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.

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.

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.

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.

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.

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.

Effects of methyl oleate and microparticle-enhanced cultivation on echinocandin B fermentation titer.

Echinocandin B (ECB) is a key precursor of antifungal agent Anidulafungin, which has demonstrated clinical efficacy in patients with invasive candidiasis. In this study, the effects of microparticle-enhanced cultivation and methyl oleate on echinocandin B fermentation titer were investigated. The results showed that the titer was significantly influenced by the morphological type of mycelium, and mycelium pellet was beneficial to improve the titer of this secondary metabolism. First, different carbon sources were chosen for the fermentation, and methyl oleate achieved the highest echinocandin B titer of 2133 ± 50 mg/L, which was two times higher than that of the mannitol. The study further investigated the metabolic process of the fermentation, and the results showed that L-threonine concentration inside the cell could reach 275 mg/L at 168 h with methyl oleate, about 2.5 times higher than that of the mannitol. Therefore, L-threonine may be a key precursor of echinocandin B. In the end, a new method of adding microparticles for improving the mycelial morphology was used, and the addition of talcum powder (20 g/L, diameter of 45 µm) could make the maximum titer of echinocandin B reach 3148 ± 100 mg/L.

Mutagenesis of echinocandin B overproducing Aspergillus nidulans capable of using starch as main carbon source.

Echinocandin B, a kind of antimycotic with cyclic lipo-hexapeptides, was produced by fermentation with Aspergillus nidulans using fructose as main carbon source. The objective of this study was to screen a high-yield mutant capable of using cheap starch as main carbon source by atmospheric and room temperature plasma (ARTP) treatment in order to decrease the production cost of echinocandin B. A stable mutant A. nidulans ZJB19033, which can use starch as optimal carbon source instead of expensive fructose, was selected from two thousands isolates after several cycles of ARTP mutagenesis. To further increase the production of echinocandin B, the optimization of fermentation medium was performed by response surface methodology (RSM), employing Plackett-Burman design (PBD) followed by Box-Behnken design (BBD). The optimized fermentation medium provided the optimal yield of echinocandin B, 2425.9 ± 43.8 mg/L, 1.3-fold compared to unoptimized medium. The results indicated that the mutant could achieve high echinocandin B production using cheap starch as main carbon source, and the cost of carbon sources in fermentation medium reduced dramatically by about 45%.

Engineering a Pichia pastoris nitrilase whole cell catalyst through the increased nitrilase gene copy number and co-expressing of ER oxidoreductin 1.

1-Cyanocyclohexaneacetic acid (1-CHAA) is a critical intermediate for the synthesis of the antiepileptic agent gabapentin. Previously, our group has established a novel manufacturing route for 1-CHAA through bioconversion catalyzed by an Escherichia coli (E. coli) nitrilase whole cell catalyst. However, the nitrilase expressed in E. coli has several drawbacks such as a low level of reusability, which hampered its industrial application. Herein, we investigated the potential of using the methylotrophic yeast Pichia pastoris (P. pastoris) for producing the nitrilase whole cell catalyst. To achieve strains with high catalytic activities, we investigated the effects of the promoter choice, expressing cassette copy number, and co-expression of chaperone on the production of nitrilase. Our results demonstrated that the strain harboring the multicopy integrations of nitrilase gene under the control of the alcohol oxidase 1 (AOX1) promoter and co-expressing of ER oxidoreductin 1 (ERO1) exhibited an 18-fold enhancement in the nitrilase activity compared with the strain containing a single integration of nitrilase gene under the control of glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter. This optimized P. pastoris strain, compared with the E. coli nitrilase whole cell catalyst, shows greatly improved levels of reusability and thermostability while has a similar high-substrate tolerance.

Recent advances in the improvement of enzyme thermostability by structure modification.

Thermostability is considered to be an important parameter to measure the feasibility of enzymes for industrial applications. Generally, higher thermostability makes an enzyme more competitive and desirable in industry. However, most natural enzymes show poor thermostability, which restricts their application. Protein structure modification is a desirable method to improve enzyme properties. In recent years, tremendous progress has been achieved in protein thermostability engineering. In this review, we provide a systemic overview on the approaches of protein structure modification for the improvement of enzyme thermostability during the last decade. Structure modification approaches, including the introduction of non-covalent interactions and covalent bonds, increase of proline and/or decrease in glycine, reinforcement of subunit-subunit interactions, introduction of glycosylation sites, truncation and cyclization have been highlighted.

Integrated strategy of temperature shift and mannitol feeding for enhanced production of echinocandin B by Aspergillus nidulans CCTCC M2012300.

The production of echinocandin B (ECB) by Aspergillus nidulans CCTCC M2012300 was improved by integrating the temperature-shift and fed-batch control strategies. The kinetic characteristics of batch cultures were analyzed at different culture temperatures, and then a two-stage temperature control strategy was established. In the first 6 days, the temperature was maintained at 30 °C to obtain the maximal cell growth rate; subsequently, 25 °C was used to gain a high ECB formation rate. On the basis of temperature control, the ECB productivity was increased to 143.3 mg/(L day), which was a 1.3-fold improvement compared with the optimal constant-temperature cultivations. The influences of fed-batch cultures were further investigated. A maximal ECB productivity of 170.8 mg/(L day) was obtained through a three-stage mannitol pulse-feeding strategy, which was another 1.2-fold improvement than that of the batch fermentation. This is the first report of the use of a two-stage temperature control fed-batch strategy in ECB fermentation. This strategy was simple and economical to operate and may provide new guidance for the industrial-scale production of ECB.

Enhanced catalytic stability and reusability of nitrilase encapsulated in ethyleneamine-mediated biosilica for regioselective hydrolysis of 1-cyanocycloalkaneacetonitrile.

Nitrilase-catalyzed regioselective hydrolysis of 1-cyanocyclohexaneacetonitrile (1-CHAN) is a green and efficient approach for the preparation of 1-cyanocyclohexaneacetic acid (1-CHAA), a key precursor for the synthesis of gabapentin. Here, a mesoporous biosilica particles prepared by the ethyleneamine-mediated silicification have been used as carrier for the encapsulation of nitrilase from Acidovorax facilis (NitA). The silica-encapsulated NitA (NitA@silica) with triethylenetetramine as an initiator showed the highest immobilization efficiency (98.3%) and specific activity (672.6 U/g). Both free and encapsulated NitA were optimally active at 40 °C and pH 7.0, however, the encapsulated enzyme exhibited wider optimum temperature range, and enhanced thermal stability compared with the free enzyme. The kinetic parameters Km and Vmax for free and encapsulated NitA were calculated to be 141 mM and 9.97 mM min-1, and 280 mM and 9.02 mM min-1, respectively. The encapsulated NitA showed good reusability and retained about 94.2% of its initial activity even after 16 cycles of reaction. Also, the storage experiments revealed high activity maintenance of encapsulated NitA after 17-day storage at 4 °C. A preparative scale regioselective hydrolysis of 1-CHAN to 1-CHAA with encapsulated NitA as biocatalyst was carried out in a 2 L stirred bioreactor. The concentration of 1-CHAA reached 152 g/L after 8 h reaction and the conversion was 90.9%. These results showed that the encapsulation of NitA in ethyleneamine-mediated biosilica is an efficient and simple way for preparation of stable nitrilase and have a great potential for application in enzymatic production of carboxylic acids.

Highly efficient conversion of 1-cyanocycloalkaneacetonitrile using a "super nitrilase mutant".

Nitrilase is the member of carbon-nitrogen hydrogen hydrolase superfamily, which has been widely used for the hydrolysis of nitriles into corresponding carboxylic acids. But most nitrilases are plagued by product inhibition in the industrial application. In this study, a "super nitrilase mutant" of nitrilase with high activity, thermostability and improved product tolerance from Acidovorax facilis ZJB09122 was characterized. Then, an efficient process was developed by employing the whole cell of recombinant E. coli for the conversion of high concentration of 1-cyanocyclohexylacetonitrile-to-1-cyanocyclohexaneacetic acid, an important intermediate of gabapentin. Under the optimized conditions, the higher substrate concentrations such as 1.3 M, 1.5 M and 1.8 M could be hydrolyzed by 13.58 g DCW/L with outstanding productivity (> 740 g/L/day). This study developed a highly efficient bioprocess for the preparation of 1-cyanocyclohexaneacetic acid which has the great potential for industrial application.

Conjugation of Agrobacterium radiobacter epoxide hydrolase with ficoll: Catalytic, kinetic and thermodynamic analysis.

Epoxide hydrolase-mediated biocatalysis has a great prospective in the biosynthesis of optically pure epoxides. The present work targets toward the thermo-stabilization of epoxide hydrolase by covalent conjugation with polysaccharide. An epoxide hydrolase from Agrobacterium radiobacter (ArEH) was modified by covalent coupling to seven oxidized polysaccharides with different chemical structures and characteristics. Among all conjugates, ArEH with ficoll exhibited both the highest specific activity (494 U/mg) and half-life at 60 °C (t1/2 = 183 min). The conjugated enzyme also displayed wider optimum pH and temperature ranges, higher catalytic number (kcat), and catalytic efficiency (kcat/Km) as compared to its native counterpart. The conjugation significantly decreased the enthalpy of activation (ΔH*), free energy of transition state binding (ΔG*E-T), and free energy of activation (ΔG*) for epichlorohydrin hydrolysis. Moreover, the conjugated ArEH showed enhanced thermal stability as evidenced by its longer half-life (t1/2), lower thermal deactivation constant (kd), and higher D values at 50-70 °C. Furthermore, the enzymatic activity of conjugated ArEH elevated significantly by Ca2+ and it showed increased tolerance against Co2+, Fe3+ and EDTA inhibitors. Finally, fluorescence and circular dichroism spectroscopic analysis were performed to confirm the noticeable conformational changes in ArEH structure after conjugation with ficoll, which could be responsible for the observed catalytic and stability enhancement.