Youran Li

Cloning, expression, characterization, and biocatalytic investigation of a novel bacilli thermostable type I pullulanase from Bacillus sp. CICIM 263.

The pulA1 gene, encoding a novel thermostable type I pullulanase PulA1 from Bacillus sp. CICIM 263, was identified from genomic DNA. The open reading frame of the pulA1 gene was 2655 base pairs long and encoded a polypeptide (PulA1) of 885 amino acids with a calculated molecular mass of 100,887 Da. The pulA1 gene was expressed in Escherichia coli and Bacillus subtilis. Recombinant PuLA1 showed optimal activity at pH 6.5 and 70 °C. The enzyme demonstrated moderate thermostability as PuLA1 maintained more than 88% of its acitivity when incubated at 70 °C for 1 h. The enzyme could completely hydrolyze pullulan to maltotriose, and hydrolytic activity was also detected with glycogen, starch and amylopection, but not with amylose, which is consistent with the property of type I pullulanase. PulA1 may be suitable for industrial applications to improve the yields of fermentable sugars for bioethanol production.

High-efficient production of citric acid by Aspergillus niger from high concentration of substrate based on the staged-addition glucoamylase strategy

Citric acid (CA), an important platform-compound, has attracted much attention because of its broad applications and huge market demand. To solve high residual sugar at the fermentation end, we put forwarded strategy of pre-saccharification and then fermentation. Results showed that the residual total sugar decreased by 10.4% and the productivity increased by 4.0% and initially high glucose inhibited cell growth. Furthermore, commercial glucoamylase with high low-pH stability was proposed to staged-add in the fermentation process, which timely compensated enzyme loss, ensuring the glucose supply rate. The fermentation productivity was evidently enhanced by 13.3% with residual total sugar decreasing by 31.3%, simplifying the subsequent product separation and extraction process. Our results confirmed that staged-addition glucoamylase strategy was feasible to effective production of CA.

Construction and application of multi-host integrative vector system for xylose-fermenting yeast.

The xylose-fermenting yeasts (CTG clade yeasts, e.g. Scheffersomyces stipitis, Spathaspora passalidarum, Candida amazonensis and Candida jeffriesii) have the potential to be superior platforms for the conversion of lignocellulosic hydrolysate into fuel-grade ethanol and other chemical products. Here, a genetic expression system compatible with the genetic coding characteristics of CTG clade yeasts was constructed for use in xylose-fermenting yeasts. The pRACTH-gfpm plasmid based on an 18S rDNA shuttle vector was capable of stable integration into the genomes of a wide range of heterologous hosts. Green fluorescent protein was transformed and functionally expressed in S. stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and Saccharomyces cerevisiae under control of the SpADH1 promoter and SpCYC1 terminator. Finally, the expression system was useful in multiple yeast hosts for construction of the plasmid pRACTH-ldh. Scheffersomyces stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and S. cerevisiae were enabled to produce lactate from glucose or xylose by pRACTH-based expression of a heterologous lactate dehydrogenase. Among them, C. amazonensis (pRACTH-ldh) exhibited the highest lactate fermentation capacity, which reached a maximum of 44 g L-1 of lactate with a yield of 0.85 g lactate/g xylose.

A novel constructed SPT15 mutagenesis library of Saccharomyces cerevisiae by using gTME technique for enhanced ethanol production

During the last few years, the global transcription machinery engineering (gTME) technique has gained more attention as an effective approach for the construction of novel mutants. Genetic strategies (molecular biology methods) were utilized to get mutational for both genes (SPT15 and TAF23) basically existed in the Saccharomyces cerevisiae genome via screening the gTME approach in order to obtain a new mutant S. cerevisiae diploid strain. The vector pYX212 was utilized to transform these genes into the control diploid strain S. cerevisiae through the process of mating between haploids control strains S. cerevisiae (MAT-a [CICC 1374]) and (MAT-α [CICC 31144]), by using the oligonucleotide primers SPT15-EcoRI-FW/SPT15-SalI-RV and TAF23-SalI-FW/TAF23-NheI-RV, respectively. The resultant mutants were examined for a series of stability tests. This study showed how strong the effect of using strategic gTME with the importance of the modification we conducted in Error Prone PCR protocol by increasing MnCl2 concentration instead of MgCl2. More than ninety mutants we obtained in this study were distinguished by a high level production of bio-ethanol as compared to the diploid control strain.

Efficient production of citric acid in segmented fermentation using Aspergillus niger based on recycling of a pellet-dispersion strategy

Citric acid (CA), as the most important platform compound, has attracted much attention because of its broad applications in many areas. Here, we constructed a segmentation fermentation process which effectively separated cell growth and CA production. Through a pellet-dispersion strategy, the CA yield decline caused by the mycelium structure of Aspergillus niger was solved. In particular, CA production was significantly increased from 70.3 g L−1 to 97.0 g L−1. Furthermore, the segmentation fermentation conditions were optimized to improve the CA production. Evidence showed that the process was stable during ten batches of recycling fermentation in 5 L five-conjoined-fermenters, from which the CA yield even increased by 1.4 g L−1 (by 1.26%) compared with the control. All of these results confirm that the strategy could enhance productivity and avoid the traditional long process (about 30 days) of seed culturing (including spore preparation). It could be also applied in other fermentation industries involving filamentous fungi.

Development of an inducible secretory expression system in Bacillus licheniformis based on an engineered xylose operon

The xylose operon can be an efficient biological component for regulatory expression uses in Bacillus licheniformis. However, its characteristic susceptibility to carbon catabolite repression (CCR) makes its application inconvenient. In this study, plasmids harboring the wild-type operons from three Bacillus species were constructed and introduced into B. licheniformis. These plasmids ensured secretory expression of maltogenic α-amylase (BLMA) in B. licheniformis under strict regulation. The glucose-mediated CCR was then alleviated by engineering the xylose operon of the expression system. Evidence showed that mutations in the highly conserved nucleotides of the identified catabolite responsive element (cre) consensus sequence prevented association of the regulator CcpA with DNA, thus resulting in an increase in BLMA activity of up to 12-fold. Furthermore, features of this engineered system for inducible expression were investigated. Induction in mid-log phase using 10 g/L xylose at 37 °C was found to be beneficial for promoting the accumulation of recombinant product, and the maximum yield of BlmMA reached 715.4 U/mL. This study contributes to the industrial application of the generally recognized as safe (GRAS) workhorse B. licheniformis.