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Xiaoyuan Wang - One of the best experts on this subject based on the ideXlab platform.
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Improving L-Threonine production in Escherichia coli by elimination of transporters ProP and ProVWX.
Microbial cell factories, 2021Co-Authors: Shuaiwen Wang, Yu Fang, Zhen Wang, Shuyan Zhang, Liangjia Wang, Yong Guo, Xiaoyuan WangAbstract:BACKGROUND Betaine, an osmoprotective compatible solute, has been used to improve L-Threonine production in engineered Escherichia coli L-Threonine producer. Betaine supplementation upregulates the expression of zwf encoding glucose-6-phosphate dehydrogenase, leading to the increase of NADPH, which is beneficial for L-Threonine production. In E. coli, betaine can be taken through ProP encoded by proP or ProVWX encoded by proVWX. ProP is a H+-osmolyte symporter, whereas ProVWX is an ABC transporter. ProP and ProVWX mediate osmotic stress protection by transporting zwitterionic osmolytes, including glycine betaine. Betaine can also be synthesized in E. coli by enzymes encoded by betABIT. However, the influence of ProP, ProVWX and betABIT on L-Threonine production in E. coli has not been investigated. RESULTS In this study, the influence of ProP, ProVWX and betABIT on L-Threonine production in E. coli has been investigated. Addition of betaine slightly improved the growth of the L-Threonine producing E. coli strain TWF001 as well as the L-Threonine production. Deletion of betABIT retarded the growth of TWF001 and slightly decreased the L-Threonine production. However, deletion of proP or/and proVWX significantly increased the L-Threonine production. When proP was deleted, the L-Threonine production increased 33.3%; when proVWX was deleted, the L-Threonine production increased 40.0%. When both proP and proVWX were deleted, the resulting strain TSW003 produced 23.5 g/l L-Threonine after 36 h flask cultivation. The genes betABIT, proC, fadR, crr and ptsG were individually deleted from TSW003, and it was found that further absence of either crr (TWS008) or ptsG (TWS009) improved L-Threonine production. TSW008 produced 24.9 g/l L-Threonine after 36 h flask cultivation with a yield of 0.62 g/g glucose and a productivity of 0.69 g/l/h. TSW009 produced 26 g/l L-Threonine after 48 h flask cultivation with a yield of 0.65 g/g glucose and a productivity of 0.54 g/l/h, which is 116% increase compared to the control TWF001. CONCLUSIONS In this study, L-Threonine-producing E. coli strains TSW008 and TSW009 with high L-Threonine productivity were developed by regulating the intracellular osmotic pressure. This strategy could be used to improve the production of other products in microorganisms.
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Expression regulation of multiple key genes to improve L-Threonine in Escherichia coli.
Microbial cell factories, 2020Co-Authors: Lei Zhao, Jun Yang, Yu Fang, Lifei Zhu, Ding Zhixiang, Chenhui Wang, Ma Wenjian, Xiaoyuan WangAbstract:Escherichia coli is an important strain for L-Threonine production. Genetic switch is a ubiquitous regulatory tool for gene expression in prokaryotic cells. To sense and regulate intracellular or extracellular chemicals, bacteria evolve a variety of transcription factors. The key enzymes required for L-Threonine biosynthesis in E. coli are encoded by the thr operon. The thr operon could coordinate expression of these genes when L-Threonine is in short supply in the cell. The thrL leader regulatory elements were applied to regulate the expression of genes iclR, arcA, cpxR, gadE, fadR and pykF, while the threonine-activating promoters PcysH, PcysJ and PcysD were applied to regulate the expression of gene aspC, resulting in the increase of L-Threonine production in an L-Threonine producing E. coli strain TWF001. Firstly, different parts of the regulator thrL were inserted in the iclR regulator region in TWF001, and the best resulting strain TWF063 produced 16.34 g L-Threonine from 40 g glucose after 30 h cultivation. Secondly, the gene aspC following different threonine-activating promoters was inserted into the chromosome of TWF063, and the best resulting strain TWF066 produced 17.56 g L-Threonine from 40 g glucose after 30 h cultivation. Thirdly, the effect of expression regulation of arcA, cpxR, gadE, pykF and fadR was individually investigated on L-Threonine production in TWF001. Finally, using TWF066 as the starting strain, the expression of genes arcA, cpxR, gadE, pykF and fadR was regulated individually or in combination to obtain the best strain for L-Threonine production. The resulting strain TWF083, in which the expression of seven genes (iclR, aspC, arcA, cpxR, gadE, pykF, fadR and aspC) was regulated, produced 18.76 g L-Threonine from 30 g glucose, 26.50 g L-Threonine from 40 g glucose, or 26.93 g L-Threonine from 50 g glucose after 30 h cultivation. In 48 h fed-batch fermentation, TWF083 could produce 116.62 g/L l‐threonine with a yield of 0.486 g/g glucose and productivity of 2.43 g/L/h. The genetic engineering through the expression regulation of key genes is a better strategy than simple deletion of these genes to improve L-Threonine production in E. coli. This strategy has little effect on the intracellular metabolism in the early stage of the growth but could increase L-Threonine biosynthesis in the late stage.
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Transcriptomic analysis of an L‐threonine producing Escherichia coli TWF001
Biotechnology and applied biochemistry, 2020Co-Authors: Lei Zhao, Xiaoyuan Wang, Hailing Zhang, Guoqiang Han, Ma WenjianAbstract:Wild-type Escherichia coli usually does not accumulate L-Threonine, but E. coli strain TWF001 could produce 30.35 g/L L-Threonine after 23-H fed-batch fermentation. To understand the mechanism for the high yield of L-Threonine production in TWF001, transcriptomic analyses of the TWF001 cell samples collected at the logarithmic and stationary phases were performed, using the wild-type E. coli strain W3110 as the control. Compared with W3110, 1739 and 2361 genes were differentially transcribed in the logarithmic and stationary phases, respectively. Most genes related to the biosynthesis of L-Threonine were significantly upregulated. Some key genes related to the NAD(P)H regeneration were upregulated. Many genes relevant to glycolysis and TCA cycle were downregulated. The key genes involved in the L-Threonine degradation were downregulated. The gene rhtA encoding the L-Threonine exporter was upregulated, whereas the genes sstT and tdcC encoding the L-Threonine importer were downregulated. The upregulated genes in the glutamate pathway might form an amino-providing loop, which is beneficial for the high yield of L-Threonine production. Many genes encoding the 30S and 50S subunits of ribosomes were also upregulated. The findings are useful for gene engineering to increase L-Threonine production in E. coli.
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Deletion of arcA, iclR, and tdcC in Escherichia coli to improve L-Threonine production.
Biotechnology and applied biochemistry, 2019Co-Authors: Ding Zhixiang, Lifei Zhu, Yu Fang, Jianli Wang, Xiaoyuan WangAbstract:L-Threonine is an important amino acid supplemented in food, medicine, or feed. Starting from glucose, L-Threonine production in Escherichia coli involves the glycolysis, TCA cycle, and the L-Threonine biosynthetic pathway. In this study, how the L-Threonine production in an L-Threonine producing E. coli TWF001 is controlled by the three regulators ArcA, Cra, and IclR, which control the expression of genes involved in the glycolysis and TCA cycle, has been investigated. Ten mutant strains were constructed from TWF001 by different combinations of deletion or overexpression of arcA, cra, iclR, and tdcC. L-Threonine production was increased in the mutants TWF015 (ΔarcAΔcra), TWF016 (ΔarcAPcra::Ptrc), TWF017 (ΔarcAΔiclR), TWF018 (ΔarcAΔiclRΔtdcC), and TWF019 (ΔarcAΔcraΔiclRΔtdcC). Among these mutant strains, the highest L-Threonine production (26.0 g/L) was obtained in TWF018, which was a 109.7% increase compared with the control TWF001. In addition, TWF018 could consume glucose more efficiently than TWF001 and produce less acetate. The results suggest that deletion of arcA, iclR, and tdcC could efficiently increase L-Threonine production in E. coli.
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Deletion of regulator-encoding genes fadR , fabR and iclR to increase L-Threonine production in Escherichia coli
Applied microbiology and biotechnology, 2019Co-Authors: Jun Yang, Lei Zhao, Yu Fang, Chenhui Wang, Jianli Wang, Xiaoyuan WangAbstract:Previously, we have developed an L-Threonine-producing Escherichia coli strain TWF006 in which the regulator-encoding gene iclR was deleted. In this study, further modifications were performed on TWF006 to increase L-Threonine yield. Firstly, the regulator-encoding gene fadR was deleted in TWF006, and the resulting strain TWF031 produced 18.86 g L-Threonine from 30 g glucose after 24-h cultivation. Secondly, the regulator-encoding genes fabR and lacI in TWF031 were deleted, and the resulting strain TWF033 produced 19.21 g L-Threonine from 30 g glucose after 24-h cultivation. Thirdly, additional copies of aceBA and fadBA were inserted into the lacZ locus of TWF033 and the native promoter of acs was replaced by the Ptac-trc; the resulting strain TWF038 produced 20.3 g L-Threonine from 30 g glucose after 24-h cultivation. Finally, the genes ppnK, thrA*BC-rhtC, aspC, and ppc were inserted into the chromosome of TWF038; the resulting strain TWF044 produced 21.64 g L-Threonine from 30 g glucose, or 28.49 g L-Threonine from 40 g glucose after 24-h cultivation. After 48-h fed-batch fermentation, TWF044 produced 103.89 g/l L-Threonine. The results suggest that coupling the fatty acid degradation and L-Threonine biosynthesis pathway via the glyoxylate shunt could efficiently increase L-Threonine production in E. coli.
Bernard Sève - One of the best experts on this subject based on the ideXlab platform.
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Tissue localization of threonine oxidation in pigs
British Journal of Nutrition, 1997Co-Authors: Nathalie Le Floc'h, Jean-noёl Thibault, Bernard SèveAbstract:Two experiments were designed to determine the tissue distribution of threonine oxidation through the threonine dehydrogenase (EC 1.1.1.103) pathway in pigs. The first experiment was conducted on eleven Piarain X Large White piglets. The piglets were slaughtered at 5, 12 or 20 kg after 1 h of infusion with ~-[U-~~C]threonine (55 kBqkg) mixed with unlabelled threonine (100 m&). In the second experiment, four Pihain X Large White and four Large White piglets (10 kg body weight) were infused with ~-[l-”C]threonine (50 mglkg) mixed with 50 mgkg unlabelled threonine for 1 h, then killed for tissue sampling. In the two experiments, threonine dehydrogenase specific activity and threonine and glycine specific radioactivities and enrichments were measured in several tissues and in plasma. The higher level of labelling of threonine in the pancreas than in the liver suggested either a lower protein degradation rate or a faster rate of threonine transport in the liver than in the pancreas. Threonine dehydrogenase activity was found only in the liver and the pancreas. Whereas liver and pancreas threonine dehydmgenase specific activities were similar, glycine specif~c radioactivity and enrichment were 12- to 14-fold higher in the pancreas than in the liver. This is probably the consequence of a higher production rate of glycine from sources other than threonine (protein degradation, de novo synthesis from serine) in the liver than in the pancreas. Our results showed that Large White pigs could oxidize more threonine than Pi6train X Large White pigs. This could be related to the difference in growth performance and dietary N efficiency for protein deposition between these two genotypes. Threonine: Amino acids: Genotype: Pigs
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In vivo threonine oxidation in growing pigs fed on diets with graded levels of threonine
British Journal of Nutrition, 1996Co-Authors: N. Le Floc’h, Christiane Obled, Bernard SèveAbstract:Threonine oxidation to glycine was investigated in vivo in twelve growing pigs (27.4 kg live weight) fed on one of the following three diets with graded levels of threonine supply : a low-threonine diet (LT), a control well-balanced diet (C) or a high-threonine diet (HT), during 10 h constant infusion of L-[1- 13 C]threonine and [2- 3 H]glycine in the cranial vena cava and [1- 14 C]glycine in the portal vein. 13 C-threonine and glycine enrichments and [ 3 H]glycine and [ 14 C]glycine specific radioactivities (SR) were determined at plateau in peripheral venous plasma, liver and pancreas. Glycine production rates calculated from plasma [2- 3 H]glycine or [1- 14 C]glycine SR gave similar values suggesting that [1- 14 C]glycine SR could be used in order to estimate whole-body glycine flux. The high pancreas [1- 13 C]glycine enrichment provided evidence that the pancreas may be, with the liver, a major site of threonine oxidation to glycine. Moreover, the present findings suggest that threonine transport into the liver could be the limiting step of threonine oxidation in this tissue when dietary threonine supply is low. Total threonine oxidation to glycine, calculated from plasma values of enrichment and specific radioactivity, was low and constant when the estimated absorbed threonine was lower than 4 g/d and increased for higher amounts of absorbed threonine.
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In vivo threonine oxidation rate is dependent on threonine dietary supply in growing pigs fed low to adequate levels
Journal of Nutrition, 1995Co-Authors: Nathalie Le Floc'h, Christiane Obled, Bernard SèveAbstract:Threonine oxidation was examined in 12 growing pigs fed a well-balanced control diet or a threonine-deficient diet supplemented (Glu) or not (LT) with glutamic acid during constant infusion of L-[1- 13 C]-threonine, [1- 14 C]glycine and [1- 14 C]α-ketobutyrate for 10 h. During these infusions, liver glycine enrichment was significantly lower than plasma enrichment. Moreover, the pancreas to plasma glycine enrichment ratio was higher than the liver to plasma ratio (70-89%), showing that an important part of glycine de novo synthesis in pancreas occurred through the threonine dehydrogenase (TDG) pathway. These results imply that calculation of threonine oxidation into glycine should be made with the assumption of both hepatic and extrahepatic oxidation. Plateau values of plasma threonine, glycine and α-ketobutyrate enrichments and specific radio activities allowed estimations of threonine oxidation through the TDG and threonine dehydratase (TDH) pathways. Threonine oxidation into glycine was 12.16 ± 2.06, 2.89 ± 0.61 and 2.13 ± 0.44 μmol/(kg.h), respectively, in pigs fed the control, LT and Glu diets, and threonine oxidation into α-ketobutyrate was 1.80 ± 0.31, 0.88 ± 0.02 and 0.55 ± 0.06 μmol/(kg.h) for the control, LT and Glu groups, respectively. Total threonine oxidation rates were 75 and 81% lower in the LT and Glu groups, respectively, than in the control group. Liver TDG and TDH activity measured in vitro were not affected by either the level of dietary threonine supply the addition of glutamic acid. On the basis of plasma data, it may be concluded that the addition of glutamic acid to a threonine-deficient diet had no significant effect on threonine oxidation but did reduce the rate of threonine release from protein breakdown. Oxidation appears to be related to plasma threonine concentration.
Ichiro Chibata - One of the best experts on this subject based on the ideXlab platform.
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Stereochemistry of the Conversions of L-Threonine and d-Threonine into 2-Oxobutanoate by the L-Threonine and d-Threonine Dehydratases of Serratia marcescens
FEBS Journal, 2005Co-Authors: David H. G. Crout, Maria Virgínia Mendes Gregório, Saburo Komatsubara, Masahiko Kisumi, Urs Muller, Ichiro ChibataAbstract:1 dl[3-2H]Threonine, l-[3-2H]threonine, (2RS,3S)-2-amino[3-2H1] butanoic acid, (2RS,3S) 2-amino[3-3H1]butanoic acid and dl-2-amino[3-14C]butanoic acid were synthesised. 2 l-[3-2-H]Threonine was converted into l-[4-2H1]isoleucine by Serratia marcescens strain IHr313. 3 (2RS,3S)-2-Amino[3-2-H1]butanoic acid was converted into l-[4-4-H1]isoleucine by S. marcescens strain 149. 4 Analysis by 220-MHz NMR spectroscopy of the labelled l-isoleucine produced showed that the same diastereotopic hydrogen at C-4 was labelled in each experiment, proving that, during the conversion of L-Threonine into 2-oxobutanoate mediated by biosynthetic L-Threonine dehydratase, the hydroxyl group at C-3 was replaced by hydrogen with retention of configuration. 5 S. marcescens strain 149 lacks L-Threonine dehydratase but possesses an inducible d-threonine dehydratase. This strain converted dl-[3-2H]threonine into dl-[4-2H1]isoleucine with the deuterium located in the diastereotopic C-4 hydrogen derived from the 3 pro-R hydrogen of 2-aminobutanoic acid. 6 This result proved that during the conversion of d-threonine into 2-oxobutanoate mediated by d-threonine dehydratase, the hydroxyl group at C-3 is replaced by hydrogen with retention of configuration. 7 These results also prove that the protonation at C-3 of the proposed enamine intermediate in the transformations catalysed by threonine dehydratase is under enzymatic control. 8 The present results, taken in conjunction with the independent assignment of the signals in the 220-MHz NMR spectrum of l-isoleucine due to the diastereotopic protons at C-4, prove that during the ethyl migration step in l-isoleucine biosynthesis, the configuration at the migrating centre is retained.
Hideaki Yamada - One of the best experts on this subject based on the ideXlab platform.
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The GLY1 Gene of Saccharomyces Cerevisiae Encodes a Low‐Specific L‐threonine Aldolase that Catalyzes Cleavage of L‐allo‐Threonine and L‐threonine to Glycine
FEBS Journal, 1997Co-Authors: Shinji Nagata, Tohru Dairi, Haruo Misono, Sakayu Shimizu, Hideaki YamadaAbstract:The GLY1 gene of Saccharomyces cerevisiae is required for the biosynthesis of glycine for cell growth [McNeil, J. B., McIntosh, E. V., Taylor, B. V., Zhang, F.-R., Tang, S. & Bognar, A. L. (1994)J. Biol. Chem. 269, 9155–9165], but its gene product has not been identified. We have found that the GLY1 protein is similar in primary structure to L-allo-threonine aldolase of Aeromonas jandiae DK-39, which stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The GLY1 gene was amplified by PCR, with a designed ribosome-binding site, cloned into pUC118, and expressed in Escherichia coli cells. The enzyme was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The enzyme has a molecular mass of about 170 kDa and consists of four subunits identical in molecular mass. The enzyme contains 2 mol pyridoxal 5′-phosphate/4 mol of subunit as a cofactor, and its absorption spectrum exhibits maxima at 280 nm and 420 nm. The enzyme catalyzes the cleavage of not only L-allo-threonine to glycine but also L-Threonine. We have termed the enzyme a low-specific L-Threonine aldolase to distinguish it from L-allo-threonine aldolase.
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the gly1 gene of saccharomyces cerevisiae encodes a low specific l threonine aldolase that catalyzes cleavage of l allo threonine and l threonine to glycine
FEBS Journal, 1997Co-Authors: Shinji Nagata, Tohru Dairi, Haruo Misono, Sakayu Shimizu, Hideaki YamadaAbstract:The GLY1 gene of Saccharomyces cerevisiae is required for the biosynthesis of glycine for cell growth [McNeil, J. B., McIntosh, E. V., Taylor, B. V., Zhang, F.-R., Tang, S. & Bognar, A. L. (1994)J. Biol. Chem. 269, 9155–9165], but its gene product has not been identified. We have found that the GLY1 protein is similar in primary structure to L-allo-threonine aldolase of Aeromonas jandiae DK-39, which stereospecifically catalyzes the interconversion of L-allo-threonine and glycine. The GLY1 gene was amplified by PCR, with a designed ribosome-binding site, cloned into pUC118, and expressed in Escherichia coli cells. The enzyme was purified to homogeneity, as judged by polyacrylamide gel electrophoresis. The enzyme has a molecular mass of about 170 kDa and consists of four subunits identical in molecular mass. The enzyme contains 2 mol pyridoxal 5′-phosphate/4 mol of subunit as a cofactor, and its absorption spectrum exhibits maxima at 280 nm and 420 nm. The enzyme catalyzes the cleavage of not only L-allo-threonine to glycine but also L-Threonine. We have termed the enzyme a low-specific L-Threonine aldolase to distinguish it from L-allo-threonine aldolase.
Motoji Fujioka - One of the best experts on this subject based on the ideXlab platform.
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Serine hydroxymethyltransferase and threonine aldolase: are they identical?
The International Journal of Biochemistry & Cell Biology, 2000Co-Authors: Hirofumi Ogawa, Tomoharu Gomi, Motoji FujiokaAbstract:Abstract Serine hydroxymethyltransferase, a pyridoxal phosphate-dependent enzyme, catalyses the interconversion of serine and glycine, both of which are major sources of one-carbon units necessary for the synthesis of purine, thymidylate, methionine, and so on. Threonine aldolase catalyzes the pyridoxal phosphate-dependent, reversible reaction between threonine and acetaldehyde plus glycine. No extensive studies have been carried out on threonine aldolase in animal tissues, and it has long been believed that serine hydroxymethyltransferase and threonine aldolase are the same, i.e. one entity. This is based on the finding that rabbit liver serine hydroxymethyltransferase possesses some threonine aldolase activity. Recently, however, many kinds of threonine aldolase and corresponding genes were isolated from micro-organisms, and these enzymes were shown to be distinct from serine hydroxymethyltransferase. The experiments with isolated hepatocytes and cell-free extracts from various animals revealed that threonine is degraded mainly through the pathway initiated by threonine 3-dehydrogenase, and there is little or no contribution by threonine aldolase. Thus, although serine hydroxymethyltransferase from some mammalian livers exhibits a low threonine aldolase activity, the two enzymes are distinct from each other and mammals lack the “genuine” threonine aldolase.
