Unveiling the mechanism of efficient ß-phenylethyl alcohol conversion in wild-type Saccharomyces cerevisiae WY319 through multi-omics analysis.

ß-Phenylethanol (2-PE), as an important flavor component in wine, is widely used in the fields of flavor chemistry and food health. 2-PE can be sustainably produced through Saccharomyces cerevisiae. Although significant progress has been made in obtaining high-yield strains, as well as improving the synthesis pathways of 2-PE, there still lies a gap between these two fields to unpin. In this study, the macroscopic metabolic characteristics of high-yield and low-yield 2-PE strains were systematically compared and analyzed. The results indicated that the production potential of the high-yield strain might be contributed to the enhancement of respiratory metabolism and the high tolerance to 2-PE. Furthermore, this hypothesis was confirmed through comparative genomics. Meanwhile, transcriptome analysis at key specific growth rates revealed that the collective upregulation of mitochondrial functional gene clusters plays a more prominent role in the production process of 2-PE. Finally, findings from untargeted metabolomics suggested that by enhancing respiratory metabolism and reducing the Crabtree effect, the accumulation of metabolites resisting high 2-PE stress was observed, such as intracellular amino acids and purines. Hence, this strategy provided a richer supply of precursors and cofactors, effectively promoting the synthesis of 2-PE. In short, this study provides a bridge for studying the metabolic mechanism of high-yield 2-PE strains with the subsequent targeted strengthening of relevant synthetic pathways. It also provides insights for the synthesis of nonalcoholic products in S. cerevisiae.

Promoter screening and identification for metabolic regulation in Acremonium chrysogenum.

Acremonium chrysogenum is the major industrial producer of cephalosporin C (CPC), which is used as raw material for the production of significant cephalosporin antibiotics. Due to the lack of diverse promoter elements, the development of metabolic engineering transformation is relatively slow, resulting in a limited improvement on CPC production. In this study, based on the analysis of the transcriptome profile, 27 candidate promoters were selected to drive the expression of the reporter genes. The promoter activities of this library ranged from 0.0075 to 101 times of the control promoter PAngpdA . Simultaneously, a rapid screening method for potential bidirectional promoters was developed and 4 strong bidirectional promoters from 27 candidate options were identified and validated. Finally, the Golden Gate method was employed to combine promoter modules from the library with various target genes. Through a mixed transformation and screening process, high-yielding strains AG-6, AG-18, and AG-41 were identified, exhibiting an increase in CPC production of 30%, 35%, and 29%, respectively, compared to the control strain Ac-∆axl2:: eGFP. Therefore, the utilization of this promoter library offers a broader range of synthetic biology toolkits for the genetic engineering transformation of A. chrysogenum, thus establishing a solid foundation for the precise regulation of gene expression.

An inducible CRISPRi circuit for tunable dynamic regulation of gene expression in Saccharopolyspora erythraea.

Actinomyces are gram-positive bacteria known for their valuable secondary metabolites. Redirecting metabolic flux towards desired products in actinomycetes requires precise and dynamic regulation of gene expression. In this study, we integrated the CRISPR interference (CRISPRi) system with a cumate-inducible promoter to develop an inducible gene downregulation method in Saccharopolyspora erythraea, a prominent erythromycin-producing actinobacterium. The functionality of the cumate-inducible promoter was validated using the gusA gene as a reporter, and the successful inducible expression of the dCas9 gene was confirmed. The developed inducible CRISPRi strategy was then employed to downregulate the expression of target genes rppA in the wild-type strain NRRL2338 and sucC in the high erythromycin-producing strain E3. Through dynamic control of sucC expression, a significant enhancement in erythromycin production was achieved in strain E3. This study demonstrated the effectiveness of an inducible gene downregulation approach using CRISPRi and a cumate-inducible promoter, providing valuable insights for optimizing natural product production in actinomyces.

Enhancing and monitoring spore production in Clostridium butyricum using pH-based regulation strategy and a robust soft sensor based on back-propagation neural networks.

Clostridium butyricum is a probiotic that forms anaerobic spores and plays a crucial role in regulating gut microbiota. However, the total viable cell count and spore yield of C. butyricum in industrial production are comparatively low. To this end, we investigated the metabolic characteristics of the strain and proposed three distinct pH regulation strategies for enhancing spore production. In addition, precise measurement of fermentation parameters such as substrate concentration, total viable cell count, and spore concentration is crucial for successful industrial probiotics production. Nevertheless, online measurement of these intricate parameters in the fermentation of C. butyricum poses a considerable challenge owing to the complex, nonlinear, multivariate, and strongly coupled characteristics of the production process. Therefore, we analyzed the capacitance and conductivity acquired from a viable cell sensor as the core parameters for the fermentation process. Subsequently, a robust soft sensor was developed using a seven-input back-propagation neural network model with input variables of fermentation time, capacitance, conductivity, pH, initial total sugar concentration, ammonium ion concentration, and calcium ion concentration. The model enables the online monitoring of total viable biomass count, substrate concentrations, and spore yield, and can be extended to similar fermentation processes with pH changes as a characteristic feature.

Precise fermentation coupling with simultaneous separation strategy enables highly efficient and economical sophorolipids production.

Sophorolipids (SLs) represent highly promising biosurfactants. However, its widespread production and application encounter obstacles due to the significant costs involved. Here, an intelligent and precise regulation strategy was elucidated for the fermentation process coupled with in-situ separation production mode, to achieve cost-effective SLs production. Firstly, a mechanism-assisted data-driven model was constructed for "on-demand feeding of cells". Moreover, a strategy of step-wise oxygen supply regulation based on the demand for cell metabolic capacity was developed, which accomplished "on-demand oxygen supply of cells", to optimize the control of energy consumption. Finally, a systematic approach was implemented by integrating a semi-continuous fermentation mode with in-situ separation technology for SLs production. This strategy enhanced SLs productivity and yield, reaching 2.30 g/L/h and 0.57 g/g, respectively. These values represented a 40.2% and 18.7% increase compared to fed-batch fermentation. Moreover, the concentration of crude SLs after separation reached 793.12 g/L, facilitating downstream separation and purification processes.

Optimization of the Fermentative Production of Rhizomucor miehei Lipase in Aspergillus oryzae by Controlling Morphology.

Morphology plays an important role in the fermentation bioprocess of filamentous fungi. In this study, we investigated the controlling strategies of morphology that improved the efficiency of Rhizomucor miehei lipase (RML) production using a high-yield Aspergillus oryzae. First, the inoculated spore concentrations were optimized in seed culture, and the RML activity increased by 43.4% with the well-controlled mycelium pellets in both ideal sizes and concentrations. Then, the initial nitrogen source and agitation strategies were optimized to regulate the morphology of Aspergillus oryzae in a 5 L bioreactor, and the established stable fermentation system increased the RML activity to 232.0 U/mL, combined with an increase in total RML activity from 98,080 U to 487,179 U. Furthermore, the optimized fermentation strategy was verified by a high-yield Aspergillus oryzae and achieved an additional improvement of RML activity, up to 320.0 U/mL. Moreover, this optimized fermentation bioprocess was successfully scaled up to a 50 L bioreactor, and the RML activity reached 550.0 U/mL. This work has established a stable precision fermentation bioprocess for RML production by A. oryzae in bioreactors, and the controlling strategy developed in this study could potentially be extended to an industrial scale for RML production with high efficiency.

Cephalosporin C biosynthesis and fermentation in Acremonium chrysogenum.

Cephalosporins are currently the most widely used antibiotics in clinical practice. The main strain used for the industrial production cephalosporin C (CPC) is Acremonium chrysogenum. CPC has the advantages of possessing a broad antibacterial spectrum and strong antibacterial activity. However, the yield and titer of cephalosporins obtained from A. chrysogenum are much lower than penicillin, which is also a ß-lactam antibiotic produced by Penicillium chrysogenum. Molecular biology research into A. chrysogenum has focused on gene editing technologies, multi-omics research which has provided information on the differences between high- and low-yield strains, and metabolic engineering involving different functional genetic modifications and hierarchical network regulation to understand strain characteristics. Furthermore, optimization of the fermentation process is also reviewed as it provides the optimal environment to realize the full potential of strains. Combining rational design to control the metabolic network, high-throughput screening to improve the efficiency of obtaining high-performance strains, and real-time detection and controlling in the fermentation process will become the focus of future research in A. chrysogenum. This minireview provides a holistic and in-depth analysis of high-yield mechanisms and improves our understanding of the industrial value of A. chrysogenum. KEY POINTS: • Review of the advances in A. chrysogenum characteristics improvement and process optimization • Elucidate the molecular bases of the mechanisms that control cephalosporin C biosynthesis and gene expression in A. chrysogenum • The future development trend of A. chrysogenum to meet industrial needs.

An Efficient High-Throughput Screening of High Gentamicin-Producing Mutants Based on Titer Determination Using an Integrated Computer-Aided Vision Technology and Machine Learning.

The "design-build-test-learn" (DBTL) cycle has been adopted in rational high-throughput screening to obtain high-yield industrial strains. However, the mismatch between build and test slows the DBTL cycle due to the lack of high-throughput analytical technologies. In this study, a highly efficient, accurate, and noninvasive detection method of gentamicin (GM) was developed, which can provide timely feedback for the high-throughput screening of high-yield strains. First, a self-made tool was established to obtain data sets in 24-well plates based on the color of the cells. Subsequently, the random forest (RF) algorithm was found to have the highest prediction accuracy with an R2 value of 0.98430 for the same batch. Finally, a stable genetically high-yield strain (998 U/mL) was successfully screened out from 3005 mutants, which was verified to improve the titer by 72.7% in a 5 L bioreactor. Moreover, the verified new data sets were updated on the model database in order to improve the learning ability of the DBTL cycle.

Engineering the methyltransferase through inactivation of the genK and genL leads to a significant increase of gentamicin C1a production in an industrial strain of Micromonospora echinospora 49-92S.

In this study, a single-component high-yielding Micromonospora echinospora strain 49-92S-KL01 was constructed by deleting methyltransferase-encoding genes genK and genL. In 5-L fermentation trials, gentamicin C1a titers in the mutant strain were 3.22-fold higher than that in the parental strain (211 U/mL vs. 50 U/mL). The glycolysis pathway and tricarboxylic acid cycle fluxes were reduced by 26.8% and 26.6%, respectively, compared to the parental strain according to the metabolic flux analysis during the stationary phase, resulting in lower levels of energy supplements required for the cellular maintenance. Meanwhile, a significant enhancement in precursor (paromamine) accumulation and availability was observed in 49-92S-KL01 compared to parental strain. These results indicate that genK and genL significantly affect the synthesis of gentamicin C1a. In addition, this study provides a more rational strategy for gentamicin C1a production.

Engineering of succinyl-CoA metabolism in view of succinylation regulation to improve the erythromycin production.

As a novel protein post-translational modification (PTM), lysine succinylation is widely involved in metabolism regulation by altering the activity of catalytic enzymes. Inactivating succinyl-CoA synthetase in Saccharopolyspora erythraea HL3168 E3 was proved significantly inducing the global protein hypersuccinylation. To investigate the effects, succinylome of the mutant strain E3ΔsucC was identified by using a high-resolution mass spectrometry-based proteomics approach. PTMomics analyses suggested the important roles of succinylation on protein biosynthesis, carbon metabolism, and antibiotics biosynthesis in S. erythraea. Enzymatic experiments in vivo and in vitro were further conducted to determine the succinylation regulation in the TCA cycle. We found out that the activity of aconitase (SACE_3811) was significantly inhibited by succinylation in E3ΔsucC, which probably led to the extracellular accumulation of pyruvate and citrate during the fermentation. Enzyme structural analyses indicated that the succinylation of K278 and K373, conservative lysine residues locating around the protein binding pocket, possibly affects the activity of aconitase. To alleviate the metabolism changes caused by succinyl-CoA synthetase inactivation and protein hypersuccinylation, CRISPR interference (CRISPRi) was applied to mildly downregulate the transcription level of gene sucC in E3. The erythromycin titer of the CRISPRi mutant E3-sucC-sg1 was increased by 54.7% compared with E3, which was 1200.5 mg/L. Taken together, this work not only expands our knowledge of succinylation regulation in the TCA cycle, but also validates that CRISPRi is an efficient strategy on the metabolic engineering of S. erythraea. KEY POINTS: • We reported the first systematic profiling of the S. erythraea succinylome. • We found that the succinylation regulation on the activity of aconitase. • We enhanced the production of erythromycin by using CRISPRi to regulate the transcription of gene sucC.

Knockout and functional analysis of BSSS-related genes in Acremonium chrysogenum by novel episomal expression vector containing Cas9 and AMA1.

OBJECTIVE: The target sorB gene, related to sorbicillinoid production, and the free expression element, AMA1, were used to verify the methodological approach in Acremonium chrysogenum. RESULT: CRISPR-Cas9 episomal expression system was used to introduce a point mutation into the sorB gene and the addition of sorB donor DNA achieved complete knockout of target genes. Four BSSS (yeast bud site selection system)-related genes, axl1, axl2, bud3, and bud4 were knocked out without impact on yield, dry weight, or pH. Relationships between morphology and stress tolerance in knockout strains were analyzed. CONCLUSION: The gene-editing system used in the current study exceeded 80% efficiency and arthrospores development was found to differ from that in wild-type strain.

The arthrospore-related gene Acaxl2 is involved in cephalosporin C production in industrial Acremonium chrysogenum by the regulatory factors AcFKH1 and CPCR1.

Cephalosporin C (CPC) production is often accompanied by a typical morphological differentiation of Acremonium chrysogenum, involving the fragmentation of its hyphae into arthrospores. The type I integral plasma membrane protein Axl2 is a central component of the bud site selection system (BSSS), which was identified as the regulatory factor involved in the hyphal septation process and arthrospore formation. Using CRISPR/Cas9 technology and homologous recombination (HR), we inserted an egfp donor DNA sequence into the Acaxl2 locus, causing the generation of the deletion strain Ac-ΔAcaxl2:eGFP from Acremonium chrysogenum FC3-5-23, the industrial producer of CPC. The mycelial morphology of the deletion strain Ac-ΔAcaxl2:eGFP was mainly composed of arthrospores with a characteristic diameter of 2-8 µm, which increased from 75% at 48 h to 90% at 72 h post culture and were maintained until the end of the fermentation process. However, the deletion strain showed accelerated production of CPC, and the final titer was 5573 µg/ml, which was nearly three times higher than that of the control strain FC3-5-23. The up-regulation of genes related to the biosynthesis gene cluster in Ac-ΔAcaxl2:eGFP, especially the "late" genes, was one reason why its CPC production was higher than that of the original strain. Furthermore, compared with FC3-5-23, the more significant increase of genes involved in the BSSS (Acbud3 and Acbud4) in Ac-ΔAcaxl2:eGFP in the late stage of fermentation, may be responsible for this increase in arthrospore formation. Similarily, the transcription of the regulatory factors AcFKH1 and CPCR1 were also markedly increased at this time and may be the factors responsible for the regulation of CPC synthesis. These results indicated that Acaxl2 plays an important role in both arthrospore formation and CPC production, strongly implicating these regulatory factors as having pivotal links between mycelial morphology and secondary metabolite production in high-yielding A. chrysogenum. To the opposite, the axl2 gene knockout of wild strain CGMCC 3.3795 did not significantly influence the CPC production, which reflected the complexity of the secondary metabolic process and the differences in the function of axl2 gene in high- and low-yielding strains.

Ultrasound assisted in situ separation of sophorolipids in multi-phase fermentation system to achieve efficient production by Candida bombicola.

BACKGROUND: Sophorolipids (SLs) are regarded as one of the most promising biosurfactants. However, high production costs are the main obstacle to extended SLs application. Semi-continuous fermentation, which is based on in situ separation, is a promising technology for achieving high SLs productivity. METHODS AND RESULTS: In this study, the sedimentation mechanism of SLs was analyzed. The formation of a hydrophobic mixture of SLs and rapeseed oil was a key factor in sedimentation. And the hydrophobicity and density of the mixture determined SLs sedimentation rate. On this basis, ultrasonic enhanced sedimentation technology (UEST) was introduced, by which the sedimentation rates were increased by 46.9%-485.4% with different ratio of rapeseed oil to SLs. UEST-assisted real-time in situ separation and semi-continuous fermentation were performed. SLs productivity and yield were 2.15 g L-1  h-1 and 0.58 g g-1 , respectively, simultaneously the loss ratio of cells, glucose, and rapeseed oil were significantly reduced. CONCLUSIONS: This study provides a new horizon for optimization of the SLs fermentation process.

Advances in sustainable approaches utilizing orange peel waste to produce highly value-added bioproducts.

Orange peel waste (OPW), a discarded part of orange fruit, is a rich source of essential constituents that can be transformed into highly value-added bioproducts. OPW is being generated in million tonnes globally and returns to the environment without complete benefit. Thus, a high volume of annually produced OPW in the industry requires effective valorization. In this regard, limited data is available that summarizes the broader spectrum for the sustainable fate of OPW to produce value-added bioproducts. The main objective of this treatise is to explore the sustainable production of bioproducts from OPW. Therefore, this review covers all the aspects of OPW, from its production to complete valorization. The review encompasses the extraction technologies employed for extracting different valuable bioactive compounds, such as: essential oil (EO), pectin, and carotenoids, from OPW. Furthermore, the suitability of bioconversion technologies (digestion/fermentation) in transforming OPW to other useful bioproducts, such as: biochemicals (lactic acid and succinic acid), biopolysaccharides (xanthan and curdlan gum), and bioenergy (biomethane and bioethanol) is discussed. Also, it includes the concept of OPW-based biorefineries and their development that shall play a definite role in future to cover demands for: food, chemicals, materials, fuels, power, and heat. Lastly, this review focuses on OPW-supplemented functional food products such as: beverages, yogurts, and extruded products. In conclusion, insights provided in this review maximize the potential of OPW for commercial purposes, leading to a safe, and waste-free environment.

Kinetically modelled approach of xanthan production using different carbon sources: A study on molecular weight and rheological properties of xanthan.

The present study emphasizes improving the overall yield, productivity and quality of xanthan by Xanthomonas campestris using different carbon sources via optimizing the fermentation media and kinetic modelling work. After optimization, six carbon sources and one nitrogen source were selected for xanthan production in 5 L bioreactor. Kinetic modelling was applied to assess the experimental fermentation data and to check its influence on scale-up production. In this work, xanthan production reached 40.65 g/L with a growth-associated rate constant (α) of 2.831, and highest specific growth rate (µm) of 0.37/h while using maltose as the sole carbon source. Furthermore, rheological properties were determined, and Herschel-Bulkley model was employed to assess the experimental data. Interestingly, xanthan obtained from sucrose and glucose showed the highest yield stress (τ0) of 12.50 ± 0.31 and 7.17 ± 0.21. Moreover, the highest xanthan molecular weight of 3.53 × 107 and 3.25 × 107 g/mol were also found with sucrose and glucose. At last, the proposed mechanism of sugar metabolism and xanthan biosynthesis pathway were described. Conclusively, maltose appeared as the best carbon source for maximum xanthan production: while sucrose and glucose gave qualitatively best results. In short, this systematically modelled approach maximizes the potential output and provides a solid base for continuous cultivation of xanthan at large-scale production.

System optimization of an embedding protocol to immobilize cells from Candida bombicola to improve the efficiency of sophorolipids production.

This study introduces the implication of immobilization technology in the fermentation process of sophorolipids (SLs) production by Candida bombicola. Firstly, an evaluation system was established for the performance of embedding immobilization and subsequently applied to guide the optimization of operating conditions for sodium alginate immobilization. Correspondingly, the SLs titer increased from 11.4 g/L to 14.6 g/L. Secondly, polyvinyl alcohol was introduced for composite embedding to improve the stability of immobilized beads. Then exogenous addition of 1.5% diatomite further enhanced the fermentation performance of immobilized cells, thereby increasing the SLs titer to 35.9 g/L, which was 2.1 times higher than the original immobilized cells method. Finally, the immobilized cells were tested for three repeated batches of SLs fermentation. Compared to the free cells fermentation, the SLs productivity and substrate conversion rate were increased by 35.5% and 9.1%, respectively. The obtained results showed high potential for application on an industrial scale.

Development of a novel noninvasive quantitative method to monitor Siraitia grosvenorii cell growth and browning degree using an integrated computer-aided vision technology and machine learning.

The rapid, accurate and noninvasive detection of biomass and plant cell browning can provide timely feedback on cell growth in plant cell culture. In this study, Siraitia grosvenorii suspension cells were taken as an example, a phenotype analysis platform was successfully developed to predict the biomass and the degree of cell browning based on the color changes of cells in computer-aided vision technology. First, a self-made laboratory system was established to obtain images. Then, matrices were prepared from digital images by a self-developed high-throughput image processing tool. Finally, classification models were used to judge different cell types, and then a semi-supervised classification to predict different degrees of cell browning. Meanwhile, regression models were developed to predict the plant cell mass. All models were verified with a good agreement by biological experiments. Therefore, this method can be applied for low-cost biomass estimation and browning degree quantification in plant cell culture.

A P300 Brain-Computer Interface Paradigm Based on Electric and Vibration Simple Command Tactile Stimulation.

This paper proposed a novel tactile-stimuli P300 paradigm for Brain-Computer Interface (BCI), which potentially targeted at people with less learning ability or difficulty in maintaining attention. The new paradigm using only two types of stimuli was designed, and different targets were distinguished by frequency and spatial information. The classification algorithm was developed by introducing filters for frequency bands selection and conducting optimization with common spatial pattern (CSP) on the tactile evoked EEG signals. It features a combination of spatial and frequency information, with the spatial information distinguishing the sites of stimuli and frequency information identifying target stimuli and disturbances. We investigated both electrical stimuli and vibration stimuli, in which only one target site was stimulated in each block. The results demonstrated an average accuracy of 94.88% for electrical stimuli and 95.21% for vibration stimuli, respectively.

[New opportunities and challenges for hybrid data and model driven bioprocess optimization and scale-up].

Currently, biomanufacturing technology and industry are receiving worldwide attention. However, there are still great challenges on bioprocess optimization and scale-up, including: lacing the process detection methods, which makes it difficult to meet the requirement of monitoring of key indicators and parameters; poor understanding of cell metabolism, which arouses problems to rationally achieve process optimization and regulation; the reactor environment is very different across the scales, resulting in low efficiency of stepwise scale-up. Considering the above key issues that need to be resolved, here we summarize the key technological innovations of the whole chain of fermentation process, i.e., real-time detection-dynamic regulation-rational scale-up, through case analysis. In the future, bioprocess design will be guided by a full lifecycle in-silico model integrating cellular physiology (spatiotemporal multiscale metabolic models) and fluid dynamics (CFD models). This will promote computer-aided design and development, accelerate the realization of large-scale intelligent production and serve to open a new era of green biomanufacturing.

Metabolic engineering coupled with adaptive evolution strategies for the efficient production of high-quality L-lactic acid by Lactobacillus paracasei.

The main indicators for industrial production of high-quality lactic acid at elevated temperatures are high titer, productivity, yield, and optical purity. However, no such strains have been reported to meet all these requirements simultaneously. In this study, a high optical purity L-lactic acid producing strain is developed through the CRISPR-Cas9 gene editing platform. Further, adaptive evolution was used to breed and select a high-performance strain (NCBIO01-M2-ldhL1-HT) that could efficiently produce L-lactic acid at a high temperature of 45℃. This strain produced 221.0 g/L of L-lactic acid in open fermentation with high initial glucose concentration. Also, L-lactic acid productivity and yield was above 7.5 g/L/h and 0.96 g/g respectively, as well as the optical purity of L-lactic acid in the fermentation broth exceeded 99.1%. In short, this breeding strain possess high potential to be considered for the commercial production of polymer-grade L-lactic acid.