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The gastrointestinal tract is a complex niche and the main port of entry of many pathogens that trigger a wide range of diseases like inflammatory bowel disease (IBD) and colon cancer. Antibodies are effective for treating such diseases, but a system capable of local delivery at the site of the pathology, thus avoiding systemic side effects, is not yet available. Here we report a novel recombinant scFvSIgA1 protein produced by Lactococcus lactis, anchored to the bacterial membrane, which retains its full immuno-recognizing potential. This scFv fragment employed was specific for a colon cancer epitope, epithelial glycoprotein protein-2 (EGP-2). Accordingly L. lactis expressing this chimeric protein was capable of binding cells expressing this epitope. Expression of specific antibodies on bacteria may allow local delivery of anticancer agents produced by such bacteria in conjunction with the antibody and provides a new avenue in the quest for targeted drug delivery.

The tyrosine decarboxylase operon of Lactobacillus brevis IOEB9809 contains, adjacent to the tyrosine decarboxylase gene, a gene for TyrP, a putative tyrosine transporter. The two genes potentially form a proton motive tyrosine decarboxylation pathway. The putative tyrosine transporter gene of L. brevis was expressed in Lactococcus lactis and functionally characterized using right-side-out membranes. The transporter very efficiently catalyzes homologous tyrosine-tyrosine exchange and heterologous exchange between tyrosine and its decarboxylation product tyramine. Tyrosine-tyramine exchange was shown to be electrogenic. In addition to the exchange mode, the transporter catalyzes tyrosine uniport but at a much lower rate. Analysis of the substrate specificity of the transporter by use of a set of 19 different tyrosine substrate analogues showed that the main interactions between the protein and the substrates involve the amino group and the phenyl ring with the para hydroxyl group. The carboxylate group that is removed in the decarboxylation reaction does not seem to contribute to the affinity of the protein for the substrates significantly. The properties of the TyrP protein are those typical for precursor-product exchangers that operate in proton motive decarboxylation pathways. It is proposed that tyrosine decarboxylation in L. brevis results in proton motive force generation by an indirect proton pumping mechanism.

Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The hdcA gene, coding for the histidine decarboxylase of L. hilgardii IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the hdcA locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in Lactococcus lactis. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.

Many organisms have the ability to form spores, a remarkable phase in their life cycles. Compared with vegetative cells, spores have several advantages (e.g. resistance to toxic compounds, temperature, desiccation and radiation) making them well suited to various applications. The applications of spores that first spring to mind are bio-warfare and the related, but more positive, field of biological control. Although they are often considered metabolically inert, spores can also be used as biocatalysts. Other uses for spores are found in the fields of probiotics, tumour detection and treatment, biosensing and in the "war against drugs".

Spores, although often considered metabolically inert, catalyze a variety of reactions. The use of spores instead of mycelium for bioconversions has several advantages. In this paper, we describe the difference in susceptibility of mycelium and spores against toxic substrates and products. A higher resistance of spores toward the toxic effects of bioconversion substrates and products is an advantage that has not been studied in detail until now. This paper shows that spores of Penicillium digitatum ATCC 201167 are on average over 2.5 times more resistant than mycelium toward the toxicity of substrates, intermediates, and products of the geraniol bioconversion pathway. Furthermore, the higher resistance of spores to citral was shown as an advantage in its biotransformation by P. digitatum. Using three different approaches the toxicity of the compounds were tested. The order of toxicity toward P. digitatum was, starting with the most toxic, citral > nerol/geraniol > geranic acid > methylheptenone >> acetaldehyde., (Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 685-690, 2002.)

A novel lyase, combining hydratase and aldolase activity, that converts citral into methylheptenone and acetaldehyde, was purified from spores of Penicillium digitatum. Remarkably, citral lyase activity was induced 118-fold by incubating nongerminating spores with the substrate, citral. This cofactor independent hydratase/aldolase, was purified and found to be a monomeric enzyme of 31 kDa. Citral lyase has a Km of 0.058 mm and a Vmax of 52.6 U.mg-1. Enzyme activity was optimal at 20 degrees C and pH 7.6. The enzyme has a strong preference for the trans isomer of citral (geranial). Citral lyase also converts other alpha,beta-unsaturated aldehydes (farnesal, methyl-crotonaldehyde, decenal and cinnemaldehyde).

Spores of Penicillium digitatum ATCC 201167 transform geraniol, nerol, citral, and geranic acid into methylheptenone. Spore extracts of P. digitatum convert geraniol and nerol NAD+-dependently into citral. Spore extract also converts citral NAD+-dependently into geranic acid. Furthermore, a novel enzymatic activity, citral lyase, which cofactor-independently converts citral into methylheptenone and acetaldehyde, was detected. These result show that spores of P. digitatum convert geraniol via a novel biotransformation pathway. This is the first time a biotransformation pathway in fungal spores has been substantiated by biochemical studies. Geraniol and nerol are converted into citral by citrol dehydrogenase activity. The citral formed is subsequently deacetylated by citral lyase activity, forming methylheptenone. Moreover, citral is converted reversibly into geranic acid by citral dehydrogenase activity.

Under alkaline conditions, amino acids or proteins catalyze the deacetylation of citral, a major aroma component, resulting in methylheptenone and acetaldehyde formation. 3-Hydroxycitronellal is an intermediate in this reaction. Amino acids also catalyze the cis-trans isomerization of the pure isomers of citral, geranial, and neral. Most likely the amino acids are involved in stabilizing intermediates of the isomerization and deacetylation reaction of citral. On the basis of the findings presented, some consequences for the application of citral, or its isomers, in food are discussed.

Zygosaccharomyces rouxii is an important yeast in the formation of flavor in soy sauce. In this study, we investigated the separate effects of exogenous threonine, cystathionine, and the branched-chain amino acids on the metabolism of Z. rouxii. The addition of these amino acids had significant effects on both Z. rouxii growth and glycerol and higher alcohol production. It also seemed that Z. rouxii displayed the Crabtree effect, which was independent of the added amino acids. Furthermore, we investigated the regulation of the metabolism of alpha-ketobutyrate, which is a key-intermediate in Z. rouxii amino acid metabolism. Threonine and cystathionine were introduced separately to stimulate the formation rate of alpha-ketobutyrate and the branched-chain amino acids to inhibit its conversion rate. Enzyme activities showed that these amino acids had a significant effect on the formation and conversion rate of alpha-ketobutyrate but that the alpha-ketobutyrate pool size in Z. rouxii was in balance all the time. The latter was confirmed by the absence of alpha-ketobutyrate accumulation.