Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway inE. coli

Abstract

25One-carbon (C1) compounds, such as methanol, have recently gained attention as 26 alternative low-cost and non-food feedstocks for microbial bioprocesses. Considerable 27 research efforts are thus currently focused on the generation of synthetic 28 methylotrophs by transferring methanol assimilation pathways into established 29 bacterial production hosts. In this study, we used an iterative combination of dry and 30 wet approaches to design, implement and optimize this metabolic trait in the most 31 common chassis, E. coli. Through in silico modeling, we designed a new route that 32 "mixed and matched" two methylotrophic enzymes: a bacterial methanol 33 dehydrogenase (Mdh) and a dihydroxyacetone synthase (Das) from yeast. To identify 34 the best combination of enzymes to introduce into E. coli, we built a library of 266 35 pathway variants containing different combinations of Mdh and Das homologues and 36 screened it using high-throughput 13 C-labeling experiments. The highest level of 37 incorporation, 22% of labeled methanol carbon into the multi-carbon compound PEP, 38 was obtained using a variant composed of a Mdh from A. gerneri and a codon-39 optimized version of P. angusta Das. Finally, the activity of this new synthetic pathway 40 was further improved by engineering strategic metabolic targets identified using omics 41 and modelling approaches. The final synthetic strain had 1.5 to 5.9 times higher 42 methanol assimilation in intracellular metabolites and proteinogenic amino acids than 43 the starting strain did. Broadening the repertoire of methanol assimilation pathways is 44 one step further toward synthetic methylotrophy in E. coli. 45

Engineering and adaptive laboratory evolution of Escherichia coli for improving methanol utilization based on a hybrid methanol assimilation pathway

“…DHA can be further converted into dihydroxyacetone phosphate (DHAP) by the endogenous DHA kinase of E. coli (Figure 1). Previously, De Simone et al showed that a strain expressing mdh from A. garner and das from P. angusta exhibited a higher growth rate in minimal medium with methanol and xylose than that with xylose alone (De Simone et al, 2020). In that case, Xu5P was mainly derived from xylose, thus, it was not clear whether the cyclic XuMP pathway would be functional in sugar-free conditions.…”

“…The hybrid methanol assimilation pathway can generate key glycolytic intermediate glyceraldehyde 3-phosphate (GAP) with only two enzymatic steps and the energy efficiency of the pathway is equal to the RuMP pathway when fructose-6-phosphate aldolase (Fsa) and transaldolase (Tal) are used for xylulose 5-phosphate (Xu5P) generation (Supplementary Table S1). Recently, De Simone et al demonstrated the first example of heterologous expression of Mdh and Das in E. coli and showed that methanol can be integrated into several intermediates in central metabolism when cells were cultured with methanol and xylose as co-substrates (De Simone et al, 2020). However, the engineered strain showed low incorporation of 13 C-methanol into the pentose phosphate pool and proteinogenic amino acids, indicating that the regeneration of Xu5P should be further improved to increase the efficiency of the cyclic XuMP pathway.…”