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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [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]

Microbial preference for glucose implies incomplete and/or slow utilization of lignocellulose hydrolysates, which is caused by the regulatory mechanism named carbon catabolite repression (CCR). In this study, a 2,3-butanediol (2,3-BD) producing Klebsiella oxytoca strain was engineered to eliminate glucose repression of xylose utilization. The crp(in) gene, encoding the mutant cyclic adenosine monophosphate (cAMP) receptor protein CRP(in), which does not require cAMP for functioning, was characterized and overexpressed in K. oxytoca. The engineered recombinant could utilize a mixture of glucose and xylose simultaneously, without CCR. The profiles of sugar consumption and 2,3-BD production by the engineered recombinant, in glucose and xylose mixtures, were examined and showed that glucose and xylose could be consumed simultaneously to produce 2,3-BD. This study offers a metabolic engineering strategy to achieve highly efficient utilization of sugar mixtures derived from the lignocellulosic biomass for the production of bio-based chemicals using enteric bacteria. [ABSTRACT FROM AUTHOR]