A Novel Method for Creating Heterologous Lethal Antibiotic Producers by Screening from Combi-OGAB Library with Various Promoters in a Biosynthetic Gene Cluster.

In this study, we devised a novel method to create heterologous producers of lethal antibiotics against host bacteria. Heterologous producers cannot be created when antibiotics are toxic to host bacteria. To overcome this challenge, we developed a novel method involving construction of a combinatorial library with various promoters and screening based on the production. To realize this, we utilized Combi-OGAB (Combinatorial Ordered Gene Assembly in Bacillus subtilis), which technology can effectively construct diverse combinatorial library and accelerate screening procedures. B. subtilis and Gramicidin S were selected as the host bacterium and the targeted antibiotic, respectively. The screened producer from Combi-OGAB screening cycles achieved >30-fold productivity over the lethal level. These results suggest that our strategy has the potential to maximize the phenotypic resistance of host bacteria to create heterologous lethal antibiotic producers.

Online SFE-SFC-MS/MS colony screening: A high-throughput approach for optimizing (-)-limonene production.

Conventional analysis of microbial bioproducers requires the extraction of metabolites from liquid cultures, where the culturing steps are time consuming and greatly limit throughput. To break through this barrier, the current study aims to directly evaluate microbial bioproduction colonies by way of supercritical fluid extraction-supercritical fluid chromatography-triple quadrupole mass spectrometry (SFE-SFC-MS/MS). The online SFE-SFC-MS/MS system offers great potential for high-throughput analysis due to automated metabolite extraction without any need for pretreatment. This is the first report of SFE-SFC-MS/MS as a method for direct colony screening, as demonstrated in the high-throughput screening of (-)-limonene bioproducers. Compared with conventional analysis, the SFE-SFC-MS/MS system enables faster and more convenient screening of highly productive strains.

Simple and Rapid Non-ribosomal Peptide Synthetase Gene Assembly Using the SEAM-OGAB Method.

Recombination of biosynthetic gene clusters including those of non-ribosomal peptide synthetases (NRPSs) is essential for understanding the mechanisms of biosynthesis. Due to relatively huge gene cluster sizes ranging from 10 to 150 kb, the prevalence of sequence repeats, and inability to clearly define optimal points for manipulation, functional characterization of recombinant NRPSs with maintained activity has been hindered. In this study, we introduce a simple yet rapid approach named "Seamed Express Assembly Method (SEAM)" coupled with Ordered Gene Assembly in Bacillus subtilis (OGAB) to reconstruct fully functional plipastatin NRPS. This approach is enabled by the introduction of restriction enzyme sites as seams at module borders. SEAM-OGAB is then first demonstrated by constructing the ppsABCDE NRPS (38.4 kb) to produce plipastatin, a cyclic decapeptide in B. subtilis. The introduced amino acid level seams do not hinder the NRPS function and enable successful production of plipastatin at a commensurable titer. It is challenging to modify the plipastatin NRPS gene cluster due to the presence of three long direct-repeat sequences; therefore, this study demonstrates that SEAM-OGAB can be readily applied towards the recombination of various NRPSs. Compared to previous NRPS gene assembly methods, the advantage of SEAM-OGAB is that it readily enables the shuffling of NRPS gene modules, and therefore, chimeric NRPSs can be rapidly constructed for the production of novel peptides. This chimeric assembly application of SEAM-OGAB is demonstrated by swapping plipastatin NRPS and surfactin NRPS modules to produce two novel lipopeptides in B. subtilis.

Machine learning discovery of missing links that mediate alternative branches to plant alkaloids.

Engineering the microbial production of secondary metabolites is limited by the known reactions of correctly annotated enzymes. Therefore, the machine learning discovery of specialized enzymes offers great potential to expand the range of biosynthesis pathways. Benzylisoquinoline alkaloid production is a model example of metabolic engineering with potential to revolutionize the paradigm of sustainable biomanufacturing. Existing bacterial studies utilize a norlaudanosoline pathway, whereas plants contain a more stable norcoclaurine pathway, which is exploited in yeast. However, committed aromatic precursors are still produced using microbial enzymes that remain elusive in plants, and additional downstream missing links remain hidden within highly duplicated plant gene families. In the current study, machine learning is applied to predict and select plant missing link enzymes from homologous candidate sequences. Metabolomics-based characterization of the selected sequences reveals potential aromatic acetaldehyde synthases and phenylpyruvate decarboxylases in reconstructed plant gene-only benzylisoquinoline alkaloid pathways from tyrosine. Synergistic application of the aryl acetaldehyde producing enzymes results in enhanced benzylisoquinoline alkaloid production through hybrid norcoclaurine and norlaudanosoline pathways.

Mechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloids.

Previous studies have utilized monoamine oxidase (MAO) and L-3,4-dihydroxyphenylalanine decarboxylase (DDC) for microbe-based production of tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) precursor to opioid analgesics. In the current study, a phylogenetically distinct Bombyx mori 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) is identified to bypass MAO and DDC for direct production of 3,4-dihydroxyphenylacetaldehyde (DHPAA) from L-3,4-dihydroxyphenylalanine (L-DOPA). Structure-based enzyme engineering of DHPAAS results in bifunctional switching between aldehyde synthase and decarboxylase activities. Output of dopamine and DHPAA products is fine-tuned by engineered DHPAAS variants with Phe79Tyr, Tyr80Phe and Asn192His catalytic substitutions. Balance of dopamine and DHPAA products enables improved THP biosynthesis via a symmetrical pathway in Escherichia coli. Rationally engineered insect DHPAAS produces (R,S)-THP in a single enzyme system directly from L-DOPA both in vitro and in vivo, at higher yields than that of the wild-type enzyme. However, DHPAAS-mediated downstream BIA production requires further improvement.

Author Correction: Mechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloids.

In the original version of this Article, the abbreviation of 3,4-dihydroxyphenylacetaldehyde synthase presented in the first paragraph of the Discussion section was given incorrectly as DYPAA. The correct abbreviation for this enzyme is DHPAAS. This error has been corrected in both the PDF and HTML versions of the Article.

Improved measurement of absolute mRNA quantity without reverse transcription.

Gene expression studies using microarrays have provided important insights into understanding the mechanisms of transcriptional regulation in a variety of biological and disease phenomena. In a previous study, we developed Photo-DEAN, a universal-microarray-based RNA quantification method that enabled reverse transcription-free multiplex measurement of the absolute amount of RNA. Photo-DEAN promotes high-throughput and bias-less transcriptome analysis without the need for common controls or additional complicated normalization steps. In this study, we empirically identified two conditions (individual specificity and uniform duplex stability) necessary for in silico design of probe sequences, allowing the Photo-DEAN method to accurately measure the absolute amount of target RNA in total RNA. We then demonstrated that using the modified probe design conditions, the Photo-DEAN method successfully measured the absolute amount of pgi mRNA spiked into E. coli total RNA. The measurement was performed at five different sites in the coding region of pgi mRNA, exhibiting no significant site dependence. Theoretical considerations suggested that probe sequences longer than the previously used 30-bases better satisfy the necessary design conditions.

Heterologous production of free dihomo-γ-linolenic acid by Aspergillus oryzae and its extracellular release via surfactant supplementation.

Free dihomo-γ-linolenic acid (DGLA) and its desaturated form, free arachidonic acid (ARA) are polyunsaturated free fatty acids (FFAs). They are useful raw materials to produce eicosanoid pharmaceuticals. In this study, we aimed at their production by the oleaginous filamentous fungus Aspergillus oryzae via metabolic engineering. Three genes encoding enzymes involved in the synthesis of DGLA and ARA, were isolated from the filamentous fungus Mortierella alpina that produces ARA in a triacylglycerol form. These genes were concatenated to promoters and terminators of highly expressed genes of A. oryzae, and the concatenated DNA fragments were further concatenated with each other to generate a single DNA fragment in the form of a biosynthetic gene cluster. By homologous recombination, the resulting DNA fragment was integrated to the chromosome of the A. oryzae acyl-CoA synthetase gene disruptant whose FFA productivity was enhanced at 9.2-fold more than the wild-type strain. The DNA-integrated disruptant produced free DGLA but did not produce free ARA. Thus, focusing on free DGLA, after removal of the gene for converting DGLA to ARA, the constructed strain produced free DGLA at 145 mg/l for 5 d. Also, by supplementing Triton X-100 surfactant at 1% to the culture, over 80% of free DGLA was released from cells without inhibiting the growth. Consequently, the constructed strain will be useful for attempting production of free DGLA-derived eicosanoids because it bypasses excision of free DGLA from triacylglycerols by lipase. To our knowledge, this is the first report on microbial production of free DGLA and its extracellular release.

Biomimetic strategy for constructing Clostridium thermocellum cellulosomal operons in Bacillus subtilis.

BACKGROUND: Enzymatic conversion of lignocellulosic biomass into soluble sugars is a major bottleneck in the plant biomass utilization. Several anaerobic organisms cope these issues via multiple-enzyme complex system so called 'cellulosome'. Hence, we proposed a "biomimic operon" concept for making an artificial cellulosome which can be used as a promising tool for the expression of cellulosomal enzymes in Bacillus subtilis. RESULTS: According to the proteomic analysis of Clostridium thermocellum ATCC27405 induced by Avicel or cellobiose, we selected eight highly expressed cellulosomal genes including a scaffoldin protein gene (cipA), a cell-surface anchor gene (sdbA), two exoglucanase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ). Arranging these eight genes in two different orders, we constructed two different polycistronic operons using the ordered gene assembly in Bacillus method. This is the first study to express the whole CipA along with cellulolytic enzymes in B. subtilis. Each operon was successfully expressed in B. subtilis RM125, and the protein complex assembly, cellulose-binding ability, thermostability, and cellulolytic activity were demonstrated. The operon with a higher xylanase activity showed greater saccharification on complex cellulosic substrates such as Napier grass than the other operon. CONCLUSIONS: In this study, a strategy for constructing an efficient cellulosome system was developed and two different artificial cellulosomal operons were constructed. Both operons could efficiently express the cellulosomal enzymes and exhibited cellulose saccharification. This strategy can be applied to different industries with cellulose-containing materials, such as papermaking, biofuel, agricultural compost, mushroom cultivation, and waste processing industries.

Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli.

BACKGROUND: N-Butanol has favorable characteristics for use as either an alternative fuel or platform chemical. Bio-based n-butanol production using microbes is an emerging technology that requires further development. Although bio-industrial microbes such as Escherichia coli have been engineered to produce n-butanol, reactive oxygen species (ROS)-mediated toxicity may limit productivity. Previously, we show that outer-membrane-targeted tilapia metallothionein (OmpC-TMT) is more effective as an ROS scavenger than human and mouse metallothioneins to reduce oxidative stress in the host cell. RESULTS: The host strain (BUT1-DE) containing the clostridial n-butanol pathway displayed a decreased growth rate and limited n-butanol productivity, likely due to ROS accumulation. The clostridial n-butanol pathway was co-engineered with inducible OmpC-TMT in E. coli (BUT3-DE) for simultaneous ROS removal, and its effect on n-butanol productivity was examined. The ROS scavenging ability of cells overexpressing OmpC-TMT was examined and showed an approximately twofold increase in capacity. The modified strain improved n-butanol productivity to 320 mg/L, whereas the control strain produced only 95.1 mg/L. Transcriptomic analysis revealed three major KEGG pathways that were significantly differentially expressed in the BUT3-DE strain compared with their expression in the BUT1-DE strain, including genes involved in oxidative phosphorylation, fructose and mannose metabolism and glycolysis/gluconeogenesis. CONCLUSIONS: These results indicate that OmpC-TMT can increase n-butanol production by scavenging ROS. The transcriptomic analysis suggested that n-butanol causes quinone malfunction, resulting in oxidative-phosphorylation-related nuo operon downregulation, which would diminish the ability to convert NADH to NAD+ and generate proton motive force. However, fructose and mannose metabolism-related genes (fucA, srlE and srlA) were upregulated, and glycolysis/gluconeogenesis-related genes (pfkB, pgm) were downregulated, which further assisted in regulating NADH/NAD+ redox and preventing additional ATP depletion. These results indicated that more NADH and ATP were required in the n-butanol synthetic pathway. Our study demonstrates a potential approach to increase the robustness of microorganisms and the production of toxic chemicals through the ability to reduce oxidative stress.

Method of preparing an equimolar DNA mixture for one-step DNA assembly of over 50 fragments.

In the era of synthetic biology, techniques for rapidly constructing a designer long DNA from short DNA fragments are desired. To realize this, we attempted to establish a method for one-step DNA assembly of unprecedentedly large numbers of fragments. The basic technology is the Ordered Gene Assembly in Bacillus subtilis (OGAB) method, which uses the plasmid transformation system of B. subtilis. Since this method doesn't require circular ligation products but needs tandem repeat ligation products, the degree of deviation in the molar concentration of the material DNAs is the only determinant that affects the efficiency of DNA assembly. The strict standardization of the size of plasmids that clone the DNA block and the measurement of the block in the state of intact plasmid improve the reliability of this step, with the coefficient of variation of the molar concentrations becoming 7%. By coupling this method with the OGAB method, one-step assembly of more than 50 DNA fragments becomes feasible.

Fermentation approach for enhancing 1-butanol production using engineered butanologenic Escherichia coli.

In this study, engineered butanologenic Escherichia coli T5 constructed by the OGAB method was used for 1-butanol production. The results showed the feasibility of the artificial butanologenic operon, (Promoter Pr)-thil-crt-bcd-etfB-etfA-hbd-adhe1-adhe, where the 1-butanol titer, specific BuOH yield, and BuOH yield were 4.50 mg/L, 4.50 mg-BuOH/g cell, and 0.35 mg-BuOH/g-glucose, respectively. Fermentation conditions of anaerobic, low initial concentrations of carbon sources, low oxidation state of carbon source, pH of 6, addition of glutathione and citrate, had been shown for efficiently improving the 1-butanol production. The premise behind these fermentation approaches can be categorized into two lines of reasoning, either elevated the availability of acetyl-CoA or lowered the intracellular redox state. By comparing the fermentation conditions tested in this study, pH has been shown to be the most efficiency strategies for 1-butanol production while the replacement of glucose with glycerol provides the highest improvement in butanol yield.

Termination of the structural confusion between plipastatin A1 and fengycin IX.

Plipastatin A1 and fengycin IX were experimentally proven to be identical compounds, while these had been considered as diastereomers due to the permutation of the enantiomeric pair of Tyr in most papers. The (1)H NMR spectrum changed to become quite similar to that of plipastatin A1, when the sample which provided resembled spectrum of fengycin IX was treated with KOAc followed by LH-20 gel filtration. Our structural investigations disclosed that the structures of these molecules should be settled into that of plipastatin A1 by Umezawa (L-Tyr4 and D-Tyr10).

Rearrangement of gene order in the phaCAB operon leads to effective production of ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] in genetically engineered Escherichia coli.

Ultrahigh-molecular-weight poly[(R)-3-hydroxybutyrate] [UHMW-P(3HB)] synthesized by genetically engineered Escherichia coli is an environmentally friendly bioplastic material which can be processed into strong films or fibers. An operon of three genes (organized as phaCAB) encodes the essential proteins for the production of P(3HB) in the native producer, Ralstonia eutropha. The three genes of the phaCAB operon are phaC, which encodes the polyhydroxyalkanoate (PHA) synthase, phaA, which encodes a 3-ketothiolase, and phaB, which encodes an acetoacetyl coenzyme A (acetoacetyl-CoA) reductase. In this study, the effect of gene order of the phaCAB operon (phaABC, phaACB, phaBAC, phaBCA, phaCAB, and phaCBA) on an expression plasmid in genetically engineered E. coli was examined in order to determine the best organization to produce UHMW-P(3HB). The results showed that P(3HB) molecular weights and accumulation levels were both dependent on the order of the pha genes relative to the promoter. The most balanced production result was achieved in the strain harboring the phaBCA expression plasmid. In addition, analysis of expression levels and activity for P(3HB) biosynthesis enzymes and of P(3HB) molecular weight revealed that the concentration of active PHA synthase had a negative correlation with P(3HB) molecular weight and a positive correlation with cellular P(3HB) content. This result suggests that the level of P(3HB) synthase activity is a limiting factor for producing UHMW-P(3HB) and has a significant impact on P(3HB) production.

Construction and manipulation of giant DNA by a genome vector.

Since the entire sequence of a number of genome came into determination, current studies are gradually focusing on unveiling global networks of gene products, RNA, protein, and metabolites that support real-life activities. Our understanding of whole gene networks will be brought about by use of not only a few recombinant genes but also more number of genes at a time, or the genome. Genomes should be likely handled freely; however, there exist certain barriers in handling between genes and genomes. They are intrinsic fragility of giant DNA in test tube and the size limit of conventional cloning vector systems relying on prevailing cloning host Escherichia coli. A eubacterium, Bacillus subtilis has been offered as a replacement for particular large DNA or genomes, relying on inherent ability to take up DNA given outside and integrate it into its own genome via homologous recombination. The Bacillus GenoMe (BGM) vector derived from the 4,200-kbp genome of B. subtilis 168 has been demonstrated to accommodate fairly large DNAs and is highlighted by the successful stable cloning of a whole 3,500-kbp genome of the nonpathogenic, unicellular photosynthetic bacterium Synechocystis and any sequence-known DNAs. In the chapter, highlighted are clear differences in cloning concept and actual manipulation from other conventional ones, focusing methodological aspects as plainly as possible. We may also indicate that B. subtilis provides other opportunities for assembly of a large number of DNA fragments, in unbelievably high efficiency. The new workhorse described here exhibits technical breakthroughs leading to the new concept for designing the desired genomes even from scratch. The novel system not only offers unprecedented opportunities for addressing important contemporary issues in biotechnology, but also gives rise to new ideas of thinking among versatile field of biology.

Bottom-up genome assembly using the Bacillus subtilis genome vector.

We established a protocol to construct complete recombinant genomes from their small contiguous DNA pieces and obtained the genomes of mouse mitochondrion and rice chloroplast using a B. subtilis genome (BGM) vector. This method allows the design of any recombinant genomes, valuable not only for fundamental research in systems biology and synthetic biology but also for various applications in the life sciences.

Conjugational transfer system to shuttle giant DNA cloned by Bacillus subtilis genome (BGM) vector.

The Bacillus subtilis GenoMe (BGM) vector was designed as a versatile vector for the cloning of giant DNA segments. Cloned DNA in the BGM can be retrieved to a plasmid using our Bacillus recombinational transfer (BReT) method that takes advantage of competent cell transformation. However, delivery of the plasmid to a different B. subtilis strain by the normal transformation method is hampered by DNA size-related inefficiency. Therefore, we designed a novel method, conjugational plasmid-mediated DNA retrieval and transfer (CReT) from the BGM vector, and investigated conjugational transmission to traverse DNA between cells to circumvent the transformation-induced size limitation. pLS20, a 65-kb plasmid capable of conjugational transfer between B. subtilis strains, was modified to retrieve DNA cloned in the BGM vector by homologous recombination during normal culture. As the plasmid copy number was estimated to be 3, the retrieval plasmid was selected using increased numbers of marker genes derived from the retrieved DNA. We applied this method to retrieve Synechocystis genome segments up to 90 kb in length. We observed retrieved plasmid transfers between B. subtilis strains by conjugation in the absence of structural alterations in the DNA fragment. Our observations extend DNA transfer protocols over previously exploited size ranges.

Production of the non-ribosomal peptide plipastatin in Bacillus subtilis regulated by three relevant gene blocks assembled in a single movable DNA segment.

Methods that allow the assembly of genes in one single DNA segment are of great use in bioengineering and synthetic biology. The biosynthesis of plipastatin, a lipopeptide antibiotic synthesized non-ribosomally by Bacillus subtilis 168, requires three gene blocks at different genome loci, i.e. the peptide synthetase operon ppsABCDE (38-kb), degQ (0.6kb), and sfp (1.0kb). We applied a DNA assembly protocol in B. subtilis, named ordered gene assembly in B. subtilis (OGAB) method, to incorporate those three gene blocks into a one-unit plasmid via one ligation-reaction. High yields of correct assembly, above 87%, allowed us to screen for the plasmid that produced plipastatin at a level approximately 10-fold higher than in the wild-type. In contrast to that recombinogenic technologies used in E. coli require repetitive assembly steps and/or several selection markers, our method features high fidelity and efficiency, is completed in one ligation using only one selection marker associating with plasmid vector, and is applicable to DNA fragments larger than 40kb.

Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis.

We attempted to optimize the production of zeaxanthin in Escherichia coli by reordering five biosynthetic genes in the natural carotenoid cluster of Pantoea ananatis. Newly designed operons for zeaxanthin production were constructed by the ordered gene assembly in Bacillus subtilis (OGAB) method, which can assemble multiple genes in one step using an intrinsic B. subtilis plasmid transformation system. The highest level of production of zeaxanthin in E. coli (820 microg/g [dry weight]) was observed in the transformant with a plasmid in which the gene order corresponds to the order of the zeaxanthin metabolic pathway (crtE-crtB-crtI-crtY-crtZ), among a series of plasmids with circularly permuted gene orders. Although two of five operons using intrinsic zeaxanthin promoters failed to assemble in B. subtilis, the full set of operons was obtained by repressing operon expression during OGAB assembly with a p(R) promoter-cI repressor system. This result suggests that repressing the expression of foreign genes in B. subtilis is important for their assembly by the OGAB method. For all tested operons, the abundance of mRNA decreased monotonically with the increasing distance of the gene from the promoter in E. coli, and this may influence the yield of zeaxanthin. Our results suggest that rearrangement of biosynthetic genes in the order of the metabolic pathway by the OGAB method could be a useful approach for metabolic engineering.