Regulatory control circuits for stabilizing long-term anabolic product formation in yeast

Abstract

Engineering living cells for production of chemicals, enzymes and therapeutics can burden cells due to use of limited native co-factor availability and/or expression burdens, totalling a fitness deficit compared to parental cells encoded through long evolutionary trajectories to maximise fitness. Ultimately, this discrepancy puts a selective pressure against fitness-burdened engineered cells under prolonged bioprocesses, and potentially leads to complete eradication of highperforming engineered cells at the population level. Here we present the mutation landscapes of fitness-burdened yeast cells engineered for vanillin-β-glucoside production. Next, we design synthetic control circuits based on transcriptome analysis and biosensors responsive to vanillin-βglucoside pathway intermediates in order to stabilize vanillin-β-glucoside production over ~55 generations in sequential passage experiments. Furthermore, using biosensors with two different modes of action we identify control circuits linking vanillin-β-glucoside pathway flux to various essential cellular functions, and demonstrate control circuits robustness and 92% higher vanillin-βglucoside production, including 5-fold increase in total vanillin-β-glucoside pathway metabolite accumulation, in a fed-batch fermentation compared to vanillin-β-glucoside producing cells without control circuits.

Integrating continuous hypermutation with high‐throughput screening for optimization of cis,cis‐muconic acid production in yeast

“…For such purposes, transferring the biosensor read-out from fluorescence to growth would be beneficial from a technical and scalability point of view (Zhong et al, 2020). For such a purpose, the coupling of CCM to growth using synthetic control circuits founded on BenM, or the recently developed vanillin biosensor, could be useful for genome-wide searches of nucleotide polymorphisms and chromosomal re-arrangements limiting PCA efflux and/or further boosting PCA metabolic flux respectively (Ambri et al, 2020;D'Ambrosio et al, 2020). Another aspect to consider is to diversify the selection criteria beyond the stringent one used in this study (Fig.…”

“…However, the build-up and secretion of the CCM pathway intermediate PCA remain rate-limiting for high CCM production (Suastegui et al, 2016;Leavitt et al, 2017;Snoek et al, 2018;Wang et al, 2020). This observation is propelled by the fact that PCA accumulation supposedly is not limited to suboptimal catalytic activity or expression of the downstream CCM pathway enzyme, PCA decarboxylase, as PCA accumulation also remains a persistent issue for microbial biosynthesis of other PCA-derived chemicals and nutraceuticals without decarboxylase requirements (Strucko et al, 2015(Strucko et al, , 2017D'Ambrosio et al, 2020). Yet, no attempts for directed evolution of the heterologous enzymes towards increased PCAderived production has to our knowledge been performed.…”

Integrating continuous hypermutation with high-throughput screening for optimization of cis,cis-muconic acid production in yeast

“…For such purposes, transferring the biosensor read-out from fluorescence to growth would be beneficial from a technical and scalability point of view (Zhong et al, 2020). For such a purpose, the coupling of CCM to growth using synthetic control circuits founded on BenM, or the recently developed vanillin biosensor, could be useful for genome-wide searches of nucleotide polymorphisms and chromosomal rearrangements limiting PCA efflux and/or further boosting PCA metabolic flux, respectively (Ambri et al, 2020;D'Ambrosio et al, 2020).…”

“…KpAroY variants, we next investigated if PCA titers for the pathways expressing PCA decarboxylase variants were perturbed. Importantly, PCA titers of no higher than 250-300 mg/L are known to cause a significant fitness burden to S. cerevisiae (D'Ambrosio et al, 2020), making it critical to investigate if the observed increases in CCM titers for the 6 different PCA decarboxylase variants would support catalytic activities to lower PCA below fitness-burdening PCA levels. Indeed, of the KpAroY variants supporting increased CCM titers, KpAroY.B_P146T also showed significantly reduced PCA titers (77%, p < 0.01)( Figure 3D), further highlighting this variant as a new and catalytically improved PCA decarboxylase.…”

“…However, the build-up and secretion of the CCM pathway intermediate PCA remains rate-limiting for high CCM production (Suastegui et al, 2016;Leavitt et al, 2017;Snoek et al, 2018;Wang et al, 2020). This observation is propelled by the fact that PCA accumulation supposedly is not limited to suboptimal catalytic activity or expression of the downstream CCM pathway enzyme, PCA decarboxylase, as PCA accumulation also remains a persistent issue for microbial biosynthesis of other PCA-derived chemicals and nutraceuticals without decarboxylase requirements (Strucko et al, 2015(Strucko et al, , 2017D'Ambrosio et al, 2020). Yet, no attempts for directed evolution of the heterologous enzymes towards increased PCA-derived production has to our knowledge been performed.…”