Production Of Industrial Enzymes Research Articles

Production of grape pomace extracts with enhanced antioxidant and prebiotic activities through solid-state fermentation by Aspergillus niger and Aspergillus oryzae

Grape pomace is a winery by-product that accumulates in high amounts in winemaking industry, which usually found difficulties regarding its disposal. Nonetheless, it has a great proportion of polyphenols which can be recovered to valorise this biomass. Filamentous fungi produce hydrolytic enzymes, which can assist in the liberation of polyphenols. Grape pomace was fermented in solid-state by Aspergillus niger and Aspergillus oryzae with the aim of facilitating the aqueous extraction of polyphenols with antioxidant activity by on-site enzyme production. Both fungi increased the antioxidant activity of the extracts, reaching maximum values of 73.7 ± 0.8 (A. niger) and 109.2 ± 0.5 (A. oryzae) mmol of Trolox equivalents/100 g of grape pomace. During fermentations, relevant enzymes were produced at high yields, A. niger produced a balanced profile of enzymes (cellulase, tannase, and pectinase), while A. oryzae switches to cellulase or tannase selective induction according to the fermentation conditions. Positive correlations were found between enzyme production, polyphenols recovery, and antioxidant activity. The extracts obtained after fermentation promoted the growth of Lactobacillus casei cultures. Therefore, the solid-state fermentation was effective for the simultaneous production of relevant industrial enzymes and grape pomace extracts with antioxidant and prebiotic properties, which have potential as functional food additives.

Cloning and Heterologous Expression of a Novel Xylanase Gene TAX1 from Trichoderma atroviride and Its Application in the Deconstruction of Corn Stover.

Xylanase plays a vital role in the efficient utilization of xylan, which accounts for up to 30% of plant dry matter. However, the production cost of xylanase remains high, and the enzymatic characteristics of xylanases of most microorganisms are not suitable for industrial production. Therefore, it is of great significance to discover and develop new and efficient xylanases. In this study, the xylanase gene TAX1 (672 bp cDNA) was cloned from Trichoderma atroviride 3.3013 and expressed in Pichia pastoris. The TAX1 gene encoded a 223-amino acid protein (TAX1) with a molecular weight of 24.2 kDa which showed high similarity to glycoside hydrolase family 11. Enzyme activity assay verified that the recombinant xylanase TAX1 had optimal activity (215.3 IU/mL) at 50°C and pH 6.0. Stable working conditions were measured as pH 4.0-7.0 and 40-60°C. By adding Zn2+, the relative enzymatic activity of recombinant TAX1 was enhanced by 26%. The recombinant xylanase showed high activity toward birchwood xylan and corn stover. The Km and Kcat for xylan and corn stover were 0.36 mg/mL and 0.204 S-1 and 0.48 mg/mL and 0.149 S-1, respectively. The enzymatic activity of the TAX1 produced by P. pastoris was about 2.4-4 times higher that directly isolated from T. atroviride, so engineered P. pastoris for xylanase production could be an ideal candidate for industrial enzyme production.

Increase of a Fibrinolytic Enzyme Production through Promoter Replacement of aprE3-5 from Bacillus subtilis CH3-5.

Bacillus subtilis CH3-5 isolated from cheonggukjang secretes a 28 kDa protease with a strong fibrinolytic activity. Its gene, aprE3-5, was cloned and expressed in a heterologous host (Jeong et al., 2007). In this study, the promoter of aprE3-5 was replaced with other stronger promoters (Pcry3A, P10, PSG1, PsrfA) of Bacillus spp. using PCR. The constructed chimeric genes were cloned into pHY300PLK vector, and then introduced into B. subtilis WB600. The P10 promoter conferred the highest fibrinolytic activity, i.e., 1.7-fold higher than that conferred by the original promoter. Overproduction of the 28 kDa protease was confirmed using SDS-PAGE and fibrin zymography. RT-qPCR analysis showed that aprE3-5 expression was 2.0-fold higher with the P10 promoter than with the original promoter. Change of the initiation codon from GTG to ATG further increased the fibrinolytic activity. The highest aprE3-5 expression was observed when two copies of the P10 promoter were placed in tandem upstream of the ATG initiation codon. The construct with P10 promoter and ATG and the construct with two copies of P10 promoter in tandem and ATG exhibited 117% and 148% higher fibrinolytic activity, respectively, than that exhibited by the construct containing P10 promoter and GTG. These results confirmed that significant overproduction of a fibrinolytic enzyme can be achieved by suitable promoter modification, and this approach may have applications in the industrial production of AprE3-5 and related fibrinolytic enzymes.

Valorization of Lignocellulosic Wastes to Produce Phytase and Cellulolytic Enzymes from a Thermophilic Fungus, Thermoascus aurantiacus SL16W, under Semi-Solid State Fermentation.

Agricultural wastes are lignocellulosic biomasses that contain high mineral and nutrient contents. This waste can be used as a raw material in industrial enzyme production by microbial fermentation. Phytase is an important enzyme used in animal feed to enhance the amount of phosphorus available for the growth and overall health improvement of monogastric animals. Fungi offer high potential as an effective source in the production of various extracellular enzymes. In this study, the production of lignocellulolytic enzymes (endoglucanase and xylanase) and phytase by a thermophilic fungus, namely Thermoascus aurantiacus strain SL16W, was evaluated using sixteen different Thai agricultural forms of waste under conditions of high temperature (45 °C). Semi-solid state fermentation was used in the production experiments. The results of this study reveal that the highest phytase activity (58.6 U/g substrate) was found in rice bran, whereas the highest degrees of activity of endoglucanase and xylanase were observed in wheat bran and red tea leaves at 19 and 162 U/g substrate, respectively. Consequently, the optimal conditions for phytase production of this fungus using rice bran were investigated. The results indicate that the highest phytase yield (58.6 to 84.1 U/g substrate) was observed in rice bran containing 0.5% ammonium sulfate as a nitrogen source with 10 discs of inoculum size at a cultivation period of 9 days at 45 °C and moisture content of 95%. Notably, the phytase yield increased by 1.71-fold, while endoglucanase and xylanase were also increased by 1.69- and 1.12-fold, respectively. Furthermore, the crude enzyme obtained from the optimal condition was extracted. The crude enzyme extract was then separately added to red tea leaves, rice straw, corncobs, palm residue, and peanut husks. Subsequently, total reducing sugar and phosphorus contents were determined. The results indicate that the highest level of reducing sugar (122.6 mg/L) and phosphorus content (452.6 mg/L) (p < 0.05) were obtained in palm residue at 36 and 48 h, respectively, after the addition of the crude enzyme extract. This study has provided valuable information on a potentially eco-friendly way to valorize agricultural waste into value-added products as industrial enzymes.

An overview of Trichoderma reesei co-cultures for the production of lignocellulolytic enzymes.

Biorefineries are core facilities for implementing a sustainable circular bioeconomy. These facilities rely on microbial enzymes to hydrolyze lignocellulosic substrates into fermentable sugars. Fungal co-cultures mimic the process of natural biodegradation and have been shown to increase certain enzyme activities. Trichoderma reesei and its many mutant strains are major cellulase producers and are heavily utilized as a source of carbohydrate-active enzymes. Several reports have demonstrated that T. reesei co-cultures present higher enzyme activities compared with its monocultures, especially in the context of β-glucosidase activity. The performance of T. reesei during co-culturing has been assessed with several fungal partners, including Aspergillus niger, one of the most recurrent partners. Various aspects of co-cultivation still need further investigation, especially regarding the molecular interactions between fungi in controlled environments and the optimization of the resulting enzyme cocktails. Since plenty of genetic and physiological data on T. reesei is available, the species is an outstanding candidate for future co-culture investigations. Co-cultures are still a developing field for industrial enzyme production, and many aspects of the technique need further improvement before real applications. KEY POINTS: • T. reesei co-cultures are an alternative for producing lignocellulolytic enzymes. • Several reports suggest an increase in certain enzyme activities in co-cultures. • More in-depth investigations of co-cultures are necessary for advancing this field.

CRISPR/Cas-based genome editing in Aspergillus niger

Aspergillus niger is a vital industrial workhouse widely used for the production of organic acids and industrial enzymes. This fungus is a crucial cell factory due to its innate tolerance to a diverse range of abiotic conditions, high production titres, robust growth during industrial scale fermentation, and status as a generally recognized as safe (GRAS) organism. Rapid development of synthetic biology and systems biology not only offer powerful approaches to unveil the molecular mechanisms of A. niger productivity, but also provide more new strategies to construct and optimize the A. niger cell factory. As a new generation of genome editing technology, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) system brings a revolutionary breakthrough in targeted genome modification for A. niger. In this review, we focus on current advances to the CRISPR/Cas genome editing toolbox, its application on gene modification and gene expression regulation in this fungal. Moreover, the future directions of CRISPR/Cas genome editing in A. niger are highlighted.

Impact of activation of neotrehalosadiamine/kanosamine biosynthetic pathway on the metabolism of Bacillus subtilis.

The pentose phosphate (PP) pathway is one of the major sources of cellular NADPH. A B. subtilis zwf mutant that lacks glucose-6-phosphate dehydrogenase (the enzyme that catalyzes the first step of the PP pathway) showed inoculum-dose-dependent growth. This growth defect was suppressed by glcP disruption, which causes the upregulation of an autoinducer neotrehalosadiamine (NTD)/kanosamine biosynthetic pathway. A metabolome analysis showed that the stimulation of NTD/kanosamine biosynthesis caused significant accumulation of TCA cycle intermediates and NADPH. Because the major malic enzyme YtsJ concomitantly generates NADPH through malate-to-pyruvate conversion, de novo NTD/kanosamine biosynthesis can result in an increase in the intracellular NADPH pool via the accumulation of malate. In fact, a zwf mutant grew in malate-supplemented medium. Artificial induction of glcP in the zwf mutant caused a reduction in the intracellular NADPH pool. Moreover, the correlation between the expression level of the NTD/kanosamine biosynthesis operon ntdABC and the intracellular NADPH pool was confirmed. Our results suggest that NTD/kanosamine has the potential to modulate the carbon-energy metabolism through an autoinduction mechanism.ImportanceAutoinducers enable bacteria to sense cell density and to coordinate collective behavior. NTD/kanosamine is an autoinducer produced by B. subtilis and several close relatives, although its physiological function remains unknown. The most important finding of this study was the significance of de novo NTD/kanosamine biosynthesis in the modulation of the central carbon metabolism in B. subtilis We showed that NTD/kanosamine biosynthesis caused an increase in the NADPH pool via the accumulation of TCA cycle intermediates. These results suggest a possible role for NTD/kanosamine in the carbon-energy metabolism. As Bacillus species are widely used for the industrial production of various useful enzymes and compounds, the NTD/kanosamine biosynthetic pathway might be utilized to control metabolic pathways in these industrial strains.

Production of Industrial Enzymes via Pichia pastoris as a Cell Factory in Bioreactor: Current Status and Future Aspects.

Industrial enzymes have been widely preferred in various industries such as chemical production, food & beverage, pharmaceutical, textile, cosmetics, etc. due to the advancements in recent years. They are considered more economic than using whole cells and more environmental-friendly than chemical alternatives. Since the demand for industrial enzymes has been rising, the development of production strategies has been gathered speed. In this respect, the efficiency of Pichia pastoris (P. pastoris) as a host for heterologous protein expression has proved and gained attention due to its great potential for large-scale studies. Especially high-cell density fermentation of P. pastoris is a well-studied and efficient method. Moreover, the improvements in the state of art gene-editing tools have broadened the possibilities of strain improvement for P. pastoris. This review summarized the role of P. pastoris as a cell factory by accentuating the accomplishments in biocatalyst production. Moreover, the benefits and challenges of the most relevant expression systems named Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), P. pastoris and recent evolvements and future directions were revealed in detail. Subsequently, offers for prospects and the latest evolvements to enhance the recombinant protein production were discussed.

Artificial AmyR::XlnR transcription factor induces α-amylase production in response to non-edible xylan-containing hemicellulosic biomass

Filamentous fungi belonging to the Aspergillus genus are one of the most favored microorganisms for industrial enzyme production because they can secrete large amounts of proteins into the culture medium. α-Amylase, an enzyme produced by Aspergillus species, is important for food and industrial applications. The production of α-amylase is induced by starch, mainly obtained from the edible biomass; however, the increasing demand for foods is limiting the application of the latter. Therefore, it is expected that using the non-edible biomass, such as rice straw, could improve the competition for industrial application starch containing resources. The transcription factor AmyR activates the transcription of amylolytic enzyme genes, while the transcription factor XlnR activates the transcription of xylanolytic enzyme genes in response to xylose. In this study, we aimed to construct an artificial AmyR::XlnR transcription factor (AXTF) by replacing the DNA-binding domain (1–159 amino acids) of XlnR with that (1–68 aa) of AmyR, which is capable of inducing amylolytic enzyme production in response to xylan-containing hemicellulosic biomass. The chimeric transcription factor AXTF was constructed and expressed using the gapA promoter in the amyR-deficient mutant strain SA1. When the AXTF strain was cultured in the minimal medium containing xylose as the carbon source, the amyB, amyF, agdB, and agdE transcription levels were 41.1-, 11.3-, 37.9-, and 23.7-fold higher, respectively, than those of the wild-type strain. The α-amylase and α-glucosidase activities in the culture supernatant of the AXTF strain grown with xylose for 48 h were 696.6 and 536.1 U/mL, respectively, while these activities were not detected in the culture supernatant of the wild-type and SA1 strains. When rice straw hydrolysate was used as a carbon source, the α-amylase and α-glucosidase activities were 590.2 and 362.7 U/mL, respectively. Thus, we successfully generated an Aspergillus nidulans strain showing amylolytic enzyme production in response to non-edible xylan-containing hemicellulosic biomass by transforming it with the chimeric transcription factor AXTF. Furthermore, the use of genes encoding engineered transcription factors is advantageous because introducing such genes into an industrial Aspergillus strain has similar simultaneous effects on multiple amylase genes controlled by AmyR.

Protein expression and secretion by filamentous fungi

In the search for optimal platforms for protein expression and secretion, filamentous fungi in principle provide some of the best microbial cell factories. They are inherently endowed with the ability to secrete proteins. Fungi belonging to Aspergillus and Trichoderma species are well-studied for industrial production of proteins and enzymes. Our understanding of these organisms at the level of transcription, translation, post-translational processing and the secretory pathways has improved significantly in recent years. Despite this, the ability of these fungal secretion platforms has not yet been able to match their intrinsic secretion capacity to produce foreign proteins. Details of the molecular mechanisms of the secretory pathways in filamentous fungi are emerging. This knowledge can be gainfully employed to enhance protein production in filamentous fungi, particularly in the secretion of heterologous proteins of value.

Prototyping Yarrowia lipolytica for industrial production of hyperthermophilic enzymes- a case of β-glucosidase (CelB) from Pyrococcus furiosus

Hyperthermozymes have recently gained significant recognition as prospective industrial biocatalysts. However, the necessity to ease their large scale synthesis in a cost-effective manner often challenges the viability of industrial bioprocesses. In the present study, Yarrowia lipolytica, because of its high secretion capacity and robust growth, was assessed for its efficacy to express and secrete one of the widely used hyperthermozyme, CelB (β-glucosidase) of Pyrococcus furiosus. Preliminary expression studies using preproLIP2 and preXPR2 as signal peptides indicated that preproLIP2 was more efficient in targeting CelB outside the cell with enzyme yields of 0.42 ± 0.016 U/mL. To further improve the extracellular CelB yields, the preproLIP2-CelB expression cassette was integrated in one, two or three copies at different genomic loci of Y. lipolytica. Though variances in CelB expression were observed across the integration sites, its constitutive expression did not hamper the cell fitness. Also, with increasing CelB copy number linear increase in expression was observed reaching the highest enzyme yields of ∼5 U/mL for Po1f_LP3 strain, 12 fold higher than the base strain (Po1g_LP). Interestingly, comparable enzyme yields were achieved when rich YPD medium for cultivating Y. lipolytica was replaced with a low-cost SPD medium. Such high titres observed in shake-flask cultivation demonstrates the potential of Y. lipolytica for fermentative production of hyperthermozymes.

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris

BackgroundPichia pastoris (Komagataella phaffii) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of P. pastoris to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in P. pastoris, with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains.ResultsPotential bottlenecks for expression of E. coli AppA phytase in P. pastoris were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by ERV2 that has not been characterised in P. pastoris was used to improve the expression of the phytase, indicating its role as an alternative pathway to ERO1. Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the AOX1 promoter.DiscussionThe microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in P. pastoris contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.

Transcriptional analysis of the lichenase-like gene cel12A of the filamentous fungus Stachybotrys atra BP-A and its relevance for lignocellulose depolymerization.

To rationally optimize the production of industrial enzymes by molecular means requires previous knowledge of the regulatory circuits controlling the expression of the corresponding genes. The genus Stachybotrys is an outstanding producer of cellulose-degrading enzymes. Previous studies isolated and characterized the lichenase-like/non-typical cellulase Cel12A of S. atra (AKA S. chartarum) belonging to glycosyl hydrolase family 12 (GH12). In this study, we used RT-qPCR to determine the pattern of expression of cel12A under different carbon sources and initial ambient pH. Among the carbon sources examined, rice straw triggered a greater increase in the expression of cel12A than 1% lactose or 0.1% glucose, indicating specific induction by rice straw. In contrast, cel12A was repressed in the presence of glucose even when combined with this inducer. The proximity of 2 adjacent 5'-CTGGGGTCTGGGG-3' CreA consensus target sites to the translational start site of cel12A strongly suggests that the carbon catabolite repression observed is directly mediated by CreA. Ambient pH did not have a significant effect on cel12A expression. These findings present new knowledge on transcriptional regulatory networks in Stachybotrys associated with cellulose/hemicellulose depolymerization. Rational engineering of CreA to remove CCR could constitute a novel strategy for improving the production of Cel12A.

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

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

Biodiversity and biotechnological potential of marine derived actinobacteria from Black Sea coastal zone

Actinobacteria are economically and biotechnologically valuable prokaryotes. It is crucial that new group of actinobacteria from unexplored habitats, marine environmental conditions, will be pursued as sources of novel bioactive secondary metabolites. Therefore, actinobacterial strains were isolated from the Black Sea sediments and screened for production of antimicrobial metabolites and industrial enzymes. The isolates were identified with sequencing analysis by using 16S rDNA regions. Antimicrobial activity was determined by disc diffusion method by using extracts of fermentation broth inoculated with the isolates against seven pathogenic organisms. According to results, the majority of isolates belonged to Streptomyces genus. Their anticandidal activity and ability to produce cellulase enzyme were remarkable. In addition, it was determined that the isolates which not shown antimicrobial activity produced at least one industrial enzyme. As a result, the potential of isolates to be used as an antibiotic and enzyme producer in the industrial field is quite high.

High-level heterologous protein production using an attenuated selection marker in Aspergillus sojae.

Filamentous fungi, including Aspergillus sojae, are essential for the industrial production of enzymes. Although multi-copy introduction of a gene encoding the protein of interest is useful for increasing protein production, this method has not been established in the case of filamentous fungi. In this study, we aimed to establish an efficient system for multi-copy chromosomal integration and high-level expression of a heterologous gene in A. sojae using an attenuated selectable marker. Consequently, by truncating the promoter region of selectable markers, we efficiently introduced multiple copies of a heterologous gene and enhanced the rate of high-level protein-production in the strains. Since the multi-copy strains obtained in this study maintained high productivity even in a non-selective medium, this system could be applicable for industrial protein production.

Characterization of Metagenome-Assembled Genomes and Carbohydrate-Degrading Genes in the Gut Microbiota of Tibetan Pig.

Tibetan pig is an important domestic mammal, providing products of high nutritional value for millions of people living in the Qinghai-Tibet Plateau. The genomes of mammalian gut microbiota encode a large number of carbohydrate-active enzymes, which are essential for the digestion of complex polysaccharides through fermentation. However, the current understanding of microbial degradation of dietary carbohydrates in the Tibetan pig gut is limited. In this study, we produced approximately 145 gigabases of metagenomic sequence data for the fecal samples from 11 Tibetan pigs. De novo assembly and binning recovered 322 metagenome-assembled genomes taxonomically assigned to 11 bacterial phyla and two archaeal phyla. Of these genomes, 191 represented the uncultivated microbes derived from novel prokaryotic taxa. Twenty-three genomes were identified as metagenomic biomarkers that were significantly abundant in the gut ecosystem of Tibetan pigs compared to the other low-altitude relatives. Further, over 13,000 carbohydrate-degrading genes were identified, and these genes were more abundant in some of the genomes within the five principal phyla: Firmicutes, Bacteroidetes, Spirochaetota, Verrucomicrobiota, and Fibrobacterota. Particularly, three genomes representing the uncultivated Verrucomicrobiota encode the most abundant degradative enzymes in the fecal microbiota of Tibetan pigs. These findings should substantially increase the phylogenetic diversity of specific taxonomic clades in the microbial tree of life and provide an expanded repertoire of biomass-degrading genes for future application to microbial production of industrial enzymes.

Reducing the cell lysis to enhance yield of acid-stable alpha amylase by deletion of multiple peptidoglycan hydrolase-related genes in Bacillus amyloliquefaciens

Bacillus amyloliquefaciens is a major industrial host for extracellular protein production, with great potential in the enzyme industry. However, the strain has accelerated the autolysis drawback in the process of secreting extracellular enzymes, which can significantly lower the density of cells and decrease the product yield. To identify target genes, we employed comparative transcriptome sequencing and KEGG analysis to indicate the increased expression of peptidoglycan hydrolase-regulated genes from the exponential phase to the apoptotic phase of growth; this was further confirmed by quantitative RT-PCR. By deleting lytD, lytE, and sigD genes, cell lysis was reduced and the production of acid-stable Bacillus licheniformis alpha-amylase was enhanced. After 36 h of culture, multiple deletion mutant BA ΔSDE had significantly more viable cells compared to the control strain BA Δupp, and flow cytometry analysis indicated that 48.43% and 64.03% of the cells were lysed in cultures of BA ΔSDE and BA Δupp, respectively. In a 2-L fed-batch fermenter, viable cell number of the triple deletion mutant BA ΔSDE increased by 2.79 Log/cfu/mL, and the activity of acid-stable alpha-amylase increased by 48.4%, compared to BA Δupp. Systematic multiple peptidoglycan hydrolases deletion relieved the autolysis and increased the production of industrial enzymes, and provided a useful strategy for guiding efforts to manipulate the genomes of other B. amyloliquefaciens used for chassis host.

Proteomic analysis on Aspergillus strains that are useful for industrial enzyme production.

A simple intracellular proteomic study was conducted to investigate the biological activities of Aspergillus niger during industrial enzyme production. A strain actively secreting a heterologous enzyme was compared to a reference strain. In total, 1824 spots on 2-D gels were analyzed using MALDI-TOF MS, yielding 343 proteins. The elevated levels of UPR components, BipA, PDI, and calnexin, and proteins related to ERAD and ROS reduction, were observed in the enzyme-producer. The results suggest the occurrence of these responses in the enzyme-producers. Major glycolytic enzymes, Fba1, EnoA, and GpdA, were abundant but at a reduced level relative to the reference, indicating a potential repression of the glycolytic pathway. Interestingly, it was observed that a portion of over-expressed heterologous enzyme accumulated inside the cells and digested during fermentation, suggesting the secretion capacity of the strain was not enough for completing secretion. Newly identified conserved-proteins, likely in signal transduction, and other proteins were also investigated. Abbreviations: 2-D: two-dimensional; UPR: unfolded protein response; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; PDI: protein disulfide-isomerase; ROS: reactive oxygen species; RESS: Repression under Secretion Stress; CSAP: Conserved Small Abundant Protein; TCTP: translationally controlled tumor protein.

Fungal Cell Biomass From Enzyme Industry as a Sustainable Source of Hydrocolloids

Industrial enzyme production yields vast amounts of cell biomass as by-product that is treated as waste, which brings costs and causes an environmental burden. Nonetheless with the current drive towards resource efficiency, conversion of the cell biomass into value-added products would provide an economically viable alternative for the industry. The biomass is composed of fungal cells, containing potentially valuable heteropolysaccharides and glycoproteins, but a technology is lacking to valorize them. For the first time, we characterized the composition of the biomass, extracted biopolymers by aqueous and alkaline treatments, and studied their behavior as novel hydrocolloids in multiphase systems. Alkaline extraction yielded a carbohydrate content of 37.5% and an amino acid content of 27.6%, whereas water extraction yielded 6.6% and 61.3%, respectively. Emulsions prepared with alkaline extracts were relatively stable during a 15-day storage period. We demonstrated that the costly waste management in enzyme production can be replaced by sustainable fractioning, and these fractions can be valorized.