scholarly journals Sustainable Production of N-methylphenylalanine by Reductive Methylamination of Phenylpyruvate Using Engineered Corynebacterium glutamicum

2021 ◽  
Vol 9 (4) ◽  
pp. 824
Author(s):  
Anastasia Kerbs ◽  
Lynn Schwardmann ◽  
Melanie Mindt ◽  
Volker F. Wendisch
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Corynebacterium Glutamicum ◽  
Xanthomonas Campestris ◽  
De Novo ◽  
Xylose Isomerase ◽  
Sustainable Production ◽  
Volumetric Productivity ◽  
Genes Encoding ◽  
Branched Chain

N-alkylated amino acids occur widely in nature and can also be found in bioactive secondary metabolites such as the glycopeptide antibiotic vancomycin and the immunosuppressant cyclosporine A. To meet the demand for N-alkylated amino acids, they are currently produced chemically; however, these approaches often lack enantiopurity, show low product yields and require toxic reagents. Fermentative routes to N-alkylated amino acids like N-methyl-l-alanine or N-methylantranilate, a precursor of acridone alkaloids, have been established using engineered Corynebacterium glutamicum, which has been used for the industrial production of amino acids for decades. Here, we describe metabolic engineering of C. glutamicum for de novo production of N-methylphenylalanine based on reductive methylamination of phenylpyruvate. Pseudomonas putida Δ-1-piperideine-2-carboxylate reductase DpkA containing the amino acid exchanges P262A and M141L showed comparable catalytic efficiencies with phenylpyruvate and pyruvate, whereas the wild-type enzyme preferred the latter substrate over the former. Deletion of the anthranilate synthase genes trpEG and of the genes encoding branched-chain amino acid aminotransferase IlvE and phenylalanine aminotransferase AroT in a strain engineered to overproduce anthranilate abolished biosynthesis of l-tryptophan and l-phenylalanine to accumulate phenylpyruvate. Upon heterologous expression of DpkAP262A,M141L, N-methylphenylalanine production resulted upon addition of monomethylamine to the medium. In glucose-based minimal medium, an N-methylphenylalanine titer of 0.73 ± 0.05 g L−1, a volumetric productivity of 0.01 g L−1 h−1 and a yield of 0.052 g g−1 glucose were reached. When xylose isomerase gene xylA from Xanthomonas campestris and the endogenous xylulokinase gene xylB were expressed in addition, xylose as sole carbon source supported production of N-methylphenylalanine to a titer of 0.6 ± 0.04 g L−1 with a volumetric productivity of 0.008 g L−1 h−1 and a yield of 0.05 g g−1 xylose. Thus, a fermentative route to sustainable production of N-methylphenylalanine by recombinant C. glutamicum has been established.

scholarly journals Mechanism of De Novo Branched-Chain Amino Acid Synthesis as an Alternative Electron Sink in Hypoxic Aspergillus nidulans Cells

2010 ◽  
Vol 76 (5) ◽  
pp. 1507-1515 ◽  
Author(s):  
Motoyuki Shimizu ◽  
Tatsuya Fujii ◽  
Shunsuke Masuo ◽  
Naoki Takaya
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aspergillus Nidulans ◽  
De Novo ◽  
Acid Synthesis ◽  
Branched Chain Amino Acids ◽  
Amino Acid Synthesis ◽  
Lactate Dehydrogenase Activity ◽  
Branched Chain Amino Acid ◽  
Branched Chain

ABSTRACT Although branched-chain amino acids are synthesized as building blocks of proteins, we found that the fungus Aspergillus nidulans excretes them into the culture medium under hypoxia. The transcription of predicted genes for synthesizing branched-chain amino acids was upregulated by hypoxia. A knockout strain of the gene encoding the large subunit of acetohydroxy acid synthase (AHAS), which catalyzes the initial reaction of the synthesis, required branched-chain amino acids for growth and excreted very little of them. Pyruvate, a substrate for AHAS, increased the amount of hypoxic excretion in the wild-type strain. These results indicated that the fungus responds to hypoxia by synthesizing branched-chain amino acids via a de novo mechanism. We also found that the small subunit of AHAS regulated hypoxic branched-chain amino acid production as well as cellular AHAS activity. The AHAS knockout resulted in higher ratios of NADH/NAD+ and NADPH/NADP+ under hypoxia, indicating that the branched-chain amino acid synthesis contributed to NAD+ and NADP+ regeneration. The production of branched-chain amino acids and the hypoxic induction of involved genes were partly repressed in the presence of glucose, where cells produced ethanol and lactate and increased levels of lactate dehydrogenase activity. These indicated that hypoxic branched-chain amino acid synthesis is a unique alternative mechanism that functions in the absence of glucose-to-ethanol/lactate fermentation and oxygen respiration.

scholarly journals Molecular analysis of the role of two aromatic aminotransferases and a broad-specificity aspartate aminotransferase in the aromatic amino acid metabolism ofPyrococcus furiosus

Archaea ◽  
2002 ◽  
Vol 1 (2) ◽  
pp. 133-141 ◽  
Author(s):  
Donald E. Ward ◽  
William M. de Vos ◽  
John van der Oost
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Aspartate Aminotransferase ◽  
Pyrococcus Furiosus ◽  
Aromatic Amino Acids ◽  
Significant Activity ◽  
Transamination Reaction ◽  
Genes Encoding ◽  
Branched Chain ◽  
Aromatic Amino Acid Metabolism

The genes encoding aromatic aminotransferase II (AroAT II) and aspartate aminotransferase (AspAT) fromPyrococcus furiosushave been identified, expressed inEscherichia coliand the recombinant proteins characterized. The AroAT II enzyme was specific for the transamination reaction of the aromatic amino acids, and uses α-ketoglutarate as the amino acceptor. Like the previously characterized AroAT I, AroAT II has highest efficiency for phenylalanine (kcat/Km= 923 s–1mM–1). Northern blot analyses revealed that AroAT I was mainly expressed when tryptone was the primary carbon and energy source. Although the expression was significantly lower, a similar trend was observed for AroAT II. These observations suggest that both AroATs are involved in amino acid degradation. Although AspAT exhibited highest activity with aspartate and α-ketoglutarate (kcat~105 s–1), it also showed significant activity with alanine, glutamate and the aromatic amino acids. With aspartate as the amino donor, AspAT catalyzed the amination of α-ketoglutarate, pyruvate and phenylpyruvate. No activity was detected with either branched-chain amino acids or α-keto acids. The AspAT gene (aspC) was expressed as a polycistronic message as part of thearooperon, with expression observed only when the aromatic amino acids were absent from the growth medium, indicating a role in the biosynthesis of the aromatic amino acids.

scholarly journals Dynamic Co-Cultivation Process of Corynebacterium glutamicum Strains for the Fermentative Production of Riboflavin

Fermentation ◽  
2021 ◽  
Vol 7 (1) ◽  
pp. 11
Author(s):  
Fernando Pérez-García ◽  
Arthur Burgardt ◽  
Dina R. Kallman ◽  
Volker F. Wendisch ◽  
Nadav Bar
Keyword(s):  
Corynebacterium Glutamicum ◽  
Xanthomonas Campestris ◽  
Product Formation ◽  
Volumetric Productivity ◽  
Riboflavin Production ◽  
Xylose Consumption ◽  
Single Strain ◽  
Sugar Mixtures ◽  
Fed Batch Fermentation ◽  
Overexpressing Strain

Residual streams from lignocellulosic processes contain sugar mixtures of glucose, xylose, and mannose. Here, the industrial workhorse Corynebacterium glutamicum was explored as a research platform for the rational utilization of a multiple sugar substrate. The endogenous manA gene was overexpressed to enhance mannose utilization. The overexpression of the xylA gene from Xanthomonas campestris in combination with the endogenous xylB gene enabled xylose consumption by C. glutamicum. Furthermore, riboflavin production was triggered by overexpressing the sigH gene from C. glutamicum. The resulting strains were studied during batch fermentations in flasks and 2 L lab-scale bioreactors separately using glucose, mannose, xylose, and a mixture of these three sugars as a carbon source. The production of riboflavin and consumption of sugars were improved during fed-batch fermentation thanks to a dynamic inoculation strategy of manA overexpressing strain and xylAB overexpressing strain. The final riboflavin titer, yield, and volumetric productivity from the sugar mixture were 27 mg L−1, 0.52 mg g−1, and 0.25 mg L−1 h−1, respectively. It reached a 56% higher volumetric productivity with 45% less by-product formation compared with an equivalent process inoculated with a single strain overexpressing the genes xylAB and manA combined. The results indicate the advantages of dynamic multi strains processes for the conversion of sugar mixtures.

scholarly journals Action of growth hormone in vitro on the net uptake and incorporation into protein of amino acids in muscle from intact rabbits given protein-deficient diets

1976 ◽  
Vol 35 (1) ◽  
pp. 1-10 ◽  
Author(s):  
M. R. Turner ◽  
P. J. Reeds ◽  
K. A. Munday
Keyword(s):  
Amino Acids ◽  
Growth Hormone ◽  
Protein Synthesis ◽  
Amino Acid ◽  
De Novo ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Amino Acid Incorporation ◽  
Acid Incorporation

1. Net amino acid uptake, and incorporation into protein have been measured in vitro in the presence and absence of porcine growth hormone (GH) in muscle from intact rabbits fed for 5 d on low-protein (LP), protein-free (PF) or control diets.2. In muscle from control and LP animals GH had no effect on the net amino acid uptake but stimulated amino acid incorporation into protein, although this response was less in LP animals than in control animals.3. In muscle from PF animals, GH stimulated both amino acid incorporation into protein and the net amino acid uptake, a type of response which also occurs in hypophysectomized animals. The magnitude of the effect of GH on the incorporation of amino acids into protein was reduced in muscle from PF animals.4. The effect of GH on the net amino acid uptake in PF animals was completely blocked by cycloheximide; the uptake effect of GH in these animals was dependent therefore on de novo protein synthesis.5. It is proposed that in the adult the role of growth hormone in protein metabolism is to sustain cellular protein synthesis when there is a decrease in the level of substrate amino acids, similar to that which occurs during a short-term fast or when the dietary protein intake is inadequate.

scholarly journals De Novo Synthesis of Amino Acids by the Ruminal Bacteria Prevotella bryantii B14,Selenomonas ruminantium HD4, and Streptococcus bovis ES1

1998 ◽  
Vol 64 (8) ◽  
pp. 2836-2843 ◽  
Author(s):  
Cengiz Atasoglu ◽  
Carmen Valdés ◽  
Nicola D. Walker ◽  
C. James Newbold ◽  
R. John Wallace
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
De Novo ◽  
Dependent Manner ◽  
Streptococcus Bovis ◽  
De Novo Synthesis ◽  
Ruminal Bacteria ◽  
Total N ◽  
Selenomonas Ruminantium ◽  
Prevotella Bryantii

ABSTRACT The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B14, Selenomonas ruminantiumHD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing 15NH4Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii, S. ruminantium, andS. bovis, respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii, peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.

scholarly journals Rhizobium leguminosarum Has a Second General Amino Acid Permease with Unusually Broad Substrate Specificity and High Similarity to Branched-Chain Amino Acid Transporters (Bra/LIV) of the ABC Family

2002 ◽  
Vol 184 (15) ◽  
pp. 4071-4080 ◽  
Author(s):  
A. H. F. Hosie ◽  
D. Allaway ◽  
C. S. Galloway ◽  
H. A. Dunsby ◽  
P. S. Poole
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rhizobium Leguminosarum ◽  
Binding Protein ◽  
Amino Acid Transporters ◽  
Acid Uptake ◽  
Amino Acid Permease ◽  
Branched Chain Amino Acid ◽  
General Amino Acid Permease ◽  
Branched Chain

ABSTRACT Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (BraRl). Characterization of the solute specificity of BraRl shows it to be the second general amino acid permease of R. leguminosarum. Although BraRl has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (l-glutamate, l-arginine, and l-histidine), in addition to neutral amino acids (l-alanine and l-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be α-amino acids. Consistent with this, BraRl is the first ABC transporter to be shown to transport γ-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by BraRl does not appear to be stereospecific as d amino acids cause significant inhibition of uptake of l-glutamate and l-leucine. Unlike all other solutes tested, l-alanine uptake is not dependent on solute binding protein BraCRl. Therefore, a second, unidentified solute binding protein may interact with the BraDEFGRl membrane complex during l-alanine uptake. Overall, the data indicate that BraRl is a general amino acid permease of the HAAT family. Furthermore, BraRl has the broadest solute specificity of any characterized bacterial amino acid transporter.

Consistency and Change

2019 ◽  
Author(s):  
Alan Kelly
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Living Organism ◽  
Peptide Bond ◽  
The Body ◽  
Blood Proteins ◽  
Genes Encoding ◽  
Fundamental Phenomenon ◽  
P Proteins

Proteins are, in my view, the most impressive molecules in food. They influence the texture, crunch, chew, flow, color, flavor, and nutritional quality of food. Not only that, but they can radically change their properties and how they behave depending on the environment and, critically for food, in response to processes like heating. Even when broken down into smaller components they are important, for example giving cheese many of its critical flavor notes. Indeed, I would argue that perhaps the most fundamental phenomenon we encounter in cooking or processing food is the denaturation of proteins, as will be explained shortly. Beyond food, the value of proteins and their properties is widespread across biology. Many of the most significant molecules in our body and that of any living organism (including plants and animals) are proteins. These include those that make hair and skin what they are, as well as the hemoglobin that transports oxygen around the body in our blood. Proteins are built from amino acids, a family of 20 closely related small molecules, which all have in chemical terms the same two ends (chemically speaking, an amino end and an acidic end, hence the name) but differ in the middle. This bit in the middle varies from amino acid to amino acid, from simple (a hydrogen atom in the case of glycine, the simplest amino acid) to much more complex structures. Amino acids can link up very neatly, as the amino end of one can form a bond (called a peptide bond) with the acid end of another, and so forth, so that chains of amino acids are formed that, when big enough (more than a few dozen amino acids), we call proteins. Our bodies produce thousands of proteins for different functions, and the instructions for which amino acids combine to make which proteins are essentially what the genetic code encrypted in our DNA specifies. We hear a lot about our genes encoding the secrets of life, but what that code spells is basically P-R-O-T-E-I-N. Yes, these are very important molecules!

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