Table 2.

Production of fuels and chemicals using native methylotrophic yeasts.

Product	Strain	Metabolic engineering strategy	Substrate	Culture condition	Product titer/yield	Ref.
Free fatty acids (FFA)	P. pastoris	Blocking FFA consumption (ΔFAA1, ΔFAA2, and ΔPOX1), enhancing acetyl-CoA (Overexpression of mmACL, bbXFPK, ckPTA) and NADPH (overexpression scIDP2), improving methanol assimilation (overexpression of DAS2)	Methanol	Fed-batch using methanol as the sole carbon source in minimal media (MM), methanol concentration was kept below 20 g/l	23 g/l	[30]
Fatty alcohol	P. pastoris	Heterologous expression of mmCAR, npgA, scADH5 Overexpression of FaCoAR in FFA overproducing strain	Methanol	Fed-batch using methanol as the sole carbon source in MM, methanol concentration was kept below 20 g/l	2 g/l	[30]
FFA	O. polymorpha	ΔFAA	Methanol	Shake flask using 10 g/l methanol in MM	Growth defects	[31]
			Methanol, xylose, and glucose	Shake flask using MM with 20 g/l glucose, 10 g/l xylose and 10 g/l methanol	1.2 g/l
FFA	O. polymorpha	ΔFAA; enhancing methanol assimilation (overexpression of FBP1and RPE); enhancing acetyl-coA (overexpression of mmACL) enhancing NADPH (overexpression of ZWF1and ScIDP2); ALE-derived activation of LPL1and IZH3	Methanol	Fed-batch fermenter with initial culture using 10 g/l methanol in MM, continuous feeding of methanol after 24 h	15.9 g/l	[32]
			Methanol and glucose	Fed-batch fermenter with initial culture using 20 g/l glucose in MM followed by continuous feeding of methanol after 15 h	9.1 g/l
			Methanol and glycerol	Fed-batch fermenter with initial culture using 20 g/l glycerol in MM followed by continuous feeding of methanol after 24 h	13.2 g/l
Ricinoleic acid	P. pastoris	Δ12 desaturase, Heterologous expression of cpFAH and cpDGAT1	Methanol andglycerol	Fed-batch fermenter, Initial growth on glycerol followed by methanol induction and addition of 0.5% methanol every 24 h	495 μg/ml	[33]
γ- Linoleic acid (GLA)	P. pastoris	Heterologous expression of Δ6-Desaturase under methanol oxidation promoter (MOX)	Methanol	Fed-batch fermenter using 2% methanol in MM	11.9% GLA of Total fatty acids	[35]
6-methylsalicylic acid	P. pastoris	Heterologous expression of npgA, 6-MSAS	Methanol and glycerol	Fed-batch fermenter with initial growth on glycerol in MM followed by methanol induction for 20 h	2.2 g/l	[27]
Citrinin	P. pastoris	Heterologous expression of pksCT, npgA; mpl6; mpl7 overexpression of mpl1, mpl2 and mpl4	Methanol	Fed-batch fermenter with 0.5% methanol added after every 24 h	0.6 mg/l	[28]
D-lactic acid	P. pastoris	Heterologous expression of d-LDH	Methanol	Test tube fermentation using methanol as the sole carbon source in YPM (Yeast extract-peptone-Mannitol medium) for 96 h	3.48 g/l	[38]
β-alanine	P. pastoris	Heterologous expression of multiple copies of ADCs; overexpression of aspartate dehydrogenase	Methanol and glycerol	Fed-batch with two-stage fermentation using 40 g/l of glycerol for the growth phase followed by the production phase maintaining 3 g/l of methanol	5.6 g/l	[39]
α-alkenes	P. pastoris	ΔFAA; Heterologous expression of UndB	Methanol	Shake flask containing 20 g/l methanol in MM	1.6 mg/l	[36]
Monacolin J	P. pastoris	Expression of monacolin J and lovastatin biosynthesis pathway; distribution of the pathway in two strains to use as co-cultures	Methanol and glycerol	Fed-batch with initial growth using glycerol followed by methanol induction	593.9 mg/l	[29]
Lovastatin	P. pastoris	Expression of lovastatin biosynthesis pathway; distribution of the pathway in two strains to use as co-cultures	Methanol and glycerol	Fed-batch with initial growth using glycerol followed by methanol induction	250.8 mg/l	[29]
Malic acid	P. pastoris	Introduction of malic acid producing genes	Methanol	Fed-batch fermentation using methanol and yeast extract in MM	2.79 g/l	[41]
Glutathione	H. polymorpha DL-1	Overexpression of GSH2 and MET4	Methanol	Fed-Batch using methanol in MM	250 mg/l	[134]
Lycopene	P. pastoris	Heterologous expression of genes encoding crtB, crtE, and crtI; Overexpression of HMG1	Glycerol and methanol	Fed-batch fermenter using two-stage cultivation with initial growth on glycerol in BSM (Basal salt medium) followed by methanol induction	714 mg/l	[24]
Nootkatone	P. pastoris	Heterologous expression of HPO, CPR, and valence synthase; Overexpression of ADH, HMG1	Glucose and methanol	Fed-batch fermenter, biphasic cultivation using glucose and methanol in BSM, the addition of dodecane overlay to prevent evaporation	208 mg/l	[25]
Dammarenediol-II	P. pastoris	Heterologous expression of PgDDS; overexpression of ERG1; repression of ERG7	Methanol and squalene	BMMY media with 0.5% methanol supplementation every 24 h, Exogenous addition of squalene	1.073 mg/g DCW	[26]