Glarea lozoyensis is an important industrial fungus that produces the pneumocandin B 0 , which is used for the synthesis of antifungal drug caspofungin. However, because of the limitations and complications of traditional genetic tools, G. lozoyensis strain engineering has been hindered. In this study, we established an efficient CRISPR/Cas9-based gene editing tool in G. lozoyensis SIPI1208. With this method, gene mutagenesis efficiency in the target locus can be up to 80%, which enables the rapid gene knockout. According to the reports, GloF and Ap-HtyE, proline hydroxylases involved in pneumocandin and Echinocandin B biosynthesis, respectively, can catalyze the proline to generate different ratios of trans-3-hydroxy-L-proline to trans-4-hydroxy-L-proline. Heterologous expression of Ap-HtyE in G. lozoyensis decreased the ratio of pneumocandin C 0 to (pneumocandin B 0 + pneumocandin C 0 ) from 33.5% to 11% without the addition of proline to the fermentation medium. Furthermore, the gloF was replaced by ap-htyE to study the production of pneumocandin C 0 . However, the gene replacement has been hampered by traditional gene tools since gloF and gloG, two contiguous genes indispensable in the biosynthesis of pneumocandins, are cotranscribed into one mRNA. With the CRISPR/Cas9 strategy, ap-htyE was knocked in and successfully replaced gloF, and results showed that the knock-in strain retained the ability to produce pneumocandin B 0 , but the production of pneumocandin C 0 was abolished. Thus, this strain displayed a competitive advantage in the industrial production of pneumocandin B 0 . In summary, this study showed that the CRISPR/Cas9-based gene editing tool is efficient for manipulating genes in G. lozoyensis.

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The compound FR901379, a sulfated echinocandin produced by the filamentous fungus Coleophoma empetri F-11899, is an important intermediate for the synthesis of the antifungal drug micafungin. In this study, we established an efficient clustered regularly interspaced short palindromic repeats/Cas9-based gene editing tool for the industrial production strain C. empetri SIPI1284. With this method, the efficiency of gene mutagenesis in the target locus is up to 84%, which enables the rapid gene disruption for the analysis of FR901379 biosynthetic genes. Next, we verified the putative functional genes of the FR901379 biosynthetic gene cluster via gene disruption and gene complementation in vivo. These core functional genes included the nonribosomal peptide synthetase gene (CEnrps), the fatty-acyl-AMP ligase gene (CEligase) responsible for the formation of the activated form of palmitic acid and its transfer to CEnrps, four nonheme mononuclear iron oxygenase genes (CEoxy1, CEoxy2, CEoxy3, and CEoxy4) responsible for the synthesis of nonproteinogenic amino acids, l-homotyrosine biosynthesis genes (CEhtyA-D), two cytochrome P450 enzyme genes (CEp450-1 and CEp450-2), and a transcription regulator gene (CEhyp). In addition, by screening the whole genome, we identified two unknown genes (CEp450-3 and CEsul) responsible for the sulfonyloxy group of FR901379, which were separated from the core FR901379 biosynthetic cluster. Furthermore, during gene disruptions in the research, we obtained a series of FR901379 analogues and elucidated the relationship between the groups and antifungal activities.

Chloro-1-(2,4-dichlorophenyl) ethanol ( 3) is a chiral intermediate in the synthesis of luliconazole ((R)-E-1). Here, we report a novel biopreparation of 3 by bioreduction of 2-chloro-1-(2,4-dichlorophenyl) ethanone (2) using recombinant Escherichia coli expressing LK08, a ketoreductase mutant from Lactobacillus kefiri, as a biocatalyst. The reaction conditions for the biotransformation including pH, temperature, and concentration of isopropanol and NADP + , as well as the amount of recombinant E. coli cells, were optimized to improve the process productivity. When the enzymatic process was carried out on a 300 g scale under the optimized conditions, the ketone 2 was fully converted to chiral alcohol 3 with a product ee value of >99%. Furthermore, 3 was isolated and used to chemically synthesize luliconazole with 38% yield and 99% ee. This study presents an efficient and cost-effective chemoenzymatic process for the production of (R)-E-1.

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