EMS and adaptive evolutionary engineering improved micro-aerobic growth and isobutanol tolerance of S.cerevisiae
Our strain development strategy is outlined in Fig.1. We used methods of adaptive laboratory evolution as generalized protocols, where ethyl methane sulfonate (EMS) was used on wild-type strain W303-1A to obtain a randomly mutagenized and genetically diverse initial population. The resulting population was used for 15 days Erlenmeyer flasks selections under higher glucose (100 g/L) and isobutanol (16 g/L) conditions throughout the cultivations. Individual mutant colonies are randomly selected from the final population and tested for their isobutanol tolerance using spot assay test. Meanwhile, relative viability rates were also determined. A strain designated as EMS39 with higher tolerance toward both glucose and isobutanol was identified. As shown in Fig.2a, both wild-type and the evolved strain EMS39 population could grow in control medium at all their dilutions (from 100 to 10-4). However, wild type could barely grow at its 100 dilution in YPD with 16 g/L isobutanol (stress containing medium). Additionally, the evolved strain EMS39 could grow nearly at all of its dilutions (100 to 10-4 ) in YPD with 16 g/L isobutanol, which demonstrated its higher resistance properties against the stress factor. As shown in Fig.2b, the evolved strain EMS39 conferred a significantly improved cellular viability (over the course of 60 hours of culturing) above that of the control strain, even at concentrations as high as 20 g/L isobutanol. All these results indicated that the evolved strain EMS39 might be a predominant strain with higher tolerance toward isobutanol. So we used the evolved strain EMS39 as a platform to produce isobutanol.
Application of the evolved strain EMS39 as a platform for isobutanol production
Metabolic engineering of the evolved strain EMS39 as a platform for isobutanol production were carried out. To over-express ILV2, ILV5 and ARO10, 2μ plasmids YEplac195-PGK1p-ILV2, YEplac112-PGK1p-ILV5 and YEplac181-TDH3p-cox4-ARO10 were transformed into the evolved strain EMS39, wild type strain W303-1A and strain HZAL-7 using LiAc/ssDNA/PEG methods, the resulting strains were designated as strain EMS39V2V5A10, strain W303-1AV2V5A10 and strain HZAL-7V2V5A10, respectively. And in order to increase the integrated copy number of ILV3 (encoding enzyme dihydroxyacid dehydratase limiting the isobutanol pathway in S.cerevisiae), δ-integration method was used to over-express ILV3 in EMS39V2V5A10, W303-1AV2V5A10 and HZAL-7V2V5A10. And the resulting strains were donated as strain EMS39V2δV3V5A10, strain W303-1AV2δV3V5A10 and strain HZAL-7V2δV3V5A10, respectively. To determine whether the increased isobutanol tolerance of the evolved strain EMS39 could improve isobutanol yield, we examined performances of strain EMS39V2δV3V5A10, strain W303-1AV2δV3V5A10 and strain HZAL-7V2δV3V5A10 in micro-aerobic batch fermentation in YPD medium with 40 g L-1 glucose and 130 g L-1 glucose as carbon source in shaker flasks with OD600=0.5 and 3.0, as the initial inoculums size, respectively. And strain W303-1AδHis3 carrying plasmids YEplac181, YEplac195 and YEplac112 was used as the control strain.
As shown in Fig.3a and b, the control strain has the lowest growth rate and glucose consumption rate and glucose consumption was complete at 48h. Growth rate of strain EMS39V2δV3V5A10 was slightly higher than that of the control strain. And strain EMS39V2δV3V5A10 used up glucose at 32h. Meanwhile, growth rate of strain W303-1AV2δV3V5A10 and strain HZAL-7V2δV3V5A10 were higher than that of strain EMS39V2δV3V5A10. In addition, strain W303-1AV2δV3V5A10 and strain HZAL-7V2δV3V5A10 consumed glucose faster than strain EMS39V2δV3V5A10.
Isobutanol concentrations in the media at 24h, 28h, 32h, 36h and 48h after the start of cultivation were determined. As shown in Fig3.c, the control strain and strain W303-1A V2δV3V5A10 generated 0.064 g L-1 and 0.299 g L-1 isobutanol at 48h, respectively. While strain EMS39 V2δV3V5A10 generated 0.404 g L-1 isobutanol at 32h. And strain HZAL-7 V2δV3V5A10 produced 0.384 g L-1 isobutanol at 48h. Isobutanol titers of strain EMS39V2δV3V5A10 increased by 35.1% and 5.2% compared with that of strain W303-1AV2δV3V5A10 and strain HZAL-7V2δV3V5A10, respectively. These data suggested that the increased isobutanol tolerance of the evolved strain EMS39 was useful for improving isobutanol titers.
To further gain insights into fermentation characteristics of the evolved strain EMS39 and strain EMS39V2δV3V5A10 in higher glucose concentration, we carried out fermentations in YPD medium with 130 g L-1 glucose in shaker flasks with OD600=3, as the initial inoculums size. As shown in Fig.4a and Fig.4b, the growth rate and glucose consumption of strain EMS39YEplac181YEplac195YEplac112 were markedly higher and faster than that of the other four strains. While strain EMS39 V2δV3V5A10 resulted in lower growth rate and glucose consumption rate than strain EMS39YEplac181YEplac195YEplac112. In addition, there were no obviously differences between the control strain and strain W303-1AV2δV3V5A10 in the growth rate and glucose consumption. Finally, the growth rate and glucose consumption rate of strain HZAL-7 V2δV3V5A10 were slightly lower than that of the control strain.
As shown in Fig.4c, the control strain and strain EMS39YEplac181YEplac195YEplac112 produced 0.807 g L-1 isobutanol at 36h and 1.33 g L-1 isobutanol at 32h, respectively. The increased isobutanol titers in strain EMS39YEplac181YEplac195YEplac112 further indicated that increased isobutanol tolerance was useful for improving isobutanol titers. Meanwhile, strain EMS39 V2δV3V5A10 generated 2.79 g L-1 isobutanol at 24h. These results indicated that over-expression of ILV2, ILV5, ILV3 and ARO10 could increase isobutanol yield markedly. But after 24h, isobutanol titers of strain EMS39 V2δV3V5A10 decreased slightly. This perhaps due to the exhaustion of glucose. In addition, we found that strain W303-1AV2δV3V5A10 and strain HZAL-7V2δV3V5A10 gained 4.20 g L-1 and 3.45 g L-1 isobutanol at 48h, respectively. These results suggested that over-expression of ILV2, ILV5, ILV3 and ARO10 in strain W303-1A could markedly improve isobutanol titers. But over-expression of BAT2 and deletion of PDC6 is not useful for increasing isobutanol titers in strain W303-1A V2δV3V5A10.
Ethanol was one of the main byproducts in isobutanol fermentation in yeast. As shown in Fig.3d, the control strain and strain W303-1AV2δV3V5A10 generated 3.45g L-1 ethanol at 48h and 3.51 g L-1 ethanol at 36h, respectively. And the strain EMS39 V2δV3V5A10 produced 4.34 g L-1 ethanol at 32h. While strain HZAL-7 V2δV3V5A10 produced 4.38 g L-1 ethanol at 36h. The increased ethanol titers of strain EMS39 V2δV3V5A10 might be resulted from higher tolerance toward alcohols. While increased ethanol titers of strain HZAL-7 V2δV3V5A10 might be resulted from the deletion of PDC6. As illustrated in Fig.4d, the control strain produced 48.3 g L-1 ethanol at 48h. Strain EMS39YEplac181 YEplac195YEplac112 and strain EMS39V2δV3V5A10 generated 49.7 g L-1 and 46.2 g L-1 ethanol at 28h, respectively. Meanwhile, strain W303-1A V2δV3V5A10 and strain HZAL-7 V2δV3V5A10 produced 47.0 g L-1 ethanol at 36h and 45.2 g L-1 ethanol at 48h, respectively. Ethanol titers of strain EMS39 YEplac181YEplac195YEplac112 and strain EMS39V2δV3V5A10 increased than that of strain W303-1A V2δV3V5A10 and strain HZAL-7V2δV3V5A10 in the first 32 h fermentation. These data suggested that the evolved strain EMS39 also might be a predominant strain for producing ethanol.
The highest isobutanol yields and productivities of these strains were also calculated (as shown in Table4). The highest isobutanol yields of strain W303-1AV2δV3V5A10, strain EMS39V2δV3V5A10, strain EMS39YEplac181YEplac195YEplac112 and strain HZAL-7V2δV3V5A10 were 31.7, 21.0, 10.2 and 26.2 mg per g glucose, respectively. And strain EMS39V2δV3V5A10 has the highest isobutanol productivity (0.116 g L-1 h-1). Meanwhile, ethanol yields of strain W303-1AV2δV3V5A10, strain EMS39V2δV3V5A10, strain EMS39YEplac181YEplac195YEplac112 and strain HZAL-7V2δV3V5A10 were 0.354, 0.347, 0.382 and 0.343 g per g glucose, respectively. All these results suggested that strain EMS39V2δV3V5A10 had advantages in isobutanol fermentation.
Prelimiary investigation on the genetic basis of improved phenotype of evolved strain EMS39
To identified the genetic basis of improved phenotype in the evolved strain EMS39, whole genome resequence of the evolved strain EMS39 were carried out. More than 59 genes had mutations (including nucleotides insertions, nucleotides deletions and base changes) in their ORF or in upstream and downstream regulatory regions of genes. As shown in Table S1, twenty-six genes ( including GPR1, WSC2, APC1, CLB5, COS4, COS6, SOK2, FLO1,FLO5,FLO9, ASG1, AAD4, MTL1, MSS11, BUD27, PAF1, EPL1, TIR1, FIG2, RPL14A, RPS28B, SRP40, NGR1, NCL1, FAB1 and ENT1) had multi mutations in their ORF. These gene are involved in cell growth, Ras-cAMP pathway, MAPK signaling pathway, cell cycle, turnover of plasma membrane, pseudohyphal differentiation, flocculation, oxidative stress response, cell integrity signaling, basal transcription factors, translation initiation and elongation, autophagy, cell wall mannoprotein and adhesion, component of ribosomal subunits, ribosome assembly and function, RNA binding protein, RNA processing, phosphatidylinositol signaling system and endocytosis and so on. And six gens (including COS4, SOD2, SPT6, RPL14A, PSR1, RAD2 ) had multi-nucleotides insertions or deletions in their upstream regulatory regions. They are related to turnover of plasma membrane, protecting cells against oxygen toxicity and oxidative stress, transcription, chromatin maintenance, and RNA processing, component of ribosomal subunit, ribosome assembly and function, nucleotide excision repair and so on. Fourteen genes (including WSC2, CDC20, CLB6, TUP1, SMC4, FLO1, TAF6, NIP1, RDH54, RPL14A, RPS28B, PSR1, NGR1, NCL1) have mutations in the downstream regulatory regions. These results suggested that isobutanol tolerancerequired synergism of polygenic. But further investigations need to be carried out to explore mutations that can lead to improved isobutanol tolerance in S.cerevisiae.
EMS39V2δV3V5A10 resulted in transcription perturbations
In order to investigate cellular transcription profile changes in strain EMS39V2δV3V5A10 in micro-aerobic fermentation with 130 g L-1, samples were taken at 24 h for RNA sequencing, and strain W303-1AV2δV3V5A10 was used as the control strain. RNA-Seq-based transcriptomic analysis revealed cellular transcription profile changes resulting from EMS39. The volcano plots of differentially expressed genes (DEGs) for W303-1AV2δV3V5A10 vs EMS39V2δV3V5A10 were shown in Fig.5. Compared with strain W303-1AV2δV3V5A10, 401 differentially expressed genes (DEGs) were identified in strain EMS39 V2δV3V5A10 (fold change > 2, P value < 0.05), including 160 up-regulated and 241 down-regulated.
Firstly, we reconstructed the central carbon metabolic network based on the RPKM values of genes involved in this network (Fig. 6 and Table S2). Glucose is phosphorylated by hexose–glucose kinase after uptake, and then enters the glycolytic pathway. The high-affinity glucose transporter genes HXT6 and HXT7 in strain EMS39V2δV3V5A10 were up-regulated by 8.1-fold and 11.5-fold compared with that in W303-1A V2δV3V5A10, respectively. The up-regulation of HXT6 and HXT7 in strain EMS39V2δV3V5A10 might promote its glucose assimilation ability. Transcriptional levels of gene TPS2 and TSL1 in strain EMS39V2δV3V5A10 increased by 8.1-fold 5.7-fold, respectively. The increased transcriptional level of TSL1 and TPS1 might resulted in accumulation of trehalose, which could increase the stability of the cells and stimulates the secretion of heat shock proteins . SOL4 and SOL1 were up-regulated in strain EMS39V2δV3V5A10. These two genes encode 6-phosphogluconolactonase in the pentose phosphate pathway. The enhanced pentose phosphate pathway activity perhaps might provide a large amount of nicotinamide adenine dinucleotide phosphate (NADPH) for isobutanol biosynthesis. FBP1, encoding fructose 1,6-bisphosphatase, was found up-regulated by 16.4-fold. ERR2, encoding a phosphopyruvate hydratase, was down-regulated by 17.2-fold. PCK1, encoding phosphoenolpyruvate carboxykinase, was up-regulated by 17.9-fold. The perturbations of FBP1, ERR2 and PCK1 perhaps could promote regeneration of 2-phosphoglycerate. It was reported that ADH1 knockdowns conferred increased tolerance toward both isobutanol and 1-butanol. While our result indicated that alcohol dehydrogenase genes ADH1 and ADH5 were up-regulated by 2.8-fold and 3.0-fold in strain EMS39V2δV3V5A10, respectively. But alcohol dehydrogenase gene ADH4 was down-regulated by more than 42-fold. ADH1 is required for the reduction of acetaldehyde to ethanol, while ADH4 is involved in the degradation of ethanol and thereby contribute to ethanol detoxification to ensure cell survival. We found that FPKM values of ADH1 increased by 78.1-fold compared with that of ADH4. Hence, the enhanced transcription of ADH1 in strain EMS39V2δV3V5A10 might confer it higher ethanol biogenesis. MPC3, encoding the highly conserved subunit of mitochondrial pyruvate carrier, was up-regulated by 33.2-fold in strain EMS39V2δV3V5A10. The up-regulation of MPC3 might promote pyruvate uptake into mitochondrial matrix. CIT1 was found up-regulated by 6.7-fold in strain EMS39V2δV3V5A10. Cit1p catalyzes the first reaction of the TCA cycle that is condensation of acetyl-CoA and oxaloacetate to form citrate. And Cit1p functions as a rate-limiting enzyme of the TCA cycle . IDP2, encoding cytosolic NADP-specific isocitrate dehydrogenase Idp2 in the TCA cycle, was also up-regulated in strain EMS39V2δV3V5A10. The up-regulation of genes CIT1 and IDP2 might enhance the activity of TCA cycle, Under anaerobic conditions, the TCA cycle can work as a reducing cycle, reducing the excess NADH. Additionally, MLS1, encoding malate synthase in the glyoxylate cycle, was also up-regulated by 9.4-fold in strain EMS39V2δV3V5A10. The up-regulation of MLS1 perhaps could improve utilization of acetyl CoA. GLO1 and CYB2, encoding glyoxalase I and L-lactate dehydrogenase, were up-regulated by 5.9-fold and 6.9-fold in strain EMS39V2δV3V5A10, respectively. The enhanced transcription of GLO1 and CYB2 perhaps could improve pyruvate biogenesis. Transcriptional levels of ILV2, ILV3 and ADH7 were up-regulated by 3.3-fold, 2.1-fold and 8.8-fold in strain EMS39V2δV3V5A10, respectively. The up-regulations of ILV2, ILV3 and ADH7 conferred strain EMS39V2δV3V5A10 higher isobutanol titers. But transcriptional level of ARO10 decreased by more than 90-fold. This indicated that there still existed transcription unbalance of genes involved in isobutanol biosynthesis.
Secondly, the RPKM values of genes related to transporters of the plasma membrane and mitochondrial membrane were analyzed. PMA2, encoding plasma membrane H+-ATPase, involved in pumping protons out of the cell and it was the regulator of cytoplasmic pH and plasma membrane potential. The down-regulation of PMA2 might decrease the pumping protons out of the cell and increase pH gradient across the membrane in strain EMS39V2δV3V5A10. JEN1, encoding monocarboxylate/proton symporter Jen1p of the plasma membrane, was up-regulated by 6.4-fold in strain EMS39V2δV3V5A10. Jen1p mediates high-affinity uptake of carbon sources lactate, pyuvate, and acetate, and also of the micronutrient selenite. And transport activity of Jen1p is dependent on the pH gradient across the membrane. The increased pH gradient across the membrane, which was due to the down-regulation of PMA2 in strain EMS39V2δV3V5A10, perhaps resulted in up-regulation of JEN1. STL1, encoding glycerol proton symporter of the plasma membrane, was up-regulated by 5.3-fold in strain EMS39V2δV3V5A10. This gene was subject to glucose-induced inactivation and was strongly but transiently induced when cells are subjected to osmotic shock. The up-regulation of STL1 in strain EMS39V2δV3V5A10 might confer it higher osmotic tolerance. While PHO84, PHO89, FRE1, FRE7, FET4, MRS3, ZRG17, IZH2, ZRT1 and ZRT2 were down-regulated in strain EMS39V2δV3V5A10. The down-regulation of PHO84 and PHO89 might decrease the phosphate ion transmembrane transport and polyphosphate metabolism. The down-regulation of FRE1, FRE7, FET4, MRS3 might maintain iron ion homeostasis in cytoplasmic and mitochondria. And the down-regulation of ZRG17, IZH2, ZRT1 and ZRT2 might maintain zinc ion homeostasis in endoplasmic reticulum and cytoplasmic. In addition, the down-regulated genes related to iron ion homeostasis and zinc ion homeostasis perhaps indicated the decreased synthesis of iron ion-containing amino acids and zinc ion-containing amino acids in strain EMS39V2δV3V5A10. SFC1, encoding mitochondrial succinate-fumarate transporter Sfc1, was up-regulated by 17.5-fold in strain EMS39V2δV3V5A10. Sfc1 is involved in transporting succinate into and fumarate out of the mitochondrion. We also found that UPS2, UPS3 and ODC1 were up-regulated by 4.9-fold, 3.4-fold and 3.8-fold in strain EMS39V2δV3V5A10, respectively. UPS2, encoding mitochondrial intermembrane space protein, was involved in phospholipid metabolism. ODC1, encoding 2-oxodicarboxylate transporter on mitochondrial inner membrane, exports 2-oxoadipate and 2-oxoglutarate from the mitochondrial matrix to the cytosol for lysine and glutamate biosynthesis and lysine catabolism. The up-regulation of UPS2 perhaps could improve activity of phospholipid metabolism. And the up-regulation of ODC1 perhaps could improve lysine and glutamate biosynthesis and lysine catabolism. TPC1, encoding mitochondrial membrane transporter, was down-regulated by 3.2-fold in strain EMS39V2δV3V5A10. The down- regulation of TPC1 might decreased the uptake of the essential cofactor thiamine pyrophosphate (ThPP) into mitochondria.
Thirdly, other groups up-regulated DEGs in strain EMS39V2δV3V5A10 are related to thiamine metabolism (i.e. THI12, THI5, THI11,THI13), heat shock proteins (such as HSP26, HSP30 and HSP10), response to oxidative stress (such as STF2 ), oxidative phosphorylation (such as QCR9, QCR8, COX7, QCR10, COX12, COX6, RIP1 and ATP20), cytochrome c (i.e. CYC1 , CYC7, PET100), some transcription factors (such as MET28, HAP4 and MET32), adenylylsulfate kinase (i.e. MET14) and SNARE- and chaperone-binding protein (i.e.BTN2) (Table S2 ). Thiamine and its phosphate compounds are required by all organisms to participate in various cellular metabolism. It was reported that the production of thiamine played an important role in the stress of oxidative stress[34,38]. The up-regulation of genes involved in thiamine metabolism and response to oxidative stress in strain EMS39 V2δV3V5A10 might improve its oxidative stress tolerance. The up-regulation of genes involved in heat shock proteins, SNARE- and chaperone-binding protein might improve isobutanol tolerance and ethanol tolerance of strain EMS39 V2δV3V5A10. The up-regulation of genes involved in oxidative phosphorylation and cytochrome c might supply cells with more energy. Another group of genes MET5, MET13, MET14, MHT1 and YCT1 were up-regulated in strain EMS39 V2δV3V5A10. These genes are involved in sulfur assimilation and are associated with methionine/cysteine metabolism. Now the roles of amino acids in the alcohol tolerance regulatory network is not yet clear. It was speculated that the synthesis of sulfur-containing amino acids was increased in order to increase the sulfur reserve in advance to ensure the subsequent synthesis of glutathione (GSH) . Other groups down-regulated DEGs in strain EMS39V2δV3V5A10 are involved in biotin metabolism (i.e. BIO3, BIO4, BIO5), the vacuolar transporter chaperone complex (i.e.VTC1), ribosome biogenesis (such as NHP2, CBF5, NOP58, NOP1, GAR1, NOG2, NOP4, NOG1, UTP14, EMG1, UTP5, DIP2, UTP21, PWP2, UTP10, UTP13, KRE33, UTP4, RIX7, UTP8), purine metabolism (i.e. ADE17, AAH1, ADE4), Vitamin B6 metabolism(i.e. BUD17 and SNO1), putative aryl alcohol dehydrogenase (i.e. AAD16) and drug metabolism (i.e.IMD4, GUA1, IMD2, IMD3). Down-regulation of genes involved in ribosome biogenesis, purine metabolism, vitamin metabolism, putative aryl alcohol dehydrogenase and drug metabolism in strains with higher tolerance toward alcohols were found by previous report [35,40,41]. Intriguingly, down regulation of biotin metabolism and regulatory subunit of the vacuolar transporter chaperone (VTC) complex coding gene VTC1 were only found in our study. Biotin function as cofactors of carboxylase involved in carbon dioxide transfer during respiration. The down-regulated biotin metabolism in strain EMS39V2δV3V5A10 indicated the decreased activity of carboxylase. The down-regulated gene VTC1 perhaps indicated decreased membrane trafficking and DNA replication stress in strain EMS39V2δV3V5A10 .
To further explore the transcriptome perturbations caused by EMS39, we conducted differential expression analysis based on the RNA-Seq data. Gene Ontology (GO) (Additional file 4) and KEGG (Additional file 5) enrichment analysis were conducted to identify the functions of DEGs. The results of GO enrichment show that biological processes related to gluconeogenesis (GO:0006094), cell division (GO:0051301), DNA integration (GO:0015074), rRNA processing (GO:0006364), ribosome biogenesis (GO:0042254), pseudouridine synthesis (GO:0001522) and zinc ion transmembrane transport (GO:0071577) were enriched in the down-regulated DEGs (Additional file 4). And biological processes related to trehalose biosynthetic process (GO:0005992) were enriched in the up-regulated DEGs. In addition, cellular components related to ribonucleoprotein complex (GO:0030529), phosphopyruvate hydratase complex (GO:0000015), vacuolar transporter chaperone complex (GO:0033254) and intracellular part (GO:0044424) were enriched in the down-regulated DEGs. Furthermore, molecular functions related to RNA binding (GO:0003723), pseudouridine synthase activity (GO:0009982), aspartic-type endopeptidase activity (GO:0004190), IMP dehydrogenase activity (GO:0003938), hydrogen ion transmembrane transporter activity (GO:0015078), phosphopyruvate hydratase activity (GO:0004634), zinc ion transmembrane transporter activity (GO:0005385) and L-serine ammonia-lyase activity (GO:0003941) were enriched in the down-regulated DEGs. According to the KEGG enrichment result (Additional file 5), apoptosis (ko04215), sulfur metabolism (ko00920), oxidative phosphorylation (ko00190, ko04260) and thiamine metabolism (ko00730) were enriched in the up-regulated DEGs in strain EMS39 V2δV3V5A10. Regarding the enrichment analysis of the down-regulated DEGs, the biological processes related to ribosome biogenesis, biotin metabolism, drug metabolism and purine metabolism were enriched in strain EMS39 V2δV3V5A10. In previous studies, GO analysis revealed the following GO terms to be overrepresented in the DEGs in alcohol-tolerant strains: gluconeogenesis, cell division, DNA integration, rRNA processing, ribosome biogenesis, sulfur metabolism, oxidative phosphorylation, vitamin metabolism, electron transport and trehalose biosynthetic process[35, 37, 40-42]. Intriguingly, down-regulation of genes involved in zinc ion transmembrane transport and vacuolar transporter chaperone complex were only found in this study.