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

2019 ◽  
Vol 20 (8) ◽  
pp. 2020 ◽  
Author(s):  
Ying-Yu Wang ◽  
Feng Zhang ◽  
Jian-Zhong Xu ◽  
Wei-Guo Zhang ◽  
Xiu-Lai Chen ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Glutamate Dehydrogenase ◽  
Redox State ◽  
Leucine Biosynthesis ◽  
Lysinibacillus Sphaericus ◽  
Coenzyme Binding ◽  
Binding Domains ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
By Products

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.

scholarly journals The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation.

1995 ◽  
Vol 15 (1) ◽  
pp. 52-57 ◽  
Author(s):  
Y Hu ◽  
T G Cooper ◽  
G B Kohlhaw
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Glutamate Dehydrogenase ◽  
Specific Activity ◽  
Yeast Cells ◽  
Homologous Sequence ◽  
Biosynthetic Pathways ◽  
Lacz Expression ◽  
Branched Chain Amino Acid ◽  
Branched Chain

The Leu3 protein of Saccharomyces cerevisiae has been shown to be a transcriptional regulator of genes encoding enzymes of the branched-chain amino acid biosynthetic pathways. Leu3 binds to upstream activating sequences (UASLEU) found in the promoters of LEU1, LEU2, LEU4, ILV2, and ILV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of alpha-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when alpha-isopropylmalate is absent. Following identification of a UASLEU-homologous sequence in the promoter of GDH1, the gene encoding NADP(+)-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UASLEU element. We then show that Leu3 is required for full activation of the GDH1 gene. First, the expression of a GDH1-lacZ fusion gene is three- to sixfold lower in a strain lacking the LEU3 gene than in an isogenic LEU3+ strain. Expression is restored to near-normal levels when the leu3 deletion cells are transformed with a LEU3-bearing plasmid. Second, a significant decrease in GDH1-lacZ expression is also seen when the UASLEU of the GDH1-lacZ construct is made nonfunctional by mutation. Third, the steady-state level of GDH1 mRNA decreases about threefold in leu3 null cells. The decrease in GDH1 expression in leu3 null cells is reflected in a diminished specific activity of NADP(+)-dependent glutamate dehydrogenase. We also demonstrate that the level of GDH1-lacZ expression correlates with the cells' ability to generate alpha-isopropylmalate and is lowest in cells unable to produce alpha-isopropylmalate. We conclude that GDH1, which plays an important role in the assimilation of ammonia in yeast cells, is, in part, activated by a Leu3-alpha-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.

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

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2102 ◽  
Author(s):  
Li-Yan Feng ◽  
Jian-Zhong Xu ◽  
Wei-Guo Zhang
Keyword(s):  
Corynebacterium Glutamicum ◽  
Branched Chain Amino Acids ◽  
Gene Products ◽  
Branched Chain Amino Acid ◽  
Synthesis Activity ◽  
Branched Chain ◽  
Aromatic Aminotransferase ◽  
First Time ◽  
Branched Chain Aminotransferase

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.

scholarly journals Linking Central Metabolism with Increased Pathway Flux: l-Valine Accumulation by Corynebacterium glutamicum

2002 ◽  
Vol 68 (5) ◽  
pp. 2246-2250 ◽  
Author(s):  
Eva Radmacher ◽  
Adela Vaitsikova ◽  
Udo Burger ◽  
Karin Krumbach ◽  
Hermann Sahm ◽  
...  
Keyword(s):  
Amino Acid ◽  
Corynebacterium Glutamicum ◽  
Acid Synthesis ◽  
Acetohydroxy Acid Synthase ◽  
Amino Acid Synthesis ◽  
Branched Chain Amino Acid ◽  
In The Wild ◽  
Valine Biosynthesis ◽  
Branched Chain ◽  
Pathway Flux

ABSTRACT Mutants of Corynebacterium glutamicum were made and enzymatically characterized to clone ilvD and ilvE, which encode dihydroxy acid dehydratase and transaminase B, respectively. These genes of the branched-chain amino acid synthesis were overexpressed together with ilvBN (which encodes acetohydroxy acid synthase) and ilvC (which encodes isomeroreductase) in the wild type, which does not excrete l-valine, to result in an accumulation of this amino acid to a concentration of 42 mM. Since l-valine originates from two pyruvate molecules, this illustrates the comparatively easy accessibility of the central metabolite pyruvate. The same genes, ilvBNCD, overexpressed in an ilvA deletion mutant which is unable to synthesize l-isoleucine increased the concentration of this amino acid to 58 mM. A further dramatic increase was obtained when panBC was deleted, making the resulting mutant auxotrophic for d-pantothenate. When the resulting strain, C. glutamicum 13032ΔilvAΔpanBC with ilvBNCD overexpressed, was grown under limiting conditions it accumulated 91 mM l-valine. This is attributed to a reduced coenzyme A availability and therefore reduced flux of pyruvate via pyruvate dehydrogenase enabling its increased drain-off via the l-valine biosynthesis pathway.

scholarly journals THE PRODUCT OF THE LEU-3 CISTRON AS A REGULATORY ELEMENT FOR THE PRODUCTION OF THE LEUCINE BIOSYNTHETIC ENZYMES OF NEUROSPORA

Genetics ◽  
1973 ◽  
Vol 74 (3) ◽  
pp. 443-459
Author(s):  
Joseph C Polacco ◽  
S R Gross
Keyword(s):  
Enzyme Production ◽  
Amino Acid Metabolism ◽  
Acid Metabolism ◽  
Regulatory Element ◽  
Branched Chain Amino Acids ◽  
Regulatory Activity ◽  
Leucine Biosynthesis ◽  
Branched Chain Amino Acid ◽  
Branched Chain ◽  
Isopropylmalate Dehydrogenase

ABSTRACT A class of intracistronic (or closely linked) partial reversions of leu-3 mutations has been found to be conditionally constitutive with respect to the synthesis of isopropylmalate isomerase (specified by the leu-2 cistron) and β-isopropylmalate dehydrogenase (specified by the leu-1 cistron), two of the enzymes of leucine biosynthesis in Neurospora. The intermediate level of enzyme production by these leu-3cc mutants is independent of the obligatory inducer effector, α-isopropylmalate, but dependent upon the presence of the branched-chain amino acids, isoleucine, valine and leucine. The properties of leu-3+, leu-3 and leu-3cc in heterokaryons indicate that the transnuclear regulatory activity of the leu-3 product varies specifically as a function of available effector molecules. The information presented suggests that the leu-3 cistron is responsible not only for the production of a "positive" regulatory substance necessary for the expression of the leu-1 and leu-2 cistrons, but that it probably serves also a coordinating role in the expression of many of the genes involved in branched-chain amino acid metabolism.

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