Anaerobic digestion integrated with microbial electrolysis cell to enhance biogas production and upgrading in situ.

Anaerobic digestion (AD) is an effective and applicable technology for treating organic wastes to recover bioenergy, but it is limited by various drawbacks, such as long start-up time for establishing a stable process, the toxicity of accumulated volatile fatty acids and ammonia nitrogen to methanogens resulting in extremely low biogas productivities, and a large amount of impurities in biogas for upgrading thereafter with high cost. Microbial electrolysis cell (MEC) is a device developed for electrosynthesis from organic wastes by electroactive microorganisms, but MEC alone is not practical for production at large scales. When AD is integrated with MEC, not only can biogas production be enhanced substantially, but also upgrading of the biogas product performed in situ. In this critical review, the state-of-the-art progress in developing AD-MEC systems is commented, and fundamentals underlying methanogenesis and bioelectrochemical reactions, technological innovations with electrode materials and configurations, designs and applications of AD-MEC systems, and strategies for their enhancement, such as driving the MEC device by electricity that is generated by burning the biogas to improve their energy efficiencies, are specifically addressed. Moreover, perspectives and challenges for the scale up of AD-MEC systems are highlighted for in-depth studies in the future to further improve their performance.

Systems metabolic engineering of Corynebacterium glutamicum to assimilate formic acid for biomass accumulation and succinic acid production.

Formate as an ideal mediator between the physicochemical and biological realms can be obtained from electrochemical reduction of CO2 and used to produce bio-chemicals. Yet, limitations arise when employing natural formate-utilizing microorganisms due to restricted product range and low biomass yield. This study presents a breakthrough: engineered Corynebacterium glutamicum strains (L2-L4) through modular engineering. L2 incorporates the formate-tetrahydrofolate cycle and reverse glycine cleavage pathway, L3 enhances NAD(P)H regeneration, and L4 reinforces metabolic flux. Metabolic modeling elucidates C1 assimilation, guiding strain optimization for co-fermentation of formate and glucose. Strain L4 achieves an OD600 of 0.5 and produces 0.6 g/L succinic acid. 13C-labeled formate confirms C1 assimilation, and further laboratory evolution yields 1.3 g/L succinic acid. This study showcases a successful model for biologically assimilating formate in C. glutamicum that could be applied in C1-based biotechnological production, ultimately forming a formate-based bioeconomy.

Making the biochemical conversion of lignocellulose more robust.

Lignocellulose is an alternative to fossil resources, but its biochemical conversion is not economically competitive. While decentralized processing can reduce logistical cost for this feedstock, sugar platforms need to be developed with energy-saving pretreatment technologies and cost-effective cellulases, and products must be selected correctly. Anaerobic fermentation with less energy consumption and lower contamination risk is preferred, particularly for producing biofuels. Great effort has been devoted to producing cellulosic ethanol, but CO2 released with large quantities during ethanol fermentation must be utilized in situ for credit. Unless titer and yield are improved substantially, butanol cannot be produced as an advanced biofuel. Microbial lipids produced through aerobic fermentation with low yield and intensive energy consumption are not affordable as feedstocks for biodiesel production.

Intracellular accumulation of c-di-GMP and its regulation on self-flocculation of the bacterial cells of Zymomonas mobilis.

Zymomonas mobilis is an emerging chassis for being engineered to produce bulk products due to its unique glycolysis through the Entner-Doudoroff pathway with less ATP produced for lower biomass accumulation and higher product yield. When self-flocculated, the bacterial cells are more productive, since they can self-immobilize within bioreactors for high density, and are more tolerant to stresses for higher product titers, but this morphology needs to be controlled properly to avoid internal mass transfer limitation associated with their strong self-flocculation. Herewith we explored the regulation of cyclic diguanosine monophosphate (c-di-GMP) on self-flocculation of the bacterial cells through activating cellulose biosynthesis. While ZMO1365 and ZMO0919 with GGDEF domains for diguanylate cyclase activity catalyze c-di-GMP biosynthesis, ZMO1487 with an EAL domain for phosphodiesterase activity catalyzes c-di-GMP degradation, but ZMO1055 and ZMO0401 contain the dual domains with phosphodiesterase activity predominated. Since c-di-GMP is synthesized from GTP, the intracellular accumulation of this signal molecule through deactivating phosphodiesterase activity is preferred for activating cellulose biosynthesis to flocculate the bacterial cells, because such a strategy exerts less perturbance on intracellular processes regulated by GTP. These discoveries are significant for not only engineering unicellular Z. mobilis strains with the self-flocculating morphology to boost production but also understanding mechanism underlying c-di-GMP biosynthesis and degradation in the bacterium.

Evolution of cyclic di-GMP signalling on a short and long term time scale.

Diversifying radiation of domain families within specific lineages of life indicates the importance of their functionality for the organisms. The foundation for the diversifying radiation of the cyclic di-GMP signalling network that occurred within the bacterial kingdom is most likely based in the outmost adaptability, flexibility and plasticity of the system. Integrative sensing of multiple diverse extra- and intracellular signals is made possible by the N-terminal sensory domains of the modular cyclic di-GMP turnover proteins, mutations in the protein scaffolds and subsequent signal reception by diverse receptors, which eventually rewires opposite host-associated as well as environmental life styles including parallel regulated target outputs. Natural, laboratory and microcosm derived microbial variants often with an altered multicellular biofilm behaviour as reading output demonstrated single amino acid substitutions to substantially alter catalytic activity including substrate specificity. Truncations and domain swapping of cyclic di-GMP signalling genes and horizontal gene transfer suggest rewiring of the network. Presence of cyclic di-GMP signalling genes on horizontally transferable elements in particular observed in extreme acidophilic bacteria indicates that cyclic di-GMP signalling and biofilm components are under selective pressure in these types of environments. On a short and long term evolutionary scale, within a species and in families within bacterial orders, respectively, the cyclic di-GMP signalling network can also rapidly disappear. To investigate variability of the cyclic di-GMP signalling system on various levels will give clues about evolutionary forces and discover novel physiological and metabolic pathways affected by this intriguing second messenger signalling system.

Developing high-dimensional machine learning models to improve generalization ability and overcome data insufficiency for mixed sugar fermentation simulation.

Biorefinery can be promoted by building accurate machine learning models. This work proposed a strategy to enhance model's generalization ability and overcome insufficient data conditions for mixed sugar fermentation simulation. Multiple inputs single output models, using initial glucose, initial xylose, and time together as inputs, have higher generalization ability than single input single output models with time as sole input in predicting glucose, xylose, ethanol, or biomass separately. Multiple inputs multiple outputs models, integrating outputs, enhanced model accuracy and resulted in an average R2 at 0.99. To overcome data insufficiency conditions, consensus yeast (CY) model, through consolidating data from 4 yeasts, obtained R2 at 0.90. By adjusting the pretrained CY model, the model can save more than 50% data and get R2 at 0.95 and 0.93 for yeast and bacterial fermentation simulation. The strategy can expand the application range and save costs of data curation for ANN models.

Engineering Corynebacterium glutamicum for efficient production of succinic acid from corn stover pretreated by concentrated-alkali under steam-assistant conditions.

Corynebacterium glutamicum was developed for efficient production of succinic acid from corn stover (CS) pretreated by concentrated-alkali under steam-assistant (CASA) conditions. First, C. glutamicum was engineered by 1) blocking the by-products pathways (deletion of ldh, pta-ackA, and cat), 2) enhancing the carbon flux to succinate (overexpression of pyc and ppc), and 3) releasing the end-product inhibition (overexpression of Ncgl0275). The recombinant strain produced 117.8 g/L succinate in fed-batch fermentation. Second, to fully utilize xylose in lignocellulosic hydrolysate, two xylose utilization pathways-the isomerase pathway and the Weimberg pathway-were introduced into the recombinant strain. Third, CS was pretreated by CASA with a higher sugars yield and a lower black liquid. Finally, 64.16 g/L of succinic acid was obtained from 150 g/L CASA-pretreated CS by engineered C. glutamicum. These results showed a succinate high-producing C. glutamicum strain using glucose and xylose simultaneously as well as an effective and environmentally acceptable pretreatment strategy.

Regulation of biofilm formation in Zymomonas mobilis to enhance stress tolerance by heterologous expression of pfs and luxS.

Zymomonas mobilis is a potential alternative of Saccharomyces cerevisiae to produce cellulosic ethanol with strengths in cofactor balance, but its lower tolerance to inhibitors in the lignocellulosic hydrolysate restricts the application. Although biofilm can improve bacteria stress tolerance, regulating biofilm formation in Z. mobilis is still a challenge. In this work, we constructed a pathway by heterologous expressing pfs and luxS from Escherichia coli in Z. mobilis to produce AI-2 (autoinducer 2), a universal quorum-sensing signal molecule, to control cell morphology for enhancing stress tolerance. Unexpectedly, the results suggested that neither endogenous AI-2 nor exogenous AI-2 promoted biofilm formation, while heterologous expression of pfs can significantly raise biofilm. Therefore, we proposed that the main factor in assisting biofilm formation was the product accumulated due to heterologous expression of pfs, like methylated DNA. Consequently, ZM4::pfs produced more biofilm, which presented an enhanced tolerance to acetic acid. All these findings provide a novel strategy to improve the stress tolerance of Z. mobilis by enhancing biofilm formation for efficient production of lignocellulosic ethanol and other value-added chemical products.

Benchmarking of long-read sequencing, assemblers and polishers for yeast genome.

BACKGROUND: The long reads of the third-generation sequencing significantly benefit the quality of the de novo genome assembly. However, its relatively high single-base error rate has been criticized. Currently, sequencing accuracy and throughput continue to improve, and many advanced tools are constantly emerging. PacBio HiFi sequencing and Oxford Nanopore Technologies (ONT) PromethION are two up-to-date platforms with low error rates and ultralong high-throughput reads. Therefore, it is urgently needed to select the appropriate sequencing platforms, depths and genome assembly tools for high-quality genomes in the era of explosive data production. METHODS: We performed 455 (7 assemblers with 4 polishing pipelines or without polishing on 13 subsets with different depths) and 88 (4 assemblers with or without polishing on 11 subsets with different depths) de novo assemblies of Yeast S288C on high-coverage ONT and HiFi datasets, respectively. The assembly quality was evaluated by Quality Assessment Tool (QUAST), Benchmarking Universal Single-Copy Orthologs (BUSCO) and the newly proposed Comprehensive_score (C_score). In addition, we applied four preferable pipelines to assemble the genome of nonreference yeast strains. RESULTS: The assembler plays an essential role in genome construction, especially for low-depth datasets. For ONT datasets, Flye is superior to other tools through C_score evaluation. Polishing by Pilon and Medaka improve accuracy and continuity of the preassemblies, respectively, and their combination pipeline worked well in most quality metrics. For HiFi datasets, Flye and NextDenovo performed better than other tools, and polishing is also necessary. Enough data depth is required for high-quality genome construction by ONT (>80X) and HiFi (>20X) datasets.

Deciphering Molecular Mechanism Underlying Self-Flocculation of Zymomonas mobilis for Robust Production.

Zymomonas mobilis metabolizes sugar anaerobically through the Entner-Doudoroff pathway with less ATP generated for lower biomass accumulation to direct more sugar for product formation with improved yield, making it a suitable host to be engineered as microbial cell factories for producing bulk commodities with major costs from feedstock consumption. Self-flocculation of the bacterial cells presents many advantages, such as enhanced tolerance to environmental stresses, a prerequisite for achieving high product titers by using concentrated substrates. ZM401, a self-flocculating mutant developed from ZM4, the unicellular model strain of Z. mobilis, was employed in this work to explore the molecular mechanism underlying this self-flocculating phenotype. Comparative studies between ZM401 and ZM4 indicate that a frameshift caused by a single nucleotide deletion in the poly-T tract of ZMO1082 fused the putative gene with the open reading frame of ZMO1083, encoding the catalytic subunit BcsA of the bacterial cellulose synthase to catalyze cellulose biosynthesis. Furthermore, the single nucleotide polymorphism mutation in the open reading frame of ZMO1055, encoding a bifunctional GGDEF-EAL protein with apparent diguanylate cyclase/phosphodiesterase activities, resulted in the Ala526Val substitution, which consequently compromised in vivo specific phosphodiesterase activity for the degradation of cyclic diguanylic acid, leading to intracellular accumulation of the signaling molecule to activate cellulose biosynthesis. These discoveries are significant for engineering other unicellular strains from Z. mobilis with the self-flocculating phenotype for robust production. IMPORTANCE Stress tolerance is a prerequisite for microbial cell factories to be robust in production, particularly for biorefinery of lignocellulosic biomass to produce biofuels, bioenergy, and bio-based chemicals for sustainable socioeconomic development, since various inhibitors are released during the pretreatment to destroy the recalcitrant lignin-carbohydrate complex for sugar production through enzymatic hydrolysis of the cellulose component, and their detoxification is too costly for producing bulk commodities. Although tolerance to individual stress has been intensively studied, the progress seems less significant since microbial cells are inevitably suffering from multiple stresses simultaneously under production conditions. When self-flocculating, microbial cells are more tolerant to multiple stresses through the general stress response due to enhanced quorum sensing associated with the morphological change for physiological and metabolic advantages. Therefore, elucidation of the molecular mechanism underlying such a self-flocculating phenotype is significant for engineering microbial cells with the unique multicellular morphology through rational design to boost their production performance.

Omics analysis reveals mechanism underlying metabolic oscillation during continuous very-high-gravity ethanol fermentation by Saccharomyces cerevisiae.

During continuous very-high-gravity (VHG) ethanol fermentation with Saccharomyces cerevisiae, the process exhibits sustained oscillation in residual glucose, ethanol, and biomass, raising a question: how do yeast cells respond to this phenomenon? In this study, the oscillatory behavior of yeast cells was characterized through transcriptome and metabolome analysis for one complete oscillatory period. By analyzing the accumulation of 26 intracellular metabolites and the expression of 90 genes related to central carbon metabolism and stress response, we confirmed that the process oscillation was attributed to intracellular metabolic oscillation with phase difference, and the expression of HXK1, HXT1,2,4, and PFK1 was significantly different from other genes in the Embden-Meyerhof-Parnas pathway, indicating that glucose transport and phosphorylation could be key nodes for regulating the intracellular metabolism under oscillatory conditions. Moreover, the expression of stress response genes was triggered and affected predominately by ethanol inhibition in yeast cells. This progress not only contributes to the understanding of mechanisms underlying the process oscillation observed for continuous VHG ethanol fermentation, but also provides insights for understanding unsteady state that might develop in other continuous fermentation processes operated under VHG conditions to increase product titers for robust production.

Measurement of Cellulase and Xylanase Activities in Trichoderma reesei.

The microbial cellulase system is responsible for the generation of glucose from cellulose. Cellulases are comprised of at least three major groups of enzymes, namely endoglucanases, exoglucanases, and ß-glucosidases. On the other hand, xylanases function in the degradation of hemicellulose and work synergistically with cellulases for the degradation of lignocellulosic biomass. Here, we describe the most commonly used methods for the activity measurement of cellulases and xylanases.

Genome sequencing, assembly, and annotation of the self-flocculating microalga Scenedesmus obliquus AS-6-11.

BACKGROUND: Scenedesmus obliquus belongs to green microalgae and is widely used in aquaculture as feed, which is also explored for lipid production and bioremediation. However, genomic studies of this microalga have been very limited. Cell self-flocculation of microalgal cells can be used as a simple and economic method for harvesting biomass, and it is of great importance to perform genome-scale studies for the self-flocculating S. obliquus strains to promote their biotechnological applications. RESULTS: We employed the Pacific Biosciences sequencing platform for sequencing the genome of the self-flocculating microalga S. obliquus AS-6-11, and used the MECAT software for de novo genome assembly. The estimated genome size of S. obliquus AS-6-11 is 172.3 Mbp with an N50 of 94,410 bp, and 31,964 protein-coding genes were identified. Gene Ontology (GO) and KEGG pathway analyses revealed 65 GO terms and 428 biosynthetic pathways. Comparing to the genome sequences of the well-studied green microalgae Chlamydomonas reinhardtii, Chlorella variabilis, Volvox carteri and Micractinium conductrix, the genome of S. obliquus AS-6-11 encodes more unique proteins, including one gene that encodes D-mannose binding lectin. Genes encoding the glycosylphosphatidylinositol (GPI)-anchored cell wall proteins, and proteins with fasciclin domains that are commonly found in cell wall proteins might be responsible for the self-flocculating phenotype, and were analyzed in detail. Four genes encoding both GPI-anchored cell wall proteins and fasciclin domain proteins are the most interesting targets for further studies. CONCLUSIONS: The genome sequence of the self-flocculating microalgal S. obliquus AS-6-11 was annotated and analyzed. To our best knowledge, this is the first report on the in-depth annotation of the S. obliquus genome, and the results will facilitate functional genomic studies and metabolic engineering of this important microalga. The comparative genomic analysis here also provides new insights into the evolution of green microalgae. Furthermore, identification of the potential genes encoding self-flocculating proteins will benefit studies on the molecular mechanism underlying this phenotype for its better control and biotechnological applications as well.

Design, Evolution, and Characterization of a Xylose Biosensor in Escherichia coli Using the XylR/xylO System with an Expanded Operating Range.

Genetically encoded biosensors are extensively utilized in synthetic biology and metabolic engineering. However, reported xylose biosensors are far too sensitive with a limited operating range to be useful for most sensing applications. In this study, we describe directed evolution of Escherichia coli XylR, and construction of biosensors based on XylR and the corresponding operator xylO. The operating range of biosensors containing the mutant XylR was increased by nearly 10-fold comparing with the control. Two individual amino acid mutations (either L73P or N220T) in XylR were sufficient to extend the linear response range to upward of 10 g/L xylose. The evolved biosensors described here are well suited for developing whole-cell biosensors for detecting varying xylose concentrations across an expanded range. As an alternative use of this system, we also demonstrate the utility of XylR and xylO as a xylose inducible system to enable graded gene expression through testing with ß-galactosidase gene and the lycopene synthetic pathway. This evolution strategy identified a less-sensitive biosensor for real applications, thus providing new insights into strategies for expanding operating ranges of other biosensors for synthetic biology applications.

Engineering the Effector Domain of the Artificial Transcription Factor to Improve Cellulase Production by Trichoderma reesei.

Filamentous fungal strains of Trichoderma reesei have been widely used for cellulase production, and great effort has been devoted to enhancing their cellulase titers for the economic biorefinery of lignocellulosic biomass. In our previous studies, artificial zinc finger proteins (AZFPs) with the Gal4 effector domain were used to enhance cellulase biosynthesis in T. reesei, and it is of great interest to modify the AZFPs to further improve cellulase production. In this study, the endogenous activation domain from the transcription activator Xyr1 was used to replace the activation domain of Gal4 of the AZFP to explore impact on cellulase production. The cellulase producer T. reesei TU-6 was used as a host strain, and the engineered strains containing the Xyr1 and the Gal4 activation domains were named as T. reesei QS2 and T. reesei QS1, respectively. Compared to T. reesei QS1, activities of filter paper and endoglucanases in crude cellulase produced by T. reesei QS2 increased 24.6 and 50.4%, respectively. Real-time qPCR analysis also revealed significant up-regulation of major genes encoding cellulase in T. reesei QS2. Furthermore, the biomass hydrolytic performance of the cellulase was evaluated, and 83.8 and 97.9% more glucose was released during the hydrolysis of pretreated corn stover using crude enzyme produced by T. reesei QS2, when compared to the hydrolysis with cellulase produced by T. reesei QS1 and the parent strain T. reesei TU-6. As a result, we proved that the effector domain in the AZFPs can be optimized to construct more effective artificial transcription factors for engineering T. reesei to improve its cellulase production.

Intracellular Redox Perturbation in Saccharomyces cerevisiae Improved Furfural Tolerance and Enhanced Cellulosic Bioethanol Production.

Furfural is a major toxic byproduct found in the hydrolysate of lignocellulosic biomass, which adversely interferes with the growth and ethanol fermentation of Saccharomyces cerevisiae. The current study was focused on the impact of cofactor availability derived intracellular redox perturbation on furfural tolerance. Here, three strategies were employed in cofactor conversion in S. cerevisiae: (1) heterologous expression of NADH dehydrogenase (NDH) from E. coli which catalyzed the NADH to NAD+ and increased the cellular sensitivity to furfural, (2) overexpression of GLR1, OYE2, ZWF1, and IDP1 genes responsible for the interconversion of NADPH and NADP+, which enhanced the furfural tolerance, (3) expression of NAD(P)+ transhydrogenase (PNTB) and NAD+ kinase (POS5) which showed a little impact on furfural tolerance. Besides, a substantial redistribution of metabolic fluxes was also observed with the expression of cofactor-related genes. These results indicated that NADPH-based intracellular redox perturbation plays a key role in furfural tolerance, which suggested single-gene manipulation as an effective strategy for enhancing tolerance and subsequently achieving higher ethanol titer using lignocellulosic hydrolysate.

Global Reprogramming of Gene Transcription in Trichoderma reesei by Overexpressing an Artificial Transcription Factor for Improved Cellulase Production and Identification of Ypr1 as an Associated Regulator.

Synthetic biology studies on filamentous fungi are providing unprecedented opportunities for optimizing this important category of microbial cell factory. Artificial transcription factor can be designed and used to offer novel modes of regulation on gene transcription network. Trichoderma reesei is commonly used for cellulase production. In our previous studies, a plasmid library harboring genes encoding artificial zinc finger proteins (AZFPs) was constructed for engineering T. reesei, and the mutant strains with improved cellulase production were selected. However, the underlying mechanism by which AZFP function remain unclear. In this study, a T. reesei Rut-C30 mutant strain T. reesei U5 bearing an AZFP named as AZFP-U5 was focused, which secretes high level protein and shows significantly improved cellulase and xylanase production comparing with its parental strain. In addition, enhanced sugar release was achieved from lignocellulosic biomass using the crude cellulase from T. reesei U5. Comparative transcriptome analysis was further performed, which showed reprogramming of global gene transcription and elevated transcription of genes encoding glycoside hydrolases by overexpressing AZFP-U5. Furthermore, 15 candidate regulatory genes which showed remarkable higher transcription levels by AZFP-U5 insertion were overexpressed in T. reesei Rut-C30 to examine their effects on cellulase biosynthesis. Among these genes, TrC30_93861 (ypr1) and TrC30_74374 showed stimulating effects on filter paper activity (FPase), but deletion of these two genes did not affect cellulase activity. In addition, increased yellow pigment production in T. reesei Rut-C30 by overexpression of gene ypr1 was observed, and changes of cellulase gene transcription were revealed in the ypr1 deletion mutant, suggesting possible interaction between pigment production and cellulase gene transcription. The results in this study revealed novel aspects in regulation of cellulase gene expression by the artificial regulators. In addition, the candidate genes and processes identified in the transcriptome data can be further explored for synthetic biology design and metabolic engineering of T. reesei to enhance cellulase production.

Potential of Zymomonas mobilis as an electricity producer in ethanol production.

BACKGROUND: Microbial fuel cell (MFC) convokes microorganism to convert biomass into electricity. However, most well-known electrogenic strains cannot directly use glucose to produce valuable products. Zymomonas mobilis, a promising bacterium for ethanol production, owns special Entner-Doudoroff pathway with less ATP and biomass produced and the low-energy coupling respiration, making Z. mobilis a potential exoelectrogen. RESULTS: A glucose-consuming MFC is constructed by inoculating Z. mobilis. The electricity with power density 2.0 mW/m2 is derived from the difference of oxidation-reduction potential (ORP) between anode and cathode chambers. Besides, two-type electricity generation is observed as glucose-independent process and glucose-dependent process. For the sake of enhancing MFC efficiency, extracellular and intracellular strategies are implemented. Biofilm removal and addition of c-type cytochrome benefit electricity performance and Tween 80 accelerates the electricity generation. Perturbation of cellular redox balance compromises the electricity output, indicating that redox homeostasis is the principal requirement to reach ideal voltage. CONCLUSION: This study identifies potential feature of electricity activity for Z. mobilis and provides multiple strategies to enhance the electricity output. Therefore, additional electricity generation will benefit the techno-economic viability of the commercial bulk production for biochemicals or biofuels in an efficient and environmentally sustainable manner.

Identification of a novel repressor encoded by the putative gene ctf1 for cellulase biosynthesis in Trichoderma reesei through artificial zinc finger engineering.

Strains from Trichoderma reesei have been used for cellulase production with a long history. It has been well known that cellulase biosynthesis by the fungal species is controlled through regulators, and elucidation of their regulation network is of great importance for engineering T. reesei with robust cellulase production. However, progress in this regard is still very limited. In this study, T. reesei RUT-C30 was transformed with an artificial zinc finger protein (AZFP) library, and the mutant T. reesei M2 with improved cellulase production was screened. Compared to its parent strain, the filter paper activity and endo-ß-glucanase activity in cellulases produced by T. reesei M2 increased 67.2% and 35.3%, respectively. Analysis by quantitative reverse transcription polymerase chain reaction indicated significant downregulation of the putative gene ctf1 in T. reesei M2, and its deletion mutants were thus developed for further studies. An increase of 36.9% in cellulase production was observed in the deletion mutants, but when ctf1 was constitutively overexpressed in T. reesei RUT-C30 under the control of the strong pdc1 promoter, cellulase production was substantially compromised. Comparative transcriptomic analysis revealed that the deletion of ctf1 upregulated transcription of gene encoding the regulator VIB1, but downregulated transcription of gene encoding another regulator RCE1, which consequently upregulated genes encoding the transcription factors XYR1 and ACE3 for the activation of genes encoding cellulolytic enzymes. As a result, ctf1 was characterized as a gene encoding a repressor for cellulase production in T. reesei RUT-C30, which is significant for further elucidating molecular mechanism underlying cellulase biosynthesis by the fungal species for rational design to develop robust strains for cellulase production. And in the meantime, AZFP transformation was validated to be an effective strategy for identifying functions of putative genes in the genome of T. reesei.

Improved cellulase production in recombinant Saccharomyces cerevisiae by disrupting the cell wall protein-encoding gene CWP2.

Budding yeast Saccharomyces cerevisiae has been widely used for heterologous protein production. However, low protein production titer and secretion levels continue to challenge its practical applications. The yeast cell wall plays important roles in yeast cell growth and environmental responses. Nevertheless, the effects of yeast cell wall proteins on heterologous protein production and secretion remain unclear. CWP2 encodes a mannoprotein that is the major component of the yeast cell wall. So far, studies on its function have been very limited. Here we show that CWP2 disruption improved extracellular cellobiohydrolase activity by 85.9%. A calcofluor white hypersensitivity assay revealed increased sensitivity of the mutant compared to the parental strain, indicating impaired cell wall integrity. However, no changes were observed in normal cell growth or growth stressed by tunicamycin and dithiothreitol, suggesting that the unfolded protein response pathway was not affected by the gene disruption. Comparative transcriptome analysis revealed changes in multiple genes involved in cell wall structure, biosynthesis, and cell wall integrity induced by CWP2 disruption, suggesting a pivotal role of Cwp2p in yeast cell wall organization. Notably, CWP2 disruption also led to elevated transcription of a large number of genes involved in ribosome biogenesis, which indicated that CWP2 is not only in yeast cell wall biosynthesis, but also in protein translation. This work reveals novel insights into the functions of CWP2 and also presents a new strategy to increase heterologous protein production in yeast strains by manipulating cell wall-related proteins.