Metagenomic gut microbiome analysis of Japanese patients with multiple chemical sensitivity/idiopathic environmental intolerance.

BACKGROUND: Although the pathology of multiple chemical sensitivity (MCS) is unknown, the central nervous system is reportedly involved. The gut microbiota is important in modifying central nervous system diseases. However, the relationship　between the gut microbiota and MCS remains unclear. This study aimed to identify gut microbiota variations associated with MCS using shotgun metagenomic sequencing of fecal samples. METHODS: We prospectively recruited 30 consecutive Japanese female patients with MCS and analyzed their gut microbiomes using shotgun metagenomic sequencing. The data were compared with metagenomic data obtained from 24 age- and sex-matched Japanese healthy controls (HC). RESULTS: We observed no significant difference in alpha and beta diversity of the gut microbiota between the MCS patients and HC. Focusing on the important changes in the literatures, at the genus level, Streptococcus, Veillonella, and Akkermansia were significantly more abundant in MCS patients than in HC (p < 0.01, p < 0.01, p = 0.01, respectively, fold change = 4.03, 1.53, 2.86, respectively). At the species level, Akkermansia muciniphila was significantly more abundant (p = 0.02, fold change = 3.3) and Faecalibacterium prausnitzii significantly less abundant in MCS patients than in HC (p = 0.03, fold change = 0.53). Functional analysis revealed that xylene and dioxin degradation pathways were significantly enriched (p < 0.01, p = 0.01, respectively, fold change = 1.54, 1.46, respectively), whereas pathways involved in amino acid metabolism and synthesis were significantly depleted in MCS (p < 0.01, fold change = 0.96). Pathways related to antimicrobial resistance, including the two-component system and cationic antimicrobial peptide resistance, were also significantly enriched in MCS (p < 0.01, p < 0.01, respectively, fold change = 1.1, 1.2, respectively). CONCLUSIONS: The gut microbiota of patients with MCS shows dysbiosis and alterations in bacterial functions related to exogenous chemicals and amino acid metabolism and synthesis. These findings may contribute to the further development of treatment for MCS. TRIAL REGISTRATION: This study was registered with the University Hospital Medical Information Clinical Trials Registry as UMIN000031031. The date of first trial registration: 28/01/2018.

A novel sorting signal for RNA packaging into small extracellular vesicles.

Extracellular vesicles (EVs) play a critical role in the transport of functional RNAs to target recipient cells in numerous physiological processes. The RNA profiles present in EVs differed significantly from those in the originating cells, suggesting selective and active loading of specific RNAs into EVs. Small EVs (sEVs) obtained by stepwise ultracentrifugation have been reported to contain non-sEV components. Analysis of sEVs separated from non-sEVs components revealed that microRNAs may not be released by sEVs. This has raised interest in other RNA types, such as mRNA, which may be functional molecules released by sEVs. However, the molecular mechanisms underlying selective loading of mRNA into sEVs remain unclear. Here, we show that the part of 3' untranslated region (UTR) sequence of RAB13 selectively enriches RNA in sEVs and serves as an RNA signal for loading into sEVs. Our results demonstrate that RAB13 is the most enriched RNA in sEVs, and this enrichment is primarily driven by its 3'UTR sequence. These findings highlight the potential of the RAB13 3'UTR sequence as an RNA signal that enables the loading of target RNA into sEVs. This technology has the potential to improve EV-based drug delivery and other applications.

Greedy reduction of Bacillus subtilis genome yields emergent phenotypes of high resistance to a DNA damaging agent and low evolvability.

Genome-scale engineering enables rational removal of dispensable genes in chassis genomes. Deviating from this approach, we applied greedy accumulation of deletions of large dispensable regions in the Bacillus subtilis genome, yielding a library of 298 strains with genomes reduced up to 1.48 Mb in size. High-throughput physiological phenotyping of these strains confirmed that genome reduction is associated with substantial loss of cell fitness and accumulation of synthetic-sick interactions. Transcriptome analysis indicated that <15% of the genes conserved in our genome-reduced strains exhibited a twofold or higher differential expression and revealed a thiol-oxidative stress response. Most transcriptional changes can be explained by loss of known functions and by aberrant transcription at deletion boundaries. Genome-reduced strains exhibited striking new phenotypes relative to wild type, including a very high resistance (increased >300-fold) to the DNA-damaging agent mitomycin C and a very low spontaneous mutagenesis (reduced 100-fold). Adaptive laboratory evolution failed to restore cell fitness, except when coupled with a synthetic increase of the mutation rate, confirming low evolvability. Although mechanisms underlying this emergent phenotype are not understood, we propose that low evolvability can be leveraged in an engineering strategy coupling reductive cycles with evolutive cycles under induced mutagenesis.

The role of p63 in embryonic external genitalia outgrowth in mice.

Embryonic external genitalia (genital tubercle [GT]) protrude from the cloaca and outgrow as cloacal development progresses. Individual gene functions and knockout phenotypes in GT development have been extensively analyzed; however, the interactions between these genes are not fully understood. In this study, we investigated the role of p63, focusing on its interaction with the Shh-Wnt/Ctnnb1-Fgf8 pathway, a signaling network that is known to play a role in GT outgrowth. p63 was expressed in the epithelial tissues of the GT at E11.5, and the distal tip of the GT predominantly expressed the ΔNp63α isoform. The GTs in p63 knockout embryos had normal Shh expression, but CTNNB1 protein and Fgf8 gene expression in the distal urethral epithelium was decreased or lost. Constitutive expression of CTNNB1 in p63-null embryos restored Fgf8 expression, accompanied by small bud structure development; however, such bud structures could not be maintained by E13.5, at which point mutant GTs exhibited severe abnormalities showing a split shape with a hemorrhagic cloaca. Therefore, p63 is a key component of the signaling pathway that triggers Fgf8 expression in the distal urethral epithelium and contributes to GT outgrowth by ensuring the structural integrity of the cloacal epithelia. Altogether, we propose that p63 plays an essential role in the signaling network for the development of external genitalia.

Genome sequences of two strains of Lactococcus lactis subsp. cremoris with the same ancestry but a different capacity to produce exopolysaccharides.

Strains of Lactococcus lactis subsp. cremoris are used to produce yogurt containing exopolysaccharides with a sticky texture. When strain G3-2 producing exopolysaccharides was grown at elevated temperatures, a spontaneous mutant EPSC, which had lost exopolysaccharides biosynthesis, was isolated. Genomes of the two strains were determined to be composed of a 2.4-Mb chromosome and up to eleven plasmids, and it was revealed that one of the plasmids encoding the gene cluster for exopolysaccharides biosynthesis was lost selectively in EPSC.

Co-Inoculation of Bacillus velezensis Strain S141 and Bradyrhizobium Strains Promotes Nodule Growth and Nitrogen Fixation.

The objective of this research was to evaluate the PGPR effect on nodulation and nitrogen-fixing efficiency of soybean (Glycine max (L.) Merr.) by co-inoculation with Bradyrhizobium diazoefficiens USDA110. Co-inoculation of Bacillus velezensis S141 with USDA110 into soybean resulted in enhanced nodulation and N2-fixing efficiency by producing larger nodules. To understand the role of S141 on soybean and USDA110 symbiosis, putative genes related to IAA biosynthesis were disrupted, suggesting that co-inoculation of USDA110 with S141ΔyhcX reduces the number of large size nodules. It was revealed that yhcX may play a major role in IAA biosynthesis in S141 as well as provide a major impact on soybean growth promotion. The disruption of genes related to cytokinin biosynthesis and co-inoculation of USDA110 with S141ΔIPI reduced the number of very large size nodules, and it appears that IPI might play an important role in nodule size of soybean-Bradyrhizobium symbiosis. However, it was possible that not only IAA and cytokinin but also some other substances secreted from S141 facilitate Bradyrhizobium to trigger bigger nodule formation, resulting in enhanced N2-fixation. Therefore, the ability of S141 with Bradyrhizobium co-inoculation to enhance soybean N2-fixation strategy could be further developed for supreme soybean inoculants.

Complete Genome Sequence of Thermophilic Bacterium Aeribacillus pallidus PI8.

Here, we report the complete genome sequence of Aeribacillus pallidus PI8, a thermophilic bacterium, isolated from soybean stem extract. The sequence was determined using Illumina and Nanopore sequencers. Bioinformatic analyses of the genome sequence revealed the presence of possible bacteriocin gene clusters.

A bacterial cell factory converting glucose into scyllo-inositol, a therapeutic agent for Alzheimer's disease.

A rare stereoisomer of inositol, scyllo-inositol, is a therapeutic agent that has shown potential efficacy in preventing Alzheimer's disease. Mycobacterium tuberculosis ino1 encoding myo-inositol-1-phosphate (MI1P) synthase (MI1PS) was introduced into Bacillus subtilis to convert glucose-6-phosphate (G6P) into MI1P. We found that inactivation of pbuE elevated intracellular concentrations of NAD+·NADH as an essential cofactor of MI1PS and was required to activate MI1PS. MI1P thus produced was dephosphorylated into myo-inositol by an intrinsic inositol monophosphatase, YktC, which was subsequently isomerized into scyllo-inositol via a previously established artificial pathway involving two inositol dehydrogenases, IolG and IolW. In addition, both glcP and glcK were overexpressed to feed more G6P and accelerate scyllo-inositol production. Consequently, a B. subtilis cell factory was demonstrated to produce 2 g L-1 scyllo-inositol from 20 g L-1 glucose. This cell factory provides an inexpensive way to produce scyllo-inositol, which will help us to challenge the growing problem of Alzheimer's disease in our aging society.

SDR9C7 catalyzes critical dehydrogenation of acylceramides for skin barrier formation.

The corneocyte lipid envelope, composed of covalently bound ceramides and fatty acids, is important to the integrity of the permeability barrier in the stratum corneum, and its absence is a prime structural defect in various skin diseases associated with defective skin barrier function. SDR9C7 encodes a short-chain dehydrogenase/reductase family 9C member 7 (SDR9C7) recently found mutated in ichthyosis. In a patient with SDR9C7 mutation and a mouse Sdr9c7-KO model, we show loss of covalent binding of epidermal ceramides to protein, a structural fault in the barrier. For reasons unresolved, protein binding requires lipoxygenase-catalyzed transformations of linoleic acid (18:2) esterified in ω-O-acylceramides. In Sdr9c7-/- epidermis, quantitative liquid chromatography-mass spectometry (LC-MS) assays revealed almost complete loss of a species of ω-O-acylceramide esterified with linoleate-9,10-trans-epoxy-11E-13-ketone; other acylceramides related to the lipoxygenase pathway were in higher abundance. Recombinant SDR9C7 catalyzed NAD+-dependent dehydrogenation of linoleate 9,10-trans-epoxy-11E-13-alcohol to the corresponding 13-ketone, while ichthyosis mutants were inactive. We propose, therefore, that the critical requirement for lipoxygenases and SDR9C7 is in producing acylceramide containing the 9,10-epoxy-11E-13-ketone, a reactive moiety known for its nonenzymatic coupling to protein. This suggests a mechanism for coupling of ceramide to protein and provides important insights into skin barrier formation and pathogenesis.

Bradyrhizobium diazoefficiens USDA110 PhaR functions for pleiotropic regulation of cellular processes besides PHB accumulation.

BACKGROUND: Bradyrhizobium diazoefficiens USDA110 nodulates soybeans for nitrogen fixation. It accumulates poly-3-hydroxybutyrate (PHB), which is of physiological importance as a carbon/energy source for survival during starvation, infection, and nitrogen fixation conditions. PHB accumulation is orchestrated by not only the enzymes for PHB synthesis but also PHB-binding phasin proteins (PhaPs) stabilizing the PHB granules. The transcription factor PhaR controls the phaP genes. RESULTS: Inactivation of phaR led to decreases in PHB accumulation, less cell yield, increases in exopolysaccharide (EPS) production, some improvement in heat stress tolerance, and slightly better growth under microaerobic conditions. Changes in the transcriptome upon phaR inactivation were analyzed. PhaR appeared to be involved in the repression of various target genes, including some PHB-degrading enzymes and others involved in EPS production. Furthermore, in vitro gel shift analysis demonstrated that PhaR bound to the promoter regions of representative targets. For the phaP1 and phaP4 promoter regions, PhaR-binding sites were determined by DNase I footprinting, allowing us to deduce a consensus sequence for PhaR-binding as TGCRNYGCASMA (R: A or G, Y: C or T, S: C or G, M: A or C). We searched for additional genes associated with a PhaR-binding sequence and found that some genes involved in central carbon metabolism, such as pdhA for pyruvate dehydrogenase and pckA for phosphoenolpyruvate carboxykinase, may be regulated positively and directly by PhaR. CONCLUSIONS: These results suggest that PhaR could regulate various genes not only negatively but also positively to coordinate metabolism holistically in response to PHB accumulation.

A novel method for transforming the thermophilic bacterium Geobacillus kaustophilus.

BACKGROUND: Bacterial strains of the genus Geobacillus grow at high temperatures of 50-75 °C and could thus be useful for biotechnological applications. However, genetic manipulation of these species is difficult because the current techniques for transforming Geobacillus species are not efficient. In this study, we developed an easy and efficient method for transforming Geobacillus kaustophilus using the conjugative plasmid pLS20cat. RESULTS: We constructed a transformation system comprising (i) a mobilizable Bacillus subtilis-G. kaustophilus shuttle plasmid named pGK1 that carries the elements for selection and replication in Geobacillus, and (ii) a pLS20cat-harboring B. subtilis donor strain expressing the dam methylase gene of Escherichia coli and the conjugation-stimulating rapLS20 gene of pLS20cat. This system can be used to efficiently introduce pGK1 into G. kaustophilus by mobilization in a pLS20cat-dependent way. Whereas the thermostable kanamycin marker and Geobacillus replication origin of pGK1 as well as expression of dam methylase in the donor were indispensable for mobilization, ectopic expression of rapLS20 increased its efficiency. In addition, the conditions of the recipient influenced mobilization efficiency: the highest mobilization efficiencies were obtained using recipient cells that were in the exponential growth phase. Furthermore, elimination of the origin of transfer from pLS20cat enhanced the mobilization. CONCLUSIONS: We describe a novel method of plasmid mobilization into G. kaustophilus recipient from B. subtilis donor depending on the helper function of pLS20cat, which enables simple, rapid, and easy transformation of the thermophilic Gram-positive bacterium.

Rapid conjugative mobilization of a 100 kb segment of Bacillus subtilis chromosomal DNA is mediated by a helper plasmid with no ability for self-transfer.

BACKGROUND: The conjugative plasmid, pLS20, isolated from Bacillus subtilis natto, has an outstanding capacity for rapid self-transfer. In addition, it can function as a helper plasmid, mediating the mobilization of an independently replicating co-resident plasmid. RESULTS: In this study, the oriT sequence of pLS20cat (oriTLS20) was eliminated to obtain the plasmid, pLS20catΔoriT. This resulted in the complete loss of the conjugative transfer of the plasmid but still allowed it to mobilize a co-resident mobilizable plasmid. Moreover, pLS20catΔoriT was able to mobilize longer DNA segments, up to 113 kb of chromosomal DNA containing oriTLS20, after mixing the liquid cultures of the donor and recipient for only 15 min. CONCLUSIONS: The chromosomal DNA mobilization mediated by pLS20catΔoriT will allow us to develop a novel genetic tool for the rapid, easy, and repetitive mobilization of longer DNA segments into a recipient chromosome.

Characterization and mutation analysis of a halotolerant serine protease from a new isolate of Bacillus subtilis.

OBJECTIVES: A bacterial halotolerant enzyme was characterized to understand the molecular mechanism of salt adaptation and to explore its protein engineering potential. RESULTS: Halotolerant serine protease (Apr_No16) from a newly isolated Bacillus subtilis strain no. 16 was characterized. Multiple alignments with previously reported non-halotolerant proteases, including subtilisin Carlsberg, indicated that Apr_No16 has eight acidic or polar amino acid residues that are replaced by nonpolar amino acids in non-halotolerant proteases. Those residues were hypothesized to be one of the primary contributors to salt adaptation. An eightfold mutant substituted with Ala residues exhibited 1.2- and 1.8-fold greater halotolerance at 12.5% (w/v) NaCl than Apr_No16 and Carlsberg, respectively. Amino acid substitution notably shifted the theoretical pI of the eightfold mutant, from 6.33 to 9.23, compared with Apr_No16. The resulting protein better tolerated high salt conditions. CONCLUSIONS: Changing the pI of a bacterial serine protease may be an effective strategy to improve the enzyme's halotolerance.

Genome Sequence of Bacillus velezensis S141, a New Strain of Plant Growth-Promoting Rhizobacterium Isolated from Soybean Rhizosphere.

Bacillus velezensis strain S141 is a plant growth-promoting rhizobacterium isolated from soybean (Glycine max) rhizosphere that enhances soybean growth, nodulation, and N2 fixation efficiency by coinoculation with Bradyrhizobium diazoefficiens USDA110. The S141 genome was identified to comprise a 3,974,582-bp-long circular DNA sequence encoding at least 3,817 proteins.

Homology modeling and prediction of the amino acid residues participating in the transfer of acetyl-CoA to arylalkylamine by the N-acetyltransferase from Chryseobacterium sp.

OBJECTIVES: To predict the amino acid residues playing important roles in acetyl-CoA and substrate binding and to study the acetyl group transfer mechanism of Chryseobacterium sp. 5-3B N-acetyltransferase (5-3B NatA). RESULTS: A 3-dimensional homology model of 5-3B NatA was constructed to compare the theoretical structure of this compound with the structures of previously reported proteins belonging to the bacterial GCN5 N-acetyltransferase family. Homology modeling of the 5-3B NatA structure and a characterization of the enzyme's kinetic parameters identified the essential amino acid residues involved in binding and acetyl-group transfer. 126Leu, 132Leu, and 135Lys were implicated in the binding of phosphopantothenic acid, and 100Tyr and 131Lys in that of adenosyl biphosphate. The data supported the participation of 83Glu and 133Tyr in catalyzing acetyl-group transfer to L-2-phenylglycine. CONCLUSIONS: 5-3B NatA catalyzes the enantioselective N-acetylation of L-2-phenylglycine via a ternary complex comprising the enzyme, acetyl-CoA, and the substrate.

Isolation and identification of Bifidobacterium species from feces of captive chimpanzees.

Recently, gut-dwelling bifidobacteria from chimpanzees, which are phylogenetically close to humans and have feeding habits similar to humans, have been frequently investigated. Given this, we speculated that like humans, chimpanzees would have a unique diversity of bifidobacteria. We herein describe a taxonomically novel member of bifidobacteria isolated from fecal samples of captive chimpanzees. Bifidobacteria were detected in all fecal samples by quantitative polymerase chain reaction. A Bifidobacterium pseudolongum-like species, which could not be detected using B. pseudolongum-specific primers targeting the groEL gene sequence, was dominant in the feces of five chimpanzees. Seven bifidobacterial strains were isolated from this group of five chimpanzees, and all isolates were identified as B. pseudolongum. B. pseudolongum has previously often been isolated from non-primate animals as well as humans; however, here we demonstrate its presence in a nonhuman primate species.

Taurine does not affect the composition, diversity, or metabolism of human colonic microbiota simulated in a single-batch fermentation system.

Accumulating evidence suggests that dietary taurine (2-aminoethanesulfonic acid) exerts beneficial anti-inflammatory effects in the large intestine. In this study, we investigated the possible impact of taurine on human colonic microbiota using our single-batch fermentation system (Kobe University Human Intestinal Microbiota Model; KUHIMM). Fecal samples from eight humans were individually cultivated with and without taurine in the KUHIMM. The results showed that taurine remained largely undegraded after 30 h of culturing in the absence of oxygen, although some 83% of the taurine was degraded after 30 h of culturing under aerobic conditions. Diversity in bacterial species in the cultures was analyzed by 16S rRNA gene sequencing, revealing that taurine caused no significant change in the diversity of the microbiota; both operational taxonomic unit and Shannon-Wiener index of the cultures were comparable to those of the respective source fecal samples. In addition, principal coordinate analysis indicated that taurine did not alter the composition of bacterial species, since the 16S rRNA gene profile of bacterial species in the original fecal sample was maintained in each of the cultures with and without taurine. Furthermore, metabolomic analysis revealed that taurine did not affect the composition of short-chain fatty acids produced in the cultures. These results, under these controlled but artificial conditions, suggested that the beneficial anti-inflammatory effects of dietary taurine in the large intestine are independent of the intestinal microbiota. We infer that dietary taurine may act directly in the large intestine to exert anti-inflammatory effects.

Bacillus subtilis IolQ (DegA) is a transcriptional repressor of iolX encoding NAD+-dependent scyllo-inositol dehydrogenase.

BACKGROUND: Bacillus subtilis is able to utilize at least three inositol stereoisomers as carbon sources, myo-, scyllo-, and D-chiro-inositol (MI, SI, and DCI, respectively). NAD+-dependent SI dehydrogenase responsible for SI catabolism is encoded by iolX. Even in the absence of functional iolX, the presence of SI or MI in the growth medium was found to induce the transcription of iolX through an unknown mechanism. RESULTS: Immediately upstream of iolX, there is an operon that encodes two genes, yisR and iolQ (formerly known as degA), each of which could encode a transcriptional regulator. Here we performed an inactivation analysis of yisR and iolQ and found that iolQ encodes a repressor of the iolX transcription. The coding sequence of iolQ was expressed in Escherichia coli and the gene product was purified as a His-tagged fusion protein, which bound to two sites within the iolX promoter region in vitro. CONCLUSIONS: IolQ is a transcriptional repressor of iolX. Genetic evidences allowed us to speculate that SI and MI might possibly be the intracellular inducers, however they failed to antagonize DNA binding of IolQ in in vitro experiments.

Biotransformation of ferulic acid to protocatechuic acid by Corynebacterium glutamicum ATCC 21420 engineered to express vanillate O-demethylase.

Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA) is a lignin-derived phenolic compound abundant in plant biomass. The utilization of FA and its conversion to valuable compounds is desired. Protocatechuic acid (3,4-dihydroxybenzoic acid, PCA) is a precursor of polymers and plastics and a constituent of food. A microbial conversion system to produce PCA from FA was developed in this study using a PCA-producing strain of Corynebacterium glutamicum F (ATCC 21420). C. glutamicum strain F grown at 30 °C for 48 h utilized 2 mM each of FA and vanillic acid (4-hydroxy-3-methoxybenzoic acid, VA) to produce PCA, which was secreted into the medium. FA may be catabolized by C. glutamicum through proposed (I) non-ß-oxidative, CoA-dependent or (II) ß-oxidative, CoA-dependent phenylpropanoid pathways. The conversion of VA to PCA is the last step in each pathway. Therefore, the vanillate O-demethylase gene (vanAB) from Corynebacterium efficiens NBRC 100395 was expressed in C. glutamicum F (designated strain FVan) cultured at 30 °C in AF medium containing FA. Strain C. glutamicum FVan converted 4.57 ± 0.07 mM of FA into 2.87 ± 0.01 mM PCA after 48 h with yields of 62.8% (mol/mol), and 6.91 mM (1064 mg/L) of PCA was produced from 16.0 mM of FA after 12 h of fed-batch biotransformation. Genomic analysis of C. glutamicum ATCC 21420 revealed that the PCA-utilization genes (pca cluster) were conserved in strain ATCC 21420 and that mutations were present in the PCA importer gene pcaK.

A new-generation of Bacillus subtilis cell factory for further elevated scyllo-inositol production.

BACKGROUND: A stereoisomer of inositol, scyllo-inositol (SI), has been regarded as a promising therapeutic agent for Alzheimer's disease. However, this compound is relatively rare, whereas another stereoisomer of inositol, myo-inositol (MI) is abundant in nature. Bacillus subtilis 168 has the ability to metabolize inositol stereoisomers, including MI and SI. Previously, we reported a B. subtilis cell factory with modified inositol metabolism that converts MI into SI in the culture medium. The strain was constructed by deleting all genes related to inositol metabolism and overexpressing key enzymes, IolG and IolW. By using this strain, 10 g/l of MI initially included in the medium was completely converted into SI within 48 h of cultivation in a rich medium containing 2% (w/v) Bacto soytone. RESULTS: When the initial concentration of MI was increased to 50 g/l, conversion was limited to 15.1 g/l of SI. Therefore, overexpression systems of IolT and PntAB, the main transporter of MI in B. subtilis and the membrane-integral nicotinamide nucleotide transhydrogenase in Escherichia coli respectively, were additionally introduced into the B. subtilis cell factory, but the conversion efficiency hardly improved. We systematically determined the amount of Bacto soytone necessary for ultimate conversion, which was 4% (w/v). As a result, the conversion of SI reached to 27.6 g/l within 48 h of cultivation. CONCLUSIONS: The B. subtilis cell factory was improved to yield a SI production rate of 27.6 g/l/48 h by simultaneous overexpression of IolT and PntAB, and by addition of 4% (w/v) Bacto soytone in the conversion medium. The concentration of SI was increased even in the stationary phase perhaps due to nutrients in the Bacto soytone that contribute to the conversion process. Thus, MI conversion to SI may be further optimized via identification and control of these unknown nutrients.