ABSTRACT
BACKGROUND: Computational mining of useful enzymes and biosynthesis pathways is a powerful strategy for metabolic engineering. Through systematic exploration of all conceivable combinations of enzyme reactions, including both known compounds and those inferred from the chemical structures of established reactions, we can uncover previously undiscovered enzymatic processes. The application of the novel alternative pathways enables us to improve microbial bioproduction by bypassing or reinforcing metabolic bottlenecks. Benzylisoquinoline alkaloids (BIAs) are a diverse group of plant-derived compounds with important pharmaceutical properties. BIA biosynthesis has developed into a prime example of metabolic engineering and microbial bioproduction. The early bottleneck of BIA production in Escherichia coli consists of 3,4-dihydroxyphenylacetaldehyde (DHPAA) production and conversion to tetrahydropapaveroline (THP). Previous studies have selected monoamine oxidase (MAO) and DHPAA synthase (DHPAAS) to produce DHPAA from dopamine and oxygen; however, both of these enzymes produce toxic hydrogen peroxide as a byproduct. RESULTS: In the current study, in silico pathway design is applied to relieve the bottleneck of DHPAA production in the synthetic BIA pathway. Specifically, the cytochrome P450 enzyme, tyrosine N-monooxygenase (CYP79), is identified to bypass the established MAO- and DHPAAS-mediated pathways in an alternative arylacetaldoxime route to DHPAA with a peroxide-independent mechanism. The application of this pathway is proposed to result in less formation of toxic byproducts, leading to improved production of reticuline (up to 60 mg/L at the flask scale) when compared with that from the conventional MAO pathway. CONCLUSIONS: This study showed improved reticuline production using the bypass pathway predicted by the M-path computational platform. Reticuline production in E. coli exceeded that of the conventional MAO-mediated pathway. The study provides a clear example of the integration of pathway mining and enzyme design in creating artificial metabolic pathways and suggests further potential applications of this strategy in metabolic engineering.
Subject(s)
Benzylisoquinolines , Escherichia coli , Metabolic Engineering , Metabolic Engineering/methods , Benzylisoquinolines/metabolism , Escherichia coli/metabolism , Escherichia coli/genetics , Cytochrome P-450 Enzyme System/metabolism , Biosynthetic Pathways , Computer Simulation , Tetrahydropapaveroline/metabolism , 3,4-Dihydroxyphenylacetic Acid/metabolism , 3,4-Dihydroxyphenylacetic Acid/analogs & derivativesABSTRACT
Advancements in synthetic biology have facilitated the microbial production of valuable plant metabolites. However, constructing complete biosynthetic pathways within a single host organism remains challenging. To solve this problem, modular co-culture systems involving host organisms with partial pathways have been developed. We focused on Escherichia coli, a general host for metabolite production, and Pichia pastoris (Komagataella phaffii), a novel synthetic biology host due to its high expression of biosynthetic enzymes. Previously, we reported the co-culture of E. coli cells, which produce reticuline (an important intermediate for various alkaloids) from glycerol, with P. pastoris cells, which produce the valuable alkaloid stylopine from reticuline. However, Pichia cells inhibited E. coli growth and reticuline production. Therefore, we aimed to improve this co-culture system. We investigated the pre-culture time before co-culture to enhance E. coli growth and reticuline production. Additionally, we examined the optimal concentration of Pichia cells inoculated for co-culture and methanol addition during co-culture for the continuous expression of biosynthetic enzymes in Pichia cells. We successfully established an improved co-culture system that exhibited an 80-fold increase in productivity compared to previous methods. This enhanced system holds great potential for the rapid and large-scale production of various valuable plant metabolites.
Subject(s)
Escherichia coli , Pichia , Escherichia coli/genetics , Coculture Techniques , Pichia/genetics , Recombinant Proteins/metabolismABSTRACT
Colonic luminal aromatic amines have been historically considered to be derived from dietary source, especially fermented foods; however, recent studies indicate that the gut microbiota serves as an alternative source of these amines. Herein, we show that five prominent genera of Firmicutes (Blautia, Clostridium, Enterococcus, Ruminococcus, and Tyzzerella) have the ability to abundantly produce aromatic amines through the action of aromatic amino acid decarboxylase (AADC). In vitro cultivation of human fecal samples revealed that a significant positive correlation between aadc copy number of Ruminococcus gnavus and phenylethylamine (PEA) production. Furthermore, using genetically engineered Enterococcus faecalis-colonized BALB/cCrSlc mouse model, we showed that the gut bacterial aadc stimulates the production of colonic serotonin, which is reportedly involved in osteoporosis and irritable bowel syndrome. Finally, we showed that human AADC inhibitors carbidopa and benserazide inhibit PEA production in En. faecalis.
Subject(s)
Carbidopa , Gastrointestinal Microbiome , Animals , Aromatic-L-Amino-Acid Decarboxylases/genetics , Aromatic-L-Amino-Acid Decarboxylases/metabolism , Benserazide/pharmacology , Humans , Mice , Phenethylamines , Serotonin/metabolismABSTRACT
Transporters have been used in the production of plant metabolites in microorganisms. This study introduced a tobacco multidrug and toxic compound extrusion transporter, NtJAT1, into alkaloid-producing Escherichia coli cells. NtJAT1 expression enhanced alkaloid production secretion into the medium by 14 folds. Our findings further demonstrate the usefulness of the transport-engineering approach.
Subject(s)
Alkaloids , Nicotiana , Alkaloids/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Plants/metabolism , Nicotiana/genetics , Nicotiana/metabolismABSTRACT
Peptidyl-prolyl cis-trans isomerase (PPIase, EC 5.2.1.8) catalyzes the racemization reaction of proline residues on a polypeptide chain. This enzyme is also known to function as a molecular chaperon to stabilize protein conformation during the folding process. In this study, we noted FK506 binding protein (FKBP)-type PPIase from a hyperthemophilic archaeon Thermococcus sp. strain KS-1 (PPIase KS-1) to improve the solubility of Pseudomonas putida aromatic amino acid decarboxylase (AADC) that is an indispensable enzyme for fermentative production of plant isoquinoline alkaloids. AADC fused N-terminally with the PPIase KS-1 (PPIase KS-1-AADC), which was synthesized utilizing Escherichia coli host, showed improved solubility and, consequently, the cell-free extract from the recombinant strain exhibited 2.6- to 3.4-fold elevated AADC activity than that from the control strain that expressed the AADC gene without PPIase KS-1. On the other hand, its thermostability was slightly decreased by fusing PPIase KS-1. The recombinant E. coli cells expressing the PPIase KS-1-AADC gene produced dopamine and phenylethylamine from L-dopa and phenylalanine by two- and threefold faster, respectively, as compared with the control strain. We further demonstrated that the efficacy of PPIase KS-1-AADC in solubility and activity enhancement was a little but obviously higher than that of AADC fused N-terminally with NusA protein, which has been assumed to be the most effective protein solubilizer. These results suggest that PPIase KS-1 can be used as one of the best choices for producing heterologous proteins as active forms in E. coli.
ABSTRACT
BACKGROUND: Plants produce a variety of specialized metabolites, many of which are used in pharmaceutical industries as raw materials. However, certain metabolites may be produced at markedly low concentrations in plants. This problem has been overcome through metabolic engineering in recent years, and the production of valuable plant compounds using microorganisms such as Escherichia coli or yeast cells has been realized. However, the development of complicated pathways in a single cell remains challenging. Additionally, microbial cells may experience toxicity from the bioactive compounds produced or negative feedback effects exerted on their biosynthetic enzymes. Thus, co-culture systems, such as those of E. coli-E. coli and E. coli-Saccharomyces cerevisiae, have been developed, and increased production of certain compounds has been achieved. Recently, a co-culture system of Pichia pastoris (Komagataella phaffii) has gained considerable attention due to its potential utility in increased production of valuable compounds. However, its co-culture with other organisms such as E. coli, which produce important intermediates at high concentrations, has not been reported. RESULTS: Here, we present a novel co-culture platform for E. coli and P. pastoris. Upstream E. coli cells produced reticuline from a simple carbon source, and the downstream P. pastoris cells produced stylopine from reticuline. We investigated the effect of four media commonly used for growth and production of P. pastoris, and found that buffered methanol-complex medium (BMMY) was suitable for P. pastoris cells. Reticuline-producing E. coli cells also showed better growth and reticuline production in BMMY medium than that in LB medium. De novo production of the final product, stylopine from a simple carbon source, glycerol, was successful upon co-culture of both strains in BMMY medium. Further analysis of the initial inoculation ratio showed that a higher ratio of E. coli cells compared to P. pastoris cells led to higher production of stylopine. CONCLUSIONS: This is the first report of co-culture system established with engineered E. coli and P. pastoris for the de novo production of valuable compounds. The co-culture system established herein would be useful for increased production of heterologous biosynthesis of complex specialized plant metabolites.
Subject(s)
Coculture Techniques/methods , Escherichia coli/growth & development , Metabolic Engineering/methods , Saccharomycetales/growth & developmentABSTRACT
Microorganisms can be metabolically engineered to produce specialized plant metabolites. However, these methods are limited by low productivity and intracellular accumulation of metabolites. We sought to use transport engineering for producing reticuline, an important intermediate in the alkaloid biosynthetic pathway. In this study, we established a reticuline-producing Escherichia coli strain into which the multidrug and toxic compound extrusion transporter Arabidopsis AtDTX1 was introduced. AtDTX1 was selected due to its suitable expression in E. coli and its reticuline-transport activity. Expression of AtDTX1 enhanced reticuline production by 11-fold, and the produced reticuline was secreted into the medium. AtDTX1 expression also conferred high plasmid stability and resulted in upregulation or downregulation of several genes associated with biological processes, including metabolic pathways for reticuline biosynthesis, leading to the production and secretion of high levels of reticuline. The successful employment of a transporter for alkaloid production suggests that the proposed transport engineering approach may improve the biosynthesis of specialized metabolites via metabolic engineering.
ABSTRACT
We have constructed an Escherichia coli-based platform producing (S)-reticuline, an important intermediate of benzylisoquinoline alkaloids (BIAs), using up to 14 genes. (S)-reticuline was produced from a simple carbon source such as glucose and glycerol via L-DOPA, which is synthesized by hydroxylation of L-tyrosine, one of the rate-limiting steps of the reaction. There are three kinds of enzymes catalyzing tyrosine hydroxylation: tyrosinase (TYR), tyrosine hydroxylase (TH), and 4-hydroxyphenylacetate 3-monooxygenase (HpaBC). Here, to further improve (S)-reticuline production, we chose eight from these three kinds of tyrosine hydroxylation enzymes (two TYRs, four THs, and two HpaBCs) derived from various organisms, and examined which enzyme was optimal for (S)-reticuline production in E. coli. TH from Drosophila melanogaster was the most suitable for (S)-reticuline production under the experimental conditions tested. We improved the productivity by genome integration of a gene set for L-tyrosine overproduction, introducing the regeneration pathway of BH4, a cofactor of TH, and methionine addition to enhance the S-adenosylmethionine supply. As a result, the yield of (S)-reticuline reached up to 384 µM from glucose in laboratory-scale shake flask. Furthermore, we found three inconsistent phenomena: an inhibitory effect due to additional gene expression, conflicts among the experimental conditions, and interference of an upstream enzyme from an additional downstream enzyme. Based on these results, we discuss future perspectives and challenges of integrating multiple enzyme genes for material production using microbes. Graphical abstract The optimal tyrosine hydroxylation enzyme for (S)-reticuline production in Escherichia coli KEY POINTS: ⢠There are three types of enzymes catalyzing tyrosine hydroxylation reaction: tyrosinase, tyrosine hydroxylase, and 4-hydroxyphenylacetate 3-monooxygenase. ⢠Tyrosine hydroxylase from Drosophila melanogaster exhibited the highest activity and was suitable for (S)-reticuline production in E. coli. ⢠New insights were provided on constructing an alkaloid production system with multi-step reactions in E. coli.
Subject(s)
Benzylisoquinolines , Escherichia coli , Animals , Drosophila melanogaster , Escherichia coli/genetics , Escherichia coli/metabolism , Hydroxylation , Tyrosine/metabolismABSTRACT
Natural products from plants are useful as lead compounds in drug discovery. Plant benzylisoquinoline alkaloids (BIAs) exhibit various pharmaceutical activities. Although unidentified BIAs are expected to be of medicinal value, sufficient quantities of such BIAs, for biological assays, are sometimes difficult to obtain due to their low content in natural sources. Here, we showed that high productivity of BIAs in engineered Escherichia coli could be exploited for drug discovery. First, we improved upon the previous microbial production system producing (S)-reticuline, an important BIA intermediate, to obtain yields of around 160 mg/L, which was 4-fold higher than those of the previously reported highest production system. Subsequently, we synthesised non-natural BIAs (O-sulphated (S)-reticulines) by introducing human sulphotransferases into the improved (S)-reticuline production system. Analysis of human primary cells treated with these BIAs demonstrated that they affected a biomarker expression in a manner different from that by the parent compound (S)-reticuline, suggesting that simple side-chain modification altered the characteristic traits of BIA. These results indicated that highly productive microbial systems might facilitate the production of scarce or novel BIAs and enable subsequent evaluation of their biological activities. The system developed here could be applied to other rare natural products and might contribute to the drug-discovery process as a next-generation strategy.
Subject(s)
Alkaloids/biosynthesis , Drug Discovery , Escherichia coli/metabolism , Metabolic Engineering/methods , Sulfates/metabolism , Alkaloids/metabolism , Animals , Benzylisoquinolines/metabolism , Drosophila melanogaster/enzymology , Drosophila melanogaster/genetics , Escherichia coli/genetics , Organisms, Genetically Modified , Tyrosine 3-Monooxygenase/genetics , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Regio- and 1,2- cis-stereoselective chemical glycosylation of unprotected glycosyl acceptors has been in great demand for the efficient synthesis of natural glycosides. However, simultaneously regulating these selectivities has been a longstanding problem in synthetic organic chemistry. In nature, glycosyl transferases catalyze regioselective 1,2- cis-glycosylations via the SNi mechanism, yet no useful chemical glycosylations based on this mechanism have been developed. In this paper, we report a highly regio- and 1,2- cis-stereoselective SNi-type glycosylation of 1,2-anhydro donors and unprotected sugar acceptors using p-nitrophenylboronic acid (10e) as a catalyst in the presence of water under mild conditions. Highly controlled regio- and 1,2- cis-stereoselectivities were achieved via the combination of boron-mediated carbohydrate recognition and the SNi-type mechanism. Mechanistic studies using the KIEs and DFT calculations were consistent with a highly dissociative concerted SNi mechanism. This glycosylation method was applied successfully to the direct glycosylation of unprotected natural glycosides and the efficient synthesis of a complex oligosaccharide with minimal protecting groups.
ABSTRACT
Recently, a "human gut microbial gene catalogue," which ranks the dominance of microbe genus/species in human fecal samples, was published. Most of the bacteria ranked in the catalog are currently publicly available; however, the growth media recommended by the distributors vary among species, hampering physiological comparisons among the bacteria. To address this problem, we evaluated Gifu anaerobic medium (GAM) as a standard medium. Forty-four publicly available species of the top 56 species listed in the "human gut microbial gene catalogue" were cultured in GAM, and out of these, 32 (72%) were successfully cultured. Short-chain fatty acids from the bacterial culture supernatants were then quantified, and bacterial metabolic pathways were predicted based on in silico genomic sequence analysis. Our system provides a useful platform for assessing growth properties and analyzing metabolites of dominant human gut bacteria grown in GAM and supplemented with compounds of interest.
Subject(s)
Bacteria/growth & development , Bacteria/metabolism , Fatty Acids, Volatile/metabolism , Fermentation , Gastrointestinal Microbiome , Anaerobiosis , Bacteria/genetics , Computer Simulation , Culture Techniques , DNA, Bacterial/genetics , GenomicsABSTRACT
Benzylisoquinoline alkaloids (BIAs) are a group of plant secondary metabolites that have been identified as targets for drug discovery because of their diverse pharmaceutical activities. Well-known BIAs are relatively abundant in plants and have therefore been extensively studied. However, although unknown BIAs are also thought to have valuable activities, they are difficult to obtain because the raw materials are present at low abundance in nature. We have previously reported the fermentative production of an important intermediate (S)-reticuline from dopamine using Escherichia coli. However, the yield is typically limited. Here, we improved production efficiency by combining in vivo tetrahydropapaveroline production in E. coli with in vitro enzymatic synthesis of (S)-reticuline. Finally, 593 mg of pure (S)-reticuline was obtained from 1 L of the reaction mixture. Because this bacterial-based method is simple, it could be widely used for production of (S)-reticuline and related BIAs, thereby facilitating studies of BIAs for drug discovery.
Subject(s)
Benzylisoquinolines/chemistry , Bioreactors/microbiology , Escherichia coli/metabolism , Laboratories , Benzylisoquinolines/metabolism , Dopamine/metabolism , Tetrahydropapaveroline/metabolismABSTRACT
Opiates such as morphine and codeine are mainly obtained by extraction from opium poppies. Fermentative opiate production in microbes has also been investigated, and complete biosynthesis of opiates from a simple carbon source has recently been accomplished in yeast. Here we demonstrate that Escherichia coli serves as an efficient, robust and flexible platform for total opiate synthesis. Thebaine, the most important raw material in opioid preparations, is produced by stepwise culture of four engineered strains at yields of 2.1 mg l(-1) from glycerol, corresponding to a 300-fold increase from recently developed yeast systems. This improvement is presumably due to strong activity of enzymes related to thebaine synthesis from (R)-reticuline in E. coli. Furthermore, by adding two genes to the thebaine production system, we demonstrate the biosynthesis of hydrocodone, a clinically important opioid. Improvements in opiate production in this E. coli system represent a major step towards the development of alternative opiate production systems.
Subject(s)
Analgesics, Opioid/metabolism , Escherichia coli/genetics , Fermentation , Organisms, Genetically Modified/genetics , Papaver/genetics , Thebaine/metabolism , Acetyltransferases/genetics , Benzylisoquinolines/metabolism , Codeine/biosynthesis , Coptis/genetics , Escherichia coli/metabolism , Glycerol/metabolism , Hydrocodone/metabolism , Methyltransferases/genetics , Methyltransferases/metabolism , Morphine/biosynthesis , Organisms, Genetically Modified/metabolism , Oxidoreductases/genetics , Oxycodone/metabolismABSTRACT
Sake is made from steamed rice, malted rice, and water. Sake production begins with the preparation of a small-scale starter (moto); the quality of moto significantly influences the flavor and richness of sake. In the traditional starter, yamahai-moto, the growth of naturally occurring lactic acid bacteria represses the putrefactive micro-organisms, whereas in the modern starter, sokujo-moto, this is achieved by adding lactic acid. In this study, the successive change in bacterial flora of yamahai-moto was analyzed by pyrosequencing 16S ribosomal RNA genes. Lactobacillus was dominant throughout the process (93-98%). Nitrate-reducing bacteria that have been generally assumed to be the first colonizers of yamahai-moto were scarcely found in the early stage, but Lactobacillus acidipiscis dominated. Lactobacillus sakei drastically increased in the middle stage. This is the first report, though one case study, to show how the early stage microbiota in Japanese yamahai-moto is varyingly controlled without nitrate-reducing bacteria using next-generation sequencing.
Subject(s)
Alcoholic Beverages/microbiology , Food Microbiology , Lactobacillaceae/genetics , Microbiota/genetics , Oryza/metabolism , Phylogeny , Alcoholic Beverages/analysis , Bacterial Load , Ethanol/metabolism , Fermentation , High-Throughput Nucleotide Sequencing , Lactobacillaceae/classification , Lactobacillaceae/metabolism , RNA, Ribosomal, 16S/geneticsABSTRACT
Regioselective and 1,2-cis-α-stereoselective glycosylations using 1α,2α-anhydro glycosyl donors and diol glycosyl acceptors in the presence of a glycosyl-acceptor-derived boronic ester catalyst. The reactions proceed smoothly to give the corresponding 1,2-cis-α-glycosides with high stereo- and regioselectivities in high yields without any further additives under mild reaction conditions. In addition, the present glycosylation method was successfully applied to the synthesis of an isoflavone glycoside.
Subject(s)
Boron/chemistry , Catalysis , Esters , Glycosylation , StereoisomerismABSTRACT
Tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) found in diverse pharmaceutical compounds, is used as a starting material for the production of BIA. THP also has various neurobiological properties but is difficult to synthesize. Therefore, a simple method for THP production is desired. Recent studies have shown that microbes, especially bacteria, can serve as platforms for synthesizing these complex compounds; however, because bacteria lack organelles, the designed synthetic pathway cannot be compartmentalized. Thus, the metabolic flow is frequently inhibited or disrupted by undesirable reactions. Indeed, in the first attempt to synthesize THP using a single strain of engineered Escherichia coli, the yield was quite low (<5â µM), mainly because of the oxidation of THP by tyrosinase, an essential enzyme in our production system. To circumvent these problems, we constructed a stepwise (R,S)-THP production system, in which the dopamine-producing step and the subsequent THP-producing step were separated. The yield of (R,S)-THP reached 1.0â mM (287â mg/L), the highest yielding BIA production method using a microbe reported to date. Furthermore, we demonstrated that (R,S)-THP produced by stepwise fermentation is useful for the production of reticuline, an important BIAs intermediate. Based on these observations, applying the stepwise fermentation method is discussed.
Subject(s)
Fermentation , Metabolic Engineering , Monophenol Monooxygenase/genetics , Tetrahydropapaveroline/chemical synthesis , Escherichia coli/genetics , Escherichia coli/metabolism , Monophenol Monooxygenase/metabolism , Tetrahydropapaveroline/metabolismABSTRACT
We previously found that juvenile pituitary adenylate cyclase-activating polypeptide (PACAP)-knockout (PACAP(-/-)) mice reared in an enriched environment (EE) for 4 weeks showed attenuated hyperactivity, jumping behavior, impairments in social interaction, and depression-like behavior. The present study examined the effects of EE on memory function and memory-related protein levels in PACAP(-/-) mice. Eight-week-old PACAP(-/-) mice displayed fear memory dysfunction in a contextual fear conditioning test and cognitive impairments in a novel object recognition test. Rearing of 4-week-old PACAP(-/-) mice in an EE for 4 weeks ameliorated these memory impairments. The beneficial effects of EE were also observed 2 weeks after a return to housing in a standard environment (SE). This suggests that the effects of EE on impaired memory are long-lasting. In both wild-type and PACAP(-/-) mice, EE increased the protein levels of the NMDA receptor NR2B subunit, phospho-ERK, phospho-CaMKII, and brain-derived neurotrophic factor (BDNF) in the hippocampus, and decreased neurotrophin-3 levels, whereas it did not affect nerve growth factor and glial cell-derived neurotrophic factor levels. Increased levels of NR2B, phospho-ERK, phospho-CaMKII and BDNF were not observed 2 weeks after a return to housing in a SE. These findings suggest that living in an EE engenders long-lasting reductions in memory impairments in PACAP(-/-) mice. The present study also implies that increases in hippocampal memory-related protein and BDNF levels are responsible for the beneficial effects of an EE, but not for the maintenance of these effects.
Subject(s)
Hippocampus/physiopathology , Housing, Animal , Memory Disorders/physiopathology , Memory Disorders/therapy , Pituitary Adenylate Cyclase-Activating Polypeptide/deficiency , Animals , Brain-Derived Neurotrophic Factor/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Conditioning, Psychological/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , Fear/physiology , Glial Cell Line-Derived Neurotrophic Factor/metabolism , Hippocampus/growth & development , Male , Mice, Inbred ICR , Mice, Knockout , Nerve Growth Factor/metabolism , Neurotrophin 3/metabolism , Phosphorylation , Pituitary Adenylate Cyclase-Activating Polypeptide/genetics , Random Allocation , Receptors, N-Methyl-D-Aspartate/metabolism , Recognition, Psychology/physiologyABSTRACT
Benzylisoquinoline alkaloids (BIAs) are pharmaceutically important compounds. We have previously devised a reticuline (BIA) production method from dopamine by using Escherichia coli; however, its productivity was relatively low (33 µM, 11 mg/L). We report here, by fine-tuning the method, higher reticuline productivity of 165 µM (54 mg/L), increasing the conversion efficiency by 8-fold. These results are important for developing an efficient route to fermentative reticuline production.
Subject(s)
Benzylisoquinolines/metabolism , Dopamine/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Metabolic Engineering/methods , FermentationABSTRACT
The processing of archetypal Japanese sushi involves microbial fermentation. The traditional sushi kaburazushi, introduced in the middle ages, is made by fermenting salted yellow tail, salted turnip, and malted rice, and is distinguished from the ancient sushi narezushi, made from fish and boiled rice. In this study, we examined changes in the microbial population during kaburazushi fermentation by pyrosequencing the 16S ribosomal RNA genes (rDNA) of the organisms in the fermentation medium. Ribosomal Database Project Classifier analysis identified 31 genera, among which Lactobacillus drastically increased during fermentation (150-fold increment over 8 d), while the relative populations of the other gram-positive bacteria (Staphylococcus and Bacillus) decreased. Basic Local Alignment Search Tool analysis revealed the dominant species to be L. sakei. This organism constituted approximately 90% of Lactobacillus and 79% of total microbiota. The taxonomic diversity and species richness (assayed by Shannon-Weiner Index and Chao 1, respectively) were not significantly different between middle-ages kaburazushi and ancient narezushi. Both types were characterized by the preferential growth of Lactobacillales.
Subject(s)
Bacteria/genetics , Fishes/microbiology , Food Microbiology , High-Throughput Nucleotide Sequencing , Sequence Analysis , Animals , Bacteria/isolation & purification , DNA, Bacterial/genetics , DNA, Ribosomal/genetics , Fermentation , Japan , MicrobiotaABSTRACT
Microbes with smaller genomes would be better chassis for analysis, design, and improvement in the fields of metabolic engineering, synthetic biology, and molecular breeding. To create an Escherichia coli strain with a smaller genome, we used a stepwise genome reduction approach. Beginning with strain MGF-01, which has a genome of 3.62 megabase pairs (Mbp), we generated two E. coli K-12 strains without any insertion sequence (IS), DGF-327 and DGF-298, with reduced genome sizes of 3.27 and 2.98 Mbp, respectively. During the strain construction, intrinsic mutations of ilvG and rph were functionally restored to accelerate initial growth after inoculation. The genomes of the two strains were sequenced, and their structures were confirmed. Both strains showed no auxotrophy, and had better growth fitness, especially in the initial phase, and better cell yield in a rich medium than the wild type K-12 strain. Transcriptome analysis revealed that ibpAB and lon, which encode a heat-shock chaperone and a protease for abnormal proteins, respectively, are down-regulated in DGF strains, compared to the ancestral strains with larger genomes. We concluded that down-regulation of the genes encoding chaperones and proteases is one of the factors that improve the fitness of DGF strains. The DGF strains with fewer genes and better cell yield will be good hosts for applications.