Self-regulated efficient production of L-threonine via an artificial quorum sensing system in engineered Escherichia coli.

Imbalance in carbon flux distribution is one of the most important factors affecting the further increase in the yield of high value-added natural products in microbial metabolic engineering. Meanwhile, the most common inducible expression systems are difficult to achieve industrial-scale production due to the addition of high-cost or toxic inducers during the fermentation process. Quorum sensing system, as a typical model for density-dependent induction of gene expression, has been widely applied in synthetic biology. However, there are currently few reports for efficient production of microbial natural products by using quorum sensing system to self-regulate carbon flux distribution. Here, we designed an artificial quorum sensing system to achieve efficient production of L-threonine in engineered Escherichia coli by altering the carbon flux distribution of the central metabolic pathways at specific periods. Under the combination of switch module and production module, the system was applied to divide the microbial fermentation process into two stages including growth and production, and improve the production of L-threonine by self-inducing the expression of pyruvate carboxylase and threonine extracellular transporter protease after a sufficient amount of cell growth. The final strain TWF106/pST1011, pST1042pr could produce 118.2 g/L L-threonine with a yield of 0.57 g/g glucose and a productivity of 2.46 g/(L· h). The establishment of this system has important guidance and application value for the production of other high value-added chemicals in microorganisms by self-regulation.

Construction of an Escherichia coli Strain Lacking Fimbriae by Deleting 64 Genes and Its Application for Efficient Production of Poly(3-Hydroxybutyrate) and l-Threonine.

Escherichia coli contains 12 chaperone-usher operons for biosynthesis and assembly of various fimbriae. In this study, each of the 12 operons was deleted in E. coli MG1655, and the resulting 12 deletion mutants all grew better than the wild type, especially in the nutrient-deficient M9 medium. When the plasmid pBHR68 containing the key genes for polyhydroxyalkanoate production was introduced into these 12 mutants, each mutant synthesized more polyhydroxyalkanoate than the wild-type control. These results indicate that the fimbria removal in E. coli benefits cell growth and polyhydroxyalkanoate production. Therefore, all 12 chaperone-usher operons, including 64 genes, were deleted in MG1655, resulting in the fimbria-lacking strain WQM026. WQM026 grew better than MG1655, and no fimbria structures were observed on the surface of WQM026 cells. Transcriptomic analysis showed that in WQM026 cells, the genes related to glucose consumption, glycolysis, flagellar synthesis, and biosynthetic pathways of some key amino acids were upregulated, while the tricarboxylic acid cycle-related genes were downregulated. When pBHR68 was introduced into WQM026, huge amounts of poly-3-hydroxybutyrate were produced; when the plasmid pFW01-thrA*BC-rhtC, containing the key genes for l-threonine biosynthesis and transport, was transferred into WQM026, more l-threonine was synthesized than with the control. These results suggest that this fimbria-lacking E. coli WQM026 is a good host for efficient production of polyhydroxyalkanoate and l-threonine and has the potential to be developed into a valuable chassis microorganism. IMPORTANCE In this study, we investigated the interaction between the biosynthesis and assembly of fimbriae and intracellular metabolic networks in E. coli. We found that eliminating fimbriae could effectively improve the production of polyhydroxyalkanoate and l-threonine in E. coli MG1655. These results contribute to understanding the necessity of fimbriae and the advantages of fimbria removal for industrial microorganisms. The knowledge gathered from this study may be applied to the development of superior chassis microorganisms.

Improving L-threonine production in Escherichia coli by elimination of transporters ProP and ProVWX.

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.

An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis.

Phage infection is common during the production of L-threonine by E. coli, and low L-threonine production and glucose conversion percentage are bottlenecks for the efficient commercial production of L-threonine. In this study, 20 antiphage mutants producing high concentration of L-threonine were obtained by atmospheric and room temperature plasma (ARTP) mutagenesis, and an antiphage E. coli variant was characterized that exhibited the highest production of L-threonine Escherichia coli ([E. coli] TRFC-AP). The elimination of fhuA expression in E. coli TRFC-AP was responsible for phage resistance. The biomass and cell growth of E. coli TRFC-AP showed no significant differences from those of the parent strain (E. coli TRFC), and the production of L-threonine (159.3 g L-1 ) and glucose conversion percentage (51.4%) were increased by 10.9% and 9.1%, respectively, compared with those of E. coli TRFC. During threonine production (culture time of 20 h), E. coli TRFC-AP exhibited higher activities of key enzymes for glucose utilization (hexokinase, glucose phosphate dehydrogenase, phosphofructokinase, phosphoenolpyruvate carboxylase, and PYK) and threonine synthesis (glutamate synthase, aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase) compared to those of E. coli TRFC. The analysis of metabolic flux distribution indicated that the flux of threonine with E. coli TRFC-AP reached 69.8%, an increase of 16.0% compared with that of E. coli TRFC. Overall, higher L-threonine production and glucose conversion percentage were obtained with E. coli TRFC-AP due to increased activities of key enzymes and improved carbon flux for threonine synthesis.

Rebalancing microbial carbon distribution for L-threonine maximization using a thermal switch system.

In metabolic engineering, unbalanced microbial carbon distribution has long blocked the further improvement in yield and productivity of high-volume natural metabolites. Current studies mostly focus on regulating desired biosynthetic pathways, whereas few strategies are available to maximize L-threonine efficiently. Here, we present a strategy to guarantee the supply of reduced cofactors and actualize L-threonine maximization by regulating cellular carbon distribution in central metabolic pathways. A thermal switch system was designed and applied to divide the whole fermentation process into two stages: growth and production. This system could rebalance carbon substrates between pyruvate and oxaloacetate by controlling the heterogenous expression of pyruvate carboxylase and oxaloacetate decarboxylation that responds to temperature. The system was tested in an L-threonine producer Escherichia coli TWF001, and the resulting strain TWF106/pFT24rp overproduced L-threonine from glucose with 111.78% molar yield. The thermal switch system was then employed to switch off the L-alanine synthesis pathway, resulting in the highest L-threonine yield of 124.03%, which exceeds the best reported yield (87.88%) and the maximum available theoretical value of L-threonine production (122.47%). This inducer-free genetic circuit design can be also developed for other biosynthetic pathways to increase product conversion rates and shorten production cycles.

Engineering Halomonas bluephagenesis for L-Threonine production.

Halophilic Halomonas bluephagenesis (H. bluephagenesis), a chassis for cost-effective Next Generation Industrial Biotechnology (NGIB), was for the first time engineered to successfully produce L-threonine, one of the aspartic family amino acids (AFAAs). Five exogenous genes including thrA*BC, lysC* and rhtC encoding homoserine dehydrogenase mutant at G433R, homoserine kinase, L-threonine synthase, aspartokinase mutant at T344M, S345L and T352I, and export transporter of threonine, respectively, were grouped into two expression modules for transcriptional tuning on plasmid- and chromosome-based systems in H. bluephagenesis, respectively, after pathway tuning debugging. Combined with deletion of import transporter or/and L-threonine dehydrogenase encoded by sstT or/and thd, respectively, the resulting recombinant H. bluephagenesis TDHR3-42-p226 produced 7.5 g/L and 33 g/L L-threonine when grown under open unsterile conditions in shake flasks and in a 7 L bioreactor, respectively. Engineering H. bluephagenesis demonstrates strong potential for production of diverse metabolic chemicals.

Expression regulation of multiple key genes to improve L-threonine in Escherichia coli.

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

Transcriptomic analysis of an l-threonine-producing Escherichia coli TWF001.

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.

Rational engineering of amide synthetase enables bioconversion to diverse xiamenmycin derivatives.

XimA is a unique amide synthetase that belongs to the ANL superfamily of adenylating enzymes, but with a special structural fold. In order to improve the enzyme promiscuity, we engineered XimA by site-directed mutagenesis at a specific position based on our theoretical model of XimA. Thus, we were able to produce diverse benzopyran derivatives with up to 15 different l-form and d-form amino acid substitutions, catalyzed by several XimA variants. Molecular docking and molecular dynamics simulations conducted for various XimA systems provide further structural insights into the substitution effects of the phenylalanine-201 as an active site residue on protein dynamics and enzyme catalysis.

Developing an l-threonine-producing strain from wild-type Escherichia coli by modifying the glucose uptake, glyoxylate shunt, and l-threonine biosynthetic pathway.

Wild-type Escherichia coli MG1655 usually does not accumulate l-threonine. In this study, the effects of 13 genes related to the glucose uptake, glycolysis, TCA cycle, l-threonine biosynthesis, or their regulation on l-threonine accumulation in E. coli MG1655 were investigated. Sixteen E. coli mutant strains were constructed by chromosomal deletion or overexpression of one or more genes of rsd, ptsG, ptsH, ptsI, crr, galP, glk, iclR, and gltA; the plasmid pFW01-thrA*BC-rhtC harboring the key genes for l-threonine biosynthesis and secretion was introduced into these mutants. The analyses on cell growth, glucose consumption, and l-threonine production of these recombinant strains showed that most of these strains could accumulate l-threonine, and the highest yield was obtained in WMZ016/pFW01-thrA*BC-rhtC. WMZ016 was derived from MG1655 by deleting crr and iclR and enhancing the expression of gltA. WMZ016/pFW01-thrA*BC-rhtC could produce 17.98 g/L l-threonine with a yield of 0.346 g/g glucose, whereas the control strain MG1655/pFW01-thrA*BC-rhtC could only produce 0.68 g/L l-threonine. In addition, WMZ016/pFW01-thrA*BC-rhtC could tolerate the high concentration of glucose and produced no detectable by-products; therefore, it should be an ideal platform strain for further development. The results indicate that manipulating the glucose uptake and TCA cycle could efficiently increase l-threonine production in E. coli.

Optimized medium via statistical approach enhanced threonine production by Pediococcus pentosaceus TL-3 isolated from Malaysian food.

BACKGROUND: Threonine is an essential amino acid that is extensively used in livestock industry as feed supplement due to its pronounced effect in improving the growth performance of animals. Application of genetically engineered bacteria for amino acid production has its share of controversies after eosinophils myalgia syndrome outbreak in 1980s. This has urged for continuous search for a food grade producer as a safer alternative for industrial amino acid production. Lactic acid bacteria (LAB) appear as an exceptional candidate owing to their non-pathogenic nature and reputation of Generally Recognized as Safe (GRAS) status. Recently, we have identified a LAB, Pediococcus pentosaceus TL-3, isolated from Malaysian food as a potential threonine producer. Thus, the objective of this study was to enhance the threonine production by P. pentosaceus TL-3 via optimized medium developed by using Plackett-Burman design (PBD) and central composite design (CCD). RESULTS: Molasses, meat extract, (NH4)2SO4, and MnSO4 were identified as the main medium components for threonine production by P. pentosaceus TL-3. The optimum concentration of molasses, meat extract, (NH4)2SO4 and MnSO4 were found to be 30.79 g/L, 25.30 g/L, 8.59 g/L, and 0.098 g/L respectively based on model obtained in CCD with a predicted net threonine production of 123.07 mg/L. The net threonine production by P. pentosaceus TL-3 in the optimized medium was enhanced approximately 2 folds compared to the control. CONCLUSIONS: This study has revealed the potential of P. pentosaceus TL-3 as a safer alternative to produce threonine. Additionally, the current study has identified the key medium components affecting the production of threonine by P. pentosaceus TL-3, followed by optimization of their concentrations by means of statistical approach. The findings of this study could act as a guideline for the future exploration of amino acid production by LAB.

Increasing L-threonine production in Escherichia coli by overexpressing the gene cluster phaCAB.

L-Threonine is an important branched-chain amino acid and could be applied in feed, drugs, and food. In this study, L-threonine production in an L-threonine-producing Escherichia coli strain TWF001 was significantly increased by overexpressing the gene cluster phaCAB from Ralstonia eutropha. TWF001/pFW01-phaCAB could produce 96.4-g/L L-threonine in 3-L fermenter and 133.5-g/L L-threonine in 10-L fermenter, respectively. In addition, TWF001/pFW01-phaCAB produced 216% more acetyl-CoA, 43% more malate, and much less acetate than the vector control TWF001/pFW01, and meanwhile, TWF001/pFW01-phaCAB produced poly-3-hydroxybutyrate, while TWF001/pFW01 did not. Transcription analysis showed that the key genes in the L-threonine biosynthetic pathway were up-regulated, the genes relevant to the acetate formation were down-regulated, and the gene acs encoding the enzyme which converts acetate to acetyl-CoA was up-regulated. The results suggested that overexpression of the gene cluster phaCAB in E. coli benefits the enhancement of L-threonine production.

Construction of a novel Escherichia coli expression system: relocation of lpxA from chromosome to a constitutive expression vector.

The selective marker in the plasmid-based expression system is usually a gene that encodes an antibiotic-resistant protein; therefore, the antibiotic has to add to maintain the plasmid when growing the bacteria. This antibiotic addition would lead to increase of production cost and the environment contamination. In this study, a novel Escherichia coli expression system, the lpxA deletion mutant harboring an lpxA-carrying vector, was developed. To develop this system, three plasmids pCas9Cre, pTF-A-UD, and pRSFCmlpxA were constructed. The plasmid pCas9Cre produces enzymes Cas9, λ-Red, and Cre and can be cured by growing at 42 °C; pTF-A-UD contains several DNA fragments required for deleting the chromosomal lpxA and can be cured by adding isopropyl-D-thiogalactopyranoside; pRSFCmlpxA contains the lpxA mutant lpxA123 and CamR. When E. coli were transformed with these three plasmids, the chromosomal lpxA and the CamR in pRSFCmlpxA can be efficiently removed, resulting in an E. coli lpxA mutant harboring pRSFlpxA. The lpxA is essential for the growth of E. coli; its relocation from chromosome to a constitutive expression vector is an ideal strategy to maintain the vector without antibiotic addition. The lpxA123 in pRSFlpxA can complement the deletion of the chromosomal lpxA and provide a strong selective pressure to maintain the plasmid pRSFlpxA. This study provides an experimental evidence that this novel expression system is convenient and efficient to use and can be used to improve L-threonine biosynthesis in the wild type E. coli MG1655 and an L-threonine producing E. coli TWF006.

Deletion of arcA, iclR, and tdcC in Escherichia coli to improve l-threonine production.

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.

Deletion of regulator-encoding genes fadR, fabR and iclR to increase L-threonine production in Escherichia coli.

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.

Fermentative production of the unnatural amino acid L-2-aminobutyric acid based on metabolic engineering.

BACKGROUND: L-2-aminobutyric acid (L-ABA) is an unnatural amino acid that is a key intermediate for the synthesis of several important pharmaceuticals. To make the biosynthesis of L-ABA environmental friendly and more suitable for the industrial-scale production. We expand the nature metabolic network of Escherichia coli using metabolic engineering approach for the production of L-ABA. RESULTS: In this study, Escherichia coli THR strain with a modified pathway for threonine-hyperproduction was engineered via deletion of the rhtA gene from the chromosome. To redirect carbon flux from 2-ketobutyrate (2-KB) to L-ABA, the ilvIH gene was deleted to block the L-isoleucine pathway. Furthermore, the ilvA gene from Escherichia coli W3110 and the leuDH gene from Thermoactinomyces intermedius were amplified and co-overexpressed. The promoter was altered to regulate the expression strength of ilvA* and leuDH. The final engineered strain E. coli THR ΔrhtAΔilvIH/Gap-ilvA*-Pbs-leuDH was able to produce 9.33 g/L of L-ABA with a yield of 0.19 g/L/h by fed-batch fermentation in a 5 L bioreactor. CONCLUSIONS: This novel metabolically tailored strain offers a promising approach to fulfill industrial requirements for production of L-ABA.

An in silico approach in identification of drug targets in Leishmania: A subtractive genomic and metabolic simulation analysis.

Leishmaniasis is one of the major health issue in developing countries. The current therapeutic regimen for this disease is less effective with lot of adverse effects thereby warranting an urgent need to develop not only new and selective drug candidates but also identification of effective drug targets. Here we present subtractive genomics procedure for identification of putative drug targets in Leishmania. Comprehensive druggability analysis has been carried out in the current work for identified metabolic pathways and drug targets. We also demonstrate effective metabolic simulation methodology to pinpoint putative drug targets in threonine biosynthesis pathway. Metabolic simulation data from the current study indicate that decreasing flux through homoserine kinase reaction can be considered as a good therapeutic opportunity. The data from current study is expected to show new avenue for designing experimental strategies in search of anti-leishmanial agents.

Why Threonine Is an Essential Amino Acid in Mammals and Birds: Studies at the Enzyme Level.

The only pathway for the synthesis of essential amino acids in vertebrates is reversible transamination of their keto analogs with glutamic acid. At the same time, it is commonly accepted that such essential amino acids as lysine and threonine are not involved in transamination and, therefore, cannot be synthesized from their keto analogs. However, using radiolabeled isotopes, synthesis of threonine was demonstrated in rat liver and in a reaction mixture containing chicken liver threonine dehydrogenase. In the review, we discuss why threonine is an essential amino acid in mammals and birds based on the pathways of threonine biosynthesis in these two classes of vertebrates.

Effects of betaine supplementation on L-threonine fed-batch fermentation by Escherichia coli.

Betaine can act as a stress protectant, methyl donor, or enzyme stabilizer in vitro for the biosynthesis of structurally complex compounds. The performances of betaine type and concentration on the metabolic processes of Escherichia coli JLTHR in a 5-L fermentor were investigated. The results showed that the maximum L-threonine production of 127.3 g/L and glucose conversion percentage of 58.12% was obtained fed with the glucose solution containing 2 g/L betaine hydrochloride, which increased by 14.5 and 6.87% more compared to that of the control, respectively. This study presents an analysis of the metabolic fluxes of E. coli JLTHR for the production of L-threonine with betaine supplementation. When betaine was fed into the fermentation culture medium, the metabolic flux entering into the pentose phosphate pathway (HMP) and biosynthesis route of L-threonine increased by 57.3 and 10.1%, respectively. In conclusion, exogenous addition of betaine was validated to be a feasible and efficacious approach to improve L-threonine production.

Increasing L-threonine production in Escherichia coli by engineering the glyoxylate shunt and the L-threonine biosynthesis pathway.

L-threonine is an important amino acid that can be added in food, medicine, or feed. Here, the influence of glyoxylate shunt on an L-threonine producing strain Escherichia coli TWF001 has been studied. The gene iclR was deleted, and the native promoter of the aceBA operon was replaced by the trc promoter in the chromosome of TWF001, the resulting strainTWF004 could produce 0.39 g L-threonine from1 g glucose after 36-h flask cultivation. Further replacing the native promoter of aspC by the trc promoter in the chromosome of TWF004 resulted in the strain TWF006. TWF006 could produce 0.42 g L-threonine from 1 g glucose after 36-h flask cultivation. Three key genes in the biosynthetic pathway of L-threonine, thrA * (a mutated thrA), thrB, and thrC were overexpressed in TWF006, resulting the strain TWF006/pFW01-thrA * BC. TWF006/pFW01-thrA * BC could produce 0.49 g L-threonine from 1 g glucose after 36-h flask cultivation. Next, the genes asd, rhtA, rhtC, or thrE were inserted into the plasmid TWF006/pFW01-thrA * BC, and TWF006 was transformed with these plasmids, resulting the strains TWF006/pFW01-thrA * BC-asd, TWF006/pFW01-thrA * BC-rhtA, TWF006/pFW01-thrA * BC-rhtC, and TWF006/pFW01-thrA * BC-thrE, respectively. These four strains could produce more L-threonine than the control strain, and the highest yield was produced by TWF006/pFW01-thrA * BC-asd; after 36-h flask cultivation, TWF006/pFW01-thrA * BC-asd could produce 15.85 g/l L-threonine, i.e., 0.53 g L-threonine per 1 g glucose, which is a 70% increase relative to the control strain TWF001. The results suggested that the combined engineering of glyoxylate shunt and L-threonine biosynthesis pathway could significantly increase the L-threonine production in E. coli.