Engineering redox cofactor regeneration for improved pentose fermentation in Saccharomyces cerevisiae

Pentose fermentation to ethanol with recombinant Saccharomyces cerevisiae is slow and has a low yield. A likely reason for this is that the catabolism of the pentoses D-xylose and L-arabinose through the corresponding fungal pathways creates an imbalance of redox cofactors. The process, although redox neutral, requires NADPH and NAD+, which have to be regenerated in separate processes. NADPH is normally generated through the oxidative part of the pentose phosphate pathway by the action of glucose-6-phosphate dehydrogenase (ZWF1). To facilitate NADPH regeneration, we expressed the recently discovered gene GDP1, which codes for a fungal NADP+-dependent D-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) (EC 1.2.1.13), in an S. cerevisiae strain with the D-xylose pathway. NADPH regeneration through an NADP-GAPDH is not linked to CO2 production. The resulting strain fermented D-xylose to ethanol with a higher rate and yield than the corresponding strain without GDP1; i.e., the levels of the unwanted side products xylitol and CO2 were lowered. The oxidative part of the pentose phosphate pathway is the main natural path for NADPH regeneration. However, use of this pathway causes wasteful CO2 production and creates a redox imbalance on the path of anaerobic pentose fermentation to ethanol because it does not regenerate NAD+. The deletion of the gene ZWF1 (which codes for glucose-6-phosphate dehydrogenase), in combination with overexpression of GDP1 further stimulated D-xylose fermentation with respect to rate and yield. Through genetic engineering of the redox reactions, the yeast strain was converted from a strain that produced mainly xylitol and CO2 from D-xylose to a strain that produced mainly ethanol under anaerobic conditions.     

The role of glutathione reductase in the interplay between oxidative stress response and turnover of cytosolic NADPH in Kluyveromyces lactis

The phosphoglucose isomerase mutant of the respiratory yeast Kluyveromyces lactis (rag2) is forced to metabolize glucose through the oxidative pentose phosphate pathway and shows an increased respiratory chain activity and reactive oxygen species production. We have proved that the K. lactis rag2 mutant is more resistant to oxidative stress (OS) than the wild type, and higher activities of glutathione reductase (GLR) and catalase contribute to this phenotype. Resistance to OS of the rag2 mutant is reduced when the gene encoding GLR is deleted. The reduction is higher when, in addition, catalase activity is inhibited. In K. lactis, catalase activity is induced by peroxide-mediated OS but GLR is not. We have found that the increase of GLR activity is correlated with that of glucose-6-phosphate dehydrogenase (G6PDH) activity that produces NADPH. G6PDH is positively regulated by an active respiratory chain and GLR plays a role in the reoxidation of the NADPH from the pentose phosphate pathway in these conditions. Cytosolic NADPH is also used by mitochondrial external alternative dehydrogenases. Neither GLR overexpression nor induction of the OS response restores growth on glucose of the rag2 mutant when the mitochondrial reoxidation of cytosolic NADPH is blocked.     

酿酒酵母中NAD激酶同功酶对偶数位不饱和脂肪酸β-氧化的影响

ATP-NAD激酶利用ATP,催化NAD磷酸化,生成NADP,而ATP-NADH激酶则催化NAD和NADH发生磷酸化。酿酒酵母细胞内存在三种NAD激酶同功酶Utr1p、Pos5p和Yef1p,它们都是ATP-NADH激酶,对细胞内NADP(H)的供应起到重要作用。酵母偶数位双键不饱和脂肪酸的β-氧化依赖于过氧化物酶体基质内的NADPH。通过构建NAD激酶基因的单、双基因缺失株,并验证它们和对照菌株对不饱和脂肪酸的氧化、利用能力,证实NAD激酶同功酶,尤其是Pos5p,对过氧化物酶体基质内NADP(H)的供应起着重要作用,并推测NADP可以从过氧化物酶体膜外转运至过氧化物酶体基质内。     

Connection between the Rag4 glucose sensor and the KlRgt1 repressor in Kluyveromyces lactis

The RAG4 gene encodes for the sole transmembrane glucose sensor of Kluyveromyces lactis. A rag4 mutation leads to a fermentation-deficient phenotype (Rag- phenotype) and to a severe defect in the expression of the major glucose transporter gene RAG1. A recessive extragenic suppressor of the rag4 mutation has been identified. It encodes a protein (KlRgt1) 31% identical to the Saccharomyces cerevisiae Rgt1 regulator of the HXT genes (ScRgt1). The Klrgt1 null mutant displays abnormally high levels of RAG1 expression in the absence of glucose but still presents an induction of RAG1 expression in the presence of glucose. KlRgt1 is therefore only a repressor of RAG1. As described for ScRgt1, the KlRgt1 repressor function is controlled by phosphorylation in response to high glucose concentration and this phosphorylation is dependent on the sensor Rag4 and the casein kinase Rag8. However, contrary to that observed with ScRgt1, KlRgt1 is always bound to the RAG1 promoter. This article reveals that the key components of the glucose-signaling pathway are conserved between S. cerevisiae and K. lactis, but points out major differences in Rgt1 regulation and function that might reflect different carbon metabolism of these yeasts.     

Fermentation characteristics and protein expression patterns in a recombinant Escherichia coli mutant lacking phosphoglucose isomerase for poly(3-hydroxybutyrate) production

For the efficient production of poly(3-hydroxybutyrate) (PHB) using recombinant Escherichia coli, it is of primal importance to overproduce NADPH, which is necessary for the PHB synthetic pathway. In order to overproduce NADPH in the pentose phosphate (PP) pathway, a recombinant E. coli was constructed in which the phosphoglucose isomerase ( pgi) gene was knocked out to force the carbon flow into the PP pathway. The fermentation characteristics of the recombinant E. coli mutant lacking pgi were then investigated to determine the effect of overproduction of NADPH on efficient PHB production. It was found that, compared with the parent strain ( E. coli JM109), growth of the E. coli mutant lacking pgi ( E. coli DF11) is repressed due to NADPH overproduction in the PP pathway. Furthermore, repressed cell growth can be recovered to some extent by introducing a NADPH-consuming pathway, such as the PHB synthetic pathway. Efficient PHB production using such recombinant E. coli (DF11/pAeKG1) could be attained by appropriately controlling the glucose concentration in the fermentor. Total gene expression was investigated at the protein level by two-dimensional electrophoresis. Out of 22 differentially expressed proteins, 12 were identified with the aid of MALDI-TOF mass spectrometry. Variations in the accumulation of PHB in the recombinant pgi mutant carrying phb (E. coli DF11/pAeKG1) corresponded to the expression of proteins encoded by rpsA, znuA, fabD, potD, fkpA, gapA, ynaF and ibpA. The unfavorable conditions generated by PHB accumulation in the pgi mutant carrying phb resulted in the highest expression of 30S ribosomal protein S1, which ultimately caused a further increase in soluble protein synthesis.     

Glucose utilization of strains lacking PGI1 and expressing a transhydrogenase suggests differences in the pentose phosphate capacity among Saccharomyces cerevisiae strains

Saccharomyces cerevisiae strains lacking phosphoglucose isomerase (pgi1) cannot use the pentose phosphate (PP) pathway to oxidize glucose, which has been explained by the lack of mechanism for reoxidation of the NADPH surplus. Consistent with this, the defective growth on glucose of a ENYpgi1 strain can be partially restored by expressing the Escherichia coli transhydrogenase udhA. In this work it was found that growth of V5 (wine yeast-derived) and FY1679 (isogenic to S288C) pgi1 mutants is not rescued by expression of udhA. Moreover, the flux through the PP pathway of 11 S. cerevisiae strains from various origins was estimated, by calculating the ratio between the enzymatic activity of the G6PDH and HXK, placed at the glycolysis-PP pathway branch point. The results show that ENY.WA-1A exhibited the highest ratio (1.5-3-fold) and the highest G6PDH activity. Overexpression of ZWF1 encoding the G6PDH in V5pgi1udhA did not rescue growth on glucose, suggesting that steps downstream the G6PDH might limit the PP pathway in this strain. As a whole, these data highlight a great intraspecies diversity in the PP pathway capacity among S. cerevisiae strains and suggest that a low capacity may be the prime limiting factor in glucose oxidation through this pathway.     

An approach to the hypoxic and oxidative stress responses in Kluyveromyces lactis by analysis of mRNA levels

Genome duplication, after the divergence of Saccharomyces cerevisiae from Kluyveromyces lactis along evolution, has been proposed as a mechanism of yeast evolution from strict aerobics, such as Candida albicans, to facultatives/fermentatives, such as S. cerevisiae. This feature, together with the preponderance of respiration and the use of the pentose phosphate pathway in glucose utilization, makes K. lactis a model yeast for studies related to carbon and oxygen metabolism. In this work, and based on the knowledge of the sequence of the genome of K. lactis, obtained by the Génolevures project, we have constructed DNA arrays from K. lactis including a limited amount of selected probes. They are related to the aerobiosis-hypoxia adaptation and to the oxidative stress response, and have been used to test changes in mRNA levels in response to hypoxia and oxidative stress generated by H(2)O(2). The study was carried out in both wild-type and rag2 mutant K. lactis strains in which glycolysis is blocked at the phosphoglucose isomerase step. This approach is the first analysis carried out in K. lactis for the majority of the genes selected.     

Deletion of the glucose-6-phosphate dehydrogenase gene KlZWF1 affects both fermentative and respiratory metabolism in Kluyveromyces lactis

In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Delta strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield.     

Synthetic lethal and biochemical analyses of NAD and NADH kinases in Saccharomyces cerevisiae establish separation of cellular functions

Production of NADP and NADPH depends on activity of NAD and NADH kinases. Here we characterized all combinations of mutants in yeast NAD and NADH kinases to determine their physiological roles. We constructed a diploid strain heterozygous for disruption of POS5, encoding mitochondrial NADH kinase, UTR1, cytosolic NAD kinase, and YEF1, a UTR1-homologous gene we characterized as encoding a low specific activity cytosolic NAD kinase. pos5 utr1 is a synthetic lethal combination rescued by plasmid-borne copies of the POS5 or UTR1 genes or by YEF1 driven by the ADH1 promoter. Respiratory-deficient and oxidative damage-sensitive defects in pos5 mutants were not made more deleterious by yef1 deletion, and a quantitative growth phenotype of pos5 and its arginine auxotrophy were repaired by plasmid-borne POS5 but not UTR1 or ADH1-driven YEF1. utr1 haploids have a slow growth phenotype on glucose not exacerbated by yef1 deletion but reversed by either plasmid-borne UTR1 or ADH1-driven YEF1. The defect in fermentative growth of utr1 mutants renders POS5 but not POS5-dependent mitochondrial genome maintenance essential because rho-utr1 derivatives are viable. Purified Yef1 has similar nucleoside triphosphate specificity but substantially lower specific activity and less discrimination in favor of NAD versus NADH phosphorylation than Utr1. Low expression and low intrinsic NAD kinase activity of Yef1 and the lack of phenotype associated with yef1 suggest that Utr1 and Pos5 are responsible for essentially all NAD/NADH kinase activity in vivo. The data are compatible with a model in which there is no exchange of NADP, NADPH, or cytoplasmic NAD/NADH kinase between nucleocytoplasmic and mitochondrial compartments, but the cytoplasm is exposed to mitochondrial NAD/NADH kinase during the transit of the molecule.     

Metabolic flux screening of Saccharomyces cerevisiae single knockout strains on glucose and galactose supports elucidation of gene function

New methods for an extended physiological characterization of yeast at a microtiter plate scale were applied to 27 deletion mutants of Saccharomyces cerevisiae cultivated on glucose and galactose as sole carbon sources. In this way, specific growth rates, specific rates of glucose consumption and ethanol production were determined. Flux distribution, particularly concerning branching into the pentose phosphate pathway was determined using a new (13)C-labelling method using MALDI-ToF-mass spectrometry. On glucose, the growth was predominantly fermentative whereas on galactose respiration was more active with correspondingly lower ethanol production. Some deletion strains showed unexpected behavior providing very informative data about the function of the corresponding gene. Deletion of malic enzyme gene, MAE1, did not show any significant phenotype when grown on glucose but a drastically increased branching from glucose 6-phosphate into the pentose phosphate pathway when grown on galactose. This allows the conclusion that MAE1 is important for the supply of NADPH during aerobic growth on galactose.     

Transformation of Kluyveromyces fragilis

For the transformation of the yeast species Kluyveromyces fragilis, we have constructed a vector containing a bacterial kanamycin resistance (Kmr) gene, the TRP1 gene of Saccharomyces cerevisiae, and an autonomously replicating sequence of Kluyveromyces lactis called KARS2 . By utilizing the method based on treatment by alkali cations and with the Kmr gene as the selective marker, a wild-type strain of K. fragilis was transformed to resistance against the antibiotic G418 . In the transformed cell the plasmid replicates autonomously. The same plasmid could also be used to transform S. cerevisiae trp1 mutant to Trp+. Thus, KARS2 of K. lactis enables the vector to replicate in K. fragilis, K. lactis, and S. cerevisiae, whereas ARS1 of S. cerevisiae allows autonomous replication only in S. cerevisiae.     

Reoxidation of cytosolic NADPH in Kluyveromyces lactis

Saccharomyces cerevisiae and Kluyveromyces lactis are considered to be the prototypes of two distinct metabolic models of facultatively-aerobic yeasts: Crabtree-positive/fermentative and Crabtree-negative/respiratory, respectively. Our group had previously proposed that one of the molecular keys supporting this difference lies in the mechanisms involved in the reoxidation of the NADPH produced as a consequence of the activity of the pentose phosphate pathway. It has been demonstrated that a significant part of this reoxidation is carried out in K. lactis by mitochondrial external alternative dehydrogenases which use NADPH, the enzymes of S. cerevisiae being NADH-specific. Moreover, the NADPH-dependent pathways of response to oxidative stress appear as a feasible alternative that might co-exist with direct mitochondrial reoxidation.