Applying an enhanced gene expression tool, a pathway-based qRT-PCR array validated by the robust external mRNA controls, we presented unique pathways to demonstrate mechanisms of the acquired tolerance and the in situ detoxification of furfural and HMF by the tolerant ethanologenic yeast S. cerevisiae
NRRL Y-50049 based on metabolic profiling analyses and dynamics of absolute quantification of mRNA transcripts. The recent development of the tolerant yeast (Liu et al. 2008b
) and the identification and synthesis of HMF metabolic conversion product FDM (Liu et al. 2004
) are necessary to make this study possible.
The robust performance using the control gene CAB
as sole standard of a fixed manual threshold setting and thereafter the use of the master equation provided reliable and consistent absolute quantification reference for real time qRT-PCR assays. As demonstrated by this study, such a standard measurement is independent from toxic treatment, culture conditions, varied strains, and different set of reactions that allow comparison of data obtained from different conditions and sources. It has significant advantages over variable housekeeping genes used in qRT-PCR assays (Baker et al. 2005
; The External RNA Control Consortium 2005
). Such a development significantly improved reliability, reproducibility, and throughput of the qRT-PCR and simplified the conventional practice of the assay.
Redox metabolism, in the form of interconversion of the pyridine-nucleotide co-factors NADH/NAD+
, plays a key role in the metabolism of yeast. NADH is required in respiration and fermentative pathway in conversion of pyruvate to CO2
and ethanol; and NADPH is mainly required for the synthesis of amino acids and nucleotides. Furfural conversion to furan methanol and HMF to furan dimethanol are NAD(P)H-dependent reduction activities by multiple enzymes (Petersson et al. 2006
; Song and Liu 2007
; Liu et al. 2008b
; Song et al. 2009
). These reduction reactions consume NADH and NADPH. Therefore, a well maintained redox balance is critical for efficient conversion and in situ detoxification of furfural and HMF. A major source of NADPH production in yeast is through the oxidative phase of pentose phosphate pathway. Since expressions of ZWF1, SOL3, GND1
, and GND2
were up-regulated at earlier time points, pentose phosphate pathway became a dominant reprogrammed path for glucose metabolisms and inhibitor conversions for Y-50049. These genes along with enhanced expressed TDH1
are significant for NADPH regenerations to supply necessary cofactors needed for acetaldehyde conversion and reduction of furfural and HMF. We previously observed that deletion mutants of ZWF1
were highly sensitive to furfural and HMF (Gorsich et al. 2006
). Significant effect of phosphogluconate dehydrogenase in redox metabolism has been demonstrated (Bro et al. 2004
). Apparently, enriched genetic background by aforementioned genes and a well maintained redox balance through the reprogrammed expression responses of Y-50049 were likely accountable for the acquired tolerance and detoxification of the inhibitors.
Glycolysis and pentose phosphate pathway are closely related pathways for glucose metabolism. This close relationship is of such significant importance that the two pathways cannot be viewed separately in the view of yeast tolerance and detoxification of furfural and HMF. For Y-50049, after initiation stage of phosphorylation of glucose by enzyme encoding genes HXK1, HXK2, and GLK1, glucose metabolism apparently shifted toward pentose phosphate pathways than glycolysis due to significantly induced expression of ZWF1, SOL3, GND1, and GND2 and repression of glycolytic enzyme phosphoglucose isomerase pathway. Such a pathway shift appeared to be significant for Y-50049 to carry continued glucose consumption and detoxification metabolism under the inhibitor stress.
It is known that furfural and HMF inhibit yeast growth and cause delayed glucose consumption at a tolerable dose (Taherzadeh et al. 2000
; Liu et al. 2004
). The reprogrammed transcription responses and the altered metabolic pathways presented in this study can also explain such observations that yeast has an accelerated glucose conversion rate once cells recovered from the inhibitor challenges than would normally occur without furfural and HMF. As demonstrated in this study, inhibition of glucose phosphorylation, together with repression of PFK1, PFK2, PYK2
expressions was likely to be the mechanism of the delay of glycolysis by furfural and HMF. This delayed biological process in yeast is also attributed to a lack of ATP, NAD(P)H, and carbon intermediate metabolites necessary to support cell growth and reproduction (Fisk et al. 2006
; Liu 2006
). Comparing the susceptible parental strain Y12632, gene transcription levels of PGK1, ENO1, ENO2, PYK2, CDC19, PDA1
, and PDB1
encoding varied enzymes for extended pyruvate metabolisms did not show significant effect by the inhibitor challenge at earlier time points for the tolerant Y-50049. Transcription levels were low for numerous other genes in glycolysis and pentose phosphate pathways for both strains. However, most of these genes were able to recover to normal function levels at 42 h for Y-50049, whereas those for Y-12632 continued to be repressed by the furfural and HMF treatment that eventually led to no viable cell functions after 42 h.
Over 300 genes were found to be statistically significant in response to the inhibitor challenges for the ethanologenic yeast genome (Liu 2006
). Numerous members of PDR gene family and key transcription factors played significant roles in regulating global transcription response to the bioethanol conversion inhibitors (Song and Liu 2007
; Song et al. 2009
). Puzzles of many complex pathways at the genome level remain unresolved. Nonetheless, regarding the in situ detoxification of furfural and HMF metabolic pathways involving glycolysis and pentose phosphate pathway, at least three significant groups of events of the reprogrammed evolutionary engineering are accountable for the acquired tolerance of Y-50049. First, initial high abundant transcript levels of at least 16 genes were critical enablers for Y-50049 to withstand the inhibitor stress. This represents the enriched genetic characteristics of Y-50049. Second, more than a dozen genes immediately responded with induced expression, specifically, the genes with continued enhanced expression over time enabled Y-50049 to function and detoxify furfural and HMF into corresponding furan methanol with a well maintained redox balance. Third, the integrated transcription response and reprogrammed regulatory networks, including those significantly enhanced expressed and as well as many initially repressed but able to recover to normal function levels at a later time, which allowed Y-50049 to maintain a balanced biological process to complete ethanol fermentation. In the absence of such reprogrammed transcription responses at the genome level as shown by the control yeast Y-12632, continued inhibition and repression by furfural and HMF led the yeast to lost function and eventually death. Our results guide continued efforts in developing stress tolerant ethanologenic yeast for a sustainable lignocellulosic biomass-to-ethanol industry.