Overexpression of the genes PDC1 and ADH1 activates glycerol conversion to ethanol in the thermotolerant yeast Ogataea (Hansenula) polymorpha

Abstract

Conversion of byproduct from biodiesel production glycerol to high-value compounds is of great importance. Ethanol is considered a promising product of glycerol bioconversion. The methylotrophic thermotolerant yeast Ogataea (Hansenula) polymorpha is of great interest for this purpose as the glycerol byproduct contains methanol and heavy metals as contaminants, and this yeast utilizes methanol and is relatively resistant to heavy metals. Besides, O. polymorpha shows robust growth on glycerol and produces ethanol from various carbon sources. The thermotolerance of this yeast is an additional advantage, allowing increased fermentation temperature to 45-48°C, leading to increased rate of the fermentation process and a fall in the cost of distillation. The wild-type strain of O. polymorpha produces insignificant amounts of ethanol from glycerol (0.8 g/l). Overexpression of PDC1 coding for pyruvate decarboxylase enhanced ethanol production up to 3.1 g/l, whereas simultaneous overexpression of PDC1 and ADH1 (coding for alcohol dehydrogenase) led to further increase in ethanol production from glycerol. Moreover, the increased temperature of fermentation up to 45°C stimulated the production of ethanol from glycerol used as the only carbon source up to 5.0 g/l, which exceeds the data obtained by methylotrophic yeast strains reported so far.

Overexpression of the genes of glycerol catabolism and glycerol facilitator improves glycerol conversion to ethanol in the methylotrophic thermotolerant yeast Ogataea polymorpha

“…The following O. polymorpha strains were used in this study: NCYC495 leu1‐1 (wild‐type strain, WT); WT/PDC1/ADH1—previously obtained (Kata et al, ) recombinant strain with overexpression of the genes PDC1 and ADH1 ; WT/PDC1/ADH1/GPD1, WT/PDC1/ADH1/GCY1, WT/PDC1/ADH1/GUT1, and WT/PDC1/ADH1/DAK1—recombinant strains with the overexpression of the individual genes GPD1 , GCY1 , GUT1 , or DAK1 on the background of the WT/PDC1/ADH1 strain; WT/PDC1/ADH1/GCY1/DAK1 and WT/PDC1/ADH1/GPD1/GUT1—recombinant strains with the overexpression of the gene pairs GCY1/DAK1 or GPD1/GUT1 on the background of the WT/PDC1/ADH1 strain; WT/FPS1, WT/PDC1/ADH1/GCY1/DAK1/FPS1, and WT/PDC1/ADH1/GPD1/GUT1/FPS1—recombinant strains with overexpression of the heterologous gene FPS1 from K. phaffii on the background of the NCYC495, WT/PDC1/ADH1/GCY1/DAK1, or WT/PDC1/ADH1/GPD1/GUT1, respectively.…”

“…Standard cloning techniques were carried out as described (Sambrook et al, ). Plasmid and genomic DNA isolation and PCR reactions were performed as was described in our previous work (Kata et al, ). Transformation of the yeast O. polymorpha was carried out as described elsewhere (Faber, Haima, Harder, Veenhuis, & Ab, ).…”

“…Alcoholic fermentation of WT and recombinant O. polymorpha strains and determination of ethanol and glycerol content in the cultivation medium were performed as described before (Kata et al, ) except for the fact that in some experiments crude glycerol fractions from Polish biodiesel companies Orlen (Płock) and Wratislavia (Wrocław) were used instead of the pure glycerol. Experiments were performed at least thrice.…”

“…Some new microorganisms with good ability to grow and produce ethanol on crude glycerol have been recently isolated, for example, the bacterium Kluyvera cryocrescens (Choi, Hartono, Chan, & Yeo, ) and the yeast Pachysolen tannophilus (Liu, Jensen, & Workman, ). Methylotrophic and highly stress‐ and temperature‐tolerant yeast Ogataea polymorpha is also a good candidate for crude glycerol bioconversion, as in addition to glycerol conversion, it could utilize residual methanol present in the raw glycerol fraction, so we (and the other groups) have concentrated efforts on the improving of ethanol production from glycerol by this yeast (Hong et al, ; Kata, Semkiv, Ruchala, Dmytruk, & Sibirny, ). Up to date, we have intensified the last stages of ethanol production by O. polymorpha by overexpression of the genes encoding pyruvate decarboxylase ( PDC1 ) and alcohol dehydrogenase ( ADH1 ), which resulted in the increase of ethanol accumulation from glycerol (Kata et al, ).…”

“…Methylotrophic and highly stress‐ and temperature‐tolerant yeast Ogataea polymorpha is also a good candidate for crude glycerol bioconversion, as in addition to glycerol conversion, it could utilize residual methanol present in the raw glycerol fraction, so we (and the other groups) have concentrated efforts on the improving of ethanol production from glycerol by this yeast (Hong et al, ; Kata, Semkiv, Ruchala, Dmytruk, & Sibirny, ). Up to date, we have intensified the last stages of ethanol production by O. polymorpha by overexpression of the genes encoding pyruvate decarboxylase ( PDC1 ) and alcohol dehydrogenase ( ADH1 ), which resulted in the increase of ethanol accumulation from glycerol (Kata et al, ). An increase of metabolic flow through the glycerol catabolic pathway could considerably enhance the production of ethanol.…”