Time-Course Transcriptomic Analysis Reveals Molecular Insights into the Inflorescence and Flower Development of Cardiocrinum giganteum.

Cardiocrinum giganteum is an endemic species of east Asia which is famous for its showy inflorescence and medicinal bulbs. Its inflorescence is a determinate raceme and the flowers bloom synchronously. Morphological observation and time-course transcriptomic analysis were combined to study the process of inflorescence and flower development of C. giganteum. The results show that the autonomic pathway, GA pathway, and the vernalization pathway are involved in the flower formation pathway of C. giganteum. A varied ABCDE flowering model was deduced from the main development process. Moreover, it was found that the flowers in different parts of the raceme in C. giganteum gradually synchronized during development, which is highly important for both evolution and ecology. The results obtained in this work improve our understanding of the process and mechanism of inflorescence and flower development and could be useful for the flowering period regulation and breeding of C. giganteum.

Fungicide-tolerant persister formation during cryptococcal pulmonary infection.

Bacterial persisters, a subpopulation of genetically susceptible cells that are normally dormant and tolerant to bactericides, have been studied extensively because of their clinical importance. In comparison, much less is known about the determinants underlying fungicide-tolerant fungal persister formation in vivo. Here, we report that during mouse lung infection, Cryptococcus neoformans forms persisters that are highly tolerant to amphotericin B (AmB), the standard of care for treating cryptococcosis. By exploring stationary-phase indicator molecules and developing single-cell tracking strategies, we show that in the lung, AmB persisters are enriched in cryptococcal cells that abundantly produce stationary-phase molecules. The antioxidant ergothioneine plays a specific and key role in AmB persistence, which is conserved in phylogenetically distant fungi. Furthermore, the antidepressant sertraline (SRT) shows potent activity specifically against cryptococcal AmB persisters. Our results provide evidence for and the determinant of AmB-tolerant persister formation in pulmonary cryptococcosis, which has potential clinical significance.

High-level production of Aspergillus niger prolyl endopeptidase from agricultural residue and its application in beer brewing.

BACKGROUND: Prolyl endopeptidase from Aspergillus niger (AN-PEP) is a prominent serine proteinase with various potential applications in the food and pharmaceutical industries. However, the availability of efficient and low-cost AN-PEP remains a challenge owing to its low yield and high fermentation cost. RESULTS: Here, AN-PEP was recombinantly expressed in Trichoderma reesei (rAN-PEP) under the control of the cbh1 promoter and its secretion signal. After 4 days of shaking flask cultivation with the model cellulose Avicel PH101 as the sole carbon source, the extracellular prolyl endopeptidase activity reached up to 16.148 U/mL, which is the highest titer reported to date and the secretion of the enzyme is faster in T. reesei than in other eukaryotic expression systems including A. niger and Komagataella phaffii. Most importantly, when cultivated on the low-cost agricultural residue corn cob, the recombinant strain was found to secret a remarkable amount of rAN-PEP (37.125 U/mL) that is twice the activity under the pure cellulose condition. Furthermore, treatment with rAN-PEP during beer brewing lowered the content of gluten below the ELISA kit detection limit (< 10 mg/kg) and thereby, reduced turbidity, which would be beneficial for improving the non-biological stability of beer. CONCLUSION: Our research provides a promising approach for industrial production of AN-PEP and other enzymes (proteins) from renewable lignocellulosic biomass, which provides a new idea with relevant researchers for the utilization of agricultural residues.

Oral delivery of a highly stable superoxide dismutase as a skin aging inhibitor.

As an effective antioxidant enzyme, superoxide dismutase (SOD) has been widely used as a food supplement, cosmetic additive, and therapeutic agent. However, oral delivery of SOD is challenging due to its relative instability, limited bioavailability, and low absorption efficiency in the gastrointestinal (GI) tract. We addressed these issues using a highly stable superoxide dismutase (hsSOD) generated from a hot spring microbial sample. This SOD exhibited a specific activity of 5000 IU/mg while retaining its enzymatic activity under low pH environments of an artificial GI system and in the presence of surfactants and various proteolytic enzymes. The inhibitory effects of hsSOD against skin-aging was evaluated under both in vitro and in vivo experiments using fibroblast cell and D-galactose induced aging-mouse models, respectively. Effective oral delivery of hsSOD promises wide applicability in pharmaceutical and food industries.

Rapid measurement of brown tide algae using Zernike moments and ensemble learning based on excitation-emission matrix fluorescence.

Accurate real-time prediction of microalgae density has great practical significance for taking countermeasures before the advent of Harmful algal blooms (HABs), and the non-destructive and sensitive property of excitation-emission matrix fluorescence (EEMF) spectroscopy makes it applicable to online monitoring and control. In this study, an efficient image preprocessing algorithm based on Zernike moments (ZMs) was proposed to extract compelling features from EEM intensities images. The determination of the highest order of ZMs considered both reconstruction error and computational cost, then the optimal subset of preliminarily extracted 36 ZMs was screened via the BorutaShap algorithm. Aureococcus anophagefferens concentration prediction models were developed by combining BorutaShap and ensemble learning models (random forest (RF), gradient boosting decision tree (GBDT), and XGBoost). The experimental results show that BorutaShap_GBDT preserved the superior subset of ZMs, and the integration of BorutaShap_GBDT and XGBoost achieved the highest prediction accuracy. This research provides a new and promising strategy for rapidly measuring microalgae cell density.

An Ancient Respiratory System in the Widespread Sedimentary Archaea Thermoprofundales.

Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A-E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.

Isolation and Identification of Efficient Malathion-Degrading Bacteria from Deep-Sea Hydrothermal Sediment.

The genetic and metabolic diversity of deep-sea microorganisms play important roles in phosphorus and sulfur cycles in the ocean, distinguishing them from terrestrial counterparts. Malathion is a representative organophosphorus component in herbicides, pesticides, and insecticides and is analogues of neurotoxic agent. Malathion has been one of the best-selling generic organophosphate insecticides from 1980 to 2012. Most of the sprayed malathion has migrated by surface runoff to ocean sinks, and it is highly toxic to aquatic organisms. Hitherto, there is no report on bacterial cultures capable of degrading malathion isolated from deep-sea sediment. In this study, eight bacterial strains, isolated from sediments from deep-sea hydrothermal regions, were identified as malathion degradators. Two of the tested strains, Pseudidiomarina homiensis strain FG2 and Pseudidiomarina sp. strain CB1, can completely degrade an initial concentration of 500 mg/L malathion within 36 h. Since the two strains have abundant carboxylesterases (CEs) genes, malathion monocarboxylic acid (MMC α and MMC ß) and dibasic carboxylic acid were detected as key intermediate metabolites of malathion degradation, and the pathway of malathion degradation between the two strains was identified as a passage from malathion monocarboxylic acid to malathion dicarboxylic acid.

A Novel Organophosphorus Acid Anhydrolase from Deep Sea Sediment with High Degradation Efficiency for Organophosphorus Pesticides and Nerve Agent.

Organophosphorus compounds (OPCs), including highly toxic nerve agents and pesticides, have been used widely in agricultural and military applications. However, they have aroused widespread concern because they persistently pollute the environment and threaten human life. Organophosphorus acid anhydrolase (OPAA) is a promising enzyme that can detoxify OPCs. Here, a novel OPAA (OPAA114644) was isolated and characterized from deep-sea sediment (-3104 m). It exhibited excellent alkaline stability, and the loss of activity was less than 20% in the pH range 5.0-9.0, even after being incubated for 30 d at 4 °C. It also exhibited high salt tolerance, and its enzymatic activity increased by approximately fourfold in the presence of 20% NaCl (w/v). Additionally, OPAA114644 exhibited high degradation efficiency for soman, dichlorvos, paraoxon, coumaphos, and chlorpyrifos with a concentration of up to 250 mg/L, with the degradation rate being 100%, 100%, 100%, 80% and 51%, respectively, in 20 min under optimal conditions. Notably, OPAA114644 dissolved in different solutions, such as 20% NaCl, 1 mM SDS, 0.05% soap, 10% methanol, and tap water, could efficiently decontaminate the residual paraoxon on the surfaces of glasses, cotton tissues, and apples. These results indicate that OPAA114644 has excellent potential for the biodegradation and bioremediation of OPCs pollution and represents a real application of OPAA in the decontamination and detoxification of foods and clothes, and in the remediation of sites such as floors. Deep-sea sediment might also be an abundant resource for various functional microorganisms and enzymes.

Toward more efficient ergothioneine production using the fungal ergothioneine biosynthetic pathway.

BACKGROUND: Ergothioneine (ERG) is a potent histidine-derived antioxidant that confers health-promoting effects. Only certain bacteria and fungi can biosynthesize ERG, but the ERG productivity in natural producers is low. ERG overproduction through genetic engineering represents an efficient and cost-effective manufacturing strategy. RESULTS: Here, we showed that Trichoderma reesei can synthesize ERG during conidiogenesis and hyphal growth. Co-expression of two ERG biosynthesis genes (tregt1 and tregt2) from T. reesei enabled E. coli to generate 70.59 mg/L ERG at the shaking flask level after 48 h of whole-cell biocatalysis, whereas minor amounts of ERG were synthesized by the recombinant E. coli strain bearing only the tregt1 gene. By fed-batch fermentation, the extracellular ERG production reached 4.34 g/L after 143 h of cultivation in a 2-L jar fermenter, which is the highest level of ERG production reported thus far. Similarly, ERG synthesis also occurred in the E. coli strain engineered with the two well-characterized genes from N. crassa and the ERG productivity was up to 4.22 g/L after 143 h of cultivation under the above-mentioned conditions. CONCLUSIONS: Our results showed that the overproduction of ERG in E. coli could be achieved through two-enzymatic steps, demonstrating high efficiency of the fungal ERG biosynthetic pathway. Meanwhile, this work offers a more promising approach for the industrial production of ERG.

Amplicon-based sequencing and co-occurence network analysis reveals notable differences of microbial community structure in healthy and dandruff scalps.

BACKGROUND: Dandruff is a chronic, recurring, and common scalp problem that is caused by several etiopathogeneses with complex mechanisms. Management of this condition is typically achieved via antifungal therapies. However, the precise roles played by microbiota in the development of the condition have not been elucidated. Despite their omnipresence on human scalp little is known about the co-occurrence/co-exclusion network of cutaneous microbiota. RESULTS: We characterized the scalp and hair surface bacterial and fungal communities of 95 dandruff-afflicted and healthy individuals residing in China. The degree distributions of co-occurrence/co-exclusion network in fungi-bacteria and bacteria-bacteria were higher in the healthy group (P < 0.0001), whereas the betweenness values are higher in the dandruff group (P < 0.01). Meanwhile, the co-occurrence/co-exclusion network among fungi-fungi and fungi-bacteria showed that compared to the healthy group, the dandruff group had more positive links (P < 0.0001). In addition, we observed that Malassezia slooffiae, Malassezia japonica and Malassezia furfur, were more abundant in the dandruff group than in the healthy group. These microbiota were co-exclusion by either multiple bacterial genera or Malassezia sp. in healthy group. The lactic acid bacteria on the scalp and hair surface, especially the genera Lactobacillus and Lactococcus, exhibit a negative correlation with multiple bacterial genera on the scalp and hair surface. Lactobacillus plantarum and Pediococcus lactis isolated on the healthy human scalp can inhibit the growth of Staphylococcus epidermidis in vitro. CONCLUSIONS: We showed that microbial networks on scalp and hair surface with dandruff were less integrated than their healthy counterparts, with lower node degree and more positive and stronger links which were deemed to be unstable and may be more susceptible to environmental fluctuations. Lactobacillus bacteria have extensive interactions with other bacteria or fungi in the scalp and hair surface micro-ecological network and can be used as targets for improving scalp health.

Fluorescence-activated droplet sorting of PET degrading microorganisms.

Enzymes that can decompose synthetic plastics such as polyethylene terephthalate (PET) are urgently needed. Still, a bottleneck remains due to a lack of techniques for detecting and sorting environmental microorganisms with vast diversity and abundance. Here, we developed a fluorescence-activated droplet sorting (FADS) pipeline for high-throughput screening of PET-degrading microorganisms or enzymes (PETases). The pipeline comprises three steps: generation and incubation of droplets encapsulating single cells, picoinjection of fluorescein dibenzoate (FDBz) as the fluorogenic probe, and screening of droplets to obtain PET-degrading cells. We characterized critical factors associated with this method, including specificity and sensitivity for discriminating PETase from other enzymes. We then optimized its performance and compatibility with environmental samples. The system was used to screen a wastewater sample from a PET textile mill. We successfully obtained PET-degrading species from nine different genera. Moreover, two putative PETases from isolates Kineococcus endophyticus Un-5 and Staphylococcus epidermidis Un-C2-8 were genetically derived, heterologously expressed, and preliminarily validated for PET-degrading activities. We speculate that the FADS pipeline can be widely adopted to discover new plastic-degrading microorganisms and enzymes in various environments and may be utilized in the directed evolution of degrading enzymes using synthetic biology.

Development of an artificial zinc finger - Luciferase fusion protein for the rapid detection of Salmonella typhimurium.

A novel artificial Zinc finger - luciferase fusion protein was successfully developed for rapid detection of Salmonella typhimurium, a worldwide-distributed foodborne pathogen. The designed Zinc finger (ZF) protein bound specifically to a 12 bp region of the Salmonella spp invasion gene invA. While the luciferase from Gaussia princeps called Gaussia luciferase (Gluc) was for the first time fused with the artificial ZF domain to improve the detection sensitivity. The fusion protein successfully recognized and bound to the synthesized invA dsDNA with high specificity and sensitivity. The detection limit was as low as 10 fmol of dsNDA. Then, the bacteria PCR products were subsequently used to assess the zinc finger - luciferase fusion protein. The final results indicated that the ZF-Gluc fusion protein system could detect S. typhimurium as low as 1 CFU/mL in 2 h after the PCR. Therefore, this study provided us with a novel artificial zinc finger fusion protein and an efficient method to accomplish the rapid detection of the major foodborne pathogen S. typhimurium. In addition, the specific artificial ZF proteins that bund to particular dsDNA sequences could be easily designed, the ZF-Gluc might has broad application prospects in the field of rapid pathogenic bacteria detection.

Understanding the Role of Trichoderma reesei Vib1 in Gene Expression during Cellulose Degradation.

Vib1, a member of the Ndt80/PhoG-like transcription factor family, has been shown to be essential for cellulase production of Trichoderma reesei. Here, we combined transcriptomic and genetic analyses to gain mechanistic insights into the roles of Vib1 during cellulose degradation. Our transcriptome analysis showed that the vib1 deletion caused 586 genes with decreased expression and 431 genes with increased expression on cellulose. The downregulated genes were enriched for Gene Ontology terms associated with carbohydrate metabolism, transmembrane transport, oxidoreductase activity, and transcription factor activity. Of the 258 genes induced by cellulose, 229 showed no or decreased expression in Δvib1 on cellulose, including almost all (hemi)cellulase genes, crucial sugar transporter genes (IDs:69957, 3405), and the genes encoding main transcriptional activators Xyr1 and Ace3. Additionally, Vib1 also regulated the expression of genes involved in secondary metabolism. Further comparison of the transcriptomes of Δvib1 and Δxyr1 in cellulose revealed that the genes regulated by Vib1 had much overlap with Xyr1 targets especially for the gene set induced by cellulose, presumably whose expression requires the cooperativity between Vib1 and Xyr1. Genetic evidence indicated that Vib1 regulates cellulase gene expression partially via Xyr1. Our results will provide new clues for strain improvement.

Combining protein and metabolic engineering strategies for biosynthesis of melatonin in Escherichia coli.

BACKGROUND: Melatonin has attracted substantial attention because of its excellent prospects for both medical applications and crop improvement. The microbial production of melatonin is a safer and more promising alternative to chemical synthesis approaches. Researchers have failed to produce high yields of melatonin in common heterologous hosts due to either the insolubility or low enzyme activity of proteins encoded by gene clusters related to melatonin biosynthesis. RESULTS: Here, a combinatorial gene pathway for melatonin production was successfully established in Escherichia coli by combining the physostigmine biosynthetic genes from Streptomyces albulus and gene encoding phenylalanine 4-hydroxylase (P4H) from Xanthomonas campestris and caffeic acid 3-O-methyltransferase (COMT) from Oryza sativa. A threefold improvement of melatonin production was achieved by balancing the expression of heterologous proteins and adding 3% glycerol. Further protein engineering and metabolic engineering were conducted to improve the conversion of N-acetylserotonin (NAS) to melatonin. Construction of COMT variant containing C303F and V321T mutations increased the production of melatonin by fivefold. Moreover, the deletion of speD gene increased the supply of S-adenosylmethionine (SAM), an indispensable cofactor of COMT, which doubled the yield of melatonin. In the final engineered strain EcMEL8, the production of NAS and melatonin reached 879.38 ± 71.42 mg/L and 136.17 ± 1.33 mg/L in a shake flask. Finally, in a 2-L bioreactor, EcMEL8 produced 1.06 ± 0.07 g/L NAS and 0.65 ± 0.11 g/L melatonin with tryptophan supplementation. CONCLUSIONS: This study established a novel combinatorial pathway for melatonin biosynthesis in E. coli and provided alternative strategies for improvement of melatonin production.

Detection of the deep biosphere in metamorphic rocks from the Chinese continental scientific drilling.

It is generally accepted that there is a vast, well-populated biosphere in the subsurface, but the depth limit of the terrestrial biosphere has yet to be determined, largely because of the lack of access to the subsurface. Here as part of the Chinese Continental Scientific Drilling (CCSD) project in eastern China, we acquired continuous rock cores and endeavored to probe the depth limit of the biosphere and the depth-dependent distribution of microorganisms at a geologically unique site, that is, a convergent plate boundary. Microbiological analyses of ultra-high-pressure metamorphic rock cores taken from the ground surface to 5,158-meter reveal that microbial distribution was continuous up to a depth of ~4,850 m, where temperature was estimated to be ~137°C. The metabolic state of these organisms at such great depth remains to be determined. Microbial abundance, ranging from 103 to 108  cells/g, was also related to porosity, but not to the depth and rock composition. In addition, microbial diversity systematically decreased with depth. Our results support the notion that temperature is a key factor in determining the lower limit of the biosphere in the continental subsurface.

Extraordinary diversity of viruses in deep-sea sediments as revealed by metagenomics without prior virion separation.

Our current knowledge of the virosphere in deep-sea sediments remains rudimentary. Here we investigated viral diversity at both gene and genomic levels in deep-sea sediments of Southwest Indian Ocean. Analysis of 19 676 106 non-redundant genes from the metagenomic DNA sequences revealed a large number of unclassified viral groups in these samples. A total of 1106 high-confidence viral contigs were obtained after two runs of assemblies, and 217 of these contigs with sizes up to ~120 kb were shown to represent complete viral genomes. These contigs are clustered with no known viral genomes, and over 2/3 of the ORFs on the viral contigs encode no known functions. Furthermore, most of the complete viral contigs show limited similarity to known viral genomes in genome organization. Most of the classified viral contigs are derived from dsDNA viruses belonging to the order Caudovirales, including primarily members of the families Myoviridae, Podoviridae and Siphoviridae. Most of these viruses infect Proteobacteria and, less frequently, Planctomycetes, Firmicutes, Chloroflexi, etc. Auxiliary metabolic genes (AMGs), present in abundance on the viral contigs, appear to function in modulating the host ability to sense environmental gradients and community changes, and to uptake and metabolize nutrients.

Intein-mediated intracellular production of active microbial transglutaminase in Corynebacterium glutamicum.

The microbial transglutaminase (mTGase) from Streptomyces mobaraense is widely used in the food industry. However, recombinant production of mTGase is challenging because the mTGase is synthesized as an inactive zymogen, and needs to be activated by proteolytic processing. In this study, self-cleaving intein Ssp DnaB was applied to activate the mTGase in Corynebacterium glutamicum. Premature cleavage of intein Ssp DnaB also occurred, but instead of suppressing premature cleavage, this phenomenon was used to produce active mTGase in C. glutamicum. Both SDS-PAGE analysis and mTGase activity assays indicated that the premature cleavage of intein Ssp DnaB activated the mTGase intracellularly in C. glutamicum. The subsequent N-terminal amino acid sequencing and site-directed mutagenesis studies further showed that the premature cleavage activated the mTGase intracellularly, in a highly specific manner. Moreover, the growth performance of C. glutamicum was not noticeably affected by the intracellular expression of active mTGase. Finally, the mTGase was produced in a 2 L bioreactor, with activity up to 49 U/mL, the highest intracellular mTGase activity ever reported. Using premature cleavage of intein Ssp DnaB to activate mTGase in C. glutamicum, we produced high levels of intracellular active mTGase. Moreover, this approach did not require any further processing steps, such as protease treatment or lengthy incubation, greatly simplifying the production of active mTGase. This efficient and simple approach has great potential for the large-scale industrial production of active mTGase.

Interfacial Nanoinjection-Based Nanoliter Single-Cell Analysis.

Single-cell analysis offers unprecedented resolution for the investigation of cellular heterogeneity and the capture of rare cells from large populations. Here, described is a simple method named interfacial nanoinjection (INJ), which can miniaturize various single-cell assays to be performed in nanoliter water-in-oil droplets on standard microwell plates. The INJ droplet handler can adjust droplet volumes for multistep reactions on demand with high precision and excellent monodispersity, and consequently enables a wide range of single-cell assays. Importantly, INJ can be coupled with fluorescence-activated cell sorting (FACS), which is currently the most effective and accurate single-cell sorting and isolation method. FACS-INJ pipelines for high-throughput plate well-based single-cell analyses, including single-cell proliferation, drug-resistance testing, polymerase chain reaction (PCR), reverse-transcription PCR, and whole-genome sequencing are introduced. This FACS-INJ pipeline is compatible with a wide range of samples and can be extended to various single-cell analysis applications in microbiology, cell biology, and biomedical diagnostics.

Semiholoenzyme optimizes activity and stability of a hyperthermostable iron-superoxide dismutase.

Metal ion coordination is an essential step for the maturation of metalloenzymes. Generally, the metal coordination sites are thought to be fully occupied to achieve the maximum activity and stability. In this research, we compared the structural features, activity and stability of the apo-, semiholo- and holo-forms of a hyperthermostable tetrameric Fe-superoxide dismutase (SOD). Strikingly, the three forms of enzymes had similar compact tetrameric structures. Removal of iron ions destabilized subunit-subunit interactions during guanidine hydrochloride-induced unfolding. The partially metalized semiholoenzyme possessed most of the activity and identical hyperthermostability of the holoenzyme, but weaker propensity to aggregate. Furthermore, both of the iron content and activity of the semiholoenzyme were unaffected by a 200-fold excess iron ions in solutions, suggesting that conformation of the apo-subunits were forced to the close state by the iron-containing subunits. These observations suggest that fully metalized enzyme is probably nonessential for multimeric metalloenzymes and the semiholoenzyme may be a better choice. The unique properties of semiholoenzyme also provide the organisms a compromised solution to survival under metal deficiency conditions.

Identification of two integration sites in favor of transgene expression in Trichoderma reesei.

BACKGROUND: The ascomycete fungus Trichoderma reesei was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of T. reesei can generate series of strains that express different levels of the indicated transgene. The insertion site of the GOI plays an important role in the ultimate production of the targeted proteins. However, so far no systematic studies have been made to identify transgene integration loci for optimal expression of the GOI in T. reesei. Currently, only the locus of exocellobiohydrolases I encoding gene (cbh1) is widely used as a promising integration site to lead to high expression level of the GOI. No additional sites associated with efficient gene expression have been characterized. RESULTS: To search for gene integration sites that benefit for the secreted expression of GOI, the food-and-mouth disease virus 2A protein was applied for co-expression of an Aspergillus niger lipA gene and Discosoma sp. DsRed1 gene in T. reesei, by random integration of the expression cassette into the genome. We demonstrated that the fluorescent intensity of RFP (red fluorescent protein) inside of the cell was well correlated with the secreted lipase yields, based on which, we successfully developed a high-throughput screening method to screen strains with relatively higher secreted expression of the GOI (in this study, lipase). The copy number and the insertion sites of the transgene were investigated among the selected highly expressed strains. Eventually, in addition to cbh1 gene locus, two other genome insertion loci that efficiently facilitate gene expression in T. reesei were identified. CONCLUSIONS: We have successfully developed a high-throughput screening method to screen strains with optimal expression of the indicated secreted proteins in T. reesei. Moreover, we identified two optimal genome loci for transgene expression, which could provide new approach to modulate gene expression levels while retaining the indicated promoter and culture conditions.