Engineering Bacillus subtilis for the conversion of the antimetabolite 4-hydroxy-l-threonine to pyridoxine.

Until now, pyridoxine (PN), the most commonly supplemented B6 vitamer for animals and humans, is chemically synthesized for commercial purposes. Thus, the development of a microbial fermentation process is of great interest for the biotech industry. Recently, we constructed a Bacillus subtilis strain that formed significant amounts of PN via a non-native deoxyxylulose 5'-phosphate-(DXP)-dependent vitamin B6 pathway. Here we report the optimization of the condensing reaction of this pathway that consists of the 4-hydroxy-l-threonine-phosphate dehydrogenase PdxA, the pyridoxine 5'-phosphate synthase PdxJ and the native DXP synthase, Dxs. To allow feeding of high amounts of 4-hydroxy-threonine (4-HO-Thr) that can be converted to PN by B. subtilis overexpressing PdxA and PdxJ, we first adapted the bacteria to tolerate the antimetabolite 4-HO-Thr. The adapted bacteria produced 28-34mg/l PN from 4-HO-Thr while the wild-type parent produced only 12mg/l PN. Moreover, by expressing different pdxA and pdxJ alleles in the adapted strain we identified a better combination of PdxA and PdxJ enzymes than reported previously, and the resulting strain produced 65mg/l PN. To further enhance productivity mutants were isolated that efficiently take up and convert deoxyxylulose (DX) to DXP, which is incorporated into PN. Although these mutants were very efficient to convert low amount of exogenous DX, at higher DX levels they performed only slightly better. The present study uncovered several enzymes with promiscuous activity and it revealed that host metabolic pathways compete with the heterologous pathway for 4-HO-Thr. Moreover, the study revealed that the B. subtilis genome is quite flexible with respect to adaptive mutations, a property, which is very important for strain engineering.

Overexpression of a non-native deoxyxylulose-dependent vitamin B6 pathway in Bacillus subtilis for the production of pyridoxine.

Vitamin B6 is a designation for the vitamers pyridoxine, pyridoxal, pyridoxamine, and their respective 5'-phosphates. Pyridoxal 5'-phosphate, the biologically most-important vitamer, serves as a cofactor for many enzymes, mainly active in amino acid metabolism. While microorganisms and plants are capable of synthesizing vitamin B6, other organisms have to ingest it. The vitamer pyridoxine, which is used as a dietary supplement for animals and humans is commercially produced by chemical processes. The development of potentially more cost-effective and more sustainable fermentation processes for pyridoxine production is of interest for the biotech industry. We describe the generation and characterization of a Bacillus subtilis pyridoxine production strain overexpressing five genes of a non-native deoxyxylulose 5'-phosphate-dependent vitamin B6 pathway. The genes, derived from Escherichia coli and Sinorhizobium meliloti, were assembled to two expression cassettes and introduced into the B. subtilis chromosome. in vivo complementation assays revealed that the enzymes of this pathway were functionally expressed and active. The resulting strain produced 14mg/l pyridoxine in a small-scale production assay. By optimizing the growth conditions and co-feeding of 4-hydroxy-threonine and deoxyxylulose the productivity was increased to 54mg/l. Although relative protein quantification revealed bottlenecks in the heterologous pathway that remain to be eliminated, the final strain provides a promising basis to further enhance the production of pyridoxine using B. subtilis.

Complete Genome Sequence of the Prototrophic Bacillus subtilis subsp. subtilis Strain SP1.

Here, we present the complete genome sequence of the Bacillus subtilis strain SP1. This strain is a descendant of the laboratory strain 168. The strain is suitable for biotechnological applications because the prototrophy for tryptophan has been restored. Due to laboratory cultivation, the strain has acquired 24 additional sequence variations.

The origins of 168, W23, and other Bacillus subtilis legacy strains.

Bacillus subtilis is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of B. subtilis genetics. For three--NCIB 3610T, PY79, and SMY--we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more "W23 islands" in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its trp and sac loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these B. subtilis legacy strains.

From the genome sequence to the proteome and back: evaluation of E. coli genome annotation with a 2-D gel-based proteomics approach.

The ambition of systems biology to understand complex biological systems at the molecular level implies that we need to have a concrete and correct understanding of each molecular entity and its function. However, even for the best-studied organism, Escherichia coli, a large number of proteins have never been identified and characterised from wild-type cells, and/or await unravelling of their biological role. Instead, the ORF models for these proteins have been predicted by suitable algorithms and/or through comparison with known, homologous proteins from other organisms, approaches which may be prone to error. In the present study, we used a combination of 2-DE, MALDI-TOF-MS and PMF to identify 1151 different proteins in E. coli K12 JM109. Comparison of the experimental with the theoretical Mr and pI values (4000 experimental values each) allowed the identification of numerous proteins with incorrect or incomplete ORF annotations in the current E. coli genome databases. Several inconsistencies in genome annotation were verified experimentally, and up to 55 candidates await further investigation. Our findings demonstrate how an up-to-date 2-D gel-based proteomics approach can be used for improving the annotation of prokaryotic genomes. They also highlight the need for harmonization among the different E. coli genome databases.

Phosphate starvation induces the sporulation killing factor of Bacillus subtilis.

Bacillus subtilis produces and exports a peptide sporulation killing factor (SkfA) that induces lysis of sibling cells. skfA is part of the skf operon (skfA-H), which is responsible for immunity to SkfA, as well as for production and export of SkfA. Here we report that transcription of skfA is markedly induced when cells of B. subtilis are subjected to phosphate starvation. The role of PhoP in regulation of the skf operon was confirmed by in vitro gel shift assays, which showed that this operon is a new member of the PhoP regulon. A putative stem-loop structure in the skfA-skfB intergenic region is proposed to act as a stabilizer of an skfA-specific transcript.

Genome-wide transcriptional analysis of the phosphate starvation stimulon of Bacillus subtilis.

Bacillus subtilis responds to phosphate starvation stress by inducing the PhoP and SigB regulons. While the PhoP regulon provides a specific response to phosphate starvation stress, maximizing the acquisition of phosphate (P(i)) from the environment and reducing the cellular requirement for this essential nutrient, the SigB regulon provides nonspecific resistance to stress by protecting essential cellular components, such as DNA and membranes. We have characterized the phosphate starvation stress response of B. subtilis at a genome-wide level using DNA macroarrays. A combination of outlier and cluster analyses identified putative new members of the PhoP regulon, namely, yfkN (2',3' cyclic nucleotide 2'-phosphodiesterase), yurI (RNase), yjdB (unknown), and vpr (extracellular serine protease). YurI is thought to be responsible for the nonspecific degradation of RNA, while the activity of YfkN on various nucleotide phosphates suggests that it could act on substrates liberated by YurI, which produces 3' or 5' phosphoribonucleotides. The putative new PhoP regulon members are either known or predicted to be secreted and are likely to be important for the recovery of inorganic phosphate from a variety of organic sources of phosphate in the environment.

Regulatory interactions between the Pho and sigma(B)-dependent general stress regulons of Bacillus subtilis.

When Bacillus subtilis is subjected to phosphate starvation, the Pho and sigma(B)-dependent general stress regulons are activated to elicit, respectively, specific and non-specific responses to this nutrient-limitation stress. A set of isogenic mutants, with a beta-galactosidase reporter gene transcriptionally fused to the inactivated target gene, was used to identify genes of unknown function that are induced or repressed under phosphate limitation. Nine phosphate-starvation-induced (psi) genes were identified: yhaX, yhbH, ykoL and yttP were regulated by the PhoP-PhoR two-component system responsible for controlling the expression of genes in the Pho regulon, while ywmG (renamed csbD), yheK, ykzA, ysnF and yvgO were dependent on the alternative sigma factor sigma(B), which controls the expression of the general stress genes. Genes yhaX and yhbH are unique members of the Pho regulon, since they are phosphate-starvation induced via PhoP-PhoR from a sporulation-specific sigma(E) promoter or a promoter that requires the product of a sigma(E)-dependent gene. Null mutations in key regulatory genes phoR and sigB showed that the Pho and sigma(B)-dependent general stress regulons of Bacillus subtilis interact to modulate the levels at which each are activated.

The signal peptidase II (Isp) gene of Bacillus subtilis.

The gene encoding the type II signal peptidase (SPase II) of Bacillus subtilis was isolated by screening a genomic DNA library of this bacterium for the ability to increase the levels of globomycin resistance in Escherichia coli, and to complement the growth deficiency at the non-permissive temperature of E. coli strain Y815 carrying a temperature-sensitive mutation in its Isp gene for SPase II. The deduced amino acid sequence of the B. subtilis SPase II showed significant similarity with those of other known SPase II enzymes. Activity of the B. subtilis SPase II was demonstrated by a pulse-labelling experiment in E. coli. In B. subtilis, the Isp gene is flanked by the isoleucyl-tRNA synthetase (ileS) gene and the pyrimidine biosynthetic (pyr) gene cluster, which is known to map at 139 degrees of the chromosome. In the Gram-positive bacteria studied thus far, Isp appears to be the first gene in an operon. The promoter-distal gene ("orf4') of this operon specifies a hypothetical protein in bacteria and yeast.

Transcriptional regulation of the phoPR operon in Bacillus subtilis.

When Bacillus subtilis is subjected to phosphate starvation, the Pho regulon is activated by the PhoP-PhoR two-component signal transduction system to elicit specific responses to this nutrient limitation. The response regulator, PhoP, and its cognate histidine sensor kinase, PhoR, are encoded by the phoPR operon that is transcribed as a 2.7-kb bicistronic mRNA. The phoPR operon is transcribed from two sigma(A)-dependent promoters, P(1) and P(2). Under conditions where the Pho regulon was not induced (i.e., phosphate-replete conditions or phoR-null mutant), a low level of phoPR transcription was detected only from promoter P(1). During phosphate starvation-induced transition from exponential to stationary phase, the expression of the phoPR operon was up-regulated in a phosphorylated PhoP (PhoP approximately P)-dependent manner; in addition to P(1), the P(2) promoter becomes active. In vitro gel shift assays and DNase I footprinting experiments showed that both PhoP and PhoP approximately P could bind to the control region of the phoPR operon. The data indicate that while low-level constitutive expression of phoPR is required under phosphate-replete conditions for signal perception and transduction, autoinduction is required to provide sufficient PhoP approximately P to induce other members of the Pho regulon. The extent to which promoters P(1) and P(2) are activated appears to be influenced by the presence of other sigma factors, possibly the result of sigma factor competition. For example, phoPR is hyperinduced in a sigB mutant and, later in stationary phase, in sigH, sigF, and sigE mutants. The data point to a complex regulatory network in which other stress responses and post-exponential-phase processes influence the expression of phoPR and, thereby, the magnitude of the Pho regulon response.

Production of Bacillus anthracis protective antigen is dependent on the extracellular chaperone, PrsA.

Protective antigen (PA) is a component of the Bacillus anthracis lethal and edema toxins and the basis of the current anthrax vaccine. In its heptameric form, PA targets host cells and internalizes the enzymatically active components of the toxins, namely lethal and edema factors. PA and other toxin components are secreted from B. anthracis using the Sec-dependent secretion pathway. This requires them to be translocated across the cytoplasmic membrane in an unfolded state and then to be folded into their native configurations on the trans side of the membrane, prior to their release from the environment of the cell wall. In this study we show that recombinant PA (rPA) requires the extracellular chaperone PrsA for efficient folding when produced in the heterologous host, B. subtilis; increasing the concentration of PrsA leads to an increase in rPA production. To determine the likelihood of PrsA being required for PA production in its native host, we have analyzed the B. anthracis genome sequence for the presence of genes encoding homologues of B. subtilis PrsA. We identified three putative B. anthracis PrsA proteins (PrsAA, PrsAB, and PrsAC) that are able to complement the activity of B. subtilis PrsA with respect to cell viability and rPA secretion, as well as that of AmyQ, a protein previously shown to be PrsA-dependent.

Fed-batch production of recombinant beta-galactosidase using the universal stress promoters uspA and uspB in high cell density cultivations.

A high-level production system using the universal stress promoters uspA and uspB in a fed-batch cultivation based on minimal medium was designed. In development it was shown that a standard industrial fed-batch protocol could not be used for this purpose since it failed to induce the levels of product as compared to the basal level. Instead, a batch protocol followed by a low constant feed of glucose was shown to give full induction. The levels of the product protein, beta-galactosidase, corresponded to approximately 25% of the total protein. Higher levels were found using the uspA than uspB vectors where uspA showed considerably higher basal level. The data indicate that the sigma(70) regulated promoter, uspA, although affected by the alarmone guanosine tetraphosphate, ppGpp, worked partly in a similar manner to constitutive promoters. An industrial high cell density fed-batch cultivation on the basis of the suggested fed-batch protocol and the uspA promoter gave a final beta-galatosidase concentration of 7 g/L and a final cell concentration of 65 g/L. The heterogeneity in production of the individual cell was measured by fluorescence microscopy. The data show that there is a process time independent heterogeneity in production, which is suggested to be caused by heterogeneity in the substrate uptake rate of the individual cell.

Post-transcriptional regulation of the Bacillus subtilis pst operon encoding a phosphate-specific ABC transporter.

During phosphate starvation, Bacillus subtilis regulates genes in the PhoP regulon to reduce the cell's requirement for this essential substrate and to facilitate the recovery of inorganic phosphate from organic sources such as teichoic and nucleic acids. Among the proteins that are highly induced under these conditions is PstS, the phosphate-binding lipoprotein component of a high-affinity ABC-type phosphate transporter. PstS is encoded by the first gene in the pst operon, the other four members of which encode the integral membrane and cytoplasmic components of the transporter. The transcription of the pst operon was analysed using a combination of methods, including transcriptional reporter gene technology, Northern blotting and DNA arrays. It is shown that the primary transcript of the pst operon is processed differentially to maintain higher concentrations of PstS relative to other components of the transporter. The comparative studies have revealed limitations in the use of reporter gene technology for analysing the transcription of operons in which the messenger RNA transcript is differentially processed.

Limiting factors in Escherichia coli fed-batch production of recombinant proteins.

Fed-batch production of recombinant beta-galactosidase in E. coli was studied with respect to the specific growth rate at induction. The cultivations were designed to induce protein production by IPTG at a glucose feed rate corresponding to high mu = 0.5 h(-1)) or low (mu = 0.1 h(-1)) specific growth rate. Protein production rate was approximately 100% higher at the higher specific growth rate, resulting in the accumulation of beta-galactosidase up to 30% of the total cell protein. Transcription analysis showed that beta-galactosidase-specific messenger RNA was immediately formed after induction (<5 min), but the amount was the same in both cases and was thus not the initial limiting factor. The content of ribosomes, as represented by rRNA, rapidly decreased with specific growth rate from a relative level of 100%, at the high specific growth rate, to 20% at the low specific growth rate. At high specific growth rate, ribosomes were additionally degraded upon induction due to the high production level. Translation therefore seemed to be the initial limiting factor of the protein synthesis capacity. The alarmone guanosine tetraphosphate increased at both high and low feed level inductions, indicating an induction-forced starvation of charged tRNA and/or glucose. The altered physiological status was also detected by the formation of acetic acid. However, the higher production rate resulted in high-level accumulation of acetic acid, which was absent at low feed rate production. Acetic acid production is thus coupled to the high product formation rate and is proposed to be due either to a precursor drain of Krebs cycle intermediates and a time lag before induction of the glyoxalate shunt, or to single amino acid overflow, since the model product is relatively poor in glycin and alanin. In conclusion, it is proposed that production at high specific growth rate becomes precursor-limited, while production at low specific growth rate is carbon- and/or energy-limited.