Triggering the stringent response enhances synthetic methanol utilization in Escherichia coli

Regulatory interventions improve the biosynthesis of limiting amino acids from methanol carbon to improve synthetic methylotrophy in Escherichia coli

“…The exact reason for this is unknown. Previous work from our lab, aimed at improving the biosynthesis of amino acids from methanol carbon via stringent response activation, was also unsuccessful in enabling biosynthesis of isoleucine and leucine from methanol carbon (Bennett, Agee, et al, 2020). Thus, these two amino acids should be the focus of future engineering efforts.…”

“…In absolute terms, the average carbon labeling (% 13 C) improvements are much higher than those resulting solely from removal of allosteric feedback inhibition, suggesting that enzyme abundance is another regulatory limitation that currently limits synthetic E. coli methylotrophs, since these organisms cannot properly regulate the expression of essential amino acid biosynthesis genes in response to methanol, a nonnative substrate. This regulatory limitation has been the focus of other work in our lab, which have alleviated this limitation via endogenous activation of the stringent response to increase gene expression of amino acid biosynthetic operons during methylotrophic growth (Bennett, Agee, et al, 2020) and implementing formaldehyde‐responsive promoters (P frm ) to induce the expression of essential growth‐related genes in response to methanol (Rohlhill et al, 2017; Rohlhill et al, 2020). The small increases in absolute average carbon labeling (% 13 C) of histidine and threonine of the empty plasmid control (pE) compared to the previous WT control (MG1655) most likely result from the deletion of frmA , which minimizes formaldehyde dissimilation to CO 2 , thus retaining a higher percentage of methanol‐derived carbon that enters the cell.…”

“…Here, we focused to overcome the limitation of amino acid biosynthesis from methanol carbon. Based on prior studies from our lab that determined amino acid metabolism stimulates synthetic methanol utilization in E. coli (Bennett, Agee, et al, 2020; Gonzalez et al, 2018; Whitaker et al, 2017), here, we examined how global and local amino acid regulatory networks impact synthetic methylotrophy in E. coli . To emphasize the importance and difficulty of achieving and further improving the biosynthesis of limiting amino acids from methanol carbon to realize autonomous methylotrophy, we explored several independent regulatory strategies in this study.…”

“…In one study, we determined that the stimulatory effect of yeast extract could be achieved with only threonine, which was mediated by the leucine‐responsive regulatory protein (Lrp), a transcriptional regulator (Gonzalez et al, 2018). In another study, we demonstrated that stringent response activation via ppGpp overproduction or overexpression of DksA or RpoS greatly enhanced the methylotrophic phenotype of E. coli by enabling the biosynthesis of several limiting amino acids from methanol carbon (Bennett, Agee, et al, 2020). In this study, we further expand our focus on methylotrophic amino acid metabolism by targeting global and local amino acid regulatory networks.…”

“…There are several reports on efforts to engineer synthetic methylotrophs, focusing mainly on E. coli (Bennett, Agee, et al, 2020; Bennett, Dillon, et al, 2020; Bennett, Gonzalez, Whitaker, Antoniewicz, & Papoutsakis, 2018; Chen et al, 2018; Gonzalez, Bennett, Papoutsakis, & Antoniewicz, 2018; Meyer et al, 2018; Muller et al, 2015; Price, Chen, Whitaker, Papoutsakis, & Chen, 2016; Rohlhill, Gerald Har, Antoniewicz, & Papoutsakis, 2020; Rohlhill, Sandoval, & Papoutsakis, 2017; Wang et al, 2019; Whitaker et al, 2017; Woolston, King, Reiter, Van Hove, & Stephanopoulos, 2018), Corynebacterium glutamicum (Lessmeier et al, 2015; Tuyishime et al, 2018; Witthoff et al, 2015), and Saccharomyces cerevisiae (Dai et al, 2017). As illustrated in Figure 1a, the most commonly used synthetic methanol utilization pathway requires three essential enzymes: (1) methanol dehydrogenase (Mdh), which oxidizes methanol to formaldehyde, (2) hexulose phosphate synthase (Hps), which fixes formaldehyde with ribulose 5‐phosphate (Ru5P) to generate hexulose 6‐phosphate (H6P), and (3) phosphohexulose isomerase (Phi), which converts H6P to the native glycolytic intermediate fructose 6‐phosphate (F6P).…”