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Improved Utility of Pentoses from Lignocellulolytic Hydrolysate: Challenges and Perspectives for Enabling Saccharomyces cerevisiae Ronivaldo Rodrigues da Silva,*,† Catarina Prista,‡ Maria Conceiçaõ Loureiro Dias,‡ Mauricio Boscolo,† Roberto da Silva,† and Eleni Gomes†
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Instituto de Biociências, Letras e Ciências Exatas (IBILCE), Universidade Estadual Paulista (UNESP), São José do Rio Preto, São Paulo 15054-000, Brazil ‡ Linking Landscape, Environment, Agriculture and Food (LEAF), Instituto Superior de Agronomia, Universidade de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal The limitation of wild-type S. cerevisiae on overexpression of enzymes for pentose metabolism, such as D-xylose reductase (XR), xylitol dehydrogenase (XDH), xylose isomerase (XI), Larabinitol 4-dehydrogenase (LAD), and L-xylulose reductase (LXR), and specific pentose transporters has motivated investigations on non-Saccharomyces yeasts to find more efficient transporters and enzymes for the metabolism of pentoses.2 S. cerevisiae has efficient transporters and enzymes for the use of D-glucose. The challenge, therefore, is to enable this yeast to be competent in co-metabolizing hexoses and pentoses. In this sense, studies are focused on identifying transporters that transport L-arabinose and D-xylose with higher efficiency than D-glucose. D-Xylose transporters have been identified, some of which are Gxf1 and Gxs1 from Candida intermedia, Mgt05196p from Meyerozyma guilliermondii, Xyp29, Rgt2, and Xut3 from Scheffersomyces stipitis (basionym Pichia stipitis), XylE from Escherichia coli, and An25 from Neurospora crassa.2,3 However, INTRODUCTION a principal limitation of D-xylose transport from lignocellulosic hydrolysate is that most of these transporters are inhibited Recent bioenergy studies are focused on finding the use of competitively by D-glucose. L-Arabinose transport has also lignocellulolytic enzymes and yeasts for fermenting released been reported, e.g., in Kluyveromyces marxianus (KmAxt1p) sugars, especially D-glucose, D-xylose, and L-arabinose from and M. guilliermondii (PgAxt1p). However, these transporters plant biomass. is inhibited by D-glucose, D-galactose and D-xylose.4 This In these studies, Saccharomyces cerevisiae has been used as a highlights the importance of finding specific transporters that model organism for the production of second-generation (2G) are not inhibited by D-glucose. ethanol and other chemicals because it shows high tolerance to Apart from pentose transporters, metabolic engineering of ethanol and phenolic compounds and also shows stability at a yeast to enable them to produce byproducts of pentoses has 1 varying pH range. Moreover, its whole genome is completely been successful in recent years. The search for enzymes from sequenced and widely known. All of these characteristics make non-Saccharomyces species, bacteria, and filamentous fungi and it an ideal organism for protein engineering studies. However, the consequent heterologous expression of these enzymes have wild-type S. cerevisiae strains are deficient in metabolizing contributed enormously to this progress. Most investigations pentoses.2 This deficiency prompted investigations to enable to express XR-XDH in S. cerevisiae have come from the genes this yeast to metabolize D-xylose and L-arabinose. XYL1 and XYL2 (which encode for XR and XDH, A careful literature search on this subject shows a large respectively) from D-xylose fermenting S. stipitis.5 However, number of investigations related to the use of pentoses by despite XR expression, S. stipitis XR prefer NADPH, although yeast. In the current research, although much has been it is able to use NADH. Thus, redox imbalance, because XR achieved, there are still obstacles in industrial application of preferably uses NADPH as a coenzyme, while XDH is NAD+pentose-using yeasts. dependent, has limited the greater conversion of pentose to
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ethanol or carboxylic acid. A recent report showed that XR from Spathaspora passalidarum prefers NADH over NADPH,6 thus overcoming the preference for NADPH. This offers an opportunity to use this enzyme for pentose fermenting.
BRIEF CONTEXTUALIZATION: D-XYLOSE AND L-ARABINOSE FERMENTING In plant biomass, D-xylose and L-arabinose are the second and third most abundant monosaccharides.3 Thus, the lignocellulosic hydrolysate constitutes a rich mixture, which mainly includes the sugars: D-glucose, D-xylose, and L-arabinose. © XXXX American Chemical Society
Received: May 5, 2019
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DOI: 10.1021/acs.jafc.9b02809 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Scheffersomyces stipitis Aspergillus aculeatus Candida intermedia identification of the G-G/F-XXX-G motif in Candida intermedia S. cerevisiae EBY.VW4000 and AFY10X strains Kluyveromyces marxianus CBS1089 M. guilliermondii NRRL Y-2075 ( P. guilliermondii) S. cerevisiae CEN.PK113-7D strain Piromyces sp.
wild organism
B
overexpression of yeast xylulokinase Piromyces sp. xylose isomerase
mutations in D-xylose and D-glucose pathway
different S. cerevisiae strains were constructed from D-xylose-fermenting S. cerevisiae CEN.PK113-7D strain D-xylose
D-xylose
L-arabinose
S. cerevisiae strains
L-arabinose/D-xylose
transporter (KmAxt1p and PgAxt1p)
D-xylose
S. cerevisiae EBY.VW4000 and AFY10X strains
hexose transporters (Hxt7 and Gal2)
D-xylose
pentose
D-xylose
S. cerevisiae KF-7 strain was used to construct S. cerevisiae NAPX37 strain
expression system
rewiring the sugar preference of hexose transporters from S. cerevisiae EX.12 and S. stipitis RGT2
GXS1 (D-glucose/xylose symporter 1) D-glucose-xylose symporter
reductase and xylitol dehydrogenase β-glucosidase
D-xylose
transporter/enzyme
ethanol
not evaluated
not evaluated
not evaluated
ethanol
fermentative product
transport insensitive to D-glucose
ethanol production from D-xylose and D-glucose, co-consuming
these transporters can be engineered to reduce the inhibitory effect caused by other sugars
D-xylose
transport insensitive to D-glucose; the findings in this work open opportunities to modify the preference of the sugar transporter, making it possible to rewire the preference of a hexose transporter to D-xylose
D-xylose
tolerance to inhibitors derived from lignocellulosic treatment and D-xylose fermentation
advantages
Table 1. Summary of Some Recent Studies on Improvement of Pentose Metabolism: Advantages and Disadvantages disadvantages
promising results, especially for S. cerevisiae IMX1583 strain; further studies are required for application in the industrial strain and improvement on the ethanol yield
low/moderate transport velocity for D-xylose; however, this research contributes to the mechanistic understanding of pentose transport L-arabinose transport has been inhibited by D-glucose, D-galactose, and D-xylose
low/moderate transport velocity for D-xylose; however, this research contributed to the mechanistic understanding of pentose transport
transport is susceptible to high D-glucose concentrations (above 50 g/L); xylitol was obtained as a byproduct
D-xylose
5
4
3
2
1
reference
Journal of Agricultural and Food Chemistry Viewpoint
DOI: 10.1021/acs.jafc.9b02809 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry
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Some examples of investigations indicating the advantages and disadvantages about engineered strains are shown in Table 1.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: +55-17-3221-2200. E-mail:
[email protected]. br.
PERSISTENT CHALLENGES: WHAT ARE THE ADVANCES AND WHAT CAN BE IMPROVED?
ORCID
Ronivaldo Rodrigues da Silva: 0000-0002-6504-8406 Funding
Despite numerous attempts, specific uptake of these sugars has not yet been efficiently achieved. Apart from advances in C5 sugar metabolism by engineered S. cerevisiae, the co-utilization of hexoses and pentoses still remains a challenge to overcome. An enhanced rate of pentose uptake via specific transporters is necessary. In many cases, specific transporters for D-xylose and Larabinose found in non-Saccharomyces yeasts still have low affinity for pentoses2 and/or are sensitive to D-glucose inhibition, which delays assimilation and is disadvantageous for the yield of the fermentative product. Other conventional hexose transporters (Hxt2p, Hxt7p, and Gal2p) in S. cerevisiae (Table 1), although modified for better uptake of pentoses, exhibit a low/moderate rate of transport for C5 sugar3 or remain sensitive to inhibition by hexoses and whose cultivation with mixed sugar may define a diauxic growth pattern. Further advances, through directed protein evolution, have made it possible to rewire the affinity for D-xylose in some transporters, such as C. intermedia Gxs1 and S. stipitis Xut3.2 Recently, engineered transporters with reduced D-glucose inhibition have also been reported.2,3 In fact, in the past few decades, many advances have been made from the early idealizations for the use of plant biomass in the production of chemicals to the present day, where some recent research into metabolic engineering opens an avenue for enabling S. cerevisiae and other yeasts for the better use of plant biomass. However, the use of native D-xylose-fermenting yeasts, including K. marxianus, S. stipitis, and C. intermedia, have been limited as a result of the lack of availability of genomic and proteomic information. Additional information on these non-Saccharomyces yeasts may offer alternatives in this field of study. At present, it is relevant to affirm that progress in Larabinose/D-xylose metabolism is achieved with enzyme engineering of XR, XDH, XI, LAD, and LXR, and the mitigation of catabolic limitation in redox imbalance for XR− XDH activity. Other advances in relation to transport of these sugars into the cell are made. However, expression of efficient specific pentose transporters in industrial strains has not advanced as rapidly as the intracellular expression of enzymes. In fact, the extensive research performed on S. cerevisiae hxtnull strains for the expression of specific, D -glucoseuninhibitable, and high-affinity L-arabinose/D-xylose transporters into a commercial strain is still a recurring challenge to enable efficient co-utilization of C5 and C6 sugars. This hurdle is yet to be overcome. Despite these persistent challenges, there has been an incessant search for cost-effective solutions to improve the utilization of these sugars. In this sense, directed evolution has shown good results.2,3 It can be hoped that supported by scientific advancement and extensive information on hand, these challenges can be overcome in the future. For this reason, it is very important that this investigation momentum is not reduced.
The authors acknowledge the financial support provided by Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Processes 2017/06399-3, 2018/09238-3, and 2017/ 06066-4). This work was also supported by FCT through the research unit UID/AGR/04129/2013 (LEAF). Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Li, Y.-C.; Mitsumasu, K.; Gou, Z.-X.; Gou, M.; Tang, Y.-Q.; Li, G.-Y.; Wu, X.-L.; Akamatsu, T.; Taguchi, H.; Kida, K. Xylose fermentation efficiency and inhibitor tolerance of the recombinant industrial Saccharomyces cerevisiae strain NAPX37. Appl. Microbiol. Biotechnol. 2016, 100, 1531−1542. (2) Young, E. M.; Tong, A.; Bui, H.; Spofford, C.; Alper, H. S. Rewiring yeast sugar transporter preference through modifying a conserved protein motif. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 131−136. (3) Farwick, A.; Bruder, S.; Schadeweg, V.; Oreb, M.; Boles, E. Engineering of yeast hexose transporters to transport D-xylose without inhibition by D-glucose. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 5159−5164. (4) Knoshaug, E. P.; Vidgren, V.; Magalhães, F.; Jarvis, E. E.; Franden, M. A.; Zhang, M.; Singh, A. Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on L-arabinose and D-xylose. Yeast 2015, 32, 615−628. (5) Papapetridis, I.; Verhoeven, M. D.; Wiersma, S. J.; Goudriaan, M.; van Maris, A. J. A.; Pronk, J. T. Laboratory evolution for forced glucose−xylose co-consumption enables identification of mutations that improve mixed-sugar fermentation by xylose-fermenting Saccharomyces cerevisiae. FEMS Yeast Res. 2018, 18, foy056. (6) Cadete, R. M.; de las Heras, A. M.; Sandström, A. G.; Ferreira, C.; Gírio, F.; Gorwa-Grauslund, M.-F.; Rosa, C. A.; Fonseca, C. Exploring xylose metabolism in Spathaspora species: XYL1.2 from Spathaspora passalidarum as the key for efficient anaerobic xylose fermentation in metabolic engineered Saccharomyces cerevisiae. Biotechnol. Biofuels 2016, 9, 167.
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DOI: 10.1021/acs.jafc.9b02809 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
