S-adenosyl-L-methionine Production Research Articles

The SMYD3-dependent H3K4me3 status of IGF2 intensifies local Th2 differentiation in CRSwNP via positive feedback

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a heterogeneous and common upper airway disease divided into various inflammatory endotypes. Recent epidemiological findings showed a T helper 2 (Th2)-skewed dominance in CRSwNP patients. Histone modification alterations can regulate transcriptional and translational expression, resulting in abnormal pathogenic changes and the occurrence of diseases. Trimethylation of histone H3 lysine 4 (H3K4me3) is considered an activator of gene expression through modulation of accessibility for transcription, which is closely related to CRSwNP. H3K4me3 levels in the human nasal epithelium may change under Th2-biased inflammatory conditions, resulting in exaggerated local nasal Th2 responses via the regulation of naïve CD4+ T-cell differentiation. Here, we revealed that the level of SET and MYND domain-containing protein 3 (SMYD3)-mediated H3K4me3 was increased in NPs from Th2 CRSwNP patients compared with those from healthy controls. We demonstrated that SMYD3-mediated H3K4me3 is increased in human nasal epithelial cells under Th2-biased inflammatory conditions via S-adenosyl-L-methionine (SAM) production and further found that the H3K4me3high status of insulin-like growth factor 2 (IGF2) produced in primary human nasal epithelial cells could promote naïve CD4+ T-cell differentiation into Th2 cells. Moreover, we found that SAM production was dependent on the c-Myc/methionine adenosyltransferase 2A (MAT2A) axis in the nasal epithelium. Understanding histone modifications in the nasal epithelium has immense potential utility in the development of novel classes of therapeutics targeting Th2 polarization in Th2 CRSwNP.Et9-hJKHbB2Mg3CRtXWvn_Video

Characterization and designing of an SAM riboswitch to establish a high-throughput screening platform for SAM overproduction in Saccharomyces cerevisiae.

S-adenosyl- l-methionine (SAM)is a high-value compound widely used in the treatment of various diseases. SAM can be produced through fermentation, but further enhancing the microbial production of SAM requires novel high-throughput screening methods for rapid detection and screening of mutant libraries. In this work, anSAM-OFF riboswitch capable of responding to the SAM concentration was obtained and a high-throughput platform for screening SAM overproducers was established. SAM synthase was engineered by semirational design and directed evolution, which resulted in the SAM2S203F,W164R,T251S,Y285F,S365R mutant with almost twice higher catalytic activity than the parental enzyme. The best mutant was then introduced into Saccharomyces cerevisiae BY4741, and the resulting strain BSM8 produced a sevenfold higher SAM titer in shake-flask fermentation, reaching 1.25 g L-1 . This work provides a reference for designing biosensors to dynamically detect metabolite concentrations for high-throughput screening and the construction of effective microbial cell factories.

Breeding of Saccharomyces cerevisiae with a High-Throughput Screening Strategy for Improvement of S-Adenosyl-L-Methionine Production.

S-adenosyl-l-methionine (SAM), a vital physiologically active substance in living organisms, is produced by fermentation over Saccharomyces cerevisiae. The main limitation in SAM production was the low biosynthesis ability of SAM in S. cerevisiae. The aim of this work is to breed an SAM-overproducing mutant through UV mutagenesis coupled with high-throughput selection. Firstly, a high-throughput screening method by rapid identification of positive colonies was conducted. White colonies on YND medium were selected as positive strains. Then, nystatin/sinefungin was chosen as a resistant agent in directed mutagenesis. After several cycles of mutagenesis, a stable mutant 616-19-5 was successfully obtained and exhibited higher SAM production (0.41g/L vs 1.39g/L). Furthermore, the transcript levels of the genes SAM2, ADO1, and CHO2 involved in SAM biosynthesis increased, while ergosterol biosynthesis genes in mutant 616-19-5 significantly decreased. Finally, building on the above work, S. cerevisiae 616-19-5 could produce 10.92 ± 0.2g/L SAM in a 5-L fermenter after 96h of fermentation, showing a 2.02-fold increase in the product yield compared with the parent strain. Paving the way of breeding SAM-overproducing strain has improved the good basis for SAM industrial production.

Improving ATP availability by sod1 deletion with a strategy of precursor feeding enhanced S-adenosyl-L-methionine accumulation in Saccharomyces cerevisiae

S-adenosyl-L-methionine (SAM), used in diverse pharmaceutical applications, was biosynthesized from L-methionine (L-met) and adenosine triphosphate (ATP). This study aims to increase the accumulation of SAM in Saccharomyces cerevisiae by promoting ATP availability. Strain ΔSOD1 was obtained from the parent strain WT15–33 (CCTCC M 2021915) by deleting gene sod1, which improved the supply of ATP. The SAM content in strain ΔSOD1 exhibited a 22.3% improvement compared to the parent strain, which reached 93.6 mg g−1. The transformation of NADH (reduced nicotinamide adenine dinucleotide) and the relative expression of ATPase essential genes were investigated, respectively. The results showed that the lack of gene sod1 benefited the generation of ATP, which positively regulated the synthesis of SAM. Besides that, the production of SAM was further enhanced by improving substrate assimilation. With the infusion of 1.44 g L−1L-met and 0.60 g L−1 adenosine at 24 h (h) and 0 h following fermentation, the optimum medium could produce 1.54 g L−1 SAM. Based on the regulations mentioned above, the SAM concentration of strain ΔSOD1 enhanced from 7.3 g L−1 to 10.1 g L−1 in a 5-L fermenter in 118 h. This work introduces a novel idea for the biosynthesis of ATP and SAM, and the strain ΔSOD1 has the potential for industrial production.

The multiple effects of REG1 deletion and SNF1 overexpression improved the production of S-adenosyl-l-methionine in Saccharomyces cerevisiae

BackgroundSaccharomyces cerevisiae is often used as a cell factory for the production of S-adenosyl-l-methionine (SAM) for diverse pharmaceutical applications. However, SAM production by S. cerevisiae is negatively influenced by glucose repression, which is regulated by a serine/threonine kinase SNF1 complex. Here, a strategy of alleviating glucose repression by deleting REG1 (encodes the regulatory subunit of protein phosphatase 1) and overexpressing SNF1 (encodes the catalytic subunit of the SNF1 complex) was applied to improve SAM production in S. cerevisiae. SAM production, growth conditions, glucose consumption, ethanol accumulation, lifespan, glycolysis and amino acid metabolism were analyzed in the mutant strains.ResultsThe results showed that the multiple effects of REG1 deletion and/or SNF1 overexpression exhibited a great potential for improving the SAM production in yeast. Enhanced the expression levels of genes involved in glucose transport and glycolysis, which improved the glucose utilization and then elevated the levels of glycolytic intermediates. The expression levels of ACS1 (encoding acetyl-CoA synthase I) and ALD6 (encoding aldehyde dehydrogenase), and the activity of alcohol dehydrogenase II (ADH2) were enhanced especially in the presence of excessive glucose levels, which probably promoted the conversion of ethanol in fermentation broth into acetyl-CoA. The gene expressions involved in sulfur-containing amino acids were also enhanced for the precursor amino acid biosynthesis. In addition, the lifespan of yeast was extended by REG1 deletion and/or SNF1 overexpression. As expected, the final SAM yield of the mutant YREG1ΔPSNF1 reached 8.28 g/L in a 10-L fermenter, which was 51.6% higher than the yield of the parent strain S. cerevisiae CGMCC 2842.ConclusionThis study showed that the multiple effects of REG1 deletion and SNF1 overexpression improved SAM production in S. cerevisiae, providing new insight into the application of the SNF1 complex to abolish glucose repression and redirect carbon flux to nonethanol products in S. cerevisiae.

Identification of novel metabolic engineering targets for S-adenosyl-L-methionine production in Saccharomyces cerevisiae via genome-scale engineering

S-Adenosyl-L-methionine (SAM) is an important intracellular metabolite and widely used for treatment of various diseases. Although high level production of SAM had been achieved in yeast, novel metabolic engineering strategies are needed to further enhance SAM production for industrial applications. Here genome-scale engineering (GSE) was performed to identify new targets for SAM overproduction using the multi-functional genome-wide CRISPR (MAGIC) system, and the effects of these newly identified targets were further validated in industrial yeast strains. After 3 rounds of FACS screening and characterization, numerous novel targets for enhancing SAM production were identified. In addition, transcriptomic and metabolomic analyses were performed to investigate the molecular mechanisms for enhanced SAM accumulation. The best combination (upregulation of SNZ3, RFC4, and RPS18B) improved SAM productivity by 2.2-fold and 1.6-fold in laboratory and industrial yeast strains, respectively. Using GSE of laboratory yeast strains to guide industrial yeast strain engineering presents an effective approach to design microbial cell factories for industrial applications.

Increasing glycolysis by deletion of kcs1 and arg82 improved S-adenosyl-l-methionine production in Saccharomyces cerevisiae

Reprogramming glycolysis for directing glycolytic metabolites to a specific metabolic pathway is expected to be useful for increasing microbial production of certain metabolites, such as amino acids, lipids or considerable secondary metabolites. In this report, a strategy of increasing glycolysis by altering the metabolism of inositol pyrophosphates (IPs) for improving the production of S-adenosyl-l-methionine (SAM) for diverse pharmaceutical applications in yeast is presented. The genes associated with the metabolism of IPs, arg82, ipk1 and kcs1, were deleted, respectively, in the yeast strain Saccharomyces cerevisiae CGMCC 2842. It was observed that the deletions of kcs1 and arg82 increased SAM by 83.3 % and 31.8 %, respectively, compared to that of the control. In addition to the improved transcription levels of various glycolytic genes and activities of the relative enzymes, the levels of glycolytic intermediates and ATP were also enhanced. To further confirm the feasibility, the kcs1 was deleted in the high SAM-producing strain Ymls1ΔGAPmK which was deleted malate synthase gene mls1 and co-expressed the Acetyl-CoA synthase gene acs2 and the SAM synthase gene metK1 from Leishmania infantum, to obtain the recombinant strain Ymls1Δkcs1ΔGAPmK. The level of SAM in Ymls1Δkcs1ΔGAPmK reached 2.89 g L−1 in a 250-mL flask and 8.86 g L−1 in a 10-L fermentation tank, increasing 30.2 % and 46.2 %, respectively, compared to those levels in Ymls1ΔGAPmK. The strategy of increasing glycolysis by deletion of kcs1 and arg82 improved SAM production in yeast.

Lipid Composition of Sheffersomyces stipitis M12 Strain Grown on Glycerol as a Carbon Source.

SUMMARYResearch backgroundIn this study the content and composition of lipids in ergosterol-reduced Sheffersomyces stipitis M12 strain grown on glycerol as a carbon source is determined. Blocking the ergosterol synthesis route in yeast cells is a recently proposed method for increasing S-adenosyl-l-methionine (SAM) production.Experimental approachThe batch cultivation of M12 yeast was carried out under aerobic conditions in a laboratory bioreactor with glycerol as carbon source, and with pulsed addition of methionine. Glycerol and SAM content were monitored by high-performance liquid chromatography, while fatty acid composition of different lipid classes, separated by solid phase extraction, was determined by gas chromatography.Results and conclusionDespite the reduced amount of ergosterol in yeast cells, thanks to the reorganized lipid metabolism, M12 strain achieved high biomass yield and SAM production. Neutral lipids prevailed (making more than 75% of total lipids), but their content and composition differed significantly in the two tested types of yeast. Unsaturated and C18 fatty acids prevailed in both the M12 strain and wild type. In all fractions except free fatty acids, the index of unsaturation in M12 strain was lower than in the wild strain. Our tested strain adjusts itself by changing the content of lipids (mainly phospholipids, sterols and sterol esters), and with desaturation adjustments, to maintain proper functioning and fulfil increased energy needs.Novelty and scientific contributionReorganization of S. stipitis lipid composition caused by blocking the metabolic pathway of ergosterol synthesis was presented. A simple scheme of actual lipid metabolism during active SAM production in S. stipitis, grown on glycerol was constructed and shown. This fundamental knowledge of lipid metabolic pathways will be a helpful tool in improving S. stipitis as an expression host and a model organism, opening new perspectives for its applied research.

Engineering Pichia pastoris to improve S-adenosyl- l-methionine production using systemsmetabolic strategies.

S-adenosyl-l-methionine (SAM) is a highly valued chemical that can be used as a dietary supplement and has been used to treat depression, osteoarthritis, and liver problems as well. We adopted systems metabolic engineering strategies to improve SAM production in a high-producing strain (GS115/DS56). First, the cystathionine β-synthase gene CYS4 was downregulated using a weak promoter PG12 to reduce the removal of homocysteine from SAM cycle, thus leading to a 48.8% increase in the SAM titer (1.68 g/L) from the strain G12-CBS, while preventing cysteine auxotrophy induced by deletion of this essential gene. Subsequently, the SAM titer of G12-CBS was improved to 13.01 g/L in 15-L fed-batch fermentation using the optimal l-methionine feeding strategy. Finally, based on comparative transcriptomics, five genes were chosen and overexpressed for further enhancement of SAM production. Among them, GDH2 and ACS2 exhibited positive effects, and the additional overexpression of GDH2 led to a 52.3% increase of titer (2.71 g/L) in shake flask culture. Therefore, the engineered Pichia pastoris strains can be utilized in industrial production of SAM using a simple and cost-effective process, and these approaches could be employed for improving the production of other chemicals by P. pastoris.

Improved S-adenosyl-l-methionine production in Saccharomyces cerevisiae using tofu yellow serofluid

S-adenosyl-l-methionine (SAM) has been attracting increasing attention because of its significance in the pharmaceutical industry; however, the high cost of this compound limits its application. Tofu yellow serofluid exhibits high nutritional value and is not costly; therefore, it can be utilized as a substrate in the fermentation industry. In the current study, Saccharomyces cerevisiae was cultured in the tofu yellow serofluid fermentation medium for the SAM biosynthesis. The optimum tofu yellow serofluid fermentation medium contained 70 g/L of glucose, 30 % of yellow serofluid, 20 g/L of l-methionine, and 2.5 g/L of ammonium citrate. Under these conditions, the optimum feeding strategy was established. The results revealed that the dry cell weight (DCW) reached 123.1 g/L, the maximum production of SAM was 16.14 g/L, the highest SAM productivity reached 1.048 g/L/h, and SAM content was determined at 131.1 mg/g DCW. Furthermore, addition of tofu yellow serofluid reduced the average cost of SAM by 31.9 % to compare with the culture process without addition of tofu yellow serofluid. Thus, the tofu yellow serofluid fermentation medium improved the production of SAM and significantly reduced the production costs.

Efficient production of S-adenosyl-l-methionine from dl-methionine in metabolic engineered Saccharomyces cerevisiae.

S-Adenosyl-l-methionine (SAM) is an important small molecule compound widely used in treating various diseases. Although l-methionine is generally used, the low-cost dl-methionine is more suitable as the substrate for industrial production of SAM. However, d-methionine is inefficient for SAM formation due to the substrate-specificity of SAM synthetase. In order to increase the utilization efficiency of dl-methionine, intracellular conversion of d-methionine to l-methionine was investigated in the type strain Saccharomyces cerevisiae BY4741 and an industrial strain S. cerevisiae HDL. Firstly, via disruption of HPA3 encoding d-amino acid-N-acetyltransferase, d-methionine was accumulated in vivo and no N-acetyl-d-methionine production was observed. Further, codon-optimized d-amino acid oxidase (DAAO) gene from Trigonopsis variabilis (Genbank MK280686) and l-phenylalanine dehydrogenase gene (l-PheDH) from Rhodococcus jostii (Genbank MK280687) were introduced to convert d-methionine to l-methionine, SAM concentration and content was increased by 110% and 72.1% in BY4741 (plasmid borne) and increased by 38.2% and 34.1% in HDL (genome integrated), by feeding 0.5 g/L d-methionine. Using the recently developed CRISPR tools, the DAAO and l-PheDH expression cassettes were integrated into the HPA3 and SAH1 loci while SAM2 expression was integrated into the SPE2 and GLC3 loci of HDL, and the resultant strain HDL-R2 accumulated 289% and 192% more SAM concentration and content, respectively, by feeding 0.5 g/L dl-methionine. Further, in a 10 L fed-batch fermentation process, 10.3 g/L SAM were accumulated with the SAM content of 242 mg/g dry cell weight by feeding 16 g/L dl-methionine. The strategies used here provided a promising approach to enhance SAM production using low-cost dl-methionine.

13C-metabolic flux analysis of ethanol-assimilating Saccharomyces cerevisiae for S-adenosyl-l-methionine production

BackgroundSaccharomyces cerevisiae is a host for the industrial production of S-adenosyl-l-methionine (SAM), which has been widely used in pharmaceutical and nutritional supplement industries. It has been reported that the intracellular SAM content in S. cerevisiae can be improved by the addition of ethanol during cultivation. However, the metabolic state in ethanol-assimilating S. cerevisiae remains unclear. In this study, 13C-metabolic flux analysis (13C-MFA) was conducted to investigate the metabolic regulation responsible for the high SAM production from ethanol.ResultsThe comparison between the metabolic flux distributions of central carbon metabolism showed that the metabolic flux levels of the tricarboxylic acid cycle and glyoxylate shunt in the ethanol culture were significantly higher than that of glucose. Estimates of the ATP balance from the 13C-MFA data suggested that larger amounts of excess ATP was produced from ethanol via increased oxidative phosphorylation. The finding was confirmed by the intracellular ATP level under ethanol-assimilating condition being similarly higher than glucose.ConclusionsThese results suggest that the enhanced ATP regeneration due to ethanol assimilation was critical for the high SAM accumulation.

Improving the productivity of S-adenosyl-l-methionine by metabolic engineering in an industrial Saccharomyces cerevisiae strain

S-Adenosyl-l-methionine (SAM) is an important metabolite having prominent roles in treating various diseases. In order to improve the production of SAM, the regulation of three metabolic pathways involved in SAM biosynthesis were investigated in an industrial yeast strain ZJU001. GLC3 encoded glycogen-branching enzyme (GBE), SPE2 encoded SAM decarboxylase, as well as ERG4 and ERG6 encoded key enzymes in ergosterol biosynthesis, were knocked out in ZJU001 accordingly. The results indicated that blocking of either glycogen pathway or SAM decarboxylation pathway could improve the SAM accumulation significantly in ZJU001, while single disruption of either ERG4 or ERG6 gene had no obvious effect on SAM production. Moreover, the double mutant ZJU001-GS with deletion of both GLC3 and SPE2 genes was also constructed, which showed further improvement of SAM accumulation. Finally, SAM2 was overexpressed in ZJU001-GS to give the best SAM-producing recombinant strain ZJU001-GS-SAM2, in which 12.47g/L SAM was produced by following our developed pseudo-exponential fed-batch cultivation strategy, about 81.0% increase comparing to its parent strain ZJU001. The present work laid a solid base for large-scale SAM production with the industrial Saccharomyces cerevisiae strain.

Progress in the microbial production of S-adenosyl-l-methionine

S-Adenosyl-L-methionine (SAM), which exists in all living organisms, serves as an activated group donor in a range of metabolic reactions, including trans-methylation, trans-sulfuration and trans-propylamine. Compared with its chemical synthesis and enzyme catalysis production, the microbial production of SAM is feasible for industrial applications. The current clinical demand for SAM is constantly increasing. Therefore, vast interest exists in engineering the SAM metabolism in cells for increasing product titers. Here, we provided an overview of updates on SAM microbial productivity improvements with an emphasis on various strategies that have been used to enhance SAM production based on increasing the precursor and co-factor availabilities in microbes. These strategies included the sections of SAM-producing microbes and their mutant screening, optimization of the fermentation process, and the metabolic engineering. The SAM-producing strains that were used extensively were Saccharomyces cerevisiae, Pichia pastoris, Candida utilis, Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Corynebacterium glutamicum, and Escherichia coli, in addition to others. The optimization of the fermentation process mainly focused on the enhancement of the methionine, ATP, and other co-factor levels through pulsed feeding as well as the optimization of nitrogen and carbon sources. Various metabolic engineering strategies using precise control of gene expression in engineered strains were also highlighted in the present review. In addition, some prospects on SAM microbial production were discussed.

Metabolome analysis of Saccharomyces cerevisiae and optimization of culture medium for S-adenosyl-L-methionine production.

S-Adenosyl-l-methionine (SAM) is a fine chemical used as a nutritional supplement and a prescription drug. It is industrially produced using Saccharomyces cerevisiae owing to its high SAM content. To investigate the optimization of culture medium components for higher SAM production, metabolome analysis was conducted to compare the intracellular metabolite concentrations between Kyokai no. 6 (high SAM-producing) and laboratory yeast S288C (control) under different SAM production conditions. Metabolome analysis and the result of principal component analysis showed that the rate-limiting step for SAM production was ATP supply and the levels of degradation products of adenosine nucleotides were higher in Kyokai 6 strain than in the S288C strain under the l-methionine supplemented condition. Analysis of ATP accumulation showed that the levels of intracellular ATP in the Kyokai 6 strain were also higher compared to those in the S288C strain. Furthermore, as expected from metabolome analysis, the SAM content of Kyokai 6 strain cultivated in the medium without yeast extract increased by 2.5-fold compared to that in the additional condition, by increasing intracellular ATP level with inhibited cell growth. These results suggest that high SAM production is attributed to the enhanced ATP supply with l-methionine condition and high efficiency of intracellular ATP consumption.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0210-3) contains supplementary material, which is available to authorized users.

Characterization of a S-adenosyl-l-methionine (SAM)-accumulating strain of Scheffersomyces stipitis.

S-adenosyl-l-methionine (SAM) is an important molecule in the cellular metabolism of mammals. In this study, we examined several of the physiological characteristics of a SAM-accumulating strain of the yeast Scheffersomyces stipitis (M12), including SAM production, ergosterol content, and ethanol tolerance. S. stipitis M12 accumulated up to 52.48 mg SAM/g dry cell weight. Proteome analyses showed that the disruption of C-24 methylation in ergosterol biosynthesis, a step mediated by C-24 sterol methyltransferase (Erg6p), results in greater SAM accumulation by S. stipitis M12 compared to the wild-type strain. A comparative proteome-wide analysis identified 25 proteins that were differentially expressed by S. stipitis M12. These proteins are involved in ribosome biogenesis, translation, the stress response, ubiquitin-dependent catabolic processes, the cell cycle, ethanol tolerance, posttranslational modification, peroxisomal membrane stability, epigenetic regulation, the actin cytoskeleton and cell morphology, iron and copper homeostasis, cell signaling, and energy metabolism.

13C-metabolic flux analysis in S-adenosyl-l-methionine production by Saccharomyces cerevisiae

S-Adenosyl-L-methionine (SAM) is a major biological methyl group donor, and is used as a nutritional supplement and prescription drug. Yeast is used for the industrial production of SAM owing to its high intracellular SAM concentrations. To determine the regulation mechanisms responsible for such high SAM production, (13)C-metabolic flux analysis ((13)C-MFA) was conducted to compare the flux distributions in the central metabolism between Kyokai no. 6 (high SAM-producing) and S288C (control) strains. (13)C-MFA showed that the levels of tricarboxylic acid (TCA) cycle flux in SAM-overproducing strain were considerably increased compared to those in the S228C strain. Analysis of ATP balance also showed that a larger amount of excess ATP was produced in the Kyokai 6 strain because of increased oxidative phosphorylation. These results suggest that high SAM production in Kyokai 6 strains could be attributed to enhanced ATP regeneration with high TCA cycle fluxes and respiration activity. Thus, maintaining high respiration efficiency during cultivation is important for improving SAM production.

Enhancing precursors availability in Pichia pastoris for the overproduction of S-adenosyl-l-methionine employing molecular strategies with process tuning

S-Adenosyl-l-methionine (SAM) is an important metabolite having prominent role in treating various diseases. Due to increasing demand of SAM, improvement in its production is essential. For this purpose, S-adenosyl-l-methionine synthetase gene (sam2) was overexpressed in the present study, and we studied the effect of coexpression of methionine permease (mup1) and adenylate kinase (adk1) genes. From the recombinant strains expressing individual genes, we observed that SAM2 synthetase is the primary limiting factor and its overexpression is essential to increase the SAM productivity. Coexpression of mup1 with sam2 did not enhance SAM production, while coexpression of adk1 with sam2 improved SAM production, clearly indicating that ATP is the primary limiting precursor in SAM production. However, coexpression of all three genes synergistically improved SAM productivity with better L-methionine (L-met) conversion efficiency in every stage, and it was 77% more compared to overexpressing sam2 alone. Sparging pure oxygen reduced cultivation time. Feeding nitrogen source and additional L-met during induction phase enhanced SAM yield by 38.4% and 55.1%, respectively. Moreover, building up biomass before induction resulted in 145% increase in specific yield and 83% higher L-met conversion efficiency. This is the first report on increasing both the precursors L-met and ATP availability through molecular strategies using microorganisms for the production of SAM.

Selection and Characteristics of Fermented Salted Seafood (jeotgal)-Originated Strains with Excellent S-adenosyl-L-methionine (SAM) Production and Probiotics Efficacy.

This study is executed to develop probiotics which produce S-adenosyl-L-methionine (SAM), a methyl group donor of the 5-methyltetrahydrofolate methylation reaction within the animal cell. SAM is an essential substance for the synthesis, activation, and metabolism of hormones, neurotransmitters, nucleic acids, phospholipids, and cell membranes of animals. The SAM is also known as a nutritional supplement to improve brain functions of the human. In this study, the SAM-producing strains are identified in 18 types of salted fish, and then, the strains with excellent SAM productions are being identified, with 1 strain in the Enterococcus genus and 9 strains in the Bacillus genus. Strains with a large amount of SAM production include the lactic acid bacteria such as En. faecium and En. durans, En. sanguinicola, as well as various strains in the Bacil-lus genus. The SAM-overproducing strains show antibacterial activities with certain harmful microbes in addition to the weak acid resistances and strong bile resistances, indicating characteristics of probiotics. It is possible that the jeotgal-originated beneficial strains with overproducing SAM can be commercially utilized in order to manufacture SAM enriched foods.

Comparative performance of S-adenosyl-l-methionine biosynthesis and degradation in Pichia pastoris using different promoters and novel consumption inhibitors

The yeast Pichia pastoris is a widely used host for recombinant protein expression, and has recently been engineered for whole-cell biocatalysis. The inducible PAOX and constitutive PGAP promoters are commonly employed. In this study, the S-adenosyl-l-methionine (SAM) biosynthesis and degradation efficiency of two P. pastoris strains were compared, and novel inhibitors that suppress SAM degradation were characterized. The strains exhibited clear physiological differences. PGAP-Pichia showed higher transcription and activity of SAM synthetase, and the rapid cell growth led to higher levels of spermidine synthesis from SAM. In contrast, PAOX-Pichia synthesized higher levels of glutathione from SAM, and this strain responded to hydrogen peroxide formation during methanol utilization. Aristeromycin proved an efficient inhibitor of SAM degradation in PAOX-Pichia; 0.02mg/L led to a 36.36% reduction in the ratio of glutathionine:SAM, and SAM accumulation was enhanced by 7.74% to 11.83g/L. Ethanol was an even more efficient inhibitor of SAM consumption in PGAP-Pichia; 8g/L resulted in a 73.68% decrease in the ratio of SPD:SAM, and SAM production was elevated by 54.55% to 0.17g/L/h.