[Design and practice of biological reaction engineering flipped classroom in applied undergraduate colleges].

This study aims to explore and refine the teaching aspects of a flipped classroom approach for biological reaction engineering. The study encompasses three iterations of teaching practice, focusing on key elements such as theme content selection, implementation process, evaluation and effectiveness. By integrating relevant industry and societal topics with course's professional knowledge, students are encouraged to independently collect data, analyze and discuss findings, and present their work in group. Comprehensive literacy of students is assessed through discussion reports, defense reports, utilization of new tools, and team cooperation. Analysis of student performance reveals that the design and implementation of the flipped classroom approach significantly enhances student motivation to learn, improves scores, and supports the achievement of course objectives. Therefore, the methodology presented in this study may serve as a reference for implementing teaching reforms in core courses in applied undergraduate colleges, thereby fostering well-round individuals with strong theoretical foundation, innovative analytical skills, and excellent teamwork abilities.

Advances in the adenylation domain: discovery of diverse non-ribosomal peptides.

Non-ribosomal peptide synthetases are mega-enzyme assembly lines that synthesize many clinically useful compounds. As a gatekeeper, they have an adenylation (A)-domain that controls substrate specificity and plays an important role in product structural diversity. This review summarizes the natural distribution, catalytic mechanism, substrate prediction methods, and in vitro biochemical analysis of the A-domain. Taking genome mining of polyamino acid synthetases as an example, we introduce research on mining non-ribosomal peptides based on A-domains. We discuss how non-ribosomal peptide synthetases can be engineered based on the A-domain to obtain novel non-ribosomal peptides. This work provides guidance for screening non-ribosomal peptide-producing strains, offers a method to discover and identify A-domain functions, and will accelerate the engineering and genome mining of non-ribosomal peptide synthetases. KEY POINTS: • Introducing adenylation domain structure, substrate prediction, and biochemical analysis methods • Advances in mining homo polyamino acids based on adenylation domain analysis • Creating new non-ribosomal peptides by engineering adenylation domains.

[Construction of the curriculum for developing the ability to solving complex engineering problems in the field of biotechnology industry].

Accurately understanding the features and connotations of complex engineering problems is an important prerequisite for setting graduation requirements, constructing curriculum and designing teaching contents. By discussing the characteristics of complex engineering problems in the biological industry, this paper explored the demands for undergraduates in Yangtze river delta region, summarized the typical jobs and their requirements, and expounded the connotation of complex engineering problems contained in various typical tasks. On this basis, a gradual curriculum system was constructed, which included multiple stages of conceiving, formation and application, to cultivate the ability to solve complex engineering problems in the major of bioengineering. The curriculum coordinated the implementation of deep integration of industry and education, research feed back course construction, course team and advanced courses building up, professional associations covered all crews and students, supporting the ability training of solving complex engineering problems.

Separation of Heat-Stable Antifungal Factor From Lysobacter enzymogenes Fermentation Broth via Photodegradation and Macroporous Resin Adsorption.

Heat-stable antifungal factor (HSAF) is produced by the fermentation of Lysobacter enzymogenes, which is known for its broad-spectrum antifungal activity and novel mode of action. However, studies on the separation of HSAF have rarely been reported. Herein, alteramide B (the main byproduct) was removed firstly from the fermentation broth by photodegradation to improve the purity of HSAF. Then, the separation of HSAF via adsorption by macroporous adsorption resins (MARs) was evaluated and NKA resin showed highest static adsorption and desorption performances. After optimizing the static and dynamic adsorption characteristics, the content of HSAF in the purified product increased from 8.67 ± 0.32% (ethyl acetate extraction) to 31.07 ± 1.12% by 3.58-fold. These results suggest that the developed strategy via photodegradation and macroporous resin adsorption is an effective process for the separation of HSAF, and it is also a promising method for the large-scale preparation of HSAF for agricultural applications.

[Development of a practical innovation course for biological engineering major under the background of Engineering Education Certification].

According to the teaching philosophy of the outcome-based education, this study elaborates the development of a practical innovation course for biological engineering major after five runs of teaching practice and continuous improvement. It mainly includes the methods for selection of teaching subjects, implementation of teaching process, process assessment, evaluation and improvement. Based on the performance and achievements of three grades of students majored in bioengineering, we found that the logic and methods of the practical innovation course could greatly stimulate the motivation of students for learning, as well as their scores. Therefore, the logic and methods described in this study may serve as a reference for the reforms of practical training courses of engineering major under the background of Engineering Education Certification.

Efficient biosynthesis of exopolysaccharide from Jerusalem artichoke using a novel strain of Bacillus velezensis LT-2.

This study focused on the non-grain biorefining of Jerusalem artichoke (JA) for exopolysaccharide (EPS) efficient production by using Bacillus velezensis LT-2. Results showed that LT-2 could directly utilize JA tuber power (JATP) for EPS production, and its EPS yield reached 11.47 ± 0.33 g/L in the simultaneous saccharification and fermentation (SSF) mode. Furthermore, the SSF mode shortened the fermentation period by 26.67% and reduced the fermentation cost by 79.41% due to the improved substrate utilization and the avoidance of inhibition effects of a high fructose concentration. Transcriptome sequencing results showed that inulin could accelerate nucleotide-sugars biosynthesis, induce EPS synthetic gene cluster transcription, and strengthen the electron transport respiratory chain and the transporter systems, thereby ensuring EPS efficient synthesis. This work exhibited a productive non-grain and environmentally friendly fermentation strategy for EPS biosynthesis, which promoted the JA industry development and created new prospects for high-value industrial products biosynthesis by using JATP.

Production exopolysaccharide from Kosakonia cowanii LT-1 through solid-state fermentation and its application as a plant growth promoter.

Exopolysaccharide has the activity of promoting plants growth. Herein, several agro-industrial residues were used for EPS biosynthesis through solid-state fermentation (SSF) using an EPS producing stain Kosakonia cowanii LT-1. Then the influences of cane molasses powder (CMP), NaNO3 and temperature on EPS biosynthesis through SSF were studied using response surface methodology. Maximum EPS yield (41.62 mg/gds) was obtained in a mixed substrate composed of cane bagasse and broadbean seed capsule (2:1, w/w) supplemented with CMP (5.09%, w/w) and NaNO3 (4.16‰, w/w), which achieved about 30.43% cost savings for substrates. Transcriptomic analysis explained the mechanism of NaNO3 promoting EPS production. Repeated batch SSF was performed eight times with 10% substrate as the seeds of the next SSF cycle successfully. Therefore, the fermented SSF substrate containing K. cowanii LT-1 and its EPS was applied in maize growth, which could promote seed germination rate and growth vigor of maize. Hence, fermented SSF substrates would be an ideal candidate for application as a plant growth promoter in agriculture field.

Efficient expression of chondroitinase ABC I for specific disaccharides detection of chondroitin sulfate.

Chondroitinase ABC I (ChSase ABC I) is a key enzyme of chondroitin sulfate (CS) degradation and widely used for CS detection in the medicine filed. However, the recombinant ChSase ABC I was weakly expressed in Escherichia coli because the forms of it were mostly inclusion bodies. In this study, a signal peptide (pelB) was used for the soluble form expression of ChSase ABC I in E. coli. Then the culture condition for ChSase ABC I expression was optimized through response surface methodology. Results revealed that the expression level of ChSase ABC I in a 7.5 L fermentor (29.03 mL-1) was approximately 1.65-fold higher than that of the shake flask level (17.55 mL-1). The enzymatic properties and kinetic constants of recombinant ChSase ABC I were also studied. Recombinant ChSase ABC I was also used to detect the specific disaccharides content of CS from different sources. This study not only eliminates the problem of the enzyme expressed as an inclusion body, but also solves the current problem of expensive ChSase ABC. In a word, it would be an ideal strategy for ChSase ABC high-efficiency expression and a great method to detect specific disaccharides of CS in biomedical field.

Development of sugarcane resource for efficient fermentation of exopolysaccharide by using a novel strain of Kosakonia cowanii LT-1.

This work focuses on the development of non-food fermentation for the cost-effective biosynthesis of exopolysaccharide (EPS) by using a new strain of Kosakonia cowanii LT-1. This novel strain more efficiently utilizes sucrose for EPS production than other glycosyl donors. Comparative transcriptomic analysis is used to understand EPS synthesis promotion and the effects of sucrose on EPS biosynthesis. We speculate that ATP-binding cassette transporter, phosphotransferase, and two-component systems may be the most essential factors for EPS biosynthesis. The enhanced oxidative phosphorylation increases the synthesis rate of ATP to satisfy the energy demands for EPS production with sucrose as the substrate. Sugarcane juice, a cheap raw material, could improve the EPS yield in batch fermentation and achieve approximately 29.66% cost savings for substrate. Our work presents a promising non-food fermentation approach for the synthesis of high-value industrial products.

Two-step economical welan gum production by Sphingomonas sp. HT-1 from sugar industrial by-products.

A two-step fermentation strategy using glucose mother liquor (GML) for cell growth and xylose mother liquor (XML) for welan gum synthesis was used to alleviate uneconomic welan gum fermentation. This study revealed: (1) optimal initial GML concentration was 11.7g/L (10g/L sugars contained); (2) optimal XML feeding strategy was pseudo-exponential fed-batch and feeding time was 12thh-54thh, amounting to 25.7g/L XML (20g/L sugars contained); and (3) in a 7.5-L bioreactor, welan gum concentration was 22.68±0.50g/L and its yield reached 0.756g/g sugars with trace residual sugars. Compared with the cost of batch fermentation using glucose as sole carbon source, the final carbon source costs decreased by 61.40% and the welan gum yield increased by 50%. GML and XML can be used as inexpensive carbon sources for welan gum production with higher yield, giving them industrial application potential to produce value-added chemicals.

Green synthesis of isomaltulose from cane molasses by Bacillus subtilis WB800-pHA01-palI in a biologic membrane reactor.

A green process and environmentally benign process is highly desirable in the development of enzymatic catalysis. In this work, the shuttle plasmid pHA01 was constructed and the sucrose isomerase (SIase) was expressed in Bacillus subtilis WB800. The optimal nitrogen and carbon sources for SIase expression were yeast extract (15g/L) and un-pretreated cane molasses (UCM, 20g/L), respectively. After the UCM fed, the whole cell activity reached 5.2U/mL in a 7.5L fermentor. Optimum catalytic temperature and pH of whole cell were 35°C and 5.5, respectively. Although the biologic membrane reactor (BMR) system consecutively worked for 12 batches, the sucrose conversion remained higher than 90%, indicating the BMR system had a greater operational stability. Furthermore, isomaltulose production using the BMR system with low-cost cane molasses as its substrate not only reduces the production cost and mediates environmental pollution, but also solves the genetic background problem of the non-food-grade strains.

Efficient production of lactulose from whey powder by cellobiose 2-epimerase in an enzymatic membrane reactor.

In this study, the gene encoding cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) was successfully expressed in Bacillus subtilis WB800. After the fermentation medium optimization, the activity of recombinant strain was 4.5-fold higher than the original medium in a 7.5L fermentor. The optimal catalytic pH and temperature of crude CsCE were 7.0 and 80°C, respectively. An enzymatic synthesis of lactulose was developed using cheese-whey lactose as its substrate. The maximum conversion rate of whey powder obtained was 58.5% using 7.5 U/mL CsCE. The enzymatic membrane reactor system exhibited a great operational stability, confirmed with the higher lactose conversion (42.4%) after 10 batches. To our best knowledge, this is the first report of lactulose synthesis in food grade strain, which improve the food safety, and we not only realize the biological production of lactulose, but also make good use of industrial waste, which have positive impact on environment.

Enhancement of welan gum production in Sphingomonas sp. HT-1 via heterologous expression of Vitreoscilla hemoglobin gene.

Welan gum is a microbial polysaccharide produced by Sphingomonas sp. Its production is limited by the dissolved oxygen levels in the highly viscous fermentation. A strategy of heterologous expression of the Vitreoscilla hemoglobin gene in Sphingomonas sp. HT-1 was investigated to alleviate oxygen limitation and improve the yield of welan gum. Ultimately, the welan gum production increased from 25.3g/L to 34.6g/L, whereas the rheological behavior of welan gum solutions remained virtually unchanged. The transcriptional levels of the key genes in the electron transfer chain, TCA cycle and welan gum synthesis pathway, as well as ATP level revealed that the VHb expression in Sphingomonas sp. HT-1 enhanced welan gum biosynthesis by improving respiration and ATP supply. This study would pave the genetic manipulation way for enhancing welan gum yield, and it's also of great importance for the industrial applications of welan gum under harsh conditions.

Modified nanoporous titanium dioxide as a novel carrier for enzyme immobilization.

ε-Poly-L-lysine (EPL)-modified mesoporous titanium dioxide (M-TiO2) was assembled through the electrostatic attraction between EPL and M-TiO2. Through modification, the M-TiO2 surface tends to form multilayered and complex architectures, which can be used as artificial matrices to change the microenvironment of carriers for enzyme immobilization. The modified M-TiO2 was characterized through scanning electron microscopy, Fourier transform infrared spectroscopy, zeta potential analysis, and thermogravimetric analysis. All of the immobilized enzymes with negative charges display strong storage stability, thermal stability, and good reusability. Results indicate that EPL can self-assemble onto the surface of M-TiO2 and form a considerable number of active coatings. Our results also demonstrate that this simple and novel method can be potentially used to immobilize negatively charged enzymes for biosensor applications.

An innovative method for immobilizing sucrose isomerase on ε-poly-L-lysine modified mesoporous TiO2.

Sucrose isomerase (SIase) is the key enzyme in the enzymatic synthesis of isomaltulose. Mesoporous titanium dioxide (M-TiO2) and ε-poly-L-lysine-functionalized M-TiO2 (EPL-M-TiO2) were prepared as carriers for immobilizing SIase. SIase was effectively immobilized on EPL-M-TiO2 (SI-EPL-M-TiO2) with an enzyme activity of 39.41 U/g, and the enzymatic activity recovery rate up to 93.26%. The optimal pH and temperature of immobilized SIase were 6.0 and 30° C, respectively. SI-EPL-M-TiO2 was more stable in pH and thermal tests than SIase immobilized on M-TiO2 and free SIase. K(m) of SI-EPL-M-TiO2 was 204.92 mmol/L, and vmax was 45.7 µmol/L/s. Batch catalysis reaction of sucrose by SI-EPL-M-TiO2 was performed under the optimal conditions. The half-life period of SI-EPL-M-TiO2 under continuous reaction was 114 h, and the conversion rate of sucrose after 16 batches consistently remained at around 95%, which indicates that SI-EPL-M-TiO2 has good operational stability. Thus, SI-EPL-M-TiO2 can be used as a biocatalyst in food industries.

Efficient production of D-tagatose using a food-grade surface display system.

D-tagatose, a functional sweetener, is commonly transformed from D-galactose by L-arabinose isomerase (L-AI). In this study, a novel type of biocatalyst, L-AI from Lactobacillus fermentum CGMCC2921 displayed on the spore surface of Bacillus subtilis 168, was developed for producing D-tagatose. The anchored L-AI, exhibiting the relatively high bioactivity, suggested that the surface display system using CotX as the anchoring protein was successfully constructed. The stability of the anchored L-AI was significantly improved. Specifically, the consolidation of thermal stability representing 87% of relative activity was retained even at 80 °C for 30 min, which remarkably favored the production of D-tagatose. Under the optimal conditions, the robust spores can convert 75% D-galactose (100 g/L) into D-tagatose after 24 h, and the conversion rate remained at 56% at the third cycle. Therefore, this biocatalysis system, which could express the target enzyme on the food-grade vector, was an alternative method for the value-added production of D-tagatose.