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Bacillus subtilis is capable of degrading fructosamines. The phosphorylation and the cleavage of the resulting fructosamine 6-phosphates is catalyzed by the frlD and frlB gene products, respectively. This study addresses the physiological importance of the frlBONMD genes (formerly yurPONML), revealing the necessity of their expression for growth on fructosamines and focusing on the complex regulation of the corresponding transcription unit. In addition to the known regulation by the global transcriptional regulator CodY, the frl genes are repressed by the convergently transcribed FrlR (formerly YurK). The latter causes repression during growth on substrates other than fructosamines. Additionally, we identified in the first intergenic region of the operon an FrlR binding site which is centrally located within a 38-bp perfect palindromic sequence. There is genetic evidence that this sequence, in combination with FrlR, contributes to the remarkable decrease in the transcription downstream of the first gene of the frl operon.Amadori products (fructosamines), the first stable intermediates of the Maillard reaction, result from nonenzymatic glycation of amino acids or proteins with reducing sugars such as glucose. As Amadori products are found in heated food and cause several diseases in connection with diabetes and aging (4), enzymes catalyzing the degradation of Amadori products are of interest to industry and medicine for food processing and diagnostic purposes.There are two enzymatic mechanisms for the deglycation of Amadori products (reviewed in reference 7): fructosyl-amino acid oxidases (known as Amadoriases) and fructosamine kinases. The former deglycate by means of oxidation, generating the corresponding amino acid, glucosone, and H 2 O 2 . Amadoriases have been found in Aspergillus and Penicillium spp. (38, 42) but also in bacteria such as Arthrobacter and Corynebacterium spp. (9, 32). Fructosamine kinases phosphorylate the Amadori products prior to cleavage. Mammalian enzymes add the phosphate at C-3, and the product subsequently undergoes autocatalytic degradation (20). However, Escherichia coli and Bacillus subtilis kinases phosphorylate at C-6; further processing of such intermediates needs a second deglycase enzyme catalyzing the cleavage of the fructosamine 6-phosphates to generate, for example, glucose 6-phosphate and a free amine.E. coli grows on fructosyl-lysine as the sole carbon and nitrogen source, and expression of frlD (encoding the kinase) and frlB (encoding the deglycating protein) is induced by fructosyl-lysine (39). In B. subtilis, frlD and frlB belong to a gene cluster which additionally comprises three genes coding for putative transporters (frlONM) as well as the convergently oriented putative repressor gene frlR (Fig. 1). Substrate specificities of FrlD and FrlB are quite different than those of the enzymes of E. coli, as B. subtilis acts on ␣-glycated amino acids rather than on ε-glycated lysine (catalytic efficiencies Ͼ30-fold higher), the latter being the preferred substrate of the E...

BackgroundIn white biotechnology biocatalysis represents a key technology for chemical functionalization of non-natural compounds. The plasmid-born overproduction of an alcohol dehydrogenase, an L-alanine-dependent transaminase and an alanine dehydrogenase allows for redox self-sufficient amination of alcohols in whole cell biotransformation. Here, conditions to optimize the whole cell biocatalyst presented in (Bioorg Med Chem 22:5578–5585, 2014), and the role of L-alanine for efficient amine functionalization of 1,10-decanediol to 1,10-diaminodecane were analyzed.ResultsThe enzymes of the cascade for amine functionalization of alcohols were characterized in vitro to find optimal conditions for an efficient process. Transaminase from Chromobacterium violaceum, TaCv, showed three-fold higher catalytic efficiency than transaminase from Vibrio fluvialis, TaVf, and improved production at 37°C. At 42°C, TaCv was more active, which matched thermostable alcohol dehydrogenase and alanine dehydrogenase and improved the 1,10-diaminodecane production rate four-fold. To study the role of L-alanine in the whole cell biotransformation, the L-alanine concentration was varied and 1,10.diaminodecane formation tested with constant 10 mM 1,10- decanediol and 100 mM NH4Cl. Only 5.6% diamine product were observed without added L-alanine. L-alanine concentrations equimolar to that of the alcohol enabled for 94% product formation but higher L-alanine concentrations allowed for 100% product formation. L-alanine was consumed by the E. coli biocatalyst, presumably due to pyruvate catabolism since up to 16 mM acetate accumulated. Biotransformation employing E. coli strain YYC202/pTrc99a-ald-adh-taCv, which is unable to catabolize pyruvate, resulted in conversion with a selectivity of 42 mol-%. Biotransformation with E. coli strains only lacking pyruvate oxidase PoxB showed similar reduced amination of 1,10-decanediol indicating that oxidative decarboxylation of pyruvate to acetate by PoxB is primarily responsible for pyruvate catabolism during redox self-sufficient amination of alcohols using this whole cell biocatalyst.ConclusionThe replacement of the transaminase TaVf by TaCv, which showed higher activity at 42°C, in the artificial operon ald-adh-ta improved amination of alcohols in whole cell biotransformation. The addition of L-alanine, which was consumed by E. coli via pyruvate catabolism, was required for 100% product formation possibly by providing maintenance energy. Metabolic engineering revealed that pyruvate catabolism occurred primarily via oxidative decarboxylation to acetate by PoxB under the chosen biotranformation conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-014-0189-x) contains supplementary material, which is available to authorized users.