Rational engineering of xylanase hyper-producing system in Trichoderma reesei for efficient biomass degradation

Background Filamentous fungus Trichoderma reesei has been widely used as a workhorse for cellulase and xylanase productions. Xylanase has been reported as the crucial accessory enzyme in the degradation of lignocellulose for higher accessibility of cellulase. In addition, the efficient hydrolysis of xylan needs the co-work of multiple xylanolytic enzymes, which rise an increasing demand for the high yield of xylanase for efficient biomass degradation. Results In this study, a xylanase hyper-producing system in T. reesei was established by tailoring two transcription factors, XYR1 and ACE1, and homologous overexpression of the major endo-xylanase XYNII. The expressed xylanase cocktail contained 5256 U/mL xylanase activity and 9.25 U/mL β-xylosidase (pNPXase) activity. Meanwhile, the transcription level of the xylanolytic genes in the strain with XYR1 overexpressed was upregulated, which was well correlated with the amount of XYR1-binding sites. In addition, the higher expression of associated xylanolytic enzymes would result in more efficient xylan hydrolysis. Besides, 2310–3085 U/mL of xylanase activities were achieved using soluble carbon source, which was more efficient and economical than the traditional strategy of xylan induction. Unexpectedly, deletion of ace1 in C30OExyr1 did not give any improvement, which might be the result of the disturbed function of the complex formed between ACE1 and XYR1. The enzymatic hydrolysis of alkali pretreated corn stover using the crude xylanase cocktails as accessory enzymes resulted in a 36.64% increase in saccharification efficiency with the ratio of xylanase activity vs FPase activity at 500, compared to that using cellulase alone. Conclusions An efficient and economical xylanase hyper-producing platform was developed in T. reesei RUT-C30. The novel platform with outstanding ability for crude xylanase cocktail production would greatly fit in biomass degradation and give a new perspective of further engineering in T. reesei for industrial purposes.

A homologous overexpression system to study roles of drug transporters in Candida glabrata

Considering the relevance of drug transporters belonging to ABC and MFS superfamilies in pathogenic Candida species, there has always been a need to have an overexpression system where these membrane proteins for functional analysis could be expressed in a homologous background. We could address this unmet need by constructing a highly drug-susceptible Candida glabrata strain deleted in seven dominant ABC transporters genes such as CgSNQ2, CgAUS1, CgCDR1, CgPDH1, CgYCF1, CgYBT1 and CgYOR1 and introduced a GOF mutation in transcription factor (TF) CgPDR1 leading to a hyper-activation of CgCDR1 locus. The expression system was validated by overexpressing four GFP tagged ABC (CgCDR1, CgPDH1, CaCDR1 and ScPDR5) and an MFS (CgFLR1) transporters genes facilitated by an engineered expression plasmid to integrate at the CgCDR1 locus. The properly expressed and localized transporters were fully functional, as was revealed by their several-fold increased drug resistance, growth kinetics, localization studies and efflux activities. The present homologous system will facilitate in determining the role of an individual transporter for its substrate specificity, drug efflux, pathogenicity and virulence traits without the interference of other major transporters.

Homologous Overexpression of Genes in Cordyceps militaris Improves the Production of Polysaccharide

Background Cordyceps polysaccharides have been used around the globe for its bioactivity for millennia. However, the study and medicinal of Cordyceps militaris polysaccharides has been hampered by the low in natural abundances. Recently, the genetic engineered C. militaris developed for production of exopolysaccharides (EPS) had received extensive attention.Results In this study, based on the biosynthetic pathway and metabolization mechanism of exopolysaccharides, the crucial biosynthetic genes of Cordyceps polysaccharides were introduced by Agrobacterium transformation to provide a high flux of EPS. 21 mutants of C. militaris were identified through antibiotic screening and DNA sequencing. The maximum yield of EPS produced by mutant CM-pgm-H was 4.63 ± 0.23 g/L, while the yield of wild-type strain was 3.43 ± 0.26 g/L. And the data obtained in the present study indicated that the yield of EPS produced by the engineered strain treated with co-overexpression of phosphoglucomutase and UDP-glucose 6-dehydrogenase genes achieved 6.11 ± 0.21 g/L, which was increased by 78.13% compared with the wild-type strain.Conclusions CM-pgm-H obtained the highest EPS content than that of mutants glucokinase, UDP-glucose pyrophosphorylase, UDP-glucose 6-dehydrogenase. It indicated that the content of protein phosphoglucomutase was the most critical influencing factor on the CP production in C.militaris. Furthermore, the EPS production of CM-ugdh-pgm-M was significantly improved 1.78-fold by co-overexpression. It anticipated that our engineering strategies will play an important role in the development of C. militaris for sustainable production of Cordyceps polysaccharides.

The N-terminal domain of rhamnosyltransferase EpsF influences exopolysaccharide chain length determination in Streptococcus thermophilus 05-34

Glycosyltransferases are key enzymes involved in the assembly of repeating units of exopolysaccharides (EPS). A glycosyltransferase generally consists of the N-terminal and the C-terminal domain, however, the functional role of these domains in EPS biosynthesis remains largely unknown. In this study, homologous overexpression was employed to investigate the effects of EpsFN, a truncated form of rhamnosyltransferase EpsF with only the N-terminal domain, on EPS biosynthesis in Streptococcus thermophilus 05-34. Reverse transcription qPCR and Western blotting analysis confirmed the successful expression of epsFN in 05-34 at the transcription and translation level, respectively. Further analysis showed that the monosaccharide composition and yield of EPS were not affected by the overexpression of epsFN, whereas the molecular mass decreased by 5-fold. Accordingly, the transcription levels of genes involved in EPS biosynthesis, including chain-length determination gene epsC, were down-regulated by 5- to 6-fold. These results indicated that the N-terminal domain of EpsF alone could influence the molecular mass of EPS, probably via lowering the concentration of sugar precursors, which may lead to decreased expression of genes responsible for chain-length determination.

Genetic Modification of Mucor circinelloides to Construct Stearidonic Acid Producing Cell Factory

Stearidonic acid (SDA; 18:4, n-3) is the delta 15-desaturase product of gamma linolenic acid (GLA; 18:3, n-6) and delta 6-desaturase product of alpha linolenic acid (ALA; 18:3, n-3). Construction of engineered oleaginous microbes have been attracting significant interest in producing SDA because of its nutritional value and pharmaceutical applications. Mucor circinelloides is a GLA producing filamentous fungus, which can be a useful tool to produce SDA. This study has, therefore, overexpressed the delta-15 desaturase (D15D) gene from Mortierella alpina in this fungus to construct a SDA-producing cell factory. To produce SDA in M. circinelloides, the homologous overexpression of D15D gene was analyzed. When the gene was overexpressed in M. circinelloides CBS 277.49, up to 5.0% SDA was accumulated in this strain. According to current knowledge, this is the first study describing the construction of a SDA-producing cell factory by overexpression of D15D gene in oleaginous fungus M. circinelloides. A new scope for further research has been established by this work to improve SDA production in this fungus, specifically in its high lipid-producing strain, WJ11.