Production of non‐proteinogenic amino acids from α‐keto acid precursors with recombinant Corynebacterium glutamicum

Abstract

In the present work, Corynebacterium glutamicum was metabolically engineered for the enantioselective synthesis of non-proteinogenic amino acids as valuable building blocks for pharmaceuticals and agrochemicals. The novel bio-catalytic activity of C. glutamicum was obtained by heterologous expression of the branched chain aminotransferase IlvE from Escherichia coli. Upon this modification, the recombinant cells converted the α-keto acid precursor 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (HOAE) into the corresponding amino acid 2-(3-hydroxy-1-adamantyl)-(2S)-amino ethanoic acid (HAAE). Similarly, also L-tert-leucine could be obtained from trimethyl pyruvate indicating a broader applicability of the novel strategy. In both cases, the amino group donor glutamate was supplied from the endogenous metabolism of the recombinant producer. Hereby, the uptake of the precursor and secretion of the product was supported by an enhanced cell permeability through treatment of ethambutol, which inhibits arabinosyl transferases involved in cell wall biosynthesis. The excretion of HAAE into the reaction medium was linked to the secretion of glutamate, indicating a similar mechanism for the export of both compounds. On the other hand, the efflux of L-tert-leucine appeared to be driven by active transport. Subsequent bioprocess engineering enabled HAAE and L-tert-leucine to be produced at a rate of 0.21 and 0.42 mmol (g dry cells)⁻¹ h⁻¹, respectively up to a final product titer of 40 mM. Beyond the given examples, integrated metabolic and cell envelop engineering might extend the production of a variety of other non-proteinogenic amino acids as well as chiral amines by C. glutamicum.

Bromination of L-tryptophan in a Fermentative Process With Corynebacterium glutamicum

“…Thus, C. glutamicum was engineered for production of carboxylic acids such as pyruvate (Wieschalka et al, 2012) and succinate (Litsanov et al, 2012), oxoacids such as 2-ketoisovalerate (Krause et al, 2010) and 2-ketoisocaproate (Bückle-Vallant et al, 2014), alcohols such as ethanol (Inui et al, 2004a), isobutanol (Blombach et al, 2011), and n -propanol (Siebert and Wendisch, 2015), polymers such as polyhydroxyalkanoate (Ma et al, 2018). As industrial amino acid producer C. glutamicum is ideal for fermentative production of various other nitrogenous compounds such as the cyclic amino acid pipecolic acid (Pérez-García et al, 2016), the ω-amino acids γ-aminobutyrate (Kim et al, 2013; Jorge et al, 2016; Pérez-García et al, 2016) and 5-aminovalerate (Rohles et al, 2016; Jorge et al, 2017), the diamines putrescine (Schneider and Wendisch, 2010) and cadaverine (Tateno et al, 2009; Kim et al, 2018) and alkylated and hydroxylated amino acids such as N -methylalanine (Mindt et al, 2018) and 5-hydroxy-isoleucine (Wendisch, 2019). Noteworthy, several excellent C. glutamicum producer strains have been developed for production of muconic acid (Becker et al, 2018), phenylpropanoids (Kallscheuer and Marienhagen, 2018), para -hydroxybenzoic acid (Purwanto et al, 2018), and protocatechuate (Wendisch et al, 2016; Lee and Wendisch, 2017).…”

Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being

“…The top low-price bulk amino acids for use in food and feed are l-lysine [6], l-glutamate [3], l-tryptophan [9], and l-methionine [41,58]. In addition, C. glutamicum has been successfully engineered to produce amino acids with a higher value, mainly for pharmaceutical and medical applications, including branched chain and non-proteinogenic derivatives [59] (Table 1).…”

Amino Acid and Peptide‐Based Antiviral Agents

“…Due to the above, enantiomerically pure l ‐ tert ‐leucine is produced on an industrial scale and is commercially available. There exist a relatively wide range of enzymes producing enantiomerically pure l ‐ tert ‐leucine, this includes: aminotransferases, [28–30] lipase, [31] hydantoinases, [31] and penicillin G acylase [31] . The most extensively studied in this field are amidotransferases converting trimethylpyruvate into l ‐ tert ‐leucine using glutamate as an amino group donor and leucine dehydrogenases converting trimethylpyruvate in assistance of NADP coenzyme (Scheme 4).…”