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.

Abstract Background The use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purification. As the field of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purification protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express. The current study describes a plasmid construct that allowed expression of barnase in Escherichia coli under co-expression of its native inhibitor barstar. Results The pure enzyme without the inhibitor barstar was exported to the extracellular space through the periplasm and then purified from the cell-free supernatant. Cation exchange chromatography was employed as a primary purification step. This was followed by hydrophobic interaction chromatography which resulted in a concentrated fraction of active enzyme. Although current levels of volumetric activity achieved are quite meagre (4 Kunitz units mL− 1), in principle its application to plasmid DNA purification could be proved. Currently, this is capable of processing small amounts (13 g) of bacterial biomass for plasmid production. Conclusions The current work focusses on the downstream purification strategies for a recombinant RNase and sets a framework for higher scale production if specific productivity is increased by optimal hosts and/or re-engineered plasmids. Also important is to curtail the massive enzyme loss during purification by cation exchange chromatography. Application of even a relatively small amount of recombinant RNase would contribute to greatly reducing the initial RNA levels in alkaline lysates thereby augmenting further downstream plasmid purification steps.

Coenzyme Q 10 (CoQ10) serves as an electron carrier in aerobic respiration and has become an interesting target for biotechnological production due to its antioxidative effect and benefits in supplementation to patients with various diseases. For the microbial production, so far only bacteria have been used that naturally synthesize CoQ10 or a related CoQ species. Since the whole pathway involves many enzymatic steps and has not been fully elucidated yet, the set of genes required for transfer of CoQ10 synthesis to a bacterium not naturally synthesizing CoQ species remained unknown. Here, we established CoQ10 biosynthesis in the non-ubiquinone-containing Gram-positive Corynebacterium glutamicum by metabolic engineering. CoQ10 biosynthesis involves prenylation and, thus, requires farnesyl diphosphate as precursor. A carotenoid-deficient strain was engineered to synthesize an increased supply of the precursor molecule farnesyl diphosphate. Increased farnesyl diphosphate supply was demonstrated indirectly by increased conversion to amorpha-4,11-diene. To provide the first CoQ10 precursor decaprenyl diphosphate (DPP) from farnesyl diphosphate, DPP synthase gene ddsA from Paracoccus denitrificans was expressed. Improved supply of the second CoQ10 precursor, para -hydroxybenzoate (pHBA), resulted from metabolic engineering of the shikimate pathway. Prenylation of pHBA with DPP and subsequent decarboxylation, hydroxylation, and methylation reactions to yield CoQ10 was achieved by expression of ubi genes from Escherichia coli . CoQ10 biosynthesis was demonstrated in shake-flask cultivation and verified by liquid chromatography mass spectrometry analysis. To the best of our knowledge, this is the first report of CoQ10 production in a non-ubiquinone-containing bacterium.

Abstract l‐amino acid oxidases (LAAOs) are flavoenzymes that catalyze the oxidative deamination of l‐amino acids to the corresponding α‐keto acids, ammonia, and hydrogen peroxide. Here, we show the overexpression, purification, and the characterization of LAAO4 from the fungus Hebeloma cylindrosporum in the yeast Pichia pastoris with a 9His‐tag and compare this with the recently characterized 6His‐hcLAAO4 expressed in E. coli. The expression of the enzyme with an ER‐signal sequence in P. pastoris resulted in a glycosylated, secreted protein. The enzymatic activity without activation was higher after expression in P. pastoris compared to E. coli. Due to treatment with acidic pH, a striking increase of activity could be detected for both expression systems resulting in similar specific activities after acid activation. Regarding the substrate spectrum, temperature stability, Km, and vmax values, hcLAAO4 showed very few differences when produced in these two expression systems. A higher yield of hcLAAO4 could be obtained by fermentation.

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.

Brominated compounds such as 7-bromo-L-tryptophan (7-Br-Trp) occur in Nature. Many synthetic and natural brominated compounds have applications in the agriculture, food, and pharmaceutical industries, for example, the 20S-proteasome inhibitor TMC-95A that may be derived from 7-Br-Trp. Mild halogenation by cross-linked enzyme aggregates containing FAD-dependent halogenase, NADH-dependent flavin reductase, and alcohol dehydrogenase as well as by fermentation with recombinant Corynebacterium glutamicum expressing the genes for the FAD-dependent halogenase RebH and the NADH-dependent flavin reductase RebF from Lechevalieria aerocolonigenes have recently been developed as green alternatives to more hazardous chemical routes. In this study, the fermentative production of 7-Br-Trp was established. The fermentative process employs an L-tryptophan producing C. glutamicum strain expressing rebH and rebF from L. aerocolonigenes for halogenation and is based on glucose, ammonium and sodium bromide. C. glutamicum tolerated high sodium bromide concentrations, but its growth rate was reduced to half-maximal at 0.09g L-1 7-bromo-L-tryptophan. This may be, at least in part, due to inhibition of anthranilate phosphoribosyltransferase by 7-Br-Trp since anthranilate phosphoribosyltransferase activity in crude extracts was half-maximal at about 0.03 g L-1 7-Br-Trp. Fermentative production of 7-Br-Trp by recombinant C. glutamicum was scaled up to a working volume of 2 L and operated in batch and fed-batch mode. The titers were increased from batch fermentation in CGXII minimal medium with 0.3 g L-1 7-Br-Trp to fed-batch fermentation in HSG complex medium, where up to 1.2 g L-1 7-Br-Trp were obtained. The product isolated from the culture broth was characterized by NMR and LC-MS and shown to be 7-Br-Trp.

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.

The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E.coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one- or two-step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often-overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.

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.

Patchoulol is a sesquiterpene alcohol and an important natural product for the perfume industry. Corynebacterium glutamicum is the prominent host for the fermentative production of amino acids with an average annual production volume of ~6 million tons. Due to its robustness and well established large-scale fermentation, C. glutamicum has been engineered for the production of a number of value-added compounds including terpenoids. Both C40 and C50 carotenoids, including the industrially relevant astaxanthin, and short-chain terpenes such as the sesquiterpene valencene can be produced with this organism. In this study, systematic metabolic engineering enabled construction of a patchoulol producing C. glutamicum strain by applying the following strategies: (i) construction of a farnesyl pyrophosphate-producing platform strain by combining genomic deletions with heterologous expression of ispA from Escherichia coli; (ii) prevention of carotenoid-like byproduct formation; (iii) overproduction of limiting enzymes from the 2-c-methyl-d-erythritol 4-phosphate (MEP)-pathway to increase precursor supply; and (iv) heterologous expression of the plant patchoulol synthase gene PcPS from Pogostemon cablin. Additionally, a proof of principle liter-scale fermentation with a two-phase organic overlay-culture medium system for terpenoid capture was performed. To the best of our knowledge, the patchoulol titers demonstrated here are the highest reported to date with up to 60 mg L−1 and volumetric productivities of up to 18 mg L−1 d−1.

The dicarboxylic acid glutarate is an important building-block gaining interest in the chemical and pharmaceutical industry. Here, a synthetic pathway for fermentative production of glutarate by the actinobacterium Corynebacterium glutamicum has been developed. The pathway does not require molecular oxygen and operates via lysine decarboyxylase followed by two transamination and two NAD-dependent oxidation reactions. Using a genome-streamlined L-lysine producing strain as basis, metabolic engineering was performed to enable conversion of L-lysine to glutarate in a five-step synthetic pathway comprising lysine decarboxylase, putrescine transaminase and γ-aminobutyraldehyde dehydrogenase from Escherichia coli and GABA/5AVA amino transferase and succinate/glutarate semialdehyde dehydrogenase either from C. glutamicum or from three Pseudomonas species. Loss of carbon via formation of the by-products cadaverine and N-acetylcadaverine was avoided by deletion of the respective acetylase and export genes. As the two transamination reactions in the synthetic glutarate biosynthesis pathway yield L-glutamate, biosynthesis of L-glutamate by glutamate dehydrogenase was expected to be obsolete and, indeed, deletion of its gene gdh increased glutarate titers by 10%. Glutarate production by the final strain was tested in bioreactors (n = 2) in order to investigate stability and reliability of the process. The most efficient glutarate production from glucose was achieved by fed-batch fermentation (n = 1) with a volumetric productivity of 0.32 g L-1 h-1, an overall yield of 0.17 g g-1 and a titer of 25 g L-1.

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.

Streptomyces is a genus of Gram-positive bacteria that grow in various environments, and its shape resembles filamentous fungi. The most interesting property of Streptomyces is the ability to produce bioactive secondary metabolites, such as antifungals, antivirals, antitumorals, and especially antibiotics. In submersed culture Streptomyces and other filamentous microorganisms can grow as free mycelium, pellets or intermediate growth forms. All growth forms can be beneficial for product formation depending on the microbial strain and the product of interest. Here, the effects of addition of particles on the growth and product formation of two Streptomyces strains were investigated. Streptomyces coelicolor was used as a model organism for the production of the antibiotic actinorhodin and Streptomyces avidinii was used to produce streptavidin. In all experiments, the addition of broken and porous SiO2 particles (120–200µm) to the culture medium led to an increased productivity. In order to evaluate the particle effect on actinorhodin production three different media with and without the addition of particles were compared. The productivity was strongly influenced by the choice of media and was further improved by the addition of particles. The enhancement factor between un-supplemented and supplemented cultures varied between 85% by using a complex medium and 160% in a minimal medium. The highest product concentration of 855mg actinorhodin L−1 was measured after 12 days in a complex medium by addition of 5g glass particles L−1. In contrast, the final concentration of streptavidin was not affected by the supplementation of glass beads (diameter: 0.25–0.5mm) to cultures of Streptomyces avidinii. Nevertheless, a kinetic effect occurred upon supplementation by particles, allowing an acceleration of product formation. The impact of the particle addition to the Streptomyces cultures on the sustainability of the bioprocess was proven by using the E-factor. The E-factor denotes the amount of waste generated per product equivalent. This evaluation shows the beneficial effects of the particle addition in both production systems. Due to the fact that the particle addition leads to a decreased E-factor a life cycle assessment should be performed to further evaluate the particle effect on the sustainability of the complete process chain.

The cyclic amino acid ectoine is a compatible solute serving as a protective substance against osmotic stress. Ectoine finds various applications due to its moisturizing effect. To avoid the disadvantages of the prevailing so-called "bacterial milking ectoine production process" caused by the high salt concentration, low salt fermentation strategies are sought after. As l-lysine and ectoine biosynthesis share l-aspartate-semialdehyde as common precursor, l-lysine producing strains can be converted to ectoine producing strains. Corynebacterium glutamicum, which is used for l-lysine production in the million-ton-scale, was engineered for ectoine production by heterologous expression of the ectoine biosynthesis operon ectABC from Chromohalobacter salexigens. Derepression of glucose metabolism by deletion of the regulatory gene sugR and avoiding l-lactate formation by deletion of the lactate dehydrogenase gene ldhA increased ectoine productivity. In bioreactor fed-batch cultivations an ectoine titer of 22gL-1 and a volumetric productivity of 0.32gL-1h-1 were obtained. The ectoine yield of 0.16gg-1, to the best of our knowledge, exceeded previously reported yields. Moreover, ectoine production from the alternative carbon sources glycerol, glucosamine, xylose, arabinose, and soluble starch was achieved.

Corynebacterium glutamicum is used for the million-ton scale production of amino acids and has recently been engineered for production of the cyclic non-proteinogenic amino acid L-pipecolic acid (L-PA). In this synthetic pathway L-lysine was converted to L-PA by oxidative deamination, dehydration and reduction by L-lysine 6-dehydrogenase (deaminating) from Silicibacter pomeroyi and pyrroline 5-carboxylate reductase from C. glutamicum. However, production of L-PA occurred as by-product of L-lysine production only. Here, the author show that abolishing L-lysine export by the respective gene deletion resulted in production of L-PA as major product without concomitant lysine production while the specific growth rate was reduced due to accumulation of high intracellular lysine concentrations. Increasing expression of the genes encoding L-lysine 6-dehydrogenase and pyrroline 5-carboxylate reductase in C. glutamicum strain PIPE4 increased the L-PA titer to 3.9 g L-1 , and allowed faster growth and, thus, a higher volumetric productivity of 0.08 ± 0.00 g L-1 h-1 respectively. Secondly, expression of heterologous genes for utilization of glycerol, xylose, glucosamine, and starch in strain PIPE4 enabled L-PA production from these alternative carbon sources. Third, in a glucose/sucrose-based fed-batch fermentation with C. glutamicum PIPE4 L-PA was produced to a titer of 14.4 g L-1 with a volumetric productivity of 0.21 g L-1 h-1 and an overall yield of 0.20 g g-1 .

Streptavidin is a tetrameric protein with an extremely high affinity to biotin and different biotin-like peptide-tags. This characteristic causes its widespread use in biotechnology. Streptavidin is produced by the fermentation of wild type Streptomyces avidinii or by recombinant Streptomyces lavendulae, Escherichia coli, and Bacillus subtilis strains. However, little is known about the influence of power input and oxygen supply as well as feeding strategies on the production of streptavidin by S. avidinii. This paper provides a systematic analysis of the effect of rotary frequency of the stirrer, leading to a plateau-like streptavidin formation behaviour between 400 and 700 min−1. This plateau was characterized by specific power inputs between 79 and 107 W L−1 and corresponding maximal product concentrations of 6.90 μM in 6 days. Lower as well as higher rotary frequencies were not beneficial. Subsequently, a linear fed-batch procedure could be established reproducibly yielding 39.20 μM streptavidin in 14 days, characterized by a constant productivity of 114 nM h−1. Fed-batch procedures based on dissolved oxygen were less efficient. The linear feeding strategy presented in this paper led to the highest streptavidin concentration ever reported and exceeded the maximal product level given in the literature drastically by a factor of 8.5.

Streptavidin - a protein secreted by the filamentous bacterium Streptomyces avidinii - is applied in a variety of methods, leading to numerous studies on its heterologous production. Development and characterization of a novel expression system for streptavidin genes by Hansenula polymorpha is described utilizing different target gene variants along with the two methanol-inducible promoters PMOX and PFMD. Extracellular product concentrations were higher for cultivation at 30 instead of 37°C. The best performing strain carrying the full-length streptavidin gene under control of PFMD was characterized in the bioreactor applying a synthetic medium and oxygen-controlled feeding of glucose. Derepression resulted in an extracellular concentration of 1.31±0.07μM of tetrameric streptavidin after 48h (27.3nMh(-1)). Feeding of glycerol improved biomass formation, but lowered the product concentration. By combining derepression and methanol induction the final extracellular streptavidin concentration increased to 11.42±0.22μM (approx. 751mgL(-1)), yielding a productivity of 52.5nMh(-1). Despite supplementing biotin the proportion of biotin-blocked binding sites in the supernatant dropped from 54.4±5.0 % after 18h to 17.2±6.5 % towards the end of glucose feeding to a final value of 1.1±3.8 %, indicating a highly bioactive product. Thus, H. polymorpha proved to be a suitable host for the production of streptavidin.