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Cryptococcus terricola is a promising oleaginous yeast for biodiesel production from starch through consolidated bioprocessing.

ABSTRACT: Starch is considered a potential feedstock for biofuel production, particularly in light of the large-scale landfilling of food waste and other starchy materials worldwide. Lipid accumulation by oleaginous yeast is a promising method for biodiesel production from starch. However, most oleaginous yeasts are grown on monosaccharides or oligosaccharides because they cannot directly utilize starch. We therefore investigated the starch-assimilation ability of 1,200 yeasts. We found that Cryptococcus terricola could be used for fuel production through consolidated bioprocessing. C. terricola JCM 24523 exhibited the highest lipid content of 61.96% on medium with 5% starch at 10 days. Fatty acid methyl ester analysis showed that this strain produced high proportions of C16:0 and C18 fatty acids when grown on starch, which are ideal for use in biodiesel. Considering the yield and cost, lipids derived from starch using C. terricola would be a promising alternative source for biodiesel production.

SUBMITTER: Tanimura A

PROVIDER: S-EPMC3998020 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Cryptococcus terricola is a promising oleaginous yeast for biodiesel production from starch through consolidated bioprocessing.

Tanimura Ayumi A Takashima Masako M Sugita Takashi T Endoh Rikiya R Kikukawa Minako M Yamaguchi Shino S Sakuradani Eiji E Ogawa Jun J Ohkuma Moriya M Shima Jun J

Scientific reports 20140424

Starch is considered a potential feedstock for biofuel production, particularly in light of the large-scale landfilling of food waste and other starchy materials worldwide. Lipid accumulation by oleaginous yeast is a promising method for biodiesel production from starch. However, most oleaginous yeasts are grown on monosaccharides or oligosaccharides because they cannot directly utilize starch. We therefore investigated the starch-assimilation ability of 1,200 yeasts. We found that Cryptococcus ...[more]

PMID: 24758986

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Project description:Natural yeast with superior fermentative traits can serve as a platform for the development of recombinant strains that can be used to improve the sustainability of bioethanol production from starch. This process will benefit from a consolidated bioprocessing (CBP) approach where an engineered strain producing amylases directly converts starch into ethanol. The yeast Saccharomyces cerevisiae L20, previously selected as outperforming the benchmark yeast Ethanol Red, was here subjected to a comparative genomic investigation using a dataset of industrial S. cerevisiae strains. Along with Ethanol Red, strain L20 was then engineered for the expression of α-amylase amyA and glucoamylase glaA genes from Aspergillus tubingensis by employing two different approaches (delta integration and CRISPR/Cas9). A correlation between the number of integrated copies and the hydrolytic abilities of the recombinants was investigated. L20 demonstrated important traits for the construction of a proficient CBP yeast. Despite showing a close relatedness to commercial wine yeast and the benchmark Ethanol Red, a unique profile of gene copy number variations (CNVs) was found in L20, mainly encoding membrane transporters and secretion pathway proteins but also the fermentative metabolism. Moreover, the genome annotation disclosed seven open reading frames (ORFs) in L20 that are absent in the reference S288C genome. Genome engineering was successfully implemented for amylase production. However, with equal amylase gene copies, L20 proved its proficiency as a good enzyme secretor by exhibiting a markedly higher amylolytic activity than Ethanol Red, in compliance to the findings of the genomic exploration. The recombinant L20 dT8 exhibited the highest amylolytic activity and produced more than 4 g/L of ethanol from 2% starch in a CBP setting without the addition of supplementary enzymes. Based on the performance of this strain, an amylase/glucoamylase ratio of 1:2.5 was suggested as baseline for further improvement of the CBP ability. Overall, L20 showed important traits for the future construction of a proficient CBP yeast. As such, this work shows that natural S. cerevisiae strains can be used for the expression of foreign secreted enzymes, paving the way to strain improvement for the starch-to-bioethanol route.

Project description:BACKGROUND: The recalcitrant nature of hemicellulosic materials and the high cost in depolymerization are the primary obstacles preventing the use of xylan as feedstock for fuel and chemical production. Consolidated bioprocessing, incorporating enzyme-generating, biomass-degrading and bioproduct-producing capabilities into a single microorganism, could potentially avoid the cost of the dedicated enzyme generation in the process of xylan utilization. In this study, we engineered Escherichia coli strains capable of exporting three hemicellulases to the broth for the succinate production directly from beechwood xylan. RESULTS: Xylanases were extracellular environment-directed by fusing with OsmY. Subsequently, twelve variant OsmY fused endoxylanase-xylosidase combinations were characterized and tested. The combination of XynC-A from Fibrobacter succinogenes S85 and XyloA from Fusarium graminearum which appeared to have optimal enzymatic properties was identified as the best choice for xylan hydrolysis (0.18 ± 0.01 g/l protein in the broth with endoxylanase activity of 12.14 ± 0.34 U/mg protein and xylosidase activity of 92 ± 3 mU/mg protein at 8 h after induction). Further improvements of hemicellulases secretion were investigated by lpp deletion, dsbA overexpression and expression level optimization. With co-expression of ?-arabinofuranosidase, the engineered E. coli could hydrolyze beechwood xylan to pentose monosaccharides. The hemicellulolytic capacity was further integrated with a succinate-producing strain to demonstrate the production of succinate directly from xylan without externally supplied hydrolases and any other organic nutrient. The resulting E. coli Z6373 was able to produce 0.37 g/g succinate from xylan anaerobically equivalent to 76% of that from xylan acid hydrolysates. CONCLUSIONS: This report represents a promising step towards the goal of hemicellulosic chemical production. This engineered E. coli expressing and secreting three hemicellulases demonstrated a considerable succinate production on the released monosaccharides from xylan. The ability to use lower-cost crude feedstock will make biological succinate production more economically attractive.

Project description:BackgroundThe biomanufacturing of D-glucaric acid has attracted increasing interest because it is one of the top value-added chemicals produced from biomass. Saccharomyces cerevisiae is regarded as an excellent host for D-glucaric acid production.ResultsThe opi1 gene was knocked out because of its negative regulation on myo-inositol synthesis, which is the limiting step of D-glucaric acid production by S. cerevisiae. We then constructed the biosynthesis pathway of D-glucaric acid in S. cerevisiae INVSc1 opi1Δ and obtained two engineered strains, LGA-1 and LGA-C, producing record-breaking titers of D-glucaric acid: 9.53 ± 0.46 g/L and 11.21 ± 0.63 g/L D-glucaric acid from 30 g/L glucose and 10.8 g/L myo-inositol in fed-batch fermentation mode, respectively. However, LGA-1 was preferable because of its genetic stability and its superior performance in practical applications. There have been no reports on D-glucaric acid production from lignocellulose. Therefore, the biorefinery processes, including separated hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF) and consolidated bioprocessing (CBP) were investigated and compared. CBP using an artificial microbial consortium composed of Trichoderma reesei (T. reesei) Rut-C30 and S. cerevisiae LGA-1 was found to have relatively high D-glucaric acid titers and yields after 7 d of fermentation, 0.54 ± 0.12 g/L D-glucaric acid from 15 g/L Avicel and 0.45 ± 0.06 g/L D-glucaric acid from 15 g/L steam-exploded corn stover (SECS), respectively. In an attempt to design the microbial consortium for more efficient CBP, the team consisting of T. reesei Rut-C30 and S. cerevisiae LGA-1 was found to be the best, with excellent work distribution and collaboration.ConclusionsTwo engineered S. cerevisiae strains, LGA-1 and LGA-C, with high titers of D-glucaric acid were obtained. This indicated that S. cerevisiae INVSc1 is an excellent host for D-glucaric acid production. Lignocellulose is a preferable substrate over myo-inositol. SHF, SSF, and CBP were studied, and CBP using an artificial microbial consortium of T. reesei Rut-C30 and S. cerevisiae LGA-1 was found to be promising because of its relatively high titer and yield. T. reesei Rut-C30 and S. cerevisiae LGA-1were proven to be the best teammates for CBP. Further work should be done to improve the efficiency of this microbial consortium for D-glucaric acid production from lignocellulose.

Dataset Information

Cryptococcus terricola is a promising oleaginous yeast for biodiesel production from starch through consolidated bioprocessing.

Publications

Cryptococcus terricola is a promising oleaginous yeast for biodiesel production from starch through consolidated bioprocessing.

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