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

Abstract

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

Unravelling Formaldehyde Metabolism in Bacteria: Road towards Synthetic Methylotrophy

“…It seems that FrmA is not the unique GD-Faldh in E. coli, as it was shown that production of formate also occurs in the ∆frmA E. coli strain. It was suggested that a promiscuous alcohol dehydrogenase could replace GD-Faldh activity in the conversion of formaldehyde to S-formyl-GSH, which is then converted to formate by Fgh (Figure 2) [101]. Moreover, two paralogous genes encode Fgh: frmB and yeiG.…”

“…In recent times, the development of novel assimilation pathways has received increasing interest, with some of these promising alternative pathways described here. The dihydroxyacetone (DHA) synthase (DAS) pathway is a cyclic formaldehyde assimilation pathway designed using in silico modelling [101]. It was built starting with the tool Find-Path to identify the most efficient pathway for E. coli to consume methanol [101,153].…”

“…The dihydroxyacetone (DHA) synthase (DAS) pathway is a cyclic formaldehyde assimilation pathway designed using in silico modelling [101]. It was built starting with the tool Find-Path to identify the most efficient pathway for E. coli to consume methanol [101,153]. This resulted in two equally promising hits: a RuMP-based pathway and a hybrid metabolic pathway involving bacterial Mdh from Acinetobacter gerneri and yeast DAS from Pichia angusta, the latter referred to as the aforementioned DAS pathway (Table 4, Figure 5) [101].…”

“…It was built starting with the tool Find-Path to identify the most efficient pathway for E. coli to consume methanol [101,153]. This resulted in two equally promising hits: a RuMP-based pathway and a hybrid metabolic pathway involving bacterial Mdh from Acinetobacter gerneri and yeast DAS from Pichia angusta, the latter referred to as the aforementioned DAS pathway (Table 4, Figure 5) [101]. DAS is a transketolase that catalyzes the condensation of formaldehyde with Xu5P yielding GAP and DHA in the XuMP pathway present in methylotrophic yeasts [101].…”

“…This resulted in two equally promising hits: a RuMP-based pathway and a hybrid metabolic pathway involving bacterial Mdh from Acinetobacter gerneri and yeast DAS from Pichia angusta, the latter referred to as the aforementioned DAS pathway (Table 4, Figure 5) [101]. DAS is a transketolase that catalyzes the condensation of formaldehyde with Xu5P yielding GAP and DHA in the XuMP pathway present in methylotrophic yeasts [101]. A library of 266 variants containing different combinations of Mdh and DAS homologues was built and screened using high-throughput 13 C-labeling experiments [101].…”