Coupling metabolic addiction with negative autoregulation to improve strain stability and pathway yield

Abstract

Metabolic addiction, an organism that is metabolically addicted with a compound to maintain its growth fitness, is an underexplored area in metabolic engineering.Microbes with heavily engineered pathways or genetic circuits tend to experience metabolic burden leading to degenerated or abortive production phenotype during long-term cultivation or scale-up. A promising solution to combat metabolic instability is to tie up the end-product with an intermediary metabolite that is essential to the growth of the producing host. Here we present a simple strategy to improve both metabolic stability and pathway yield by coupling chemical addiction with negative autoregulatory genetic circuits. Naringenin and lipids compete for the same precursor with inversed pathway yield in oleaginous yeast. Negative autoregulation of the lipogenic pathways, enabled by CRISPRi and fatty acid-inducible promoters, repartitioned malonyl-CoA to favor flavonoid synthesis and increased naringenin production by 74.8%. With flavonoid-sensing hybrid promoters to control leucine synthesis, this flavonoid addiction phenotype confers a selective growth advantage to the naringenin-producing cell. The engineered yeast persisted 90.9% of naringenin titer up to 324 generations. Cells without flavonoid addiction regained growth fitness but lost 94.5% of the naringenin titer after cell passage beyond 300 generations.Metabolic addiction and negative autoregulation may be generalized as basic tools to eliminate metabolic heterogeneity, improve strain stability and pathway yield.

Engineering Yarrowia lipolytica as a Chassis for De Novo Synthesis of Five Aromatic-Derived Natural Products and Chemicals

“…Yarrowia lipolytica , as a “generally regarded as safe” (GRAS) yeast, 1 has been extensively engineered for the production of oleochemicals, fuels, and commodity chemicals. 2 − 5 The abundant acetyl-CoA and malonyl-CoA precursors in Y. lipolytica have been harnessed for synthesizing plant secondary metabolites in recent years, including flavonoids, 6 − 9 polyketides, 10 , 11 polyunsaturated fatty acids, 12 , 13 and isoprenoids. 14 − 17 A large collection of customized genetic toolkits, including Golden-gate cloning, 18 − 20 genome integration, 6 , 21 , 22 CRISPR-Cas9/Cpf1 genome editing, 23 − 25 transposons, 26 auxotrophic markers, 27 and promoter libraries 28 − 30 have accelerated our ability to perform targeted genetic manipulations.…”

A roadmap to engineering antiviral natural products synthesis in microbes

“…And genetically-encoded biosensors may empower us to analyze and monitor cellular process with temporal and spatial resolutions [ 24 ]. More importantly, metabolic addiction allows us to link cell growth to an end product that may selectively enrich overproduction subpopulation and improve community-level production performance [ 25 , 26 •• ]. Bioprocess control with feedback cascades and feeding pattern will be critical to improve the titer and productivity [ 27 ].…”

Engineering the oleaginous yeast Yarrowia lipolytica for high-level resveratrol production

“…These metabolic traits could be particularly relevant for the production of shikimate pathway-derived compounds and plant natural products requiring aromatic amino acids and malonyl-CoA-derived building blocks, such as phenylpropanoids. Indeed, this yeast was recently shown as an attractive host for the biosynthesis of several plant flavonoids, including naringenin, eriodictyol, taxifolin, and other aromatic compounds ( Lv et al, 2019 , 2020 ; Palmer et al, 2020 ; Gu et al, 2020a , 2020b ; Shang et al, 2020 ; Wei et al, 2020 ). Resveratrol in particular has also been produced in Y. lipolytica , first described by DuPont and more recently in studies exploring the ability of the non-conventional yeast as production chassis for different polyketides and aromatics ( Supplementary Table S1 ) ( Huang et al, 2006 ; Palmer et al, 2020 ; Gu et al, 2020a ).…”