Developing methylotrophic microbial platforms for a methanol-based bioindustry

Abstract

Methanol, a relatively cheap and renewable single-carbon feedstock, has gained considerable attention as a substrate for the bio-production of commodity chemicals. Conventionally produced from syngas, along with emerging possibilities of generation from methane and CO2, this C1 substrate can serve as a pool for sequestering greenhouse gases while supporting a sustainable bio-economy. Methylotrophic organisms, with the inherent ability to use methanol as the sole carbon and energy source, are competent candidates as platform organisms. Accordingly, methanol bioconversion pathways have been an attractive target for biotechnological and bioengineering interventions in developing microbial cell factories. This review summarizes the recent advances in methanol-based production of various bulk and value-added chemicals exploiting the native and synthetic methylotrophic organisms. Finally, the current challenges and prospects of streamlining these methylotrophic platforms are discussed.

Keywords: bio-based chemical production; metabolic engineering; methanol; methylotrophs; microbial cell factories; synthetic biology.

FIGURE 1

Schematic summary of engineering native and synthetic methylotrophs. (A) Engineering scheme of native methylotrophs for chemical production from methanol, (B) Engineering scheme of synthetic methylotrophs for chemical production from methanol. ALE; adaptive laboratory evolution.

FIGURE 2

Methanol metabolism via RuMP pathway. Compounds targeted for production represented in green. Overexpressed genes or enzymes are presented in red. GABA, gama-aminobutyric acid; Aox, alcohol oxidase; hps, hexulose-6-phosphate synthase; phi, 6-phospho-3-hexuloisomerase; rpe, ribose 5-phosphate epimerase; rpi, ribose 5-phosphate isomerase; CL, 4-coumaroyl CoA ligase; CHS, chalcone synthase; lysC, aspartokinase; dapAB, dihydrodipicolinate synthase and reductase; cadA, L-lysine decarboxylase; hd, homoserine dehydrogenase; thrC, threonine synthase; atoB, acetyl-CoA acetyltransferase; adc, acetoacetate decarboxylase; ctfAB, coenzyme A transferase; hbd, 3-hydroxylbutyryl-CoA dehydrogenase; crt, crotonase; ter, trans-2-enoyl-CoA reductase; adhE, aldehyde/alcohol dehydrogenase; gad, glutamate decarboxylase.

FIGURE 3

Methanol metabolism via Serine pathway. Targeted compounds represented in green. Overexpressed genes or enzymes are presented in red. mdh, methanol dehydrogenase; flds, formaldehyde dehydrogenase; fdh, formate dehydrogenase; mcr, malonyl-CoA reductase; mvaK, D, mevalonate kinase and decarboxylase; fni, isopentenyl-diphosphate delta-isomerase; ERG20, farnesyl pyrophosphate synthase; ZZSl, α-humulene synthase; hmgs, hydroxymethylglutaryl-CoA synthase; hmgr, hydroxymethylglutaryl-CoA reductase; pha, thiolase; cad, cis-aconitate decarboxylase; ldh, lactate dehydrogenase; vio A-E, violacein operon.

FIGURE 4

Methanolic carbon assimilation in synthetic pathways. Key enzymes are presented in red. GAA, glycoaldehyde assimilation pathway; GAPA, glycolaldehyde-allose 6-phosphate pathway; MCC, methanol condensation cycle; FLS, formolase pathway; rGlyP, reductive glycine pathway; SACA, synthetic acetyl-CoA pathway; HACL, 2-Hydroxyacyl-CoA lyase pathway; KHB, ketohydroxy butyrate pathway. Faldh, formaldehyde dehydrogenase; Sdh, serine dehydratase; Pps, phosphoenolpyruvate synthase; Agt, alanine-glyoxylate transaminase; LtaE, threonine aldolase; RhmA, 2-keto-3-deoxy-L-rhamnonate aldolase; Hat, HOB aminotransferase; Hsk, homoserine kinase; Ts, threonine synthase; Gals, benzoylformate decarboxylase; Pkt, phosphoketolase; DeoC, 2-deoxy-D-ribose-5-phosphate aldolase; RpiB, ribose-5-phosphate isomerase B; AlsE, D-allulose-6-phosphate 3-epimerase; Pta, phosphate acetyltransferase; FLS, formolase; Dhak, dihydroxyacetone kinase; GCS system, Glycine cleavage system; Acr, acetyl CoA reductase; HACL, 2-hydroxyacyl-CoA-lyase; KHGA, 2-keto-4-hydroxyglutarate aldolase; PDC, pyruvate decarboxylase; DhaT, 1,3-propanediol oxidoreductase.