Engineering the β-ketoacyl-acyl carrier protein synthase gene in the synthesis of cell wall mycolic acids in Mycobacterium neoaurum for enhancing the bioconversion of steroidal intermediates from phytosterols

Background: The bioconversion of phytosterols into high value-added steroidal intermediates is the cornerstone in steroid pharmaceutical industry. However, the low transportation efficiency of hydrophobic substrates into mycobacterial cells severely limits the transformation. In this study, a robust and stable modification of the cell wall strikingly enhanced the cell permeability for the high production of steroids. Results: The interference of the nonessential β-ketoacyl-acyl carrier protein synthase KasB resulted in a disturbed proportion and a shortened length of mycolic acids (MAs), thus leading to a remarkable improvement of cell permeability. The yield of 9 α -hydroxy-4-androstene-3,17-dione (9-OHAD) was increased by 137.7% in the vegetative cell transformation. Ultimately, the 9-OHAD productivity in an industrial used resting cell system was reached 0.1135 g/L/h (10.9 g/L 9-OHAD from 20 g/L phytosterol) and the conversion time was shortened by 33%. In addition, a similar self-enhancement effect (34.5%) was realized in the 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain. Conclusions : The modification of kasB resulted in a meaningful change in the cell wall mycolic acids. Deletion of the kasB gene remarkably improved the cell permeability, leading to a self-enhancement of the steroidal intermediate conversion. The results showed a high efficiency and feasibility of this construction strategy.

than 400 kinds of steroid drugs for a wide range of diseases are selling with an annual sale of 100 billion dollars [1]. Modifying the mycobacterial metabolic pathway for accumulating high value-added steroid intermediates is the most important step of the latest upgraded semi-synthetic route in steroidal pharmaceutical industry [2]. By the conversion of low value-added phytosterols, environment friendly extracts from the vegetable oil processing waste, sustainable pine tree bioresource and waste products in papermaking [3], C19 steroids (androst-4-ene-3,17-dione, AD; boldenone, BD; 9α-hydroxy-androst-4-ene-3,17-dione, 9-OHAD) [4,5] and C22 steroids (22-hydroxy-23,24-bisnorchol-4-ene-3-one, 4-HBC) [6] can be respectively accumulated. Then, almost all kinds of steroid drugs, including adrenocortical and progestational hormones, can be produced by the combinational chemical modifications [7]. For instance, 9-OHAD is a core intermediate and has been used as a cost-effective precursor to synthesize C21 adrenocortical hormone drugs [7]. However, the unsatisfying yield and productivity of the currently used strains has prompted researchers to intensively investigate more efficient and stable strategies for the biosynthesis of important steroidal intermediates [8,9].
Sterols can be catabolized as the sole carbon and energy source for maintaining the balance of basic physiological metabolism in mycobacteria [8]. The uptake of sterols in cells may be divided into two distinguished stages: (I) the mass transfer stage of sterol molecules and particles to cell surface and (II) the diffusion stage of sterols across the cell wall and membrane. Stage I is mainly depends on the direct contact with the substrates dispersed in the extracellular environment. Early studies on material transfers demonstrated that in the presence of hydroxypropyl-βcyclodextrin [10], the use of biocompatible water-immiscible organic phase [11] could largely improve the solubilization of sterol substrates in the transformation system. As a result, the cells contacted with the sterols more efficiently. The substrate transfer was enhanced and the conversion productivity was increased accordingly. In addition, the β-cyclodextrin possibly improved the permeability due to the alteration of mycobacterial cell wall structure [12]. Thus, with the addition of glycine and vancomycin, which were inhibitors to the synthesis of mycobacterial cell wall, the cell permeability displayed a marked improvement [13]. However, these strategies employing massive additives are seldom used in the industrial process because of the high costs and low effects. It is noteworthy that most of the aforementioned methods possibly lead to some defects of the cell wall. The mycobacteria cell wall contains extremely rich mycolic acids [14]. This component accounts for 40%-60% of the cell dry weight and are probably responsible for the crucial cell permeability characteristic [15,16]. Rational modifications of the mycolic acid biosynthesis pathway might be reasonable ways to alter the permeability performance of the steroidal conversion microbial cell factories.
Then, a long mero chain can be obtained by the repetitive reductive cycles due to the catalysis of multienzyme fatty acid synthase II complex (FAS-II). Additional elongation cycles are subsequently catalyzed by the two β-ketoacyl-ACP synthase KasA and KasB. After the mero-chain and α-chain are coupled together by the acyl-AMP ligase FadD32 and the polyketide synthase Pks13 and then deoxidized by the mycolate reductase CmrA, the mature mycolate (trehalose monomycolate, TMM) can be synthetized in the mycobacterial cytoplasm. Next, the TMM is transported to the cell periplasm and participates in the subsequent assembly of mycolic acid-related structures, including the polar TDM and mycolic acid methyl esters in the core mycolyl-arabinogalactan-peptidoglycan (MAMEs-AG-PG) complex of cell wall [16].
The FAS-I synthesis gene fas is required in M. smegmatis [ 18] and M. tuberculosis [19] and the fatty acid synthase II (FASII) enzymes InhA [20], MabA [21], HadB [22], and KasA [23] are also required. The inactivation of these indispensable genes could lead to the lysis of mycobacterial cells [20][21][22][23]. The disruption of nonessential genes possibly caused some stable defects only in the cell wall. Thus, the loss of the dispensable genes, such as hadA, hadC and kasB in the mero-mycolic acid synthesis pathway, are worth investigation in the model steroid transformation cells [15,17].
The biotransformation process is a rate-limiting step in the microbes producing steroid intermediates. It usually takes 120 to 144 h to realize a satisfactory conversion rate of the substrate to target steroid intermediates in the microbes [4,5,24]. However, it only takes about 48 to 72 h in most of other prokaryotic microorganisms [25][26][27]. The long conversion time is primarily attributed to the low permeability of sterol substrates into the cell wall [28]. Promoting the substrate to enter microbial cells by modifying the cell wall may shorten the time required by the bioconversion process and improve the integral production capacity of mycobacterial cells.
Increasing the sterol biotransformation efficiency in M. neoaurum through a systemic cell wall engineering technique was rarely reported [28]. The disruption of the genes involved in mycolic acid synthesis in mycobacterial cells was not directly assessed. In the study, the annotated nonessential mycolic acid synthetic genes were inactivated individually. The modification which significantly altered the sterol conversion was further investigated. The result revealed the roles of accessory genes in the formation of mycolic acids and provided an alternative evolution strategy for the microbial transformation of steroidal intermediates.

Strains, plasmids and primers
All strains used in this study are described below ( was constructed by Li-Qin Xu [6]. Others were all derived from the above three M. neoaurum strains. Common plasmids (Additional file 1: Table S1) and primers (Additional file 1: Table S2) were used for constructing the mutants.

Media and culture conditions
Media and culture conditions were the same as the previously described conditions [28,29]. E. coli DH5α was inoculated at 37 °C in 5 mL of Luria-Bertani (LB) medium.

Construction of genetically modified strains
Target gene-deleted strains were obtained through allelic homologous recombination in mycobacteria as previously described [31]. p2NIL and pGOAL19 were used for the construction of the homologous recombination plasmids (Additional file 1: Table S1). The knockout-plasmids p19-gene was transferred into mycobacterial cells via electroporation.
To complement the deficient-gene function, the complete gene sequence was firstly amplified from the wild type strain and then inserted into the pMV261 to create a recombinant pMV261-gene plasmid, which could be used to overexpress the carried gene in multiple copies. Moreover, the expression cassette of the target gene containing a heat shock promoter hsp60 was obtained from the recombinant p261gene through double-digestion and then integrated into the pMV306 to create a complemental plasmid p306-gene. The constructed plasmid p306-gene could be integrated into chromosomal DNA in single copy to complement the disrupted gene function.

Analysis of cell permeability and steroid uptake performance
The permeability change of cell envelope was estimated by measuring the fluorescence intensity of cells labeled by fluorescein diacetate (FDA, Aladdin Reagents (Shanghai) Co., Ltd., Shanghai, China) according to previous procedures with some minor amendments [32]. The cells were suspended in 4.
Briefly, 50 mg (in wet weight) of mycobacterial cells were collected at 12,000 g for The extracted mycolic acids were analyzed by silica gel TLC plates in a solvent system (chloroform: methanol, 90:10, v/v). The keto-MA spots on preparative silica gel TLC were purified for MALDI-TOF-MS (Xevo G2, Waters, Ltd., MA) analysis as described [15].

Sterol bioconversion and the extraction and analysis of steroidal intermediates
Both vegetative cells and resting cells were determined to assess the sterol conversion capability [28,30]. Firstly, the vegetative cell biotransformation medium

Disruption of the mycolic acid synthesis genes disturbed the sterol conversion
Mycolic acids, as the main cell wall constituent, are generally synthesized in the cytoplasm (Fig. 1a) [16,17]. The interference with the nonessential gene, such as the (3R)-hydroxyacyl-ACP dehydratase hadA and methyl mycolic acid synthase 1 mmaA1, etc., involved in the synthesis of mycolic acids might reduce the tightness of cell wall and lead to a stable change in cell permeability. For further studies, the genes involved in the synthesis of mycolic acids were preliminarily evaluated by the comparative transcriptome analysis between the wild type strain and its primary derivative 9-OHAD-producing strain (MnΔkstD1) [31]. The transcription of the annotated genes showed discrete variations, revealing that the mycolic acid synthesis might not be dramatically disturbed during the bioconversion of sterols (Fig. 1b, Additional file 1: Table S3).
Next, some dispensable genes were screened through the targeted deletion of the mycolic acid synthesis pathway. Interestingly, the inactivation of most of the accessary genes resulted in a slight alteration of sterol conversion rate in all the strains except the kasB-deleted strain (Fig. 1c). As expected, the deletion of the gene remarkably increased the sterol conversion by 143% at the 72-h sampling time. Early studies demonstrated that the kasB was a nonessential gene responsible for the extension to full-length mero-mycolic acids in M. tuberculosis. The loss of the gene caused the synthesis of shorter mycolates [15], indicating that a meaningful permeability change might occur in the mutant strain.  Table S4). The allelic homologous recombination was employed to delete the kasB cassette in the wild type M. neoaurum. A 1171-bp upstream sequence and 1111-bp downstream sequence were amplified to construct the plasmid vector for gene knockout (Additional file 2: Fig. S2). PCR and electrophoresis analysis results of the kasB region in genomic DNA confirmed the occurrence of allelic replacement in M. neoaurum (Fig. 2a).
In mycobacteria, kasA and kasB encode two distinct fatty acid synthase II complexes. KasA is responsible for the initial elongation of mycolic acids less than 40 carbons, whereas KasB is involved in the extension from 40 carbons to 54 carbons [17]. The MnΔkasB mutant strain and the complemented strain MnΔkasB+kasB were generated for subsequent experiments. The deletion of kasB led to an obvious alteration of cell growth in the presence of cholesterol and the MnΔkasB strain growth was much faster than that of its parental wild type strain and the complemented strain (Fig. 2b). Subsequently, the permeability of kasBdeficient strain was assessed through determining the fluorescence intensity of the cells after labeling with fluorescein diacetate (FDA). The MnΔkasB mutant strain had the more permeable cell wall. This wild type property could be restored in the mutant strain upon the introduction of the complete functional kasB gene (Fig. 2c).
To further confirm this, the analog of cholesterol, cholest-4-en-3-one was employed as a label to check for the cell permeability to steroids [28]. The analysis indicated that the improved the cell wall permeability indeed resulted in a significant enhancement in the uptake of cholest-4-en-3-one in the kasB-deficient strain (Fig.   2d). The improvement might be interpreted as a chain effect caused by the enhanced cell permeability. These results further confirmed that the observed enhancement of sterol conversion and utilization was probably attributed to the improved cell permeability through the inactivation of KasB function.

Deletion of kasB changed the composition of cell wall mycolic acids
Previous studies demonstrated that KasB was dispensable for normal mycobacterial growth in M. marinum and M. smegmatis [23,34]. The KasB in M. neoaurum was proved to play a similar role in mycobacterial growth. The mechanism for the alternation of cell permeability with respect to the kasB deficiency in M. neoaurum remains unclear. Notably, KasB is responsible for the extension of mero-mycolic acid carbon chain [15]. This function indicated that the increased permeability was likely attributed to the changed KasB-responsible cell wall mycolic acid synthesis in the mutant strain. All of these promoted us to investigate the changes in the mycolic acid composition and length of the kasB-deficient M. neoaurum.
In the TLC analysis results, the polar TMM and TDM showed no obvious difference, whereas the mycolic acid methyl esters (MAMEs) displayed a slight decrease in the kasB mutant strain ( Fig. 3a; Additional file 2: Fig. S3). The relative abundances of the α-MA, methoxy-MA and keto-MA were respectively 25.1%, 23.5%, and 51.4% in the kasB mutant strain and 23.5%, 22.6%, and 53.9% in its parental strain (Fig. 3b).
The decrease in keto-MA content was similar to the trend of the kasB-deleted M. tuberculosis [ 15]. Next, the keto-MA spot was purified and analyzed by MALDI-TOF MS. The spectrogram showed a changed mycolic acid length and the molecule weight of the most abundant keto-MA in ΔkasB strain was reduced by 42 (Fig. 3c).
The length of keto-MA in the kasB mutant was three carbon units shorter than that of the wild type [15]. Despite the inactivation of the kasB slightly changed the mycolate length, the cell wall permeability of the mutant strain was strikingly altered. As a result, the process that sterol substrates entered the cell was accelerated significantly.

Loss of kasB led to a remarkable improvement in steroid intermediate productivity
To determine the effect of altered MAMEs and permeability on the production of steroidal intermediates in the kasB mutant strain, the gene was deleted in the previously constructed 9-OHAD-producing strain MnΔkstD1ΔkstD2ΔkstD3 (WI). The growth speed of the mutant strain WIΔkasB was not decreased under the sterol-free culture conditions (Additional file 2: Fig. S4). In addition, the cell morphology of mutant strain was unaffected apparently (Fig. 4a). These results indicated that despite the deficiency of kasB, the stability of cellular structure could be still maintained in M. neoaurum. In view of the enhanced uptake of sterols resulted from the altered cell permeability, the accumulation capability of target steroids was preliminary analyzed. The vegetative cell transformation led to a remarkably increased 9-OHAD yield in the WIΔkasB strain compared to its parental strain (Fig.   4b). The deletion of kasB increased the target steroid by 137.7% from 0.61 g/L to 1.45 g/L after 72-h conversion. However, the increase precipitously declined to 28% at 96 h, revealing that the bioconversion maybe highly depended on the sterol supply (Additional file 2: Fig. S5).
Next, a resting cell bioconversion system widely applied in the industry was used to further assess the enhancement effect of C19 steroid intermediate 9-OHAD generated by the kasB deletion (Fig. 4c). The highest increase was detected in WIΔkasB strain after 72-h transformation with the production of 9.8 g/L, which was 48.5% higher than that of its parental WI strain (6.6 g/L). Ultimately, the kasBdefected strain yielded 10.9 g/L 9-OHAD with a molar yield of 69.5%, whereas its parental strain only produced 8.9 g/L with a molar yield of 56.7%. In addition, if the bioconversion time was extended by 48 h, the 9-OHAD production of WI strain would increase to about 10.3 g/L, which was still lower than that of the kasB mutant strain.
In other words, the modification of kasB gene shortened the conversion time by more than 33%. The screened kasB stably remodeled the cell wall mycolic acid component, thus resulting in an increase of 22.5% in the production of C19 steroidal 9-OHAD.
Similarly, an obvious improvement in the target intermediate was detected in the kasB-deficient vegetative cell (Additional file 2: Fig. S6), indicating that the strategy of disrupting the mycolic acid synthesis might be efficient for the stable evolution towards target steroidal producer. Accordingly, the assessment of resting cells showed that the 4-HBC production in the WIIIΔkasB strain was increased by 34.5% from 5.8 g/L to 7.8 g/L after 96-h conversion (Fig. 4d). In addition, the 4-HBC yield was improved by 37.5% from 6.4 g/L to 8.8 g/L after 120-h biotransformation [28].