Morphological Engineering of Filamentous Fungi: Research Progress and Perspectives.

Filamentous fungi are important cell factories for the production of high-value enzymes and chemicals for the food, chemical, and pharmaceutical industries. Under submerged fermentation, filamentous fungi exhibit diverse fungal morphologies that are influenced by environmental factors, which in turn affect the rheological properties and mass transfer of the fermentation system, and ultimately the synthesis of products. In this review, we first summarize the mechanisms of mycelial morphogenesis and then provide an overview of current developments in methods and strategies for morphological regulation, including physicochemical and metabolic engineering approaches. We also anticipate that rapid developments in synthetic biology and genetic manipulation tools will accelerate morphological engineering in the future.

Signal peptide replacement of Ag43 enables an efficient bacterial cell surface display of receptor-binding domain of coronavirus.

To enable an efficient bacterial cell surface display with effective protein expression and cell surface loading ability via autotransporter for potential vaccine development applications, the inner membrane protein translocation efficiency was investigated via a trial-and-error strategy by replacing the original unusual long signal peptide of E. coli Ag43 with 11 different signal peptides. The receptor-binding domain (RBD) of coronavirus was used as a neutral display substrate to optimize the expression conditions, and the results showed that signal peptides from PelB, OmpC, OmpF, and PhoA protein enhance the bacterial cell surface display efficiency of RBD. In addition, the temperature has also a significant effect on the autodisplay efficiency of RBD. Our data provide further technical basis for the biotechnological application of Ag43 as a bacterial surface display carrier system and further potential application in vaccine development.

Geographical differentiation of garlic based on HS-GC-IMS combined with multivariate statistical analysis.

Garlic is famous for its unique flavor and health benefits. An effective means of authenticating garlic's origin is through the implementation of the Protected Geographical Indication (PGI) scheme. However, the prevalence of fraudulent behavior raises concerns regarding the reliability of this system. In this study, garlic samples from six distinct production areas (G1: Cangshan garlic, G2: Qixian garlic, G3: Dali single clove garlic, G4: Jinxiang garlic, G5: Yongnian garlic, and G6: Badong garlic) underwent analysis using HS-GC-IMS. A total of 26 VOCs were detected in the samples. The differences in VOCs among the different garlic samples were visually presented in a two-dimensional topographic map and fingerprint map. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were employed to demonstrate the capacity of the HS-GC-IMS method for effectively distinguishing garlic samples from different geographical sources. Further screening based on the p-value and VIP score threshold identified 12 different aroma substances, which can be utilized for the identification of garlic from different producing areas. The fusion of HS-GC-IMS with multivariate statistical analysis proved to be a rapid, intuitive, and efficient approach for identifying and categorizing garlic VOCs, offering a novel strategy for ascertaining garlic origin and ensuring quality control.

[Control of acetic acid metabolism of recombinant Yarrowia lipolytica for efficient succinic acid production].

Succinic acid is a high value-added organic acid widely used in food, chemical and pesticide industries. As a new robust non-conventional yeast, Yarrowia lipolytica attracts more and more attention due to its potential for industrial applications. Previously, we obtained a succinic acid-producing strain through gene deletion of succinic acid dehydrogenase subunit encoding gene Ylsdh5, resulting in the strain of PGC01003. However, the recombinant strain produced large amount of acetic acid due to imbalance between glycolysis and TCA cycle which hindered the efficient production of succinic acid. PDH bypass was interfered to decrease the overflow of acetic acid and produce succinic acid under natural pH. Acetic acid was reduced to 4.6 g/L through heterologous expression of acetyl coenzyme A synthase from Salmonella enteric, which was 75.4% of the control strain. Deletion of CoA-transferase gene Ylach1 eliminated acetate formation and improved succinic acid production, and the resulting strain produced as high as 7.0 g/L succinic acid. Our study provides foundation for further construction of efficient cell factory of succinic acid production.

Engineering of unconventional yeast Yarrowia lipolytica for efficient succinic acid production from glycerol at low pH.

Yarrowia lipolytica is considered as a potential candidate for succinic acid production because of its innate ability to accumulate citric acid cycle intermediates and its tolerance to acidic pH. Previously, a succinate-production strain was obtained through the deletion of succinate dehydrogenase subunit encoding gene Ylsdh5. However, the accumulation of by-product acetate limited further improvement of succinate production. Meanwhile, additional pH adjustment procedure increased the downstream cost in industrial application. In this study, we identified for the first time that acetic acid overflow is caused by CoA-transfer reaction from acetyl-CoA to succinate in mitochondria rather than pyruvate decarboxylation reaction in SDH negative Y. lipolytica. The deletion of CoA-transferase gene Ylach eliminated acetic acid formation and improved succinic acid production and the cell growth. We then analyzed the effect of overexpressing the key enzymes of oxidative TCA, reductive carboxylation and glyoxylate bypass on succinic acid yield and by-products formation. The best strain with phosphoenolpyruvate carboxykinase (ScPCK) from Saccharomyces cerevisiae and endogenous succinyl-CoA synthase beta subunit (YlSCS2) overexpression improved succinic acid titer by 4.3-fold. In fed-batch fermentation, this strain produced 110.7g/L succinic acid with a yield of 0.53g/g glycerol without pH control. This is the highest succinic acid titer achieved at low pH by yeast reported worldwide, to date, using defined media. This study not only revealed the mechanism of acetic acid overflow in SDH negative Y. lipolytica, but it also reported the development of an efficient succinic acid production strain with great industrial prospects.

Robust succinic acid production from crude glycerol using engineered Yarrowia lipolytica.

BACKGROUND: Integrating waste management with fuels and chemical production is considered to address the food waste problem and oil crisis. Approximately, 600 million tonnes crude glycerol is produced from the biodiesel industry annually, which is a top renewable feedstock for succinic acid production. To meet the increasing demand for succinic acid production, the development of more efficient and cost-effective production methods is urgently needed. Herein, we have proposed a new strategy for integration of both biodiesel and SA production in a biorefinery unit by construction of an aerobic yeast Yarrowia lipolytica with a deletion in the gene coding succinate dehydrogenase subunit 5. RESULTS: Robust succinic acid production by an engineered yeast Y. lipolytica from crude glycerol without pre-treatment was demonstrated. Diversion of metabolic flow from tricarboxylic acid cycle led to the success in generating a succinic acid producer Y. lipolytica PGC01003. The fermentation media and conditions were optimized, which resulted in 43 g L(-1) succinic acid production from crude glycerol. Using the fed-batch strategy in 2.5 L fermenter, up to 160 g L(-1) SA was yielded, indicating the great industrial potential. CONCLUSIONS: Inactivation of SDH5 in Y. lipolytica Po1f led to succinic acid accumulation and secretion significantly. To our best knowledge, this is the highest titer obtained in fermentation on succinic acid production. In addition, the performance of batch and fed-batch fermentation showed high tolerance and yield on biodiesel by-product crude glycerol. All these results indicated that PGC01003 is a promising microbial factorial cell for the highly efficient strategy solving the environmental problem in connection with the production of value-added product.

Secretory production of antimicrobial peptides in Escherichia coli using the catalytic domain of a cellulase as fusion partner.

Antimicrobial peptides (AMPs) are small molecules which serve as essential components of the innate immune system in various organisms. AMPs possess a broad spectrum of antimicrobial activities. However, the scaled production of such peptides in Escherichia coli faces many difficulties because of their small size and toxicity to the host. Here, we described a new fusion strategy to extracellularly produce significant amounts of these antimicrobial peptides in recombinant E. coli at significant amount. Employing the catalytic domain of a cellulase (Cel-CD) from Bacillus subtilis KSM-64 as the fusion partner, five recombinant antimicrobial peptides were confirmed to accumulate in the culture medium at concentrations ranging from 184 mg/L to 297 mg/L. The radical diffusion experiment demonstrated that the released model antimicrobial peptide, bombinin, had antibacterial activities against both E. coli and Staphylococcus aureus. This strategy will be suitable for the production of antimicrobial peptides and other toxicity proteins.

Exploring medium-chain-length polyhydroxyalkanoates production in the engineered yeast Yarrowia lipolytica.

Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are a large class of biopolymers that have attracted extensive attention as renewable and biodegradable bio-plastics. They are naturally synthesized via fatty acid de novo biosynthesis pathway or ß-oxidation pathway from Pseudomonads. The unconventional yeast Yarrowia lipolytica has excellent lipid/fatty acid catabolism and anabolism capacity depending of the mode of culture. Nevertheless, it cannot naturally synthesize PHA, as it does not express an intrinsic PHA synthase. Here, we constructed a genetically modified strain of Y. lipolytica by heterologously expressing PhaC1 gene from P. aeruginosa PAO1 with a PTS1 peroxisomal signal. When in single copy, the codon optimized PhaC1 allowed the synthesis of 0.205 % DCW of PHA after 72 h cultivation in YNBD medium containing 0.1 % oleic acid. By using a multi-copy integration strategy, PHA content increased to 2.84 % DCW when the concentration of oleic acid in YNBD was 1.0 %. Furthermore, when the recombinant yeast was grown in the medium containing triolein, PHA accumulated up to 5.0 % DCW with as high as 21.9 g/L DCW, which represented 1.11 g/L in the culture. Our results demonstrated the potential use of Y. lipolytica as a promising microbial cell factory for PHA production using food waste, which contains lipids and other essential nutrients.

Mixed food waste as renewable feedstock in succinic acid fermentation.

Mixed food waste, which was directly collected from restaurants without pretreatments, was used as a valuable feedstock in succinic acid (SA) fermentation in the present study. Commercial enzymes and crude enzymes produced from Aspergillus awamori and Aspergillus oryzae were separately used in hydrolysis of food waste, and their resultant hydrolysates were evaluated. For hydrolysis using the fungal mixture comprising A. awamori and A. oryzae, a nutrient-complete food waste hydrolysate was generated, which contained 31.9 g L(-1) glucose and 280 mg L(-1) free amino nitrogen. Approximately 80-90 % of the solid food waste was also diminished. In a 2.5 L fermentor, 29.9 g L(-1) SA was produced with an overall yield of 0.224 g g(-1) substrate using food waste hydrolysate and recombinant Escherichia coli. This is comparable to many similar studies using various wastes or by-products as substrates. Results of this study demonstrated the enormous potential of food waste as renewable resource in the production of bio-based chemicals and materials via microbial bioconversion.

The effect of cyclodextrins on the ethanol tolerance of microorganisms suggests potential application.

Cyclodextrins (CDs) are used in food, pharmaceutical, and chemical industries, as well as agriculture and environmental engineering. Cyclodextrin glucanotransferase (CGTase) is an important industrial extracellular enzyme which is used to produce CDs and oligosaccharides. We previously developed a novel yeast-surface CGTase expression system which was used for the production of CDs from starch. In the present study, we showed that the presence of CDs may increase the ethanol tolerance of microorganisms. The cell numbers of Saccharomyces cerevisiae and Escherichia coli in the presence of ß-cyclodextrin and ethanol were 1,000-fold and 10-fold higher than that without CDs. The yeast strain with the immobilized CGTase produced 13 g CDs/l and 1.8 g ethanol/l when it was incubated in yeast medium supplemented with 4% starch. The effect of CDs on microorganisms suggests a potential application for the co-production of CDs and ethanol.

A novel strategy for succinate and polyhydroxybutyrate co-production in Escherichia coli.

Based on the metabolic analysis, a succinate and polyhydroxybutyrate (PHB) co-production pathway was designed and engineered in Escherichia coli MG1655. Batch cultivation of the engineered E. coli revealed that it was able to accumulate both extracellular succinate and intracellular PHB simultaneously. PHB accumulation not only improved succinate production, but also reduced pyruvate and acetate secretion. With a consumption of 45 g l(-1) glucose, E. coli QZ1112 was shown to accumulate 24.6 g l(-1) succinate and 4.95 g l(-1) PHB in batch fermentation. The PHB content reached 41.3 wt.% of its cell dry weight, which suggested that the cell debris can be used as value added by-product.

Global transcription engineering of brewer's yeast enhances the fermentation performance under high-gravity conditions.

Global transcription engineering was developed as a tool to reprogram gene transcription for eliciting new phenotypes important for technological applications (Science 2006, 314(5805):1565-1568). A recent report indicated that the beneficial growth advantage of yeast cells expressing the SPT15-300 mutation is the result of enhanced uptake and/or improved utilization of leucine and thus was seen only on defined media with low concentrations of leucine (Appl Environ Microbiol 2009, 75(19):6055-6061). Further investigation towards a leucine-prototrophic strain of industrial lager brewer's yeast indicated that integration one copy of SPT15-300 in SPT15 allele, however, did lead to an increased ethanol tolerance on complex rich medium at high gravity fermentation condition. Under brewing conditions, the SPT15-300 mutant produced 80.78 g/L ethanol from 200 g/L carbohydrates after 384 h, almost twice as much as that of the wild-type strain. The results convinced us that the effect of global regulator modification of yeast is at multi-genes level and is extremely complicated.

Optimization of an ethanol production medium in very high gravity fermentation.

Concentrations of Mg(2+), glycine, yeast extract, biotin, acetaldehyde and peptone were optimized by a uniform design process for ethanol production by Saccharomyces cerevisiae. Using non-linear step-wise regression analysis, a predictive mathematical model was established. Concentrations of Mg(2+) and peptone were identified as the critical factors: 50 mM Mg(2+) and 1.5% (w/v) peptone in the medium increased the final ethanol titre from 14.2% (v/v) to 17% (v/v) in 48 h.