Improvement of Nemadectin Production by Overexpressing the Regulatory Gene nemR and Nemadectin Biosynthetic Gene Cluster in Streptomyces Cyaneogriseus

Abstract Nemadectin, a 16-member macrocyclic lactone antiparasitic antibiotic, is produced by Streptomyces cyaneogriseus subspecies noncyanogenus. Moxidectin, a C-23 oximate derivative of nemadectin, is widely used as a pesticide due to its broad-spectrum, highly efficient, and safe anthelmintic activity. NemR, a LAL family regulator, is encoded by nemR and is involved in nemadectin biosynthesis in S. cyaneogriseus. In this report, gene disruption and complementation experiments showed that nemR plays a positive role in the biosynthesis of nemadectin. The transcription level of nemadectin biosynthetic genes in the nemR knockout strain was significantly decreased compared with those in the wild-type strain MOX-101. However, overexpression of nemR under the control of native or strong constitutive promoters resulted in the opposite, increasing the production of nemadectin by 56.5 or 73.5%, respectively, when compared with MOX-101. In addition, the gene cluster of nemadectin biosynthesis was further cloned and overexpressed using a CRISPR method, which significantly increase nemadectin yield by 108.6% (509 mg/L) when compared with MOX-101.

The preliminary research on nemadectin was mainly focused on strain breeding and optimization of fermentation technology. In 2009, the yield of the strain was increased to 172 mg/L by ultraviolet (UV) mutagenesis combined with the screening of precursor resistance. 11 In 2014, the fermentation process of nemadectin was optimized by investigating various factors, such as time, temperature, and dissolved oxygen. 12 In 2015, Li et al reported a method for improving nemadectin production by screening a mutant strain DC18-01 and adding the precursor sodium acetate for one or more times in the middle and later stages of fermentation. 13 Although the Keywords ► nemadectin ► overexpression ► biosynthesis gene cluster ► Streptomyces cyaneogriseus biosynthetic gene cluster of nemadectin has been identified, and its complete sequence is also available, 14 the study on genetic modification to nemadectin-producing strain remains largely unknown. In 2019, the function of the gene nemR was characterized, which is a positive regulatory gene encoding a LAL family transcriptional regulator within the nemadectin biosynthesis gene cluster of the strain NMWT1. 15 The low fermentation yield of nemadectin resulted in the high production cost of moxidectin. In this study, the LAL family regulatory gene nemR, which serves as an activator for nemadectin biosynthesis, was confirmed and was employed to improve nemadectin production by overexpression of nemR in the strain MOX-101. The nemadectin biosynthesis gene cluster in MOX-101 was successfully cloned and used to increase nemadectin production by duplicating of the biosynthesis gene cluster in S. cyaneogriseus.

Strains, Plasmids, and Primers
Strains and plasmids used in this study are listed in ►Table 1.
Primers are listed in ►Table 2.

Construction of Gene Deletion, Complementation, and Overexpression Strains
The construction process for the DnemR mutant with deletion of nemR is as follows. A 1.5-kb fragment upstream of the nemR start codon and a 1.5-kb fragment downstream of the nemR stop codon were amplified from MOX-101 genomic DNA (gDNA) using the primer pairs nemR-up-F/R and nemR-down-F/R, respectively. Two polymerase chain reaction (PCR) fragments were spliced together through overlapping PCR using the primers nemR-up-F and nemRdown-R. The resulting product was cloned into a 3.1-kb DNA fragment that was amplified from pSET152 using the primers pSET152-F/R to obtain the nemR-deleted plasmid pDnemR using the seamless ligation method (ClonExpress II One Step Cloning kit) (►Fig. 2A). The plasmid pDnemR was transferred from S17-1 into MOX-101 by intergeneric conjugation. After selecting single-crossover recombinant strains and double-crossover recombinant strains, the resulting mutant with deletion of nemR was named DnemR.
The nemR complementation strain DnemR/pnemR was constructed as follows. Using the gDNA of MOX-101 as the template, a 3.7-kb DNA fragment containing the gene nemR and its promoter region was amplified by PCR with the primers nemR-N-F/R. The PCR product was inserted into the XbaI/BamHI site of pSET152 to generate the integrative plasmid pnemR (►Fig. 2B). The sequence of nemR in pnemR was confirmed by DNA sequencing. After conjugal transfer, pnemR was transferred into the DnemR strain to obtain the nemR complementation strain DnemR/pnemR. As a control, the control plasmid pSET152 was transferred into MOX-101 to generate the strain MOX-101/pSET152.

Construction of Nemadectin Biosynthetic Gene Cluster Overexpression Strain
Cloning nemadectin biosynthetic gene cluster was conducted following the CRISPR-TAR method reported previously. 18 The nemadectin biosynthetic gene cluster MOX was divided into two parts, namely, MOX1 (50 kb) and MOX2 (41.7 kb). MOX1 and MOX2 were cloned respectively, and then two modules were spliced to generate the complete nemadectin biosynthetic gene cluster.
CRISPR-digested gDNA was prepared as follows. First, four gRNA minigenes encoding gRNA were amplified from the plasmid pKCCas9 (tipAp) by the primers gRNA-MOX1-up-F/gRNA-R, gRNA-MOX1-down-F/gRNA-R, gRNA-MOX2up-F/gRNA-R, and gRNA-MOX2-down-F/gRNA-R designed at each end of MOX1 and MOX2 gene clusters, respectively. Next, the resulting products were transcribed in vitro by MEGAScriptTMT7 Kit (Thermo Fisher Scientific, China) to obtain gRNAs used to guide Cas9 nuclease to cleave the gDNA of MOX-101. These gRNAs were purified using MEGAClear Kit (Thermo Fisher Scientific, China). Then, the gDNA isolated by the phenol-chloroform method was digested in vitro overnight with the help of the Cas9 nuclease and gRNAs.
Plasmids pCL-M1, pCL-M2, and pCL-M were constructed to capture MOX1, MOX2, and MOX gene clusters, respectively.     Digested genome DNAs and linearized capture plasmids were introduced into Saccharomyces cerevisiae VL6-48 to clone the biosynthetic gene cluster MOX1 and MOX2. After verifying transformants, positive plasmids pCL-MOX1 and pCL-MOX2 were transferred into Escherichia coli EPI300 for enrichment. Positive plasmids pCL-MOX1 and pCL-MOX2 were collected by E.Z.N.A.BAC/PAC DNA (Omega Bio-Tek, China) and digested with restriction enzyme SwaI to obtain gene clusters MOX1 and MOX2, respectively. The obtained gene clusters and the linearized capture plasmid pCL-M were introduced into the yeast to clone the biosynthetic gene cluster MOX. After confirming the transformant, the positive plasmid pCL-MOX was transferred into E. coli EPI300 for enrichment. Plasmids pCL01, pCL-MOX1, pCL-MOX2, and pCL-MOX were digested with the restriction enzyme KpnI to verify if the captured gene clusters were correct. The correct plasmid pCL-MOX was transferred from S17-1 into MOX-101 to generate the engineering strain MOX-101/pCL-MOX, in which the nemadectin biosynthesis gene cluster was overexpressed. As a control, the control plasmid pCL01 was transferred into MOX-101 to generate the strain MOX-101/pCL01.

Analytical Method
To analyze nemadectin yield, 1.0 mL culture broth (collected for 4, 6, and 8 days) was extracted with 3.0 mL methanol for 30 minutes and centrifuged at 10,000 Â g for 5 minutes. The supernatant was analyzed by high-pressure liquid chromatography (HPLC) with a Hypersil C18 column (5 μm, 4.6 mm Â 150 mm). The column was maintained at 30°C with mobile phases of methanol:water (85:15 [vol/vol]) at a flow rate of 1.0 mL/min and the product was UV-detected at 240 nm using an Agilent 1260 HPLC system.

Transcriptional Assay by Real-time Polymerase Chain Reaction Analysis
First, total RNA was isolated from the strains and used as the template to synthesize cDNA samples. Fermentation broths (10 mL) of MOX-101 and DnemR collected at day 4, 6, and 8 were centrifuged at 10,000 Â g for 5 minutes, and pellets were ground into powder in liquid nitrogen. Next, an Ultrapure RNA Kit (CWBIO, China) was used for total RNA extraction according to the user manual provided by the manufacturer. The obtained RNA sample was treated with RNase-free DNase I (Takara, China) to remove the remaining gDNA. To obtain cDNA samples, reverse transcription (RT) was performed using M-MLV Reverse Transcriptase (Promega, United States).
Then, a cotranscriptional experiment utilizing RT-PCR was employed to investigate whether two nemadectin biosynthetic structural or regulatory genes spanning a short intergenic region and transcribed in the same direction were cotranscribed. Primers used to amplify the cDNA sample of MOX-101 were nemA1-2A2-F/R, nemA2C-F/R, nemA3E-F/R, and nemA4A3-F/R. gDNA and RNA samples without RT served as the positive and negative controls, respectively, in the PCR analyses.
Then, quantitative RT-PCR (qRT-PCR) was performed by TB Green TM Premix Ex Taq TM II (Takara, China) according to the manufacturer's instructions. qRT-PCR experiments were performed to assay samples collected for 4, 6, and 8 days of fermentation. One gene was selected from each transcriptional unit for transcriptional analysis. Five primer pairs, including nemG-F/R, nemF-F/R, nemA1-2-F/R, nemA3-F/R, and nemA4-F/R, were used in the qRT-PCR tests. Transcriptional levels of the tested genes were normalized using hrdB (TU94_24720) as the internal control. 19 Each qRT-PCR analysis was repeated three times, and the error bar represents the standard deviation.

NemR Functions as an Activator in the Biosynthesis of Nemadectin
To study the function of nemR in nemadectin biosynthesis, we constructed the DnemR mutant strain with deletion of the nemR gene using MOX-101 as the parental strain (►Fig. 3A). Two strains were cultured in the fermentation medium, and broths were collected for 4, 6, and 8 days. The result showed that nemadectin production was significantly decreased in DnemR with the deletion of the nemR gene. Nemadectin yield in DnemR was decreased by approximately 80% in comparison with the parental strain MOX-101 (►Fig. 3B). To further verify the function of NemR, we performed a complementation experiment in which the complemented plasmid pnemR was introduced into the DnemR mutant (►Fig. 3A). In pnemR, nemR was driven by the native promoter. The result showed that the complementation of nemR could restore the yield level of nemadectin to that of the parental strain (►Fig. 3C), revealing that the decline in nemadectin yield was mainly due to nemR inactivation. These results demonstrated that NemR acts as an activator for nemadectin biosynthesis in S. cyaneogriseus.

qRT-PCR Analysis of Transcriptional Levels of the Nemadectin Biosynthetic Gene Cluster
A transcriptional assay was performed to further study the function of the NemR protein in nemadectin production. The gene cluster for nemadectin biosynthesis contains 10 structural genes and one regulatory gene (►Fig. 4A). The result of the cotranscriptional experiment suggested that there were five cotranscriptional units altogether in the gene cluster of nemadectin biosynthesis, including nemG, nemF, nemA1-1-C, nemA3-D, and nemA4 (►Fig. 4B).
The transcriptional assay result indicated that nemR deletion strongly affects the transcription of nemadectin biosynthetic genes. As shown in ►Fig. 4C, compared with the parental strain MOX-101, transcriptional levels of all nemadectin biosynthetic genes in the knockout strain DnemR were significantly decreased. Among these structural genes, transcriptional levels of nemG/F and nemA1-1/A1-2/A2/C/D/E/A3/A4 in DnemR were only approximately 30% and less than 3% of those in MOX-101. These results suggested that nemR might play a positive regulatory role in nemadectin production by enhancing the expression of the nemadectin biosynthetic gene cluster.

Effect of Overexpression of nemR on Nemadectin Production
To enhance nemadectin yield, pnemR under the control of the native promoter was transferred into MOX-101 to generate the overexpression strain MOX-101/pnemR. As a control, the control plasmid pSET152 was transferred into MOX-101 to generate MOX-101/pSET152. Three strains, MOX-101, MOX-101/pnemR, and MOX-101/pSET152, were cultured for 4, 6, and 8 days, and broths were collected to detect the fermentation products by HPLC analysis. The result showed that compared with the original strain MOX-101, MOX-101/pnemR improved nemadectin production by 56.5%, while the effect of MOX-101/pSET152 on nemadectin production was similar to that of MOX-101 (►Fig. 5A). These results suggested that the increase in production of MOX-101/pnemR was due to an extra copy of the gene nemR.
To confirm whether the improvement in nemadectin yield was caused by the improved expression of nemadectin biosynthetic genes, a transcriptional assay was performed by qRT-PCR analysis to determine the transcriptional levels of the genes in MOX-101 and MOX-101/pnemR. The result indicated that the transcription levels of the nemadectin biosynthetic genes in MOX-101/pnemR were higher than those in MOX-101 (►Fig. 5B). Among these genes, the transcription of nemG/F and nemA1-1/A3/A4 improved above 30 and 100%, respectively.

Effect of Overexpression of Nemadectin Biosynthetic Gene Cluster on Nemadectin Production
The result of the transcriptional assay in MOX-101 and MOX-101/pnemR suggested that the improvement in nemadectin yield was caused by the increase in transcription levels of nemadectin biosynthetic genes. Then we attempted to directly overexpress the nemadectin biosynthetic gene cluster to increase its yield.
In the early stage of the experiment, several attempts were made to clone a complete nemadectin biosynthetic gene cluster, but all failed because of its large size ($90.9 kb). Therefore, two modules, MOX1 (50 kb) and MOX2 (41.7 kb), of the complete biosynthetic gene cluster were cloned respectively, and then the two modules were spliced together to obtain the complete nemadectin biosynthetic gene cluster MOX (►Fig. 6A). Plasmids pCL01, pCL-MOX1, pCL-MOX2, and pCL-MOX were verified by the restriction enzyme KpnI digestion, which showed that gene clusters MOX1, MOX2, and MOX were successfully cloned (►Fig. 6B). The plasmid pCL-MOX, containing the complete nemadectin biosynthetic gene cluster, was transferred into MOX-101 to generate the overexpression strain MOX-101/pCL-MOX. As a control, the control plasmid pCL01 was transferred into MOX-101 to generate MOX-101/pCL01. Three strains, MOX-101, MOX-101/pCL01,  and MOX-101/pCL-MOX, were cultured for 4, 6, and 8 days. The result showed that MOX-101/pCL-MOX significantly increased the production of nemadectin by 108.6% (509 mg/L) compared with the MOX-101 (►Fig. 6C).

Discussion
Strain improvement is very important for the industrialization of microbial medicine. Previously, random selection, such as UV mutagenesis and atmospheric and room temperature plasma mutation, and rational breeding were used to increase nemadectin yield. 11,20 Overexpression of transcriptional regulators is an effective and commonly used method to improve antibiotic production, especially when regulators were driven by strong constitutive promoters, such as ermEp Ã . [21][22][23] In 2019, a positive transcriptional regulator, NemR, involved in nemadectin biosynthesis was reported. 15 In this study, we overexpressed nemR in MOX-101 under the control of both native and strong constitutive promoters. The result showed that the yield of nemadectin was considerably improved compared with that of MOX-101 owing to the increase in transcription levels of nemadectin biosynthetic genes. Therefore, we attempted to directly overexpress the nemadectin biosynthetic gene cluster to increase its yield. This is the first report that an extra copy of the complete nemadectin biosynthesis gene cluster was introduced and overexpressed in a nemadectin-producing strain and the production was more than doubled.
Overexpression of nemR or nemadectin biosynthesis gene cluster increased the transcriptional level of nemadectin biosynthesis-related genes and thus improved nemadectin production. This revealed that the low fermentation yield of nemadectin may be due to the low transcriptional level of nemadectin biosynthesis-related genes. These findings helped us to elucidate nemadectin biosynthesis, and provide approaches to enhance nemadectin production by modifying new positive regulatory genes or further increasing the copy number of the nemadectin biosynthetic gene cluster in MOX-101/pCL-MOX.