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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. [ABSTRACT FROM AUTHOR]

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 (P) and AlperBB (P)). The strength of P is twice stronger than P. 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 P 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 %. [ABSTRACT FROM AUTHOR]

[Display omitted] • Comprehensive analysis on the superiority of Y. lipolytica. • Up-to-date overview of synthetic biology tools in Y. lipolytica. • First review on synthesis of diverse natural products by Y. lipolytica. • In-depth discussion on challenges and future perspectives for the production of natural products. 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. [ABSTRACT FROM AUTHOR]

The efficient valorization of lignin-derived vanillyl alcohol for the production of value-added biovanillin with enzyme or whole-cell based biotransformation is attracting increasing attention. In this study, a recombinant Escherichia coli HMFO was explored for the production of vanillin through the selective aerobic oxidation of vanillyl alcohol. The resting cells of this strain indicated a high vanillin tolerance and good biocatalytic activity over a temperature range of 10–35 °C. The vanillyl alcohol oxidation process produced vanillin as the sole product, indicating excellent reaction selectivity. Vanillyl alcohol substrate of 250 mM was converted to vanillin under optimal conditions with 96 % yield. In addition, 449.2 mM vanillin was obtained in a fed-batch experiment, providing a productivity of 1.2 g/L per hour and 90 % yield. The production of biovanillin could be further improved by using enzyme engineering and process engineering methods. [Display omitted] • Highly vanillin-tolerant recombinant E.coli HMFO was used for vanillin synthesis. • Vanillin was biosynthesized from lignin-based vanillyl alcohol with E.coli HMFO. • The sole product of vanillin from vanillyl alcohol oxidation was obtained. • Vanillyl alcohol (250 mM) was converted to vanillin with a yield of 96 %. [ABSTRACT FROM AUTHOR]

Lipid accumulation of Schizochytrium sp. can be induced by stress condition, but this stress-induction usually reduce cell growth and cause oxidative damage, which can eventually lower the lipid yield. Here, adaptive laboratory evolution (ALE) combined high salinity was performed to enhance the antioxidant system and lipid accumulation. The final strain ALE150, which was obtained after 150 days, showed a maximal cell dry weight (CDW) of 134.5 g/L and lipid yield of 80.14 g/L, representing a 32.7 and 53.31% increase over the starting strain, respectively. Moreover, ALE150 exhibited an overall higher total antioxidant capacity (T-AOC) and lower reactive oxygen species (ROS) levels than the starting strain. Furthermore, the regulatory mechanisms responsible for the improved performance of ALE150 were analyzed by transcriptomic analysis. Genes related to the antioxidant enzymes and central carbon metabolism were up-regulation. Moreover, the metabolic fluxes towards the fatty acid synthase (FAS) and polyketide synthase (PKS) pathways were also changed. [ABSTRACT FROM AUTHOR]

[Display omitted] • Alternative strategy for nervonic acid-enriched plant oil production is developed. • Orthogonal plant and non-plant nervonic acid synthetic pathways are constructed. • MoLPAAT from Malania oleifera is identified to be specificity for nervonic acid. • The highest titer of nervonic acid at 13.56 g/L is achieved in Yarrowia lipolytica. Nervonic acid has proven efficacy in brain development and the prevention of neurodegenerative diseases. Here, an alternative and sustainable strategy for nervonic acid-enriched plant oil production was established. Different β-ketoacyl-CoA synthases and heterologous Δ15 desaturase were co-expressed, combined with the deletion of the β-oxidation pathway to construct orthogonal plant and non-plant nervonic acid biosynthesis pathways in Yarrowia lipolytica. A "block-pull-restrain" strategy was further applied to improve the supply of stearic acid as the precursor of the non-plant pathway. Then, lysophosphatidic acid acyltransferase from Malania oleifera (Mo Lpaat) was identified, which showed specificity for nervonic acid. Endogenous LPAAT was exchanged by MoLPAAT resulted in 17.10 % nervonic acid accumulation. Finally, lipid metabolism was engineered and cofactor supply was increased to boost the lipid accumulation in a stable null-hyphal strain. The final strain produced 57.84 g/L oils with 23.44 % nervonic acid in fed-batch fermentation, which has the potential to substitute nervonic acid-enriched plant oil. [ABSTRACT FROM AUTHOR]

As one of the most important environmental factors, oxygen is particularly important for synthesis of n-3 polyunsaturated fatty acids (n-3 PUFA) in microalgae. In general, a higher oxygen supply is beneficial for cell growth but obstructs PUFA synthesis. The generation of reactive oxygen species (ROS) under aerobic conditions, which leads to the peroxidation of lipids and especially PUFA, is an inevitable aspect of life, but is often ignored in fermentation processes. Irritability, microalgal cells are able to activate a number of anti-oxidative defenses, and the lipid profile of many species is reported to be altered under oxidative stress. In this review, the effects of oxygen on the PUFA synthesis, sources of oxidative damage, and anti-oxidative defense systems of microalgae were summarized and discussed. Moreover, this review summarizes the published reports on microalgal biotechnology involving direct/indirect oxygen regulation and new bioreactor designs that enable the improved production of PUFA. [ABSTRACT FROM AUTHOR]

Yarrowia lipolytica , a non-conventional oleaginous yeast with special traits, has attracted increasing interest for producing value-added products. Generally, the DNA fragments of these heterologous metabolic pathways are constructed via the classic restriction digestion and ligation method. In contrast, the one-step in vivo pathway assembly method has been only rarely applied to Y. lipolytica . Here, with arachidonic acid biosynthesis as a case study, a one-step in vivo pathway assembly and integration method was used for engineering Y. lipolytica . Using rDNA as integrative locus, this study showed that there was a relation between the assembly efficiency and the length of overlapping region. Especially, with an overlap up to 1 kb, the method was able to rapidly assemble the arachidonic acid biosynthesis pathway (nearly 10 kb) into the chromosome with high efficiency (nearly 23%). Meanwhile, the pathway assembled in Y. lipolytica demonstrated long-term genetic stability and the engineered strain exhibited robust growth. Furthermore, this study demonstrated that the codon-optimized genes from Mortierella alpina can function efficiently in Y. lipolytica : a high level arachidonic acid production (0.4% of total fatty acids) was produced in the engineered strain. To our knowledge, this is the first time that this method is applied to Y. lipolytica for functional polyunsaturated fatty acids production. This method represents a powerful tool with potential for facilitating engineering applications in non-conventional yeasts. [ABSTRACT FROM AUTHOR]