Protein-protein interaction and site prediction using transfer learning.

The advanced language models have enabled us to recognize protein-protein interactions (PPIs) and interaction sites using protein sequences or structures. Here, we trained the MindSpore ProteinBERT (MP-BERT) model, a Bidirectional Encoder Representation from Transformers, using protein pairs as inputs, making it suitable for identifying PPIs and their respective interaction sites. The pretrained model (MP-BERT) was fine-tuned as MPB-PPI (MP-BERT on PPI) and demonstrated its superiority over the state-of-the-art models on diverse benchmark datasets for predicting PPIs. Moreover, the model's capability to recognize PPIs among various organisms was evaluated on multiple organisms. An amalgamated organism model was designed, exhibiting a high level of generalization across the majority of organisms and attaining an accuracy of 92.65%. The model was also customized to predict interaction site propensity by fine-tuning it with PPI site data as MPB-PPISP. Our method facilitates the prediction of both PPIs and their interaction sites, thereby illustrating the potency of transfer learning in dealing with the protein pair task.

Sub-genomic RNAi-assisted strain evolution of filamentous fungi for enhanced protein production.

Genetic engineering at the genomic scale provides a rapid means to evolve microbes for desirable traits. However, in many filamentous fungi, such trials are daunted by low transformation efficiency. Differentially expressed genes under certain conditions may contain important regulatory factors. Accordingly, although manipulating these subsets of genes only can largely reduce the time and labor, engineering at such a sub-genomic level may also be able to improve the microbial performance. Herein, first using the industrially important cellulase-producing filamentous fungus Trichoderma reesei as a model organism, we constructed suppression subtractive hybridization (SSH) libraries enriched with differentially expressed genes under cellulase induction (MM-Avicel) and cellulase repression conditions (MM-Glucose). The libraries, in combination with RNA interference, enabled sub-genomic engineering of T. reesei for enhanced cellulase production. The ability of T. reesei to produce endoglucanase was improved by 2.8~3.3-fold. In addition, novel regulatory genes (tre49304, tre120391, and tre123541) were identified to affect cellulase expression in T. reesei. Iterative manipulation using the same strategy further increased the yield of endoglucanase activity to 75.6 U/mL, which was seven times as high as that of the wild type (10.8 U/mL). Moreover, using Humicola insolens as an example, such a sub-genomic RNAi-assisted strain evolution proved to be also useful in other industrially important filamentous fungi. H. insolens is a filamentous fungus commonly used to produce catalase, albeit with similarly low transformation efficiency and scarce knowledge underlying the regulation of catalase expression. By combining SSH and RNAi, a strain of H. insolens producing 28,500 ± 288 U/mL of catalase was obtained, which was 1.9 times as high as that of the parent strain.IMPORTANCEGenetic engineering at the genomic scale provides an unparalleled advantage in microbial strain improvement, which has previously been limited only to the organisms with high transformation efficiency such as Saccharomyces cerevisiae and Escherichia coli. Herein, using the filamentous fungus Trichoderma reesei as a model organism, we demonstrated that the advantage of suppression subtractive hybridization (SSH) to enrich differentially expressed genes and the convenience of RNA interference to manipulate a multitude of genes could be combined to overcome the inadequate transformation efficiency. With this sub-genomic evolution strategy, T. reesei could be iteratively engineered for higher cellulase production. Intriguingly, Humicola insolens, a fungus with even little knowledge in gene expression regulation, was also improved for catalase production. The same strategy may also be expanded to engineering other microorganisms for enhanced production of proteins, organic acids, and secondary metabolites.

Mining and rational design of psychrophilic catalases using metagenomics and deep learning models.

A complete catalase-encoding gene, designated soiCat1, was obtained from soil samples via metagenomic sequencing, assembly, and gene prediction. soiCat1 showed 73% identity to a catalase-encoding gene of Mucilaginibacter rubeus strain P1, and the amino acid sequence of soiCAT1 showed 99% similarity to the catalase of a psychrophilic bacterium, Pedobacter cryoconitis. soiCAT1 was identified as a psychrophilic enzyme due to the low optimum temperature predicted by the deep learning model Preoptem, which was subsequently validated through analysis of enzymatic properties. Experimental results showed that soiCAT1 has a very narrow range of optimum temperature, with maximal specific activity occurring at the lowest test temperature (4 °C) and decreasing with increasing reaction temperature from 4 to 50 °C. To rationally design soiCAT1 with an improved temperature range, soiCAT1 was engineered through site-directed mutagenesis based on molecular evolution data analyzed through position-specific amino acid possibility calculation. Compared with the wild type, one mutant, soiCAT1S205K, exhibited an extended range of optimum temperature ranging from 4 to 20 °C. The strategies used in this study may shed light on the mining of genes of interest and rational design of desirable proteins. KEY POINTS: • Numerous putative catalases were mined from soil samples via metagenomics. • A complete sequence encoding a psychrophilic catalase was obtained. • A mutant psychrophilic catalase with an extended range of optimum temperature was engineered through site-directed mutagenesis.

Production of cello-oligosaccharides from corncob residue by degradation-synthesis reactions.

The cellulose-rich corncob residue (CCR) is an abundant and renewable agricultural biomass that has been under-exploited. In this study, two strategies were compared for their ability to transform CCR into cello-oligosaccharides (COS). The first strategy employed the use of endo-glucanases. Although selected endo-glucanases from GH9, GH12, GH45, and GH131 could release COS with degrees of polymerization from 2 to 4, the degrading efficiency was low. For the second strategy, first, CCR was efficiently depolymerized to glucose and cellobiose using the cellulase from Trichoderma reesei. Then, using these simple sugars and sucrose as the starting materials, phosphorylases from different microorganisms were combined to generate COS to a level up to 100.3 g/L with different patterns and degrees of polymerization. Using tomato as a model plant, the representative COS obtained from BaSP (a sucrose phosphorylase from Bifidobacterium adolescens), CuCbP (a cellobiose phosphorylase from Cellulomonas uda), and CcCdP (a cellodextrin phosphorylase from Clostridium cellulosi) were shown to be able to promote plant growth. The current study pointed to an approach to make use of CCR for production of the value-added COS. KEY POINTS: • Sequential use of cellulase and phosphorylases effectively generated cello-oligosaccharides from corncob residue. • Cello-oligosaccharides patterns varied in accordance to cellobiose/cellodextrin phosphorylases. • Spraying cello-oligosaccharides promoted tomato growth.

Targeting deoxynivalenol for degradation by a chimeric manganese peroxidase/glutathione system.

The manganese peroxidase (MnP) can degrade multiple mycotoxins including deoxynivalenol (DON) efficiently; however, the lignin components abundant in foods and feeds were discovered to interfere with DON catalysis. Herein, using MnP from Ceriporiopsis subvermispora (CsMnP) as a model, it was demonstrated that desired catalysis of DON, but not futile reactions with lignin, in the reaction systems containing feeds could be achieved by engineering MnP and supplementing with a boosting reactant. Specifically, two successive strategies (including the fusion of CsMnP to a DON-recognizing ScFv and identification of glutathione as a specific targeting enhancer) were combined to overcome the lignin competition, which together resulted into elevation of the degradation rate from 2.5% to as high as 82.7% in the feeds. The method to construct a targeting MnP and fortify it with an additional enhancer could be similarly applied to catalyze the many other mycotoxins with yet unknown responsive biocatalysts.

Theoretical insights into the mechanism underlying aflatoxin B1 transformation by the BsCotA-methyl syringate system.

Global concern exists regarding the contamination of food and animal feed with aflatoxin B1 (AFB1), which poses a threat to the health of both humans and animals. Previously, we found that a laccase from Bacillus subtilis (BsCotA) effectively detoxified AFB1 in a reaction mediated by methyl syringate (MS), although the underlying mechanism has not been determined. Therefore, our primary objective of this study was to explore the detoxification mechanism employed by BsCotA. First, the enzyme and mediator dependence of AFB1 transformation were studied using the BsCotA-MS system, which revealed the importance of MS radical formation during the oxidation process. Aflatoxin Q1 (AFQ1) resulting from the direct oxidation of AFB1 by BsCotA, was identified using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results of UPLC-MS/MS and density functional theory calculations indicated that the products included AFQ1, AFB1-, and AFD1-MS-coupled products in the BsCotA-MS system. The toxicity evaluations revealed that the substances derived from the transformation of AFB1 through the BsCotA-MS mechanism exhibited markedly reduced toxicity compared to AFB1. Finally, we proposed a set of different AFB1-transformation pathways generated by the BsCotA-MS system based on the identified products. These findings greatly enhance the understanding of the AFB1-transformation mechanism of the laccase-mediator system.

MaSMG7-Mediated Degradation of MaERF12 Facilitates Fusarium oxysporum f. sp. cubense Tropical Race 4 Infection in Musa acuminata.

Modern plant breeding relies heavily on the deployment of susceptibility and resistance genes to defend crops against diseases. The expression of these genes is usually regulated by transcription factors including members of the AP2/ERF family. While these factors are a vital component of the plant immune response, little is known of their specific roles in defense against Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) in banana plants. In this study, we discovered that MaERF12, a pathogen-induced ERF in bananas, acts as a resistance gene against Foc TR4. The yeast two-hybrid assays and protein-protein docking analyses verified the interaction between this gene and MaSMG7, which plays a role in nonsense-mediated RNA decay. The transient expression of MaERF12 in Nicotiana benthamiana was found to induce strong cell death, which could be inhibited by MaSMG7 during co-expression. Furthermore, the immunoblot analyses have revealed the potential degradation of MaERF12 by MaSMG7 through the 26S proteasome pathway. These findings demonstrate that MaSMG7 acts as a susceptibility factor and interferes with MaERF12 to facilitate Foc TR4 infection in banana plants. Our study provides novel insights into the biological functions of the MaERF12 as a resistance gene and MaSMG7 as a susceptibility gene in banana plants. Furthermore, the first discovery of interactions between MaERF12 and MaSMG7 could facilitate future research on disease resistance or susceptibility genes for the genetic improvement of bananas.

Identification and Characterization of the Determinants of Copper Resistance in the Acidophilic Fungus Acidomyces richmondensis MEY-1 Using the CRISPR/Cas9 System.

Copper (Cu) homeostasis has not been well documented in filamentous fungi, especially extremophiles. One of the main obstacles impeding their characterization is the lack of a powerful genome-editing tool. In this study, we applied a CRISPR/Cas9 system for efficient targeted gene disruption in the acidophilic fungus Acidomyces richmondensis MEY-1, formerly known as Bispora sp. strain MEY-1. Using this system, we investigated the basis of Cu tolerance in strain MEY-1. This strain has extremely high Cu tolerance among filamentous fungi, and the transcription factor ArAceA (A. richmondensis AceA) has been shown to be involved in this process. The ArAceA deletion mutant (ΔArAceA) exhibits specific growth defects at Cu concentrations of ≥10 mM and is transcriptionally more sensitive to Cu than the wild-type strain. In addition, the putative metallothionein ArCrdA was involved in Cu tolerance only under high Cu concentrations. MEY-1 has no Aspergillus nidulans CrpA homologs, which are targets of AceA-like transcription factors and play a role in Cu tolerance. Instead, we identified the Cu-transporting P-type ATPase ArYgA, homologous to A. nidulans YgA, which was involved in pigmentation rather than Cu tolerance. When the ΔArYgA mutant was grown on medium supplemented with Cu ions, the black color was completely restored. The lack of CrpA homologs in A. richmondensis MEY-1 and its high tolerance to Cu suggest that a novel Cu detoxification mechanism differing from the AceA-CrpA axis exists. IMPORTANCE Filamentous fungi are widely distributed worldwide and play an important ecological role as decomposers. However, the mechanisms of their adaptability to various environments are not fully understood. Various extremely acidophilic filamentous fungi have been isolated from acidic mine drainage (AMD) with extremely low pH and high heavy metal and sulfate concentrations, including A. richmondensis. The lack of genetic engineering tools, particularly genome-editing tools, hinders the study of these acidophilic and heavy metal-resistant fungi at the molecular level. Here, we first applied a CRISPR/Cas9-mediated gene-editing system to A. richmondensis MEY-1. Using this system, we identified and characterized the determinants of Cu resistance in A. richmondensis MEY-1. The conserved roles of the Cu-binding transcription factor ArAceA in Cu tolerance and the Cu-transporting P-type ATPase ArYgA in the Cu-dependent production of pigment were confirmed. Our findings provide insights into the molecular basis of Cu tolerance in the acidophilic fungus A. richmondensis MEY-1. Furthermore, the CRISPR/Cas9 system used here would be a powerful tool for studies of the mechanisms of adaptability of acidophilic fungi to extreme environments.

Review of glucose oxidase as a feed additive: production, engineering, applications, growth-promoting mechanisms, and outlook.

The regulation and prohibition of antibiotics used as growth promoters (AGP) in the feed field are increasing because they cause antimicrobial resistance and drug residue issues and threaten community health. Recently, glucose oxidase (GOx) has attracted increasing interest in the feed industry as an alternative to antibiotics. GOx specifically catalyzes the production of gluconic acid (GA) and hydrogen peroxide (H2O2) by consuming molecular oxygen, and plays an important role in relieving oxidative stress, preserving health, and promoting animal growth. To expand the application of GOx in the feed field, considerable efforts have been made to mine new genetic resources. Efforts have also been made to heterologously overexpress relevant genes to reduce production costs and to engineer proteins by modifying enzyme properties, both of which are bottleneck problems that limit industrial feed applications. Herein, the: different sources, diverse biochemical properties, distinct structural features, and various strategies of GOx engineering and heterologous overexpression are summarized. The mechanism through which GOx promotes growth in animal production, including the improvement of antioxidant capacity, maintenance of intestinal microbiota homeostasis, and enhancement of gut function, are also systematically addressed. Finally, a new perspective is provided for the future development of GOx applications in the feed field.

Engineering Escherichia coli for efficient assembly of heme proteins.

BACKGROUND: Heme proteins, such as hemoglobin, horseradish peroxidase and cytochrome P450 (CYP) enzyme, are highly versatile and have widespread applications in the fields of food, healthcare, medical and biological analysis. As a cofactor, heme availability plays a pivotal role in proper folding and function of heme proteins. However, the functional production of heme proteins is usually challenging mainly due to the insufficient supply of intracellular heme. RESULTS: Here, a versatile high-heme-producing Escherichia coli chassis was constructed for the efficient production of various high-value heme proteins. Initially, a heme-producing Komagataella phaffii strain was developed by reinforcing the C4 pathway-based heme synthetic route. Nevertheless, the analytical results revealed that most of the red compounds generated by the engineered K. phaffii strain were intermediates of heme synthesis which were unable to activate heme proteins. Subsequently, E. coli strain was selected as the host to develop heme-producing chassis. To fine-tune the C5 pathway-based heme synthetic route in E. coli, fifty-two recombinant strains harboring different combinations of heme synthesis genes were constructed. A high-heme-producing mutant Ec-M13 was obtained with negligible accumulation of intermediates. Then, the functional expression of three types of heme proteins including one dye-decolorizing peroxidase (Dyp), six oxygen-transport proteins (hemoglobin, myoglobin and leghemoglobin) and three CYP153A subfamily CYP enzymes was evaluated in Ec-M13. As expected, the assembly efficiencies of heme-bound Dyp and oxygen-transport proteins expressed in Ec-M13 were increased by 42.3-107.0% compared to those expressed in wild-type strain. The activities of Dyp and CYP enzymes were also significantly improved when expressed in Ec-M13. Finally, the whole-cell biocatalysts harboring three CYP enzymes were employed for nonanedioic acid production. High supply of intracellular heme could enhance the nonanedioic acid production by 1.8- to 6.5-fold. CONCLUSION: High intracellular heme production was achieved in engineered E. coli without significant accumulation of heme synthesis intermediates. Functional expression of Dyp, hemoglobin, myoglobin, leghemoglobin and CYP enzymes was confirmed. Enhanced assembly efficiencies and activities of these heme proteins were observed. This work provides valuable guidance for constructing high-heme-producing cell factories. The developed mutant Ec-M13 could be employed as a versatile platform for the functional production of difficult-to-express heme proteins.

Development of an efficient protein expression system in the thermophilic fungus Myceliophthora thermophila.

BACKGROUND: Thermophilic fungus Myceliophthora thermophila has been widely used in industrial applications due to its ability to produce various enzymes. However, the lack of an efficient protein expression system has limited its biotechnological applications. RESULTS: In this study, using a laccase gene reporting system, we developed an efficient protein expression system in M. thermophila through the selection of strong constitutive promoters, 5'UTRs and signal peptides. The expression of the laccase was confirmed by enzyme activity assays. The results showed that the Mtpdc promoter (Ppdc) was able to drive high-level expression of the target protein in M. thermophila. Manipulation of the 5'UTR also has significant effects on protein expression and secretion. The best 5'UTR (NCA-7d) was identified. The transformant containing the laccase gene under the Mtpdc promoter, NCA-7d 5'UTR and its own signal peptide with the highest laccase activity (1708 U/L) was obtained. In addition, the expression system was stable and could be used for the production of various proteins, including homologous proteins like MtCbh-1, MtGh5-1, MtLPMO9B, and MtEpl1, as well as a glucoamylase from Trichoderma reesei. CONCLUSIONS: An efficient protein expression system was established in M. thermophila for the production of various proteins. This study provides a valuable tool for protein production in M. thermophila and expands its potential for biotechnological applications.

Recombinant expression of IGF-1 and LR3 IGF-1 fused with xylanase in Pichia pastoris.

Insulin-like growth factor-1 (IGF-1) is a pleiotropic protein hormone and has become an attractive therapeutic target because of its multiple roles in various physiological processes, including growth, development, and metabolism. However, its production is hindered by low heterogenous protein expression levels in various expression systems and hard to meet the needs of clinical and scientific research. Here, we report that human IGF-1 and its analog Long R3 IGF-1 (LR3 IGF-1) are recombinant expressed and produced in the Pichia pastoris (P. pastoris) expression system through being fused with highly expressed xylanase XynCDBFV. Furthermore, purified IGF-1 and LR3 IGF-1 display excellent bioactivity of cell proliferation compared to the standard IGF-1. Moreover, higher heterologous expression levels of the fusion proteins XynCDBFV-IGF-1 and XynCDBFV-LR3 IGF-1 are achieved by fermentation in a 15-L bioreactor, reaching up to about 0.5 g/L XynCDBFV-IGF-1 and 1 g/L XynCDBFV-TEV-LR3 IGF-1. Taken together, high recombinant expression of bioactive IGF-1 and LR3 IGF-1 is acquired with the assistance of xylanase as a fusion partner in P. pastoris, which could be used for both clinical and scientific applications. KEY POINTS: • Human IGF-1 and LR3 IGF-1 are produced in the P. pastoris expression system. • Purified IGF-1 and LR3 IGF-1 show bioactivity comparable to the standard IGF-1. • High heterologous expression of IGF-1 and LR3 IGF-1 is achieved by fermentation in a bioreactor.

Metabolic engineering of Komagataella phaffii for synergetic utilization of glucose and glycerol.

Komagataella phaffii GS115 is a proven heterologous expression system and has recently been exploited for the production of value-added biochemicals from glucose through metabolic engineering. A major challenge for high-level biochemical production is the appropriate distribution of carbon flux between cell growth and product biosynthesis. In this study, we report the development of a synergetic glucose and glycerol coutilization strategy for K. phaffii, potentially enabling this strain to consume glycerol for growth while conserving more glucose for product formation. First, several potential genes encoding mediator proteins and transcriptional factors that were considered to be associated with carbon catabolite repression in K. phaffii were screened, and deletion of gss1, a glucose sensor, appeared to be able to eliminate the glucose-induced repression of glycerol utilization in a mixed glucose-glycerol medium. Transcriptome comparisons between the parent strain and the Δgss1 mutant were then performed, and the glycerol-metabolism genes that were subjected to glucose regulation were identified. Second, coutilization of glucose and glycerol in K. phaffii was achieved by overexpressing genes relevant to glycerol metabolism, namely, gt1, gut1, and gut2. Furthermore, knockout or knockdown of pfk and zwf genes resulted in a reduction of carbon flux from glucose towards glycolysis and the pentose phosphate pathway. With these efforts, the cell metabolism of the final strain was divided into growth and production modules. This study describes a promising strategy to address the challenge of carbon flux distribution in K. phaffii, and would be valuable in engineering this strain as a versatile fermentation platform for biochemical production.

Enhancing the Thermostability of Phytase to Boiling Point by Evolution-Guided Design.

The good thermostability of enzymes is an important basis for their wide application in industry. In this study, the phytase APPA from Yersinia intermedia was designed by evolution-guided design. Through the collection of homologous sequences in the NCBI database, we obtained a sequence set composed of 5,569 sequences, counted the number and locations of motif N-X-T/S, and selected the sites with high frequency in evolution as candidate sites for experiments. Based on the principle that N-glycosylation modification sites are located on the protein surface, 13 mutants were designed to optimize the number and location of N-glycosylation sites. Through experimental verification, 7 single mutants with improved thermostability were obtained. The best mutant, M14, with equal catalytic efficiency as the wild-type was obtained through combined mutation. The half-life (t1/2) value of mutant M14 was improved from 3.32 min at 65°C to 25 min of at 100°C, allowing it to withstand boiling water treatment, retaining approximately 75% initial activity after a 10-min incubation at 100°C. Differential scanning calorimetry analysis revealed that while the mutants' thermodynamic stability was nearly unchanged, their kinetic stability was greatly improved, and the combined mutant exhibited strong refolding ability. The results of a in vitro digestibility test indicated that the application effect of mutant M14 was about 4.5 times that of wild-type APPA, laying a foundation for its industrial application. IMPORTANCE Due to the harsh reaction conditions of industrial production, the relative instability of enzymes limits their application in industrial production, such as for food, pharmaceuticals, and feed. For example, the pelleting process of feed includes a brief high temperature (80 to 85°C), which requires the enzyme to have excellent thermostability. Therefore, a simple and effective method to improve the thermostability of enzymes has important practical value. In this study, we make full use of the existing homologous sequences (5,569) in the database to statistically analyze the existence frequency of N-X-T/S motifs in this large sequence space to design the phytase APPA with improved thermostability and a high hit rate (~50%). We obtained the best combination mutant, M14, that can tolerate boiling water treatment and greatly improved its kinetic stability without damaging its specific activity. Simultaneously, we proved that its performance improvement is due to its enhanced refolding ability, which comes from N-glycan modification rather than amino acid replacement. Our results provide a feasible and effective method for the modification of enzyme thermostability.

Identification and Mutation Analysis of Nonconserved Residues on the TIM-Barrel Surface of GH5_5 Cellulases for Catalytic Efficiency and Stability Improvement.

Exploring the potential functions of nonconserved residues on the outer side of α-helices and systematically optimizing them are pivotal for their application in protein engineering. Based on the evolutionary structural conservation analysis of GH5_5 cellulases, a practical molecular improvement strategy was developed. Highly variable sites on the outer side of the α-helices of the GH5_5 cellulase from Aspergillus niger (AnCel5A) were screened, and 14 out of the 34 highly variable sites were confirmed to exert a positive effect on the activity. After the modular combination of the positive mutations, the catalytic efficiency of the mutants was further improved. By using CMC-Na as the substrate, the catalytic efficiency and specific activity of variant AnCel5A_N193A/T300P/D307P were approximately 2.0-fold that of AnCel5A (227 ± 21 versus 451 ± 43 ml/s/mg and 1,726 ± 19 versus 3,472 ± 42 U/mg, respectively). The half-life (t1/2) of variant AnCel5A_N193A/T300P/D307P at 75°C was 2.36 times that of AnCel5A. The role of these sites was successfully validated in other GH5_5 cellulases. Computational analyses revealed that the flexibility of the loop 6-loop 7-loop 8 region was responsible for the increased catalytic performance. This work not only illustrated the important role of rapidly evolving positions on the outer side of the α-helices of GH5_5 cellulases but also revealed new insights into engineering the proteins that nature left as clues for us to find. IMPORTANCE A comprehensive understanding of the residues on the α-helices of the GH5_5 cellulases is important for catalytic efficiency and stability improvement. The main objective of this study was to use the evolutionary conservation and plasticity of the TIM-barrel fold to probe the relationship between nonconserved residues on the outer side of the α-helices and the catalytic efficiency of GH5_5 cellulases by conducting structure-guided protein engineering. By using a four-step nonconserved residue screening strategy, the functional role of nonconserved residues on the outer side of the α-helices was effectively identified, and a variant with superior performance and capability was constructed. Hence, this study proved the effectiveness of this strategy in engineering GH5_5 cellulases and provided a potential competitor for industrial applications. Furthermore, this study sheds new light on engineering TIM-barrel proteins.

Engineering a carbohydrate-binding module to increase the expression level of glucoamylase in Pichia pastoris.

BACKGROUND: Glucoamylase is an important industrial enzyme for the saccharification of starch during sugar production, but the production cost of glucoamylase is a major limiting factor for the growth of the starch-based sugar market. Therefore, seeking strategies for high-level expression of glucoamylase in heterologous hosts are considered as the main way to reduce the enzyme cost. RESULTS: ReGa15A from Rasamsonia emersonii and TlGa15B-GA2 from Talaromyces leycettanus have similar properties. However, the secretion level of ReGa15A was significantly higher than TlGa15B-GA2 in Pichia pastoris. To explore the underlying mechanisms affecting the differential expression levels of glucoamylase in P. pastoris, the amino acid sequences and three-dimensional structures of them were compared and analyzed. First, the CBM region was identified by fragment replacement as the key region affecting the expression levels of ReGa15A and TlGa15B-GA2. Then, through the substitution and site-directed mutation of the motifs in the CBM region, three mutants with significantly increased expression levels were obtained. The eight-point mutant TlGA-M4 (S589D/Q599A/G600Y/V603Q/T607I/V608L/N609D/R613Q), the three-point mutant TlGA-M6 (Q599A/G600Y/V603Q) and the five-point mutant TlGA-M7 (S589D/T607I/V608L/N609D/R613Q) have the same specific activity with the wild-type, and the enzyme activity and secretion level have increased by 4-5 times, respectively. At the same time, the expression levels were 5.8-, 2.0- and 2.4-fold higher than that of wild type, respectively. Meanwhile, the expression of genes related to the unfolded protein responses (UPR) in the endoplasmic reticulum (ER) did not differ significantly between the mutants and wild type. In addition, the most highly expressed mutant, TlGA-M7 exhibits rapidly and effectively hydrolyze raw corn starch. CONCLUSIONS: Our results constitute the first demonstration of improved expression and secretion of a glucoamylase in P. pastoris by introducing mutations within the non-catalytic CBM. This provides a novel and effective strategy for improving the expression of recombinant proteins in heterologous host expression systems.

Metabolic engineering of Pichia pastoris for myo-inositol production by dynamic regulation of central metabolism.

BACKGROUND: The methylotrophic budding yeast Pichia pastoris GS115 is a powerful expression system and hundreds of heterologous proteins have been successfully expressed in this strain. Recently, P. pastoris has also been exploited as an attractive cell factory for the production of high-value biochemicals due to Generally Recognized as Safe (GRAS) status and high growth rate of this yeast strain. However, appropriate regulation of metabolic flux distribution between cell growth and product biosynthesis is still a cumbersome task for achieving efficient biochemical production. RESULTS: In this study, P. pastoris was exploited for high inositol production using an effective dynamic regulation strategy. Through enhancing native inositol biosynthesis pathway, knocking out inositol transporters, and slowing down carbon flux of glycolysis, an inositol-producing mutant was successfully developed and low inositol production of 0.71 g/L was obtained. The inositol production was further improved by 12.7% through introduction of heterologous inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP) which catalyzed the rate-limiting steps for inositol biosynthesis. To control metabolic flux distribution between cell growth and inositol production, the promoters of glucose-6-phosphate dehydrogenase (ZWF), glucose-6-phosphate isomerase (PGI) and 6-phosphofructokinase (PFK1) genes were replaced with a glycerol inducible promoter. Consequently, the mutant strain could be switched from growth mode to production mode by supplementing glycerol and glucose sequentially, leading to an increase of about 4.9-fold in inositol formation. Ultimately, the dissolved oxygen condition in high-cell-density fermentation was optimized, resulting in a high production of 30.71 g/L inositol (~ 40-fold higher than the baseline strain). CONCLUSIONS: The GRAS P. pastoris was engineered as an efficient inositol producer for the first time. Dynamic regulation of cell growth and inositol production was achieved via substrate-dependent modulation of glycolysis and pentose phosphate pathways and the highest inositol titer reported to date by a yeast cell factory was obtained. Results from this study provide valuable guidance for engineering of P. pastoris for the production of other high-value bioproducts.

Biodegradation of nitriles derived from glucosinolates in rapeseed meal by BnNIT2: a nitrilase from Brassica napus with wide substrate specificity.

Nitriles derived from glucosinolates (GSLs) in rapeseed meal (RSM) can cause lesions on animal liver and kidneys. Nitrilase converts nitriles to carboxylic acids and NH3, eliminating their toxicity. Here we describe a nitrilase, BnNIT2, from Brassica napus (optimal temperature, 45 °C; pH, 7.0) that is stable at 40 °C and has a wide substrate specificity. Recombinant BnNIT2 converted the three main nitriles from GSLs (3-hydroxy-4-pentenenitrile, 3-butenenitrile, and 4-pentenenitrile), with the highest specific activity (58.6 U/mg) for 4-pentenenitrile. We used mutagenesis to improve the thermostability of BnNIT2; the resulting mutant BnNIT2-H90M had an ~ 14.5% increase in residual activity at 50 °C for 1 h. To verify the functionality of BnNIT2, GSLs were extracted from RSM and converted into nitriles at pH 5.0 in the presence of Fe2+. Then, BnNIT2 was used to degrade the nitriles from GSLs; ultimately, ~ 80% of nitriles were removed. Thus BnNIT2 is a potential enzyme for detoxification of RSM. KEY POINTS: • Functional identification of the plant nitrilase BnNIT2. • Identified a mutant, H90M, with improved thermostability. • BnNIT2 was capable of degrading nitriles from transformed GSLs.

Identification of WxL and S-Layer Proteins from Lactobacillus brevis with the Ability to Bind Cellulose and Xylan.

Xylanase releases xylo-oligosaccharides from dietary xylan, which stimulate the growth of the gut bacteria lactobacilli. Many lactobacilli adhere to dietary fibers, which may facilitate the assimilation of xylo-oligosaccharides and help them gain competence in the gut, but the underlying mechanisms remain elusive. Herein we report, from the highly abundant transcripts of Lactobacillus brevis cultured in wheat arabinoxylan supplemented with a xylanase, the identification of genes encoding four putative cell-surface WxL proteins (Lb630, Lb631, Lb632, and Lb635) and one S-layer protein (Lb1325) with either cellulose- or xylan-binding ability. The repetitively occurring WxL proteins were encoded by a gene cluster, among which Lb630 was chosen for further mutational studies. The analysis revealed three aromatic residues (F30, W61, and W156) that might be involved in the interaction of the protein with cellulose. A homology search in the genome of Enterococcus faecium identified three WxL proteins with conserved counterparts of these three aromatic residues, and they were also found to be able to bind cellulose and xylan. The findings suggested a role of the cell-surface WxL and S-layer proteins in assisting the cellular adhesion of L. brevis to plant cell wall polysaccharides.

Synergetic Fermentation of Glucose and Glycerol for High-Yield N-Acetylglucosamine Production in Escherichia coli.

N-acetylglucosamine (GlcNAc) is an amino sugar that has been widely used in the nutraceutical and pharmaceutical industries. Recently, microbial production of GlcNAc has been developed. One major challenge for efficient biosynthesis of GlcNAc is to achieve appropriate carbon flux distribution between growth and production. Here, a synergistic substrate co-utilization strategy was used to address this challenge. Specifically, glycerol was utilized to support cell growth and generate glutamine and acetyl-CoA, which are amino and acetyl donors, respectively, for GlcNAc biosynthesis, while glucose was retained for GlcNAc production. Thanks to deletion of the 6-phosphofructokinase (PfkA and PfkB) and glucose-6-phosphate dehydrogenase (ZWF) genes, the main glucose catabolism pathways of Escherichia coli were blocked. The resultant mutant showed a severe defect in glucose consumption. Then, the GlcNAc production module containing glucosamine-6-phosphate synthase (GlmS*), glucosamine-6-phosphate N-acetyltransferase (GNA1*) and GlcNAc-6-phosphate phosphatase (YqaB) expression cassettes was introduced into the mutant, to drive the carbon flux from glucose to GlcNAc. Furthermore, co-utilization of glucose and glycerol was achieved by overexpression of glycerol kinase (GlpK) gene. Using the optimized fermentation medium, the final strain produced GlcNAc with a high stoichiometric yield of 0.64 mol/mol glucose. This study offers a promising strategy to address the challenge of distributing carbon flux in GlcNAc production.