Abstract
The use of methanol as carbon source for biotechnological processes has recently attracted great interest due to its relatively low price, high abundance, high purity, and the fact that it is a non-food raw material. In this study, methanol-based production of 5-aminovalerate (5AVA) was established using recombinant Bacillus methanolicus strains. 5AVA is a building block of polyamides and a candidate to become the C5 platform chemical for the production of, among others, δ-valerolactam, 5-hydroxy-valerate, glutarate, and 1,5-pentanediol. In this study, we test five different 5AVA biosynthesis pathways, whereof two directly convert L-lysine to 5AVA and three use cadaverine as an intermediate. The conversion of L-lysine to 5AVA employs lysine 2-monooxygenase (DavB) and 5-aminovaleramidase (DavA), encoded by the well-known Pseudomonas putida cluster davBA, among others, or lysine α-oxidase (RaiP) in the presence of hydrogen peroxide. Cadaverine is converted either to γ-glutamine-cadaverine by glutamine synthetase (SpuI) or to 5-aminopentanal through activity of putrescine oxidase (Puo) or putrescine transaminase (PatA). Our efforts resulted in proof-of-concept 5AVA production from methanol at 50°C, enabled by two pathways out of the five tested with the highest titer of 0.02 g l–1. To our knowledge, this is the first report of 5AVA production from methanol in methylotrophic bacteria, and the recombinant strains and knowledge generated should represent a valuable basis for further improved 5AVA production from methanol.
Highlights
The worldwide amino acid market is progressively growing at 5.6% annual rate and is estimated to reach US$25.6 billion by 2022, with amino acids used for animal feed production being its largest component (Wendisch, 2020)
The E. coli strain DH5α was used as general cloning host, and B. methanolicus strains MGA3 and M168-20 were used as expression hosts
Due to the fact that B. methanolicus is a thermophile, a typical issue concerning implementation of biosynthetic pathways from heterologous hosts is the lack of thermostability of the transferred enzymes
Summary
The worldwide amino acid market is progressively growing at 5.6% annual rate and is estimated to reach US$25.6 billion by 2022, with amino acids used for animal feed production being its largest component (Wendisch, 2020). MGA3 produces 0.4 g l−1 of Llysine in high cell density fed-batch fermentations (Brautaset et al, 2010; Table 1); this titer was improved nearly 30fold up to 11 g l−1 by plasmid-based overexpression of a gene coding for aspartokinase, a key enzyme controlling the synthesis of aspartate-derived amino acids (Jakobsen et al, 2009). Through application of a classical mutagenesis technique, a derivative of B. methanolicus MGA3 (M168-20) was constructed, which produces 11 g l−1 of L-lysine in high cell density methanol-controlled fed-batch fermentations (Brautaset et al, 2010); the L-lysine overproduction being caused among others by mutation in the hom-1 gene coding for homoserine dehydrogenase (Hom) and in the putative lysine 2,3-aminomutase gene (locus tag BMMGA3_02505). The mutation in hom-1 leads to the loss of catalytic activity of homoserine dehydrogenase and redirection of metabolic flux toward the L-lysine pathway and its accumulation (Nærdal et al, 2011, 2017)
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