Improving xylitol production at elevated temperature with engineered Kluyveromyces marxianus through over-expressing transporters

doi:10.1016/j.biortech.2014.10.150

Bioresource Technology

Volume 175, January 2015, Pages 642-645

https://doi.org/10.1016/j.biortech.2014.10.150 Get rights and content

Highlights

•
Glucose/xylose facilitator 1 improved xylitol production in K. marxianus YZJ074.
•
At 42 °C, YZJ074 produced 99.29 g/L xylitol with productivity of 4.14 g/L/h.
•
At 45 °C, YZJ074 produced 101.30 g/L xylitol with productivity 2.81 g/L/h.
•
YZJ074 produced 312.05 g/L xylitol through fed-batch fermentation.
•
Direct non-sterilized substrate feeding in fed-batch fermentation.

Abstract

Three transporter genes including Kluyveromyces marxianus aquaglyceroporin gene (KmFPS1), Candida intermedia glucose/xylose facilitator gene (CiGXF1) or glucose/xylose symporter gene (CiGXS1) were over-expressed in K. marxianus YZJ017 to improve xylitol production at elevated temperatures. The xylitol production of YZJ074 that harbored CiGXF1 was improved to 147.62 g/L in Erlenmeyer flask at 42 °C. In fermenter, 99.29 and 149.60 g/L xylitol were produced from 99.55 and 151.91 g/L xylose with productivity of 4.14 and 3.40 g/L/h respectively at 42 °C. Even at 45 °C, YZJ074 could produce 101.30 g/L xylitol from 101.41 g/L xylose with productivity of 2.81 g/L/h. Using fed-batch fermentation through repeatedly adding non-sterilized substrate directly, YZJ074 could produce 312.05 g/L xylitol which is the highest yield reported to date. The engineered strains YZJ074 which can produce xylitol at elevated temperatures is an excellent foundation for xylitol bioconversion.

Introduction

d-Xylose is one of the primary hydrolysis products of lignocellulosic biomass and is the second most abundant fermentable material possessing the potential of producing value-added products (Hickert et al., 2013, Xiong et al., 2011, Zhang et al., 2010). Xylitol is the reduction product of xylose and a five-carbon sugar alcohol which is as sweet as sucrose but renders less calories than sucrose (Guo et al., 2013, Prakash et al., 2011). Additionally, xylitol does not require insulin for its metabolic regulation and is therefore suitable for diabetic patients (Zhang et al., 2014). Xylitol is also classified as one of the most promising chemicals among the 12 bio-based chemicals by the US Department of Energy and thereby serves as a key economic driver of the biorefinery concept (Zhang et al., 2014). For these reasons, xylitol production has attracted worldwide attention in recent years. Fermentation at elevated temperatures has certain advantages such as reducing cooling costs, increasing fermentation rate, and a reduced risk of contamination (Abdel-Banat et al., 2010). Thermo-tolerant Kluyveromyces marxianus is considered to be a ‘generally regarded as safe’ (GRAS) microorganism and has advantages like short generation time and a high growth rate at elevated temperatures (0.86–0.99 h⁻¹ at 40 °C) (Zhang et al., 2013, Zhang et al., 2011). Therefore, genetically engineered K. marxianus could be a valuable candidate for xylitol production.

In this study, the constructed strains could produce xylitol efficiently under aerobic conditions and the productivity was significantly improved at elevated temperatures. The K. marxianus strain YZJ017 (Zhang et al., 2014) wherein the xylitol dehydrogenase (XDH) gene (KmXYL2) was disrupted and the Neurospora crassa xylose reductase (XR) gene (NcXYL1) was over-expressed, was used as the host for construction of further engineered strains. Three transporter genes which encoding the aquaglyceroporin of K. marxianus (KmFPS1), the glucose/xylose facilitator (CiGXF1) and glucose/xylose symporter (CiGXS1) of Candida intermedia were transformed into YZJ017. Xylitol production as well as the productivity of K. marxianus strains over-expressing above genes was evaluated under various conditions.

Section snippets

Microorganisms and fermentation conditions

K. marxianus YZJ017 was constructed in our laboratory from K. marxianus YLUA005 (Zhang et al., 2014). K. marxianus NBRC1777 and C. intermedia NBRC10601 were obtained from NITE Biological Resource Center (NBRC, Tokyo, Japan). Synthetic dropout (SD) medium (yeast nitrogen base without amino acids 6.7 g/L, glucose 20 g/L) supplemented with appropriate amino acids was used to select the transformants. Yeast extract/peptone dextrose (YPD) medium (yeast extract 10 g/L, bacteriological peptone 20 g/L,

Effect of KmFPS1 over-expression

Although the yield of xylitol producing in YZJ017 at elevated temperature using glycerol as co-substrate was very high in our previous study, only 100.38 g/L xylose could be used (Zhang et al., 2014). Further improvement is necessary for economical industrious applications. Previous study showed that, in Saccharomyces cerevisiae, compared to the parental strain, less xylitol was produced when FPS1p gene was disrupted while xylitol was produced at a faster rate and a higher yield when a FPS1 was

Conclusions

The result that K. marxianus YZJ074 accumulated 1.49-fold more xylitol than its parental strain suggested that the xylose uptake was a limiting step during xylitol production in this yeast. Glycerol is the best co-substrate in this study and the glycerol drastic glut on the market would reduce the industrial xylitol production cost. So far, the highest productivity and production of xylitol obtained by YZJ074 with fermenter showed the high fermentative potential of thermo-tolerant yeast K.

Acknowledgements

This work was supported by a Grant-in-Aid from the National High Technology Research and Development Program (2012AA02A708), the National Natural Science Foundation of China (31070028 and 31270149), and National Basic Research Program of China (2011CBA00801). The authors do not have any possible conflicts of interest.

References (16)

X.X. Guo et al.
A novel pathway construction in Candida tropicalis for direct xylitol conversion from corncob xylan
Bioresour. Technol.
(2013)
L.R. Hickert et al.
Simultaneous saccharification and co-fermentation of un-detoxified rice hull hydrolysate by Saccharomyces cerevisiae ICV D254 and Spathaspora arborariae NRRL Y-48658 for the production of ethanol and xylitol
Bioresour. Technol.
(2013)
S. Karlgren et al.
Conditional osmotic stress in yeast: a system to study transport through aquaglyceroporins and osmostress signaling
J. Biol. Chem.
(2005)
S. Pal et al.
Studies on xylitol production by metabolic pathway engineered Debaryomyces hansenii
Bioresour. Technol.
(2013)
G. Prakash et al.
Microbial production of xylitol from d-xylose and sugarcane bagasse hemicellulose using newly isolated thermotolerant yeast Debaryomyces hansenii
Bioresour. Technol.
(2011)
F. Sun et al.
Organosolv pretreatment by crude glycerol from oleochemicals industry for enzymatic hydrolysis of wheat straw
Bioresour. Technol.
(2008)
M. Xiong et al.
Alteration of xylose reductase coenzyme preference to improve ethanol production by Saccharomyces cerevisiae from high xylose concentrations
Bioresour. Technol.
(2011)
J. Zhang et al.
Xylitol production at high temperature by engineered Kluyveromyces marxianus
Bioresour. Technol.
(2014)

There are more references available in the full text version of this article.

Cited by (51)

Expanding sugar alcohol industry: Microbial production of sugar alcohols and associated chemocatalytic derivatives
2023, Biotechnology Advances
Sugar alcohols are polyols that are widely employed in the production of chemicals, pharmaceuticals, and food products. Chemical synthesis of polyols, however, is complex and necessitates the use of hazardous compounds. Therefore, the use of microbes to produce polyols has been proposed as an alternative to traditional synthesis strategies. Many biotechnological approaches have been described to enhancing sugar alcohols production and microbe-mediated sugar alcohol production has the potential to benefit from the availability of inexpensive substrate inputs. Among of them, microbe-mediated erythritol production has been implemented in an industrial scale, but microbial growth and substrate conversion rates are often limited by harsh environmental conditions. In this review, we focused on xylitol, mannitol, sorbitol, and erythritol, the four representative sugar alcohols. The main metabolic engineering strategies, such as regulation of key genes and cofactor balancing, for improving the production of these sugar alcohols were reviewed. The feasible strategies to enhance the stress tolerance of chassis cells, especially thermotolerance, were also summarized. Different low-cost substrates like glycerol, molasses, cellulose hydrolysate, and CO₂ employed for producing these sugar alcohols were presented. Given the value of polyols as precursor platform chemicals that can be leveraged to produce a diverse array of chemical products, we not only discuss the challenges encountered in the above parts, but also envisioned the development of their derivatives for broadening the application of sugar alcohols.
Improving glycerol utilization during high-temperature xylitol production with Kluyveromyces marxianus using a transient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system
2022, Bioresource Technology
Citation Excerpt :
The growth and xylitol-producing ability were determined. The NADPH in YZB152, YZB230, YZB231, YZB232, YZB240, YZB367, and YZB392 cultured with YPG were also deternimed according to the previously reported method (Zhang et al., 2015). Seed culture (250 mL) that was obtained after culturing the yeast in YPD medium at 37 °C was inoculated into a 5-L modular benchtop fermenter (Baoxing Corp., Shanghai, China) containing 2.5 L of fermentation medium at natural pH. For comparative analysis, analytical grade glycerol (30 g/L) and xylose (100 g/L) were used as carbon sources, while yeast extract (10 g/L) and peptone (20 g/L) were used as nitrogen sources.
Glycerol is an ideal co-substrate for xylitol production with Kluyveromyces marxianus. This study demonstrated that K. marxianus catabolizes glycerol through the Gut1-Gut2 pathway instead of the previously speculated NADPH-dependent Gcy1-Dak1 pathway using the transient clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9) system. Additionally, Utr1p was demonstrated to mediate NADPH generation through NADH phosphorylation. YZB392, which was constructed by integrating Utr1 into the Ypr1 site in the strain overexpressing NcXyl1 and CiGxf1 and harboring disrupted Xyl2, exhibited enhanced glycerol utilization for xylitol production (from 2.50- to 3.30- g/L after consuming 1 g/L glycerol). Fed-batch fermentation at 42 °C with YZB392 yielded 322.07 g/L xylitol, which is the highest known xylitol titer obtained via biological method. Feeding crude glycerol, xylose mother liquor, and corn steep liquor powder into a bioreactor resulted in the production of 235.69 g/L xylitol. This study developed a platform for xylitol production from industrial by-products.
Ergosterol production at elevated temperatures by Upc2-overexpressing Kluyveromyces marxianus using Jerusalem artichoke tubers as feedstock
2022, Bioresource Technology
Ergosterol is an important precursor in the pharmaceutical industry for the production of numerous drugs. In this study, Kluyveromyces marxianus that showed more potential for ergosterol production than some other yeasts was reported. The effects of transcription factors UPC2, MOT3, and ROX1 of K. marxianus on ergosterol synthesis were explored, and a Upc2-overexpressing strain produced 1.78 times more ergosterol (167.33 mg/L) than the wild-type strain (60.04 mg/L). A total of 239.98 mg/L ergosterol was produced when glucose was replaced with fructose to limit ethanol production. Enhanced aeration increased ergosterol titer from 63.09 mg/L to 128.46 mg/L at 42 °C. The ergosterol titer reached 304.37 mg/L in a shake flask at 37 °C, or 1124.38 and 948.32 mg/L at 37 °C and 42 °C, respectively, in a 5 L bioreactor, using Jerusalem artichoke tubers as the sole carbon source. This study establishes a platform for ergosterol biosynthesis using inexpensive materials.
Scaling up xylitol bioproduction: Challenges to achieve a profitable bioprocess
2022, Renewable and Sustainable Energy Reviews
Xylitol is a GRAS (Generally Recognized as Safe) polyol commonly used in the food industry and able to promote several benefits to the health. In addition, it can also be used as a building block molecule for the manufacture of different high-value chemicals. Currently, the commercial production of xylitol occurs by chemical route through the catalytic hydrogenation of xylose from lignocellulosic biomass. Since this is an expensive process due to the severe reactional conditions employed, the biotechnological route for xylitol production, which comprises the biological conversion of xylose into xylitol, emerges as a potential lower-cost alternative to obtain this polyol due to the milder process conditions required. However, the biotechnological route still presents important bottlenecks and challenges that impairs the process scaling up. Modern strategies and technologies that can potentially improve xylitol bioproduction include adaptive evolution of microbial strains to enhance their tolerance to inhibitors and the xylose uptake rate during the fermentation step; development of engineered microorganisms to result in higher xylose-to-xylitol bioconversion yields; as well as xylitol purification techniques to improve the recovery yields. Moreover, techno-economic analysis of the overall production chain is essential to identify the process viability for large-scale implementation as well as the steps requiring improvements. These are some key factors discussed in this review, which aims to provide insights for the development of a more economically competitive, less energy demanding and scalable new technology for xylitol production.
Technology toward biochemicals precursors and bioplastic production
2022, A-Z of Biorefinery: A Comprehensive View
In this chapter, some building-block chemicals that play important roles in an industrial application are summarized. These building blocks are usually referred as bio-based chemicals that are produced by biological routes using substrates as pure sugars or even renewable biomass including cellulosic and lignocellulosic materials. C3 (glycerol and 3-hydroxy propionic acid), C4 (succinic acid, malic acid, itaconic acid, glucaric acid, glutamic acid, and l-aspartic acid), C5 (xylitol and levulinic acid), and C6 (sorbitol) bio-based chemicals have been selected for discussion due to their capability to be utilized in broad applications and to replace existing petroleum-based chemicals in commercialized scales in high volume. Biological techniques including metabolic engineering either by an inactivation or overexpression of interested genes or both, metabolic evolution, and enzymatic catalysis that are related to construct efficient microbial strains and even to optimize parameters affecting suitable fermentation conditions for developed microorganisms are employed for sustainable productions of these bio-based chemicals.
Transporter engineering promotes the co-utilization of glucose and xylose by Candida glycerinogenes for D-xylonate production
2021, Biochemical Engineering Journal
Sugar transporter-mediated sugar transport is the first rate-limiting step in the control of yeast metabolism. Almost all known wild-type sugar transporters do not have the ability to co-transport glucose and xylose, which makes yeast less available to mixed sugar substrates (e.g. lignocellulose hydrolysate) and leads to lower product titers. Here, we achieved co-utilization of glucose and xylose by the tolerant yeast Candida glycerinogenes and facilitated D-xylonate production through rational modification of the hexose transporter (transporter engineering). Specifically, a hexose transporter mutant CgHxt4.2A.S (CgHxt4^{K9A, N351S, K538A}) with high membrane localization stability (MLS) and co-transport of glucose and xylose was obtained by functional characterization, homology modeling, molecular docking and rational mutagenesis. Ultimately, this mutant enhanced the utilization of sugars (especially xylose) by C. glycerinogenes and increased the titer of D-xylonate in sugarcane bagasse hydrolysate by 49.3%. Our study provides a promising strategy for the future efficient utilization of cheaper lignocellulosic hydrolysates by recombinant yeast for the production of high-value chemicals.

View all citing articles on Scopus

¹: These authors contributed equally to this work.

View full text

Article preview

Bioresource Technology

Highlights

Abstract

Introduction

Section snippets

Microorganisms and fermentation conditions

Effect of KmFPS1 over-expression

Conclusions

Acknowledgements

References (16)

A novel pathway construction in Candida tropicalis for direct xylitol conversion from corncob xylan

Bioresour. Technol.

Simultaneous saccharification and co-fermentation of un-detoxified rice hull hydrolysate by Saccharomyces cerevisiae ICV D254 and Spathaspora arborariae NRRL Y-48658 for the production of ethanol and xylitol

Bioresour. Technol.

Conditional osmotic stress in yeast: a system to study transport through aquaglyceroporins and osmostress signaling

J. Biol. Chem.

Studies on xylitol production by metabolic pathway engineered Debaryomyces hansenii

Bioresour. Technol.

Microbial production of xylitol from d-xylose and sugarcane bagasse hemicellulose using newly isolated thermotolerant yeast Debaryomyces hansenii

Bioresour. Technol.

Organosolv pretreatment by crude glycerol from oleochemicals industry for enzymatic hydrolysis of wheat straw

Bioresour. Technol.

Alteration of xylose reductase coenzyme preference to improve ethanol production by Saccharomyces cerevisiae from high xylose concentrations

Bioresour. Technol.

Xylitol production at high temperature by engineered Kluyveromyces marxianus

Bioresour. Technol.

Cited by (51)

Expanding sugar alcohol industry: Microbial production of sugar alcohols and associated chemocatalytic derivatives

Improving glycerol utilization during high-temperature xylitol production with Kluyveromyces marxianus using a transient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system

Ergosterol production at elevated temperatures by Upc2-overexpressing Kluyveromyces marxianus using Jerusalem artichoke tubers as feedstock

Scaling up xylitol bioproduction: Challenges to achieve a profitable bioprocess

Technology toward biochemicals precursors and bioplastic production

Transporter engineering promotes the co-utilization of glucose and xylose by Candida glycerinogenes for D-xylonate production

Short CommunicationImproving xylitol production at elevated temperature with engineered Kluyveromyces marxianus through over-expressing transporters

Highlights

Abstract

Introduction

Section snippets

Microorganisms and fermentation conditions

Effect of KmFPS1 over-expression

Conclusions

Acknowledgements

Bioresour. Technol.

Bioresour. Technol.

J. Biol. Chem.

Bioresour. Technol.

Bioresour. Technol.

Bioresour. Technol.

Bioresour. Technol.

Bioresour. Technol.

Short Communication
Improving xylitol production at elevated temperature with engineered Kluyveromyces marxianus through over-expressing transporters