Simultaneous monitoring of each component on degradation of blended bioplastic using gas chromatography-mass spectrometry.

The increasing interest in bioplastics, with regard to future environmental issues, has rendered research on bioplastic biodegradation highly important. However, only a few tools directly monitor the degradation of bioplastics without measuring the levels of gaseous products, such as carbon dioxide. Classical nonquantitative methods, such as clear zone tests on solid plates, and less-sensitive weight-loss experiments in liquid media measured using a precision scale, are still employed to screen the microbial players associated with bioplastic degradation and monitor the biodegradation rates. However, the simultaneous monitoring of the degradation of each component of blended bioplastics has not been previously reported. In the present study, to provide information regarding the degradation rates and compositional changes of different bioplastics in a blend in a time-dependent manner, we simultaneously monitored and quantified the degradation of four bioplastics, polyhydroxybutyrate (PHB), polybutylene succinate (PBS), polycaprolactone (PCL), and poly(butylene adipate-co-terephthalate) (PBAT), by Bacillus sp. JY36 using gas chromatography-mass spectrometry (GC-MS) analysis after fatty acid methyl ester (FAME) derivatization. Our results demonstrate the feasibility of using the GC-MS-based method described here to obtain comprehensive data regarding blended bioplastics and their degradation. Moreover, our findings indicate that this method may support classical analytic tools for assessing bioplastic biodegradation.

6,8-Diprenylorobol induces apoptosis in human colon cancer cells via activation of intracellular reactive oxygen species and p53.

6,8-Diprenylorobol is a natural compound mainly found in Glycyrrhiza uralensis fisch and Maclura tricuspidata, which has been used traditionally as food and medicine in Asia. So far, the antiproliferative effect of 6,8-diprenylorobol has not been studied yet in colon cancer. In this study, we aimed to evaluate the antiproliferative effects of 6,8-diprenylorobol in LoVo and HCT15, two kinds of human colon cancer cells. 6,8-Diprenylorobol inhibited the proliferation of LoVo and HCT15 cells in a dose- and time-dependent manner. A 40 µM of 6,8-diprenylorobol for 72 h reduced both of cell viability under 50%. After treatment of 6,8-diprenylorobol (40 and 60 µM) for 72 h, late apoptotic cell portion in LoVo and HCT15 cells were 24, 70% and 13, 90%, respectively, which was confirmed by checking DNA fragmentation in both cells. Mechanistically, 6,8-diprenylorobol activated p53 and its phosphorylated form (Ser15, Ser20, and Ser46) expression but suppressed Akt and mitogen-activated protein kinases (MAPKs) phosphorylation in LoVo and HCT15 cells. Interestingly, 6,8-diprenylorobol induced the generation of intracellular reactive oxygen species (ROS), which was attenuated with N-acetyl cysteine (NAC) treatment. Compared to the control, 60 µM of 6,8-diprenylorobol caused to increase ROS level to 210% in LoVo and HCT15, which was reduced into 161% and 124%, respectively with NAC. Furthermore, cell viability and apoptotic cell portion by 6,8-diprenylorobol was recovered by incubation with NAC. Taken together, these results indicate that 6,8-diprenylorobol has the potential antiproliferative effect against LoVo and HCT15 colon cancer cells through activation of p53 and generation of ROS.

Improvement of cadaverine production in whole cell system with baker's yeast for cofactor regeneration.

Cadaverine, 1,5-diaminopentane, is one of the most promising chemicals for biobased-polyamide production and it has been successfully produced up to molar concentration. Pyridoxal 5'-phosphate (PLP) is a critical cofactor for inducible lysine decarboxylase (CadA) and is required up to micromolar concentration level. Previously the regeneration of PLP in cadaverine bioconversion has been studied and salvage pathway pyridoxal kinase (PdxY) was successfully introduced; however, this system also required a continuous supply of adenosine 5'-triphosphate (ATP) for PLP regeneration from pyridoxal (PL) which add in cost. Herein, to improve the process further a method of ATP regeneration was established by applying baker's yeast with jhAY strain harboring CadA and PdxY, and demonstrated that providing a moderate amount of adenosine 5'-triphosphate (ATP) with the simple addition of baker's yeast could increase cadaverine production dramatically. After optimization of reaction conditions, such as PL, adenosine 5'-diphosphate, MgCl2, and phosphate buffer, we able to achieve high production (1740 mM, 87% yield) from 2 M L-lysine. Moreover, this approach could give averaged 80.4% of cadaverine yield after three times reactions with baker's yeast and jhAY strain. It is expected that baker's yeast could be applied to other reactions requiring an ATP regeneration system.

Simultaneous monitoring of the bioconversion from lysine to glutaric acid by ethyl chloroformate derivatization and gas chromatography-mass spectrometry.

Glutaric acid is a precursor of a plasticizer that can be used for the production of polyester amides, ester plasticizer, corrosion inhibitor, and others. Glutaric acid can be produced either via bioconversion or chemical synthesis, and some metabolites and intermediates are produced during the reaction. To ensure reaction efficiency, the substrates, intermediates, and products, especially in the bioconversion system, should be closely monitored. Until now, high performance liquid chromatography (HPLC) has generally been used to analyze the glutaric acid-related metabolites, although it demands separate time-consuming derivatization and non-derivatization analyses. To substitute for this unreasonable analytical method, we applied herein a gas chromatography - mass spectrometry (GC-MS) method with ethyl chloroformate (ECF) derivatization to simultaneously monitor the major metabolites. We determined the suitability of GC-MS analysis using defined concentrations of six metabolites (l-lysine, cadaverine, 5-aminovaleric acid, 2-oxoglutaric acid, glutamate, and glutaric acid) and their mass chromatograms, regression equations, regression coefficient values (R2), dynamic ranges (mM), and retention times (RT). This method successfully monitored the production process in complex fermentation broth.

Metabolic engineering of Corynebacterium glutamicum for the production of glutaric acid, a C5 dicarboxylic acid platform chemical.

Corynebacterium glutamicum was metabolically engineered for the production of glutaric acid, a C5 dicarboxylic acid that can be used as platform building block chemical for nylons and plasticizers. C. glutamicum gabT and gabD genes and Pseudomonas putida davT and davD genes encoding 5-aminovalerate transaminase and glutarate semialdehyde dehydrogenase, respectively, were examined in C. glutamicum for the construction of a glutaric acid biosynthesis pathway along with P. putida davB and davA genes encoding lysine 2-monooxygenase and delta-aminovaleramidase, respectively. The glutaric acid biosynthesis pathway constructed in recombinant C. glutamicum was engineered by examining strong synthetic promoters PH30 and PH36, C. glutamicum codon-optimized davTDBA genes, and modification of davB gene with an N-terminal His6-tag to improve the production of glutaric acid. It was found that use of N-terminal His6-tagged DavB was most suitable for the production of glutaric acid from glucose. Fed-batch fermentation using the final engineered C. glutamicum H30_GAHis strain, expressing davTDA genes along with davB fused with His6-tag at N-terminus could produce 24.5 g/L of glutaric acid with low accumulation of l-lysine (1.7 g/L), wherein 5-AVA accumulation was not observed during fermentation.

Enhanced production of glutaric acid by NADH oxidase and GabD-reinforced bioconversion from l-lysine.

Glutaric acid is a promising alternative chemical to phthalate plasticizer since it can be produced by the bioconversion of lysine. Though, recent studies have enabled the high-yield production of its precursor, 5-aminovaleric acid (AMV), glutaric acid production via the AMV pathway has been limited by the need for cofactors. Introduction of NAD(P)H oxidase (Nox) with GabTD enzyme remarkably diminished the demand for oxidized nicotinamide adenine dinucleotide (NAD+ ). Supply of oxygen through vigorous shaking had a significant effect on the conversion of AMV with a reduced requirement of NAD + . A high conversion rate was achieved in Nox coupled GabTD reaction under optimized expression vector, terrific broth (TB), and pH 8.5 at high cell density. Supplementary expression of GabD resulted in the production of 353 ± 35 mM glutaric acid with 88.3 ± 8.7% conversion from 400 mM AMV. Moreover, the reaction with a higher concentration of AMV could produce 528 ± 21 mM glutaric acid with 66.0 ± 2.7% conversion. In addition, the co-biotransformation strategy of GabTD and DavBA whole cells could produce 282 mM glutaric acid with 70.8% conversion from lysine, compared to the 111 mM glutaric acid yield from the combined GabTD-DavBA system.

Effect of PelB signal sequences on Pfe1 expression and ω-hydroxyundec-9-enoic acid biotransformation in recombinant Escherichia coli.

ω-Hydroxyundec-9-enoic acid (ω-HUA) was reported as a valuable medium-chain fatty acid with industrial potentials. For bioconversion of ricinoleic acid to ω-HUA, in this study, an alcohol dehydrogenase (Adh) from Micrococcus luteus, a Baeyer-Villiger monooxygenase (BVMO) from Pseudomonas putida KT2440 and an esterase (Pfe1) from Pseudomonas fluorescens SIK WI were overexpressed in Escherichia coli BL21(DE3). In order to enhance accessibility of Pfe1 to the (E)-11-(heptanoyloxy) undec-9-enoic acid (11-HOUA) substrate, a truncated PelB signal sequence without the recognition site of signal peptidase (tPelB) was attached to the N-terminus of Pfe1, resulting in the construction of E. coli AB-tPE strain expressing Adh, BVMO and the tPelB-Pfe1 fusion protein. A batch-type biotransformation of ricinoleic acid by E. coli AB-tPE resulted in 1.8- and 2.2-fold increases in ω-HUA conversion yield and productivity, respectively, relative to those for the control strain without the PelB sequence (AB-E). By fed-batch-type biotransformation with glycerol feeding, the AB-tPE strain produced 23.7 mM (equivalent to 4.7 g/L) of ω-HUA with 60.8%(mol/mol) of conversion yield and 1.2 mM/h of productivity, which were 13.2, 2.9, and 2.6 times higher than those in a batch-type biotransformation using the AB-E strain. In conclusion, combination of the truncated PelB-Pfe1 fusion and fed-batch process with glycerol feeding provided the highest efficiency of ω-HUA biotransformation, of which strategies might be applicable for biotransformation of hydrophobic substances.

Nanoparticulation improves bioavailability of Erlotinib.

OBJECTIVES: Nanoparticulation using fat and supercritical fluid (NUFSTM) is a drug delivery platform technology enabling efficient and effective formulation of poorly soluble drugs. We performed experiments to examine whether NUFS™ could improve poor bioavailability and reduce fed-fasted bioavailability variances of erlotinib (Ert). METHODS: NUFS-Ert was prepared using NUFS™ technology; its physical properties were characterized, and drug release was measured. Furthermore, in vitro and in vivo efficacy tests and pharmacokinetic analysis were performed. RESULTS: NUFS-Ert nanoparticles had an average size of 250 nm and were stable for 2 months at 40 °C, 4 °C, and room temperature. The dissolution rate of NUFS-Ert increased in bio-relevant dissolution media. NUFS-Ert was more potent in inhibiting EGF signaling and in suppressing the proliferation of A549, a human non-small cell lung cancer cell line. Furthermore, A549 xenografts in BALB/c nude mice treated with NUFS-Ert regressed more efficiently than those in the mice treated with vehicle or Tarceva®. In addition, experimental lung metastasis was more efficiently inhibited by NUFS-Ert than by Tarceva®. The relative bioavailability of NUFS-Ert compared with that of Tarceva® was 550% and the ratio of the area under the concentration-time curve (AUC) of fed state to the AUC of fasted state was 1.8 for NUFS-Ert and 5.8 for Tarceva®. CONCLUSIONS: NUFS-Ert could improve poor bioavailability and reduce fed-fasted bioavailability variances of Ert. NUFS-Ert was more efficacious than Tarceva®.

Combinatorial application of two aldehyde oxidoreductases on isobutanol production in the presence of furfural.

Furfural is a toxic by-product formulated from pretreatment processes of lignocellulosic biomass. In order to utilize the lignocellulosic biomass on isobutanol production, inhibitory effect of the furfural on isobutanol production was investigated and combinatorial application of two oxidoreductases, FucO and YqhD, was suggested as an alternative strategy. Furfural decreased cell growth and isobutanol production when only YqhD or FucO was employed as an isobutyraldehyde oxidoreductase. However, combinatorial overexpression of FucO and YqhD could overcome the inhibitory effect of furfural giving higher isobutanol production by 110% compared with overexpression of YqhD. The combinatorial oxidoreductases increased furfural detoxification rate 2.1-fold and also accelerated glucose consumption 1.4-fold. When it compares to another known system increasing furfural tolerance, membrane-bound transhydrogenase (pntAB), the combinatorial aldehyde oxidoreductases were better on cell growth and production. Thus, to control oxidoreductases is important to produce isobutanol using furfural-containing biomass and the combinatorial overexpression of FucO and YqhD can be an alternative strategy.

Overview on the biotechnological production of L-DOPA.

L-DOPA (3,4-dihydroxyphenyl-L-alanine) has been widely used as a drug for Parkinson's disease caused by deficiency of the neurotransmitter dopamine. Since Monsanto developed the commercial process for L-DOPA synthesis for the first time, most of currently supplied L-DOPA has been produced by the asymmetric method, especially asymmetric hydrogenation. However, the asymmetric synthesis shows critical limitations such as a poor conversion rate and a low enantioselectivity. Accordingly, alternative biotechnological approaches have been researched for overcoming the shortcomings: microbial fermentation using microorganisms with tyrosinase, tyrosine phenol-lyase, or p-hydroxyphenylacetate 3-hydroxylase activity and enzymatic conversion by immobilized tyrosinase. Actually, Ajinomoto Co. Ltd commercialized Erwinia herbicola fermentation to produce L-DOPA from catechol. In addition, the electroenzymatic conversion system was recently introduced as a newly emerging scheme. In this review, we aim to not only overview the biotechnological L-DOPA production methods, but also to briefly compare and analyze their advantages and drawbacks. Furthermore, we suggest the future potential of biotechnological L-DOPA production as an industrial process.

Development of engineered Escherichia coli whole-cell biocatalysts for high-level conversion of L-lysine into cadaverine.

A whole-cell biocatalytic system for the production of cadaverine from L-lysine has been developed. Among the investigated lysine decarboxylases from different microorganisms, Escherichia coli LdcC showed the best performance on cadaverine synthesis when E. coli XL1-Blue was used as the host strain. Six different strains of E. coli expressing E. coli LdcC were investigated and recombinant E. coli XL1-Blue, BL21(DE3) and W were chosen for further investigation since they showed higher conversion yield of lysine into cadaverine. The effects of substrate pH, substrate concentrations, buffering conditions, and biocatalyst concentrations have been investigated. Finally, recombinant E. coli XL1-Blue concentrated to an OD(600) of 50, converted 192.6 g/L (1317 mM) of crude lysine solution, obtained from an actual lysine manufacturing process, to 133.7 g/L (1308 mM) of cadaverine with a molar yield of 99.90 %. The whole-cell biocatalytic system described herein is expected to be applicable to the development of industrial bionylon production process.

Customized optimization of cellulase mixtures for differently pretreated rice straw.

Lignocellulose contains a large amount of cellulose but is recalcitrant to enzymatic hydrolysis, which yields sugars for fuels or chemicals. Various pretreatment methods are used to improve the enzymatic digestibility of cellulose in lignocellulose. Depending on the lignocellulose types and pretreatment methods, biomass compositions and physical properties significantly vary. Therefore, customized enzyme mixtures have to be employed for the efficient hydrolysis of pretreated lignocellulose. Here, using three recombinant model enzymes consisting of endoglucanase, cellobiohydrolase, and xylanase with a fixed amount of ß-glucosidase, the optimal formulation of enzyme mixtures was designed for two differently pretreated rice straws (acid-pretreated or alkali-pretreated rice straw) by the mixture design methodology. As a result, different optimal compositions for the enzyme mixtures were employed depending on the type of pretreatment of rice straw. These results suggest that customized enzyme mixtures for pretreated lignocellulosic biomass are necessary to obtain increased sugar yields and should be considered in the industrial utilization of lignocellulose.

Biotransformation of lysine into cadaverine using barium alginate-immobilized Escherichia coli overexpressing CadA.

In this study, Escherichia coli cells overexpressing lysine decarboxylase (CadA) were used for cadaverine production. Barium alginate was selected as a matrix for immobilization of E. coli YH91. Free cells and immobilized cells (IC) were characterized for their physiochemical properties, and the optimum pH and temperature were determined as 6 and 37 °C, respectively. Immobilized cells were three times more thermally stable compared to free cells at the optimum temperature and had a half-life (t 1/2) of 131 h. The free cells lost most of lysine decarboxylase activity after nine cycles, but in contrast immobilized cells retained 56% of their residual activity even after the 18th cycle. The immobilized cells gave a maximum production of cadaverine (75.8 g/L) with 84% conversion.

Increasing lipid production in Chlamydomonas reinhardtii through genetic introduction for the overexpression of glyceraldehyde-3-phosphate dehydrogenase.

Microalgae, valued for their sustainability and CO2 fixation capabilities, are emerging as promising sources of biofuels and high-value compounds. This study aimed to boost lipid production in C. reinhardtii by overexpressing chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the Calvin cycle and glycolysis, under the control of a nitrogen-inducible NIT1 promoter, to positively impact overall carbon metabolism. The standout transformant, PNG#7, exhibited significantly increased lipid production under nitrogen starvation, with biomass rising by 44% and 76% on days 4 and 16, respectively. Fatty acid methyl ester (FAME) content in PNG#7 surged by 2.4-fold and 2.1-fold, notably surpassing the wild type (WT) in lipid productivity by 3.4 and 3.7 times on days 4 and 16, respectively. Transcriptome analysis revealed a tenfold increase in transgenic GAPDH expression and significant upregulation of genes involved in fatty acid and triacylglycerol synthesis, especially the gene encoding acyl-carrier protein gene (ACP, Cre13. g577100. t1.2). In contrast, genes related to cellulose synthesis were downregulated. Single Nucleotide Polymorphism (SNP)/Indel analysis indicated substantial DNA modifications, which likely contributed to the observed extensive transcriptomic and phenotypic changes. These findings suggest that overexpressing chloroplast GAPDH, coupled with genetic modifications, effectively enhances lipid synthesis in C. reinhardtii. This study not only underscores the potential of chloroplast GAPDH overexpression in microalgal lipid synthesis but also highlights the expansive potential of metabolic engineering in microalgae for biofuel production.

Optimization and mechanism analysis of photosynthetic EPA production in Nannochloropsis salina: Evaluating the effect of temperature and nitrogen concentrations.

Microalgae, recognized as sustainable and eco-friendly photosynthetic microorganisms, play a pivotal role in converting CO2 into value-added products. Among these, Nannochloropsis salina (Microchloropsis salina) stands out, particularly for its ability to produce eicosapentaenoic acid (EPA), a crucial omega-3 fatty acid with significant health benefits such as anti-inflammatory properties and cardiovascular health promotion. This study focused on optimizing the cultivation conditions of Nannochloropsis salina to maximize EPA production. We thoroughly investigated the effects of varying temperatures and nitrogen (NaNO3) concentrations on biomass, total lipid content, and EPA proportions. We successfully identified optimal conditions at an initial NaNO3 concentration of 1.28 g.L-1 and a temperature of 21 °C. This condition was further validated by response surface methodology, which resulted in the highest EPA productivity reported in batch systems (14.4 mg.L-1.day-1). Quantitative real-time PCR and transcriptomic analysis also demonstrated a positive correlation between specific gene expressions and enhanced EPA production. Through a comprehensive lipid analysis and photosynthetic pigment analysis, we deduced that the production of EPA in Nannochloropsis salina seemed to be produced by the remodeling of chloroplast membrane lipids. These findings provide crucial insights into how temperature and nutrient availability influence fatty acid composition in N. salina, offering valuable guidance for developing strategies to improve EPA production in various microalgae species.

Establishment of efficient 5-hydroxyvaleric acid production system by regenerating alpha-ketoglutaric acid and its application in poly(5-hydroxyvaleric acid) production.

5-hydroxyvaleric acid (5-HV) is a versatile C5 intermediate of bio-based high-value chemical synthesis pathways. However, 5-HV production faces a few shortcomings involving the supply of cofactors, especially α-ketoglutaric acid (α-KG). Herein, we established a two-cell biotransformation system by introducing L-glutamate oxidase (GOX) to regenerate α-KG. Additionally, the catalase KatE was adapted to inhibit α-KG degradation by the H2O2 produced during GOX reaction. We searched for the best combination of genes and vectors and optimized the biotransformation conditions to maximize GOX effectiveness. Under the optimized conditions, 5-HV pathway with GOX showed 1.60-fold higher productivity than that of without GOX, showing 11.3 g/L titer. Further, the two-cell system with GOX and KatE was expanded to produce poly(5-hydroxyvaleric acid) (P(5HV)), and it reached at 412 mg/L of P(5HV) production and 20.5% PHA contents when using the biotransformation supernatant. Thus, the two-cell biotransformation system with GOX can potentially give the practical and economic alternative of 5-HV production using bio-based methods. We also propose direct utilization of 5-HV from bioconversion for P(5HV) production.

Enhanced theanine production with reduced ATP supply by alginate entrapped Escherichia coli co-expressing γ-glutamylmethylamide synthetase and polyphosphate kinase.

L-theanine is an amino acid with a unique flavor and many therapeutic effects. Its enzymatic synthesis has been actively studied and γ-Glutamylmethylamide synthetase (GMAS) is one of the promising enzymes in the biological synthesis of theanine. However, the theanine biosynthetic pathway with GMAS is highly ATP-dependent and the supply of external ATP was needed to achieve high concentration of theanine production. As a result, this study aimed to investigate polyphosphate kinase 2 (PPK2) as ATP regeneration system with hexametaphosphate. Furthermore, the alginate entrapment method was employed to immobilize whole cells containing both gmas and ppk2 together resulting in enhanced reusability of the theanine production system with reduced supply of ATP. After immobilization, theanine production was increased to 239 mM (41.6 g/L) with a conversion rate of 79.7% using 15 mM ATP and the reusability was enhanced, maintaining a 100% conversion rate up to the fifth cycles and 60% of conversion up to eighth cycles. It could increase long-term storage property for future uses up to 35 days with 75% activity of initial activity. Overall, immobilization of both production and cofactor regeneration system could increase the stability and reusability of theanine production system.

Effect of pH on the metabolic flux of Klebsiella oxytoca producing 2,3-butanediol in continuous cultures at different dilution rates.

The efficiency of the bioconversion process and the achievable end-product concentration decides the economic feasibility of microbial 2,3-butanediol (2,3-BDO) production. In 2,3-BDO production, optimization of culture condition is required for cell growth and metabolism. Also, the pH is an important factor that influences microbial performance. For different microorganisms and substrates, it has been shown that the distribution of the metabolites in 2,3-BDO fermentation is greatly affected by pH, and the optimum pH for 2,3-BDO production seems dependently linked to the particular strain and the substrate employed. Quantification analysis of intracellular metabolites and metabolic flux analysis (MFA) were used to investigate the effect of pH on the Klebsiella oxytoca producing 2,3-BDO and other organic acids. The main objectives of MFA are the estimation of intracellular metabolic fluxes and the identification of rate-limiting step and the key enzymes. This study was conducted under continuous aerobic conditions at different dilution rates (0.1, 0.2, and 0.3 h(-1)) and different pH values (pH 5.5 and 7.0) for the steady-state experimental data. In order to obtain the flux distribution, the extracellular specific rates were calculated from the experimental data using the metabolic network model of K. oxytoca. Intracellular metabolite concentration profiles were generated using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Enhanced production of glutaric acid by biocatalyst-recycled bioconversion process integrated with in situ product recovery by adsorption.

Product inhibition caused by organic acids is a serious issue in establishing economical biochemical production systems. Herein, for enhanced production of glutaric acid by overcoming product inhibition triggered by glutaric acid, a whole-cell bioconversion system equipped with biocatalyst recycling process and in situ product recovery by adsorption was developed successfully. From the whole-cell bioconversion reaction, we found that both dissociated and undissociated forms of glutaric acid acted as an inhibitor in the whole-cell bioconversion reaction, wherein bioconversion was hindered beyond 200 mM glutaric acid regardless of reaction pH. Therefore, as the promising solution for the inhibition issue by glutaric acid, the biocatalyst-recycled bioconversion process integrated with in situ product recovery by adsorption was introduced in the whole-cell bioconversion. As a result, 592 mM glutaric acid was produced from 1000 mM 5-aminovaleric acid with 59.2% conversion. We believe that our system will be a promising candidate for economically producing organic acids with high titer.

Complementation of reducing power for 5-hydroxyvaleric acid and 1,5-pentanediol production via glucose dehydrogenase in Escherichia coli whole-cell system.

One of the key intermediates, 5-hydroxyvaleric acid (5-HV), is used in the synthesis of polyhydroxyalkanoate monomer, δ-valerolactone, 1,5-pentanediol (1,5-PDO), and many other substances. Due to global environmental problems, eco-friendly bio-based synthesis of various platform chemicals and key intermediates are socially required, but few previous studies on 5-HV biosynthesis have been conducted. To establish a sustainable bioprocess for 5-HV production, we introduced gabT encoding 4-aminobutyrate aminotransferase and yqhD encoding alcohol dehydrogenase to produce 5-HV from 5-aminovaleric acid (5-AVA), through glutarate semialdehyde in Escherichia coli whole-cell reaction. As, high reducing power is required to produce high concentrations of 5-HV, we newly introduced glucose dehydrogenase (GDH) for NADPH regeneration system from Bacillus subtilis 168. By applying GDH with D-glucose and optimizing the parameters, 5-HV conversion rate from 5-AVA increased from 47% (w/o GDH) to 82% when using 200 mM (23.4 g/L) of 5-AVA. Also, it reached 56% conversion in 2 h, showing 56 mM/h (6.547 g/L/h) productivity from 200 mM 5-AVA, finally reaching 350 mM (41 g/L) and 14.6 mM/h (1.708 g/L/h) productivity at 24 h when 1 M (117.15 g/L) 5-AVA was used. When the whole-cell system with GDH was expanded to produce 1,5-PDO, its production was also increased 5-fold. Considering that 5-HV and 1,5-PDO production depends heavily on the reducing power of the cells, we successfully achieved a significant increase in 5-HV and 1,5-PDO production using GDH.