Integrated pathway mining and selection of an artificial CYP79-mediated bypass to improve benzylisoquinoline alkaloid biosynthesis.

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.

Complete Genome Sequence of Sphingopyxis macrogoltabida Strain 203N (NBRC 111659), a Polyethylene Glycol Degrader.

We determined the complete genome sequence of Sphingopyxis macrogoltabida strain 203N, a polyethylene glycol degrader. Because the PacBio assembly (285× coverage) seemed to be full of nucleotide-level mismatches, the Newbler assembly of MiSeq mate-pair and paired-end data was used for finishing and the PacBio assembly was used as a reference. The PacBio assembly carried 414 nucleotide mismatches over 5,953,153 bases of the 203N genome.

Complete Genome Sequence of Sphingopyxis terrae Strain 203-1 (NBRC 111660), a Polyethylene Glycol Degrader.

The complete genome sequence of Sphingopyxis terrae strain 203-1, which is capable of growing on polyethylene glycol, was determined. The genome consisted of a chromosome with a size of 3.98 Mb and a plasmid with a size of 4,328 bp. The strain was deposited to the National Institute of Technology and Evaluation (Tokyo, Japan) under the number NBRC 111660.

Phylogenetics of family Enterobacteriaceae and proposal to reclassify Escherichia hermannii and Salmonella subterranea as Atlantibacter hermannii and Atlantibacter subterranea gen. nov., comb. nov.

Multilocus sequence analysis based on hypervariable housekeeping proteins was utilized to differentiate closely related species in the family Enterobacteriaceae. Of 150 housekeeping proteins, the top 10 hypervariable proteins were selected and concatenated to obtain distance data. Distances between concatenated proteins within the family were 0.9-41.2%, whereas the 16S rRNA and atpD-gyrB-infB-rpoB concatenated sequence (4MLSA) distances were 0.8-6.0% and 0.9-22.1%, respectively. These data indicate that phylogenetic analysis by concatenation of hypervariable proteins is a powerful tool for discriminating species in the family Enterobacteriaceae. To confirm the discriminatory power of the 10 chosen concatenated hypervariable proteins (C10HKP), phylogenetic trees based on C10HKP, 4MLSA, and the 16S rRNA gene were constructed. Comparison of average bootstrap values among C10HKP, 4MLSA and 16S rRNA genes indicated that the C10HKP tree was the most reliable. Location via the C10HKP tree was consistent with existing assignments for almost all species in the family Enterobacteriaceae. However, the C10HKP tree suggested that several species (including Enterobacter massiliensis, Escherichia vulneris, Escherichia hermannii, and Salmonella subterranea) should be reassigned to different clusters than those defined in previous analyses. Furthermore, E. hermannii and S. subterranea appeared to fall onto a branch independent from those occupied by the other Enterobacteriaceae. Therefore, we propose Atlantibacter gen. nov., such that E. hermannii and S. subterranea would be transferred to genus Atlantibacter as Atlantibacter hermannii, comb. nov. and Atlantibacter subterranea. comb. nov., respectively.

Genetic basis of the highly efficient yeast Kluyveromyces marxianus: complete genome sequence and transcriptome analyses.

BACKGROUND: High-temperature fermentation technology with thermotolerant microbes has been expected to reduce the cost of bioconversion of cellulosic biomass to fuels or chemicals. Thermotolerant Kluyveromyces marxianus possesses intrinsic abilities to ferment and assimilate a wide variety of substrates including xylose and to efficiently produce proteins. These capabilities have been found to exceed those of the traditional ethanol producer Saccharomyces cerevisiae or lignocellulose-bioconvertible ethanologenic Scheffersomyces stipitis. RESULTS: The complete genome sequence of K. marxianus DMKU 3-1042 as one of the most thermotolerant strains in the same species has been determined. A comparison of its genomic information with those of other yeasts and transcriptome analysis revealed that the yeast bears beneficial properties of temperature resistance, wide-range bioconversion ability, and production of recombinant proteins. The transcriptome analysis clarified distinctive metabolic pathways under three different growth conditions, static culture, high temperature, and xylose medium, in comparison to the control condition of glucose medium under a shaking condition at 30°C. Interestingly, the yeast appears to overcome the issue of reactive oxygen species, which tend to accumulate under all three conditions. CONCLUSIONS: This study reveals many gene resources for the ability to assimilate various sugars in addition to species-specific genes in K. marxianus, and the molecular basis of its attractive traits for industrial applications including high-temperature fermentation. Especially, the thermotolerance trait may be achieved by an integrated mechanism consisting of various strategies. Gene resources and transcriptome data of the yeast are particularly useful for fundamental and applied researches for innovative applications.

Complete Genome Sequences of Sulfurospirillum Strains UCH001 and UCH003 Isolated from Groundwater in Japan.

Sulfurospirillum strains UCH001 and UCH003 were isolated from anaerobic cis-1,2-dichloroethene-dechlorinating microbial consortia derived from groundwater in Japan. Here, we report the complete genome sequences of strains UCH001 and UCH003.

Complete Genome Sequence of a Dimethyl Sulfide-Utilizing Bacterium, Acinetobacter guillouiae Strain 20B (NBRC 110550).

Acinetobacter guillouiae strain 20B can utilize dimethyl sulfide (DMS) as the sole sulfur source and degrade chloroethylenes. We report here the complete 4,648,418-bp genome sequence for this strain, which contains 4,367 predicted coding sequences (CDSs), including a well-characterized DMS degradative operon.

Complete Genome Sequence of Polychlorinated Biphenyl Degrader Comamonas testosteroni TK102 (NBRC 109938).

Comamonas testosteroni TK102 (NBRC 109938; JCM 19603) can utilize biphenyl as a sole carbon source and degrade polychlorinated biphenyls (PCBs). The complete nucleotide sequence of the TK102 genome was determined. TK102 possesses several integrative and conjugative element-like regions, and one of them carries biphenyl-degradative genes.

Complete Genome Sequence of an Alkane Degrader, Alcanivorax sp. Strain NBRC 101098.

Alcanivorax sp. strain NBRC 101098 was isolated from seawater in Japan. Strain NBRC 101098 is able to degrade various types of n-alkanes. Here, we report the complete genome of strain NBRC 101098.

The Complete Genome Sequence of Pseudomonas putida NBRC 14164T Confirms High Intraspecies Variation.

Pseudomonas putida has attracted much interest for its environmental, industrial, biotechnological, and clinical importance. Here, we report the complete genome sequence of the type strain P. putida NBRC 14164. This genome sequence will assist to further elucidate the molecular mechanisms of the characteristic traits among strains belonging to the species P. putida.

Complete Genome Sequence of the Carbazole Degrader Pseudomonas resinovorans Strain CA10 (NBRC 106553).

Pseudomonas resinovorans strain CA10 can grow on carbazole as its sole carbon and nitrogen source. Here, we report the complete nucleotide sequence of the CA10 genome (a 6,285,863-bp chromosome and a 198,965-bp plasmid). CA10 carries a larger number of genes that are potentially responsible for aromatic hydrocarbon metabolism than do other previously sequenced Pseudomonas spp.