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 malonyl-CoA with inversed pathway yield in oleaginous yeast. Negative autoregulation of the lipogenic pathways, enabled by CRISPRi and fatty acid-inducible promoters, repartitions malonyl-CoA to favor flavonoid synthesis and increased naringenin production by 74.8%. With flavonoid-sensing transcriptional activator FdeR and yeast hybrid promoters to control leucine synthesis and cell grwoth fitness, this amino acid feedforward metabolic circuit confers a flavonoid addiction phenotype that selectively enrich the naringenin-producing pupulation in the leucine auxotrophic yeast. 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 in long-term and large-scale bioproduction.
Highlights
Metabolic heterogeneity has been found to play an essential role in determining cellular performance and pathway efficiency (Xiao et al, 2016; Ceroni et al, 2018; Rugbjerg et al, 2018)
The engineered cell still contained more than 35% (w/w) of oil (Beopoulos et al, 2011), indicating a substantial portion of malonylCoA was used for lipid synthesis
Consistent with this study, mitigating lipogenesis competition has been reported as effective strategies to improve malonyl-CoA-derived end product synthesis in oleaginous yeast (Wu et al, 2014; Yang et al, 2015)
Summary
Metabolic heterogeneity has been found to play an essential role in determining cellular performance and pathway efficiency (Xiao et al, 2016; Ceroni et al, 2018; Rugbjerg et al, 2018). A large portfolio of engineering work has been dedicated to mitigate lipogenesis or redirect lipid synthesis for heterologous chemical production (Ledesma-Amaro and Nicaud, 2016; Markham et al, 2018), but the heavily engineered strains, even the chromosomally-integrated cell lines, are often difficult to maintain high performance during long-term cultivations (Roth et al, 2009; Xu et al, 2017; Wei et al, 2019) To solve this challenge, we took advantage of the transcriptional activity of metabolite-responsive promoters (Skjoedt et al, 2016; D'Ambrosio and Jensen, 2017; Wan et al, 2019), aiming to develop an end-product addiction circuit to rewire cell metabolism in Y. lipolytica. These results highlight the importance of applying dynamic population control and microbial cooperation to improve the community-level metabolic performance
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