Abstract
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.
Highlights
L-Carnitine [(R)-3-hydroxy-4-trimethylaminobutyrate] is an essential compound in the intermediary metabolism of eukaryotes, which is involved in the transport of activated longchain fatty acids and products of peroxisomal β-oxidation into the mitochondria for subsequent completion of β-oxidation (Vaz and Wanders, 2002)
We have recently developed a genetically encoded biosensor that responds in a dose-dependent manner to external L-carnitine in a concentration range of 0.1–100 μM by expression of the fluorescent reporter mVenus (Kugler et al, 2020)
The selection of the potential enzymes for the L-carnitine biosynthetic pathway in this study was based on two sets of N. crassa genes that were previously considered for a transfer of the pathway to heterologous hosts (Kang et al, 2013; Franken et al, 2015)
Summary
L-Carnitine [(R)-3-hydroxy-4-trimethylaminobutyrate] is an essential compound in the intermediary metabolism of eukaryotes, which is involved in the transport of activated longchain fatty acids and products of peroxisomal β-oxidation into the mitochondria for subsequent completion of β-oxidation (Vaz and Wanders, 2002). Carnitine can be used as dietary supplement for the treatment of this deficiency (Flanagan et al, 2010; El-Hattab and Scaglia, 2015), and as feed additive to improve livestock performance (Rehman et al, 2017; Ringseis et al, 2018a,b; Li L. et al, 2019), and as L-carnitine or acyl-L-carnitine esters in further pharmaceutical applications (DiNicolantonio et al, 2013; Chen et al, 2014; Song et al, 2017; Parvanova et al, 2018; Veronese et al, 2018) It is marketed as food additive and dietary supplement for improving athletic performance and weight management as positive effects on physical performance and weight loss under conditions with disorders have been observed (Fielding et al, 2018), while an improvement in healthy individuals or athletes is debated (Gnoni et al, 2020). The carnitine market of US$ ∼170 million in 2018 is expected to grow by 4.8% annually (Grand View Research, 2019; The Insight Partners, 2020)
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