Enzymatic production of L-serine with a feedback control system for formaldehyde addition

Serine hydroxymethyltransferase (SHMT) in the form of crude extract from a recombinant strain of Klebsiella aerogenes was used for the production of L-serine from glycine and formaldehyde (HCHO). A stirred tank bio-reactor with a continuous feed of HCHO (37%) was employed. Since the performance of the serine bioreactor was heavily dependent on how HCHO was fed, an automatic feedback control system was developed for HCHO delivery utilizing the phenomenon of formol titration. This control procedure was based on the following circumstance: as a bioconversion proceeded, if the rate of HCHO feed was balanced by the rate of serine synthesis so that HCHO concentration was maintained near zero, then there was no pH change in the bioreactor. Once the rate of HCHO addition exceeded that of serine synthesis, the HCHO concentration built up and the excess HCHO reacted with the amino group of an amino acid (e.g. glycine or serine) to produce a Schiff base and a proton which lowered the pH. A pH controller detected and relayed this pH change to the on-off switch of the HCHO feed pump. Thus, HCHO infusion stopped when the pH was lower than the set point, which was the initial pH of the reaction. With this control system, the maximum concentration of HCHO that was reached in the bioreactor was only 1mM-3.3mM depending on the pH and amino acid composition in the bioreactor. Moreover, a decrease in pH also signaled the use of a slower feed rate at which HCHO was to be, delivered once the pH resumed its initial value after excess HCHO was consumed by the reaction. Employing this control system, we have optimized the performance of the serine bioreactor to give a serine titer of 450 g/L with an 88% molar conversion of glycine at a volumetric serine productivity of 8.9 g/L/h.     

L-serine production by a methionine-auxotrophic mutant of methylotrophic Pseudomonas

A methionine-auxotropic mutant deficient in homocysteine transmethylation activity was induced from a methylotrophic L-serine-producing derivatives of Pseudomonas MS31. This mutant grown with limited L-methionine had more than 1.7-fold higher serine hydroxymethyltransferase (SHMT) activity than its parent strain. The elevated SHMT activity significantly contributed to the improvement of L-serine accumulation from glycine and methanol. Under the optimum conditions, this mutant accumulated up to 23.9 mg/ml of L-serine. The yield coefficient L-serine from consumed glycine was 89% (mol/mol). The maximum conversion rate of added glycine (19 mg/ml) to L-serine was 77% (mol/mol).     

Molecular cloning and biochemical characterization of Leishmania donovani serine hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT) catalyzes the inter conversion of serine and tetrahydrofolate (H(4)-folate) to form glycine and 5,10-methylene H(4)-folate and generates one-carbon fragments for the synthesis of nucleotides, methionine, thymidylate, choline, etc. In spite of being an indispensable enzyme of the thymidylate cycle, SHMT in Leishmania donovani remains uncharacterized. The study of L. donovani SHMT (ldSHMT) becomes important as this gene is preferentially expressed in the amastigote stage of parasite, which resides in human macrophages. Here we report cloning, expression and purification of a catalytically active ldSHMT. The homogeneity of recombinant protein was analyzed by denaturing gel electrophoresis and protein was found to be 95% pure having yield of 1mg/l. The recombinant protein is a tetramer of 216kDa as evidenced by gel filtration chromatography and uses serine and tetrahydrofolate as substrates with Km of 1.6 and 2.4mM, respectively. Further biochemical studies revealed that pH optimum of ldSHMT is 7.8 and enzyme is thermally stable up to 45 degrees C. ldSHMT was found sensitive towards denaturants as manifested by loss of enzyme activity at the concentration of 1M urea or 0.25M guanidine hydrochloride. This is the first report of purification and characterization of recombinant SHMT from any protozoan source. Studies on recombinant ldSHMT will help in evaluating this enzyme as potential drug target.     

Enzymatic synthesis of L-tryptophan from hair acid hydrolysis industries wastewater with tryptophan synthase

An effective method for production of L-tryptophan from hair acid hydrolysis wastewater (HHW) containing L-serine was developed by recombinant tryptophan synthase. This study provides us with an alternative HHW utilization strategy. Tryptophan synthase could efficiently convert L-serine contained in HHW to L-tryptophan at pH 8.0, 40°C and Tween-80 of 0.04%. The enzyme also showed high tolerance to ammonium chloride, a component in HHW. In a scale up study, L-serine conversion rate reach 95.1% with a final L-tryptophan concentration of 33.2 g l(-1).     

13C nuclear magnetic resonance detection of interactions of serine hydroxymethyltransferase with C1-tetrahydrofolate synthase and glycine decarboxylase complex activities in Arabidopsis.

In C3 plants, serine synthesis is associated with photorespiratory glycine metabolism involving the tetrahydrofolate (THF)-dependent activities of the glycine decarboxylase complex (GDC) and serine hydroxymethyl transferase (SHMT). Alternatively, THF-dependent serine synthesis can occur via the C1-THF synthase/SHMT pathway. We used 13C nuclear magnetic resonance to examine serine biosynthesis by these two pathways in Arabidopsis thaliana (L.) Heynh. Columbia wild type. We confirmed the tight coupling of the GDC/ SHMT system and observed directly in a higher plant the flux of formate through the C1-THF synthase/SHMT system. The accumulation of 13C-enriched serine over 24 h from the GDC/SHMT activities was 4-fold greater than that from C1-THF synthase/SHMT activities. Our experiments strongly suggest that the two pathways operate independently in Arabidopsis. Plants exposed to methotrexate and sulfanilamide, powerful inhibitors of THF biosynthesis, reduced serine synthesis by both pathways. The results suggest that continuous supply of THF is essential to maintain high rates of serine metabolism. Nuclear magnetic resonance is a powerful tool for the examination of THF-mediated metabolism in its natural cellular environment.     

Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism

Serine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gln, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylenetetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond.     

Production of L-serine by Sarcina albida.

Conditions for the production of microbial L-serine hydroxymethyltransferase and for the conversion of glycine to L-serine were studied. A number of microorganisms were screened for their abilities to form and accululate L-serine from glycine, and Sarcina albida was selected as the best organism. Enzyme activity in this organism as high as 0.12 U/ml could be produced in shaken cultures at 30 degrees C in a medium containing glucose, ammonium sulfate, glycine, yeast extract, and inorganic salts. L-Serine was produced most efficiently by shaking cells at 30 degrees C in a reaction mixture containing 20% glycine, 5 X 10(-3) M formaldehyde, and 3 X 10(-4) M pyridoxal phosphate in yields of 22 mg of broth in 5 days. L-Serine was easily isolated in 84% yields by ion-exchange resin.     

L-Serine Production by Temperature-sensitive Mutants of Methanol-utilizing Bacterium Pseudomonas MS 31

Temperature-sensitive mutants producing L-serine efficiently from glycine were obtained from the facultative methylotroph Pseudomonas MS 31. Forty-five mutant strains showed adequate growth on methanol at 30°C but little or no growth at 37°C. Fourteen of these mutants produced L- serine more efficiently than the wild-type strain. The typical mutant strain ts 162 showed a high conversion rate in glycine-to-L-serine when the cultivation temperature was changed from a permissive (30°C) to non-permissive state (38?42°C) together with the addition of glycine and methanol after adequate growth. The mutant strain accumulated 6.8 mg L-serine from 12 mg glycine per ml culture under optimum conditions. The reduction of L-serine degrading activity in the mutant strain seemed to contribute to the high productivity of L-serine.     

Production of L-serine by Sarcina albida.

Conditions for the production of microbial L-serine hydroxymethyltransferase and for the conversion of glycine to L-serine were studied. A number of microorganisms were screened for their abilities to form and accululate L-serine from glycine, and Sarcina albida was selected as the best organism. Enzyme activity in this organism as high as 0.12 U/ml could be produced in shaken cultures at 30 degrees C in a medium containing glucose, ammonium sulfate, glycine, yeast extract, and inorganic salts. L-Serine was produced most efficiently by shaking cells at 30 degrees C in a reaction mixture containing 20% glycine, 5 X 10(-3) M formaldehyde, and 3 X 10(-4) M pyridoxal phosphate in yields of 22 mg of broth in 5 days. L-Serine was easily isolated in 84% yields by ion-exchange resin.     

Production of L-serine by the methanol utilizing bacterium,Pseudomonas 3ab

Summary A bacterium capable of growth on methanol and some organic acids as sole source of carbon and energy has been isolated and designated Pseudomonas 3ab. This facultative methylotrophic organism apparently utilizes the serine pathway of formaldehyde fixation.When methanol was used as the sole carbon source for growth, L-serine production by Pseudomonas 3ab occurred upon the addition of glycine and methanol at the end of the exponential growth phase. The maximum yield of L-serine (4.7 g/l) was obtained when 20 g/l glycine and 8 g/l methanol were added and the pH of the culture medium was changed to 8.5.Although Pseudomonas 3ab is unable to grow on L-serine or glycine, it is very active in decomposing these amino acids. The degradation of L-serine and glycine has been shown to be pH-dependent with a minimum at pH 8.5–9.0.     

A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor

To develop a feasible enzymatic process for d-tagatose production, a thermostable l-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 degrees C in the immobilized enzyme system and pH 7.5 and 60 degrees C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 degrees C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor.h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L.h) and a conversion yield of 46%.     

Flux of the L-serine metabolism in rabbit, human, and dog livers. Substantial contributions of both mitochondrial and peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase.

L-Serine metabolism in rabbit, dog, and human livers was investigated, focusing on the relative contributions of the three pathways, one initiated by serine dehydratase, another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Under quasi-physiological in vitro conditions (1 mM L-serine and 0.25 mM pyruvate), flux through serine dehydratase accounted for only traces, and that through SPT/AGT substantially contributed no matter whether the enzyme was located in peroxisomes (rabbit and human) or largely in mitochondria (dog). As for flux through serine hydroxymethyltransferase and GCS, the conversion of serine to glycine occurred fairly rapidly, followed by GCS-mediated slow decarboxylation of the accumulated glycine. The flux through GCS was relatively high in the dog and low in the rabbit, and only in the dog was it comparable with that through SPT/AGT. An in vivo experiment with L-[3-3H,14C]serine as the substrate indicated that in rabbit liver, gluconeogenesis from L-serine proceeds mainly via hydroxypyruvate. Because an important role in the conversion of glyoxylate to glycine has been assigned to peroxisomal SPT/AGT from the studies on primary hyperoxaluria type 1, these results suggest that SPT/AGT in this organelle plays dual roles in the metabolism of glyoxylate and serine.     

SHMT2 promotes the tumorigenesis of renal cell carcinoma by regulating the m6A modification of PPAT

ObjectiveSerine hydroxymethyltransferase 2 (SHMT2) is the first rate-limiting enzyme for serine/glycine biosynthesis and one carbon metabolism. Here, we explore the underlying mechanism of how SHMT2 functions in renal cell carcinoma (RCC) initiation.MethodsIn this study, SHMT2 expression was assessed in RCC tissues. In vitro experiments were performed to investigate the functional role of SHMT2. The detailed mechanisms of SHMT2-mediated PPAT were addressed.ResultsIncreased SHMT2 facilitated RCC cell proliferation by inducing the G1/S phase transition. And SHMT2 promoted the expression of PPAT. Mechanism dissection revealed that SHMT2 enhanced the m6A modification through the endogenous methyl donor SAM mediated by SHMT2 via serine/glycine one carbon metabolic networks. SHMT2-catalyzed serine/glycine conversion regulated PPAT expression in an m6A-IGF2BP2-dependent manner. SHMT2 promoted RCC cell proliferation by upregulating PPAT expression.ConclusionsSHMT2 promotes RCC tumorigenesis by increasing PPAT expression. Thus, SHMT2 may be a novel potential therapeutic target for RCC.     

Glycine and serine catabolism in non-photosynthetic higher plant cells: their role in C1 metabolism

Glycine and serine are two interconvertible amino acids that play an important role in C1 metabolism. Using 13C NMR and various 13C-labelled substrates, we studied the catabolism of each of these amino acids in non-photosynthetic sycamore cambial cells. On one hand, we observed a rapid glycine catabolism that involved glycine oxidation by the mitochondrial glycine decarboxylase (GDC) system. The methylenetetra- hydrofolate (CH2-THF) produced during this reaction did not equilibrate with the overall CH2-THF pool, but was almost totally recycled by the mitochondrial serine hydroxymethyltransferase (SHMT) for the synthesis of one serine from a second molecule of glycine. Glycine, in contrast to serine, was a poor source of C1 units for the synthesis of methionine. On the other hand, catabolism of serine was about three times lower than catabolism of glycine. Part of this catabolism presumably involved the glycolytic pathway. However, the largest part (about two-thirds) involved serine-to-glycine conversion by cytosolic SHMT, then glycine oxidation by GDC. The availability of cytosolic THF for the initial SHMT reaction is possibly the limiting factor of this catabolic pathway. These data support the view that serine catabolism in plants is essentially connected to C1 metabolism. The glycine formed during this process is rapidly oxidized by the mitochondrial GDC-SHMT enzymatic system, which is therefore required in all plant tissues.     

Crystal structure of binary and ternary complexes of serine hydroxymethyltransferase from Bacillus stearothermophilus: insights into the catalytic mechanism

Serine hydroxymethyltransferase (SHMT), a member of the alpha-class of pyridoxal phosphate-dependent enzymes, catalyzes the reversible conversion of serine to glycine and tetrahydrofolate to 5,10-methylene tetrahydrofolate. We present here the crystal structures of the native enzyme and its complexes with serine, glycine, glycine, and 5-formyl tetrahydrofolate (FTHF) from Bacillus stearothermophilus. The first structure of the serine-bound form of SHMT allows identification of residues involved in serine binding and catalysis. The SHMT-serine complex does not show any significant conformational change compared with the native enzyme, contrary to that expected for a conversion from an "open" to "closed" form of the enzyme. However, the ternary complex with FTHF and glycine shows the reported conformational changes. In contrast to the Escherichia coli enzyme, this complex shows asymmetric binding of the FTHF to the two monomers within the dimer in a way similar to the murine SHMT. Comparison of the ternary complex with the native enzyme reveals the structural basis for the conformational change and asymmetric binding of FTHF. The four structures presented here correspond to the various reaction intermediates of the catalytic pathway and provide evidence for a direct displacement mechanism for the hydroxymethyl transfer rather than a retroaldol cleavage.     

假单胞菌N-13中丝氨酸羟甲基转移酶的酶学性质研究

从产L-丝氨酸菌株假单胞菌N-13中纯化了丝氨酸羟甲基转移酶,并对其性质进行了研究.结果表明,丝氨酸羟甲基转移酶酶活力在pH=7.0～9.0间稳定,最适宜pH=8.0;酶的最适温度为35℃,在30～40℃水浴30 min酶活力未见明显下降.磷酸吡哆醛的最适添加浓度为25 μmol·L-1.研究了不同金属离子对酶活力的影...     

Identification of glyA (encoding serine hydroxymethyltransferase) and its use together with the exporter ThrE to increase L-threonine accumulation by Corynebacterium glutamicum

L-threonine can be made by the amino acid-producing bacterium Corynebacterium glutamicum. However, in the course of this process, some of the L-threonine is degraded to glycine. We detected an aldole cleavage activity of L-threonine in crude extracts with an activity of 2.2 nmol min(-1) (mg of protein)(-1). In order to discover the molecular reason for this activity, we cloned glyA, encoding serine hydroxymethyltransferase (SHMT). By using affinity-tagged glyA, SHMT was isolated and its substrate specificity was determined. The aldole cleavage activity of purified SHMT with L-threonine as the substrate was 1.3 micromol min(-1) (mg of protein)(-1), which was 4% of that with L-serine as substrate. Reduction of SHMT activity in vivo was obtained by placing the essential glyA gene in the chromosome under the control of P(tac), making glyA expression isopropylthiogalactopyranoside dependent. In this way, the SHMT activity in an L-threonine producer was reduced to 8% of the initial activity, which led to a 41% reduction in glycine, while L-threonine was simultaneously increased by 49%. The intracellular availability of L-threonine to aldole cleavage was also reduced by overexpressing the L-threonine exporter thrE. In C. glutamicum DR-17, which overexpresses thrE, accumulation of 67 mM instead of 49 mM L-threonine was obtained. This shows that the potential for amino acid formation can be considerably improved by reducing its intracellular degradation and increasing its export.     

The role of SHMT2 in modulating lipid metabolism in hepatocytes via glycine-mediated mTOR activation

Serine hydroxymethyltransferase 2 (SHMT2) converts serine into glycine in the mitochondrial matrix, transferring a methyl group to tetrahydrofolate. SHMT2 plays an important role in the maintenance of one-carbon metabolism. Previously, we found a negative correlation between the serine concentration and the progression of fatty liver disease (FLD). However, little is known about the role of SHMT2 in hepatic lipid metabolism. We established SHMT2 knockdown (KD) mouse primary hepatocytes using RNA interference to investigate the role of SHMT2 in lipid metabolism. SHMT2 KD hepatocytes showed decreased lipid accumulation with reduced glycine levels compared to the scramble cells, which was restored upon reintroducing SHMT2. SHMT2 KD hepatocytes showed downregulation of the mTOR/PPAR? pathway with decreased gene expression related to lipogenesis and fatty acid uptake. Pharmacological activation of mTOR or PPAR? overexpression blocked the inhibitory effect of SHMT2 KD on lipid accumulation. We also showed that glycine activated mTOR/PPAR? signaling and identified glycine as a mediator of SHMT2-responsive lipid accumulation in hepatocytes. In conclusion, silencing SHMT2 in hepatocytes ameliorates lipid accumulation via the glycine-mediated mTOR/PPAR? pathway. Our findings underscore the possibility of SHMT2 as a therapeutic target of FLD.

     

Ethanol production from alkaline peroxide pretreated enzymatically saccharified wheat straw

Wheat straw used in this study contained 44.24 +/- 0.28% cellulose and 25.23 +/- 0.11% hemicellulose. Alkaline H(2)O(2) pretreatment and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to fermentable sugars. The maximum yield of monomeric sugars from wheat straw (8.6%, w/v) by alkaline peroxide pretreatment (2.15% H(2)O(2), v/v; pH 11.5; 35 degrees C; 24 h) and enzymatic saccharification (45 degrees C, pH 5.0, 120 h) by three commercial enzyme preparations (cellulase, beta-glucosidase, and xylanase) using 0.16 mL of each enzyme preparation per g of straw was 672 +/- 4 mg/g (96.7% yield). During the pretreatment, no measurable quantities of furfural and hydroxymethyl furfural were produced. The concentration of ethanol (per L) from alkaline peroxide pretreated enzyme saccharified wheat straw (66.0 g) hydrolyzate by recombinant Escherichia coli strain FBR5 at pH 6.5 and 37 degrees C in 48 h was 18.9 +/- 0.9 g with a yield of 0.46 g per g of available sugars (0.29 g/g straw). The ethanol concentration (per L) was 15.1 +/- 0.1 g with a yield of 0.23 g/g of straw in the case of simultaneous saccharification and fermentation by the E. coli strain at pH 6.0 and 37 degrees C in 48 h.     

A serine hydroxymethyltransferase from marine bacterium Shewanella algae: Isolation,purification, characterization and l-serine production

Currently, l-serine is mainly produced by enzymatic conversion, in which serine hydroxymethyltransferase (SHMT) is the key enzyme, suggesting the importance of searching for a SHMT with high activity. Shewanella algae, a methanol-utilizing marine bacterium showing high SHMT activity, was selected based on screening bacterial strains and comparison of the activities of SHMTs. A glyA was isolated from the S. algae through thermal asymmetric interlaced PCR (TAIL-PCR) and it encoded a 417 amino acid polypeptide. The SaSHMT, encoded by the glyA, showed the optimal activity at 50 °C and pH 7.0, and retained over 45% of its maximal activity after incubation at 40 °C for 3 h. The enzyme showed better stability under alkaline environment (pH 6.5–9.0) than Hyphomicrobium methylovorum GM2's SHMT (pH 6.0–7.5). The SaSHMT can produce 77.76 mM of l-serine by enzymatic conversion, with the molecular conversion rate in catalyzing glycine to l-serine being 1.41-fold higher than that of Escherichia coli. Therefore, the SaSHMT has the potential for industrial applications due to its tolerance of alkaline environment and a relatively high enzymatic conversion rate.