Whole-cell bioconversion for producing thymoquinone by engineered Saccharomyces cerevisiae.

Thymoquinone, extracted from the black seeds of Nigella sativa, is a natural substance with highly beneficial effects against various human diseases. In this study, we aimed to construct a Saccharomyces cerevisiae strain that, produce thymoquinone from thymol, a relatively inexpensive substrate. To achieve this, cytochrome P450 from Origanum vulgare was expressed in S. cerevisiae for the bioconversion of thymol to thymoquinone, with the co-expression of cytochrome P450 reductase (CPR) from Arabidopsis thaliana, ATR1. Additionally, flexible linkers were used to connect these two enzymes. Furthermore, modifications were performed to expand the endoplasmic reticulum (ER) space, leading to increased thymoquinone production. After integrating the genes into the chromosome and optimizing the media components, a significant improvement in the thymol-to-thymoquinone conversion rate and yield were achieved. This study represents a possibility of the production of thymoquinone, a bioactive ingredient of a plant, using an engineered microbial cell.

Medium Chain Length Polyhydroxyalkanoate Production by Engineered Pseudomonas gessardii Using Acetate-formate as Carbon Sources.

Production of medium chain length polyhydroxyalkanoate (mcl-PHA) was attempted using Pseudomonas gessardii NIBRBAC000509957, which was isolated from Sunchang, Jeollabuk-do, Republic of Korea (35°24'27.7"N, 127°09'13.0"E) and effectively utilized acetate and formate as carbon sources. We first evaluated the utilization of acetate as a carbon source, revealing optimal growth at 5 g/L acetate. Then, formate was supplied to the acetate minimal medium as a carbon source to enhance cell growth. After overexpressing the acetate and formate assimilation pathway enzymes, this strain grew at a significantly higher rate in the medium. As this strain naturally produces PHA, it was further engineered metabolically to enhance mcl-PHA production. The engineered strain produced 0.40 g/L of mcl-PHA with a biomass content of 30.43% in fed-batch fermentation. Overall, this strain can be further developed to convert acetate and formate into valuable products.

Development of an Ephedrine In-House Matrix Reference Material and Its Application to Doping Analysis.

Biomatrix-based reference materials (RMs) improve the quality of laboratory test results by better representing actual samples. However, a matrix RM of ephedrine (EP) for threshold substances that require accurate analysis results has not yet been developed. Therefore, this study aimed to develop an in-house matrix RM for EP and subsequently apply it to analytical procedures. During the development of the in-house matrix EP RM, the system underwent homogeneity and stability studies. Additionally, it was subjected to interlaboratory comparison study in 11 laboratories, including 10 World Anti-Doping Agency (WADA)-accredited laboratories and our laboratory. Stability testing revealed no significant changes in the RM characteristics. For homogeneity, 10 random batches out of 200 were analyzed to confirm the uniformity within and between bottles. These results, combined with data from 11 laboratories, ensured retroactive validation. The traceability value of the in-house matrix EP RM was assigned as 9.83 ± 0.57 µg/mL (k = 2) by interlaboratory comparison studies and traceable uncertain evaluation. The feasibility of this method as a single calibration standard was confirmed in two laboratories. This substance is reliable and consistent for quality control during EP quantification, ensuring accurate and trustworthy outcomes. Consequently, this study establishes a framework and guidelines for producing in-house matrix RMs and serves as a reference for generating similar matrix RMs in other contexts.

Enhancing the soluble expression of α-1,2-fucosyltransferase in E. coli using high-throughput flow cytometry screening coupled with a split-GFP.

2'-Fucosyllactose (2'-FL), one of the major human milk oligosaccharides, was produced in several engineered microorganisms. However, the low solubility of α-1,2-fucosyltransferase (α1,2-FucT) often becomes a bottleneck to produce maximum amount of 2'-FL in the microorganisms. To overcome this solubility issue, the following studies were conducted to improve the soluble expression of α1,2-FucT. Initially, hydrophobic amino acids in the hydrophilic region of the 6 α-helices were mutated, adhering to the α-helix rule. Subsequently, gfp11 was fused to the C-terminal of futC gene encoding α1,2-FucT (FutC), enabling selection of high-fluorescence mutants through split-GFP. Each mutant library was screened via fluorescence activated cell sorting (FACS) to separate soluble mutants for high-throughput screening. As a result, L80C single mutant and A121D/P124A/L125R triple mutant were found, and a combined quadruple mutant was created. Furthermore, we combined mutations of conserved sequences (Q150H/C151R/Q239S) of FutC, which showed positive effects in the previous studies from our lab, with the above quadruple mutants (L80C/A121D/P124A/L125R). The resulting strain produced approximately 3.4-fold higher 2'-FL titer than that of the wild-type, suggesting that the conserved sequence mutations are an independent subset of the mutations that further improve the solubility of the target protein acquired by random mutagenesis using split-GFP.

Simultaneous Correlative Interferometer Technique for Direction Finding of Signal Sources.

In this paper, we propose a novel simultaneous Correlative Interferometer (CI) technique that elaborately estimates the Direction of Arrival (DOA) of multiple source signals incident on an antenna array. The basic idea of the proposed technique is that the antenna-array-based receiver compares the phase of the received signal with one of the candidates at each time sample and jointly exploits these multiple time samples to estimate the DOAs of multiple signal sources. The proposed simultaneous CI-based DOA estimation technique collectively utilizes multiple time-domain samples and can be regarded as a generalized version of the conventional CI algorithm for the case of multiple time-domain samples. We first thoroughly review the conventional CI algorithm to comprehensively explain the procedure of the direction-finding algorithm that adopts the phase information of received signals. We also discuss several technical issues of conventional CI-based DOA estimation techniques that are originally proposed for the case of a single time-domain sample. Then, we propose a simultaneous CI-based DOA estimation technique with multi-sample diversity as a novel solution for the case of multiple time-domain samples. We clearly compare the proposed simultaneous CI technique with the conventional CI technique and we compare the existing Multiple Signal Classification (MUSIC)-based DOA estimation technique with the conventional CI-based technique by using the DOA spectrum as well. To the best of our knowledge, the simultaneous CI-based DOA estimation technique that effectively utilizes the characteristics of multiple signal sources over multiple time-domain samples has not been reported in the literature. Through extensive computer simulations, we show that the proposed simultaneous CI technique significantly outperforms both the conventional CI technique in terms of DOA estimation even in harsh environments and with various antenna array structures. It is worth noting that the proposed simultaneous CI technique results in much better performance than the classical MUSIC algorithm, which is one of the most representative subspace-based DOA estimation techniques.

Heterologous overproduction of oviedomycin by refactoring biosynthetic gene cluster and metabolic engineering of host strain Streptomyces coelicolor.

BACKGROUND: Oviedomycin is one among several polyketides known for their potential as anticancer agents. The biosynthetic gene cluster (BGC) for oviedomycin is primarily found in Streptomyces antibioticus. However, because this BGC is usually inactive under normal laboratory conditions, it is necessary to employ systematic metabolic engineering methods, such as heterologous expression, refactoring of BGCs, and optimization of precursor biosynthesis, to allow efficient production of these compounds. RESULTS: Oviedomycin BGC was captured from the genome of Streptomyces antibioticus by a newly constructed plasmid, pCBA, and conjugated into the heterologous strain, S. coelicolor M1152. To increase the production of oviedomycin, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized in an in vitro setting to refactor the native promoters within the ovm BGC. The target promoters of refactoring were selected based on examination of factors such as transcription levels and metabolite profiling. Furthermore, genome-scale metabolic simulation was applied to find overexpression targets that could enhance the biosynthesis of precursors or cofactors related to oviedomycin production. The combined approach led to a significant increase in oviedomycin production, reaching up to 670 mg/L, which is the highest titer reported to date. This demonstrates the potential of the approach undertaken in this study. CONCLUSIONS: The metabolic engineering approach used in this study led to the successful production of a valuable polyketide, oviedomycin, via BGC cloning, promoter refactoring, and gene manipulation of host metabolism aided by genome-scale metabolic simulation. This approach can be also useful for the efficient production of other secondary molecules encoded by 'silent' BGCs.

Production of S-methyl-methionine using engineered Saccharomyces cerevisiae sake K6.

S-methyl-methionine (SMM), also known as vitamin U, is an important food supplement produced by various plants. In this study, we attempted to produce it in an engineered microorganism, Saccharomyces cerevisiae, by introducing an MMT gene encoding a methionine S-methyltransferase from Arabidopsis thaliana. The S. cerevisiae sake K6 strain, which is a Generally Recognized as Safe (GRAS) strain, was chosen as the host because it produces a significant amount of S-adenosylmethionine (SAM), a precursor of SMM. To increase SMM production in the host, MHT1 and SAM4 genes encoding homocysteine S-methyltransferase were knocked out to prevent SMM degradation. Additionally, MMP1, which encodes S-methyl-methionine permease, was deleted to prevent SMM from being imported into the cell. Finally, ACS2 gene encoding acetyl-CoA synthase was overexpressed, and MLS1 gene encoding malate synthase was deleted to increase SAM availability. Using the engineered strain, 1.92 g/L of SMM was produced by fed-batch fermentation. ONE-SENTENCE SUMMARY: Introducing a plant-derived MMT gene encoding methionine S-methyltransferase into engineered Saccharomyces cerevisiae sake K6 allowed microbial production of S-methyl-methionine (SMM).

Quorum sensing-based metabolic engineering of the precursor supply in Streptomyces coelicolor to improve heterologous production of neoaureothin.

Streptomyces are important industrial bacteria that produce pharmaceutically valuable polyketides. However, mass production on an industrial scale is limited by low productivity, which can be overcome through metabolic engineering and the synthetic biology of the host strain. Recently, the introduction of an auto-inducible expression system depending on microbial physiological state has been suggested as an important tool for the industrial-scale production of polyketides. In this study, titer improvement by enhancing the pool of CoA-derived precursors required for polyketide production was driven in a quorum sensing (QS)-dependent manner. A self-sustaining and inducer-independent regulatory system, named the QS-based metabolic engineering of precursor pool (QMP) system, was constructed, wherein the expression of genes involved in precursor biosynthesis was regulated by the QS-responsive promoter, scbAp. The QMP system was applied for neoaureothin production in a heterologous host, Streptomyces coelicolor M1152, and productivity increased by up to 4-fold. In particular, the engineered hyperproducers produced high levels of neoaureothin without adversely affecting cell growth. Overall, this study showed that self-regulated metabolic engineering mediated by QS has the potential to engineer strains for polyketide titer improvement.

Production of 2'-fucosyllactose using ⍺1,2-fucosyltransferase from a GRAS bacterial strain.

2'-Fucosyllactose (2'-FL) is a major oligosaccharide found in human breast milk. It is produced from GDP-L-fucose and D-lactose by ⍺1,2-fucosyltransferase (⍺1,2-fucT), but the enzyme has been identified mostly in pathogens. In this study, an ⍺1,2-fucT was isolated from a Generally Recognized as Safe (GRAS) Bacillus megaterium strain. The enzyme was successfully expressed in metabolically-engineered Escherichia coli. Furthermore, replacement of non-conserved amino acid residues with conserved ones in the protein led to an increase in the rate of 2'-FL production. As a result, fed-batch fermentation of E. coli produced 30 g/L of 2'-FL from glucose and lactose. Thus, the overproduction of 2'-FL using a novel enzyme from a GRAS bacteria strain was successfully demonstrated.

Development and validation of a method for analyzing the sialylated glycopeptides of recombinant erythropoietin in urine using LC-HRMS.

Erythropoietin (EPO) is a glycoprotein hormone that stimulates red blood cell production. It is produced naturally in the body and is used to treat patients with anemia. Recombinant EPO (rEPO) is used illicitly in sports to improve performance by increasing the blood's capacity to carry oxygen. The World Anti-Doping Agency has therefore prohibited the use of rEPO. In this study, we developed a bottom-up mass spectrometric method for profiling the site-specific N-glycosylation of rEPO. We revealed that intact glycopeptides have a site-specific tetra-sialic glycan structure. Using this structure as an exogenous marker, we developed a method for use in doping studies. The profiling of rEPO N-glycopeptides revealed the presence of tri- and tetra-sialylated N-glycopeptides. By selecting a peptide with a tetra-sialic acid structure as the target, its limit of detection (LOD) was estimated to be < 500 pg/mL. Furthermore, we confirmed the detection of the target rEPO glycopeptide using three other rEPO products. We additionally validated the linearity, carryover, selectivity, matrix effect, LOD, and intraday precision of this method. To the best of our knowledge, this is the first report of a doping analysis using liquid chromatography/mass spectrometry-based detection of the rEPO glycopeptide with a tetra-sialic acid structure in human urine samples.

Minicell-forming Escherichia coli mutant with increased chemical production capacity and tolerance to toxic compounds.

Minicell, a small spherical form of bacterium produced by abnormal fission, possesses cytoplasmic constituents similar to those of the parental cell, except for genomic DNA. E. coli strains were engineered to produce minicells and value-added chemicals. Minicell-forming mutants showed enhanced tolerance to toxic chemicals and a higher intracellular NADH/NAD+ ratio than the wild-type. When toxic chemicals such as isobutanol, isobutyraldehyde, and isobutyl acetate were produced in this mutant, the titers increased by 67 %, 175 %, and 214 %, respectively. In addition, morphological changes and membrane dispersion mechanisms in minicell-forming mutants improved lycopene production by 259 %. This increase in production capacity was more pronounced when biomass hydrolysate was used as the substrate. Isobutanol and lycopene production also increased by 92 % and 295 %, respectively, on using the substrate in the mutant. It suggests that minicell-forming mutants are an excellent platform for biochemical production.

Recent progress in the engineering of C1-utilizing microbes.

The global climate crisis has led to the transition toward the sustainable production of chemicals and fuels with a low carbon footprint. Microbial utilization of one-carbon (C1) substrates, such as carbon dioxide, carbon monoxide, methane, formate, and methanol, may be a promising replacement for the current fossil fuel-based industry. However, natural C1-utilizing microbes are currently unsuitable for industrial applications because of their slow growth and low carbon conversion efficiency, which results in low productivity and yield. Here, we review the recent achievements in engineering C1-utilizing microbes with improved carbon assimilation efficiency and describe the development of synthetic microorganisms by introducing natural C1 assimilation pathways in non-C1-utilizing microbes. Finally, we outline the future directions for realizing the industrial potential of C1-utilizing microbes.

Escherichia coli minicells with targeted enzymes as bioreactors for producing toxic compounds.

Formed by aberrant cell division, minicells possess functional metabolism despite their inability to grow and divide. Minicells exhibit not only superior stability when compared with bacterial cells but also exceptional tolerance-characteristics that are essential for a de novo bioreactor platform. Accordingly, we engineered minicells to accumulate protein, ensuring sufficient production capability. When tested with chemicals regarded as toxic against cells, the engineered minicells produced titers of C6-C10 alcohols and esters, far surpassing the corresponding production from bacterial cells. Additionally, microbial autoinducer production that is limited in expanding bacterial population was conducted in the minicells. Because bacterial population growth was nonexistent, the minicells produced autoinducers in constant amounts, which allowed precise control of the bacterial population having autoinducer-responsive gene circuits. When bacterial population growth was nonexistent, the minicells produced autoinducers in constant amounts, which allowed precise control of the bacterial population having autoinducer-based gene circuits with the minicells. This study demonstrates the potential of minicells as bioreactors suitable for products with known limitations in microbial production, thus providing new possibilities for bioreactor engineering.

Comparative genomic analysis of Streptomyces rapamycinicus NRRL 5491 and its mutant overproducing rapamycin.

Streptomyces rapamycinicus NRRL 5491 is a well-known producer of rapamycin, a secondary metabolite with useful bioactivities, including antifungal, antitumor, and immunosuppressive functions. For the enhanced rapamycin production, a rapamycin-overproducing strain SRMK07 was previously obtained as a result of random mutagenesis. To identify genomic changes that allowed the SRMK07 strain's enhanced rapamycin production, genomes of the NRRL 5491 and SRMK07 strains were newly sequenced in this study. The resulting genome sequences of the wild-type and SRMK07 strains showed the size of 12.47 Mbp and 9.56 Mbp, respectively. Large deletions were observed at both end regions of the SRMK07 strain's genome, which cover 17 biosynthetic gene clusters (BGCs) encoding secondary metabolites. Also, genes in a genomic region containing the rapamycin BGC were shown to be duplicated. Finally, comparative metabolic network analysis using these two strains' genome-scale metabolic models revealed biochemical reactions with different metabolic fluxes, which were all associated with NADPH generation. Taken together, the genomic and computational approaches undertaken in this study suggest biological clues for the enhanced rapamycin production of the SRMK07 strain. These clues can also serve as a basis for systematic engineering of a production host for further enhanced rapamycin production.

Enriching intracellular macrolides in Escherichia coli improved the sensitivity of bioluminescent sensing systems.

A repressor protein MphR and an enhanced green fluorescent protein (eGFP) were used to construct a bioluminescent sensing system for macrolide analysis in Escherichia coli host cells. We deleted TolC, an efflux pump for macrolides in E. coli, to promote the intracellular accumulation of macrolides. The binding constant (K1/2) of the sensing system constructed in an E. coli strain was decreased up to 33-fold with deleted TolC, and its sensitivity to the macrolides erythromycin, azithromycin, roxithromycin, and pikromycin was increased. The limit of detection of the bioluminescent sensing system for serum azithromycin was 4.1 nM. The ability to detect serum azithromycin concentrations was confirmed by analyzing photographs using ImageJ software. We also developed a novel sensing system for the immune suppressor FK506, another macrolide that is frequently prescribed. Deleting TolC also significantly improved the sensitivity of this sensing system. Bioluminescent sensing systems constructed in TolC mutants were sensitive to various macrolides, indicating their potential for clinical application with hand-held devices.

Systems metabolic engineering of Streptomyces venezuelae for the enhanced production of pikromycin.

Pikromycin is an important precursor of drugs, for example, erythromycin. Hence, systems metabolic engineering for the enhanced pikromycin production can contribute to the development of pikromycin-related drugs. In this study, metabolic genes in Streptomyces venezuelae were systematically engineered for enhanced pikromycin production. For this, a genome-scale metabolic model of S. venezuelae was reconstructed and simulated, which led to the selection of 11 metabolic gene targets. These metabolic genes, including four overexpression targets and seven knockdown targets, were individually engineered first. Next, two overexpression targets and two knockdown targets were selected based on the 11 strains' production performances to engineer two to four of these genes together for the potential synergistic effects on the pikromycin production. As a result, the NM1 strain with AQF52_RS24510 (methenyltetrahydrofolate cyclohydrolase/methylenetetrahydrofolate dehydrogenase) overexpression and AQF52_RS30320 (sulfite reductase) knockdown showed the best production performance among all the 22 strains constructed in this study. Fed-batch fermentation of the NM1 strain produced 295.25 mg/L of pikromycin, by far the best production titer using the native producer S. venezuelae, to the best of our knowledge. The systems metabolic engineering strategy demonstrated herein can also be applied to the overproduction of other secondary metabolites using S. venezuelae.

Poly-3-hydroxybutyrate production in acetate minimal medium using engineered Methylorubrum extorquens AM1.

Acetate is regarded as a sustainable microbial feedstock that is synthesized from biowastes such as synthesis gas (syngas), carbon dioxide, lignocellulose, or organic waste. In this study, Methylorubrum extorquens AM1 was engineered to improve the production of bioplastic poly-3-hydroxybutyrate (PHB) using acetate as the sole carbon source. To utilize acetate as a carbon source and methanol as an energy source, acs encoding acetyl-CoA synthetase and fdh from Burkholderia stabilis were overexpressed, while ftfL involved in the assimilation of methanol into formyl-tetrahydrofolate was deleted. The yields of biomass and PHB from acetate significantly improved, and the growth rate and PHB content of the bacteria increased. In addition, sustainability of the PHB production was demonstrated using acetate derived from carbon dioxide and syngas. This study shows that biopolymers could be synthesized efficiently using acetate as the sole carbon source through metabolic engineering and the supply of energy cofactors.

Strategies for Biosynthesis of C1 Gas-derived Polyhydroxyalkanoates: A review.

Biosynthesis of polyhydroxyalkanoates (PHAs) from C1 gases is highly desirable in solving problems such as climate change and microplastic pollution. PHAs are biopolymers synthesized in microbial cells and can be used as alternatives to petroleum-based plastics because of their biodegradability. Because 50% of the cost of PHA production is due to organic carbon sources and salts, the utilization of costless C1 gases as carbon sources is expected to be a promising approach for PHA production. In this review, strategies for PHA production using C1 gases through fermentation and metabolic engineering are discussed. In particular, autotrophs, acetogens, and methanotrophs are strains that can produce PHA from CO2, CO, and CH4. In addition, integrated bioprocesses for the efficient utilization of C1 gases are introduced. Biorefinery processes from C1 gas into bioplastics are prospective strategies with promising potential and feasibility to alleviate environmental issues.

Multi-Odor Discrimination by Rat Sniffing for Potential Monitoring of Lung Cancer and Diabetes.

The discrimination learning of multiple odors, in which multi-odor can be associated with different responses, is important for responding quickly and accurately to changes in the external environment. However, very few studies have been done on multi-odor discrimination by animal sniffing. Herein, we report a novel multi-odor discrimination system by detection rats based on the combination of 2-Choice and Go/No-Go (GNG) tasks into a single paradigm, in which the Go response of GNG was replaced by 2-Choice, for detection of toluene and acetone, which are odor indicators of lung cancer and diabetes, respectively. Three of six trained rats reached performance criterion, in 12 consecutive successful tests within a given set or over 12 sets with a success rate of over 90%. Through a total of 1300 tests, the trained animals (N = 3) showed multi-odor sensing performance with 88% accuracy, 87% sensitivity and 90% specificity. In addition, a dependence of behavior response time on odor concentrations under given concentration conditions was observed, suggesting that the system could be used for quantitative measurements. Furthermore, the animals' multi-odor sensing performance has lasted for 45 days, indicating long-term stability of the learned multi-odor discrimination. These findings demonstrate that multi-odor discrimination can be achieved by rat sniffing, potentially providing insight into the rapid, accurate and cost-effective multi-odor monitoring in the lung cancer and diabetes.