De novo tryptophanase-based indole production by metabolically engineered Corynebacterium glutamicum.

Indole has an increasing interest in the flavor and fragrance industry. It is used in dairy products, tea drinks, and fine fragrances due to its distinct floral odor typical of jasmine blossoms. The current production of indole based on isolation from coal tar is non-sustainable and its isolation from plants is often unprofitable due to low yields. To offer an alternative to the conventional production, biosynthesis of indole has been studied recently. A glucose-based indole production was achieved by employing the Corynebacterium glutamicum tryptophan synthase α-subunit (TrpA) or indole-3-glycerol phosphate lyase (IGL) from wheat Triticum aestivum in a genetically-engineered C. glutamicum strain. In addition, a highly efficient bioconversion process using C. glutamicum heterologously expressing tryptophanase gene (tnaA) from Providencia rettgeri as a biocatalyst was developed. In this work, de novo indole production from glucose was enabled by expressing the P. rettgeri tnaA in a tryptophan-producing C. glutamicum strain. By metabolic engineering of a C. glutamicum shikimate accumulating base strain, tryptophan production of 2.14 ± 0.02 g L-1 was achieved. Introduction of the tryptophanase form P. rettgeri enabled indole production, but to low titers, which could be improved by sequestering indole into the water-immiscible solvent tributyrin during fermentation and a titer of 1.38 ± 0.04 g L-1 was achieved. The process was accelerated by decoupling growth from production increasing the volumetric productivity about 4-fold to 0.08 g L-1 h-1. KEY POINTS: • Efficient de novo indole production via tryptophanases from glucose • Increased indole titers by product sequestration and improved precursor supply • Decoupling growth from production accelerated indole production.

Fermentative Indole Production via Bacterial Tryptophan Synthase Alpha Subunit and Plant Indole-3-Glycerol Phosphate Lyase Enzymes.

Indole is produced in nature by diverse organisms and exhibits a characteristic odor described as animal, fecal, and floral. In addition, it contributes to the flavor in foods, and it is applied in the fragrance and flavor industry. In nature, indole is synthesized either from tryptophan by bacterial tryptophanases (TNAs) or from indole-3-glycerol phosphate (IGP) by plant indole-3-glycerol phosphate lyases (IGLs). While it is widely accepted that the tryptophan synthase α-subunit (TSA) has intrinsically low IGL activity in the absence of the tryptophan synthase ß-subunit, in this study, we show that Corynebacterium glutamicum TSA functions as a bona fide IGL and can support fermentative indole production in strains providing IGP. By bioprospecting additional bacterial TSAs and plant IGLs that function as bona fide IGLs were identified. Capturing indole in an overlay enabled indole production to titers of about 0.7 g L-1 in fermentations using C. glutamicum strains expressing either the endogenous TSA gene or the IGL gene from wheat.

l-Serine Biosensor-Controlled Fermentative Production of l-Tryptophan Derivatives by Corynebacterium glutamicum.

l-Tryptophan derivatives, such as hydroxylated or halogenated l-tryptophans, are used in therapeutic peptides and agrochemicals and as precursors of bioactive compounds, such as serotonin. l-Tryptophan biosynthesis depends on another proteinogenic amino acid, l-serine, which is condensed with indole-3-glycerophosphate by tryptophan synthase. This enzyme is composed of the α-subunit TrpA, which catalyzes the retro-aldol cleavage of indole-3-glycerol phosphate, yielding glyceraldehyde-3-phosphate and indole, and the ß-subunit TrpB that catalyzes the ß-substitution reaction between indole and l-serine to water and l-tryptophan. TrpA is reported as an allosteric actuator, and its absence severely attenuates TrpB activity. In this study, however, we showed that Corynebacterium glutamicum TrpB is catalytically active in the absence of TrpA. Overexpression of C. glutamicumtrpB in a trpBA double deletion mutant supported growth in minimal medium only when exogenously added indole was taken up into the cell and condensed with intracellularly synthesized l-serine. The fluorescence reporter gene of an l-serine biosensor, which was based on the endogenous transcriptional activator SerR and its target promoter PserE, was replaced by trpB. This allowed for l-serine-dependent expression of trpB in an l-serine-producing strain lacking TrpA. Upon feeding of the respective indole derivatives, this strain produced the l-tryptophan derivatives 5-hydroxytryptophan, 7-bromotryptophan, and 5-fluorotryptophan.

Production of indole by Corynebacterium glutamicum microbial cell factories for flavor and fragrance applications.

BACKGROUND: The nitrogen containing aromatic compound indole is known for its floral odor typical of jasmine blossoms. Due to its characteristic scent, it is frequently used in dairy products, tea drinks and fine fragrances. The demand for natural indole by the flavor and fragrance industry is high, yet, its abundance in essential oils isolated from plants such as jasmine and narcissus is low. Thus, there is a strong demand for a sustainable method to produce food-grade indole. RESULTS: Here, we established the biotechnological production of indole upon L-tryptophan supplementation in the bacterial host Corynebacterium glutamicum. Heterologous expression of the tryptophanase gene from E. coli enabled the conversion of supplemented L-tryptophan to indole. Engineering of the substrate import by co-expression of the native aromatic amino acid permease gene aroP increased whole-cell biotransformation of L-tryptophan to indole by two-fold. Indole production to 0.2 g L-1 was achieved upon feeding of 1 g L-1 L-tryptophan in a bioreactor cultivation, while neither accumulation of side-products nor loss of indole were observed. To establish an efficient and robust production process, new tryptophanases were recruited by mining of bacterial sequence databases. This search retrieved more than 400 candidates and, upon screening of tryptophanase activity, nine new enzymes were identified as most promising. The highest production of indole in vivo in C. glutamicum was achieved based on the tryptophanase from Providencia rettgeri. Evaluation of several biological aspects identified the product toxicity as major bottleneck of this conversion. In situ product recovery was applied to sequester indole in a food-grade organic phase during the fermentation to avoid inhibition due to product accumulation. This process enabled complete conversion of L-tryptophan and an indole product titer of 5.7 g L-1 was reached. Indole partitioned to the organic phase which contained 28 g L-1 indole while no other products were observed indicating high indole purity. CONCLUSIONS: The bioconversion production process established in this study provides an attractive route for sustainable indole production from tryptophan in C. glutamicum. Industrially relevant indole titers were achieved within 24 h and indole was concentrated in the organic layer as a pure product after the fermentation.

Metabolic Engineering of Pseudomonas putida for Fermentative Production of l-Theanine.

N-alkylated amino acids are intermediates of natural biological pathways and can be found incorporated in peptides or have physiological roles in their free form. The N-ethylated amino acid l-theanine shows taste-enhancing properties and health benefits. It naturally occurs in green tea as major free amino acid. Isolation of l-theanine from Camilla sinensis shows low efficiency, and chemical synthesis results in a racemic mixture. Therefore, biochemical approaches for the production of l-theanine gain increasing interest. Here, we describe metabolic engineering of Pseudomonas putida KT2440 for the fermentative production of l-theanine from monoethylamine and carbon sources glucose, glycerol, or xylose using heterologous enzymes from Methylorubrum extorquens for l-theanine production and heterologous enzymes from Caulobacter crescentus for growth with xylose. l-Theanine (15.4 mM) accumulated in shake flasks with minimal medium containing monoethylamine and glucose, 15.2 mM with glycerol and 7 mM with xylose. Fed-batch bioreactor cultures yielded l-theanine titers of 10 g L-1 with glucose plus xylose, 17.2 g L-1 with glycerol, 4 g L-1 with xylose, and 21 g L-1 with xylose plus glycerol, respectively. To the best of our knowledge, this is the first l-theanine process using P. putida and the first compatible with the use of various alternative carbon sources.

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

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.

Fermentative N-Methylanthranilate Production by Engineered Corynebacterium glutamicum.

The N-functionalized amino acid N-methylanthranilate is an important precursor for bioactive compounds such as anticancer acridone alkaloids, the antinociceptive alkaloid O-isopropyl N-methylanthranilate, the flavor compound O-methyl-N-methylanthranilate, and as a building block for peptide-based drugs. Current chemical and biocatalytic synthetic routes to N-alkylated amino acids are often unprofitable and restricted to low yields or high costs through cofactor regeneration systems. Amino acid fermentation processes using the Gram-positive bacterium Corynebacterium glutamicum are operated industrially at the million tons per annum scale. Fermentative processes using C. glutamicum for N-alkylated amino acids based on an imine reductase have been developed, while N-alkylation of the aromatic amino acid anthranilate with S-adenosyl methionine as methyl-donor has not been described for this bacterium. After metabolic engineering for enhanced supply of anthranilate by channeling carbon flux into the shikimate pathway, preventing by-product formation and enhancing sugar uptake, heterologous expression of the gene anmt encoding anthranilate N-methyltransferase from Ruta graveolens resulted in production of N-methylanthranilate (NMA), which accumulated in the culture medium. Increased SAM regeneration by coexpression of the homologous adenosylhomocysteinase gene sahH improved N-methylanthranilate production. In a test bioreactor culture, the metabolically engineered C. glutamicum C1* strain produced NMA to a final titer of 0.5 g·L-1 with a volumetric productivity of 0.01 g·L-1·h-1 and a yield of 4.8 mg·g-1 glucose.

Microbial Engineering for Production of N-Functionalized Amino Acids and Amines.

N-functionalized amines play important roles in nature and occur, for example, in the antibiotic vancomycin, the immunosuppressant cyclosporine, the cytostatic actinomycin, the siderophore aerobactin, the cyanogenic glucoside linamarin, and the polyamine spermidine. In the pharmaceutical and fine-chemical industries N-functionalized amines are used as building blocks for the preparation of bioactive molecules. Processes based on fermentation and on enzyme catalysis have been developed to provide sustainable manufacturing routes to N-alkylated, N-hydroxylated, N-acylated, or other N-functionalized amines including polyamines. Metabolic engineering for provision of precursor metabolites is combined with heterologous N-functionalizing enzymes such as imine or ketimine reductases, opine or amino acid dehydrogenases, N-hydroxylases, N-acyltransferase, or polyamine synthetases. Recent progress and applications of fermentative processes using metabolically engineered bacteria and yeasts along with the employed enzymes are reviewed and the perspectives on developing new fermentative processes based on insight from enzyme catalysis are discussed.

Fermentative Production of N-Alkylated Glycine Derivatives by Recombinant Corynebacterium glutamicum Using a Mutant of Imine Reductase DpkA From Pseudomonas putida.

Sarcosine, an N-methylated amino acid, shows potential as antipsychotic, and serves as building block for peptide-based drugs, and acts as detergent when acetylated. N-methylated amino acids are mainly produced chemically or by biocatalysis, with either low yields or high costs for co-factor regeneration. Corynebacterium glutamicum, which is used for the industrial production of amino acids for decades, has recently been engineered for production of N-methyl-L-alanine and sarcosine. Heterologous expression of dpkA in a C. glutamicum strain engineered for glyoxylate overproduction enabled fermentative production of sarcosine from sugars and monomethylamine. Here, mutation of an amino acyl residue in the substrate binding site of DpkA (DpkAF117L) led to an increased specific activity for reductive alkylamination of glyoxylate using monomethylamine and monoethylamine as substrates. Introduction of DpkAF117L into the production strain accelerated the production of sarcosine and a volumetric productivity of 0.16 g L-1 h-1 could be attained. Using monoethylamine as substrate, we demonstrated N-ethylglycine production with a volumetric productivity of 0.11 g L-1 h-1, which to the best of our knowledge is the first report of its fermentative production. Subsequently, the feasibility of using rice straw hydrolysate as alternative carbon source was tested and production of N-ethylglycine to a titer of 1.6 g L-1 after 60 h of fed-batch bioreactor cultivation could be attained.

Xylose as preferred substrate for sarcosine production by recombinant Corynebacterium glutamicum.

The aim of this work was to study the fermentative production of the N-methylated amino acid sarcosine by C. glutamicum. Characterization of the imine reductase DpkA from Pseudomonas putida revealed that it catalyses N-methylamination of glyoxylate to sarcosine. Heterologous expression of dpkA in a C. glutamicum strain engineered for glyoxylate overproduction enabled fermentative production of sarcosine from sugars and monomethylamine. Glucose-based fermentation reached sarcosine production titers of 2.4 ±â€¯0.1 g L-1. Sarcosine production based on the second generation feedstocks xylose and arabinose led to higher product titers of 2.7 ±â€¯0.1 g L-1 and 3.4 ±â€¯0.3 g L-1, respectively, than glucose-based production. Optimization of production conditions with xylose and potassium acetate blends increased sarcosine titers to 8.7 ±â€¯0.2 g L-1 with a yield of 0.25 g g-1. This is the first example in which a C. glutamicum process using lignocellulosic pentoses is superior to glucose-based production.

Fermentative Production of N-Methylglutamate From Glycerol by Recombinant Pseudomonas putida.

N-methylated amino acids are present in diverse biological molecules in bacteria, archaea and eukaryotes. There is an increasing interest in this molecular class of alkylated amino acids by the pharmaceutical and chemical industries. N-alkylated amino acids have desired functions such as higher proteolytic stability, enhanced membrane permeability and longer peptide half-lives, which are important for the peptide-based drugs, the so-called peptidomimetics. Chemical synthesis of N-methylated amino acids often is limited by incomplete stereoselectivity, over-alkylation or the use of hazardous chemicals. Here, we describe metabolic engineering of Pseudomonas putida KT2440 for the fermentative production of N-methylglutamate from simple carbon sources and monomethylamine. P. putida KT2440, which is generally recognized as safe and grows with glucose and the alternative feedstock glycerol as sole carbon and energy source, was engineered for the production of N-methylglutamate using heterologous enzymes from Methylobacterium extorquens. About 3.9 g L-1 N-methylglutamate accumulated within 48 h in shake flask cultures with minimal medium containing monomethylamine and glycerol. A fed-batch cultivation process yielded a N-methylglutamate titer of 17.9 g L-1.

One-step process for production of N-methylated amino acids from sugars and methylamine using recombinant Corynebacterium glutamicum as biocatalyst.

N-methylated amino acids are found in Nature in various biological compounds. N-methylation of amino acids has been shown to improve pharmacokinetic properties of peptide drugs due to conformational changes, improved proteolytic stability and/or higher lipophilicity. Due to these characteristics N-methylated amino acids received increasing interest by the pharmaceutical industry. Syntheses of N-methylated amino acids by chemical and biocatalytic approaches are known, but often show incomplete stereoselectivity, low yields or expensive co-factor regeneration. So far a one-step fermentative process from sugars has not yet been described. Here, a one-step conversion of sugars and methylamine to the N-methylated amino acid N-methyl-L-alanine was developed. A whole-cell biocatalyst was derived from a pyruvate overproducing C. glutamicum strain by heterologous expression of the N-methyl-L-amino acid dehydrogenase gene from Pseudomonas putida. As proof-of-concept, N-methyl-L-alanine titers of 31.7 g L-1 with a yield of 0.71 g per g glucose were achieved in fed-batch cultivation. The C. glutamicum strain producing this imine reductase enzyme was engineered further to extend this green chemistry route to production of N-methyl-L-alanine from alternative feed stocks such as starch or the lignocellulosic sugars xylose and arabinose.

Biotechnological production of mono- and diamines using bacteria: recent progress, applications, and perspectives.

Common plastics such as polyamides are derived typically from petroleum or natural gas. Fossil-based polyamide production often involves toxic precursors or intermediates. By contrast, bio-based polyamides offer a realistic alternative. Bio-based routes to monomeric precursors of polyamides such as diamines, dicarboxylic acids, and omega-amino acids have been developed. Recent advances in the metabolic engineering of the biotechnologically relevant Escherichia coli and Corynebacterium glutamicum for the production of monoalkylamines such as omega-amino acids as well as diamines are presented.