High-temperature ethanol production by a series of recombinant xylose-fermenting Kluyveromyces marxianus strains

Highlights

• Alteration of coenzyme specificity from NAD⁺ to NADP⁺ of KmXDH was achieved.

• We examined high-temperature characteristics of four engineered K. marxianus strains.

• Activities of enzymes expressed in DMB5 and DMB13 strains did not decrease at 40 °C.

• The most efficient glucose/xylose co-fermentation at 40 °C was observed in DMB13.

• DMB13 showed a fast conversion of xylose to ethanol with a high ethanol yield at 40 °C.

Abstract

Thermotolerant yeast Kluyveromyces marxianus can assimilate xylose but cannot produce ethanol from xylose under anaerobic conditions. Here, we constructed two recombinant K. marxianus strains, DMB5 and DMB13, that express xylose reductase (XR), NAD⁺- or protein-engineered NADP⁺-dependent xylitol dehydrogenase (XDH), and xylulokinase (XK) from K. marxianus. These strains, together with previously reported strain DMB3-7, which expresses Scheffersomyces stipitis XR and NAD⁺-dependent XDH and Saccharomyces cerevisiae XK, were compared to evaluate enzymatic activities and ethanol productivities at 30 °C and 40 °C. Unlike the activities of xylose metabolic enzymes in DMB3-7, enzymatic activities of XR, XDH, and XK in both DMB5 and DMB13 hardly decreased even at 40 °C, suggesting that these enzymes from K. marxianus are highly thermostable. The most efficient glucose/xylose co-fermentation at 40 °C was found in DMB13; namely, DMB13 rapidly converted xylose to ethanol, especially after glucose depletion, and showed the highest ethanol yield (0.402 g/g). These findings support the view that alteration of coenzyme specificity of XDH expressed in K. marxianus will be efficacious for high-temperature ethanol production from mixed sugars containing xylose.

Introduction

Efficient microbial conversion of xylose and glucose to ethanol is essential for economical production of ethanol from lignocellulosic biomass [1]. In xylose-fermenting yeasts, including Scheffersomyces (Pichia) stipitis, xylose is reduced to xylitol by xylose reductase (XR) and then xylitol is oxidized to xylulose by xylitol dehydrogenase (XDH) [[2], [3], [4], [5]]. Conversely, bacteria or anaerobic fungi isomerize xylose directly to xylulose with xylose isomerase (XI) [[6], [7], [8]]. Xylulose is phosphorylated to xylulose 5-phosphate by xylulokinase (XK) and then enters through the non-oxidative pentose phosphate pathway and glycolysis. Saccharomyces cerevisiae is one of the most successful and widely used microorganisms for ethanol production. S. cerevisiae can produce high ethanol concentrations from glucose but cannot utilize xylose for growth and ethanol fermentation due to its lack of an active catabolic pathway for xylose. Therefore, a number of researchers have introduced genes encoding XR-XDH or XI from other organisms in combination with endogenous XK encoding gene into S. cerevisiae to generate xylose-utilizing S. cerevisiae strains (reviewed in [9,10]). Nevertheless, the xylose-fermenting ability of recombinant S. cerevisiae is still low compared to its glucose-fermenting ability.

Recombinant S. cerevisiae that expresses the XR-XDH pathway has been reported to exhibit a higher xylose consumption rate and ethanol productivity than recombinant S. cerevisiae that expresses the XI pathway [11,12]. However, the XR-XDH pathway generates an imbalance of redox cofactors between XR and XDH [13]. Generally, XR prefers NADPH over NADH, whereas XDH uses NAD⁺ as a coenzyme [14,15]. Consequently, recombinant S. cerevisiae strains expressing XR and XDH accumulate a considerable amount of xylitol and produce little ethanol during xylose fermentation [13]. To improve the cofactor balance and reduce xylitol production, protein engineering strategies by site-directed mutagenesis have been applied to alter coenzyme specificity for NADPH-preferred XR and/or NAD⁺-dependent XDH (reviewed in [9,10]). Several XR mutants that prefer NADH rather than NADPH have been created in S. stipitis [16,17], Candida tenuis [18], Hansenula polymorpha [19], and Candida utilis [20]. XDH mutants that use NADP⁺ have also been generated in S. stipitis [21] and Galactocandida mastotermitis [18]. S. cerevisiae strains harboring these mutated enzymes (NADH-preferring XR and/or NADP⁺-dependent XDH) exhibit higher ethanol production and lower xylitol yield from xylose compared with a reference strain harboring native enzymes. In the case of NADP⁺-dependent XDH, for instance, a quadruple ARSdR mutant (D207A/I208R/F209S/N211R) with complete reversal of coenzyme specificity toward NADP⁺ was generated using XDH derived from S. stipitis [21]. Moreover, expression of the ARSdR mutant along with the wild-type NADPH-preferred XR improved ethanol yield and decreased xylitol yield [[22], [23], [24]].

In addition to the importance of xylose fermentability, producing ethanol at high temperature has many advantages such as reduced cooling costs and bacterial contamination [25]. It has been calculated that a 5 °C increase in fermentation temperature can greatly reduce the cost of fuel ethanol production from starchy materials with a hyperthermostable α-amylase by reducing cooling energy and would be approximately $30,000 USD/year for a 30,000-kL scale ethanol plant [25]. If the fermentation temperature increases from 30 °C to 40 °C, the cooling cost could be reduced by up to 65%. Accordingly, high-temperature fermentation (HTF) is likely to be much more cost-effective and practical for ethanol production from lignocellulosic biomass, and thermotolerant microbial strains capable of producing a substantial amount of ethanol at high temperatures are in great demand for HTF. Thermotolerant yeast Kluyveromyces marxianus is a particularly promising candidate because it utilizes xylose as a carbon source [26] and can grow at temperatures as high as 45–52 °C [27], at which its fermentation efficiency is similar to that of S. cerevisiae at 30 °C [28]. A novel thermotolerant yeast strain, K. marxianus DMB1, was isolated from sugarcane bagasse hydrolysates and classified into K. marxianus NBRC1777 clade by D1/D2 and Internal Transcribed Spacer (ITS) sequence analysis [29]. As with other K. marxianus strains, K. marxianus DMB1 utilizes xylose as a sole carbon source but cannot produce ethanol from xylose under anaerobic conditions. We previously constructed a xylose-fermenting recombinant strain of K. marxianus, DMB3-7, that expresses XR and XDH genes from S. stipitis (referred to as SsXR and SsXDH, respectively) and XK gene from S. cerevisiae (ScXK) in DMB1 [30]. At 30 °C, both xylose consumption and ethanol production of DMB3-7 were remarkably increased compared with those of wild-type DMB1 under anaerobic conditions. However, DMB3-7 also produced significant amounts of xylitol as a byproduct, with a yield of 0.44 g of xylitol/g of consumed xylose. Additionally, ethanol production of DMB3-7 was remarkably decreased at 42 °C and 45 °C compared with that at 30 °C due to decreased activities of the three enzymes (SsXR, SsXDH, and ScXK) associated with xylose metabolism [30]. K. marxianus also has three xylose-metabolizing enzymes, XR, XDH, and XK (referred to as KmXR, KmXDH, and KmXK, respectively) [[31], [32], [33]]. KmXR has sole coenzyme specificity, which is only activated with the NADPH cofactor [31]; however, KmXDH has a greater affinity for NAD⁺ than NADP⁺ [32], which is identical to SsXDH [4]. Hence, similar to SsXR and SsXDH, different coenzyme specificities between KmXR and KmXDH lead to an intracellular redox imbalance. Hong and coworkers previously reported that K. marxianus expressing NADH-preferring KmXR mutant or K. marxianus expressing NADP⁺-dependent SsXDH mutant showed increased xylose consumption and ethanol productivity [34,35]. It should also be noted that the amino acid sequence of the NAD⁺ binding domain and the putative coenzyme binding domain between SsXDH (363 amino acids) and KmXDH (354 amino acids) were found to be highly conserved [32]. Nevertheless, the effect of KmXDH (NADP⁺-dependent KmXDH mutant) modifications on the xylose fermentation ability of K. marxianus has not yet been reported.

Here, we constructed two recombinant K. marxianus strains (DMB5 and DMB13) with xylose-fermenting ability. DMB5 was engineered by chromosomal integration to overexpress genes encoding KmXR, KmXDH, and KmXK. DMB13 was generated by multiple site-directed mutagenesis to overexpress the gene encoding NADP⁺-dependent KmXDH mutant, along with NADPH-dependent KmXR and KmXK genes. These recombinant K. marxianus strains, together with previously generated recombinant strain DMB3-7, were characterized with respect to their enzymatic activity and ability to ferment glucose and xylose mixtures into ethanol at high temperature.