Analysis of FR901379 Biosynthetic Genes in Coleophoma empetri by Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Based Genomic Manipulation.

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.

CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Glarea lozoyensis and Its Application in Manipulating gloF.

Glarea lozoyensis is an important industrial fungus that produces the pneumocandin B0, 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 C0 to (pneumocandin B0 + pneumocandin C0) 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 C0. 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 B0, but the production of pneumocandin C0 was abolished. Thus, this strain displayed a competitive advantage in the industrial production of pneumocandin B0. In summary, this study showed that the CRISPR/Cas9-based gene editing tool is efficient for manipulating genes in G. lozoyensis.

Enhancement of FK520 production in Streptomyces hygroscopicus by combining traditional mutagenesis with metabolic engineering.

FK520 (ascomycin), a 23-membered macrolide with immunosuppressive activity, is produced by Streptomyces hygroscopicus. The problem of low yield and high impurities (mainly FK523) limits the industrialized production of FK520. In this study, the FK520 yield was significantly improved by strain mutagenesis and genetic engineering. First, a FK520 high-producing strain SFK-6-33 (2432.2 mg/L) was obtained from SFK-36 (1588.4 mg/L) through ultraviolet radiation mutation coupled with streptomycin resistance screening. The endogenous crotonyl-CoA carboxylase/reductase (FkbS) was found to play an important role in FK520 biosynthesis, identified with CRISPR/dCas9 inhibition system. FkbS was overexpressed in SFK-6-33 to obtain the engineered strain SFK-OfkbS, which produced 2817.0 mg/L of FK520 resulting from an increase in intracellular ethylmalonyl-CoA levels. In addition, the FK520 levels could be further increased with supplementation of crotonic acid in SFK-OfkbS. Overexpression of acetyl-CoA carboxylase (ACCase), used for the synthesis of malonyl-CoA, was also investigated in SFK-6-33, which improved the FK520 yield to 3320.1 mg/L but showed no significant inhibition in FK523 production. To further enhance FK520 production, FkbS and ACCase combinatorial overexpression strain SFK-OASN was constructed; the FK520 production increased by 44.4% to 3511.4 mg/L, and the FK523/FK520 ratio was reduced from 9.6 to 5.6% compared with that in SFK-6-33. Finally, a fed-batch culture was carried out in a 5-L fermenter, and the FK520 yield reached 3913.9 mg/L at 168 h by feeding glycerol, representing the highest FK520 yield reported thus far. These results demonstrated that traditional mutagenesis combined with metabolic engineering was an effective strategy to improve FK520 production.

Development of an Efficient and Cost-Effective Enzymatic Process for Production of (R)-[3,5-bis(trifluoromethyl)phenyl] Ethanol Using Carbonyl Reductase Derived from Leifsonia sp. S749.

(R)-[3,5-bis(trifluoromethyl) phenyl] ethanol [(R)-3,5-BTPE] is a crucial chiral intermediate for the synthesis of the NK-1 receptor antagonists aprepitant, rolapitant and fosaprepitant. The carbonyl reductase KR01 from Leifsonia sp. S749, discovered by protein sequence alignment, could convert 3',5'-bis(trifluoromethyl) acetophenone (3,5-BTAP) into (R)-3,5-BTPE with excellent activity and enantioselectivity. In order to enhance the conversion efficiency at high substrate concentrations, the reaction conditions were optimized by response surface analysis. The results showed that 600 g/L 3,5-BTAP was bioreduced to (R)-3,5-BTPE (> 99.9% enantiomeric excess) by the recombinant Escherichia coli/pET-28a (+)-KR01 whole cells, with a 98.3% conversion and 59 g/L/h productivity under the optimized reaction conditions. In addition, the recombinant E. coli cells could be repeatedly used up to seven times in the reaction mixture containing 90% isopropanol (IPA). This is the highest substrate loading and productivity for the bioreduction of 3,5-BTAP by carbonyl reductase ever reported, and this method represents an efficient and cost-effective process for production of (R)-3,5-BTPE.