Scaffoldless engineered enzyme assembly for enhanced methanol utilization

Abstract

Methanol is an important feedstock derived from natural gas and can be chemically converted into commodity and specialty chemicals at high pressure and temperature. Although biological conversion of methanol can proceed at ambient conditions, there is a dearth of engineered microorganisms that use methanol to produce metabolites. In nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction. Thus, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. Here, we report a scaffoldless, self-assembly strategy to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3-ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate (F6P). To increase methanol consumption, an "NADH Sink" was created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction. Combination of the two strategies improved in vitro F6P production by 97-fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly was also realized in vivo as the engineered enzyme assembly improved whole-cell methanol consumption rate by ninefold. This approach will ultimately allow direct coupling of enhanced F6P synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol.

Keywords: methane; methylotophs; scaffold; substrate channeling; supramolcular.

Fig. 1.

(A) A schematic of the Mdh–RuMP pathway for methanol assimilation. (B) H6P formation with increasing ratios of Mdh3–sSH3lig:Hps or Mdh3–sSH3lig–SH3–Hps enzyme complexes. Purified Mdh–sSH3lig was mixed with equal molar ratios of purified Hps ranging from 1:1–1:10. H6P was assayed according to the previously established method by Arfman (39). Error bars represent the SD of at least three replicate experiments.

Fig. S1.

Comparison of formaldehyde consumption rate by MDH (blue) and HPS (red). Virtually no consumption was detected without enzymes (orange).

Fig. 2.

(A) A schematic of the Mdh3–Hps–Phi supramolecular enzyme complex and the corresponding cascade reactions. Ldh was used as an NADH Sink to further minimize formaldehyde reduction. (B) Formaldehyde (first bar) and F6P (second bar) formation with and without SH3-tethered enzymes. Purified Mdh–sSH3lig was mixed with either Hps and Phi, SH3–Hps and Phi, or the SH3–Hps–Phi fusion. Formaldehyde (HCHO) was assayed using the Nash reagent. Error bars represent the SD of at least three replicate experiments.

Fig. S2.

SDS/PAGE pull-down assay. Pull-down assays were performed to test the functionality of the individual SH3 interaction components when fused to enzymes. Briefly, (A) MDH–sSH3lig (1) and ELP–SH3 (2) were independently expressed in BL21(DE3), and soluble lysates were collected and mixed together at a 1:1 mass ratio (1+2). One round of ELP purification was performed and the purification product (P) was assessed by SDS/PAGE and commassie blue staining. (B) The same experiment was performed with SH3–HPS and ELP–sSH3lig (4), with SH3–HPS_PHI and ELP–sSH3lig (6), and with an empty vector lysate with ELP–sSH3lig (5). One round of ELP purification was performed, and purification products (P4, P5, and P6) for each condition assayed were examined by SDS/PAGE and commassie blue staining.

Fig. S3.

H6P and F6P production by purified Mdh–sSH3lig and the SH3–Hps–Phi fusion in the presence or absence of LDH. F6P production was assayed as described earlier. The total concentration of H6P and F6P was determined by first deactivating the enzymes by acid treatment. The difference between the two is the net amount of H6P accumulated. In all cases, only a very small quantity of H6P accumulation was detected.

Fig. 3.

Characterization of the Mdh3–Hps–Phi supramolecular enzyme complex. (A) DLS was used to characterize the size of the individual enzyme components and the 1:1 Mdh3–sSH3lig to SH3–Hps–Phi enzyme conjugates using the Zetasizer Nano ZS instrument. Three measurements were made at 25 °C, and the volume average is reported. (B) TEM of enzyme complexes. The TEM micrograph for Mdh3–sSH3lig showed the previously reported pentagonal shape of B. methanolicus MGA3 Mdh3, whereas the SH3–Hps–Phi fusion proteins were mostly larger and nonuniform enzyme clusters. Complex formation was clearly demonstrated by mixing the two proteins at a 1:1 molar ratio. Error bars represent the SD of at least three replicate experiments.

Fig. 4.

(A) A schematic illustrating the concept of NADH Sink using E. coli Ldh. (B) Effects of NADH Sink on methanol (CH₃OH) conversion. Purified Mdh3–sSH3lig was mixed with increasing ratios of purified E. coli Ldh and assayed for formaldehyde (HCHO) according to the Nash method. Error bars represent the SD of at least three replicate experiments.

Fig. S4.

NADH production during methanol consumption by MDH in the presence (orange) or absence of LDH (red). NADH production was monitored by the increase in absorbance at 340 nm. A control without enzyme (blue) produced no NADH.

Fig. 5.

Combinatorial effect of enzyme assembly and NADH Sink on methanol consumption and F6P production. Purified Mdh3–sSH3lig was mixed at a 1:1 molar ratio with either Hps and Phi, SH3–Hps and Phi, or SH3–Hps–Phi with or without an equal molar of Ldh. The HCHO and F6P concentration in the absence (first bar) and presence (second bar) of Ldh are shown. Error bars represent the SD of at least three replicate experiments.

Fig. S5.

Analysis of in vivo enzyme assembly. (A) SDS/PAGE analysis of enzyme expression at different time points. The respective protein bands are highlighted. (B) Separation of enzyme complexes by sucrose gradient. (C) Formation of enzyme complexes by TEM analysis. (Scale bar: 50 nm.)

Fig. 6.

In vivo methanol consumption and utilization. Two recombinant ∆frmA strains were tested for methanol consumption for 24 h. One strain harboring Mdh3 and Hps–Phi without the docking components (Control) and a second strain harboring Mdh3–sSH3lig and SH3–Hps–Phi fusion (Assembled). Cells were grown to an OD₆₀₀ of 1, washed, and resuspended in M9 medium and 500 mM methanol and incubated at 37 °C for 24 h. Methanol consumption was calculated as the difference between the starting and final methanol concentration. Formaldehyde accumulation was measured with the Nash reagent by harvesting cells and testing the clarified supernatant. Formate formation was determined by HPLC analysis of culture supernatants. Error bars represent SD; n = 4.