Enhancing the expression of the unspecific peroxygenase in Komagataella phaffii through a combination strategy.

The unspecific peroxygenase (UPO) from Cyclocybe aegerita (AaeUPO) can selectively oxidize C-H bonds using hydrogen peroxide as an oxygen donor without cofactors, which has drawn significant industrial attention. Many studies have made efforts to enhance the overall activity of AaeUPO expressed in Komagataella phaffii by employing strategies such as enzyme-directed evolution, utilizing appropriate promoters, and screening secretion peptides. Building upon these previous studies, the objective of this study was to further enhance the expression of a mutant of AaeUPO with improved activity (PaDa-I) by increasing the gene copy number, co-expressing chaperones, and optimizing culture conditions. Our results demonstrated that a strain carrying approximately three copies of expression cassettes and co-expressing the protein disulfide isomerase showed an approximately 10.7-fold increase in volumetric enzyme activity, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. After optimizing the culture conditions, the volumetric enzyme activity of this strain further increased by approximately 48.7%, reaching 117.3 U/mL. Additionally, the purified catalytic domain of PaDa-I displayed regioselective hydroxylation of R-2-phenoxypropionic acid. The results of this study may facilitate the industrial application of UPOs. KEY POINTS: • The secretion of the catalytic domain of PaDa-I can be significantly enhanced through increasing gene copy numbers and co-expressing of protein disulfide isomerase. • After optimizing the culture conditions, the volumetric enzyme activity can reach 117.3 U/mL, using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as the substrate. • The R-2-phenoxypropionic acid can undergo the specific hydroxylation reaction catalyzed by catalytic domain of PaDa-I, resulting in the formation of R-2-(4-hydroxyphenoxy)propionic acid.

Hydrophobic substrate binding pocket remodeling of echinocandin B deacylase based on multi-dimensional rational design.

Actinoplanes utahensis deacylase (AAC)-catalyzed deacylation of echinocandin B (ECB) is a promising method for the synthesis of anidulafungin, the newest of the echinocandin antifungal agents. However, the low activity of AAC significantly limits its practical application. In this work, we have devised a multi-dimensional rational design strategy for AAC, conducting separate analyses on the substrate-binding pocket's volume, curvature, and length. Furthermore, we quantitatively analyzed substrate properties, particularly on hydrophilic and hydrophobic. Accordingly, we tailored the linoleic acid-binding pocket of AAC to accommodate the extended long lipid chain of ECB. By fine-tuning the key residues, the resulting AAC mutants can accommodate the ECB lipid chain with a lower curvature binding pocket. The D53A/I55F/G57M/F154L/Q661L mutant (MT) displayed 331 % higher catalytic efficiency than the wild-type (WT) enzyme. The MT product conversion was 94.6 %, reaching the highest reported level. Utilizing a multi-dimensional rational design for a customized mutation strategy of the substrate-binding pocket is an effective approach to enhance the catalytic efficiency of enzymes in handling complicated substrates.

Efficient production of R-2-(4-hydroxyphenoxy) propionic acid by Beauveria bassiana using biofilm-based two-stage fermentation.

In this work, a novel biofilm-based fermentation of Beauveria bassiana was employed to convert R-2- phenoxypropionic acid (R-PPA) to R-2-(4-hydroxyphenoxy) propionic acid (R-HPPA). The biofilm culture model of Beauveria bassiana produced a significantly higher R-HPPA titer than the traditional submerged fermentation method. Mannitol dosage, tryptone dosage, and initial pH were the factors that played a significant role in biofilm formation and R-HPPA synthesis. Under the optimal conditions, the maximum R-HPPA titer and productivity approached 22.2 g/L and 3.2 g/(L·d), respectively. A two-stage bioreactor combining agitation and static incubation was developed to further increase R-HPPA production. The process was optimized to achieve 100 % conversion of R-PPA, with a maximum R-HPPA titer of 50 g/L and productivity of 3.8 g/(L·d). This newly developed biofilm-based two-stage fermentation process provides a promising strategy for the industrial production of R-HPPA and related hydroxylated aromatic compounds.

Design of a cofactor self-sufficient whole-cell biocatalyst for enzymatic asymmetric reduction via engineered metabolic pathways and multi-enzyme cascade.

NAD(P)H-dependent oxidoreductases are crucial biocatalysts for synthesizing chiral compounds. Yet, the industrial implementation of enzymatic redox reactions is often hampered by an insufficient supply of expensive nicotinamide cofactors. Here, a cofactor self-sufficient whole-cell biocatalyst was developed for the enzymatic asymmetric reduction of 2-oxo-4-[(hydroxy)(-methyl)phosphinyl] butyric acid (PPO) to L-phosphinothricin (L-PPT). The endogenous NADP+ pool was significantly enhanced by regulating Preiss-Handler pathway toward NAD(H) synthesis and, in the meantime, introducing NAD kinase to phosphorylate NAD(H) toward NADP+. The intracellular NADP(H) concentration displayed a 2.97-fold increase with the strategy compared with the wild-type strain. Furthermore, a recombinant multi-enzyme cascade biocatalytic system was constructed based on the Escherichia coli chassis. In order to balance multi-enzyme co-expression levels, the strategy of modulating rate-limiting enzyme PmGluDH by RBS strengths regulation successfully increased the catalytic efficiency of PPO conversion. Finally, the cofactor self-sufficient whole-cell biocatalyst effectively converted 300 mM PPO to L-PPT in 2 h without the need to add exogenous cofactors, resulting in a 2.3-fold increase in PPO conversion (%) from 43% to 100%, with a high space-time yield of 706.2 g L-1 d-1 and 99.9% ee. Overall, this work demonstrates a technological example for constructing a cofactor self-sufficient system for NADPH-dependent redox biocatalysis.

Hyperthermophilic pretreatment significantly accelerates thermophilic composting humification through improving bacterial communities and promoting microbial cooperation.

Thermophilic composting (TC) can effectively shorten maturity period with satisfactory sanitation. However, the higher energy consumption and lower composts quality limited its widespread application. In this study, hyperthermophilic pretreatment (HP) was introduced as a novel approach within TC, and its effects on humification process and bacterial community during food waste TC was investigated from multiple perspectives. Results showed that a 4-hour pretreatment at 90 °C increased the germination index and humic acid/fulvic acid by 25.52% and 83.08%, respectively. Microbial analysis demonstrated that HP stimulated the potential functional thermophilic microbes, and significantly up-regulated the genes related to amino acid biosynthesis. Further network and correlation analysis suggested that pH was the key factor affecting bacterial communities, and higher HP temperatures help to restore bacterial cooperation and showed higher humification degree. In summary, this study contributed to a better understanding of the mechanism towards the accelerated humification by HP.

Properties and biotechnological applications of microbial deacetylase.

Deacetylases, a class of enzymes that can catalyze the hydrolysis of acetylated substrates to remove the acetyl group, used in producing various products with high qualities, are one of the most influential industrial enzymes. These enzymes are highly specific, non-toxic, sustainable, and eco-friendly biocatalysts. Deacetylases and deacetylated compounds have been widely applicated in pharmaceuticals, medicine, food, and the environment. This review synthetically summarizes deacetylases' sources, characterizations, classifications, and applications. Moreover, the typical structural characteristics of deacetylases from different microbial sources are summarized. We also reviewed the deacetylase-catalyzed reactions for producing various deacetylated compounds, such as chitosan-oligosaccharide (COS), mycothiol, 7-aminocephalosporanic acid (7-ACA), glucosamines, amino acids, and polyamines. It is aimed to expound on the advantages and challenges of deacetylases in industrial applications. Moreover, it also serves perspectives on obtaining promising and innovative biocatalysts for enzymatic deacetylation. KEYPOINTS: • The fundamental properties of microbial deacetylases of various microorganisms are presented. • The biochemical characterizations, structures, and catalyzation mechanisms of microbial deacetylases are summarized. • The applications of microbial deacetylases in food, pharmaceutical, medicine, and the environment were discussed.

A combination fermentation strategy for simultaneously increasing cellular NADP(H) level, biomass, and enzymatic activity of glufosinate dehydrogenase in Escherichia coli.

Oxidoreductase is one of the most important biocatalysts for the synthesis of various chiral compounds. However, their whole-cell activity is frequently affected by an insufficient supply of expensive nicotinamide cofactors. This study aimed to overcome such shortcomings by developing a combination fermentation strategy for simultaneously increasing intracellular NADP(H) level, biomass, and glufosinate dehydrogenase activity in E. coli. The results showed that the feeding mode of NAD(H) synthesis precursor and lactose inducer had essential effects on the accumulation level of intracellular NADPH. Adding 40 mg L-1 of L-aspartic acid to the medium increased the intracellular NADP(H) concentration by 36.3%. Under the pH-stat feeding mode and adding 0.4 g L-1 h-1 lactose, the NADP(H) concentration, biomass, and GluDH activity in the 5-L fermenter reached 445.7 µmol L-1, 21.7 gDCW L-1, and 8569.3 U L-1, respectively. As far as we know, this is the highest reported activity of GluDH in the fermentation broth. Finally, the 5000-L fermenter was successfully scaled up to use this fermentation approach. The combination fermentation strategy might serve as a useful approach for the high-activity fermentation of other NADPH-dependent oxidoreductases.

Effects of fungal agents and biochar on odor emissions and microbial community dynamics during in-situ treatment of food waste.

The effects of the co-addition of fungal agents and biochar on physicochemical properties, odor emissions, microbial community structure, and metabolic functions were investigated during the in-situ treatment of food waste. The combined addition of fungal agents and biochar decreased cumulative NH3, H2S, and VOCs emissions by 69.37%, 67.50%, and 52.02%, respectively. The predominant phyla throughout the process were Firmicutes, Actinobacteria, Cyanobacteria, and Proteobacteria. Combined treatment significantly impacted the conversion and release of nitrogen from the perspective of the variation of nitrogen content between different forms. FAPROTAX analysis revealed that the combined application of fungal agents and biochar could effectively inhibit nitrite ammonification and reduce the emission of odorous gases. This work aims to clarify the combined effect of fungal agents and biochar on odor emission and provide a theoretical basis for developing an environmentally friendly in-situ efficient biological deodorization (IEBD) technology.

Microbial production of vitamin B5: current status and prospects.

Vitamin B5, also called D-pantothenic acid (D-PA), is a necessary micronutrient that plays an essential role in maintaining the physiological function of an organism. It is widely used in: food, medicine, feed, cosmetics, and other fields. Currently, the production of D-PA in industry heavily relies on chemical processes and enzymatic catalysis. With an increasing demand on the market, replacing chemical-based production of D-PA with microbial fermentation utilizing renewable resources is necessary. In this review, the physiological role and applications of D-PA were firstly introduced, after which the biosynthesis pathways and enzymes will be summarized. Subsequently, a series of cell factory development strategies for excessive D-PA production are analyzed and discussed. Finally, the prospect of microbial production of D-PA production has been prospected.

Bacterial dynamics and functions driven by bulking agents to enhance organic degradation in food waste in-situ rapid biological reduction (IRBR).

This study investigated the effects of different bulking agents (i.e., sawdust, wheat straw, rice straw, and corncob) on bacterial structure and functions for organic degradation during food waste in-situ rapid biological reduction (IRBR) inoculated with microbial agent. Results showed that the highest organic degradation (409.5 g/kg total solid) and volatile solids removal efficiency (41.0%) were achieved when wheat straw was used, largely because the degradation of readily degradable substrates and cellulose was promoted by this bulking agent. Compared with other three bulking agents, the utilization of wheat straw was conducive to construct a more suitable environmental condition (moisture content of 18.0-28.2%, pH of 4.91-5.87) for organic degradation during IRBR process, by virtue of its excellent structural and physiochemical properties. Microbial community analysis suggested that the high-moisture environment in rice straw treatment promoted the growth of Staphylococcus and inhibited the activity of the inoculum. By contrast, lowest bacterial richness was observed in corncob treatment due to the faster water loss. Compared with these two bulking agents, sawdust and wheat straw treatment led to a more stable bacterial community structure, and the inoculated Bacillus gradually became the dominant genus (36.6-57.8%) in wheat straw treatment. Predicted metagenomics analysis showed that wheat straw treatment exhibited the highest carbohydrate metabolism activity which improved the pyruvate, amino sugar and nucleotide sugar metabolism, and thereby promoted the organic degradation and humic substrate production. These results indicated that wheat straw was a more desirable bulking agent, and revealed the potential microbial organics degradation mechanism in IRBR process.

Community scale in-situ rapid biological reduction and resource recovery of food waste.

In this study, a community-scale in-situ rapid biological reduction (IRBR) system was applied to achieve the rapid disposal and resource recovery of food waste (FW). A total of 5263 kg FW was processed in the 35 days of stably operation, during which 84.37% total mass reduction and 43.30% volatile solid removal were achieved, and the odor had been effectively controlled. Microbial sequencing results showed that aerobic and facultative thermophilic bacteria were major bacterial community, and vigorous metabolism of both carbohydrate and amino acid were maintained during the IRBR process. The final products have the potential to be recycled as organic fertilizers or bio-solid fuel to realize resource recovery. The results of economic analysis showed that the IRBR system had lower FW disposal costs due to the high automation. These results suggested that the IRBR system was an environmentally friendly, economical and practical method for the FW rapid treatment.

Effect of Moisture Distribution Changes Induced by Different Cooking Temperature on Cooking Quality and Texture Properties of Noodles Made from Whole Tartary Buckwheat.

While precooking and processing have improved the quality of gluten-free noodles, the effects of different cooking temperatures on their quality-neither gluten-free noodles nor whole Tartary buckwheat noodles-have rarely been clarified. This study investigated the key role of moisture distribution induced by different cooking temperatures in improving the noodle quality of whole Tartary buckwheat. The results showed that cooking temperatures higher than 70 °C led to a sharp increase in cooking loss, flavonoid loss and the rate of broken noodles, as well as a sharp decrease in water absorption. Moreover, the noodles cooked at 70 °C showed the lowest rate of hardness and chewiness and the highest tensile strength of all cooking temperatures from 20 °C to 110 °C. The main positive attribute of noodles cooked at 70 °C might be their high uniform moisture distribution during cooking. Cooking at 70 °C for 12 min was determined as the best condition for the quality improvement of whole Tartary buckwheat noodles. This is the first study to illustrate the importance of cooking temperatures on the quality of Tartary buckwheat noodles. More consideration must also be given to the optimal cooking conditions for different gluten-free noodles made from minor coarse cereals.

Improved production of D-pantothenic acid in Escherichia coli by integrated strain engineering and fermentation strategies.

D-pantothenic acid (D-PA) is an essential vitamin that has been widely used in medicine, food, and animal feed. Microbial production of D-PA from natural renewable resources is attractive and challenging. In this study, both strain improvements and fermentation process strategies were applied to achieve high-level D-PA production in Escherichia coli. First, a D-PA-producing strain was developed through deletion of the aceF and mdh genes combined with the overexpression of the gene ppnk. The obtained engineered E. coli DPA02/pT-ppnk accumulated 6.89 ± 0.11 g/L of D-PA in shake flask fermentation, which was 79.9 % higher than the control strain. Moreover, the cultivation process contributed greatly to D-PA production with respect to titer and productivity by betaine supplementation and dissolved oxygen (DO)-feedback feeding framework. Under optimal conditions, 68.3 g/L of D-PA, the specific productivity of 0.794 g/L h and the yield of 0.36 g/g glucose in 5 L fermenter were achieved. Overall, this research successfully exploited advanced strategies to lay the foundation for bio-based D-PA production in industrial applications.

Functional expression of an echinocandin B deacylase from Actinoplanes utahensis in Escherichia coli.

Echinocandin B deacylase (ECBD) from Actinoplanes utahensis can be applied to produce echinocandin B nucleus (ECBN), an essential intermediate of the echinocandins antifungal drugs such as anidulafungin. To date, the expression of ECBD has been limited to Streptomyces. To achieve the active expression of ECBD in Escherichia coli (E. coli), we constructed a plasmid carrying two subunits of ECBD for T7 RNA polymerase driven transcription of dicistron messenger after codon optimization. Subsequently, the introduction of peptide tags in the recombinant ECBD was adopted to reduce the formation of inclusion bodies and enhance the ECBD solubility. The peptide tags with the opposite electrostatic charge, hexa-lysine (6K) and GEGEG (GE), exhibited the best positive effect, which was verified by activity assay and structural simulation. After that, optimization of culture conditions and characterization of ECBD were conducted, the optimal pH and temperature were 7.0 and 60 °C. It is the first report concerning the functional expression of ECBD in the host E. coli. Our results reported here can provide a reference for the high-level expression of other deacylases with respect to a possible industrial application.

Fed-in-situ biological reduction treatment of food waste via high-temperature-resistant oil degrading microbial consortium.

This study aims to construct a high-temperature-resistant microbial consortium to effectively degrade oily food waste by Fed-in-situ biological reduction treatment (FBRT). Oil degrading bacteria were screened under thermophilic conditions of mineral salt medium with increased oil content. The oil degradation and emulsification ability of each stain was evaluated and their synergetic improvement was further confirmed. Consortium of Bacillus tequilensis, Bacillus licheniformis, Bacillus sonorensis and Ureibacillus thermosphaericus was selected and applicated as bacterial agents in FBRT under 55 °C. Changes in pH, moisture, bacterial community and key components of food waste were monitored for 5 days during processing. Facilitated by the bacterial consortium, FBRT gave superior total mass reduction (86.61 ± 0.58% vs. 67.25 ± 1.63%) and non-volatile solids reduction (65.91 ± 1.53% vs. 28.53 ± 2.29%) compared with negative control, the feasibility and efficiency of present FBRT providing a promising in-situ disposal strategy for rapid reduction of oily food waste.

Overproduction of D-pantothenic acid via fermentation conditions optimization and isoleucine feeding from recombinant Escherichia coli W3110.

D-pantothenic acid (D-PA), as a crucial vitamin, is widely used in food, animal feed, cosmetics, and pharmaceutical industries. In our previous work, recombinant Escherichia coli W3110 for production of D-PA was constructed through metabolic pathway modification. In this study, to enhance D-PA production, statistical optimization techniques including Plackett-Burman (PB) design and Box-Behnken design (BBD) first were adopted to optimize the culture condition. The results showed that the glucose, ß-alanine and (NH4)2SO4 have the most significant effects on D-PA biosynthesis. The response surface model based on BBD predicted that the optimal concentration is glucose 56.0 g/L, ß-alanine 2.25 g/L and (NH4)2SO4 11.8 g/L, the D-PA titer increases from 3.2 g/L to 6.73 g/L shake flask fermentation. For the fed-batch fermentation in 5 L fermenter, the isoleucine feeding strategy greatly increased the titer and productivity of D-PA. As a result, titer (31.6 g/L) and productivity (13.2 g/L·d) of D-PA were achieved, they increased by 4.66 times and 2.65 times, respectively, compared with batch culture. At the same time, the accumulation of acetate reduced from 29.79 g/L to 8.55 g/L in the fed-batch fermentation. These results demonstrated that the optimization of medium composition and the cell growth rate are important to increase the concentration of D-PA for microbial fermentation. This work laid the foundation for further research on the application of D-PA microbial synthesis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02773-0.

Identification of a novel promoter for driving antibiotic-resistant genes to reduce the metabolic burden during protein expression and effectively select multiple integrations in Pichia Pastoris.

Routine approaches for the efficient expression of heterogenous proteins in Pichia pastoris include using the strong methanol-regulated alcohol oxidase (AOX1) promoter and multiple inserts of expression cassettes. To screen the transformants harboring multiple integrations, antibiotic-resistant genes such as the Streptoalloteichus hindustanus bleomycin gene are constructed into expression vectors, given that higher numbers of insertions of antibiotic-resistant genes on the expression vector confer resistance to higher concentrations of the antibiotic for transformants. The antibiotic-resistant genes are normally driven by the strong constitutive translational elongation factor 1a promoter (PTEF1). However, antibiotic-resistant proteins are necessary only for the selection process. Their production during the heterogenous protein expression process may increase the burden in cells, especially for the high-copy strains which harbor multiple copies of the expression cassette of antibiotic-resistant genes. Besides, a high concentration of the expensive antibiotic is required for the selection of multiple inserts because of the effective expression of the antibiotic-resistant gene by the TEF1 promoter. To address these limitations, we replaced the TEF1 promoter with a weaker promoter (PDog2p300) derived from the potential promoter region of 2-deoxyglucose-6-phosphate phosphatase gene for driving the antibiotic-resistant gene expression. Importantly, the PDog2p300 has even lower activity under carbon sources (glycerol and methanol) used for the AOX1 promoter-based production of recombinant proteins compared with glucose that is usually used for the selection process. This strategy has proven to be successful in screening of transformants harboring more than 3 copies of the gene of interest by using plates containing 100 µg/ml of Zeocin. Meanwhile, levels of Zeocin resistance protein were undetectable by immunoblotting in these multiple-copy strains during expression of heterogenous proteins.Key points• PDog2p300 was identified as a novel glucose-regulated promoter.• The expression of antibiotic-resistant gene driven by PDog2p300 was suppressed during the recombinant protein expression, resulting in reducing the metabolic burden.• The transformants harboring multiple integrations were cost-effectively selected by using the PDog2p300 for driving antibiotic-resistant genes.

Efficient bio-degradation of food waste through improving the microbial community compositions by newly isolated Bacillus strains.

This study aims to screen high-degradability strains and develop a novel microbial agent for efficient food waste degradation. The effects of the novel microbial agent on organic matter degradation, enzyme activity, and bacterial succession during the in-situ reduction of food waste were evaluated and compared with other two microbial agents previously developed. Results showed that the novel agent containing four Bacillus strains received maximum organic degradation rates, volatile solid removal (46.91%) and total mass reduction (76.16%). Pyrosequencing analysis revealed that there was a significant difference in the microbial community structure of the matrix among the three biodegradation systems, and the novel agent greatly improved the stability of in-situ reduction process that Bacillus was the dominant genus (>98%) since day 4. These results indicated that the inoculant containing only Bacillus was more stable and cost-effective in FW in-situ reduction.

A integrated process for nitrilase-catalyzed asymmetric hydrolysis and easy biocatalyst recycling by introducing biocompatible biphasic system.

The whole-cell nitrilase-catalyzed asymmetric hydrolysis of nitriles is a green and efficient preparation approach for chiral carboxylic acids, but often suffers from toxicity and cell lysis from organic substrates. In this work, a novel integrated process for whole-cell nitrilase-catalyzed asymmetric hydrolysis was developed for the first time by introducing a biocompatible ionic liquid (IL)-based biphasic system. The whole-cell nitrilases displayed an outstanding stability and recyclability in the biphasic system and still retained > 85% activity even after 7 cycles reaction. A preparative-scale fed-batch hydrolysis of o-chloromandelonitrile to (R)-o-chloromandelic acid (R-CMA) was performed using the integrated process. The results revealed a yield of 91.3% and a space-time yield of 746.4 g·L-1·d-1, which are currently the highest reported values for R-CMA biosynthesis. The proposed integrated process avoids substrate inhibition, facilitates the reusability of whole-cell nitrilases, and thus shows great potential for the sustainable production of chiral carboxylic acids.

High-level production of d-pantothenic acid from glucose by fed-batch cultivation of Escherichia coli.

d-Pantothenic acid (D-PA) is an essential vitamin widely used in food, feed, chemical, and pharmaceutical industries. An Escherichia coli platform was developed for the high-level production of D-PA from glucose through fed-batch cultivation. Initially, the effects of different glucose feeding strategies D-PA synthesis were studied. It was found that D-PA production in glucose control (5 g/L) fed-batch culture reached 24.3 g/L, which was 4.09 times that in the batch culture. Next, the effect of auxotrophic amino acid (isoleucine)-limited feeding on D-PA production was investigated. The results revealed that isoleucine feeding decreased the accumulation of by-product acetic acid and promoted D-PA production significantly. Furthermore, an isoleucine feeding embedded multistage glucose supply strategy was established, and a maximum titer of 39.1 g/L was achieved. To the best of our knowledge, this levels are the highest reported so far in engineered E. coli for the D-PA production. The developed fed-batch feeding strategy may be useful for the industrial D-PA production by E. coli.