Manipulation of an α-glucosidase in the industrial glucoamylase-producing Aspergillus niger strain O1 to decrease non-fermentable sugars production and increase glucoamylase activity.

Dextrose equivalent of glucose from starch hydrolysis is a critical index for starch-hydrolysis industry. Improving glucose yield and decreasing the non]-fermentable sugars which caused by transglycosylation activity of the enzymes during the starch saccharification is an important direction. In this study, we identified two key α-glucosidases responsible for producing non-fermentable sugars in an industrial glucoamylase-producing strain Aspergillus niger O1. The results showed the transglycosylation product panose was decreased by more than 88.0% in agdA/agdB double knock-out strains than strain O1. Additionally, the B-P1 domain of agdB was found accountable as starch hydrolysis activity only, and B-P1 overexpression in ΔAΔB-21 significantly increased glucoamylase activity whereas keeping the glucoamylase cocktail low transglycosylation activity. The total amounts of the transglycosylation products isomaltose and panose were significantly decreased in final strain B-P1-3 by 40.7% and 44.5%, respectively. The application of engineered strains will decrease the cost and add the value of product for starch biorefinery.

Development of Genetic Tools in Glucoamylase-Hyperproducing Industrial Aspergillus niger Strains.

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes, particularly glucoamylase. Although a variety of genetic techniques have been successfully used in wild-type A. niger, the transformation of industrially used strains with few conidia (e.g., A. niger N1) or that are even aconidial (e.g., A. niger O1) remains laborious. Herein, we developed genetic tools, including the protoplast-mediated transformation and Agrobacterium tumefaciens-mediated transformation of the A. niger strains N1 and O1 using green fluorescent protein as a reporter marker. Following the optimization of various factors for protoplast release from mycelium, the protoplast-mediated transformation efficiency reached 89.3% (25/28) for N1 and 82.1% (32/39) for O1. The A. tumefaciens-mediated transformation efficiency was 98.2% (55/56) for N1 and 43.8% (28/64) for O1. We also developed a marker-free CRISPR/Cas9 genome editing system using an AMA1-based plasmid to express the Cas9 protein and sgRNA. Out of 22 transformants, 9 albA deletion mutants were constructed in the A. niger N1 background using the protoplast-mediated transformation method and the marker-free CRISPR/Cas9 system developed here. The genome editing methods improved here will accelerate the elucidation of the mechanism of glucoamylase hyperproduction in these industrial fungi and will contribute to the use of efficient targeted mutation in other industrial strains of A. niger.

MtTRC-1, a Novel Transcription Factor, Regulates Cellulase Production via Directly Modulating the Genes Expression of the Mthac-1 and Mtcbh-1 in Myceliophthora thermophila.

The thermophilic fungus Myceliophthora thermophila has been used to produce industrial enzymes and biobased chemicals. In saprotrophic fungi, the mechanisms regulating cellulase production have been studied, which revealed the involvement of multiple transcription factors. However, in M. thermophila, the transcription factors influencing cellulase gene expression and secretion remain largely unknown. In this study, we identified and characterized a novel cellulase regulator (MtTRC-1) in M. thermophila through a combination of functional genomics and genetic analyses. Deletion of Mttrc-1 resulted in significantly decreased cellulase production and activities. Transcriptome analysis revealed downregulation of not only the encoding genes of main cellulases but also the transcriptional regulator MtHAC-1 of UPR pathway after disruption of MtTRC-1 under cellulolytic induction conditions. Herein, we also characterized the ortholog of the yeast HAC1p in M. thermophila. We show that Mthac-1 mRNA undergoes an endoplasmic reticulum (ER) stress-induced splicing by removing a 23-nucleotide (nt) intron. Notably, the protein secretion on cellulose was dramatically impaired by the deletion of MtHAC-1. Moreover, the colonial growth on various carbon sources was defective in the absence of MtHAC-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays verified MtTRC-1 regulates the transcription of Mthac-1 and the major cellulase gene Mtcbh-1 by binding directly to the promoters in vitro and in vivo. Furthermore, DNase I footprinting assays identified the putative consensus binding site (5'-GNG/C-3'). These results revealed the importance of MtTRC-1 for positively regulating cellulase production. This finding has clarified the complex regulatory pathways involved in cellulolytic enzyme production. IMPORTANCE In the present study, we characterized a novel regulator MtTRC-1 in M. thermophila, which regulated cellulase production through direct transcriptional regulation of the Mthac-1 and Mtcbh-1 genes. Our data demonstrated that MtHAC-1 is a key factor for the cellulase secretion capacity of M. thermophila. Our data indicate that this thermophilic fungus regulates cellulase production through a multilevels network, in which the protein secretory pathway is modulated by MtHAC-1-dependent UPR pathway and the cellulase gene expression is directly regulated in parallel by transcription factors. The conservation of Mttrc1 in filamentous fungi suggests this mechanism may be exploited to engineer filamentous fungal cell factories capable of producing proteins on an industrial scale.

Development of a flow cytometry-based plating-free system for strain engineering in industrial fungi.

Recent technical advances regarding filamentous fungi have accelerated the engineering of fungal-based production and benefited basic science. However, challenges still remain and limit the speed of fungal applications. For example, high-throughput technologies tailored to filamentous fungi are not yet commonly available for genetic modification. The currently used fungal genetic manipulations are time-consuming and laborious. Here, we developed a flow cytometry-based plating-free system to directly screen and isolate the transformed protoplasts in industrial fungi Myceliophthora thermophila and Aspergillus niger. This system combines genetic engineering via the 2A peptide and the CRISPR-Cas9 system, strain screening by flow cytometry, and direct sorting of colonies for deep-well-plate incubation and phenotypic analysis while avoiding culturing transformed protoplasts in plates, colony picking, conidiation, and cultivation. As a proof of concept, we successfully applied this system to generate the glucoamylase-hyperproducing strains MtYM6 and AnLM3 in M. thermophila and A. niger, respectively. Notably, the protein secretion level and enzyme activities in MtYM6 were 17.3- and 25.1-fold higher than in the host strain. Overall, these findings suggest that the flow cytometry-based plating-free system can be a convenient and efficient tool for strain engineering in fungal biotechnology. We expect this system to facilitate improvements of filamentous fungal strains for industrial applications. KEY POINTS: • Development of a flow cytometry-based plating-free (FCPF) system is presented. • Application of FCPF system in M. thermophila and A. niger for glucoamylase platform. • Hyper-produced strains MtYM6 and AnLM3 for glucoamylase production are generated.

Synergistic effects of multiple enzymes from industrial Aspergillus niger strain O1 on starch saccharification.

BACKGROUND: Starch is one of the most important renewable polysaccharides in nature for production of bio-ethanol. The starch saccharification step facilitates the depolymerization of starch to yield glucose for biofuels production. The filamentous fungus Aspergillus niger (A. niger) is the most used microbial cell factory for production of the commercial glucoamylase. However, the role of each component in glucoamylases cocktail of A. niger O1 for starch saccharification remains unclear except glucoamylase. RESULTS: In this study, we identified the key enzymes contributing to the starch saccharification process are glucoamylase, α-amylase and acid α-amylase out of 29 glycoside hydrolases from the 6-day fermentation products of A. niger O1. Through the synergistic study of the multienzymes for the starch saccharification in vitro, we found that increasing the amount of α-amylase by 5-10 times enhanced the efficiency of starch saccharification by 14.2-23.2%. Overexpression of acid α-amylase in strain O1 in vivo increased the total glucoamylase activity of O1 cultures by 15.0%. CONCLUSIONS: Our study clarifies the synergistic effects among the components of glucoamylases cocktail, and provides an effective approach to optimize the profile of saccharifying enzymes of strain O1 for improving the total glucoamylase activity.

Simulation and experiment of soft-tissue deformation in prostate brachytherapy.

Soft-tissue deformation is one of the major reasons for the inaccurate positioning of percutaneous needle insertion process. In this article, simulations and experiments of the needle insertion soft-tissue process are both applied to study soft-tissue deformation. A needle deflection model based on the mechanics is used to calculate the needle deflection during the interaction process. The obtained needle deflection data are applied into finite element analysis process as the system input. The uniaxial tensile strength tests, compression tests, and static indentation experiments are used to obtain the soft-tissue parameters and choose the best strain-energy function to model in the simulation. Magnetic resonance imaging is used to reconstruct the prostate, establishing both prostate three-dimensional finite element model and artificial prostate model. The needle-soft tissue interaction simulation results are compared with those of the needle insertion experiment. The displacement data of the mark point in the experiment are comparable to the simulation results. It is concluded that, using this simulation method, the surgeon can predict the deformation of the tissue and the displacement of the target in advance.

Development of a new tissue-equivalent material applied to optimizing surgical accuracy.

The precision of orientation to target placement during invasive therapy is mainly influenced by tool-tissue interaction. In this study, we aim to investigate a transparent Poly (vinyl alcohol) (PVA) hydrogel as tissue-equivalent material which is used in accurate surgical insertion research. The PVA hydrogel with specified formula was prepared by means of physical and chemical crosslink. The effects of chemical composition and synthesis technique on the biomechanical property, density and micro-structure morphology of PVA materials have been investigated in detail. It can be concluded that when PVA concentration is 8 g/dl, the NaCl concentration is 4 wt.%, with mix water/DMSO solvent, prepared under 7 freeze/thaw cycles, the material has the most similar properties with kidney tissue. Experimental results demonstrate that this tissue-equivalent material could be used in the ex vivo insertion accuracy test for robot-assisted percutaneous intervention and surgical training in minimally invasive surgery (MIS).