Model-based engineering of Bacillus methanolicus towards de novo polyamine bioproduction from methanol

This study explores the one-carbon feedstock methanol to bolster sustainable bioproduction of valuable polyamines. Bacillus methanolicus MGA3, a methylotroph, stands out as a promising host due to its aptitude for employing methanol to synthesize various chemicals. Our approach used flux balance analysis (FBA) to leverage native B. methanolicus pathways for biosynthesis of the polyamines putrescine and spermidine. Despite possessing the genetic repertoire required for their production, B. methanolicus naturally secretes spermidine but not putrescine. Therefore, we created recombinant strains overexpressing endogenous and heterologous genes for putrescine biosynthesis via the arginine decarboxylase pathway, including arginine decarboxylase (speA) and agmatinase (speB). The B. methanolicus strain PUTEc, overexpressing speAB from Escherichia coli rather than native ones, achieved putrescine production of 47.5 ± 0.8 μM in shake flasks. Towards spermidine production, FBA pointed to overexpressing S-adenosylmethionine decarboxylase (speH) and spermidine synthase (speE) in the PUTEc strain. As a result, production of 83.9 ± 2.7 μM spermidine was achieved from methanol. Subsequently, the PUTEc strain underwent FBA-based screening involving precursor supplementation extracellularly in the growth medium or intracellularly by gene co-overexpression to enhance putrescine production. Overexpression of the endogenous ornithine biosynthesis pathway in the PUTEc strain yielded the highest methanol-based polyamine production in this study: 137.7 ± 1.8 μM putrescine in small-scale shake flask conditions. With further improvements in titer towards several grams per litre, this sustainable bioprocess could supply the steadily growing ∼ $500 M putrescine market. To our knowledge, this is the first proof-of-concept study towards production of putrescine and spermidine from methanol.