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L-Carnitine is a bioactive compound derived from L-lysine and S-adenosyl-L-methionine, which is closely associated with the transport of long-chain fatty acids in the intermediary metabolism of eukaryotes and sought after in the pharmaceutical, food, and feed industries. The L-carnitine biosynthesis pathway has not been observed in prokaryotes, and the use of eukaryotic microorganisms as natural L-carnitine producers lacks economic viability due to complex cultivation and low titers. While biotransformation processes based on petrochemical achiral precursors have been described for bacterial hosts, fermentative de novo synthesis has not been established although it holds the potential for a sustainable and economical one-pot process using renewable feedstocks. This study describes the metabolic engineering of Escherichia coli for L-carnitine production. L-carnitine biosynthesis enzymes from the fungus Neurospora crassa that were functionally active in E. coli were identified and applied individually or in cascades to assemble and optimize a four-step L-carnitine biosynthesis pathway in this host. Pathway performance was monitored by a transcription factor-based L-carnitine biosensor. The engineered E. coli strain produced L-carnitine from supplemented L-Nε-trimethyllysine in a whole cell biotransformation, resulting in 15.9 μM carnitine found in the supernatant. Notably, this strain also produced 1.7 μM L-carnitine de novo from glycerol and ammonium as carbon and nitrogen sources through endogenous Nε-trimethyllysine. This work provides a proof of concept for the de novo L-carnitine production in E. coli, which does not depend on petrochemical synthesis of achiral precursors, but makes use of renewable feedstocks instead. To the best of our knowledge, this is the first description of L-carnitine de novo synthesis using an engineered bacterium. Introduction

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 0.09 g 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.

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.

The biotechnologically relevant bacterium Corynebacterium glutamicum, currently used for the million ton-scale production of amino acids for the food and feed industries, is pigmented due to synthesis of the rare cyclic C50 carotenoid decaprenoxanthin and its glucosides. The precursors of carotenoid biosynthesis, isopenthenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate, are synthesized in this organism via the methylerythritol phosphate (MEP) or non-mevalonate pathway. Terminal pathway engineering in recombinant C. glutamicum permitted the production of various non-native C50 and C40 carotenoids. Here, the role of engineering isoprenoid precursor supply for lycopene production by C. glutamicum was characterized. Overexpression of dxs encoding the enzyme that catalyzes the first committed step of the MEP-pathway by chromosomal promoter exchange in a prophage-cured, genome-reduced C. glutamicum strain improved lycopene formation. Similarly, an increased IPP supply was achieved by chromosomal integration of two artificial operons comprising MEP pathway genes under the control of a constitutive promoter. Combined overexpression of dxs and the other six MEP pathways genes in C. glutamicum strain LYC3-MEP was not synergistic with respect to improving lycopene accumulation. Based on C. glutamicum strain LYC3-MEP, astaxanthin could be produced in the milligrams per gram cell dry weight range when the endogenous genes crtE, crtB, and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were coexpressed with the genes for lycopene cyclase and β-carotene hydroxylase from Pantoea ananatis and carotene C(4) oxygenase from Brevundimonas aurantiaca.