Cell surface engineering of Saccharomyces cerevisiae combined with membrane separation technology for xylitol production from rice straw hydrolysate

Abstract

Xylitol, a value-added polyol deriving from D-xylose, is widely used in both the food and pharmaceutical industries. Despite extensive studies aiming to streamline the production of xylitol, the manufacturing cost of this product remains high while demand is constantly growing worldwide. Biotechnological production of xylitol from lignocellulosic waste may constitute an advantageous and sustainable option to address this issue. However, to date, there have been few reports of biomass conversion to xylitol. In the present study, xylitol was directly produced from rice straw hydrolysate using a recombinant Saccharomyces cerevisiae YPH499 strain expressing cytosolic xylose reductase (XR), along with β-glucosidase (BGL), xylosidase (XYL), and xylanase (XYN) enzymes (co-)displayed on the cell surface; xylitol production by this strain did not require addition of any commercial enzymes. All of these enzymes contributed to the consolidated bioprocessing (CBP) of the lignocellulosic hydrolysate to xylitol to produce 5.8 g/L xylitol with 79.5 % of theoretical yield from xylose contained in the biomass. Furthermore, nanofiltration of the rice straw hydrolysate provided removal of fermentation inhibitors while simultaneously increasing sugar concentrations, facilitating high concentration xylitol production (37.9 g/L) in the CBP. This study is the first report (to our knowledge) of the combination of cell surface engineering approach and membrane separation technology for xylitol production, which could be extended to further industrial applications.

Combined Cell Surface Display of β‐d‐Glucosidase (BGL), Maltose Transporter (MAL11), and Overexpression of Cytosolic Xylose Reductase (XR) in Saccharomyces cerevisiae Enhance Cellobiose/Xylose Coutilization for Xylitol Bioproduction from Lignocellulosic Biomass

“…The recombinant yeast strain YPH499‐ Ss XR‐ Aa BGL was previously described as capable of achieving CBP of the hemicellulosic material contained in the soluble fraction of rice straw hydrolysates, as well as KP residues, to xylitol . In this work, we used this strain to perform simultaneous cofermentation of mixed sugars (i.e., glucose [60 g L −1 ]/xylose [40 g L −1 ], or cellobiose [60 g L −1 ]/xylose [40 g L −1 ], respectively) to produce xylitol (Figure ).…”

“…Genes encoding Ps HGT2, Ps SUT1, Ps LAC1, Ps LAC2, Ps LAC3, Kl LAC12, Sc HXT7, Sc MAL11, Sc MAL31, and Sc ATO were expressed in the S. cerevisiae YPH499‐XR strain as previously described . Selected yeast transformants were aerobically cultured in 5 mL of synthetic dextrose medium for 24 h and inoculated into 350 mL of “yeast extract, bacto‐peptone, d ‐glucose” medium (YPD) medium for 72 h prior to enzyme activity assays and fermentation experiments …”

“…In order to generate fermentable sugars such as glucose and xylose from lignocellulosic biomass, the addition of costly commercial enzymes is usually needed; however, this enzymatic treatment also generates oligomers of glucose such as cellobiose and xylose, which are not directly utilizable by a micro‐organism such as S. cerevisiae . In the last decade, the cell surface engineering approach has emerged as a promising option to obviate the addition of commercial enzymes . This approach consists of anchoring enzymes (e.g., those capable of degrading biomass) to the yeast cell wall (Figure ).…”

“…Cell surface engineering is a cost‐effective solution to the addition of an exogenous enzyme; moreover, the displayed enzymes can be reused in repeated batch processes . Last but not least, a consolidated bioprocessing (CBP) strategy, which integrates all biological steps such as enzyme production, saccharification, and fermentation into a single step, is also a promising approach to reduce the costs of xylitol production . Recently, the direct production of xylitol from rice straw hydrolysate as well as from woody kraft pulp (KP) by using a recombinant strain of S. cerevisiae (YPH499‐ Ss XR‐ Aa BGL) expressing a cytosolic Scheffersomyces stipitis xylose reductase ( Ss XR), along with Aspergillus aculeatus β‐glucosidase 1 ( Aa BGL), an enzyme displayed on the cell surface, has been reported .…”

A halotolerant aldose reductase from Debaryomyces nepalensis: gene isolation, overexpression and biochemical characterization

“…One of the major problems in this eld is the utilization of both hexoses and pentoses, and various efforts have been made to engineer the existing strains 18,19 and in screening novel organisms capable of utilizing both hexoses and pentoses. 20,21 We previously isolated halotolerant yeast D. nepalensis from rotten apple, which is capable of utilizing both hexoses and pentoses and tolerate both NaCl and KCl at 2 M.…”