Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway

Jia, Zhang; Zhang, Biao; Wang, Dongmei; Gao, Xiaolian; Sun, Lianhong; Hong, Jiong

doi:10.1016/j.ymben.2015.07.008

Cited by 45 publications

(28 citation statements)

References 41 publications

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“…A number of reports support the findings that K. marxianus has limited capacity to uptake C5 sugars in comparison to its ability to metabolize glucose and other C6 sugars . Genetic engineering of xylose metabolism aimed at eliminating a redox imbalance during xylose catabolism have been demonstrated and result in improved xylose uptake and K. marxianus growth .…”

Section: Resultsmentioning

confidence: 86%

“…K. marxianus was selected for screening because a number of different strains have been shown to grow on a variety of different C5 and C6 sugars and overproduction of ethyl acetate has been demonstrated . K. marxianus is also an attractive microbial host for volatile ester synthesis as it is thermotolerant to temperatures upwards of 45°C, genetic engineering tools have been demonstrated for metabolic engineering, and it is a generally regarded as safe (GRAS) organism . To demonstrate the broad utility of the assay we also investigated the extraction and quantification of longer chain esters including ethyl butyrate, isoamyl acetate, ethyl hexanoate and ethyl octanoate.…”

Section: Introductionmentioning

confidence: 99%

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High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources

Löbs

Lin

Cook

et al. 2016

Biotechnology Journal

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Advances in genome and metabolic pathway engineering have enabled large combinatorial libraries of mutant microbial hosts for chemical biosynthesis. Despite these advances, strain development is often limited by the lack of high throughput functional assays for effective library screening. Recent synthetic biology efforts have engineered microbes that synthesize acetyl and acyl esters and many yeasts naturally produce esters to significant titers. Short and medium chain volatile esters have value as fragrance and flavor compounds, while long chain acyl esters are potential replacements for diesel fuel. Here, we developed a biotechnology method for the rapid screening of microbial ester biosynthesis. Using a colorimetric reaction scheme, esters extracted from fermentation broth were quantitatively converted to a ferric hydroxamate complex with strong absorbance at 520 nm. The assay was validated for ethyl acetate, ethyl butyrate, isoamyl acetate, ethyl hexanoate, and ethyl octanoate, and achieved a z-factor of 0.77. Screening of ethyl acetate production from a combinatorial library of four Kluyveromyces marxianus strains on seven carbon sources revealed ethyl acetate biosynthesis from C5, C6, and C12 sugars. This newly adapted method rapidly identified novel properties of K. marxianus metabolism and promises to advance high throughput microbial strain engineering for ester biosynthesis.

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Section: Resultsmentioning

confidence: 86%

Section: Introductionmentioning

confidence: 99%

High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources

Löbs

Lin

Cook

et al. 2016

Biotechnology Journal

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“…Evolutionary adaptation together with genetic engineering can be a better approach to develop the sustainable and economic process (Behera et al, 2016;Kuyper et al, 2005;Liu and Hu, 2010;Zhou et al, 2012;Sharma et al, 2016). This strategy has already been implemented along with various conventional techniques including protein engineering, intergeneric hybridization, and metabolic engineering (Kuhad et al, 2011;Zhang et al, 2015). Furthermore, evolutionary adaptation has been proven a useful approach for the development of sustainable technology (Kuyper et al, 2005;Liu and Hu, 2010;Zhou et al, 2012;Pereira et al, 2015).…”

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confidence: 99%

Evolutionary Adaptation of Kluyveromyces marxianus NIRE-K3 for Enhanced Xylose Utilization

et al. 2017

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The evolutionary adaptation was approached on the thermotolerant yeast Kluyveromyces marxianus NIRE-K3 at 45°C on xylose as a sole source of carbon for enhancement of xylose uptake. After 60 cycles, evolved strain K. marxianus NIRE-K3.1 showed comparatively 3.75-and 3.0-fold higher specific growth and xylose uptake rates, respectively, than that of native strain. Moreover, the short lag phase was also observed on adapted strain. During batch fermentation with xylose concentration of 30 g l −1, K. marxianus NIRE-K3.1 could utilize about 96% of xylose in 72 h and produced 4.67 and 15.7 g l −1 of ethanol and xylitol, respectively, which were 9.72-and 4.63-fold higher than that of native strain. Similarly, specific sugar consumption rate, xylitol, and ethanol yields were 5.07-, 1.15-, and 2.44-fold higher as compared to the native strain, respectively. The results obtained after evolutionary adaptation of K. marxianus NIRE-K3 show the significant improvement in the xylose utilization, ethanol and xylitol yields, and productivities. By understanding the results obtained, the significance of evolutionary adaptation has been rationalized, since the adapted culture could be more stable and could enhance the productivity.

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“…Kluyveromyces marxianus is a 'generally regarded as safe' (GRAS) microorganism that has attracted increasing attention in bioethanol fermentation of lignocellulosic biomass due to its thermo-tolerance, high growth rate, and broad substrate spectrum [6]. K. marxianus strains can grow with a growth rate of 0.86-0.99 h − 1 at 40 °C, and some strains even grow at temperatures as high as 52 °C [7].…”

Section: Introductionmentioning

confidence: 99%

Effect of a novel alpha/beta hydrolase domain protein on tolerance of K. marxianus to lignocellulosic biomass derived inhibitors

Wang

Hong

2020

Preprint

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Background: Lignocellulosic biomass is a promising renewable and sustainable resource for biofuels and chemicals. However, the inhibitors generated during lignocellulosic biomass pretreatment hinder microbial growth and interfere with fermentation. Studies on the tolerance to inhibitors, especially multiple inhibitors, are important in the construction of robust strains that can utilize lignocellulosic biomass. Results: The expression of an uncharted α/β hydrolase (ABHD) from Kluyveromyces marxianus was found to be up-regulated in the presence of single or multiple inhibitors; this enzyme was termed KmYME in this study. Disruption of KmYME made the yeast more susceptible to inhibitors, particularly to acetic acid, furfural and 5-hydroxymethylfurfural. KmYME was located in the mitoplast of mitochondria with targeting signals in the N-terminal 1-40 amino acid residues. Under inhibitor stress, the intracellular concentration of ATP, NAD(P) in the KmYME disrupted strain decreased. In addition, the KmYME disrupted strain lost more mitochondrial membrane potential, showed increased plasma membrane permeability, a more severe redox imbalance, and increased ROS accumulation, compared with those of the non-disrupted strain in the presence of the same inhibitors. The recombinant KmYME possessed esterase/thioesterase activity and preferred short-chain pNP aliphatic esters, long-chain acyl-CoA, and succinyl-CoA as substrates. The retro-complementation results indicated that the enzymatic activity of KmYME was necessary for the resistance to inhibitors. In addition, KmYME was also required for the resistance to other stresses including ethanol, temperature, and osmotic pressure. Disruption of two possible homologous genes in S. cerevisiae also reduced its tolerance to inhibitors. Conclusion: As an uncharted α/β hydrolase, KmYME which had esterase/thioesterase activity and located in mitochondrial matrix was identified for the first time. The esterase/thioesterase activity was necessary for the tolerance to lignocellulosic biomass derived inhibitors in K. marxianus. Disruption of KmYME reduced the tolerance to inhibitors by interfering with ATP, NAD and NADP synthesis. KmYME also correlated with tolerance to other stresses such as osmotic, ethanol, and temperature. This study may provide useful information for better understanding the resistance mechanism of multiple inhibitors, which is important in the creation of a robust strain for industrial fermentation that can use cellulosic biomass as substrate.

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Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway

Cited by 45 publications

References 41 publications

High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources

High throughput, colorimetric screening of microbial ester biosynthesis reveals high ethyl acetate production from Kluyveromyces marxianus on C5, C6, and C12 carbon sources

Evolutionary Adaptation of Kluyveromyces marxianus NIRE-K3 for Enhanced Xylose Utilization

Effect of a novel alpha/beta hydrolase domain protein on tolerance of K. marxianus to lignocellulosic biomass derived inhibitors

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