INU1 Gene Research Articles

Engineering Yarrowia lipolytica for the Synthesis of Glutathione from Organic By-Products.

Tripeptide glutathione, which plays important roles in many cellular mechanisms, is also a biotechnology-oriented molecule with applications in medicine, food and cosmetic. Here, the engineering of the yeast Yarrowia lipolytica for the production of this metabolite at high titer values from various agro-industrial by-products is reported. The constitutive overexpression of the glutathione biosynthetic genes GSH1 and GSH2 encoding respectively γ-glutamylcysteine synthetase and glutathione synthetase, together with the INU1 gene from Kluyveromyces marxianus encoding inulinase yielded a glutathione titer value and a productivity of 644 nmol/mg protein and 510 µmol/gDCW, respectively. These values were obtained during bioreactor batch cultures in a medium exclusively comprising an extract of Jerusalem artichoke tuber, used as a source of inulin, and ammonium sulfate, used as a nitrogen source.

Inulinase hyperproduction by Kluyveromyces marxianus through codon optimization, selection of the promoter, and high-cell-density fermentation for efficient inulin hydrolysis

This study aimed to overexpress an inulinase gene in Kluyveromyces marxianus to achieve the inulinase overproduction and preparation of ultra-high-fructose syrup. First, the inulinase gene (INU1 gene) was overexpressed through codon optimization and selection of a suitable promoter. Then, the inulinase was overproduced by high-cell-density fermentation. Finally, ultra-high-fructose syrup was prepared. It was found that optimization of the codons of the native INU1 gene encoding inulinase from Kluyveromyces marxianus KM-0 made a recombinant strain KM-N70 carrying the optimized INU1N gene produce 251.4 U/mL of the inulinase activity. Furthermore, inulinase activity produced by a recombinant KM-KN16 strain carrying the optimized INU1N gene directed by the native TPS1 promoter from K. marxianus KM-0 reached 338.5 U/mL and expression level of the optimized INU1N gene in the recombinant KM-KN16 strain was also greatly enhanced. During a 10-L fermentation, the recombinant KM-KN16 strain could produce 374.3 U/mL of inulinase activity within 24 h, while during a high-cell-density fed-batch fermentation, the recombinant KM-KN16 strain could produce 896.1 U/mL of inulinase activity and OD600nm value of its culture reached 108. The crude inulinase preparation obtained in this study had an inulinase activity of 18,699.8 ± 736.4 U/g of the crude preparation. It was found that 90.3% of 332.4 g/L of inulin was hydrolyzed to produce 41.0 g/L of glucose and 256.0 g/L of fructose and 91.1% of 328.2 g/L of inulin in the extract of the tubers of Jerusalem artichoke was hydrolyzed to produce 48.3 g/L of glucose and 250.5 g/L of fructose by the crude inulinase preparation (75 U/g of the substrate) within 8 h. The hydrolysates contained major monosaccharides and a trace amount of trisaccharides and the monosaccharides were composed of around 85% fructose and 15% glucose. So far, any other yeasts available have produced only up to 120 U/mL of inulinase activity. Together, this made the recombinant KM-KN16 strain be the best inulinase producer at this moment. The inulinase activity of 18,699.8 ± 736.4 U/g of the crude preparation and the ultra-high-fructose syrup with 41.0 g/L of glucose and 256.0 g/L of fructose were obtained. The inulinase activity obtained in this study was the highest among all the inulinase activities produced by yeast, fungal, and bacterial strains obtained so far.

Production of high titer of citric acid from inulin

BackgroundCitric acid is considered as the most economically feasible product of microbiological production, therefore studies on cheap and renewable raw materials for its production are highly desirable. In this study citric acid was synthesized by genetically engineered strains of Yarrowia lipolytica from widely available, renewable polysaccharide – inulin. Hydrolysis of inulin by the Y. lipolytica strains was established by expressing the inulinase gene (INU1 gene; GenBank: X57202.1) with its native secretion signal sequence was amplified from genomic DNA from Kluyveromyces marxianus CBS6432. To ensure the maximum citric acid titer, the optimal cultivation strategy–repeated-batch culture was applied.ResultsThe strain Y. lipolytica AWG7 INU 8 secreted more than 200 g dm− 3 of citric acid during repeated-batch culture on inulin, with a productivity of 0.51 g dm− 3 h− 1 and a yield of 0.85 g g− 1.ConclusionsThe citric acid titer obtained in the proposed process is the highest value reported in the literature for Yarrowia yeast. The obtained results suggest that citric acid production from inulin by engineered Y. lipolytica may be a very promising technology for industrial citric acid production.

Genetic and physiological basis for antibody production by Kluyveromyces marxianus

Kluyveromyces marxianus is a thermotolerant, crabtree-negative yeast, which preferentially directs metabolism (e.g., from the tricarboxylic acid cycle) to aerobic alcoholic fermentation. Thus K. marxianus has great potential for engineering to produce various materials under aerobic cultivation conditions. In this study, we engineered K. marxianus to produce and secrete a single-chain antibody (scFv), a product that is highly valuable but has historically proven difficult to generate at large scale. scFv production was obtained with strains carrying either plasmid-borne or genomically integrated constructs using various combinations of promoters (PMDH1 or PACO1) and secretion signal peptides (KmINUss or Scα-MFss). As the wild-type K. marxianus secretes endogenous inulinase predominantly, the corresponding INU1 gene was disrupted using a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)—associated protein (CRISPR–Cas9) system to re-direct resources to scFv production. Genomic integration was used to replace INU1 with sequences encoding a fusion of the INU1 signal peptide to scFv; the resulting construct yielded the highest scFv production among the strains tested. Optimization of growth conditions revealed that scFv production by this strain was enhanced by incubation at 30 °C in xylose medium containing 200 mM MgSO4. These results together demonstrate that K. marxianus has the potential to serve as a host strain for antibody production.

Overexpression of an Inulinase Gene in an Oleaginous Yeast, Aureobasidium melanogenum P10, for Efficient Lipid Production from Inulin

In this study, in order to directly and efficiently convert inulin into a single-cell oil (SCO), an INU1 gene encoding inulinase from Kluyveromyces marxianus was integrated into the genomic DNA and actively expressed in an SCO producer Aureobasidium melanogenum P10. The transformant API41 obtained produced 28.5 U/mL of inulinase and its wild-type strain P10 yielded only 8.62 U/mL. Most (97.5%) of the inulinase produced by the transformant API41 was secreted into the culture. During a 10-L fermentation, 66.2% (w/w) lipid in the yeast cells of the transformant API41 and 14.38 g/L of cell dry weight were attained from inulin of 80.0 g/L within 120 h, high inulinase activity (23.7 U/mL) was also produced within 72 h, and the added inulin was actively hydrolyzed. This confirmed that the genetically engineered yeast of A. melanogenum P10 is suitable for direct production of lipids from inulin. The lipids produced could be used as feedstocks for biodiesel production.

Genetic Modification of the Marine-Isolated Yeast Aureobasidium melanogenum P16 for Efficient Pullulan Production from Inulin.

In this study, in order to directly and efficiently convert inulin into pullulan, the INU1 gene from Kluyveromyces maximum KM was integrated into the genomic DNA and actively expressed in the high pullulan producer Aureobasidium melanogenum P16 isolated from the mangrove ecosystem. After the ability to produce pullulan from inulin by different transformants was examined, it was found that the recombinant strain EI36, one of the transformants, produced 40.92 U/ml of inulinase activity while its wild-type strain P16 only yielded 7.57 U/ml of inulinase activity. Most (99.27 %) of the inulinase produced by the recombinant strain EI36 was secreted into the culture. During the 10-l fermentation, 70.57 ± 1.3 g/l of pullulan in the fermented medium was attained from inulin (138.0 g/l) within 108 h, high inulinase activity (42.03 U/ml) was produced within 60 h, the added inulin was actively hydrolyzed by the secreted inulinase, and most of the reducing sugars were used by the recombinant strain EI36. This confirmed that the genetically engineered yeast of A. melanogenum strain P16 was suitable for direct pullulan production from inulin.

Cloning and characterization of an inulinase gene from the marine yeast Candida membranifaciens subsp. flavinogenie W14-3 and its expression in Saccharomyces sp. W0 for ethanol production.

The INU1 gene encoding an exo-inulinase from the marine-derived yeast Candida membranifaciens subsp. flavinogenie W14-3 was cloned and characterized. It had an open reading frame of 1,536 bp long encoding an inulinase. The coding region of it was not interrupted by any intron. The cloned gene encoded 512 amino acid residues of a protein with a putative signal peptide of 23 amino acids and a calculated molecular mass of 57.8 kDa. The protein sequence deduced from the inulinase gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP FS and Q. The protein also had six conserved putative N-glycosylation sites. The deduced inulinase from the yeast strain W14-3 was found to be closely related to that from Candida kutaonensis sp. nov. KRF1, Kluyveromyces marxianus, and Cryptococcus aureus G7a. The inulinase gene with its signal peptide encoding sequence was subcloned into the pMIRSC11 expression vector and expressed in Saccharomyces sp. W0. The recombinant yeast strain W14-3-INU-112 obtained could produce 16.8 U/ml of inulinase activity and 12.5 % (v/v) ethanol from 250 g/l of inulin within 168 h. The monosaccharides were detected after the hydrolysis of inulin with the crude inulinase (the yeast culture). All the results indicated that the cloned gene and the recombinant yeast strain W14-3-INU-112 had potential applications in biotechnology.

Inulinase production by the yeast Kluyveromyces marxianus with the disrupted MIG1 gene and the over-expressed inulinase gene

Kluyveromyces marxianus has been widely used in food industries and inulinase produced by the yeast has many applications. In order to greatly enhance inulinase production by the yeast, it is very important to both derepress glucose repression and overexpress the native inulinase gene. The MIG1 gene in the yeast K. marxianus KM-0 was disrupted. It was found that a glucose-derepressed mutant (KM-69) of K. marxianus KM-0 could produce 94.6 units of inulinase activity per ml in the inulin medium within 36h. In order to further increase inulinase production, the native inulinase gene was over-expressed in the glucose-derepressed mutant. During the 10-l fermentation, the recombinant strain KM-526 yielded 133.5 units of inulinase activity per ml within very short time (24h). The results also showed that inulinase produced by the recombinant strain KM-526 could more effectively convert inulin into monosaccharides than that produced by K. marxianus KM-0. The results indicate that it is very important to delete the MIG1 gene in K. marxianus KM-0 and over-express the native INU1 gene in order to further enhance inulinase yield within the short time by this yeast and for efficient hydrolysis of inulin. The recombinant strain KM-526 and its inulinase have highly potential applications in food industries.

Cloning, characterization and heterelogous expression of the INU1 gene from Cryptococcus aureus HYA

The INU1 gene (Accession number: JX073660) encoding exo-inulinase from Cryptococcus aureus HYA was cloned and characterized. The gene had an open reading frame (ORF) of 1653bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 551 amino acid residues of a protein with a putative signal peptide of 23 amino acids and the calculated molecular mass of 59.5kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP, FS and Q. It also had two conserved putative N-glycosylation sites. The inulinase from C. aureus HYA was found to be closely related to that from Kluyveromyces marxianus and Pichia guilliermondii. The inulinase gene without the signal sequence was subcloned into pPICZaA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum inulinase activity of 16.3±0.24U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. The optimal temperature and pH for action of the enzyme were 50°C and 5.0, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin with the purified recombinant inulinase.

Both Decrease in ACL1 Gene Expression and Increase in ICL1 Gene Expression in Marine-Derived Yeast Yarrowia lipolytica Expressing INU1 Gene Enhance Citric Acid Production from Inulin

In this study, some of the ATP-citrate lyase genes (ACL1) were deleted and the copy number of the iso-citrate lyase gene (ICL1) was increased in the marine-derived yeast Yarrowia lipolytica SWJ-1b displaying the recombinant inulinase. It was found that lipid content and iso-citric acid in the transformant 30 obtained were greatly reduced and citric acid production was greatly enhanced. It was also found that the ACL1 gene expression and ATP-citrate lyase activity in the transformant 30 were declined and the ICL1 gene expression and iso-citrate lyase activity were promoted. During the 2-l fermentation, 84.0g/l of citric acid and 1.8g/l of iso-citric acid in the fermented medium were attained from 10.0% of inulin by the transformant 30 within 214h. The results showed that only 0.36% of the residual reducing sugar and 1.0% of the residual total sugar were left in the fermented medium, suggesting that 89.6% of the total sugar was used for citric acid production and cell growth by the transformant 30.

18S rDNA integration of the exo-inulinase gene into chromosomes of the high ethanol producing yeast Saccharomyces sp. W0 for direct conversion of inulin to bioethanol

Abstract Inulin and inulin-containing materials are good substrates for bioethanol production. In order to construct genetically stable recombinant yeast cells carrying the inulinase gene for bioethanol production from inulin, a new 18S rDNA integration vector was constructed in this study. After the INU1 gene encoding exo-inulinase was ligated into the 18S rDNA integration vector, transformed into uracil mutant of Saccharomyces sp. W0 and integrated into its chromosomes, the transformant MguINU1-04 obtained could produce 34.8 U cm −3 of inulinase activity within 72 h. The transformant could stably produce high activity of inulinase after five sequential batch cultivations. During 5-l fermentation, ethanol concentration in the fermentation medium containing 30.0% inulin was 14.7% (v/v) and the ethanol productivity was over 0.386 g of ethanol per g of inulin. When the tuber meal of Jerusalem artichoke (50.0%) was fermented into ethanol by the transformant MguINU1-04, 12.6% (v/v) ethanol was produced within 120 h and the ethanol productivity was over 0.331 g of ethanol per g of sugar. Therefore, inulin and inulin in the tuber meal of Jerusalem artichoke could be directly converted into high concentrations of ethanol by the engineered Saccharomyces sp. W0 carrying the inulinase gene.

Direct conversion of inulin into single cell protein by the engineered Yarrowia lipolytica carrying inulinase gene

The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the expression plasmid pINA1317 and expressed in the cells of the yeast Yarrowia lipolytica with high content of protein. The activity of the secreted inulinase was found to be 43.1 ± 0.9 U/ml after cell growth for 80 h. When the engineered yeast cells were grown in the medium containing 4.0% inulin in 2-l fermentor, crude protein in the cells and cell mass could reach 47.5% and 20.1 g/l, respectively within 72 h. Furthermore, when the engineered yeast cells were grown in the medium containing 8.0% the meal of Jerusalem artichoke tuber in 2-l fermentor, crude protein and cell mass could reach 53.7% and 20.8 g/l, respectively within 80 h. Therefore, the engineered Y. lipolytica could be further used to produce single cell protein from inulin and inulin-containing materials.

Expression of inulinase gene in the oleaginous yeast Yarrowia lipolytica and single cell oil production from inulin-containingmaterials

Yarrowia lipolytica ACA-DC 50109 has been reported to be an oleaginous yeast and significant quantities of lipids were accumulated inside the yeast cells. In this study, the INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the expression plasmid pINA1317 and expressed in the cells of the oleaginous yeast. The activity of the inulinase with 6×His tag secreted by the transformant Z31 obtained was found to be 41.7 U mL −1 after cell growth for 78 h. After optimization of the medium and cultivation conditions for single cell oil production, the transformant could accumulate 46.3% (w/w) oil from inulin in its cells and cell dry weight was 11.6 g L −1 within 78 h at the flask level. During the 2-L fermentation, the transformant could accumulate 48.3% (w/w) oil from inulin in its cells and cell dry weight was 13.3 g L −1 within 78 h while the transformant could accumulate 50.6% (w/w) oil from extract of Jerusalem artichoke tubers in its cells and cell dry weight was 14.6 g L −1 within 78 h. At the end of fermentation, most of the added sugar was utilized by the transformant cells. Over 91.5% of the fatty acids from the transformant cultivated in the extract of Jerusalem artichoke tubercles was C 16:0, C 18:1 and C 18:2, especially C 18:1 (58.5%).

Expression of the inulinase gene from the marine‐derived Pichia guilliermondii in Saccharomyces sp. W0 and ethanol production from inulin

SummaryIt has been confirmed that Saccharomyces sp. W0 can produce high concentration of ethanol. In this study, the INU1 gene cloned from the marine‐derived Pichia guilliermondii was transformed into uracil mutant of Saccharomyces sp. W0. The positive transformant Inu‐66 obtained could produce 34.2 U ml−1 of extracellular inulinase within 72 h of cultivation. It was found that 15.2 U of inulinase activity per one gram of inulin was suitable for inulin hydrolysis and ethanol production by the transformant Inu‐66. During the small‐scale fermentation, 13.7 ml of ethanol in 100 ml of medium was produced and 99.1% of the added inulin was utilized by the transformant. During the 2 l fermentation, 14.9% (v/v) of ethanol was produced from inulin and 99.5% of the added inulin was converted into ethanol, CO2 and cell mass.

Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase

The INU1 gene encoding exo-inulinase cloned from Kluyveromyces marxianus CBS 6556 was ligated into the surface display plasmid and expressed in the cells of the marine-derived yeast Yarrowia lipolytica which can produce citric acid. The expressed inulinase was immobilized on the yeast cells. The activity of the immobilized inulinase with 6 × His tag was found to be 22.6 U mg −1 of cell dry weight after cell growth for 96 h. The optimal pH and temperature of the displayed inulinase were 4.5 and 50 °C, respectively and the inulinase was stable in the pH range of 3–8 and in the temperature range of 0–50 °C. During the inulin hydrolysis, the optimal inulin concentration was 12.0% and the optimal amount of added inulinase was 181.6 U g −1 of inulin. Under such conditions, over 77.9% of inulin was hydrolyzed within 10 h and the hydrolysate contained main monosaccharides and disaccharides, and minor trisaccharides. During the citric acid production in the flask level, the recombinant yeast could produce 77.9 g L −1 citric acid and 5.3 g L −1 iso-citric acid from inulin while 68.9 g L −1 of citric acid and 4.1 g L −1 iso-citric acid in the fermented medium were attained within 312 h of the 2-L fermentation, respectively.

Inhibition of glucose on an exoinulinase from Kluyveromyces marxianus expressed in Pichia pastoris

The INU1 gene encoding an exoinlinase from Kluyveromyces marxianus KW02 was expressed in Pichia pastoris. Enzyme activity of recombinant P. pastoris in the fermentation liquid was 52.0 U/ml, which was 12 times that of the wild type. Its apparent molecular weight was 85 kDa (SDS–polyacrylamide gel electrophoresis (PAGE)) and its theoretic molecular weight of enzymic protein was 62 kDa. The optimum pH of enzyme was 4.5. Its optimum temperature was 55 °C. Fe 2+ increased the enzyme activity by 41.97%, but Mg 2+ inhibitated the enzyme activity dramatically. Glucose showed competitive inhibition on enzyme activity for hydrolysis of inulin which had a degree of polymerization (DP) not less than 10, but no inhibitory effect on enzyme activity for hydrolysis of oligo-saccharide (DP ≤ 9). It was also detected that inulinase had the synthetic activity of inulin.

Expression of an ?-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus

For expression of the alpha-galactosidase gene from Cyamopsis tetragonoloba in Kluyveromyces marxianus CBS 6556 we have used the promoter of the homologous inulinase-encoding gene (INU1). The INU1 gene has been cloned and sequenced and the coding region shows an identity of 59% with the Saccharomyces cerevisiae invertase gene (SUC2). In the 5'-flanking region of INU1 we found a sequence (TAAATCCGGGG) that perfectly matches to the MIG1 binding consensus sequence (WWWWTSYGGGG) of the S. cerevisiae GAL1, GAL4 and SUC2 genes. Using the K. marxianus INU1 promoter and prepro-signal sequence, we obtained a high alpha-galactosidase production level (153 mg/l) and a secretion efficiency of 99%. Both the production level and the secretion efficiency were significantly reduced when the INU1 pro-peptide was deleted. With either the S. cerevisiae PGK or GAL7 promoter we could obtain only low alpha-galactosidase production levels (2 mg/l).