fructose bisphosphate aldolase/ transaldolase variant of the ribulose monophosphate pathway, RuMP (4, 14, 44). The precursors (acetyl-coenzyme A, pyruvate, oxaloacetate, and 2-oxoglutarate) for lipids, amino acids, nucleic acids, and assimilation of ammonia are all synthesized from the products of the RuMP pathway (2). The dissimilation of carbon from formaldehyde to CO 2 in B. methanolicus, which may function to detoxify accumulating formaldehyde, has recently been confirmed by 13 C nuclear magnetic resonance and isotope ratio mass spectrometry (39). The theoretical yield of secreted amino acids varies depending on the percentage of formaldehyde carbon dissimilated to CO 2 (39), and therefore control of the feeding of methanol to fed-batch cultures is critical (29).Homoserine dehydrogenase mutants (HSD Ϫ ) of B. methanolicus, which are threonine plus methionine auxotrophs, have been shown to secrete substantial quantities of L-lysine when grown at 50°C in fed-batch cultures on methanol as a sole carbon source and supplied with a source of ammonia (18,29,44). B. methanolicus HSD Ϫ strain 13A52-8A66 can secrete over 60 g of L-lysine plus glutamate (glutamate) per liter, with a mass yield of 0.50 to 0.63 g/g. However, no mutants that secrete only L-lysine have been found with this approach; all simultaneously secrete significant quantities of glutamate and smaller amounts of alanine.Limited knowledge exists for most aerobic methylotrophs of the genetic basis of the regulation of pyruvate metabolism, the activity of tricarboxylic acid (TCA) cycle enzymes during rapid growth on methanol (7,13,54), and particularly regulation of the flow of carbon to amino acid biosynthesis (20,(31)(32)(33)46). To accomplish high-level L-lysine production, the percentage of carbon dissimilated to CO 2 must be minimized to generate an abundant supply of aspartate (37). In Bacillus subtilis, aspartate is synthesized from oxaloacetate by aspartate aminotransferase (glutamate:oxaloacetate transaminase) encoded by aspB, which appears to be constitutively expressed (5).The simultaneous production of glutamate and L-lysine in B. methanolicus mutants indicates a substantial flow of carbon both to aspartate and into the first three reactions of the TCA cycle catalyzed by citrate synthase (CS), aconitase, and isocitrate dehydrogenase (Fig. 1). Since many methylotrophs generate reducing power by the oxidation of formaldehyde to

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...