| The development and utilization of renewable energy has become an important measures taken by countries in the world to ensure energy security, strengthen environmental protection and to deal with climate change. Renewable energy is an important part of energy system and has several fine characteristics, such as wider distribution, great exploitation potentiality, low impact on the environment and sustainable utilization. It is conducive to the harmonious development between human and nature. With the development of society and economy and the continued growth of energy demand in the current situation, developing and utilizing renewable energy is currently the most practicable measure to combat climate change and to achieve the sustainable energy development.Biofuel ethanol is recognized as one of the very promising renewable biofuels for its excellent properties for vehicle applications. Many countries and regions,including the United States, Brazil and the European Union, are actively developing biological fuel ethanol. The biological fuel ethanol has been listed as the key field of development and support in China 'The medium and and long term Renewable Energy Development Plan' and 'Renewable energy development "12th Five Year Plan"'. The first- or 1.5-generation of fuel ethanol, which were produced from gain raw material or starch-containing non-grain feedstock include corn,wheat,or cassava and sweet potato, could not be produced in large quantity for the fewer cultivated area in China. Thus, the second-generation fuel ethanol, which was produced from abundant and cheap renewable lignocellulose biomass, is considered to be the inevitable choice for sustainable development of fuel ethanol.Generally, the production of second-generation fuel ethanol from lignocellulose biomass includes the raw material pretreatment, cellulose hydrolysis and the sugar fermentation by microorganisms. All of the above steps face many difficulties,and thus lead to the increased cost of ethanol production. This situation impedes the large-scale development and utilization of second-generation fuel ethanol and hinders its competition with traditional fossil energy. The fermentation of all the sugars in lignocellulose biomass (the majority of which are glucose and xylose) is one of the key points to improve the raw material utilization and to reduce the ethanol production cost.The yeast Saccharomyces cerevisiae has traditionally been used in industrial bioethanol production owing to its inherent numerous advantages,such as higher fermentation rate of hexose sugars, higher tolerance to ethanol, inhibitors, acidity and other conditions involved in industrial process. Thus it is also the preferred microorganism for second-generation fuel ethanol production. However, this natural ethanol producer faces at least two new rigorous challenges when the fermentation substrate is lignocellulosic hydrolysates instead of those from starch-based materials.First, natural S. cerevisiae cannot metabolize pentose sugars, particularly D-xylose,the second most abundant sugar in lignocellulosic materials, for the lack of effective initial metabolic pathway. The efficient fermentation of xylose can reduce the production cost of second-generation fuel ethanol and thus to improve its economic competitiveness. Second, the individual and synergetic negative interactions derived from the numerous inhibitory compounds, like weak acids, furan compounds and phenolic compounds, which are formed during the pretreatment and hydrolysis processes with the release of sugars,give serious negative effects on the growth and fermentation performance of S. cerevisiae, prolong fermentation period and reduce the production rate of ethanol. Although detoxification of material could be taken by biological, physical or chemical process, this will add complexity and production cost of ethanol and also result in the loss of sugar. The key point to solve this problem is to enhance the tolerance of S. cerevisiae to against the inhibitors in ethanol fermentation process. Taken together, the precondition for economic and efficient fuel ethanol production is the possession of a S. cerevisiae strain with better inhibitor tolerance and can efficiently co-ferment glucose and xylose.For this purpose, work of research has been launched on metabolic engineeringof S. cerevisiae strain for the industrialized production of ethanol from lignocellulosic materials. The main contents in this work include:1. Comprehensive evaluation and selection of S. cerevisiae strain as the chassis cell for second-generation bioethanol production.It is more convenient to choose a strain with naturally suitable properties as a chassis than to confer these properties on the strain through genetic manipulation. In this work, multi-objective synthetic evaluation were performed on 32 wild-type S.cerevisiae strains preserved in our laboratory, include: ploidy confirmation,fermentation performance on glucose, tolerance to individual stress factors (high temperature,hyper-osmotic stress,oxidative stress,and typical inhibitory compounds such as furfural, acetic acid, vanillin and ethanol), tolerance to pretreated corn stover hydrolysate and evaluation of inherent capacity for xylose utilization by integration of initial xylose metabolism pathway. The strain BSIF, isolated from tropical fruit in Thailand, was selected out of the distinctly different strains for its promising characteristics. The maximal specific growth rate of BSIF was as high as 0.65 h-1 in yeast extract peptone dextrose medium, and the ethanol yield was 0.45 g g-1 consumed glucose. Furthermore,compared with other strains,this strain exhibited superior tolerance to high temperature, hyperosmotic stress and oxidative stress;better growth performance in pretreated corn stover hydrolysate; and better xylose utilization capacity when an initial xylose metabolic pathway was introduced. All of these results indicate BSIF is an excellent chassis strain for lignocellulosic ethanol production.2. Rational metabolic engineering endows the xylose-fermentation capacity to wild-type S. cerevisiae strains.Natural wild-type S. cerevisiae strains cannot metabolize xylose for the lack of effective initial xylose metabolic pathway, but can utilize its isomer xylulose. Thus,the xylose metabolic pathway was introduced into the selected wild-type diploid S.cerevisiae strain BSIF through chromosomal integration. The XI (xylose isomerase)pathway was usually considered to be most promising in conversion of xylose to xylulose than the XR-XDH pathway for the absence of coenzyme imbalance. Three series-wound expression cassette of Ru-xylA (GenBank JF496707), which was recently isolated from the metagenomic library of bovine rumen contents and has high homology to the xylAs of Prevotella, was integrated into both alleles of PHO13 locus to reconstruct the initial xylose metabolic pathway and decrease the ATP wasting by the function of Pho13p, and the result strain's genotype is'pho13::XI'. For further improvement of the downstream metabolism of xylose, the enzymes involved in non-oxidative part of pentose phosphate pathway (PPP) was overexpressed at both alleles of GRE3 locus along with the inactivation of GRE3 which results in the yielddecrement of byproduct xylitol, the result strain's genotype is 'phol3::XI, gre3::PPP'.Xylulokinase is the key enzyme for the conversion of xylulose to xylulose 5-phosphate and is the rate limiting step in fermentation of xylulose. The native xylulokinase gene XKS1 was overexpressed moderately by replacing its native promoter with strong constitutive promoter TEF1p, and the result strain's genotype is 'pho13::XI,gre3::PPP,XK'. For further improvement of the xylose isomerase activity, three rounds integration of Ru-xylA expression cassette was performed at the?-sequences sites (reiterated chromosomal DNA sequences of yeast retrotransposon Tyl) to increase the Ru-xylA copy numbers, and the genotype of the result strain BSN3 is 'pho13::XI, 3?::XI, gre3::PPP, XK'. The fermentation performances of the recombinant strains derived in the process of rational metabolic engineering were shown in the table below. The results indicate that all the measures taken in the metabolic engineering are beneficial for improvement of xylose metabolism and confirm the successful reconstruction of xylose metabolism pathway in diploid wild-type S. cerevisiae BSIF. In the fermentation of mixed sugars (20 g L-1 glucose and 20 g L-1 xylose), the recombinant strain BSN3 consumed 18.22±0.24 g L-1 xylose and produced 14.65 g L-1 ethanol, which are more than other strains.Table. Genotype and characteristics of the recombinant strains derived in the process of rational metabolic engineering3. Adaptive evolution to improve xylose metabolism of S. cerevisiae.Rational metabolic engineering endow the diploid wild-type S. cerevisiae with the ability to metabolize xylose. However, the xylose fermentation efficiency of the result strain BSN3 was still less than expected. Generally, some unknown intracellular factors also affect the conversion rate from xylose to ethanol. For this reason, the adaptive evolution was further conducted on BSN3. BSN3 was evolved in YP medium supplied with 20 g L-1 xylose as the sole carbon source. During of adaptive incubation on xylose, the strain showed gradually improved growth with the biomass doubling time T shorten to approximately 120 min. An evolved strain XH7(pho13::XI, 3?::Xl, gre3::PPP, XK, AE) that grew fastest on xylose among the several isolated colonies was selected out for further research. As a result, the XI activity of XH7 was raised to 0.67 U mg-1 protein, which was more than two times that of BSN3. In YP medium supplied with 40 g L-1 xylose as the sole carbon source,the evolved strain XH7 consumed almost all the xylose in 26 h with ethanol yield 0.479 g g-1 consumed xylose. In glucose-xylose cofermentation (80 g L-1 glucose and 40 g L-1 xylose ), almost all the sugar was consumed by XH7 in 40 h with ethanol yield 0.451 g g-1 consumed sugar. Compared with BSN3, the xylose fermentation performance of the evolved strain XH7 was significantly improved.4. Improvement of strain tolerance to hydrolysate by adaptive evolution in leach liquor of pretreated corn stover.In fermentation with the hydrolysate from pretreated corn stover (PCS), the presence of inhibitory factors and the commonly industrial oligotrophic nitrogen source like urea exert unfavorable influence on the growth and fermentation of S.cerevisiae. Although the rise in inoculum size notably made up these negative effects,it is of great importance to improve the strain's tolerance in consideration of production cost and competitiveness. In addition, the inhibitor components and their interaction are very complicated and the tolerant mechanism of S. cerevisiae to the inhibitory factors are still not clear. The strain XH7 was continually evolved in leach liquor of pretreated corn stover (PCS liquor) supplied with 5 g L-1 urea to enhance its tolerance.During about 900 h of evolution, the culture showed gradually improved growth,the biomass doubling time (T) shortened from -7 h to 3.9 h. The evolved cell populations were spread on PCS liquor-agar plate. While hundreds of well growing colonies were obtained in the plate spreading the evolved cell populations after four days, only a few tiny colonies could observed in the plate spreading XH7. Then, about 100 big colonies were evaluated by the BioScreen system (Oy Growth Curves Ab Ltd,Helsinki, Finland). A strain grew better than others was selected and designated as XHR11 (pho13::XI, gre3::PPP, XK, 3?::XI, AE-PCS). In the PCS hydrolysate fermentation, the XHR11 exhibited better performance as expected, which may due to the higher tolerance to the inhibitors. The ?max and biomass yield of XHR11 during the glucose-consuming phase were 2.0 and 3.0 times those of XH7, and 11.96 g L-1 more xylose was consumed at the end of fermentation. The ethanol yield basing on total sugar was 21.5 % higher than that of XH7.5. Transporter expression contributes to increase the ability of co-fermentation of glucose and xyloseSugar transport across the plasma membrane is the first obligatory step of carbohydrate utilization. In natural S. cerevisiae strains, the uptake of xylose is mediated by nonspecific transporters, such as HXT1, HXT2, HXT5, HXT7 and GAL2 which are identified as affiliated with xylose-transporting proteins. However,their affinity to xylose is much lower than that of glucose. In addition, xylose uptake by the transporters is competitively inhibited by glucose. A central question in xylose fermentation by S. cerevisiae engineered for xylose fermentation is to improve the xylose uptake. The expression of efficient heterogenous pentose transporters in S.cerevisiae is beneficial to the uptake and metabolism of pentose. The transporter N360S, which is the mutant of transporter Mgt05196p cloned from the genome of M.guilliermondii ATCC 6260, is an efficient xylose transporter with self-owned intellectual property and can be successfully expressed in S. cerevisiae. Thus, the mutant transporter N360S was integrated on the chromosome of XHR11. The result recombinant strain integrated with N360S did not show better fermentation performance on xylose than that of the strain XHR11, perhaps because of the inherent potential for utilization of xylose by the host strain BSIF. However, the doubling time of strain harboring the transporter N360S further reduced when it suffered from a short evolution in YP medium supplied with 20 g L-1 xylose as the sole carbon source.Meanwhile, the evolution in xylose did not reduce the doubling time of strain XHR11.Strain LF1 (pho13::XI, 3?::XI, gre3::PPP, XK, AE-PCS, N360S, AE) was obtained after monocolony isolation and comparation. In YP medium supplied with 40 g L-1 xylose as the sole carbon source, LF1 consumed almost all the xylose in 12 h. In glucose-xylose cofermentation (80 g L-1 glucose and 40 g L-1 xylose ), almost all the sugar was consumed by LF1 in 16 h. The results were superior to those of other strains reported at present.6. The promotion of lignocellulosic ethanol industrializationSeveral kinds of lignocellulosic hydrolysates used for research were collected from different companies. Their contents of sugars and inhibitory compounds differs for the different raw materials and methods of pretreatment. LF1 performed better than strain XHR11 in fermentation with these lignocellulosic hydrolysates. In fermentation with the hydrolysate from Shandong Tranlin Group, almost all of the sugar (about 40 g L-1) was consumed by LF1 with ethanol yield 0.48 g g-1 consumed xylose, which correspond to 94.1 % of the theoretical yield. The strain LF1 has great potential for industrial production of fuel ethanol from lignocellulosic materials, and more researchs shoud be done to make the strain match the various hydrolysates from different companies. |
PDF Full Text Request