L-leucine Synthesis Research Articles

High-level and -yield production of L-leucine in engineered Escherichia coli by multistep metabolic engineering

L-leucine is an essential amino acid widely used in food and pharmaceutical industries. However, the relatively low production efficiency limits its large-scale application. In this study, we rationally developed an efficient L-leucine-producing Escherichia coli strain. Initially, the L-leucine synthesis pathway was enhanced by overexpressing feedback-resistant 2-isopropylmalate synthase and acetohydroxy acid synthase both derived from Corynebacterium glutamicum, along with two other native enzymes. Next, the pyruvate and acetyl-CoA pools were enriched by deleting competitive pathways, employing the nonoxidative glycolysis pathway, and dynamically modulating the citrate synthase activity, which significantly promoted the L-leucine production and yield to 40.69 g/L and 0.30 g/g glucose, respectively. Then, the redox flux was improved by substituting the native NADPH-dependent acetohydroxy acid isomeroreductase, branched chain amino acid transaminase, and glutamate dehydrogenase with their NADH-dependent equivalents. Finally, L-leucine efflux was accelerated by precise overexpression of the exporter and deletion of the transporter. Under fed-batch conditions, the final strain LXH-21 produced 63.29 g/L of L-leucine, with a yield and productivity of 0.37 g/g glucose and 2.64 g/(L h), respectively. To our knowledge, this study achieved the highest production efficiency of L-leucine to date. The strategies presented here will be useful for engineering E. coli strains for producing L-leucine and related products on an industrial scale.

Effect of fed-batch and chemostat cultivation processes of C. glutamicum CP for L-leucine production

ABSTRACT Most of the current industrial processes for L-leucine production are based on fermentation, usually in fed-batch operation mode. Although the culture technology has advanced in recent decades, the process still has significant drawbacks. To solve these problems, we investigated the effects of chemostat culture conditions on the production of L-leucine by Corynebacterium glutamicum CP. The dilution rate, the nitrogen source, and the carbon–nitrogen ratio of the medium were optimized. With the addition of ammonium acetate to the chemostat medium, the initial C/N ratio was adjusted to 57.6, and the L-leucine titer reached the highest level at the optimal dilution rate of 0.04 h−1. Compared with fed-batch culture, the L-leucine titer was reduced (from 53.0 to 24.8 g L−1), but the yield from glucose was increased by 10.0% (from 0.30 to 0.33 mol mol−1) and productivity was increased by 58.3% (from 1.2 to 1.9 g L−1 h−1). Moreover, the titer of the by-product L-alanine was significantly reduced (from 8.9 to 0.8 g L−1). In addition, gene expression levels and activity of key enzymes in the synthesis of L-leucine and L-alanine were analyzed to explain the difference of production performance between chemostat culture and fed-batch culture. The results indicate that chemostat culture has great potential to increase the industrial production of L-leucine compared to current fed-batch approaches.

Application of RecET-Cre/loxP system in Corynebacterium glutamicum ATCC14067 for L-leucine production.

To explore the RecET-Cre/loxP system for chromosomal replacement of promoter and its application on enhancement L-leucine production in Corynebacterium glutamicum (C. glutamicum) ATCC14067. The RecET-Cre/loxP system was used to achieve the chromosomal replacement of promoter in C. glutamicum ATCC14067 to adjust the metabolic flux involving the L-leucine synthetic pathway. First, leuAr_13032 from C. glutamicum ATCC13032 which carried two mutations was overexpressed to release enzyme feedback inhibition. Then, comparing different mutations in ilvBNC gene clusters, the results indicated that ilvBNC_CP was most effective to enhance the metabolic flux of pyruvate towards L-leucine synthesis. The promoters of pck, odx and pyk2 were overexpressed under the strong promoter Peftu or Psod to improve the supply of pyruvate. Besides, the promoter PilvBNC was employed to dynamically control the transcription level of icd due to its attenuation mechanism by responding to the concentration of L-leucine. The final engineered strain produced 14.05g L-leucine/L in flask cultivation. The RecET-Cre/loxP system is effective for gene manipulation in C. glutamicum ATCC14067. Besides, the results demonstrate the potential of C. glutamicum ATCC14067 for L-leucine production and provide new targets and strategies for strain development.

Rational modification of the carbon metabolism of Corynebacterium glutamicum to enhance L-leucine production.

L-Leucine is an essential amino acid that has wide and expanding applications in the industry. It is currently fast-growing market demand that provides a powerful impetus to further increase its bioconversion productivity and production stability. In this study, we rationally engineered the metabolic flux from pyruvate to L-leucine synthesis in Corynebacterium glutamicum to enhance both pyruvate availability and L-leucine synthesis. First, the pyc (encoding pyruvate carboxylase) and avtA (encoding alanine-valine aminotransferase) genes were deleted to weaken the metabolic flux of the tricarboxylic acid cycle and reduce the competitive consumption of pyruvate. Next, the transcriptional level of the alaT gene (encoding alanine aminotransferase) was down regulated by inserting a terminator to balance L-leucine production and cell growth. Subsequently, the genes involved in L-leucine biosynthesis were overexpressed by replacing the native promoters PleuA and PilvBNC of the leuA gene and ilvBNC operon, respectively, with the promoter Ptuf of eftu (encoding elongation factor Tu) and using a shuttle expression vector. The resulting strain WL-14 produced 28.47 ± 0.36g/L L-leucine in shake flask fermentation.

Improvement of l-Leucine Production in Corynebacterium glutamicum by Altering the Redox Flux.

The production of l-leucine was improved by the disruption of ltbR encoding transcriptional regulator and overexpression of the key genes (leuAilvBNCE) of the l-leucine biosynthesis pathway in Corynebacterium glutamicum XQ-9. In order to improve l-leucine production, we rationally engineered C. glutamicum to enhance l-leucine production, by improving the redox flux. On the basis of this, we manipulated the redox state of the cells by mutating the coenzyme-binding domains of acetohydroxyacid isomeroreductase encoded by ilvC, inserting NAD-specific leucine dehydrogenase, encoded by leuDH from Lysinibacillus sphaericus, and glutamate dehydrogenase encoded by rocG from Bacillus subtilis, instead of endogenous branched-chain amino acid transaminase and glutamate dehydrogenase, respectively. The yield of l-leucine reached 22.62 ± 0.17 g·L−1 by strain ΔLtbR-acetohydroxyacid isomeroreductase (AHAIR)M/ABNCME, and the concentrations of the by-products (l-valine and l-alanine) increased, compared to the strain ΔLtbR/ABNCE. Strain ΔLtbR-AHAIRMLeuDH/ABNCMLDH accumulated 22.87±0.31 g·L−1 l-leucine, but showed a drastically low l-valine accumulation (from 8.06 ± 0.35 g·L−1 to 2.72 ± 0.11 g·L−1), in comparison to strain ΔLtbR-AHAIRM/ABNCME, which indicated that LeuDH has much specificity for l-leucine synthesis but not for l-valine synthesis. Subsequently, the resultant strain ΔLtbR-AHAIRMLeuDHRocG/ABNCMLDH accumulated 23.31 ± 0.24 g·L−1 l-leucine with a glucose conversion efficiency of 0.191 g·g−1.

Transcriptomic and metabolomics analyses reveal metabolic characteristics of L-leucine- and L-valine-producing Corynebacterium glutamicum mutants

Industrial amino acid production strains of Corynebacterium glutamicum are usually obtained by mutagenesis. However, the genetic and metabolic characteristics and the efficient synthesis mechanism of the selected mutants are unclear. The aims of this study were (1) to determine the gene transcriptional patterns and intracellular metabolite levels of an L-leucine-producing mutant C. glutamicum CP and an L-valine-producing mutant C. glutamicum XV referring to wild type, and (2) to understand the efficient synthesis mechanism of target product of these mutants. For this purpose, transcriptomic and metabolomics analyses were combined to investigate the association between intracellular patterns and product synthesis. The high intracellular level of glucose and the low intracellular level of metabolites in the central carbon metabolism meant the glucose metabolism rate of two mutants was lower than wild type. However, the increased intracellular pentose level and gene transcription in the pentose phosphate pathway (PPP) indicated that the PPP of mutants was more active. Furthermore, the mutants showed higher intracellular level of NADPH, which was mainly generated in PPP. In the specific pathway for the synthesis of L-leucine and L-valine, the transcriptional level of most genes was upregulated in the mutants. However, the transcription of transaminase C coding gene Cgl2844 was downregulated in CP but upregulated in XV. The upregulation of Cgl2844 might benefit to the synthesis of L-valine and cause the significant decrease of intracellular level of L-alanine and L-glutamate of XV. These characteristics of the mutants provided insight into changes that could be made to systematically optimize the metabolic pathways for the production of L-leucine and L-valine.

Improved l-Leucine Production in Corynebacterium glutamicum by Optimizing the Aminotransferases.

The production of branched-chain amino acids (BCAAs) is still challenging, therefore we rationally engineered Corynebacterium glutamicum FA-1 to increase the l-leucine production by optimizing the aminotransferases. Based on this, we investigated the effects of the native aminotransferases, i.e., branched-chain amino acid aminotransferase (BCAT; encoded by ilvE) and aspartate aminotransferase (AspB; encoded by aspB) on l-leucine production in C. glutamicum. The strain FA-1△ilvE still exhibited significant growth without leucine addition, while FA-1△ilvE△aspB couldn’t, which indicated that AspB also contributes to L-leucine synthesis in vivo and the yield of leucine reached 20.81 ± 0.02 g/L. It is the first time that AspB has been characterized for l-leucine synthesis activity. Subsequently, the aromatic aminotransferase TyrB and the putative aspartate aminotransferases, the aspC, yhdR, ywfG gene products, were cloned, expressed and characterized for leucine synthesis activity in FA-1△ilvE△aspB. Only TyrB was able to synthesize l-leucine and the l-leucine production was 18.55 ± 0.42 g/L. The two putative branched-chain aminotransferase genes, ybgE and CaIlvE, were also cloned and expressed. Both genes products function efficiently in BCAAs biosynthesis. This is the first report of a rational modification of aminotransferase activity that improves the l-leucine production through optimizing the aminotransferases.

A Novel Antibiotic Mechanism of l-Cyclopropylalanine Blocking the Biosynthetic Pathway of Essential Amino Acid l-Leucine.

The unusual amino acid l-cyclopropylalanine was isolated from the mushroom Amanita virgineoides after detection in an anti-fungal screening test. l-Cyclopropylalanine was found to exhibit broad-spectrum inhibition against fungi and bacteria. The anti-fungal activity was found to be abolished in the presence of the amino acid l-leucine, but not any other amino acids, indicating that l-cyclopropylalanine may block the biosynthesis of the essential amino acid l-leucine, thereby inhibiting fungal and bacteria growth. Further biochemical studies found l-cyclopropylalanine indeed inhibits α-isopropylmalate synthase (α-IMPS), the enzyme that catalyzes the rate-limiting step in the biosynthetic pathway of l-leucine. Inhibition of essential l-leucine synthesis in fungal and bacteria organisms, a pathway absent in host organisms such as humans, may represent a novel antibiotic mechanism to counter the ever-increasing problem of drug resistance to existing antibiotics.

Surface Treatment of ZrO2 Nanoparticles with Biosafe Citric Acid and Its Utilization for the Synthesis of L-leucine Based Poly(Amide–Imide) Nanocomposites

This study aimed at preparing three nanocomposites of optically active poly(amide–imide) and zirconium dioxide (ZrO2) inorganic nanoparticles through the ultrasonic process. First, the surface of ZrO2 nanocomposites was chemically modified with bio-active citric acid in the basic media. Then, the poly(amide–imide) was reinforced with modified nanocomposites and three poly(amide–imide)/ZrO2-citric acid nanocomposites were synthesized by ultrasonic irradiation. The poly(amide–imide) was prepared by polycondensation of N-trimellitylimido-L-leucine with 4,4′-diaminodiphenylsulfone using of triphenyl phosphite and molten tetra-n-butylammonium bromide as green media. The obtained poly(amide–imide)/ZrO2-citric acid nanocomposites were characterized by different techniques.

The contest for precursors: channelling L-isoleucine synthesis in Corynebacterium glutamicum without byproduct formation.

L-Isoleucine is an essential amino acid, which is required as a pharma product and feed additive. Its synthesis shares initial steps with that of L-lysine and L-threonine, and four enzymes of L-isoleucine synthesis have an enlarged substrate specificity involved also in L-valine and L-leucine synthesis. As a consequence, constructing a strain specifically overproducing L-isoleucine without byproduct formation is a challenge. Here, we analyze for consequences of plasmid-encoded genes in Corynebacterium glutamicum MH20-22B on L-isoleucine formation, but still obtain substantial accumulation of byproducts. In a different approach, we introduce point mutations into the genome of MH20-22B to remove the feedback control of homoserine dehydrogenase, hom, and threonine dehydratase, ilvA, and we assay sets of genomic promoter mutations to increase hom and ilvA expression as well as to reduce dapA expression, the latter gene encoding the dihydrodipicolinate synthase. The promoter mutations are mirrored in the resulting differential protein levels determined by a targeted LC-MS/MS approach for the three key enzymes. The best combination of genomic mutations was found in strain K2P55, where 53 mM L-isoleucine could be obtained. Whereas in fed-batch fermentations with the plasmid-based strain, 94 mM L-isoleucine with L-lysine as byproduct was formed; with the plasmid-less strain K2P55, 109 mM L-isoleucine accumulated with no substantial byproduct formation. The specific molar yield with the latter strain was 0.188 mol L-isoleucine (mol glucose)(-1) which characterizes it as one of the best L-isoleucine producers available and which does not contain plasmids.

Synthesis of leucine micro/nanocrystals for pharmaceutical applications

We report that flower-like novel L-leucine micro/nanocrystals have been synthesized by a physical vapor deposition technique. The controllable synthesis of L-leucine micro/nanocrystals is very important for their pharmaceutical applications as drug carriers or excipients. Their morphologies have been controlled by adjusting the deposition position, deposition temperature and flux of the carrier gas. All of the micro/nanostructures have been characterized by scanning electron microscopy, transmission electron microscopy, X-ray powder diffraction, Raman spectroscopy and atomic force microscopy. A growth model for the formation mechanism of the flower-like crystal structures is proposed. The coating of leucine micro/nanocrystals for a drug model system, quercetin, is also presented. Leucine micro/nanocrystals have great potential in pharmaceutical applications such as dispersibility enhancers. This coating design concept can also be used for a variety of active pharmaceutical ingredients.

ChemInform Abstract: Three Approaches to the Synthesis of L-Leucine Selectively Labelled with Carbon-13 or Deuterium in Either Diastereotopic Methyl Group.

Three approaches to the synthesis of L-leucine selectively labelled with carbon-13 or deuterium in either diastereotopic methyl group as well as at C-3 and C-4 are described. In all three methods the stereogenic centre at C-2 was created with total stereocontrol via a one-pot, two-enzyme catalysed procedure involving hydrolysis and reductive amination of a 2-keto ester. However, the approaches vary in the synthesis of the isotopically labelled 2-keto esters and in the production of the stereogenic centre at C-4 which was achieved either via alkylation of a propionylated Evans’ auxiliary with labelled iodomethane or by the diastereoselective conjugate addition of a labelled organocopper reagent to crotonate tethered to a chiral sultam. The latter approach proved most efficient and using the (1R,2S,3R)-3-[N-phenylsulfonyl-N-(3,5-dimethyldiphenyl)aminobornan-2-ol ester 27, [5-13C]-L-leucine was prepared with >98% de at C-4 and in 49% overall yield from the first labelled intermediate 28.

Electroenzymatic strategies for deracemization, stereoinversion and asymmetric synthesis of amino acids

A combination of a selective enzymatic oxidation with an unselective electrochemical reduction step was applied for deracemization, stereoinversion and asymmetric synthesis of l-leucine (starting from racemic leucine, d-leucine or 4-methyl-2-oxovaleric acid) in a batch reactor. d-Amino acid oxidase ( d-AAO) from Trigonopsis variabilis was used as enzyme. Reaction conditions for the electrochemical and enzymatic reactions were investigated separately and finally combined to an electroenzymatic synthesis, yielding 3.5 mmol L −1 d −1 of l-leucine ( ee 91%).

Three approaches to the synthesis of L-leucine selectively labelled with carbon-13 or deuterium in either diastereotopic methyl group

Three approaches to the synthesis of L-leucine selectively labelled with carbon-13 or deuterium in either diastereotopic methyl group as well as at C-3 and C-4 are described. In all three methods the stereogenic centre at C-2 was created with total stereocontrol via a one-pot, two-enzyme catalysed procedure involving hydrolysis and reductive amination of a 2-keto ester. However, the approaches vary in the synthesis of the isotopically labelled 2-keto esters and in the production of the stereogenic centre at C-4 which was achieved either via alkylation of a propionylated Evans’ auxiliary with labelled iodomethane or by the diastereoselective conjugate addition of a labelled organocopper reagent to crotonate tethered to a chiral sultam. The latter approach proved most efficient and using the (1R,2S,3R)-3-[N-phenylsulfonyl-N-(3,5-dimethyldiphenyl)aminobornan-2-ol ester 27, [5-13C]-L-leucine was prepared with >98% de at C-4 and in 49% overall yield from the first labelled intermediate 28.

Methods for the Synthesis of L-Leucine Selectively Labelled with Carbon-13 or Deuterium in either Diastereotopic Methyl Group

Methods for the Synthesis of L-Leucine Selectively Labelled with Carbon-13 or Deuterium in either Diastereotopic Methyl Group

Methods for the Synthesis of L-Leucine Selectively Labelled with Carbon-13 or Deuterium in either Diastereotopic Methyl Group

A versatile approach is described for the enantioselective synthesis of isotopically labelled L-leucine involving the preparation of 2-oxo-4-methylpentanoic acid labelled selectively with carbon-13 or deuterium in either the pro-R or pro-S methyl group followed by a reductive amination of the ketone catalysed by leucine dehydrogenase. This strategy is applied to the total synthesis of ( 2S.4R)-[5.5.5-D 3]-leucine using CD 3I as the source of deuterium.

Enhanced formation of isoamyl alcohol in Zygosaccharomyces rouxii due to elimination of feedback inhibition of α-isopropylmalate synthase

Mutants of the ‘miso’ yeast, Zygosaccharomyces rouxii, that produced a large amount of isoamyl alcohol, an important flavour in miso fermentation, were isolated from 5,5,5-trifluoro- dl-leucine-resistant mutants, an analogue of l-leucine. One of the mutants, M21-10, produced a three-fold higher level of isoamyl alcohol than the wild-type strain MY21 in miso fermentation. The activity of α-isopropylmalate synthase, one of the enzymes used for l-leucine synthesis, in the mutant M21-10 was not inhibited by the addition of l-leucine, a feedback inhibitor.

Enhanced formation of isoamyl alcohol in Zygosaccharomyces rouxii due to elimination of feedback inhibition of alpha-isopropylmalate synthase.

Mutants of the 'miso' yeast, Zygosaccharomyces rouxii, that produced a large amount of isoamyl alcohol, an important flavour in miso fermentation, were isolated from 5,5,5-trifluoro-DL-leucine-resistant mutants, an analogue of L-leucine. One of the mutants, M21-10, produced a three-fold higher level of isoamyl alcohol than the wild-type strain MY21 in miso fermentation. The activity of alpha-isopropylamalate synthase, one of the enzymes used for L-leucine synthesis, in the mutant M21-10 was not inhibited by the addition of L-leucine, a feedback inhibitor.

Genetic Changes of Regulatory Mechanisms Occurred in Leucine and Valine Producing Mutants Derived fromBrevibacterium lactofermentum2256

The regulatory mechanisms in branched-chain amino acid synthesis were compared between 2-thiazolealanine (2-TA) resistant l-leucine and l-valine producing mutants and the 2-TA sensitive original strains of Brevibacterium lactofermentum 2256. In the original strains, sensitive to 2-TA, α-isopropylmalate (IPM) synthetase, the initial enzyme specific for l-leucine synthesis, is sensitive to feedback inhibition and to repression by l-leucine, and α-acetohydroxy acid (AHA) synthetase, the common initial enzyme for synthesis of l-isoleucine, l-valine as well as l-leucine, is sensitive to feedback inhibition by each one of these amino acids, and to repression by them all. In strain No. 218, a typical l-leucine producer resistant to 2-TA, IPM synthetase was found to be markedly desensitized and derepressed, and AHA synthetase remained unaltered. On the contrary, in strain No. 333, l-valine producer resistant to 2-TA, AHA synthetase was found to be desensitized and partially derepressed, and IPM synthetase remained unaltered. The genetic alteration of these regulatory mechanisms was discussed in connection with the accumulation pattern of amino acids.

Studies on the fermentative production of branched-chian amino acids. IV. Genetic changes of regulatory mechanisms occurred in leucine and valine producing mutants derived from Brevibacterium lactofermentum 2256.

The regulatory mechanisms in branched-chain amino acid synthesis were compared between 2-thiazolealanine (2-TA) resistant L-leucine and L-valine producing mutants and the 2-TA sensitive original strains of Brevibacterium lactofermentum 2256. In the original strains, sensitive to 2-TA, α-isopropylmalate (IPM) synthetase, the initial enzyme specific for L-leucine synthesis, is sensitive to feedback inhibition and to repression by L-leucine, and α-acetohydroxy acid (AHA) synthetase, the common initial enzyme for synthesis of L-isoleucine, L-valine as well as L-leucine, is sensitive to feedback inhibition by each one of these amino acids, and to repression by them all. In strain No. 218, a typical L-leucine producer resistant to 2-TA, IPM synthetase was found to be markedly desensitized and derepressed, and AHA synthetase remained unaltered. On the contrary, in strain No. 333, L-valine producer resistant to 2-TA, AHA synthetase was found to be desensitized and partially derepressed, and IPM synthetase remained unaltered. The genetic alteration of these regulatory mechanisms was discussed in connection with the accumulation pattern of amino acids.