De novo engineering of a bacterial lifestyle program.

Synthetic biology has shown remarkable potential to program living microorganisms for applications. However, a notable discrepancy exists between the current engineering practice-which focuses predominantly on planktonic cells-and the ubiquitous observation of microbes in nature that constantly alternate their lifestyles on environmental variations. Here we present the de novo construction of a synthetic genetic program that regulates bacterial life cycle and enables phase-specific gene expression. The program is orthogonal, harnessing an engineered protein from 45 candidates as the biofilm matrix building block. It is also highly controllable, allowing directed biofilm assembly and decomposition as well as responsive autonomous planktonic-biofilm phase transition. Coupling to synthesis modules, it is further programmable for various functional realizations that conjugate phase-specific biomolecular production with lifestyle alteration. This work establishes a versatile platform for microbial engineering across physiological regimes, thereby shedding light on a promising path for gene circuit applications in complex contexts.

Cellobiose elicits immunity in lettuce conferring resistance to Botrytis cinerea.

Cellobiose is the primary product of cellulose hydrolysis and is expected to function as a type of pathogen/damage-associated molecular pattern in evoking plant innate immunity. In this study, cellobiose was demonstrated to be a positive regulator in the immune response of lettuce, but halted autoimmunity when lettuce was exposed to concentrations of cellobiose >60 mg l-1. When lettuce plants were infected by Botrytis cinerea, cellobiose endowed plants with enhanced pre-invasion resistance by activating high ß-1,3-glucanase and antioxidative enzyme activities at the initial stage of pathogen infection. Cellobiose-activated core regulatory factors such as EDS1, PTI6, and WRKY70, as well as salicylic acid signaling, played an indispensable role in modulating plant growth-defense trade-offs. Transcriptomics data further suggested that the cellobiose-activated plant-pathogen pathways are involved in microbe/pathogen-associated molecular pattern-triggered immune responses. Genes encoding receptor-like kinases, transcription factors, and redox homeostasis, phytohormone signal transduction, and pathogenesis-related proteins were also up- or down-regulated by cellobiose. Taken together, the findings of this study demonstrated that cellobiose serves as an elicitor to directly activate disease-resistance-related cellular functions. In addition, multiple genes have been identified as potential modulators of the cellobiose-induced immune response, which could aid understanding of underlying molecular events.

Precise and reliable control of gene expression in Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a soil-borne bacterium that is known for its DNA delivery ability and widely exploited for plant transformation. Despite continued interest in improving the utility of the organism, the lack of well-characterized engineering tools limits the realization of its full potential. Here, we present a synthetic biology toolkit that enables precise and effective control of gene expression in A. tumefaciens. We constructed and characterized six inducible expression systems. Then, we optimized the one regulated by cumic acid through amplifier introduction and promoter engineering and evaluated its 15 cognate promoters. To establish fine-tunability, we constructed a series of spacers and a promoter library to systematically modulate both translational and transcriptional rates. We finally demonstrated the application of the tools by co-expressing genes with altered expression levels using a single signal. This study provides precise expression tools for A. tumefaciens, facilitating rational engineering of the bacterium for advanced plant biotechnological applications.

Extracellular protease production regulated by nitrogen and carbon sources in Trichoderma reesei.

The filamentous fungus Trichoderma reesei is an important producer of industrial enzymes, and possesses abundant extracellular protease genes based on the genome sequence data. However, the production of extracellular proteases remains poorly understood. Here, protease production was extensively investigated on different carbon (glucose and lactose) and nitrogen sources ((NH4 )2 SO4 , NaNO3 , peptone, and corn steep liquor). It was found that protease production was dominantly regulated by nitrogen sources. Organic nitrogen sources were beneficial for protease production, while the preferred nitrogen source (NH4 )2 SO4 inhibited the expression of proteases. As for carbon sources, lactose was a more effective inducer than glucose for protease production. The protease activity was further examined by protease inhibitors, which suggested that protease activity was predominantly inhibited by phenylmethanesulfonyl fluoride (PMSF) and slightly suppressed by ethylenediaminetetraacetic acid (EDTA). Moreover, proteomic analysis revealed a total of 29 extracellular proteases, including 13 serine proteases, 6 aspartic proteases, and 10 metalloproteases. In addition, seven proteases were found to be present among all conditions. These results showed the regulatory profile of extracellular protease production in Trichoderma reesei grown on various carbon and nitrogen sources, which will facilitate the development of T. reesei to be an effective workhorse for enzyme or high-value protein production in industry.

The GATA-Type Transcriptional Factor Are1 Modulates the Expression of Extracellular Proteases and Cellulases in Trichoderma reesei.

Trichoderma reesei is a biotechnologically important filamentous fungus with the remarkable ability to secrete large amounts of enzymes, whose production is strongly affected by both the carbon and nitrogen sources. While the carbon metabolism regulators are extensively studied, the regulation of enzyme production by the nitrogen metabolism regulators is still poorly understood. In this study, the GATA transcription factor Are1, which is an orthologue of the Aspergillus global nitrogen regulator AREA, was identified and characterized for its functions in regulation of both protease and cellulase production in T. reesei. Deletion of the are1 gene abolished the capability to secrete proteases, and complementation of the are1 gene rescued the ability to produce proteases. Quantitative RT-PCR analysis revealed that the transcripts of protease genes apw1 and apw2 were also significantly reduced in the Δare1 strain when grown in the medium with peptone as the nitrogen source. In addition, deletion of are1 resulted in decreased cellulase production in the presence of (NH4)2SO4. Consistent with the reduction of cellulase production, the transcription levels of the major cellulase genes, including cbh1, cbh2, egl1, and egl2, were dramatically decreased in Δare1. Sequence analysis showed that all promoter regions of the tested protease and cellulase genes contain the consensus GATA elements. However, the expression levels of the major cellulase transcription activator Xyr1 and the repressor Cre1 had no significant difference between Δare1 and the parental strain QM9414, indicating that the regulatory mechanism deserves further investigation. Taken together, these results demonstrate the important role of Are1 in the regulation of protease and cellulase production in T. reesei, although these processes depend on the kind of nitrogen sources. The findings in this study contribute to the understanding of the regulation network of carbon and nitrogen sources in filamentous fungi.

Heterologous expression of Talaromyces emersonii cellobiohydrolase Cel7A in Trichoderma reesei increases the efficiency of corncob residues saccharification.

OBJECTIVE: Improve the hydrolysis efficiency of the Trichoderma reesei cellulase system by heterologously expressing cellobiohydrolase Cel7A (Te-Cel7A) from the thermophilic fungus Talaromyces emersonii. RESULTS: Te-Cel7A was expressed in T. reesei under control of the cdna1 promoter and the generated transformant QTC14 could successfully secrete Te-Cel7A into the supernatant using glucose as carbon source. The recombinant Te-Cel7A had a temperature optimum at 65 °C and an optimal pH of 5, which were similar to those from the native host. The culture supernatant of QTC14 exhibited a 28.8% enhancement in cellobiohydrolase activity and a 65.2% increase in filter paper activity relative to that of the parental strain QP4. Moreover, the QTC14 cellulase system showed higher thermal stability than that of the parental strain QP4. In the saccharification of delignified corncob residue, the cellulose conversion of QTC14 showed 13.9% higher than that of QP4 at the end of reaction. CONCLUSIONS: The thermophilic fungus-derived cellulases could be efficiently expressed by T. reesei and the recombinant cellulases had potential applications for biomass conversion.

Production of highly efficient cellulase mixtures by genetically exploiting the potentials of Trichoderma reesei endogenous cellulases for hydrolysis of corncob residues.

BACKGROUND: Trichoderma reesei is one of the most important fungi utilized for cellulase production. However, its cellulase system has been proven to be present in suboptimal ratio for deconstruction of lignocellulosic substrates. Although previous enzymatic optimization studies have acquired different types of in vitro synthetic mixtures for efficient lignocellulose hydrolysis, production of in vivo optimized cellulase mixtures by industrial strains remains one of the obstacles to reduce enzyme cost in the biofuels production from lignocellulosic biomass. RESULTS: In this study, we used a systematic genetic strategy based on the pyrG marker to overexpress the major cellulase components in a hypercellulolytic T. reesei strain and produce the highly efficient cellulase mixture for saccharification of corncob residues. We found that overexpression of CBH2 exhibited a 32-fold increase in the transcription level and a comparable protein level to CBH1, the most abundant secreted protein in T. reesei, but did not contribute much to the cellulolytic ability. However, when EG2 was overexpressed with a 46-fold increase in the transcription level and a comparable protein level to CBH2, the engineered strain QPE36 showed a 1.5-fold enhancement in the total cellulase activity (up to 5.8 U/mL FPA) and a significant promotion of saccharification efficiency towards differently pretreated corncob residues. To assist the following genetic manipulations, the marker pyrG was successfully excised by homologous recombination based on resistance to 5-FOA. Furthermore, BGL1 was overexpressed in the EG2 overexpression strain QE51 (pyrG-excised) and a 11.6-fold increase in BGL activity was obtained. The EG2-BGL1 double overexpression strain QEB4 displayed a remarkable enhancement of cellulolytic ability on pretreated corncob residues. Especially, a nearly complete cellulose conversion (94.2%) was found for the delignified corncob residues after 48 h enzymatic saccharification. CONCLUSIONS: These results demonstrate that genetically exploiting the potentials of T. reesei endogenous cellulases to produce highly efficient cellulase mixtures is a powerful strategy to promote the saccharification efficiency, which will eventually facilitate cost reduction for lignocellulose-based biofuels.

Engineering microbial division of labor for plastic upcycling.

Plastic pollution is rapidly increasing worldwide, causing adverse impacts on the environment, wildlife and human health. One tempting solution to this crisis is upcycling plastics into products with engineered microorganisms; however, this remains challenging due to complexity in conversion. Here we present a synthetic microbial consortium that efficiently degrades polyethylene terephthalate hydrolysate and subsequently produces desired chemicals through division of labor. The consortium involves two Pseudomonas putida strains, specializing in terephthalic acid and ethylene glycol utilization respectively, to achieve complete substrate assimilation. Compared with its monoculture counterpart, the consortium exhibits reduced catabolic crosstalk and faster deconstruction, particularly when substrate concentrations are high or crude hydrolysate is used. It also outperforms monoculture when polyhydroxyalkanoates serves as a target product and confers flexible tuning through population modulation for cis-cis muconate synthesis. This work demonstrates engineered consortia as a promising, effective platform that may facilitate polymer upcycling and environmental sustainability.

Development of a novel expression platform for heterologous protein production via deleting the p53-like regulator Vib1 in Trichoderma reesei.

Trichoderma reesei is widely used as a protein production host due to its high natural capacity to secrete enzymes. Nonetheless, the complexity and abundance of secretome limit its extensive application in heterologous protein production. Here, a novel heterologous protein expression system with remarkable reduction of undesired background proteins was developed by deletion of the p53-like transcriptional factor Vib1. The vib1-deletion strain (Δvib1) exhibited a dramatic decrease in cellulase and protease secretion, whereas the growth of Δvib1 was comparable to that of the parental strain QM53, indicating that Δvib1 possesses a great potential for heterologous protein production. Therefore, the Aspergillus niger ß-glucosidase-coding gene bglA was expressed in Δvib1 and QM53 to demonstrate the feasibility of Δvib1 as the protein production host. The bglA-expression strains QVB-1 (Δvib1:bglA) and Q53B-1 (QM53:bglA) produced approximately 17.2 IU/mg and 14.7 IU/mg of ß-glucosidase activity, respectively. In addition, the ß-glucosidase activity in the supernatant of QVB-1 remained constant after 4-week incubation whereas that of Q53B-1 decreased by more than 60%. Furthermore, transcription levels of the genes involved in the unfolded protein response were relatively decreased in Δvib1 compared with that in QM53, indicating the increased protein folding capacity of the endoplasmic reticulum in Δvib1. These results demonstrate the feasibility of using T. reesei Δvib1 as the host for heterologous protein production.

A novel thermostable chitinolytic machinery of Streptomyces sp. F-3 consisting of chitinases with different action modes.

BACKGROUND: The biodegradation of chitin is an important part of the carbon and nitrogen cycles in nature. Speeding up the biotransformation of chitin substrates can not only reduce pollution, but also produce high value-added products. However, this process is strictly regulated by the catalytic efficiency of the chitinolytic machinery. Therefore, it is necessary to study the mode of action and compound mechanisms of different chitin-degrading enzymes in depth to improve the catalytic efficiency of the chitinolytic machinery. RESULTS: The thermophilic bacterium Streptomyces sp. F-3 showed comparatively high chitin degradation activities. To elucidate the mechanism underlying chitin hydrolysis, six chitin degradation-related enzymes were identified in the extracellular proteome of Streptomyces sp. F-3, including three chitinases (SsChi18A, SsChi18B, and SsChi18C) from the GH18 family, one GH19 chitinase (SsChi19A), one GH20 ß-N-acetylhexosaminidase (SsGH20A), and one lytic polysaccharide monooxygenase (SsLPMO10A) from the AA10 family. All were upregulated by chitin. The heterologously expressed hydrolases could withstand temperatures up to 70 °C and were stable at pH values of 4 to 11. Biochemical analyses displayed that these chitin degradation-related enzymes had different functions and thus showed synergistic effects during chitin degradation. Furthermore, based on structural bioinformatics data, we speculated that the different action modes among the three GH18 chitinases may be caused by loop differences in their active site architectures. Among them, SsChi18A is probably processive and mainly acts on polysaccharides, while SsChi18B and SsChi18C are likely endo-non-processive and displayed higher activity on the degradation of chitin oligosaccharides. In addition, proteomic data and synergy experiments also indicated the importance of SsLPMO10A, which could promote the activities of the hydrolases and increase the monosaccharide content in the reaction system, respectively. CONCLUSIONS: In this article, the chitinolytic machinery of a thermophilic Streptomyces species was studied to explore the structural basis for the synergistic actions of chitinases from different GH18 subfamilies. The elucidation of the degradation mechanisms of these thermophilic chitinases will lay a theoretical foundation for the efficient industrialized transformation of natural chitin.

Enhancement of Cellulase Production in Trichoderma reesei via Disruption of Multiple Protease Genes Identified by Comparative Secretomics.

The filamentous fungus Trichoderma reesei is one of the most studied cellulolytic organisms and the major producer of cellulases for industrial applications. However, undesired degradation of cellulases often happens in culture filtrates and commercial enzyme preparations. Even studies have been reported about describing proteolytic degradation of heterologous proteins in T. reesei, there are few systematic explorations concerning the extracellular proteases responsible for degradation of cellulases. In this study, the cellulase activity was observed to rapidly decrease at late cultivation stages using corn steep liquor (CSL) as the nitrogen source in T. reesei. It was discovered that this decrease may be caused by proteases. To identify the proteases, comparative secretomics was performed to analyze the concomitant proteases during the cellulase production. 12 candidate proteases from the secretome of T. reesei were identified and their encoding genes were individually deleted via homologous recombination. Furthermore, three target proteases (tre81070, tre120998, and tre123234) were simultaneously deleted by one-step genetic transformation. The triple deletion strain ΔP70 showed a 78% decrease in protease activity and a six-fold increase in cellulase activity at late fermentation stages. These results demonstrated the feasibility of improvement of cellulase production by genetically disrupting the potential protease genes to construct the T. reesei strains with low extracellular protease secretion. This dataset also provides an efficient approach for strain improvement by precise genetic engineering combined with "omics" strategy for high-production of industrial enzymes to reduce the cost of lignocellulose bioconversion.

Production of the versatile cellulase for cellulose bioconversion and cellulase inducer synthesis by genetic improvement of Trichoderma reesei.

BACKGROUND: The enzymes for efficient hydrolysis of lignocellulosic biomass are a major factor in the development of an economically feasible cellulose bioconversion process. Up to now, low hydrolysis efficiency and high production cost of cellulases remain the significant hurdles in this process. The aim of the present study was to develop a versatile cellulase system with the enhanced hydrolytic efficiency and the ability to synthesize powerful inducers by genetically engineering Trichoderma reesei. RESULTS: In our study, we employed a systematic genetic strategy to construct the carbon catabolite-derepressed strain T. reesei SCB18 to produce the cellulase complex that exhibited a strong cellulolytic capacity for biomass saccharification and an extraordinary high ß-glucosidase (BGL) activity for cellulase-inducing disaccharides synthesis. We first identified the hypercellulolytic and uracil auxotrophic strain T. reesei SP4 as carbon catabolite repressed, and then deleted the carbon catabolite repressor gene cre1 in the genome. We found that the deletion of cre1 with the selectable marker pyrG led to a 72.6% increase in total cellulase activity, but a slight reduction in saccharification efficiency. To facilitate the following genetic modification, the marker pyrG was successfully removed by homologous recombination based on resistance to 5-FOA. Furthermore, the Aspergillus niger BGLA-encoding gene bglA was overexpressed, and the generated strain T. reesei SCB18 exhibited a 29.8% increase in total cellulase activity and a 51.3-fold enhancement in BGL activity (up to 103.9 IU/mL). We observed that the cellulase system of SCB18 showed significantly higher saccharification efficiency toward differently pretreated corncob residues than the control strains SDC11 and SP4. Moreover, the crude enzyme preparation from SCB18 with high BGL activity possessed strong transglycosylation ability to synthesize ß-disaccharides from glucose. The transglycosylation product was finally utilized as the inducer for cellulase production, which provided a 63.0% increase in total cellulase activity compared to the frequently used soluble inducer, lactose. CONCLUSIONS: In summary, we constructed a versatile cellulase system in T. reesei for efficient biomass saccharification and powerful cellulase inducer synthesis by combinational genetic manipulation of three distinct types of genes to achieve the customized cellulase production, thus providing a viable strategy for further strain improvement to reduce the cost of biomass-based biofuel production.

Characterization and Strain Improvement of a Hypercellulytic Variant, Trichoderma reesei SN1, by Genetic Engineering for Optimized Cellulase Production in Biomass Conversion Improvement.

The filamentous fungus Trichoderma reesei is a widely used strain for cellulolytic enzyme production. A hypercellulolytic T. reesei variant SN1 was identified in this study and found to be different from the well-known cellulase producers QM9414 and RUT-C30. The cellulose-degrading enzymes of T. reesei SN1 show higher endoglucanase (EG) activity but lower ß-glucosidase (BGL) activity than those of the others. A uracil auxotroph strain, SP4, was constructed by pyr4 deletion in SN1 to improve transformation efficiency. The BGL1-encoding gene bgl1 under the control of a modified cbh1 promoter was overexpressed in SP4. A transformant, SPB2, with four additional copies of bgl1 exhibited a 17.1-fold increase in BGL activity and a 30.0% increase in filter paper activity. Saccharification of corncob residues with crude enzyme showed that the glucose yield of SPB2 is 65.0% higher than that of SP4. These results reveal the feasibility of strain improvement through the development of an efficient genetic transformation platform to construct a balanced cellulase system for biomass conversion.

Proteomic analysis of the biomass hydrolytic potentials of Penicillium oxalicum lignocellulolytic enzyme system.

BACKGROUND: The mining of high-performance enzyme systems is necessary to develop industrial lignocellulose bioconversion. Large amounts of cellulases and hemicellulases can be produced by Penicillium oxalicum. Hence, the enzyme system of this hypercellulolytic fungus should be elucidated to help design optimum enzyme systems for effective biomass hydrolysis. RESULTS: The cellulolytic and xylanolytic activities of an SP enzyme system prepared from P. oxalicum JU-A10 were comparatively analyzed. Results indicated that the fungus possesses a complete cellulolytic-xylanolytic enzyme system. The cellobiohydrolase- and xylanase-specific activities of this system were higher than those of two other enzyme systems, i.e., ST from Trichoderma reesei SN1 and another commercial preparation Celluclast 1.5L. Delignified corncob residue (DCCR) could be hydrolyzed by SP to a greater extent than corncob residue (CCR). Beta-glucosidase (BG) supplemented in SP increased the ability of the system to hydrolyze DCCR and CCR, and resulted in a 64 % decrease in enzyme dosage with the same glucose yield. The behaviors of the enzyme components in the hydrolysis of CCR were further investigated by monitoring individual enzyme dynamics. The total protein concentrations and cellobiohydrolase (CBH), endoglucanase (EG), and filter paper activities in the supernatants significantly decreased during saccharification. These findings were more evident in SP than in the other enzyme systems. The comparative proteomic analysis of the enzyme systems revealed that both SP and ST were rich in carbohydrate-degrading enzymes and multiple non-hydrolytic proteins. A larger number of carbohydrate-binding modules 1 (CBM1) were also identified in SP than in ST. This difference might be linked to the greater adsorption to substrates and lower hydrolysis efficiency of SP enzymes than ST during lignocellulose saccharification, because CBM1 not only targets enzymes to insoluble cellulose but also leads to non-productive adsorption to lignin. CONCLUSIONS: Penicillium oxalicum can be applied to the biorefinery of lignocellulosic biomass. Its ability to degrade lignocellulosic substrates could be further improved by modifying its enzyme system on the basis of enzyme activity measurement and proteomic analysis. The proposed strategy may also be applied to other lignocellulolytic enzyme systems to enhance their hydrolytic performances rationally.