| To exploit and utilize new energy is an important policy for human to face the shortage of fossil energy, the pollution of natural environment, and the lack of food. It has a great significance in the sustainable development of society. Fuel ethanol is recognized as a very promising new renewable biofuel. The first generation of fuel ethanol is produced using grain which exists the problem of occupying for food and farmland. Subsequently, people put forward the production of the second generation of fuel ethanol using lignocellulose materials which are the most abundant, cheap, and renewable biomass resource in the earth and fit for the large-scale production of fuel ethanol. The hydrolysis of lignocellulose materials can release a large amount of monosaccharides, including abundant glucose, and also significant xylose and L-arabinose. The utilization of all released sugar components of lignocellulose materials is a key to economically produce fuel ethanol and this is a main bottleneck that needs to be solved.S. cerevisiae is a traditional ethanol production strain and also used in the production of lignocellulosic fuel ethanol. Wild-type S. cerevisiae has excellent glucose fermentation capacity, but cannot metabolize xylose or L-arabinose. The research of pentose metabolic engineering in S. cerevisiae has been conducted for many years. S. cerevisiae has now been modified to utilize these two kinds of pentose (xylose and L-arabinose). Through multiple points, the alcohol conversion efficiency of pentose has also been improved, but there are still some bottlenecks to affect the utilization efficiency.The transport activities of pentoses are one of the limited steps for utilizing pentoses in the metabolic study of S. cerevisiae. Wild-type S. cerevisiae can use its hexose transporters to transport the two pentoses and Gal2p is the fine xylose and L-arabinose transporter, but the transport efficiency is not enough and strongly inhibited by glucose. Screening or/and modifing heterologous pentose transporters to relieve or ease glucose inhibition of S.cerevisiae is the effective strategy to improve the transport efficiency of pentose, and it can also enrich people’s perception of xylose transport mechanism. The related research has become a new study hotspot in this field. The lack of effective high-throughput screening of functional pentose transporters, the contradiction of transport efficiency and specificity of pentose transporters, and the glucose inhibition towards pentose transport are the main problems to be resolved. So far, the pentose transporters which can effectively alleviate the glucose inhibition of xylose metabolic S.cerevisiae have not yet been found. The research of L-arabinose metabolic engineering of S.cerevisiae started relatively late than that of xylose. The efficiency of ethanol transformation of L-arabinose was much lower than that of xylose, which was influenced by the transporters and more important point is the impact of various metabolic nodes. In this thesis, studies were carried out according to the problems above.1, The establishment of a high-throughput screening method of xylose transportersThrough obvious color changes, a high-throughput screening method of xylose transporters in S. cerevisiae was built based on pNPX. The principle is as follows: Xylose analogs pNPX could pass through cell membranes using transporters, and then be hydrolyzed to release group pNP by intracellularly expressed xylosidase. The free diffused pNP could exhibit yellow color under alkaline condition. So, the pNPX transport activities can be calculated according to the producing activities of pNP to indirectly quantify the capacity of xylose transport activities. A xylosidase XynB cloned from B. pumilus that could hydrolyze xylose analogs pNPX to release pNP was heterologously expressed in S. cerevisiae to exhibit an intracellular enzyme activity of 29.34±2.87 U (g DCW)-1. Extracellular enzyme activity was undetectable. The enzyme activity of intact cells was 0.21±0.01 U (g DCW)-1 and far less than the intracellular enzyme activity, which meant the enzymolysis link was not a limiting step. More than 95% of pNP existed extracellular which meant that the step of pNP effusing out of the cells was also not a limiting step. The link of pNPX transport was the only limiting step during the whole process. It was further determined that pNPX had the competitive inhibition with xylose. This method was proved and so a high-throughput screening method of xylose transporter based on pNPX in yeast strain was constructed.2. The application of this high-throughput screening method to study the functional area of xylose transporter AraEpUsing this high-throughput screening method to find out that TMS 5 is the functional area of xylose transporter AraEp. Bacterial xylose transporter AraEp from C. glutamicum was successfully expressed in S. cerevisiae, and the high expression of AraEp could increase the xylose transport activity by 184%. AraEp mutant library by error-prone PCR was constructed and then selected in 96-well plates using this high-throughput screening method. Six transformants which significantly improved the transport capacities from 15.8% to 103% were selected from about 5000 transformants. After sequence analysis, two mutants that significantly improved AraEp transport capacities held only one residue change each in TMS 5 (I178S and V179D, respectively), and it meant that this transmembrane section was one of the key regions for the transport function of AraEp.3. Cloning pentose transporters from donor strains that could simultaneously utilize glucose and pentoseSix pentose transporters were cloned from donor strains that could simultaneously utilize glucose and pentose, and then heterogenously expressed in S. cerevisiae. Through the cocultivation of glucose and pentose with the same concentration, the donor strains were verified to simultaneously utilize glucose and pentose and no obvious competitive inhibition was presence. Selecting conservative motifs of yeast sugar transporters to clone the genome sequences between the motifs, obtaining the two ends of the genome sequences by the method of chromosome walking, and then predicting the exons, we obtained two potential transporter genes tctl and tct2 from donor strain T. cutaneum. Using endogenous Gal2p as a probe, four transporters Mgt04891p, Mgt05196p, Mgt05293p, and Mgt05860p were selected from the genome database of donor strain M. guilliermondii. The amino acid sequence similarities of the six transporters with Gal2p was 47%-77%. The cloned transporters showed the wide representation in the phylogenetic tree analysis.4. The functional analysis of the heterologous pentose transportersThrough testing the intracellular accumulation of pentose, the gradient growth on hexose plates, the extracellular pH changes during the transfer process, the growth activities on xylose, the xylose metabolic activities, and also the xylose transport preference in transporter expressing hxt null strain EBY.VW4000, an efficient xylose transporter Mgt05196p was figured out. The xylose or L-arabinose transport activities were studied through the determination of intracellular xylose or L-arabinose accumulation. After analyzing, Tctlp was an L-arabinose transporter and the net accumulation of L-arabinose of Tctlp expressing strain was up to 22% of that of Gal2p expressing strain. Tct2p could transport both xylose and L-arabinose, but the efficiencies were low. Four transporters cloned from M. guilliermondii were tested to be xylose transporters. Mgt04891p and Mgt05860p were verified to transport xylose as H+ symporters, and Mgt05860p expressing strain could accumulate the highest net xylose. Compared to the Gal2p expressing strain, Mgt05860p expressing strain could increase by 116%. Mgt05196p and Mgt05293p were verified to transport xylose as facilitators, and Mgt05196p expressing strain could accumulate net xylose which was increased by 13% compared to the Gal2p expressing strain. Mgt04891p, Mgt05293p, and Mgt05860p could also show obvious L-arabinose transport activies, and Mgt05860p expressing strain held the highest net L-arabinose accumulation which was 93% higher than Gal2p expressing strain. Meanwhile, AraEp from C. glutamicum only showed a certain xylose transport activity. Stplp from T. reesei had significant xylose and L-arabinose transport activities, and the net accumulation of xylose or L-arabinose of expressing strain were up to 56% or 12% of that of Gal2p expressing strain, respectively. The test of hexose transport abilities showed that the more efficient xylose transporters could also transport hexose, and the transport efficiency and specificity of pentose transport were contradiction. Among these studied xylose transporters, Mgt05196p expressing EBY.VW4000 strain with XR-XDH pathway could obtain the highest growth and metabolic capacity on xylose, close to Gal2p expressing strain. Compared with Gal2p expressing strain, the xylose/glucose preference could increase by 50% in Mgt05196p expressing strain.5. The analysis of transport active sites in Mgt05196pUsing the large scale of site-directed mutagenesis, the transport active sites in Mgt05196p were analyzed. Using the crystal structure of xylose transporter XylEp as the template, Mgt05196p was predicted to construct the homology model and then determined the key residues for binding xylose, meanwhile, some conservative residues were selected according to the reported results of other glucose or xylose transporters. A total of 28 potential residues were screened out. The potential residues were turned into alanine (A) which was a small nonpolar amino acid to express in EBY. VW4000 strain containing the xylose XR-XDH pathway to study their function. The specific growth rates were then calculated from the growth curves to analyze xylose and glucose transport capacities of the mutants, meanwhile, the intracellular xylose accumulation was also used to determine the xylose transport activity. The change of sites D72, R164, and Y336 of Mgt05196p made the expressing strains lose both xylose and glucose transport capacities, and the change of site F333 made the expressing strain lose the xylose transport capacity. The change of sites 1202, Q326, N331, Y332, F334, and Y335 could decrease the xylose specific growth rates by 48%, 69%,90%,84%,72%, and 79%, respectively, and could decrease the amounts of intracellular xylose accumulation by 54%,54%,88%,67%,64%, and 59%. The change of sites F69, F333, and F334 could decrease the glucose specific growth rates of expressing strains by 69%,92%, and 52%, respectively. The change of sites Q199, N360, and F432 could increase the xylose specific growth rates of expressing strains; and the amount of intracellular xylose accumulation of F432A mutant could increase by 14%. The site N360 was further mutated to serine (S), the xylose specific growth rate and the intracellular xylose accumulation of expressing strains were increased by 32% and 10%, respectively. The mutant of N360 to phenylalanine (F) effectively alleviated the inhibition of glucose on xylose because the transporter mutant lost the glucose transport capacity, but retained high activity of xylose transport even glucose was present. In addition, the aromatic amino acids enriched sequence YFFYY in the conservative motif of TMS 7 had a significantly negative influence on xylose growth. The change of each amino acid could decrease the xylose growth capacity of the expressing strain.The cloned Mgt05196p was an efficient xylose transporter which had potential application value, and the mutant Mgt05196p (N360F) which held a high xylose preference and alleviated the inhibition of glucose to some extend is important in constructing the engineered strains to achieve the co-utilization of glucose and xylose. The analysis of key residues for xylose transport of Mgt05196p was helpful to illustrate the xylose transport mechanism and useful to obtain xylose transporter mutants with higher transport efficiencies.6. The construction and optimization of L-arabinose metabolic pathway in S. cerevisiaeThrough the high expression of araA, the overexpression of four key genes in PPP pathway and Ga12p, combined with the adaptive evolutionary strategy, a S. cerevisiae strain which could metabolize L-arabinose to produce ethanol was obtained. The content of L-arabinose in the lignocellulose monosaccharide components is only less than that of xylose, and so, its effective utilization is benefit for the full sugars utilization of hydrolyzed lignocellulose materials. Plasmid YIp5-ara containing the three genes araA, araB, and araD of L-arabinose initial sugar metabolic pathway from L. plantarum was integrated to the genome to express in CEN.PK102-3A. Unfortunately, the strain could not grow on L-arabinose yet. After increasing the RNA expression level of araA under promoter TEFlp (increased by 32.5±0.7-fold), the recombinant strain obtained the growth capacity on L-arabinose after incubation for 150 hours. The high expression of araA to a certain extent is a key factor for L-arabinose utilization. The L-arabinose metabolism capacity increased significantly by further high expressing the four key genes TALI, TKL1, RPE1, and RKI1 in PPP pathway, combined with the adaptive evolutionary method, and then a single colony named BSW3AP was selected out. By quantitative PCR analysis, the intracellular RNA expression of araA, araB, and araD of BSW3AP increased significantly which meant that the adaptive approach effectively promoted the exogenous genes of L-arabinose metabolic pathway to express, and improved the ability of the strain on L-arabinose metabolism. The L-arabinose fermentation capacity of BSW3AP was further improved by high expression of endogenous Gal2p transporter (resulted strain BSW3AG). It indicated that the L-arabinose transport rate of transporters was lower than the intracellular metabolic capacity. The transport link was still an important factor to limit the metabolic speed. In the anaerobic fermentation of BSW3AG on 20 g L-1 L-arabinose as the sole carbon source, the maximum specific growth rate of the strain reached 0.075 h-1, the maximum L-arabinose consumption rate was 0.61 g h-1 g-1 DCW, ethanol production rate was 0.27 g h-1 g-1 DCW, and the maximum yield of ethanol reached 0.43 g g-1. The utilization of L-arabinose effectively expanded the scope of the available substrate and had application value. |
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