Abstract
Engineering biotechnological microorganisms to use methanol as a feedstock for bioproduction is a major goal for the synthetic metabolism community. Here, we aim to redesign the natural serine cycle for implementation in E. coli. We propose the homoserine cycle, relying on two promiscuous formaldehyde aldolase reactions, as a superior pathway design. The homoserine cycle is expected to outperform the serine cycle and its variants with respect to biomass yield, thermodynamic favorability, and integration with host endogenous metabolism. Even as compared to the RuMP cycle, the most efficient naturally occurring methanol assimilation route, the homoserine cycle is expected to support higher yields of a wide array of products. We test the in vivo feasibility of the homoserine cycle by constructing several E. coli gene deletion strains whose growth is coupled to the activity of different pathway segments. Using this approach, we demonstrate that all required promiscuous enzymes are active enough to enable growth of the auxotrophic strains. Our findings thus identify a novel metabolic solution that opens the way to an optimized methylotrophic platform.
Highlights
Microbial production of commodity chemicals is limited by feedstock availability and cost
Inspecting the structure of the serine cycle (Fig. 1a and Supplementary Table S1), we identified three key shortcomings: (i) Three of the pathway reactions participate in the central metabolism of the host
Assuming formaldehyde as the substrate of the pathways, we find that: (i) the homoserine cycle requires only eight enzymes, which is half the number of enzymes needed for the serine cycle (16) and ~40% fewer enzymes than the modified serine cycle (13, Fig. 1c); unlike the other pathways, the homoserine cycle is not dependent on foreign enzymes. (ii) The homoserine cycle consumes a single ATP molecule for the production of acetyl-CoA, while the serine cycle requires 3 ATP molecules and the modified serine cycle needs 4 ATP equivalents (Fig. 1c). (iii) The homoserine cycle does not lead to the net consumption of NAD (P)H, while the other two pathways consume 2 NAD(P)H per acetyl
Summary
Microbial production of commodity chemicals is limited by feedstock availability and cost. Still, when compared to biotechnological microorganisms, such as Escherichia coli, natural methylotrophs are more difficult to manipulate and engineer (Pfeifenschneider et al, 2017) To alleviate this problem, multiple recent efforts have sought to engineer biotechnological hosts for growth on methanol via one of the naturally occurring methanol assimilation pathways (Wang et al, 2020; Whitaker et al, 2015; Zhang et al, 2017): the ribulose monophosphate (RuMP) cycle (Chen et al, 2018; He et al, 2018; Meyer et al, 2018), the dihydroxyacetone (DHA) cycle (Dai et al, 2017), and the serine cycle (Yu and Liao, 2018). The serine cycle and its modified variants are ATP-inefficient, which results in low biomass and product yields
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