Production Of L-methionine Research Articles

Comparative analysis of multi-effect evaporators in an ammonium acetate concentration process for L-methionine production

The process of treating an acetic acid aqueous solution generated during L-methionine production with ammonia and recovering a highly concentrated ammonium acetate aqueous solution using a multi-effect evaporator process was studied. The electrolyte non-random two-liquid model was applied to calculate the activity in the electrolyte solution, and the Hayden-O’Connell model was used to consider the dimerization reaction of acetic acid in the vapor phase. The dissociation equilibrium constant of acetic acid was modeled based on experimental values, and all other thermodynamic property models and parameters were used as implemented in the Aspen Plus software. Economic evaluation was carried out with various sensitivity tests, considering the loss of acetic acid and ammonia, as well as the steam cost. Evaporator operation was found to be more economical when the molar ratio of acetic acid to ammonia was equal, feed concentration was high, feed flow rate was low, and evaporator temperature was low. These results were confirmed in actual plant operations, with significant correlations between the feed flow rate and evaporator temperature. Our findings have significant implications for the development of sustainable and economical L-methionine production methods that enable the recycling of ammonium acetate.

Comparative life cycle assessment for the sustainable production of fermentation-based L-methionine

Methionine, essential for human and animal growth, faces increasing demand, contributing significantly to greenhouse gas emissions through animal digestion and nitrogen excretion. This study introduces L-methionine (L-Met) produced biologically from CJ CheilJedang Corp., offering a sustainable alternative to the conventional petroleum-based DL-methionine (DL-Met) due to its higher bioavailability and reduced environmental impact. One of the most important aspects for the sustainable lifecycle of L-Met is to reduce environmental impact during the production stage. Consequently, the aim of this study is to evaluates the environmental impact of L-Met and its environmentally improved product, L-Met Eco, through a life cycle assessment compared to DL-Met. We employed scenario analysis to consider uptake CO2 by bio-based feedstock, bioavailability of methionine, and system expansion of co-products. Our findings indicate that L-Met and L-Met Eco have lower global warming potentials of 0.92 and 0.79 kg CO2/kg Met, respectively, than DL-Met (2.60−3.32 kg CO2/kg Met). Sensitivity analysis further suggests that optimizing renewable energy use and production location can significantly enhance the sustainability of L-Met production. This comprehensive study offers valuable insights for stakeholders in the bio-based material sector, promoting environmentally sustainable practices.

Amelioration of L-methionine production by Alcaligenes faecalis ATCXT3624: Empirical optimization of culture conditions

Background: L-methionine production at the industrial scale suffers from various drawbacks, including the production and separation of the L-enantiomer of the amino acid methionine by chemical production methods. This strain exhibiting resilience to feedback inhibition by L-methionine provided a promising avenue for enhanced production. The present study has tried to uplift the production rate of the amino acid by a developed L-methionine-resistant strain Alcaligenes faecalis ATCXT 3624 through surpassing feedback inhibition. Methods: Empirical optimization of all fermentation conditions was studied by observing the effects of initial pH, temperature, inoculum age, medium volume, cell density, and different micronutrients, which enhanced L-methionine production. In addition, efforts were made to find suitable nitrogen and carbon sources. Results: Eventually, 23.8±0.22 mg/mL of L-methionine was obtained under optimized fermentation conditions, which is significantly (p<0.05) higher than that produced before optimized fermentation conditions. Conclusion: Eventually, it can be assumed that the strain effectively produced the amino acid at an enhanced rate under optimized conditions.

Optimization of the fermentative production of L-methionine by engineered Escherichia coli

As the only essential amino acid containing elemental sulphur, L-methionine has important physiological and biochemical functions in living organisms. However, the fermentative production of L-methionine has not met the requirements of industrial production because of its low production level. In this paper, the fermentation process of an efficient L-methionine producing strain E. coli W3110ΔIJAHFEBC trc-fliY trc-malY/PAM glyA-22 metF constructed previously was systematically optimized. Based on the optimal initial glucose concentration, the effects of different fed-batch fermentation processes, including DO-Stat, pH-Stat, controlling residual sugar control at different level and feeding glucose with constant rate, on L-methionine fermentation were studied. It was found that the control of glucose concentration greatly affected the fermentation process. Subsequently, an optimal fed-batch fermentation process was developed, where the L-methionine titer was increased to 31.71 g/L, the highest yield reported to date, while the fermentation time was shortened to 68 h. Meanwhile, a fermentation kinetics model under the optimal fed-batch fermentation conditions was established, which fitted well with the biosynthesis process of L-methionine. This study may facilitate further development of the fermentative production of L-methionine.

Effect of Sugar Beet Pulp on the Composition and Predicted Function of Equine Fecal Microbiota.

The purpose of this study is to determine the effect of the partial replacement of dietary hay with sugar beet pulp (SBP) on the composition and predicted function of the fecal microbiota of healthy adult horses. Fecal samples were collected daily for 12 days from six adult horses after removal from pasture, including a five-day acclimation period, and a seven-day period following the introduction of SBP into their diet, and compared to six untreated horses over a comparable period. Fecal DNA was subjected to 16S rRNA amplicon sequencing and a longitudinal analysis was performed comparing the composition and predicted function. While no significant treatment-associated changes in the richness, alpha diversity, or beta diversity were detected, random forest regression identified several high-importance taxonomic features associated with change over time in horses receiving SBP. A similar analysis of the predicted functional pathways identified several high-importance pathways, including those involved in the production of L-methionine and butyrate. These data suggest that feeding SBP to healthy adult horses acutely increases the relative abundance of several Gram-positive taxa, including Cellulosilyticum sp., Moryella sp., and Weissella sp., and mitigates the predicted functional changes associated with removal from pasture. Large-scale studies are needed to assess the protective effect of SBP on the incidence of the gastrointestinal conditions of horses.

Efficient synthesis of L-methionine by engineering the one carbon module of Escherichia coli

L-methionine, also known as L-aminomethane, is one of the eight essential amino acids required by the human body and has important applications in the fields of feed, medicine, and food. In this study, an L-methionine high-yielding strain was constructed using a modular metabolic engineering strategy based on the M2 strain (Escherichia coli W3110 ΔIJAHFEBC/PAM) previously constructed in our laboratory. Firstly, the production of one-carbon module methyl donors was enhanced by overexpression of methylenetetrahydrofolate reductase (methylenetetrahydrofolate reductase, MetF) and screening of hydroxymethyltransferase (GlyA) from different sources, optimizing the one-carbon module. Subsequently, cysteamine lyase (hydroxymethyltransferase, MalY) and cysteine internal transporter gene (fliY) were overexpressed to improve the supply of L-homocysteine and L-cysteine, two precursors of the one-carbon module. The production of L-methionine in shake flask fermentation was increased from 2.8 g/L to 4.05 g/L, and up to 18.26 g/L in a 5 L fermenter. The results indicate that the one carbon module has a significant impact on the biosynthesis of L-methionine, and efficient biosynthesis of L-methionine can be achieved through optimizing the one carbon module. This study may facilitate further improvement of microbial fermentation production of L-methionine.

Conversion of methionine biosynthesis in Escherichia coli from trans- to direct-sulfurylation enhances extracellular methionine levels

Methionine is an essential amino acid in mammals and a precursor for vital metabolites required for the survival of all organisms. Consequently, its inclusion is required in diverse applications, such as food, feed, and pharmaceuticals. Although amino acids and other metabolites are commonly produced through microbial fermentation, high-yield biosynthesis of L-methionine remains a significant challenge due to the strict cellular regulation of the biosynthesis pathway. As a result, methionine is produced primarily synthetically, resulting in a racemic mixture of D,L-methionine. This study explores methionine bio-production in E. coli by replacing its native trans-sulfurylation pathway with the more common direct-sulfurylation pathway used by other bacteria. To this end, we generated a methionine auxotroph E. coli strain (MG1655) by simultaneously deleting metA and metB genes and complementing them with metX and metY from different bacteria. Complementation of the genetically modified E. coli with metX/metY from Cyclobacterium marinum or Deinococcus geothermalis, together with the deletion of the global repressor metJ and overexpression of the transporter yjeH, resulted in a substantial increase of up to 126 and 160-fold methionine relative to the wild-type strain, respectively, and accumulation of up to 700 mg/L using minimal MOPS medium and 2 ml culture. Our findings provide a method to study methionine biosynthesis and a chassis for enhancing L-methionine production by fermentation.

Multivariate modular metabolic engineering for enhanced l-methionine biosynthesis in Escherichia coli

Backgroundl-Methionine is the only bulk amino acid that has not been industrially produced by the fermentation method. Due to highly complex and strictly regulated biosynthesis, the development of microbial strains for high-level l-methionine production has remained challenging in recent years.ResultsBy strengthening the l-methionine terminal synthetic module via site-directed mutation of l-homoserine O-succinyltransferase (MetA) and overexpression of metAfbr, metC, and yjeH, l-methionine production was increased to 1.93 g/L in shake flask fermentation. Deletion of the pykA and pykF genes further improved l-methionine production to 2.51 g/L in shake flask fermentation. Computer simulation and auxotrophic experiments verified that during the synthesis of l-methionine, equimolar amounts of l-isoleucine were accumulated via the elimination reaction of cystathionine γ-synthetase MetB due to the insufficient supply of l-cysteine. To increase the supply of l-cysteine, the l-cysteine synthetic module was strengthened by overexpression of cysEfbr, serAfbr, and cysDN, which further increased the production of l-methionine by 52.9% and significantly reduced the accumulation of the byproduct l-isoleucine by 29.1%. After optimizing the addition of ammonium thiosulfate, the final metabolically engineered strain MET17 produced 21.28 g/L l-methionine in 64 h with glucose as the carbon source in a 5 L fermenter, representing the highest l-methionine titer reported to date.ConclusionsIn this study, a high-efficiency strain for l-methionine production was derived from wild-type Escherichia coli W3110 by rational metabolic engineering strategies, providing an efficient platform for the industrial production of l-methionine.

Thorough research and modification of one-carbon units cycle for improving L-methionine production in Escherichia coli.

The online version contains supplementary material available at 10.1007/s13205-023-03625-9.

Local metabolic response of Escherichia coli to the module genetic perturbations in l-methionine biosynthetic pathway.

l-Methionine biosynthesis is through multilevel regulated and multibranched biosynthetic pathway (MRMBP). Because of the complex regulatory mechanism and the imbalanced metabolic flux between branched pathways, microbial production of l-methionine has not been commercialized. In this study, local metabolic response in MRMBP of l-methionine was investigated and various crucial genes in branched pathways were determined. In l-serine pathway, the crucial gene was serABC. In O-succinyl homoserine (OSH) pathway, which was the C4 backbone of l-methionine, metB and metL controlled the metabolic flux jointly. In l-cysteine pathway, the crucial gene cysEfbr could disturb the flux distribution of local network in l-methionine biosynthesis. However, no crucial gene for l-methionine production in 5-methyl tetrahydrofolate (CH3-THF) pathway was found. The relation between these pathways was also researched. l-Serine pathway, as the upstream pathway of l-cysteine and CH3-THF, played a crucial role in l-methionine biosynthesis. l-Cysteine pathway showed the strongest controlling force of the metabolic flux, and OSH pathway was second to l-cysteine pathway. In contrast, CH3-THF pathway was the weakest, which was probably the mainly limited steps at present and had great potential in further research. In addition, constructed W3110 IJAHFEBC/pA∗HAmL was able to produce 2.62g/L l-methionine in flask. This study is instructive for l-methionine biosynthesis and provides a new research method of biosynthesizing other metabolic products in MRMBPs.

Isolation and characterization of a novel l-Methionine producer from Mahanadi river site in Sambalpur district of Odisha, India

l-methionine is an essential amino acid for human nutrition. Its global market demand is expanding gradually. Even though large scale production of stereo-specific amino acids by fermentation was initiated with the isolation of l-glutamic acid overproducer strain in 1957, but due to several drawbacks, l-methionine fermentation in large scale was not successfully initiated. In the present study, isolation, molecular characterization as well as strain improvement have been done for l-methionine overproduction by a newly isolated bacterial strain from Sambalpur district, near Hirakud dam, Odisha. Based on the morphological and molecular characterization, the pale colored colony forming rod shaped, Gram negative, non-motile bacterium was identified as Alcaligenes faecalis strain. Mutational induction was done to develop l-methionine overproducers by N-methyl-N’-nitro-N-nitrosoguanidine (NTG) and UV rays. Finally, l-ethionine-resistant strain was developed to overcome feed-back inhibition. The mutant strain Alcaligenes faecalis ATCXT3624 appeared to be l-homoserine auxotroph in further studies. The strain could produce 20.1 ± 0.32 mg l-methionine/mL in subsequent submerged fermentation before optimization of culture conditions. The relevance of the study was discussed in relation to commercial production of l-methionine.

High-Level Production of l-Methionine by Dynamic Deregulation of Metabolism with Engineered Nonauxotroph Escherichia coli.

l-Methionine is the only sulfur-containing amino acid among the essential amino acids, and it is mainly produced by the chemical method in industry so far. The fermentation production of l-methionine by genetically engineered strains is an attractive alternative. Due to the complex metabolic mechanism and multilevel regulation of the synthesis pathway in the organism, the fermentation production of l-methionine by genetically engineered strains was still not satisfied. In this study, the biosynthesis pathway of l-methionine was regulated based on the previous studies. As the competitive pathway and an essential amino acid for cell growth, the biosynthesis pathway of l-lysine was first repaired by complementation of the lysA gene in situ on the genome and then replaced the in situ promoter with the dynamically regulated promoter PfliA to construct a nonauxotroph strain. In addition, the central metabolic pathway and l-cysteine catabolism pathway were further modified to promote the cell growth and enhance the l-methionine production. Finally, the l-methionine fermentation yield in a 5 L bioreactor reached 17.74 g/L without adding exogenous amino acids. These strategies can effectively balance the contradiction between cell growth and l-methionine production and alleviate the complexity of fermentation operation and the cost with auxotroph strains, which provide a reference for the industrial production of l-methionine by microbial fermentation.

Increased NADPH Supply Enhances Glycolysis Metabolic Flux and L-methionine Production in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important strain for the industrial production of amino acids, but the fermentation of L-methionine has not been realized. The purpose of this study is to clarify the effect of reducing power NADPH on L-methionine synthesis. Site-directed mutagenesis of zwf and gnd genes in pentose phosphate pathway relieved feedback inhibition, increased NADPH supply by 151.8%, and increased L-methionine production by 28.3%; Heterologous expression of gapC gene to introduce NADP+ dependent glyceraldehyde-3-phosphate dehydrogenase increased NADPH supply by 75.0% and L-methionine production by 48.7%; Heterologous expression of pntAB gene to introduce membrane-integral nicotinamide nucleotide transhydrogenase increased NADPH by 89.2% and L-methionine production by 35.9%. Finally, the engineering strain YM6 with a high NADPH supply was constructed, which increased the NADPH supply by 348.2% and the L-methionine production by 64.1%. The analysis of metabolic flux showed that YM6 significantly increased the glycolytic flux, including the metabolic flux of metabolites such as glycosyldehyde-3-phosphate, dihydroxyacetate phosphate, 3-phosphoglycate and pyruvate, and the significant increase of L-methionine flux also confirmed the increase of its synthesis. This study provides a research basis for the systematic metabolic engineering construction of L-methionine high-yield engineering strains.

Utilization of Various Starch Hydrolysates and Defatted Proteins by Bacillus cereus for Microbial Synthesis of Methionine in Submerged Medium

Three of the methionine-producing bacteria previously isolated from different soil ecovars in Owerri, characterized as Bacillus cereus no: DS13, RS16 and AS9, based on 16s rRNA sequencing, were screened for utilization agricultural products for production of methionine. Experimental studies on the Bacillus strains were carried out to examine the use of various starch hydrolysates from different agricultural products: wheat, sorghum, cassava, cocoyam, yam, plantain, millet, corn, potato, rice and defatted plant proteins: pigeon pea, cowpea, bambaranut, groundnut and soybean meal as source of carbon and nitrogen, respectively for the production of methionine. The result of the influence of various carbon and nitrogen sources on methionine production showed that 2.32mg/ml methionine accumulation in the culture broth of B. cereus DS13 when corn-starch hydrolysate/defatted groundnut meal was used, while B. cereus RS16 and Bacillus cereus DS13 gave the same methionine yield of 2.22mg/ml with corn-starch hydrolysate/defatted groundnut meal and sorghum hydrolysate/soyabean meal respectively. Bacillus cereus AS9 utilized wheat starch hydrolysate/soaybean meal, followed by plaintain-starch hydrolysate/bambaranut to produced 1.79mg/ml and 1.78mg/ml methionine respectively. Methionine was accumulated in the fermentation broth of the Bacillus strains after a fermentation period of 96h. This study indicates the possible utilization of agricultural products as substrates for L-methionine production in submerged medium especially when considering the limitations of producing methionine by fermentation processes.

Combining fermentation to produce O-succinyl-l-homoserine and enzyme catalysis for the synthesis of l-methionine in one pot

The biosynthetic pathway of l-methionine in microorganisms was complex and regulated at multiple levels. In this study, a two-step method for l-methionine production combined fermentation and biocatalysis was realized in one pot. The O-succinyl-l-homoserine (OSH) producing strain Escherichia coli W3110(DE3) ΔIJB∗TrcmetL/pTrc-metAfbr-Trc-thrAfbr-yjeH (ΔIJB) was constructed initially. OSH in the fermentation supernatant was then converted to l-methionine in the presence of O-succinyl-l-homoserine sulfhydrylase (OSHS) and sodium methanethiol. The titer of l-methionine could reach 21.1g/L after 88h (84h fermentation and 4h catalysis) in a two-step method (process 1). In a one-pot two-strain system (process 2), two strains ΔIJB and E.coli BL21(DE3)/pET28b-OSHS-cutinase were co-cultured, and 8.24g/L l-methionine was obtained. In another one-pot one-strain system (process 3), strain E.coli ΔIJB/pET28b-OSHS-cutinase could co-express OSHS and cutinase during ΔIJB fermentation at the same time, obtaining 13.6g/L l-methionine in a 5L fermentor after 84h. By comparing the three processes for l-methionine production based on the process 1, the simplified process in process 3 provided in this study showed potent in the large-scale production of l-methionine with convenient handling and production efficiency, but further works still need to be carried out to improve the l-methionine production.

Production of l-Methionine from 3-Methylthiopropionaldehyde and O-Acetylhomoserine by Catalysis of the Yeast O-Acetylhomoserine Sulfhydrylase

l-Methionine is an essential bioactive amino acid with high commercial value for diverse applications. Sustained attentions have been paid to efficient and economical preparation of l-methionine. In this work, a novel method for l-methionine production was established using O-acetyl-homoserine (OAH) and 3-methylthiopropionaldehyde (MMP) as substrates by catalysis of the yeast OAH sulfhydrylase MET17. The OAH sulfhydrylase gene Met17 was cloned from Saccharomyces cerevisiae S288c and overexpressed in Escherichia coli BL21. A 49 kDa MET17 was detected in the supernatant of the recombinant E. coli strain BL21-Met17 lysate with IPTG induction, which exhibited the biological activity of l-methionine biosynthesis from OAH and MMP. The recombinant MET17 was then purified from E. coli BL21-Met17 and used for in vitro biosynthesis of l-methionine. The maximal conversion rate (86%) of OAH to l-methionine catalyzed by purified MET17 was achieved by optimization of the molar ratio of OAH to MMP. The method proposed in this study provides a possible novel route for the industrial production of l-methionine.

Biochemical Changes in the Fermentation Medium During L-methionine Production by Brevibacterium roseum SXS2470

Biochemical Changes in the Fermentation Medium During L-methionine Production by Brevibacterium roseum SXS2470

Role of metAB in Methionine Metabolism and Optimal Chicken Colonization in Campylobacter jejuni.

Campylobacter jejuni is a zoonotic pathogen and is one of the leading causes of human gastroenteritis worldwide. C. jejuni IA3902 (representative of the sheep abortion clone) is genetically similar to C. jejuni W7 (representative of strain type NCTC 11168); however, there are significant differences in the ability of luxS mutants of these strains to colonize chickens. LuxS is essential for the activated methyl cycle and generates homocysteine for conversion to l-methionine. Comparative genomics identified differential distribution of the genes metA and metB, which function to convert homoserine for downstream production of l-methionine, between IA3902 and W7, which could enable a secondary pathway for l-methionine biosynthesis in a W7 ΔluxS but not in an IA3902 ΔluxS strain. To test the hypothesis that the genes metA and metB contribute to l-methionine production and chicken colonization by Campylobacter, we constructed two mutants for phenotypic comparison, the W7 ΔmetAB ΔluxS and IA3902 ΔluxS::metAB mutants. Quantitative reverse transcription-PCR and tandem mass spectrometry protein analysis were used to validate MetAB transcription and translation as present in the IA3902 ΔluxS::metAB mutant and absent in the W7 ΔmetAB ΔluxS mutant. Time-resolved fluorescence resonance energy transfer fluorescence assays demonstrated that l-methionine and S-adenosyl methionine concentrations decreased in the W7 ΔmetAB ΔluxS mutant and increased in the IA3902 ΔluxS::metAB mutant. Assessment of chicken colonization revealed that the IA3902 ΔluxS::metAB strain partially rescued the colonization defect of the IA3902 ΔluxS strain, while the W7 ΔmetAB ΔluxS strain showed significantly decreased colonization compared to that of the wild-type and the W7 ΔluxS strain. These results indicate that the ability to maintain l-methionine production in vivo, conferred by metA and metB in the absence of luxS, is critical for normal chicken colonization by C. jejuni.

Plackett-Burman design for screening of fermentation process parameters and their effects on L-methionine production

Plackett-Burman design for screening of fermentation process parameters and their effects on L-methionine production

Efficient production of L-homoserine in Corynebacterium glutamicum ATCC 13032 by redistribution of metabolic flux

L-Homoserine is a platform chemical used for the production of l-methionine and other valuable compounds. In this study, we constructed an initial l-homoserine-producing strain by metabolic engineering of Corynebacterium glutamicum ATCC 13032, including deletion of thrB, mcbR, and metD and overexpression of lysC, asd, and hom, resulting in a strain capable of producing 1.2 g/L l-homoserine and 4.8 g/L l-lysine. The metabolic flux of l-homoserine was further enhanced by overexpressing feedback-insensitive pyruvate carboxylase (encoded by pyc) from C. glutamicum ATCC 13032 and l-aspartate aminotransferase (encoded by aspC) from E. coli in order to increase oxaloacetate and l-aspartate supplies. Additionally, overexpression of brnFE, which encodes the two-component export system, improved l-homoserine production by 2-fold, suggesting involvement of the brnFE operon in l-homoserine export. Moreover, attenuated expression of dapA and icd downregulated l-lysine biosynthesis and the TCA cycle, resulting in a 25.9 % increase in l-homoserine production (up to 3.4 g/L). Furthermore, overexpression of lysCT311I, asd from C. glutamicum ATCC 13032, and the thrAS345F from E. coli K12-MG1655 resulted in l-homoserine production of 8.8 g/L and demonstrated C. glutamicum as having great potential for industrial production of l-homoserine and l-methionine.