Table 3.

Production of fuels and chemicals using native or synthetic methylotrophic bacteria.

Product	Strain	Methanol assimilation pathway	Metabolic engineering strategy	Substrate	Culture condition	Product titer/yield	Ref.
Native methylotrophic bacteria
L-glutamate	B. methanolicus	RuMP	Δhom-1	Methanol	Fed-batch fermentation with automatic control for methanol feeding	32 g/l	[42]
				Methanol and methionine	Fed-batch fermentation with automatic control for methanol along with methionine feeding	69 g/l
L-lysine	B. methanolicus	RuMP	NOA2 mutant strain	Methanol and amino acids	Fed-batch fermentation with automatic control for methanol supply; supplementation of threonine, lysine, and methionine	65 g/l	[42]
Cadaverine	B. methanolicus	RuMP	Heterologous expression of cadA and ldcC; overexpression of l-lysine synthesis pathway	Methanol	Fed-batch fermentation using methanol as the sole carbon source	11.3 g/l	[44]
5-AVA	B. methanolicus	RuMP	Expression of 5-AVA biosynthesis pathway from cadaverine and l-lysine	Methanol	Shake flask cultures using methanol as the sole carbon source	23.7 mg/l	[45]
				Methanol andcadaverine	Shake flask cultures using methanol along with supplementation of cadaverine	77 mg/l
GABA	B. methanolicus	RuMP	Heterologous expression of gadSt and gadB	Methanol	Fed-batch fermentation employing a pH shift from 6.5 to 4.6 using methanol as the sole carbon source	9 g/l	[46]
Acetoin	B. methanolicus	RuMP	Heterologous expression of alsSD operon, overexpression of malic enzyme, and isocitrate lyase	Methanol	Shake flask using methanol as the carbon source	0.42 g/l	[57]
2-HIBA	M. extorquens AM1	Serine cycle	Expression of B12-dependent mutases	Methanol	Fed-batch fermentation using methanol as the sole carbon source under nitrogen-limiting conditions	2.1 g/l	[53]
α-humulene	M. extorquens AM1	Serine cycle	Heterologous expression of α-humulene synthase and FFP; heterologous expression of the mevalonate pathway	Methanol	Fed-batch fermentation using methanol as the sole carbon source, cumate as inducer, and dodecane as an organic overlay	1.65 g/l	[64]
Itaconic acid	M. extorquens AM1	Serine cycle	Heterologous expression of cis-aconitic acid decarboxylase; repression of phaR	Methanol	Fed-batch fermentation using methanol as the sole carbon source	31.6 mg/l	[56]
Violacein	M. extorquens AM1	Serine cycle	Heterologous expression of operon VioABCDE followed by random mutagenesis	Methanol	Shake flask cultures using methanol as the sole carbon source	67.8 mg/l	[65]
				Methanol and acetate	Shake flask cultures using methanol and acetate	118 mg/l
Mesaconic acid	M. extorquens AM1	Serine cycle	Expression of yciA (thioestrase)	Methanol	Shake flask using methanol as the sole carbon source	70 mg/l	[54]
3-Hydroxypropionic acid	M. extorquens AM1	Serine cycle	Heterologous expression of mcr; enhancing the expression by promoter optimization and multicopy expression of mcr	Methanol	Shake flask using methanol as the sole carbon source	69.8 mg/l	[58]
Mevalonate	M. extorquens AM1	Serine cycle	Construction of mevalonate biosensor along with mevalonate biosynthesis pathway	Methanol	Fed-batch fermentation using methanol as the sole carbon source	2.67 g/l	[61]
Crotyl diphosphate	M. extorquens AM1	Serine cycle	Expression of THKM82V, IPK and ADH2	Methanol and crotonol	Shake flask using methanol along with supplementation of exogenous crotonal	0.60 μg/mL	[135]
Crotonic acid	M. extorquens AM1	Serine cycle	Heterologous expression of isocitrate lyase and malate synthase for activation of the glyoxylate shunt, deletion of EMC pathway genes	Methanol	Shake flask using methanol as the sole carbon source	Growth defects on sole methanol	[55]
				Acetate	Shake flasks using initial growth on acetate; supplementation of 3-nitropropionate to repress glyoxylate shunt during production phase on acetate	0.2 mg/l
PHB	M. extorquens sp. K.	Serine cycle	N/A	Methanol	Fed-batch fermentation using methanol as the sole carbon source under nitrogen and mineral salt-deficient conditions	136 g/l; 66% of total DCW	[49]
P(3HB-co-3HV-co-3HHX)	M. extorquens AM1	Serine cycle	Deletion of native phaC, heterologous expression of phaC from Aeromonas cavaie, overexpression of β-ketothiolase and acetoacetyl-CoA reductase	Methanol	Fed-batch fermentation using methanol as the sole carbon source under cobalt-deficient conditions	43.6% of DCW	[136]
PHB	M. extorquens AM1	Serine cycle	N/A	Methanol	Fed-batch fermentation with controlled methanol supplementation (0.01 g/l) in minimal media	46% of DCW	[48]
P(3HB-co-3HV)	Methylobacterium sp. GW2	Serine cycle	N/A	Methanol and valerate	Fed-batch fermentation using 0.5% methanol as carbon source; supplementation of valerate	30% of DCW	[50]
Synthetic methylotrophic bacteria
Succinic Acid	E. coli	RuMP	Heterologous expression of NAD-dependent MDH; heterologous expression of RuMP pathway	Glucose and methanol	Fed-batch fermentation using 100 g/l glucose and 6.4 g/l methanol. Methanol was used as an auxiliary substrate	68.75 g/l, 1.45% carbon derived from methanol	[113]
Ethanol	E. coli	Modified Serine cycle	Deletion of aceB, gcvP, glcB, frdB, ldhA, gcl, expression of the modified serine cycle, expression of the ethanol production pathway	LB, methanol, and xylose	Shake flask using LB medium supplemented with 30 mM xylose and 200 mM methanol	36.3 mM, 33.8% of carbon derived from methanol	[117]
Acetate	E. coli	Modified Serine cycle	ΔaceB, ΔgcvP, ΔglcB, ΔfrdB, ΔldhA, Δgcl, expression of modified serine cycle, expression of the ethanol production pathway	LB, methanol, and xylose	Shake flask using LB medium supplemented with 30 mM xylose and 200 mM methanol	37 mM, 27.2% of carbon derived from methanol	[117]
1-butanol	E. coli	RuMP	Δrpe, ΔrpiAB, expression of NAD-dependent MDH, Hps, and Phi followed by adaptive evolution, expression of the 1-butanol production pathway	Xylose and methanol	Fed-batch fermentation using 87 mM of methanol and 100 mM of xylose	2.0 g/L, 71% of carbon derived from methanol	[137]
Acetone	E. coli	RuMP	Δpgi, ΔfrmA, Expression of MDH, Hps, and Phi, Expression of non-oxidative pentose phosphate pathway; expression of acetone formation pathway	Glucose and methanol	Fed-batch fermentation using 260 mM glucose and 38.3 mM methanol	45.0 mM, 3.6% of carbon derived from methanol	[75]
	E. coli		Δpgi, ΔrpiAB, Δedd, ΔfrmA, expression of RuMP pathway followed by ALE	Glucose and methanol	Fed-batch fermentation using 36 g/l glucose and 500 mM methanol	1 g/L, 22% of carbon derived from methanol	[138]
Cadaverine	C. glutamicum	RuMP	Δald, Δfdh; heterologous expression of NAD-dependent MDH, Hps, and Phi	Ribose and methanol	Shake flask using 20 mM ribose and 200 mM	1.5 g/l, 15% of carbon derived from methanol	[119]
Naringenin	E. coli	RuMP	Expression of NAD-dependent Mdh fromBacillus stearothermophilus, Expression of Hps and Phi from B. methanolicus, Expression of Coumaroyl CoA ligase and Chalcone synthase		Co-utilization of methanol and yeast extract	3.5 mg/L 18% of carbon derived from methanol	[139]
D-allulose	E. coli	RuMP	Coupling of allulose monophosphate, RuMP, and PPP	Xylose and methanol	Fed-batch fermenter using 102 mM xylose and 97 mM methanol in the presence of yeast extract and tryptone	0.512 mM d-allulose/mM methanol	[116]
Succinic Acid	E. coli	RuMP	Expression of methanol dissimilation pathway along with RuMP pathway	Glucose, methanol, and formate	Fed-batch using 50 g/l glucose, 2 g/l formate, and 200 mM methanol. Methanol and formate were used as auxiliary substrates	63.42 g/l	[114]