Microbial Utilization of Next-Generation Feedstocks for the Biomanufacturing of Value-Added Chemicals and Food Ingredients

Abstract

Global shift to sustainability has driven the exploration of alternative feedstocks beyond sugars for biomanufacturing. Recently, C1 (CO₂, CO, methane, formate and methanol) and C2 (acetate and ethanol) substrates are drawing great attention due to their natural abundance and low production cost. The advances in metabolic engineering, synthetic biology and industrial process design have greatly enhanced the efficiency that microbes use these next-generation feedstocks. The metabolic pathways to use C1 and C2 feedstocks have been introduced or enhanced into industrial workhorses, such as Escherichia coli and yeasts, by genetic rewiring and laboratory evolution strategies. Furthermore, microbes are engineered to convert these low-cost feedstocks to various high-value products, ranging from food ingredients to chemicals. This review highlights the recent development in metabolic engineering, the challenges in strain engineering and bioprocess design, and the perspectives of microbial utilization of C1 and C2 feedstocks for the biomanufacturing of value-added products.

Keywords: C1 feedstocks; C2 feedstocks; CO2 utilization; metabolic engineering; synthetic biology.

FIGURE 1

(A) Production network among various C1 and C2 feedstocks. CO₂ and lignocellulosic biomass serve as the two ultimate carbon sources for all the liquid feedstocks. H₂ is used for the reduction of CO₂ and CO. O₂ is required for the oxidization of methane to produce methanol and formate. Gaseous feedstocks (CO₂, CO, H₂) are in circles, while liquid feedstocks (methanol, formate) are in boxes. The formats of below figures are the same in colour and shapes. (B) Free energy and the oxidation state of C1 and C2 species (Aresta et al., 2014). Synonyms: g, gas; l, liquid, s, solid; aq, aqueous.

FIGURE 2

The Calvin-Benson-Bassham (CBB) cycle and its application in metabolic engineering. (A) The simplified CBB cycle. (B) Autotrophic E. coli harnessing the CBB cycle. (C) The CBB-enabled synthetic autotrophic P. pastoris. Enzymes: RuBisCO, ribulose-1,5-bisphosphate carboxylase/oxygenase; PrkA or Prk, phosphoribulokinase; FDH, formate dehydrogenase; CA, carbonic anhydrase; PfkA/B, 6-phosphofructokinase; Zwf, glucose-6-phosphate dehydrogenase; Aox1/2, alcohol oxidase; DAS1/2, dihydroxyacetone synthase; Fld1, formaldehyde dehydrogenase; Fgh, S-formylglutathione hydrolase; PGK1, phosphoglycerate kinase; TDH3, glyceraldehyde-3-phosphate dehydrogenase; TPI1, triosephosphate isomerase; TKL1, transketolose. Metabolites: RuBP, ribulose-1,5-bisphosphate; Ru5P, ribulose-5-phosphate; 3PG, 3-phosphoglycerate; 1,3BPG, 1,3-diphosphoglycerate; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; FBP, fructose 1,6-bisphosphatase; R5P, ribose 5-phosphate. Reduced feedstocks (methanol, formate, xylose) and cofactors (ATP, NAD(P)H) are in blue. Genes/enzymes are in red.

FIGURE 3

The Wood-Ljungdahl pathway (WLP). Enzymes: ACS, acetyl-CoA synthase; CODH, carbon monoxide dehydrogenase; FDH, ormatedehydrogenase; PFOR, pyruvate:ferredoxin oxidoreductase. Metabolites/cofactors: THF, tetrahydrofolate; FD_red, reduced ferredoxins. Reduced cofactors (ATP, NAD(P)H, FD_red) are in blue. Genes/enzymes are in red.

FIGURE 4

The reductive tricarboxylic acid (rTCA) cycle. Enzymes: PyrS, pyruvate synthase; PEPC, PEP carboxylase; KGS, α-ketoglutarate synthase; ICDH, isocitrate dehydrogenase. Metabolites: PEP, phosphoenolpyruvate. Dashed arrows are multiple enzymatic reactions.

FIGURE 5

The 3-hydroxypropionate–4-hydroxybutyrate (3HP-4HB) cycle and the dicarboxylate–4-hydroxybutyrate (DC–4HB) cycle. Enzymes: AcC, acetyl-CoA carboxylase; PrC, propionyl-CoA carboxylase; PyrS, pyruvate synthase; PEPC, PEP carboxylase.

FIGURE 6

The reductive glycine pathway (rGlyP). The rGlyP has two variants: serine deaminase pathway (in orange) and glycine reductase pathway (in black). Enzymes: FDH, ormatedehydrogenase; GCS, glycine cleavage/synthase system; GlyA, serine hydroxymethyltransferase; Sda, serine deaminase; GRC, Glycine reductase complex. Metabolites: THF, tetrahydrofolate.

FIGURE 7

Methane and methanol assimilation routes. In nature, three main routes were identified: ribulose monophosphate (RuMP) cycle, xylose monophosphate (XuMP) cycle or dihydroxyacetone (DHA) cycle and Serine cycle. Enzymes: HPS, 3-hexulose-6-phosphate synthase; PHI, 6-phosphate-3-hexuloisomerase; PGDH, 6-phosphogluconate dehydrogenase; MMO, methane monooxygenase; MDH, methanol dehydrogenase; AOX, alcohol oxidase; FADH, formaldehyde dehydrogenase; FDH, formate dehydrogenase. Metabolites: H6P, hexulose 6-phosphate; F6P, fructose-6-phosphate; FBP, fructose 1,6-bisphosphatase; G6P, glucose-6-phosphate; 6PG, 6-phosphogluconate; GAP, glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate; R5P, ribose 5-phosphate; DHA, dihydroxyacetone; Xu5P, xylulose-5-phosphate; PEP, phosphoenolpyruvate; OAA, oxaloacetate.

FIGURE 8

Synthetic routes for C1-feedstock assimilation. (A) Synthetic routes of the formyl-CoA elongation (FORCE) pathways. (B) Two strain co-culture system using the FORCE pathways. (C) A synthetic CO₂ fixation pathway, the POAP cycle. Enzymes: HACL, 2-hydroxyacyl-CoA lyase; PYC, pyruvate carboxylase; OAH, oxaloacetate acetylhydrolase; ACS, acetate-CoA ligase, and PFOR, pyruvate synthase. Cofactors: FD_red, reduced ferredoxins.

FIGURE 9

Metabolic pathway for C2 feedstock. Dotted lines indicate multiple steps; letters in empty boxes indicate the single letter codes for amino acids; orange lines indicate potential routes for C2 feedstock utilization; AAC, ADP/ATP carrier protein; ACC, acetyl-CoA carboxylase; ACAT, acetyl-CoA acetyltransferase; ADH, alcohol dehydrogenase; ALD, aldehyde dehydrogenase; ALDH, Acetaldehyde dehydrogenase (EC 1.2.1.10); ACO, aconitase; ACS, acetyl-CoA synthetase; ACL, ATP-citrate lyase; AS, ATP synthase; ASCT, acetate:succinate CoA-transferase; CA, carbonic anhydrase; CAT, carnitine acetyltransferase; CRC, carnitine carrier; CTP, mitochondria citrate transporter; CS, citrate synthase, FAS, fatty acid synthase; FUM, fumarase; HK, hexokinase; IDH, isocitrate dehydrogenase; ICL, isocitrate lyase; KDG, α-ketoglutarate dehydrogenase complex; MAE, malic enzyme; MDH, malate dehydrogenase; MPC, mitochondrial pyruvate carrier; MS, malate synthase; NADK, NAD+ kinase; NADPP, NADPH phosphatase; SCS, succinyl-CoA synthetase; SDH, succinate dehydrogenase; PDC, pyruvate dehydrogenase complex; PEPCK, Phosphoenolpyruvate carboxykinase; PYC, pyruvate carboxylase; PYK, pyruvate kinase; MPC, mitochondrial pyruvate carrier; MCT, monocarboxylate transporters. The feeding point to photosynthetic product in plants and algae is shown in green.

FIGURE 10

The overview of the bioeconomy using C1 and C2 feedstocks. Gaseous feedstocks (CO₂, CO, H₂) are in circles, liquid feedstocks (methanol, formate, ethanol and acetate) are in boxes. Green and red dots refer as intermediate metabolites and by-products. Metabolic engineering strategies include deleting the competing pathways to eliminate/minimize by-products. Elution engineering refers that the mutant strains with higher fitness (rings in red) will gradually dominate in the fermentation medium supplemented with unfavorable NGFs.