Engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> to utilize xylan as a sole carbohydrate source by co-expression of an endoxylanase,xylosidase and a bacterial xylose isomerase

Xylan represents a major component of lignocellulosic biomass, and its utilization by Saccharomyces cerevisiae is crucial for the cost effective production of ethanol from plant biomass. A recombinant xylan-degrading and xylose-assimilating Saccharomyces cerevisiae strain was engineered by co-expression of the xylanase (xyn2) of Trichoderma reesei, the xylosidase (xlnD) of Aspergillus niger, the Scheffersomyces stipitis xylulose kinase (xyl3) together with the codon-optimized xylose isomerase (xylA) from Bacteroides thetaiotaomicron. Under aerobic conditions, the recombinant strain displayed a complete respiratory mode, resulting in higher yeast biomass production and consequently higher enzyme production during growth on xylose as carbohydrate source. Under oxygen limitation, the strain produced ethanol from xylose at a maximum theoretical yield of ~90 %. This study is one of only a few that demonstrates the construction of a S. cerevisiae strain capable of growth on xylan as sole carbohydrate source by means of recombinant enzymes.     

Sequence analysis of <Emphasis Type="Italic">KmXYL1</Emphasis> genes and verification of thermotolerant enzymatic activities of xylose reductase from four <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> strains

Kluyveromyces marxianus has the capability of producing xylitol from xylose because of the endogenous xylose reductase (KmXYL1) gene. In this study, we cloned KmXYL1 genes and compared amino acid sequences of xylose reductase (XR) from four K. marxianus strains (KCTC 7001, KCTC 7155, KCTC 17212, and KCTC 17555). Four K. marxianus strains showed high homologies (99%) of amino acid sequences with those from other reported K. marxianus strains and around 60% homologies with that from Scheffersomyces stipitis. For XR enzymatic activities, four K. marxianus strains exhibited thermostable XR activities up to 45°C and K. marxianus KCTC 7001 showed the highest XR activity. When reaction temperatures were increased from 30 to 45°C, NADH-dependent XR activity from K. marxianus KCTC 7001 was highly increased (46%). When xylitol fermentations were performed at 30 or 45°C, four K. marxianus strains showed very poor xylitol production capabilities regardless fermentation temperatures. Xylitol productions from four K. marxianus strains might be limited because of low xylose uptake rate or cell growth although they have high thermostable XR activities.     

Gene expression profiles of the thermotolerant yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain KKU-VN8 during high-temperature ethanol fermentation using sweet sorghum juice

Objectives

To investigate gene expression profiles of the thermotolerant yeast Saccharomyces cerevisiae strain KKU-VN8, a potential high-ethanol producer, in response to various stresses during high-temperature ethanol fermentation using sweet sorghum juice (SSJ) under optimal conditions.

Results

The maximal ethanol concentration obtained by S. cerevisiae KKU-VN8 using SSJ at 40 °C was 66.6 g/l, with a productivity of 1.39 g/l/h and a theoretical ethanol yield of 81%. Quantitative RT-PCR assays were performed to investigate the gene expression profiles of S. cerevisiae KKU-VN8. Differential expression of genes encoding heat-shock proteins (HSP82, HSP104, SSA4), genes involved in trehalose metabolism (TPS1, TPS2, NTH1) and genes involved the glycolytic pathway (ADH1, ADH2, CDC19) at various time points during fermentation was observed. The expression levels of HSP82, HSP104, SSA4, ADH1 and CDC19 were significantly higher than those of the controls (10.2-, 4-, 8-, 8.9- and 5.9-fold higher, respectively). In contrast, the expression levels of TPS1, TPS2, NTH1 and ADH2 were approx. 2-fold less than those of the controls.

Conclusions

The highly expressed genes encoding heat-shock proteins, HSP82 and SSA4, potentially play an important role in helping S. cerevisiae KKU-VN8 cope with various stresses that occur during high-temperature fermentation, leading to higher ethanol production efficiency.

     

Raw starch conversion by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> amylases

Background

Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol.

Results

The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l^-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l^-1 (0.038 and 0.028 g l^-1 h^-1), respectively, after 10 days. With a substrate load of 200 g l^-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l^-1 ethanol (0.58 g l^-1 h^-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l^-1 ethanol (0.36 g l^-1 h^-1).

Conclusions

In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l^-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases.

     

SSF of steam-pretreated wheat straw with the addition of saccharified or fermented wheat meal in integrated bioethanol production

Background

Integration of second-generation (2G) bioethanol production with existing first-generation (1G) production may facilitate commercial production of ethanol from cellulosic material. Since 2G hydrolysates have a low sugar concentration and 1G streams often have to be diluted prior to fermentation, mixing of streams is beneficial. Improved ethanol concentrations in the 2G production process lowers energy demand in distillation, improves overall energy efficiency and thus lower production cost. There is also a potential to reach higher ethanol yields, which is required in economically feasible ethanol production. Integrated process scenarios with addition of saccharified wheat meal (SWM) or fermented wheat meal (FWM) were investigated in simultaneous saccharification and (co-)fermentation (SSF or SSCF) of steam-pretreated wheat straw, while the possibility of recovering the valuable protein-rich fibre residue from the wheat was also studied.

Results

The addition of SWM to SSF of steam-pretreated wheat straw, using commercially used dried baker’s yeast, S. cerevisiae, resulted in ethanol concentrations of about 60 g/L, equivalent to ethanol yields of about 90% of the theoretical. The addition of FWM in batch mode SSF was toxic to baker’s yeast, due to the ethanol content of FWM, resulting in a very low yield and high accumulation of glucose. The addition of FWM in fed-batch mode still caused a slight accumulation of glucose, but the ethanol concentration was fairly high, 51.2 g/L, corresponding to an ethanol yield of 90%, based on the amount of glucose added.In batch mode of SSCF using the xylose-fermenting, genetically modified S. cerevisiae strain KE6-12, no improvement was observed in ethanol yield or concentration, compared with baker’s yeast, despite the increased xylose utilization, probably due to the considerable increase in glycerol production. A slight increase in xylose consumption was seen when glucose from SWM was fed at a low feed rate, after 48 hours, compared with batch SSCF. However, the ethanol yield and concentration remained in the same range as in batch mode.

Conclusion

Ethanol concentrations of about 6% (w/v) were obtained, which will result in a significant reduction in the cost of downstream processing, compared with SSF of the lignocellulosic substrate alone. As an additional benefit, it is also possible to recover the protein-rich residue from the SWM in the process configurations presented, providing a valuable co-product.

     

Mating of natural <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for improved glucose fermentation and lignocellulosic inhibitor tolerance

Natural Saccharomyces cerevisiae isolates from vineyards in the Western Cape, South Africa were evaluated for ethanol production in industrial conditions associated with the production of second-generation biofuels. The strains displayed high phenotypic diversity including the ability to grow at 45 °C and in the presence of 20% (v/v) ethanol, strain YI13. Strains HR4 and YI30 were inhibitor-tolerant under aerobic and oxygen-limited conditions, respectively. Spore-to-spore hybridization generated progeny that displayed heterosis, including increased ethanol productivity and improved growth in the presence of a synthetic inhibitor cocktail. Hybrid strains HR4/YI30#6 and V3/YI30#6 were able to grow at a high salt concentration (2 mol/L NaCl) with V3/YI30#6 also able to grow at a high temperature (45 °C). Strains HR4/YI30#1 and #3 were inhibitor-tolerant, with strain HR4/YI30#3 having similar productivity (0.36 ± 0.0036 g/L per h) as the superior parental strain, YI30 (0.35 ± 0.0058 g/L per h). This study indicates that natural S. cerevisiae strains display phenotypic variation and heterosis can be achieved through spore-to-spore hybridization. Several of the phenotypes (temperature-, osmo-, and inhibitor tolerance) displayed by both the natural strains and the generated progeny were at the maximum conditions reported for S. cerevisiae strains.     

A novel thermostable and organic solvent-tolerant lipase from <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> YB103: screening,purification and characterization

Thermostable lipases offer major biotechnological advantages over mesophilic lipases. In this study, an intracellular thermostable and organic solvent-tolerant lipase-producing strain YB103 was isolated from soil samples and identified taxonomically as Xanthomonas oryzae pv. oryzae. The lipase from X. oryzae pv. oryzae YB103 (LipXO) was purified 101.1-fold to homogeneity with a specific activity of 373.9 U/mg. The purified lipase showed excellent thermostability, exhibiting 51.1 % of its residual activity after incubation for 3 days at 70 °C. The enzyme showed optimal activity at 70 °C, suggesting it is a thermostable lipase. LipXO retained 75.1–154.1 % of its original activity after incubation in 20 % (v/v) hydrophobic organic solvents at 70 °C for 24 h. Furthermore, LipXO displayed excellent stereoselectivity (e.e._p >99 %) toward (S)-1-phenethyl alcohol in n-hexane. These unique properties of LipXO make it promising as a biocatalyst for industrial processes.     

Very high gravity ethanol and fatty acid production of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> without amino acid and vitamin

Very high gravity (VHG) fermentation is the mainstream technology in ethanol industry, which requires the strains be resistant to multiple stresses such as high glucose concentration, high ethanol concentration, high temperature and harsh acidic conditions. To our knowledge, it was not reported previously that any ethanol-producing microbe showed a high performance in VHG fermentations without amino acid and vitamin. Here we demonstrate the engineering of a xylose utilizing recombinant Zymomonas mobilis for VHG ethanol fermentations. The recombinant strain can produce ethanol up to 136 g/L without amino acid and vitamin with a theoretical yield of 90 %, which is significantly superior to that produced by all the reported ethanol-producing strains. The intracellular fatty acids of the bacterial were about 16 % of the bacterial dry biomass, with the ratio of ethanol:fatty acids was about 273:1 (g/g). The recombinant strain was achieved by a multivariate-modular strategy tackles with the multiple stresses which are closely linked to the ethanol productivity of Z. mobilis. The over-expression of metB/yfdZ operon enabled the growth of the recombinant Z. mobilis in a chemically defined medium without amino acid and vitamin; and the fatty acids overproduction significantly increased ethanol tolerance and ethanol production. The coupled production of ethanol with fatty acids of the Z. mobilis without amino acid and vitamin under VHG fermentation conditions may permit a significant reduction of the production cost of ethanol and microbial fatty acids.     

A novel strategy for production of ethanol and recovery of xylose from simulated corncob hydrolysate

Objectives

To develop a xylose-nonutilizing Escherichia coli strain for ethanol production and xylose recovery.

Results

Xylose-nonutilizing E. coli CICIM B0013-2012 was successfully constructed from E. coli B0013-1030 (pta-ack, ldhA, pflB, xylH) by deletion of frdA, xylA and xylE. It exhibited robust growth on plates containing glucose, arabinose or galactose, but failed to grow on xylose. The ethanol synthesis pathway was then introduced into B0013-2012 to create an ethanologenic strain B0013-2012PA. In shaking flask fermentation, B0013-2012PA fermented glucose to ethanol with the yield of 48.4 g/100 g sugar while xylose remained in the broth. In a 7-l bioreactor, B0013-2012PA fermented glucose, galactose and arabinose in the simulated corncob hydrolysate to 53.4 g/l ethanol with the yield of 48.9 g/100 g sugars and left 69.6 g/l xylose in the broth, representing 98.6% of the total xylose in the simulated corncob hydrolysate.

Conclusions

By using newly constructed strain B0013-2012PA, we successfully developed an efficient bioprocess for ethanol production and xylose recovery from the simulated corncob hydrolysate.

     

Genome sequence of <Emphasis Type="Italic">Candida versatilis</Emphasis> and comparative analysis with other yeast

The genome of Candida versatilis was sequenced to understand its characteristics in soy sauce fermentation. The genome size of C. versatilis was 9.7 Mb, the content of G + C was 39.74 %, scaffolds of N50 were 1,229,640 bp in length, containing 4711 gene. There were predicted 269 tRNA genes and 2201 proteins with clear function. Moreover, the genome information of C. versatilis was compared with another salt-tolerant yeast Zygosaccharomyces rouxii and the model organism Saccharomyces cerevisiae. C. versatilis and Z. rouxii genome size was close and both smaller than 12.1 for the Mb of S. cerevisiae. Using the OrthoMCL protein, three genomes were divided into 4663 groups. There were about 3326 homologous proteins in C. versatilis, Z. rouxii and S. cerevisiae.     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.     

Recalcitrance Assessment of the Agro-industrial Residues from Five <Emphasis Type="Italic">Agave</Emphasis> Species: Ionic Liquid Pretreatment,Saccharification and Structural Characterization

Agave has recently shown its potential as a bioenergy feedstock with promising features such as higher biomass productivity than leading bioenergy feedstock while at the same time being drought-resistant with low water requirements and high sugar to ethanol conversion using ionic liquid (IL) pretreatment. IL pretreatment was studied to develop the first direct side-by-side comparative recalcitrance assessment of the agro-industrial residues from five Agave species [Agave americana (AME), A. angustifolia (ANG), A. fourcroydes (FOU), A. salmiana (SAL), and A. tequilana (TEQ)] using compositional analysis, X-ray diffraction, and the lignin syringyl/guaiacyl subunit ratio (S/G) by pyrolysis molecular beam mass spectrometry (PyMBMS). Prominent calcium oxalate peaks were found only in unpretreated AME, SAL, and TEQ. The S/G ratios of all five unpretreated Agave species were between 1.27 and 1.57 while the IL-pretreated samples were from 1.39 to 1.72. The highest overall sugar production was obtained with IL-pretreated FOU with 492 mg glucose/g biomass and 157 mg xylose/g biomass at 120 °C and 3 h using 1-ethyl-3-methylimidazolium acetate ([C₂C₁Im][OAc]). An estimated theoretical ethanol yield from the studied agro-industrial residues from the five Agave species was in the range of 1060 to 5800 L ethanol/ha/year. These comparison results demonstrate the potential of the Agave spp. as a suitable biofuel feedstock which can be employed within a biorefinery scheme.     

Deletion of the <Emphasis Type="Italic">Clostridium thermocellum recA</Emphasis> gene reveals that it is required for thermophilic plasmid replication but not plasmid integration at homologous DNA sequences

A limitation to the engineering of cellulolytic thermophiles is the availability of functional, thermostable (≥?60 °C) replicating plasmid vectors for rapid expression and testing of genes that provide improved or novel fuel molecule production pathways. A series of plasmid vectors for genetic manipulation of the cellulolytic thermophile Caldicellulosiruptor bescii has recently been extended to Clostridium thermocellum, another cellulolytic thermophile that very efficiently solubilizes plant biomass and produces ethanol. While the C. bescii pBAS2 replicon on these plasmids is thermostable, the use of homologous promoters, signal sequences and genes led to undesired integration into the bacterial chromosome, a result also observed with less thermostable replicating vectors. In an attempt to overcome undesired plasmid integration in C. thermocellum, a deletion of recA was constructed. As expected, C. thermocellum ?recA showed impaired growth in chemically defined medium and an increased susceptibility to UV damage. Interestingly, we also found that recA is required for replication of the C. bescii thermophilic plasmid pBAS2 in C. thermocellum, but it is not required for replication of plasmid pNW33N. In addition, the C. thermocellum recA mutant retained the ability to integrate homologous DNA into the C. thermocellum chromosome. These data indicate that recA can be required for replication of certain plasmids, and that a recA-independent mechanism exists for the integration of homologous DNA into the C. thermocellum chromosome. Understanding thermophilic plasmid replication is not only important for engineering of these cellulolytic thermophiles, but also for developing genetic systems in similar new potentially useful non-model organisms.     

Effects of Probiotics on Survival,Growth and Digestive Enzymes Activities in Freshwater Prawn <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> (De Man 1879)

A study was conducted to examine the effects of three probiotics, Lactobacillus sporogenes, Bacillus subtilis and Saccharomyces cerevisiae on the survival, growth and digestive enzymes activities of the freshwater prawn Macrobrachium rosenbergii post larvae (PL). The probiotics, L. sporogenes (4 %), B. subtilis (3 %) and S. cerevisiae (4 %) were taken and mixed with basal diet. Diet without probiotics served as control. These probiotics diets were fed to M. rosenbergii PL for a period of 60 days. After the feeding trail, the growth parameters such as survival, weight gain, specific growth rate and protein efficiency rate were found to be significantly (P < 0.05) higher in 4 % S. cerevisiae incorporated diet fed PL when compared with control. In the case of feed conversion rate just the reverse was seen (P < 0.05) at this concentration. This indicates its superior quality among different concentrations of probiotics tested. Activities of digestive enzymes, such as protease, amylase and lipase were significantly (P < 0.05) higher at this concentration (4 % S. cerevisiae). Some of essential and non-essential amino acids also significantly elevated in probiotics supplemented diet fed prawns. This study indicated that probiotics, S. cerevisiae incorporated diets were beneficial for M. rosenbergii in terms of increasing growth, enzyme and amino acid production.     

Cloning and expression of <Emphasis Type="Italic">Perilla frutescens FAD2</Emphasis> gene and polymorphism analysis among cultivars

?12 fatty acid desaturase (FAD2) is a key enzyme for linoleic acid and linolenic acid biosynthesis. Perilla frutescens is a special oil plant species with highest linolenic acid content. In this study, based on RACE, two alleles for one FAD2 gene were isolated from P. frutescens cultivar C2: the 3956 bp PfFAD2a and the 3959 bp PfFAD2b, both with a full-length cDNA of 1526 bp, and both encoding a 382aa basic protein. The alleles have identities of over 98%, and their encoded proteins differ only by substitution of a strongly similar residue. Saccharomyces cerevisiae heterologous expression suggested that PfFAD2a/b both encode a bio-functional FAD2 enzyme. Phylogenetic analyses indicated that PfFAD2 shows the highest homologies to FAD2 genes from dicots such as Boraginaceae and Burseraceae. PfFAD2a/b expressions are mainly restricted to developing seeds. PfFAD2a/b expression in the seedling leaf is upregulated by cold (4 °C) and repressed by heat (42 °C). Each of the eight cultivars contains two alleles for one PfFAD2 and 40 SNP sites are found. One allelic gene in cultivars C1 and P1 is pseudogene because of premature stop codon mutation in 5′ coding region. All other normal PfFAD2 genes/allelic genes encode identical or very similar proteins. PfFAD2a/b expression level in developing seeds also varies among the eight cultivars. This study provides systemic molecular and functional features of PfFAD2 and enables its application in the study of plant fatty acids traits.     

Genomic analysis of a xylose operon and characterization of novel xylose isomerase and xylulokinase from <Emphasis Type="Italic">Bacillus coagulans</Emphasis> NL01

Objective

To investigate the xylose operon and properties of xylose isomerase and xylulokinase in Bacillus coagulans that can effectively ferment xylose to lactic acid.

Results

The xylose operon is widely present in B. coagulans. It is composed of four putative ORFs. Novel xylA and xylB from B. coagulans NL01 were cloned and expressed in Escherichia coli. Sequence of xylose isomerase was more conserved than that of xylulokinase. Both the enzymes exhibited maximum activities at pH 7–8 but with a high temperature maximum of 80–85 °C, divalent metal ion was prerequisite for their activation. Xylose isomerase and xylulokinase were most effectively activated by Ni²⁺ and Co²⁺, respectively.

Conclusions

Genomic analysis of xylose operon has contributed to understanding xylose metabolism in B. coagulans and the novel xylose isomerase and xylulokinase might provide new alternatives for metabolic engineering of other strains to improve their fermentation performance on xylose.