Construction of an Efficient and Robust Aspergillus terreus Cell

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Construction of an efficient and robust Aspergillus terreus cell factory for monacolin J production Xuenian Huang, Shen Tang, Linghui Zheng, Yun Teng, Yong Yang, Jinwei Zhu, and Xuefeng Lu ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00489 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 11, 2019

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ACS Synthetic Biology

Construction of an efficient and robust Aspergillus terreus cell factory for monacolin J production Xuenian Huang†, Shen Tang†,⊥, Linghui Zheng§, Yun Teng§, Yong Yang§, Jinwei Zhu§ & Xuefeng Lu†, ‡, #,* †

Shandong Provincial Key Laboratory of Synthetic Biology,

‡

Key Laboratory of

Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, No. 189 Songling Road, Qingdao 266101, China. §

Zhejiang Key Laboratory of Antifungal Drugs, Zhejiang Hisun Pharmaceutical Co.,

Ltd., Taizhou, China. #

Marine biology and Biotechnology Laboratory, Qingdao National Laboratory for

Marine Science and Technology, Wenhai Rd 1, Aoshanwei, Qingdao, China. ⊥

College of Bioscience and Bioengineering, Jiangxi Engineering Laboratory for the

Development and Utilization of Agricultural Microbial Resources, Jiangxi Agricultural University, Nanchang 330045, China. * Corresponding author: Xuefeng Lu, [email protected]

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ABSTRACT Monacolin J is a key precursor for the synthesis of the cholesterol-lowering drug simvastatin. Industrially, monacolin J is manufactured through the alkaline hydrolysis of the fungal polyketide lovastatin, which is relatively complex and environmentally unfriendly. A cell factory for monacolin J production was created by heterologously introducing lovastatin hydrolase into Aspergillus terreus in our previous study. However, residual lovastatin remained a problem for the downstream product purification. In this study, we used combined metabolic engineering strategies to create a more efficient and robust monacolin J-producing cell factory that completely lacks lovastatin residue. The complete deletion of the key gene lovF blocked the biosynthesis of lovastatin and led to a large accumulation of monacolin J without any lovastatin residue. Additionally, the overexpression of the specific transcription factor lovE under the PgpdAt promoter further increased the titer of monacolin J by 52.5% to 5.5 g L-1. Interestingly, the fermentation robustness was also significantly improved by the expression of lovE. This improvement not only avoids the process of alkaline hydrolysis but also simplifies the downstream separation process. KEYWORDS: monacolin J, lovastatin, Aspergillus terreus, LovF, transcription regulator, microbial cell factory

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Simvastatin is one of the most important cholesterol-lowering drugs, with annual sales exceeding $3 billion in 2015. Industrially, simvastatin has been produced through a multistep process that can be roughly divided into the following three parts: 1) the microbial production of lovastatin through A. terreus fermentation; 2) the alkaline hydrolysis of lovastatin to produce monacolin J; and 3) the chemical transformation of monacolin J to simvastatin (Figure 1). Lovastatin is one of the most well-known fungal polyketides, and its biosynthetic pathway has been well elucidated through genetic and biochemical characterization (Figure 1).1-4 The key intermediate, monacolin J, is synthesized by the highly reducing polyketide synthase (hrPKS) LovB, together with three tailoring enzymes, LovA, LovC and LovG. At the same time, the α-methylbutyryl side chain is synthesized by another hrPKS, LovF, and then transferred to the C-8 hydroxy group of monacolin J by the acyl transferase LovD to produce lovastatin. A biocatalysis process based on the acyltransferase LovD was developed to replace the existing semisynthetic conversion process of monacolin J to simvastatin.5-9 Therefore, monacolin J production is the only gap remaining to establish the complete bioproduction process of simvastatin. The industrial alkaline hydrolysis process of lovastatin is relatively complex, laborious, and generates pollution, as many concentrated alkali, acid and organic reagents are used (Figure 1). In fact, several earlier efforts were made to develop a monacolin J bioproduction system, including both microbial and enzymatic conversions.10-13 However, none of these improvements were applied at an industrial-scale production because of low efficiency.

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Figure 1. The industrial process for simvastatin production. The details of the alkaline hydrolysis process were provided by Hisun Pharm. SAM: S-adenosyl-L-methionine. Synthetic biology technology brings alternative strategies to produce rare natural products.14-19 Saccharomyces cerevisiae was the first reported heterologous host to produce 20 mg L-1 monacolin J via the introduction of three separate plasmids encoding A. terreus CPR, LovA, LovB, LovC, and LovG.4, 20 The monacolin J titer was further improved to 75 mg L-1 through the optimization of the biosynthetic pathways. Later, the biosynthetic pathways for monacolin J and lovastatin were assembled and optimized in Pichia pastoris, and yields of 60.0 mg L-1 monacolin J and 14.4 mg L-1 4


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lovastatin were obtained.21 And the higher yield of 593.9 mg L-1 monacolin J was achieved through the coculture of two separate P. pastoris strains that harbored the upstream and the downstream synthesis pathway respectively. Recently, we reported single-step bioproduction of monacolin J by expressing the newly identified lovastatin-hydrolyzing enzyme PcEST in an industrial lovastatinproducing A. terreus strain (Figure 1). Approximately 95% of the lovastatin was hydrolyzed in vivo, and 5.0 g L-1 monacolin J was obtained in this engineered A. terreus strain, which is a significantly higher yield than that obtained with the heterologous hosts.22 However, regarding the industrial process, the 5% of residual lovastatin in the fermentation culture is still a problem for the downstream separation, purification and quality control. In view of this problem, the monacolin J-producing A. terreus strain was further improved through introducing a more efficient PcEST mutant obtained by directional evolution, and the lovastatin residue was almost completely removed (< 0.3%) under laboratory fermentation conditions. However, the lovastatin residue was shown to reappear along with the doubling of monacolin J yield under the simulated industrial production conditions in Hisun Pharm.23 These results indicate that it is fermentation process-dependent and difficult to thoroughly solve the problem of residual lovastatin through this way, as there is a metabolic balance between the LovDcatalyzed acyl-transferring conversion of monacolin J to lovastatin and the PcESTcatalyzed hydrolyzing conversion of lovastatin to monacolin J. In this study, we present a different approach to construct a more efficient cell factory that can directly produce monacolin J without any lovastatin residue. First, the 5



biosynthesis of lovastatin in the industrial strain A. terreus HZ01 was blocked by the complete deletion of the multiple copies of the key gene lovF. Then, the pathway was further enhanced through the overexpression of the specific transcriptional regulator lovE, leading to a more efficient and robust cell factory for monacolin J production.

RESULTS AND DISCUSSION Determination of the Copy Number of the Lovastatin Synthetic Gene Cluster The industrial lovastatin-producing strain is an excellent and essential chassis cell, but the challenge lies in the complexity of the industrial strain. The analysis results of the whole genome resequencing of A. terreus HZ01, an industrial strain provided by Hisun Pharm, revealed that the sequencing depth of a particular region (NT_165939.1: 28500–75500) was 3- to 5-fold higher than that of the other regions on the same scaffold. This finding suggests that there are multiple copies of this region on the chromosome (Figure 2A). According to the reference genome sequence of A. terreus NIH2624, the genes from ATEG_09959 to ATEG_09969, which comprise the entire lovastatin biosynthetic gene cluster, are located in this multicopy region (Figure 2B). Therefore, the results of the whole genome resequencing analysis indicate that there are 3 to 5 copies of the lovastatin biosynthetic gene cluster in the chromosome of A. terreus HZ01, which is likely the main primary reason for the high productivity of lovastatin exhibited by this industrial strain.

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Figure 2. Analysis of the industrial strain A. terreus HZ01. (A) The sequencing depth of scaffold NT_16539.1. (B) Lovastatin gene cluster located in the region with high sequence depth (NT_165939.1: 28500-75500). All solid arrows represent the essential genes for the biosynthesis of lovastatin, while the dashed arrows represent irrelevant; red one, lovF, is the PKS responsible for the biosynthesis of the side chain of lovastatin; green one, lovE, is the cluster specific transcriptional factor. (C) Sensitivity analysis for three strains (HZ01, top; CIAT01, bottom left; ATCC20542, bottom right) for antifungal agents pyrithiamine, zeocin and hygromycin B. PDA: potato dextrose agar; AMM: Aspergillus minimal medium. Producing Monacolin J without Lovastatin Residues The PKS LovF responsible for the biosynthesis of the α-methylbutyryl side chain is the key enzyme in the synthesis of lovastatin from monacolin J. Therefore, theoretically, the disruption of the lovF gene can block the synthesis of lovastatin, thereby leading to the accumulation of the intermediate, monacolin J. However, the efficiency of genetargeting is very low in filamentous fungi. We also determined that the industrial strain A. terreus HZ01 is only sensitive to pyrithiamine but not to the antibiotics hygromycin B and zeocin (Figure 2C). Therefore, the pyrithiamine resistance gene ptrA may be the only selectable marker that can be used for the genetic engineering of A.terreus HZ01. Thus, completely knocking out the multiple copies of lovF is laborious and difficult. 7



To address these problems, an efficient marker-free gene-targeting system with high homologous recombination frequency and marker rescue capability was developed in A. terreus HZ01, as described previously.24 The ku80- and pyrG-deficient mutant A. terreus HZ03 (A. terreus HZ01, ∆ku80::ptrA, ∆pyrG) was constructed and used as the parental strain for the deletion of lovF in this study. The lovF gene was deleted completely with four rounds of gene-targeting and marker rescue, based on the modified Cre/loxP system (Figure S1), and A. terreus mutants with deletions of one, two, three, and all four copies of lovF were designed and labeled HZ-∆lovF1, HZ-∆lovF2, HZ-∆lovF3, and HZ-∆lovF4 respectively. This experiment indicates that there are likely four copies of the lovF genes in this industrial strain, as predicted by the abovementioned whole genome resequencing analysis. In comparison to the control strain, neither the ku80-deficient mutant A. terreus HZ02 nor the partial or complete lovF deletion mutants exhibited any significant difference in cell growth rate, conidiation or pigmentation on potato dextrose agar (PDA) plates. The lovastatin and monacolin J production of all the mutants was analyzed by shakeflask culture. There was no significant difference in the vegetative growth, mycelial morphology, pellet diameter, or culture color among the strains, indicating that the deletion of lovF in the industrial strain did not have significant adverse effects on the fermentation characteristics. Lovastatin production levels decreased with each successive lovF knock out mutation, with titers of 11.1 mM, 9.2 mM, 8.7 mM, 8.4 mM, and 0 mM for zero-four knockout mutations, respectively. Monacolin J production showed the opposite trend, increasing from 0.12 mM in the parental strain to 1.1 mM in the triple knock out strain, and 10.7 mM in the 8


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complete deletion mutant (Figure 3).These results indicated the complete deletion of lovF

can indeed block the synthesis of lovastatin and lead to the accumulation of similar amounts of monacolin J. As a result, the lovF-deficient mutant HZ-∆lovF4 is an efficient monacolin J-producing cell factory, which can directly produce monacolin J by a single-step fermentation without any lovastatin residues. This is a more practical and attractive improvement, as it not only intrinsically avoids the process of alkaline hydrolysis but also simplifies the downstream separation process.

Figure 3. Fermentation analysis of the various lovF deletion mutants of A. terreus

Improving the Production of Monacolin J by Overexpressing Regulatory Factors The LaeA protein of A. nidulans, which is a well-studied global regulator of fungal secondary metabolism, has been introduced into many kinds of fungi to improve the production of natural products.25-27 The overexpression of the laeA gene in A. terreus ATCC20542 led to a 4- to 7-fold increase in lovastatin in a previous study.25 LovE is a Zn finger (Zn2Cys6) transcription factor located in the lovastatin gene cluster, which is a specific activator for the biosynthesis pathway of lovastatin.2 The deletion of lovE can reduce lovastatin titer, whereas increasing the dosage of lovE increases the production of lovastatin in the low-yield strain A. terreus ATCC20542. Therefore, lovE is the key 9



transcriptional regulator involved in the biosynthesis of lovastatin. To further improve the production capacity of monacolin J, the fungal expression cassettes of laeA and lovE were constructed using the PgpdAt promoter, and each cassette was transformed into the monacolin J-producing strain HZ-∆lovF4-∆pyrG (Figure 4A). The transformants that integrated the expression cassettes in the ku80 loci were confirmed by genomic PCR, and the resulting mutants were designated HZ∆lovF4-laeA and HZ-∆lovF4-lovE. Two transformants for each mutant were tested for monacolin J production in shake flasks, and the monacolin J-producing strain HZ∆lovF4 was used as a control. Introducing laeA in A. terreus HZ-∆lovF4 did not improve the yield of monacolin J. All transformants of HZ-∆lovF4-laeA exhibited monacolin J production levels similar to that of the control strain, HZ-∆lovF4 (Figure 4B). These inconsistent results may be due to the different A. terreus hosts. LaeA may act as a global regulator by influencing chromatin structure, as it has sequence similarity to histone and arginine methyltransferases.28 A. terreus ATCC20542 is poor lovastatin producer, and the overexpression of laeA might have led to the reversal of the heterochromatic signature inside the cluster, thereby upregulating the entire lovastatin synthesis pathway. In contrast, the A. terreus HZ01 utilized in our case is an excellent industrial strain, and the native chromatin structure of A. terreus HZ01 is certainly beneficial to the production of lovastatin. Therefore, the production level of lovastatin caused by the overexpression of laeA was not enhanced significantly, as was the case for A. terreus ATCC20542. This finding also indicates that there are significant differences between 10


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the model strains used in the lab and the industrial strains used for application. In contrast, all of the transformants of HZ-∆lovF4-lovE produced significantly higher monacolin J titers compared with HZ-∆lovF4 (Figure 4B). The transformant HZ-∆lovF4-lovE produced 16.36 mM monacolin J in 10 days and was 52.5% higher compared with the control strain, HZ-∆lovF4 (10.73 mM). In addition, the relative transcriptions of native, overexpressed and total lovE in both strains were measured by qRT-PCR using enolase gene enoA as the control (Figure 4C). There was no significant difference in the transcription levels of native lovE between two strains HZ-∆lovF4 and HZ-∆lovF4-lovE at the same fermentation time. In HZ-∆lovF4-lovE, the transcription of overexpressed lovE was slightly increased from day 2 to day 4. Whereas, the transcription of native lovE increased by 7-8 folds from day 2 to day 4 in both strains. In total, the transcript levels of total lovE in HZ-∆lovF4-lovE was significantly higher than that in HZ-∆lovF4, 5.4 folds on day 2 and 2.2 folds on day 4. In summary, these results showed that the additional overexpression of lovE using PgpdAt promoter did significantly increase the transcript levels of lovE, and it is an effective strategy to increase the productivity of monacolin J. Different from other A. terreus strains with low yields, such a large increment achieved in the industrial strain is more significant. Besides, the more suitable and stronger promoter may be helpful to further achieve greater improvement.

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Figure 4 Construction and characterization of the A. terreus strains expressing the transcriptional factors. (A) The expression cassette of laeA or lovE was integrated in the ku80 loci of A. terreus HZ-∆lovF4-∆pyrGAn. (B) The fermentation process curves. The titer of monacolin J was determined by HPLC. (C) qRT-PCR analysis of the relative transcription of lovE in HZ-∆lovF4 and HZ-∆lovF4-lovE. Transcript levels were normalized against enoA by fold expression=2^(CTtarget-CTenoA). Total: total lovE; Native: native lovE; Overexpressed: Overexpressed lovE. Achieving Better Cell Robustness for Monacolin J Production with the A. terreus Mutant HZ-∆lovF4-lovE In industrial production, the titer of lovastatin in each batch usually varies greatly for unknown reasons and is difficult to avoid using process control. It is common that natural metabolic pathways generally utilize dynamic regulation networks to compensate for the changing conditions by altering fluxes. Secondary metabolite 12


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biosynthesis is sensitive to various environmental factors, such as carbon and nitrogen sources, pH, temperature, ventilation, and others. These external factors are transmitted to microbial cells through signal transduction, which affects biosynthetic pathways through global regulatory factors and specific transcriptional regulatory factors, and ultimately destabilizes target product yields. Therefore, genetic engineering of transcriptional regulatory systems by replacing natural genetic regulation with constitutive overexpression constructs may be an effective strategy to improve the fermentation robustness. As stated above, the A. terreus strains overexpressing transcriptional regulators laeA or lovE using PgpdAt promoter were constructed, and overexpression of lovE significantly improved the titer of monacolin J. To further assess the effect of overexpression of regulators on fermentation robustness, the production of monacolin J in the mutants HZ-∆lovF4, HZ-∆lovF4-laeA and HZ-∆lovF4-lovE on two different media was analyzed (Figure 5). The monacolin J titers of the mutants HZ-∆lovF4 and HZ-∆lovF4-laeA were significantly lower than that of HZ-∆lovF4-lovE in both media; this outcome was consistent with the abovementioned experimental results. More importantly, HZ-ΔlovF4-lovE produced similar amounts of monacolin J in two different media and was significantly different from the other two mutants. For both HZ-∆lovF4 and HZ-∆lovF4-laeA, the titers of monacolin J in medium B were approximately 40% lower compared with medium A, and the same phenomenon was observed with the lovastatin-producing strains.

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Figure 5 Comparison of monacolin J production between A. terreus HZ-∆lovF4-lovE, HZ-∆lovF4-laeA, and the parent strain HZ-∆lovF4 grown on two different media. These results, which were confirmed in multiple experiments, indicated that the overexpression of lovE using the PgpdAt promoter not only significantly increased the yield of monacolin J but also improved the fermentation robustness. And it could presumably arise from the strong constitutive promoter PgpdAt, which can lead to the stable and high expression of lovE with less environmental interference, and then LovE directly regulates the biosynthetic pathway of lovastatin and maintains it in a stable and efficient manner. The improvement of strain robustness is very important for industrial production, which can significantly reduce the production cost and the difficulty of process control. Although many strategies have been developed to improve the cellular robustness, it is still one of the major challenges for the progress of synthetic biology.29 Up to now, engineering efforts have focused on controlling natural stress response pathways to provide robustness.30-32 Accordingly, the engineering approaches adopted are mainly based on the stress related genetic devices. Even in the absence of these environmental stresses, small process deviations produce variable production titers. These are ubiquitous and unavoidable in industrial production. In this study, we found that 14


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engineering of the pathway specific regulator in an industrial strain successfully eliminated the effect on fermentation yield caused by small process deviations. Therefore, this case is also a good reference to improve the fermentation robustness, especially for other industrial strains producing secondary metabolites like lovastatin.

CONCLUSION In this study, a more efficient and robust cell factory for monacolin J production was constructed using the industrial lovastatin-producing strain A. terreus HZ01 as a chassis host. First, the presence of multiple copies of the lovastatin biosynthesis gene cluster was determined by whole genome resequencing, and then the multiple copies of the lovF gene were completely disrupted, resulting in a monacolin J-producing strain with high productivity but without any lovastatin residue. Second, the monacolin J biosynthetic pathway was further enhanced by the overexpression of the specific transcriptional regulator lovE, the productivity and fermentation robustness were both improved significantly. It also demonstrated that the engineering of transcriptional regulator is an effective strategy for the metabolic engineering of industrial fungi strains.

MATERIALS AND METHODS Strains and Media The fungal strains used or constructed in this study are listed in Table 1. The uracil auxotrophic A. terreus strains were grown on Aspergillus minimal medium (AMM, http://www.fgsc.net/methods/anidmed.html) supplemented with 0.5 g L-1 515



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fluoroorotic acid and 10 mM uracil to generate spores. Spores of other normal A. terreus strains were harvested from 7-day-old potato dextrose agar plates (PDA). The hyphae used for the protoplast transformation were grown in itaconic acid production medium (IPM: 40 g L-1 glucose, 2 g L-1 NH4NO3, 0.2 g L-1 (NH4)2HPO4, 0.4 g L-1 MgSO4, 0.02 g L-1 FeSO4, 0.04 g L-1 ZnSO4, 0.04 g L-1 CuSO4, 1 g L-1corn steep, pH 3.5) or IPM-FU medium (IPM supplemented with 0.5 g L-1 5-fluoroorotic acid and 10 mM uracil) in shake flasks. PDAS plates (PDA plates supplemented with 1.2 M D-sorbitol) and AMMS-FU plates (AMM plates supplemented with 0.5 g L-1 5-fluoroorotic acid, 10 mM uracil and 1.2 M D-sorbitol) were used for the selection of transformants. The fermentation analysis was performed in lovastatin production seed medium (LPSM: 9 g L-1 glucose, 10 g L-1 sucrose, 1 g L-1 yeast extract, 1 g L-1 peptone, 1 g L-1 sodium acetate, 0.04 g L-1 KH2PO4, 0.1 g L-1 MgSO4, 5 g L-1 soybean meal, and 1.5 g L-1 CaCO3, pH 6.8) for seed cultures and lovastatin production medium (LPM) for statin fermentation. LPM-A: 120 g L-1 glucose, 20 g L-1 sucrose, 1.5 g L-1 yeast extract (ANGEL YEAST CO.,LTD), 15 g L-1 peptone, 5 g L-1 soya peptone, 7 g L-1 sodium acetate, 0.05 g L-1 KH2PO4, 0.5 g L-1 MgSO4, 5 g L-1 soybean meal, and 5 g L-1 CaCO3, pH 6.2). LPM-B medium: 120 g L-1 glucose, 20 g L-1 sucrose, 1.5 g L-1 yeast extract (OXOID), 20 g L-1 peptone, 7 g L-1 sodium acetate, 0.05 g L-1 KH2PO4, 0.5 g L-1 MgSO4, 5 g L-1 soybean meal, and 5 g L-1 CaCO3, pH 7.2). Table 1 Fungal strains used in this study. A. terreus strains

Genotype or characteristics

Reference

HZ01

Wild type

Hisun

HZ02

HZ01, ∆ku80::ptrA

This study 16


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HZ03

HZ01, ∆ku80::ptrA, ∆pyrG; Uracil auxotrophic strain

This study

HZ-∆lovF1

HZ03, ∆lovF1:: pyrGAn-loxP; Deleted one lovF copy

This study

HZ-∆lovF1-∆pyrGAn

HZ-∆lovF1, ∆pyrGAn; Uracil auxotrophic strain

This study

HZ-∆lovF2

HZ03, ∆lovF2:: pyrGAn-loxP; Deleted two lovF copies

This study

HZ-∆lovF3

HZ03, ∆lovF3:: pyrGAn-loxP; Deleted three lovF copies

This study

HZ-∆lovF4

HZ03, ∆lovF4:: pyrGAn-loxP; Deleted all four lovF copies

This study

HZ-∆lovF4-∆pyrGAn

HZ-∆lovF4, ∆pyrGAn; Uracil auxotrophic strain

This study

HZ-∆lovF4-laeA

HZ-∆lovF4-∆pyrGAn, ∆ku80::PgpdAt-laeA-TtrpC-pyrGAn

This study

HZ-∆lovF4-lovE

HZ-∆lovF4-∆pyrGAn, ∆ku80::PgpdAt- lovE -TtrpC-pyrGAn

This study

Whole Genome Resequencing Genome resequencing was performed by Novagene (China) using the platform of Illumina HiSeq TM2000/MiSeq. On average, more than 50-fold coverage of the genome was achieved. BWA software was used for mapping the reads to the reference sequence, and SAMTOOLS software was used for counting the coverage of the reference sequence to the reads and interpreting the alignment results. Construction of A. terreus Chassis Cell A. terreus HZ01 is an industrial lovastatin-producing strain that was provided by Hisun Pharm. The ku80- /pyrG- double mutant HZ03, which is a chassis cell used for the following gene-targeting and marker rescue operations, was constructed as previously described.24 The cell growth and lovastatin productivity of HZ02 were assessed to evaluate the influences of the ku80 deletion. Deletion of the Multiple Copies of lovF The uracil auxotrophic A. terreus strain HZ03 was used as the parental strain to delete the multiple copies of lovF through gene-targeting. The selectable marker loxP-pyrGAn17



loxP was amplified from the plasmid pXH106 using the primers pyrGAn/loxP-F/ pyrGAn/loxP-R.

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For the first copy, the upstream and downstream homology arms

were amplified from the genomic DNA of HZ01 using the primer pairs U1-lovF-F/U1lovF-R and D1-lovF-F/D1-lovF-R, respectively, and then fused with loxP-pyrGAn-loxP by fusion PCR. The gene-targeting construct lovF-KO1 was amplified using the primers C1-lovF-F/C1-lovF-R and then transformed into A. terreus HZ03 using the protoplast-PEG method. The transformants HZ-∆lovF1, in which the first copy of lovF gene was disrupted, were selected on PDAS plates and purified by single spore isolation. The genotype was confirmed by genomic PCR using the primer pair U1-lovF-F/D1lovF-R. For the next round of deletion of lovF, it was necessary to perform the marker rescue in advance. The selectable marker loxP-pyrGAn-loxP of HZ-∆lovF1 was excised using the modified Cre/loxP recombination system as previously described,24, 33 resulting in the uracil auxotrophic A. terreus strain HZ-∆lovF1-∆pyrGAn. The gene-targeting DNA elements of the remaining copies were constructed as lovF-KO1 using the corresponding primers listed in table S1, and used for multiple rounds of lovF deletion. To knock out the lovF gene accurately for each round, the position of the homology arms was designed to the inside of the lovF gene gradually (Figure S1). Site-specific Integration of the Transcriptional Regulator Expression Cassettes The regulator genes laeA and lovE were amplified from the cDNA of A. nidulans and A. terreus HZ01, respectively. The DNA fragments Uku80-pyrGAn and TtrpC-Dku80 were amplified from the plasmid pXH106 using the primer pairs U-ku80-F/pyrGAn-R 18


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and TtrpC-F/D-ku80-R, respectively.24 The promoter PgpdAt was amplified from the plasmid pXH102 using the primer pair PgpdAt-F743/PgpdAt-R.34 Then, the target gene laeA or lovE was fused with three other fragments, Uku80-pyrGAn, PgpdAt and TtrpCDku80, by fusion PCR. The integration constructs were amplified from the products of the abovementioned fusion PCR using the primers C1-ku80-F/C1-ku80-R and then transferred into the uracil auxotrophic A. terreus strain HZ-∆lovF4-∆pyrGAn. The transformants that integrated the regulator expression cassettes at the ku80 loci were selected on PDAS and confirmed by PCR using the primers U-ku80-F/ D-ku80-R. Fermentation analysis of the engineered A. terreus strains Statin production was analyzed by shake flask fermentation. Approximately 2.5 × 107 spores of each strain were first inoculated in 35 mL of LPSM medium in 250-mL shake flasks for 48 h at 28 °C and 200 rpm, and 3.5 mL of seed culture was then transferred to 35 mL of LPM medium for 6 to 10 additional days of cultivation. To assess the fermentation robustness, the seed culture of strains HZ-∆lovF4, HZ-∆lovF4laeA and HZ-∆lovF4-lovE were transferred into two different lovastatin production media, LPM-A and LPM-B, respectively, and cultivated for 8 days. There are only small differences between LPM-A and LPM-B, including the source of the yeast extract, the nitrogen sources, and the pH. Three independent experiments were performed for each strain, and all assays were performed at least in duplicate. LC-MS Analysis of Statins One milliliter culture was added to a 10 mL methanol and incubated at room temperature for 2 h. The supernatant was filtered and analyzed by LC-MS for the 19



identification of lovastatin and monacolin J (Figure S2). LC separation was performed using a Thermo Accucore XL C18 column (2.1 mm × 100 mm, 5 μm) at a flow rate of 0.2 mL/min. The solvent gradient system for LC is 0-1 min 90% A, 1-26 min 90%-0% A, 26-32 min 0%-90% A (A:100% H2O with 0.1% formic acid, B: 100% ACN). HRESIMS spectra were obtained from a Dionex Ultimate 3000 system coupled Bruker Maxis Q-TOF spectrometer using positive mode electrospray ionization. HPLC Analysis of Fermentation Cultures The calibration curves of lovastatin and monacolin were developed by HPLC for the quantification (Figure S3). The extraction of 1 mL of cultures with 10 mL of alkaline methanol solution (0.1 M NaOH:methanol=1:10, v/v) was performed at room temperature for 2 h. The statins were separated on an Agilent ZORBAX SB-C18 column (4.6 ×150 mm, 5 μm) using a mixture of acetonitrile and 0.1% phosphoric acid (50:50, v/v) as the mobile phase at a flow rate of 1 mL min-1, and detected with a UV– vis detector at 237 nm. The sample injection volume was 10 μL. Quantitative real-time PCR (qRT-PCR) The fermentation cultures of HZ-∆lovF4 and HZ-∆lovF4-lovE were collected at day 2 and day 4. The mycelia were briefly washed with water and ground under liquid nitrogen. The total RNA was extracted from the obtained hypha using the TaKaRa MiniBEST Plant RNA Extraction Kit. The cDNA was synthesized using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Japan) according to the manufacturer’s protocol. qRT-PCR was carried out on a Light Cycler 480 instrument with software version 4.0 (Roche, Mannheim, Germany) using the 20


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SYBR®Premix Ex Taq™ (Perfect Real Time) (TaKaRa, Japan) following the manufacturer’s protocol and using 1:10 dilutions of cDNA samples serving as templates. The specific primers were designed respectively for the native, overexpressed and total lovE as shown in figure S4. The transcript levels of the lovE were normalized against the level of the enolase gene (enoA, ATEG_02902).35 The normalized transcript levels were calculated as fold expression = 2^( CTtarget - CTenoA). ASSOCIATED CONTENT Supporting Information. Figure S1. Schematic for the deletion of lovF Figure S2. HRESIMS spectra in positive mode of lovastatin (A) and monacolin J (B). Figure S3. Calibration curves of lovastatin (A) and monacolin J (B) developed by HPLC Figure S4. Primers for qRT-PCR analysis of the relative transcription of lovE Table S1. Primers used in this study AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] Author Contributions X.L. conceived the project and supervised the research; X.H. designed and performed the experiments; X.H., Y.Y., S.T, L.Z. Y.T., and J.Z. performed the fermentation experiments; S.T. and X.H. performed the qRT-PCR experiments; X.L., X.H., and Y.Y. analyzed all data and wrote the manuscript. 21



Nots The authors declare no conflict of interest. ACKNOWLEDGEMENTS This work was supported by the Science and Technology Service Network Initiative of Chinese Academy of Sciences (grant No. KFJ-STS-ZDTP-031), National Natural Sciences Foundation of China (grant No. 31400080), and Hisun Pharmaceutical Co., Ltd. in China.

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For Table of Contents Use Only

Construction of an efficient and robust Aspergillus terreus cell factory for monacolin J production Xuenian Huang†, Shen Tang†,⊥, Linghui Zheng§, Yun Teng§, Yong Yang§, Jinwei Zhu§ & Xuefeng Lu†, ‡, #,*

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Construction of an Efficient and Robust Aspergillus terreus Cell

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