We here present the pyc gene encoding pyruvate carboxylase (PC), and the hom-1 and hom-2 genes encoding two active homoserine dehydrogenase (HD) proteins, in methylotrophic Bacillus methanolicus MGA3. In general, both PC and HD are regarded as key targets for improving bacterial L-lysine production; PC plays a role in precursor oxaloacetate (OAA) supply while HD controls an important branch point in the L-lysine biosynthetic pathway. The hom-1 and hom-2 genes were strongly repressed by L-threonine and L-methionine, respectively. Wild-type MGA3 cells secreted 0.4 g/l L-lysine and 59 g/l L-glutamate under optimised fed batch methanol fermentation. The hom-1 mutant M168-20 constructed herein secreted 11 g/l L-lysine and 69 g/l of L-glutamate, while a sixfold higher L-lysine overproduction (65 g/l) of the previously constructed classical B. methanolicus mutant NOA2#13A52-8A66 was accompanied with reduced L-glutamate production (28 g/l) and threefold elevated pyc transcription level. Overproduction of PC and its mutant enzyme P455S in M168-20 had no positive effect on the volumetric L-lysine yield and the L-lysine yield on methanol, and caused significantly reduced volumetric L-glutamate yield and L: -glutamate yield on methanol. Our results demonstrated that hom-1 represents one key target for achieving L-lysine overproduction, PC activity plays an important role in controlling L-glutamate production from methanol, and that OAA precursor supply is not a major bottleneck for L-lysine overproduction by B. methanolicus.

Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.

Methanol is regarded as an attractive substrate for biotechnological production of value-added bulk products, such as amino acids and polyamines. In the present study, the methylotrophic and thermophilic bacterium Bacillus methanolicus was engineered into a microbial cell factory for the production of the platform chemical 1,5-diaminopentane (cadaverine) from methanol. This was achieved by the heterologous expression of the Escherichia coli genes cadA and ldcC encoding two different lysine decarboxylase enzymes, and by increasing the overall L-lysine production levels in this host. Both CadA and LdcC were functional in B. methanolicus cultivated at 50°C and expression of cadA resulted in cadaverine production levels up to 500 mg l−1 during shake flask conditions. A volume-corrected concentration of 11.3 g l−1 of cadaverine was obtained by high-cell density fed-batch methanol fermentation. Our results demonstrated that efficient conversion of L-lysine into cadaverine presumably has severe effects on feedback regulation of the L-lysine biosynthetic pathway in B. methanolicus. By also investigating the cadaverine tolerance level, B. methanolicus proved to be an exciting alternative host and comparable to the well-known bacterial hosts E. coli and Corynebacterium glutamicum. This study represents the first demonstration of microbial production of cadaverine from methanol.

No abstract

We investigated the regulation and roles of six aspartate pathway genes in L-lysine overproduction in Bacillus methanolicus: dapG, encoding aspartokinase I (AKI); lysC, encoding AKII; yclM, encoding AKIII; asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; and lysA, encoding meso-diaminopimelate decarboxylase. Analysis of the wild-type strain revealed that in vivo lysC transcription was repressed 5-fold by L-lysine and induced 2-fold by DL-methionine added to the growth medium. Surprisingly, yclM transcription was repressed 5-fold by DL-methionine, while the dapG, asd, dapA, and lysA genes were not significantly repressed by any of the aspartate pathway amino acids. We show that the L-lysine-overproducing classical B. methanolicus mutant NOA2#13A52-8A66 has-in addition to a hom-1 mutation-chromosomal mutations in the dapG coding region and in the lysA promoter region. No mutations were found in its dapA, lysC, asd, and yclM genes. The mutant dapG gene product had abolished feedback inhibition by mesodiaminopimelate in vitro, and the lysA mutation was accompanied by an elevated (6-fold) lysA transcription level in vivo. Moreover, yclM transcription was increased 16-fold in mutant strain NOA2#13A52-8A66 compared to the wild-type strain. Overexpression of wild-type and mutant aspartate pathway genes demonstrated that all six genes are important for L-lysine overproduction as tested in shake flasks, and the effects were dependent on the genetic background tested. Coupled overexpression of up to three genes resulted in additive (above 80-fold) increased L-lysine production levels.The essential amino acid L-lysine is widely used as a feed additive in animal farming, and currently Ͼ1,000,000 tons of L-lysine-HCl per year are produced worldwide by fermentation processes, using classical mutant strains of Corynebacterium glutamicum and its relatives (1,7,34). L-Lysine is a product of the aspartate pathway (Fig. 1), and several of the genes and enzymes involved are feedback regulated. The regulatory mechanisms are allosteric inhibition of the enzymes and transcriptional repression of the genes, including RNA riboswitch mechanisms at the mRNA level (15, 32). The aspartate pathway has been investigated particularly well in C. glutamicum, with the overall aim of identifying enzymes representing ratelimiting steps for L-lysine overproduction. In particular, the importance of aspartokinase (AK) in controlling L-lysine biosynthesis has been well documented for this bacterium (13) as well as for several other bacteria, including Escherichia coli (25, 30), Lactobacillus plantarum (11, 12), Bacillus subtilis (41), and Methylophilus methylotrophus (38). It has been unraveled that homoserine dehydrogenase (HD), dihydrodipicolinate synthase (DapA), dihydrodipicolinate reductase (DapB), mesodiaminopimelate decarboxylase (LysA), and L-lysine exporter (LysE) can also represent targets for achieving L-lysine overproduction (13,17,21,28,29,38). Moreover, several enzymes of cell primary metabol...