Integration of metabolism and regulation reveals rapid adaptability to growth on non-native substrates

Abstract

Engineering synthetic heterotrophy (i.e., growth on non-native substrates) is key to efficient bio-based valorization of various renewable (e.g., lignocellulosic biomass) and waste (e.g., plastics) substrates. Among these, engineering hemicellulosic pentose utilization has been well-explored in Saccharomyces cerevisiae (yeast) over several decades but genetic factors that constrain maximum growth rate remain elusive. Through a systematic analysis (flux balancing, directed evolution, functional genomics, and network modeling), we find that once global regulatory response is appropriately remodeled, wild-type-like growth profiles can be achieved with minimal metabolic engineering effort. This indicates that intrinsic yeast metabolism is highly adaptable to growth on non-native substrates. We identified that extrinsic factors – specifically, genes that direct flux of pentoses into central carbon metabolism – are rate limiting. We also find that deletion of endogenous genes to promote growth demonstrate inconsistent outcomes that are genetic-context- and condition-dependent. For the most part, these knockouts also lead to deleterious pleiotropic effects that decrease the robustness of strains against inhibitors commonly found in lignocellulosic hydrolysate. Thus, perturbation of intrinsic factors (e.g., metabolic, regulatory genes) provides incremental and inconsistent benefits at best and at worst, is detrimental. Not only do the findings and approach described here expedite the design-build-test cycle but also simplifies the engineering process. Overall, this work provides insight into the limitations and pitfalls to realizing efficient synthetic heterotrophy and provides a novel paradigm to engineer the same.

Towards universal synthetic heterotrophy using a metabolic coordinator

“…While all three approaches reached similar nal cell concentrations on each of the three substrates, the ∆GAL80 condition took roughly twice the time to do so compared with Gal3p Syn4.1 and Gal3p MC (>30 h vs. ~18 h) suggesting that de-regulation of the regulon results in reduced tness relative to strains in which the native regulatory organization is maintained. As sucrose and glucose are both capable of exerting repression on the GAL regulon 19 , subculturing all three strains from these two native substrates into complex (YP) media containing the non-native substrate xylose should therefore result in growth-coupled expression of xylose catabolic genes and rapid growth. Upon subculturing from sucrose, we observed that all three approaches enabled growth.…”

“…By placing heterologous genes for either xylose or arabinose catabolism under the control of GAL-responsive (Leloir pathway) promoters, the GAL regulon could be adapted for growth on these non-native substrates. Upon comparing this regulon-coordinated (REG) approach to growth with simple constitutive overexpression of the same catabolic genes, which we term a constitutive (CONS) approach, we found that our semi-synthetic regulon yielded superior growth rates on both xylose (µ = 0.24 h -1 vs. 0.11 h -1 ) and arabinose (µ = 0.27 h -1 vs. 0.06 h -1 ), respectively with minimal metabolic engineering 19 .…”

“…Once freed from repression, Gal4p -the master activator of the GAL regulon -binds to its cognate Upstream Activating Sequences (UAS) to directly induce transcription of galactose catabolic genes (Leloir pathway) and indirectly modulates the transcript and/or protein abundance of numerous growth-associated genes (Figure 1A) 16,17 . We previously reported the construction of a variant of sensor protein Gal3p (Gal3p Syn4.1 ) that strongly activates the GAL regulon by the pentose sugars xylose and arabinose 18,19 . By placing heterologous genes for either xylose or arabinose catabolism under the control of GAL-responsive (Leloir pathway) promoters, the GAL regulon could be adapted for growth on these non-native substrates.…”

Towards universal synthetic heterotrophy using a metabolic coordinator

“…A similar range of growth rates was observed for the xylose constructs (0.20 − 0.21 h -1 ) but the substantially reduced final cell density of two of the arrangements (ϕ-XK-XI and XK-ϕ-XI) indicates that there can be fitness consequences to expression imbalances. As no clearly superior combination could be identified to the initial arabinose and xylose designs, we decided to re-test a Consolidated approach to co-utilization on 1 % of each substrate (Figure 6E) using designs (ABD for arabinose, XI-ϕ-XK for xylose) identified as optimal in a related investigation by our lab into efficient pentose assimilation 19 .…”

“…Upon comparing this regulon-coordinated (REG) approach to growth with simple constitutive overexpression of the same catabolic genes, which we term a constitutive (CONS) approach, we found that our semi-synthetic regulon yielded superior growth rates on both xylose (µ = 0.24 h -1 vs. 0.11 h -1 ) and arabinose (µ = 0.27 h -1 vs. 0.06 h -1 ), respectively with minimal metabolic engineering 19 . Significantly, a transcriptomic comparison showed relative upregulation of genes associated with cell division and mitochondrial biogenesis in the REG strain while the CONS strain showed upregulation of stress response and starvation-associated genes 18 .…”