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

ABSTRACTOne-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 13C-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 codon-optimized 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.

Evaluation of synthetic formaldehyde and methanol assimilation pathways in Yarrowia lipolytica

Background Crude glycerol coming from biodiesel production is an attractive carbon source for biological production of chemicals. The major impurity in preparations of crude glycerol is methanol, which is toxic for most microbes. Development of microbes, which would not only tolerate the methanol, but also use it as co-substrate, would increase the feasibility of bioprocesses using crude glycerol as substrate. Results To prevent methanol conversion to CO2 via formaldehyde and formate, the formaldehyde dehydrogenase (FLD) gene was identified in and deleted from Yarrowia lipolytica. The deletion strain was able to convert methanol to formaldehyde without expression of heterologous methanol dehydrogenases. Further, it was shown that expression of heterologous formaldehyde assimilating enzymes could complement the deletion of FLD. The expression of either 3-hexulose-6-phosphate synthase (HPS) enzyme of ribulose monosphosphate pathway or dihydroxyacetone synthase (DHAS) enzyme of xylulose monosphosphate pathway restored the formaldehyde tolerance of the formaldehyde sensitive Δfld1 strain. Conclusions In silico, the expression of heterologous formaldehyde assimilation pathways enable Y. lipolytica to use methanol as substrate for growth and metabolite production. In vivo, methanol was shown to be converted to formaldehyde and the enzymes of formaldehyde assimilation were actively expressed in this yeast. However, further development is required to enable Y. lipolytica to efficiently use methanol as co-substrate with glycerol.

Growth of Mycobacteria on Carbon Monoxide and Methanol

ABSTRACT Several mycobacterial strains, such as Mycobacterium flavescens, Mycobacterium gastri, Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sole source of carbon and energy. These bacteria, except for M. tuberculosis, also utilized methanol as the sole carbon and energy source. A CO dehydrogenase (CO-DH) assay, staining by activity of CO-DH, and Western blot analysis using an antibody raised against CO-DH of Mycobacterium sp. strain JC1 (formerly Acinetobacter sp. strain JC1 [J. W. Cho, H. S. Yim, and Y. M. Kim, Kor. J. Microbiol. 23:1-8, 1985]) revealed that CO-DH is present in extracts of the bacteria prepared from cells grown on CO. Ribulose bisphosphate carboxylase/oxygenase (RubisCO) activity was also detected in extracts prepared from all cells, except M. tuberculosis, grown on CO. The mycobacteria grown on methanol, except for M. gastri, which showed hexulose phosphate synthase activity, did not exhibit activities of classic methanol dehydrogenase, hydroxypyruvate reductase, or hexulose phosphate synthase but exhibited N,N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase and RuBisCO activities. Cells grown on methanol were also found to have dihydroxyacetone synthase. Double immunodiffusion revealed that the antigenic sites of CO-DHs, RuBisCOs, and dihydroxyacetone synthases in all mycobacteria tested are identical with those of the Mycobacterium sp. strain JC1 enzymes.

Alcohol oxidase and dihydroxyacetone synthase, the abundant peroxisomal proteins of methylotrophic yeasts, assemble in different cellular compartments

Alcohol oxidase (AO) and dihydroxyacetone synthase (DHAS) constitute the bulk of matrix proteins in methylotrophic yeasts, model organisms for the study of peroxisomal assembly. Both are homooligomers; AO is a flavin-containing octamer, whereas DHAS is a thiamine pyrophosphate-containing dimer. Experiments in recent years have demonstrated that assembly of peroxisomal oligomers can occur before import; indeed the absence of chaperones within the peroxisomal matrix calls into question the ability of this compartment to assemble proteins at all. We have taken a direct pulse-chase approach to monitor import and assembly of the two major proteins of peroxisomes in Candida boidinii. Oligomers of AO are not observed in the cytosol, consistent with the proteins inability to undergo piggyback import. Indeed, oligomerization of AO can be followed within the peroxisomal matrix, directly demonstrating the capacity of this compartment for protein assembly. By contrast, DHAS quickly dimerizes in the cytosol before import. Binding and import was slowed at 15°C; the effect on AO was more dramatic. In conclusion, our data indicate that peroxisomes assemble AO in the matrix, while DHAS undergoes dimerization prior to import.

A Methylotrophic Pathway Participates in Pectin Utilization by Candida boidinii

ABSTRACT The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source. When cells were grown on 1% (wt/vol) pectin, C. boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg). The numbers of peroxisomes also increased ca. two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell). The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant. Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.