Combination of microbial and chemical synthesis for the sustainable production of ß-elemene, a promising plant-extracted anticancer compound.

Beta-elemene, a class of sesquiterpene derived from the Chinese medicinal herb Curcuma wenyujin, is widely used in clinical medicine due to its broad-spectrum antitumor activity. However, the unsustainable plant extraction prompted the search for environmentally friendly strategies for ß-elemene production. In this study, we designed a Yarrowia lipolytica cell factory that can continuously produce germacrene A, which is further converted into ß-elemene with 100% yield through a Cope rearrangement reaction by shifting the temperature to 250°C. First, the productivity of four plant-derived germacrene A synthases was evaluated. After that, the metabolic flux of the precursor to germacrene A was maximized by optimizing the endogenous mevalonate pathway, inhibiting the competing squalene pathway, and expressing germacrene A synthase gene in multiple copies. Finally, the most promising strain achieved the highest ß-elemene titer reported to date with 5.08 g/L. This sustainable and green method has the potential for industrial ß-elemene production.

A robust soft sensor based on artificial neural network for monitoring microbial lipid fermentation processes using Yarrowia lipolytica.

Microbial oils produced by Yarrowia lipolytica offer an environmentally friendly and sustainable alternative to petroleum as well as traditional lipids from animals and plants. The accurate measurement of fermentation parameters, including the substrate concentration, dry cell weight, and lipid accumulation, is the foundation of process control, which is indispensable for industrial lipid production. However, it remains a great challenge to measure the complex parameters online during the lipid fermentation process, which is nonlinear, multivariate, and characterized by strong coupling. As a type of AI technology, the artificial neural network model is a powerful tool for handling extremely complex problems, and it can be employed to develop a soft sensor to monitor the microbial lipid fermentation process of Y. lipolytica. In this study, we first analyzed and emphasized the volume of sodium hydroxide and dissolved oxygen concentration as central parameters of the fermentation process. Then, a soft sensor based on a four-input artificial neural network model was developed, in which the input variables were fermentation time, dissolved oxygen concentration, initial glucose concentration, and additional volume of sodium hydroxide. This provides the possibility of online monitoring of dry cell weight, glucose concentration, and lipid production with high accuracy, which can be extended to similar fermentation processes characterized by the addition of bases or acids, as well as changes of the dissolved oxygen concentration.

New Anti-Prelog Stereospecific Whole-Cell Biocatalyst for Asymmetric Reduction of Prochiral Ketones.

The biocatalytic asymmetric reduction of prochiral ketones for the production of enantiopure alcohols is highly desirable due to its inherent advantages over chemical methods. In this study, a new bacterial strain capable of transforming ketones to corresponding alcohols with high activity and excellent enantioselectivity was discovered in a soil sample. The strain was subsequently identified as Bacillus cereus TQ-2 based on its physiological characteristics and 16S rDNA sequence analysis. Under optimized reaction conditions, the resting cells of B. cereus TQ-2 converted acetophenone to enantioenriched (R)-1-phenylethanol with 99% enantiometric excess following anti-Prelog's rule, which is scarce in biocatalytic ketone reduction. The optimum temperature for the cells was 30 °C, and considerable catalytic activity was observed over a broad pH range from 5.0 to 9.0. The cells showed enhanced catalytic activity in the presence of 15% (v/v) glycerol as a co-substrate. The catalytic activity can also be substantially improved by adding Ca2+ or K+ ions. Moreover, the B. cereus TQ-2 cell was highly active in reducing several structurally diverse ketones and aldehydes to form corresponding alcohols with good to excellent conversion. Our study provides a versatile whole-cell biocatalyst that can be used in the asymmetric reduction of ketones for the production of chiral alcohol, thereby expanding the biocatalytic toolbox for potential practical applications.

High-yield α-humulene production in Yarrowia lipolytica from waste cooking oil based on transcriptome analysis and metabolic engineering.

BACKGROUND: α-Humulene is an important biologically active sesquiterpene, whose heterologous production in microorganisms is a promising alternative biotechnological process to plant extraction and chemical synthesis. In addition, the reduction of production expenses is also an extremely critical factor in the sustainable and industrial production of α-humulene. In order to meet the requirements of industrialization, finding renewable substitute feedstocks such as low cost or waste substrates for terpenoids production remains an area of active research. RESULTS: In this study, we investigated the feasibility of peroxisome-engineering strain to utilize waste cooking oil (WCO) for high production of α-humulene while reducing the cost. Subsequently, transcriptome analysis revealed differences in gene expression levels with different carbon sources. The results showed that single or combination regulations of target genes identified by transcriptome were effective to enhance the α-humulene titer. Finally, the engineered strain could produce 5.9 g/L α-humulene in a 5-L bioreactor. CONCLUSION: To the best of our knowledge, this is the first report that converted WCO to α-humulene in peroxisome-engineering strain. These findings provide valuable insights into the high-level production of α-humulene in Y. lipolytica and its utilization in WCO bioconversion.

Increasing the homologous recombination efficiency of eukaryotic microorganisms for enhanced genome engineering.

In recent years, eukaryotic microorganisms have been widely applied to offer many solutions for everyday life and have come to play important roles in agriculture, food, health care, and the fine-chemicals industry. However, the complex genetic background and low homologous recombination efficiency have hampered the implementation of large-scale and high-throughput gene editing in many eukaryotic microorganisms. The low efficiency of homologous recombination (HR) not only makes the modification process labor-intensive but also completely precludes the application of many otherwise very useful genome editing techniques. Thus, increasing the efficiency of HR is clearly an enabling technology for basic research and gene editing in eukaryotic microorganisms. In this review, we summarize the current strategies for enhancing the efficiency of HR in eukaryotic microorganisms (particularly yeasts and filamentous fungi), list some small molecules and candidate genes associated with homologous and non-homologous recombination, and briefly discuss the further development prospects of these strategies.

Advancing metabolic engineering of Yarrowia lipolytica using the CRISPR/Cas system.

The oleaginous yeast Yarrowia lipolytica is widely used for the production of both bulk and fine chemicals, including organic acids, fatty acid-derived biofuels and chemicals, polyunsaturated fatty acids, single-cell proteins, terpenoids, and other valuable products. Consequently, it is becoming increasingly popular for metabolic engineering applications. Multiple gene manipulation tools including URA blast, Cre/LoxP, and transcription activator-like effector nucleases (TALENs) have been developed for metabolic engineering in Y. lipolytica. However, the low efficiency and time-consuming procedures involved in these methods hamper further research. The emergence of the CRISPR/Cas system offers a potential solution for these problems due to its high efficiency, ease of operation, and time savings, which can significantly accelerate the genomic engineering of Y. lipolytica. In this review, we summarize the research progress on the development of CRISPR/Cas systems for Y. lipolytica, including Cas9 proteins and sgRNA expression strategies, as well as gene knock-out/knock-in and repression/activation applications. Finally, the most promising and tantalizing future prospects in this area are highlighted.

Metabolomics profiling reveals the mechanism of increased pneumocandin B0 production by comparing mutant and parent strains.

Metabolic profiling was used to discover mechanisms of increased pneumocandin B0 production in a high-yield strain by comparing it with its parent strain. Initially, 79 intracellular metabolites were identified, and the levels of 15 metabolites involved in six pathways were found to be directly correlated with pneumocandin B0 biosynthesis. Then by combining the analysis of key enzymes, acetyl-CoA and NADPH were identified as the main factors limiting pneumocandin B0 biosynthesis. Other metabolites, such as pyruvate, α-ketoglutaric acid, lactate, unsaturated fatty acids and previously unreported metabolite γ-aminobutyric acid were shown to play important roles in pneumocandin B0 biosynthesis and cell growth. Finally, the overall metabolic mechanism hypothesis was formulated and a rational feeding strategy was implemented that increased the pneumocandin B0 yield from 1821 to 2768 mg/L. These results provide practical and theoretical guidance for strain selection, medium optimization, and genetic engineering for pneumocandin B0 production.

CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art.

In recent years, a variety of genetic tools have been developed and applied to various filamentous fungi, which are widely applied in agriculture and the food industry. However, the low efficiency of gene targeting has for many years hampered studies on functional genomics in this important group of microorganisms. The emergence of CRISPR/Cas9 genome-editing technology has sparked a revolution in genetic research due to its high efficiency, versatility, and easy operation and opened the door for the discovery and exploitation of many new natural products. Although the application of the CRISPR/Cas9 system in filamentous fungi is still in its infancy compared to its common use in E. coli, yeasts, and mammals, the deep development of this system will certainly drive the exploitation of fungal diversity. In this review, we summarize the research progress on CRISPR/Cas9 systems in filamentous fungi and finally highlight further prospects in this area.

Effects of aeration on metabolic profiles of Mortierella alpina during the production of arachidonic acid.

To investigate the metabolic regulation against oxygen supply, comparative metabolomics was performed to explore the metabolic responses of Mortierella alpina in the process of arachidonic acid (ARA) production. More than 110 metabolites involved in Embden-Meyerhof-Parnas pathway, pentose phosphate pathway, tricarboxylic acid cycle, inositol phosphate metabolism, fatty acid biosynthesis, and amino acid metabolism were identified by gas chromatography-mass spectrometry. Samples at different aeration rates were clearly distinguished by principal components analysis and partial least squares analysis, indicating that oxygen supply had a profound effect on the metabolism of M. alpina. Eleven major metabolites were identified as potential biomarkers to be primarily responsible for the difference of metabolism. Further study of metabolic changes with the relevant pathways demonstrated that the levels of several intermediate metabolites in relation to central carbon metabolism changed remarkably via both processes and citrate and malate was supposed to play vital roles in polyunsaturated acid (PUFA) synthesis. Increase of myo-inositol and sorbitol were probably for osmo-regulation and redox balance, while enhanced phosphoric acid and pyroglutamic acid were supposed to have function in the activation of signal transduction pathway for stress resistance. The present study provides a novel insight into the metabolic responses of M. alpina to aeration rates and the metabolic characteristics during the ARA fermentation.

Constructing a synthetic constitutive metabolic pathway in Escherichia coli for (R, R)-2,3-butanediol production.

Many microorganisms could naturally produce (R, R)-2,3-butanediol ((R, R)-2,3-BD), which has unique applications due to its special chiral group and spatial configuration. But the low enantio-purity of the product hindered the development of large-scale production. In this work, a synthetic constitutive metabolic pathway for enantiomerically pure (R, R)-2,3-BD biosynthesis was constructed in Escherichia coli with vector pUC6S, which does not contain any lac sequences. The expression of this artificial constructed gene cluster was optimized by using two different strength of promoters (AlperPLTet01 (P01) and AlperBB (PBB)). The strength of P01 is twice stronger than PBB. The fermentation results suggested that the yield of (R, R)-2,3-BD was higher when using the stronger promoter. Compared with the wild type, the recombinant strain E. coli YJ2 produced a small amount of acetic acid and showed higher glucose consumption rate and higher cell density, which indicated a protection against acetic acid inhibition. In order to further increase the (R, R)-2,3-BD production by reducing the accumulation of its precursor acetoin, the synthetic operon was reconstructed by adding the strong promoter P01 in front of the gene ydjL coding for the enzyme of (R, R)-2,3-BD dehydrogenase which catalyzes the conversion of acetoin to (R, R)-2,3-BD. The engineered strain E. coli YJ3 showed a 20 % decrease in acetoin production compared with that of E. coli YJ2. After optimization the fermentation conditions, 30.5 g/L of (R, R)-2,3-BD and 3.2 g/L of acetoin were produced from 80 g/L of glucose within 18 h, with an enantio-purity over 99 %.

Constructing a synthetic metabolic pathway in Escherichia coli to produce the enantiomerically pure (R, R)-2,3-butanediol.

Enantiomerically pure (R, R)-2,3-butanediol has unique applications due to its special chiral group and spatial configuration. Currently, its chemical production route has many limitations. In addition, no native microorganisms can accumulate (R, R)-2,3-butanediol with an enantio-purity over 99%. Herein, we constructed a synthetic metabolic pathway for enantiomerically pure (R, R)-2,3-butanediol biosynthesis in Escherichia coli. The fermentation results suggested that introduction of the synthetic metabolic pathway redistributed the carbon fluxes to the neutral (R, R)-2,3-butanediol, and thus protected the strain against the acetic acid inhibition. Additionally, it showed that the traditionally used isopropyl beta-D-thiogalactoside (IPTG) induction displayed negative effect on (R, R)-2,3-butanediol biosynthesis in the recombinant E. coli, which was probably due to the protein burden. With no IPTG addition, the (R, R)-2,3-butanediol concentration reached 115 g/L by fed-batch culturing of the recombinant E. coli, with an enantio-purity over 99%, which is suitable for the pilot-scale production.

Quercetin inhibits the mTORC1/p70S6K signaling-mediated renal tubular epithelial-mesenchymal transition and renal fibrosis in diabetic nephropathy.

Quercetin is a classic flavonoid that inhibits the epithelial-mesenchymal transition (EMT) of tumor cells. However, the effects of quercetin on the EMT of renal tubular epithelial cells, a potential mechanism of renal fibrosis and important characteristic of diabetic nephropathy (DN), remain largely unknown. In the present study, we investigated the effects of quercetin on the EMT of two lines of renal tubular proximal epithelial cells (HK-2 and NRK-52E) induced with high glucose and renal fibrosis resulting from type 1 diabetes and tried to clarify the specific mechanisms underlying these effects. The in vitro results showed that the EMT of HK-2 and NRK-52E cells was induced by high glucose, and mTORC1/p70S6K was highly activated in these two cell lines cultured under high glucose. Quercetin effectively ameliorated the high glucose-induced EMT of HK-2 and NRK-52E cells and inhibited the activation of mTORC1/p70S6K. In vivo, diabetic rats showed a significant decline in renal function and severe renal fibrosis at 14 weeks after STZ injection. Furthermore, mTORC1/p70S6K was activated in the renal cortex of diabetic rats. Treatment with quercetin alleviated the decline in renal function, and the progression of renal fibrosis and inhibited mTORC1/p70S6K activation in the diabetic renal cortex. In addition, we examined the protein and mRNA levels of four transcriptional factors (snail, slug, twist and ZEB-1), which regulate E-cadherin expression at the transcriptional level both in vivo and in vitro. The results revealed that the elevated expression of snail and twist in HK-2 and NRK-52E cells cultured under high glucose and in the renal cortex of diabetic rats was inhibited by quercetin. These results demonstrated that quercetin ameliorates the EMT of HK-2 and NRK-52E cells induced by high glucose and renal fibrosis induced by diabetes, and these effects have been associated with the inhibition of the two transcriptional factors (snail and twist) and the activation of mTORC1/p70S6K.

The roles of different salts and a novel osmotic pressure control strategy for improvement of DHA production by Schizochytrium sp.

The effects of different osmotic pressure, changed by six salts (NaCl, Na2SO4, (NH4)2SO4, KH2PO4 and MSG), on cell growth and DHA synthesis by Schizochytrium sp. were investigated. Six optimal mediums were obtained to study different osmotic pressure combinations at cell growth stage and DHA synthesis stage. Results showed that cultivated cell in higher osmotic pressure condition and fermented in lower osmotic pressure condition was benefit to enhance DHA synthesis. Combination 17-6 could get the maximum cell dry weight of 56.95 g/L and the highest DHA percentage in total fatty acids of 55.21%, while combination 17-B could get the highest lipid yield of 33.47 g/L with 42.10% DHA in total fatty acids. This was the first report about the enhancement of DHA production by osmotic regulation and this work provided two novel osmotic control processes for high lipid yield and high DHA percentage in total fatty acids.

Fungal arachidonic acid-rich oil: research, development and industrialization.

Fungal arachidonic acid (ARA)-rich oil is an important microbial oil that affects diverse physiological processes that impact normal health and chronic disease. In this article, the historic developments and technological achievements in fungal ARA-rich oil production in the past several years are reviewed. The biochemistry of ARA, ARA-rich oil synthesis and the accumulation mechanism are first introduced. Subsequently, the fermentation and downstream technologies are summarized. Furthermore, progress in the industrial production of ARA-rich oil is discussed. Finally, guidelines for future studies of fungal ARA-rich oil production are proposed in light of the current progress, challenges and trends in the field.

Regulation of docosahexaenoic acid production by Schizochytrium sp.: effect of nitrogen addition.

Docosahexaenoic acid (DHA) percentage in total fatty acids (TFAs) is an important index in DHA microbial production. In this study, the change of DHA percentage in response to fermentation stages and the strategies to increase DHA percentage were investigated. Two kinds of conventional nitrogen sources, monosodium glutamate (MSG) and ammonium sulfate (AS), were tested to regulate DHA synthesis. Results showed that MSG addition could accelerate the substrate consumption rate but inhibit lipid accumulation, while AS addition could increase DHA percentage in TFAs effectively but extend fermentation period slightly. Finally, the AS addition strategy was successfully applied in 7,000-L fermentor and DHA percentage in TFAs and DHA yield reached 46.06 % and 18.48 g/L, which was 19.54 and 17.41 % higher than that of no-addition strategy. This would provide guidance for the large-scale production of the other similar polyunsaturated fatty acid, and give insight into the nitrogen metabolism in oil-producing microorganisms.

Efficient arachidonic acid-rich oil production by Mortierella alpina through a three-stage fermentation strategy.

A three-stage fermentation strategy was designed for efficient arachidonic acid (ARA)-rich oil production by Mortierella alpina. The process at different stages by changing the components of medium was investigated. In the first stage, mycelia were inoculated in a nutrient-rich medium for rapid propagation. In the second stage, mycelia were collected and then cultivated in glucose solution to achieve high cellular lipid contents. In the third stage, mycelia were cultured in a glucose-absent medium to obtain rapid ARA accumulation. Using this fermentation strategy, high dry cell weight, lipid, and ARA concentration reached 41.6, 26.6, and 11.4 g/L, respectively. The results demonstrated that mycelia propagation, lipid biosynthesis, and ARA accumulation process can be significantly spatially separated, allowing further optimization to improve the efficiency of each stage. This was the first report of using a three-stage fermentation strategy for ARA-rich oil production, and it could be applied to other similar oleaginous microorganisms to obtain high related polyunsaturated fatty acids accumulation.

Arachidonic acid-rich oil production by Mortierella alpina with different gas distributors.

Arachidonic acid (ARA)-rich oil production by Mortierella alpina is a high oxygen demand and shear-sensitive process. In the aerobic fermentation process, oxygen supply is usually a limiting factor owing to the low solubility of oxygen in the fermentation broth. Two kinds of perforated ring gas distributors and a novel microporous ceramic membrane gas distributor were designed and applied to improve oxygen supply. With the decrease of the orifice diameter of perforated ring gas distributors, dry cell weight (DCW), lipids concentration, and ARA content in total fatty acid increased from 17.86 g/L, 7.08 g/L, and 28.08 % to 25.67 g/L, 11.94 g/L, and 36.99 %, respectively. Furthermore, the effect of different dissolved oxygen (DO) on ARA-rich oil production with membrane gas distributor was also studied. The maximum DCW, lipid concentration, and ARA content using membrane gas distributor with DO controlled at 40 % reached 29.67 g/L, 16.74 g/L, and 49.53 %, respectively. The ARA titer increased from 1.99 to 8.29 g/L using the membrane gas distributor to substitute the perforated ring gas distributor. In the further experiment, a novel tubular titanium metal membrane gas distributor was successfully applied in a 7,000 L bioreactor and the results demonstrated that membrane gas distributor was industrially practical.

Harnessing oleaginous yeast to produce omega fatty acids.

Omega fatty acids are important for human health. They are traditionally extracted from animals or plants but can be alternatively produced using oleaginous yeast. Current efforts are producing yeast strains with similar fatty acid distributions and powerful lipogenesis capacity. The next step is to further make the process more competitive.

Exploiting synthetic biology platforms for enhanced biosynthesis of natural products in Yarrowia lipolytica.

With the rapid development of synthetic biology, researchers can design, modify, or even synthesize microorganisms de novo, and microorganisms endowed with unnatural functions can be considered "artificial life" and facilitate the development of functional products. Based on this concept, researchers can solve critical problems related to the insufficient supply of natural products, such as low yields, long production cycles, and cumbersome procedures. Due to its superior performance and unique physiological and biochemical characteristics, Yarrowia lipolytica is a favorable chassis cell used for green biomanufacturing by numerous researchers. This paper mainly reviews the development of synthetic biology techniques for Y. lipolytica and summarizes the recent research progress on the synthesis of natural products in Y. lipolytica. This review will promote the continued innovative development of Y. lipolytica by providing theoretical guidance for research on the biosynthesis of natural products.

Building Yarrowia lipolytica Cell Factories for Advanced Biomanufacturing: Challenges and Solutions.

Microbial cell factories have shown great potential for industrial production with the benefit of being environmentally friendly and sustainable. Yarrowia lipolytica is a promising and superior non-model host for biomanufacturing due to its cumulated advantages compared to model microorganisms, such as high fluxes of metabolic precursors (acetyl-CoA and malonyl-CoA) and its naturally hydrophobic microenvironment. However, although diverse compounds have been synthesized in Y. lipolytica cell factories, most of the relevant studies have not reached the level of industrialization and commercialization due to a number of remaining challenges, including unbalanced metabolic flux, conflict between cell growth and product synthesis, and cytotoxic effects. Here, various metabolic engineering strategies for solving the challenges are summarized, which is developing fast and extremely conducive to rational design and reconstruction of robust Y. lipolytica cell factories for advanced biomanufacturing. Finally, future engineering efforts for enhancing the production efficiency of this platform strain are highlighted.